Translational PK/PD and the first-in-human dose selection of a PD1/IL15: an engineered recombinant targeted cytokine for cancer immunotherapy.

Introduction: Interleukin 15 (IL-15) is a potential anticancer agent and numerous engineered IL-15 agonists are currently under clinical investigation. Selective targeting of IL-15 to specific lymphocytes may enhance therapeutic effects while helping to minimize toxicities.

Methods: We designed and built a heterodimeric targeted cytokine (TaCk) that consists of an anti-programmed cell death 1 receptor antibody (anti-PD-1) and an engineered IL-15.

IL-15/IL-15Rα-Fc-Fusion Protein XmAb24306 Potentiates Activity of CD3 Bispecific Antibodies through Enhancing T-Cell Expansion.

An insufficient quantity of functional T cells is a likely factor limiting the clinical activity of T-cell bispecific antibodies, especially in solid tumor indications. We hypothesized that XmAb24306 (efbalropendekin alfa), a lymphoproliferative interleukin (IL)-15/IL-15 receptor α (IL-15Rα) Fc-fusion protein, may potentiate the activity of T-cell dependent (TDB) antibodies. The activation of human peripheral T cells by cevostamab, an anti-FcRH5/CD3 TDB, or anti-HER2/CD3 TDB resulted in the upregulation of the IL-2/15Rβ (CD122) receptor subunit in nearly all CD8+ and majority of CD4+ T cells, suggesting that TDB treatment may sensitize T cells to IL-15.

Adapting Ferritin, a Naturally Occurring Protein Cage, to Modulate Intrinsic Agonism of OX40.

Ferritin is a multivalent, self-assembling protein scaffold found in most human cell types, in addition to being present in invertebrates, higher plants, fungi, and bacteria, that offers an attractive alternative to polymer-based drug delivery systems (DDS). In this study, the utility of the ferritin cage as a DDS was demonstrated within the context of T cell agonism for tumor killing. Members of the tumor necrosis factor receptor superfamily (TNFRSF) are attractive targets for the development of anticancer therapeutics.

Translatability of Stress for Predicting Deamidation and Oxidation Biotransformation on Biotherapeutics.

Biotransformation leading to single residue modifications (e.g., deamidation, oxidation) can contribute to decreased efficacy/potency, poor pharmacokinetics, and/or toxicity/immunogenicity for protein therapeutics.

Generation of a Porcine Antibody Fab Fragment Using Protein Engineering to Facilitate the Evaluation of Ocular Sustained Delivery Technology.

Treatment of age-related macular degeneration (AMD) with anti-vascular endothelial growth factor (VEGF) biologic agents has been shown to restore and maintain visual acuity for many patients afflicted with wet AMD. These agents are usually administered via intravitreal injection at a dosing interval of 4-8 weeks. Employment of long-acting delivery (LAD) technologies could improve the therapeutic outcome, ensure timely treatment, and reduce burden on patients, caregivers, and the health care system.

Engineering interferons and interleukins for cancer immunotherapy.

Cytokines are a class of potent immunoregulatory proteins that are secreted in response to various stimuli and act locally to regulate many aspects of human physiology and disease. Cytokines play important roles in cancer initiation, progression, and elimination, and thus, there is a long clinical history associated with the use of recombinant cytokines to treat cancer. However, the use of cytokines as therapeutics has been limited by cytokine pleiotropy, complex biology, poor drug-like properties, and severe dose-limiting toxicities.

Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II).

Background: The ability to site-specifically conjugate a protein to a payload of interest (e.g., a fluorophore, small molecule pharmacophore, oligonucleotide, or other protein) has found widespread application in basic research and drug development.

Direct Interfacial Y Oxidation in α by a Photoβ Subunit of Class Ia Ribonucleotide Reductase.

Proton-coupled electron transfer (PCET) is a fundamental mechanism important in a wide range of biological processes including the universal reaction catalysed by ribonucleotide reductases (RNRs) in making de novo, the building blocks required for DNA replication and repair. These enzymes catalyse the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs). In the class Ia RNRs, NDP reduction involves a tyrosyl radical mediated oxidation occurring over 35 Å across the interface of the two required subunits (β and α) involving multiple PCET steps and the conserved tyrosine triad [Y(β)-Y(α)-Y(α)].

Reconstitution of Formylglycine-generating Enzyme with Copper(II) for Aldehyde Tag Conversion.

To further our aim of synthesizing aldehyde-tagged proteins for research and biotechnology applications, we developed methods for recombinant production of aerobic formylglycine-generating enzyme (FGE) in good yield. We then optimized the FGE biocatalytic reaction conditions for conversion of cysteine to formylglycine in aldehyde tags on intact monoclonal antibodies. During the development of these conditions, we discovered that pretreating FGE with copper(II) is required for high turnover rates and yields.

Generating site-specifically modified proteins via a versatile and stable nucleophilic carbon ligation.

There is a need for facile chemistries that allow for chemo- and regioselectivity in bioconjugation reactions. To address this need, we are pioneering site-specific bioconjugation methods that use formylglycine as a bioorthogonal handle on a protein surface. Here we introduce aldehyde-specific bioconjugation chemistry, the trapped-Knoevenagel ligation.

Modulation of Y356 photooxidation in E. coli class Ia ribonucleotide reductase by Y731 across the α2:β2 interface.

Substrate turnover in class Ia ribonucleotide reductase (RNR) requires reversible radical transport across two subunits over 35 Å, which occurs by a multistep proton-coupled electron-transfer mechanism. Using a photooxidant-labeled β2 subunit of Escherichia coli class Ia RNR, we demonstrate photoinitiated oxidation of a tyrosine in an α2:β2 complex, which results in substrate turnover. Using site-directed mutations of the redox-active tyrosines at the subunit interface, Y356F(β) and Y731F(α), this oxidation is identified to be localized on Y356.

Photo-ribonucleotide reductase β2 by selective cysteine labeling with a radical phototrigger.

Photochemical radical initiation is a powerful tool for studying radical initiation and transport in biology. Ribonucleotide reductases (RNRs), which catalyze the conversion of nucleotides to deoxynucleotides in all organisms, are an exemplar of radical mediated transformations in biology. Class Ia RNRs are composed of two subunits: α2 and β2.

Deciphering radical transport in the large subunit of class I ribonucleotide reductase.

Incorporation of 2,3,6-trifluorotyrosine (F(3)Y) and a rhenium bipyridine ([Re]) photooxidant into a peptide corresponding to the C-terminus of the β protein (βC19) of Escherichia coli ribonucleotide reductase (RNR) allows for the temporal monitoring of radical transport into the α2 subunit of RNR. Injection of the photogenerated F(3)Y radical from the [Re]-F(3)Y-βC19 peptide into the surface accessible Y731 of the α2 subunit is only possible when the second Y730 is present. With the Y-Y established, radical transport occurs with a rate constant of 3 × 10(5) s(-1).

Dramatic thermal stability of virus-polymer conjugates in hydrophobic solvents.

We have developed a method for integrating the self-assembling tobacco mosaic virus capsid into hydrophobic solvents and hydrophobic polymers. The capsid was modified at tyrosine residues to display an array of linear poly(ethylene glycol) chains, allowing it to be transferred into chloroform. In a subsequent step, the capsids could be transferred to a variety of hydrophobic solvents, including benzyl alcohol, o-dichlorobenzene, and diglyme.

Dual-surface-modified bacteriophage MS2 as an ideal scaffold for a viral capsid-based drug delivery system.

With the development of covalent modification strategies for viral capsids comes the ability to convert them into modular carrier systems for drug molecules and imaging agents. With this overall goal in mind, we have used two orthogonal modification strategies to decorate the exterior surface of genome-free MS2 capsids with PEG chains, while installing 50-70 copies of a fluorescent dye inside as a drug cargo mimic. Despite the very high levels of modification, the capsids remained in the assembled state, as determined by TEM, size-exclusion chromatography, and dynamic light scattering analysis.