Role of receptor occupancy assays by flow cytometry in drug development.

The measurement of the binding of a biotherapeutic to its cellular target, receptor occupancy (RO), is increasingly important in development of biologically-based therapeutic agents. Receptor occupancy (RO) assays by flow cytometry describe the qualitative and/or quantitative assessment of the binding of a therapeutic agent to its cell surface target. Such RO assays can be as simple as measuring the number of cell surface receptors bound by an antireceptor therapeutic agent or can be designed to address more complicated scenarios such as internalization or shedding events once a receptor engages the administered therapeutic agent.

Recommendations for the development and validation of flow cytometry-based receptor occupancy assays.

Receptor occupancy measurements demonstrate the binding of a biotherapeutic agent to its extra-cellular target and represent an integral component of the pharmacodynamic (PD) portfolio utilized to advance the development and commercialization of a therapeutic agent. Coupled with traditional pharmacokinetic (PK) assessments derived from serum drug concentration, receptor occupancy data can be used to model PK/PD relationships and validate dose selection decisions throughout the drug development lifecycle. Receptor occupancy assays can be even more challenging to develop than other flow cytometric methods (e.

Out-of-Sequence Signal 3 Paralyzes Primary CD4(+) T-Cell-Dependent Immunity.

Primary T cell activation involves the integration of three distinct signals delivered in sequence: (1) antigen recognition, (2) costimulation, and (3) cytokine-mediated differentiation and expansion. Strong immunostimulatory events such as immunotherapy or infection induce profound cytokine release causing "bystander" T cell activation, thereby increasing the potential for autoreactivity and need for control. We show that during strong stimulation, a profound suppression of primary CD4(+) T-cell-mediated immune responses ensued and was observed across preclinical models and patients undergoing high-dose interleukin-2 (IL-2) therapy.

Aging predisposes to acute inflammatory induced pathology after tumor immunotherapy.

Cancer commonly occurs in the elderly and immunotherapy (IT) is being increasingly applied to this population. However, the majority of preclinical mouse tumor models assessing potential efficacy and toxicities of therapeutics use young mice. We assessed the impact of age on responses to systemic immune stimulation.

Influenza infection results in local expansion of memory CD8(+) T cells with antigen non-specific phenotype and function.

Primary viral infections induce activation of CD8(+) T cells responsible for effective resistance. We sought to characterize the nature of the CD8(+) T cell expansion observed after primary viral infection with influenza. Infection of naive mice with different strains of influenza resulted in the rapid expansion of memory CD8(+) T cells exhibiting a unique bystander phenotype with significant up-regulation of natural killer group 2D (NKG2D), but not CD25, on the CD44(high) CD8(+) T cells, suggesting an antigen non-specific phenotype.

Mechanical disruption of tumors by iron particles and magnetic field application results in increased anti-tumor immune responses.

The primary tumor represents a potential source of antigens for priming immune responses for disseminated disease. Current means of debulking tumors involves the use of cytoreductive conditioning that impairs immune cells or removal by surgery. We hypothesized that activation of the immune system could occur through the localized release of tumor antigens and induction of tumor death due to physical disruption of tumor architecture and destruction of the primary tumor in situ.

Delineation of antigen-specific and antigen-nonspecific CD8(+) memory T-cell responses after cytokine-based cancer immunotherapy.

Memory T cells exhibit tremendous antigen specificity within the immune system and accumulate with age. Our studies reveal an antigen-independent expansion of memory, but not naive, CD8(+) T cells after several immunotherapeutic regimens for cancer resulting in a distinctive phenotype. Signaling through T-cell receptors (TCRs) or CD3 in both mouse and human memory CD8(+) T cells markedly up-regulated programmed death-1 (PD-1) and CD25 (IL-2 receptor α chain), and led to antigen-specific tumor cell killing.

Regulatory and conventional CD4+ T cells show differential effects correlating with PD-1 and B7-H1 expression after immunotherapy.

Recently, our laboratory reported that secondary CD8+ T cell-mediated antitumor responses were impaired following successful initial antitumor responses using various immunotherapeutic approaches. Although immunotherapy stimulated significant increases in CD8+ T cell numbers, the number of CD4+ T cells remained unchanged. The current investigation revealed a marked differential expansion of CD4+ T cell subsets.

The synthetic triterpenoid, CDDO, suppresses alloreactive T cell responses and reduces murine early acute graft-versus-host disease mortality.

Acute graft-versus-host disease (aGVHD) still remains one of the life-threatening complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Immunomodulation of alloreactive donor T cell responses, as well as cytokine secretion is a potential therapeutic approach for the prevention of aGVHD. The synthetic triterpenoid, CDDO (2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid), exhibits potent antitumor activity and has also been shown to mediate anti-inflammatory and immunomodulatory effects.

Differential effects of proteasome inhibition by bortezomib on murine acute graft-versus-host disease (GVHD): delayed administration of bortezomib results in increased GVHD-dependent gastrointestinal toxicity.

We have recently demonstrated that the proteasome inhibitor, bortezomib, administered immediately following murine allogeneic bone marrow transplantation (BMT) resulted in marked inhibition of acute graft-versus-host disease (GVHD) with retention of graft-versus-tumor effects. We now assessed the effects of delayed bortezomib administration (5 or more days after BMT) on GVHD. Recipient C57BL/6 (H2b) mice were lethally irradiated and given transplants of bone marrow cells and splenocytes from major histocompatibility complex (MHC)-disparate BALB/c (H2d) donors.