The conjugation of biomolecules by chemoselective oxime ligation is of great interest for the site-specific modification of proteins, peptides, nucleic acids, and carbohydrates. These conjugations proceed optimally at a reaction pH of 4-5, but some biomolecules are not soluble or stable under these conditions. Aniline can be used as a nucleophilic catalyst to enhance the rate of oxime formation, but even in its presence, the reaction rate at neutral pH can be slower than desired, particularly at low reagent concentrations and/or temperature. Recently, alternative catalysts with improved properties were reported, including anthranilic acid derivatives for small molecule ligations, as well as m-phenylenediamine at high concentrations for protein conjugations. Here, we report that p-substituted anilines containing an electron-donating ring substituent are superior catalysts of oxime-based conjugations at pH 7. One such catalyst, p-phenylenediamine, was studied in greater detail. This catalyst was highly effective at neutral pH, even at the low concentration of 2 mM. In a model oxime ligation using aminooxy-functionalized PEG, catalysis at pH 7 resulted in a 120-fold faster rate of protein PEGylation as compared to an uncatalyzed reaction, and 19-fold faster than the equivalent aniline-catalyzed reaction. p-Phenylenediamine (10 mM) was also an effective catalyst under acidic conditions and was more efficient than aniline throughout the pH range 4-7. This catalyst allows efficient oxime bioconjugations to proceed under mild conditions and low micromolar concentrations, as demonstrated by the PEGylation of a small protein.

Activation of TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) can induce apoptosis in a variety of human cancer cell lines and xenografts, while lacking toxicity in normal cells. The natural ligand and agonistic antibodies show antitumor activity in preclinical models of cancer, and this had led to significant excitement in the clinical potential of these agents. Unfortunately, this optimism has been tempered by trial data that, thus far, are not showing clear signs of efficacy in cancer patients. The reasons for discrepant preclinical and clinical observations are not understood, but one possibility is that the current TRAILR2 agonists lack sufficient potency to achieve a meaningful response in patients. Toward addressing that possibility, we have developed multivalent forms of a new binding scaffold (Tn3) that are superagonists of TRAILR2 and can induce apoptosis in tumor cell lines at subpicomolar concentrations. The monomer Tn3 unit was a fibronectin type III domain engineered for high-affinity TRAILR2 binding. Multivalent presentation of this basic unit induced cell death in TRAILR2-expressing cell lines. Optimization of binding affinity, molecular format, and valency contributed to cumulative enhancements of agonistic activity. An optimized multivalent agonist consisting of 8 tandem Tn3 repeats was highly potent in triggering cell death in TRAIL-sensitive cell lines and was 1 to 2 orders of magnitude more potent than TRAIL. Enhanced potency was also observed in vivo in a tumor xenograft setting. The TRAILR2 superagonists described here have the potential for superior clinical activity in settings insensitive to the current therapeutic agonists that target this pathway.

Humans and higher primates are unique in that they lack uricase, the enzyme capable of oxidizing uric acid. As a consequence of this enzyme deficiency, humans have high serum uric acid levels. In some people, uric acid levels rise above the solubility limit resulting in crystallization in joints, acute inflammation in response to those crystals causes severe pain; a condition known as gout. Treatment for severe gout includes injection of non-human uricase to reduce serum uric acid levels. Krystexxa® is a hyper-PEGylated pig-baboon chimeric uricase indicated for chronic refractory gout that induces an immunogenic response in 91% of treated patients, including infusion reactions (26%) and anaphylaxis (6.5%). These properties limit its use and effectiveness. An innovative approach has been used to develop a therapeutic uricase with improved properties such as: soluble expression, neutral pH solubility, high E. coli expression level, thermal stability, and excellent activity. More than 200 diverse uricase sequences were aligned to guide protein engineering and reduce putative sequence liabilities. A single uricase lead candidate was identified, which showed low potential for immunogenicity in >200 human donor samples selected to represent diverse HLA haplotypes. Cysteines were engineered into the lead sequence for site specific PEGylation and studies demonstrated >95% PEGylation efficiency. PEGylated uricase retains enzymatic activity in vitro at neutral pH, in human serum and in vivo (rats and canines) and has an extended half-life. In canines, an 85% reduction in serum uric acid levels was observed with a single subcutaneous injection. This PEGylated, non-immunogenic uricase has the potential to provide meaningful benefits to patients with gout.

4-1BBL is a member of the tumor necrosis factor (TNF) superfamily and is the ligand for the TNFR superfamily receptor, 4-1BB. 4-1BB plays an immunomodulatory role in T cells and NK cells, and agonists of this receptor have garnered strong attention as potential immunotherapy agents. Broadly speaking, the structural features of TNF superfamily members, their receptors, and ligand-receptor complexes are similar. However, a published crystal structure of human 4-1BBL suggests that it may be unique in this regard, exhibiting a three-bladed propeller-like trimer assembly that is distinctly different from that observed in other family members. This unusual structure also suggests that the human 4-1BB/4-1BBL complex may be structurally unique within the TNF/TNFR superfamily, but to date no structural data have been reported. Here we report the crystal structure of the human 4-1BB/4-1BBL complex at 2.4-Å resolution. In this structure, 4-1BBL does not adopt the unusual trimer assembly previously reported, but instead forms a canonical bell-shaped trimer typical of other TNF superfamily members. The structure of 4-1BB is also largely canonical as is the 4-1BB/4-1BBL complex. Mutational data support the 4-1BBL structure reported here as being biologically relevant, suggesting that the previously reported structure is not. Together, the data presented here offer insight into structure/function relationships in the 4-1BB/4-1BBL system and improve our structural understanding of the TNF/TNFR superfamily more broadly.

Tn3 proteins are a novel class of binding molecules based on the third fibronectin type III domain of human tenascin C. Target-specific Tn3 proteins are selected from combinatorial libraries in which three surface-exposed loops have been diversified. Here, the cocrystallization of two different Tn3 proteins in complex with CD40L, a therapeutic target for immunological disease, is reported. These crystal structures are the first to be reported of Tn3 proteins and will help to reveal how these engineered molecules achieve specific recognition of a cognate target.