Selenocysteine is cotranslationally inserted into proteins by recoding the stop codon UGA from termination to selenocysteine insertion. The nucleophilic selenol group of selenocysteine endows this rare amino acid with unique chemical reactivity that allows regiospecific covalent conjugation in the presence of the other natural amino acids. Using a mammalian expression system, we generated an IgG1-derived Fc fragment with a C-terminal selenocysteine in yields comparable to conventional monoclonal antibodies and conjugated it to an electrophilic derivative of a peptidomimetic that binds with high affinity and specificity to integrin ␣4␤1. Through this conjugation, both the biological and chemical components are endowed with pharmacological advantages. We demonstrate that whereas the Fc protein increases the circulatory half-life from minutes to days and mediates transcytosis through binding to the neonatal Fc receptor, the peptidomimetic introduces cross-species binding to cell surface integrin ␣4␤1 and blocks its interaction with vascular cell adhesion molecule-1. Compared with conventional monoclonal antibodies, our technology benefits economically from combining a generic biological component with a variable chemical component.antibody engineering ͉ Fc fragment ͉ neonatal Fc receptor ͉ small synthetic molecules ͉ integrin ␣4␤1

Maintenance of naive CD8 T cells is necessary for lifelong immunocompetence but for unknown reasons requires both interleukin-7 (IL-7) and T cell receptor (TCR) signaling. We now report that naive CD8 T cells require IL-7 signaling to be intermittent, not continuous, because prolonged IL-7 signaling induces naive CD8 T cells to proliferate, produce interferon-γ (IFN-γ), and undergo IFN-γ-triggered cell death. Homeostatic TCR engagements interrupt IL-7 signaling and thereby support CD8 T cell survival and quiescence. However, CD8 T cells with insufficient TCR affinity for self-ligands receive prolonged IL-7 signaling and die during homeostasis. This study identifies the regulation of IL-7 signaling duration by homeostatic TCR engagements as the basis for in vivo CD8 T cell homeostasis.

In humans, the role and relationship between molecular pathways that lead to tissue destruction during acute allograft rejection are not fully understood. Based on studies conducted in humans, we recently hypothesized that different immune-mediated tissue destruction processes (i.e. cancer, infection, autoimmunity) share common convergent final mechanisms. We called this phenomenon the "Immunologic Constant of Rejection (ICR)." The elements of the ICR include molecular pathways that are consistently described through different immune-mediated tissue destruction processes and demonstrate the activation of interferon-stimulated genes (ISGs), the recruitment of cytotoxic immune cells (primarily through CXCR3/CCR5 ligand pathways), and the activation of immune effector function genes (IEF genes; granzymes A/B, perforin, etc.).Here, we challenge the ICR hypothesis by using a meta-analytical approach and systematically reviewing microarray studies evaluating gene expression on tissue biopsies during acute allograft rejection. We found the pillars of the ICR consistently present among the studies reviewed, despite implicit heterogeneity.Additionally, we provide a descriptive mechanistic overview of acute allograft rejection by describing those molecular pathways most frequently encountered and thereby thought to be most significant. The biological role of the following molecular pathways is described: IFN-γ, CXCR3/CCR5 ligand, IEF genes, TNF-α, IL-10, IRF-1/STAT-1, and complement pathways. The role of NK cell, B cell and T-regulatory cell signatures are also addressed.

Antibody-drug conjugates (ADC) are an emerging drug class that uses antibodies to improve cytotoxic drug targeting for cancer treatment. ADCs in current clinical trials achieve a compromise between potency and physicochemical/pharmacokinetic properties by conjugating potent cytotoxins directly to an antibody at a 4:1 or less stoichiometric ratio. Herein, we report a novel, polyacetal polymer-based platform for creating ADC that use poly-1-hydroxymethylethylene hydroxymethylformal (PHF), also known as Fleximer. The high hydrophilicity and polyvalency properties of the Fleximer polymer can be used to produce ADC with high drug loading without compromising physicochemical and pharmacokinetic properties. Using trastuzumab and a vinca drug derivative to demonstrate the utility of this platform, a novel Fleximer-based ADC was prepared and characterized in vivo. The ADC prepared had a vinca-antibody ratio of 20:1. It exhibited a high antigen-binding affinity, an excellent pharmacokinetic profile and antigen-dependent efficacy, and tumor accumulation in multiple tumor xenograft models. Our findings illustrate the robust utility of the Fleximer platform as a highly differentiated alternative to the conjugation platforms used to create ADC currently in clinical development. Cancer Res; 75(16); 3365-72. Ó2015 AACR.

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After significant effort over the last 30 years, antibody-drug conjugates (ADCs) have recently gained momentum as a therapeutic modality, and nine ADCs have been approved by the FDA to date, with additional ADCs in late stages of development. Here we introduce Dolaflexin, a novel ADC technology that overcomes key limitations of the most common ADC platforms with two key features: a higher drug-antibody ratio and a novel auristatin with a controlled bystander effect. The novel, cell permeable payload auristatin F-hydroxypropylamide undergoes metabolic conversion to the highly potent but less cell permeable auristatin F to balance the bystander effect through drug trapping within target cells. We conducted studies in mice, rats, and cynomolgus monkeys to complement in vitro characterization and contrasted the performance of Dolaflexin with regard to anti-tumor activity, pharmacokinetic properties, and safety in comparison to the ADC platform utilized in the approved ADC ado-trastuzumab emtansine. A HER2-targeted Dolaflexin ADC was shown to have a much lower threshold of antigen expression for potent cell-killing in vitro, was effective in vivo in tumors with low HER2 expression, and induced tumor regressions in a xenograft model that is resistant to adotrastuzumab emtansine.

Collectively, these results demonstrate that TNFR2-bearing CD8(+) T cells and TNFR1-bearing non-CD8(+) T cells contribute significantly to oviduct pathology following genital chlamydial infection.

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