Patient-Centric Quality Standards.

To ensure the quality, safety and efficacy of medicinal products, it is necessary to develop and execute appropriate manufacturing process and product control strategies. Traditionally, product control strategies have focused on testing known quality attributes with limits derived from levels administered in preclinical and clinical studies with an associated statistical analysis to account for variability. However, not all quality attributes have impact to the patient and those with the potential to impact safety and efficacy may not be significant when dosed at patient-centric levels.

Understanding Quality Paradigm Shifts in the Evolving Pharmaceutical Landscape: Perspectives from the USP Quality Advisory Group.

Recent changes in the pharmaceutical industry have led to significant paradigm shifts in the pharmaceutical quality environment. Globalization of the pharmaceutical industry, increasingly rapid development of novel therapies, and adoption of new manufacturing techniques have presented numerous challenges for the established regulatory framework and quality environment and are impacting the approaches utilized to ensure the quality of pharmaceutical products. Regulators, industry, and standards-setting organizations have begun to recognize the need to rely more on integrated risk-based approaches and to create more nimble and flexible standards to complement these efforts.

Strategies for Setting Patient-Centric Commercial Specifications for Biotherapeutic Products.

Commercial specifications for a new biotherapeutic product are a critical component of the product's overall control strategy that ensures safety and efficacy. This paper describes strategies for setting commercial specifications as proposed by a consortium of industry development scientists. The specifications for some attributes are guided by compendia and regulatory guidance.

PDA Biosimilars Workshop Report (September 27-28, 2018)-Getting It Right the First Time for Biosimilar Marketing Applications.

This workshop report summarizes the presentations, the breakout session outcomes, and the speaker panel discussions from the PDA Biosimilars Workshop held September 27-28, 2018, in Washington, DC. This format was deliberately selected for the workshop with the expectation of delivering a post-workshop paper on current best practices and existing challenges for sponsors. The event, co-chaired by Dr.

Expectations for Phase-Appropriate Drug Substance and Drug Product Specifications for Early-Stage Protein Therapeutics.

Early-phase specifications are established to ensure that materials used in clinical studies have appropriate product quality, reducing the risk of harm to patients. Currently, guidance is available for specification setting practices at commercial phase. With very limited data and manufacturing experience available, it is not possible to fully align to these expectations at the start of clinical trials.

First FDA approval of dual anti-HER2 regimen: pertuzumab in combination with trastuzumab and docetaxel for HER2-positive metastatic breast cancer.

On June 8, 2012, the U.S. Food and Drug Administration (FDA) approved pertuzumab (Perjeta, Genentech) for use in combination with trastuzumab (Herceptin, Genentech) and docetaxel for the treatment of patients with HER2-positive metastatic breast cancer (MBC) who have not received prior anti-HER2 therapy or chemotherapy for metastatic disease.

Phospholipase C-mediated hydrolysis of PIP2 releases ERM proteins from lymphocyte membrane.

Mechanisms controlling the disassembly of ezrin/radixin/moesin (ERM) proteins, which link the cytoskeleton to the plasma membrane, are incompletely understood. In lymphocytes, chemokine (e.g.

Considerations for the development of therapeutic monoclonal antibodies.

An increasing number of Investigational New Drug (IND) applications for therapeutic monoclonal antibodies (mAbs) have been submitted to US FDA over the past several years. Monoclonal antibodies and related products are under development for a wide range of indications. In addition, the diversity of antibody-related products is increasing including IgG2/IgG4 subclasses and engineered Fc regions to enhance or reduce antibody effector functionality.

Intramolecular regulation of phospholipase C-gamma1 by its C-terminal Src homology 2 domain.

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) is a key enzyme that governs cellular functions such as gene transcription, secretion, proliferation, motility, and development. Here, we show that PLC-gamma1 is regulated via a novel autoinhibitory mechanism involving its carboxy-terminal Src homology (SH2C) domain. Mutation of the SH2C domain tyrosine binding site led to constitutive PLC-gamma1 activation.

A new tyrosine phosphorylation site in PLC gamma 1: the role of tyrosine 775 in immune receptor signaling.

Phospholipase Cgamma (PLCgamma) is a ubiquitous gatekeeper of calcium mobilization and diacylglycerol-mediated events induced by the activation of Ag and growth factor receptors. The activity of PLCgamma is regulated through its controlled membrane translocation and tyrosine (Y) phosphorylation. Four activation-induced tyrosine phosphorylation sites have been previously described (Y472, Y771, Y783, and Y1254), but their specific roles in Ag receptor-induced PLCgamma1 activation are not fully elucidated.

A dynamic constitutive and inducible binding of c-Cbl by PLCgamma1 SH3 and SH2 domains (negatively) regulates antigen receptor-induced PLCgamma1 activation in lymphocytes.

We investigated the structural requirements for c-Cbl-mediated inhibition of Ag receptor-induced PLCgamma1 activation. Analysis of site-specific c-Cbl mutants indicated that tyrosine phosphorylation of c-Cbl was required for down-regulation of the PLCgamma1/Ca2+ pathway. Coprecipitation experiments indicated that c-Cbl and PLCgamma1 constitutively interact through a PLCgamma1 SH3 domain-dependent mechanism and that c-Cbl and PLCgamma1 can inducibly interact through the SH2(C) domain of PLCgamma1.

70Z/3 Cbl induces PLC gamma 1 activation in T lymphocytes via an alternate Lat- and Slp-76-independent signaling mechanism.

The oncoprotein 70Z/3 Cbl signals in an autonomous fashion or through blockade of endogenous c-Cbl, a negative regulator of signaling. The mechanism of 70Z/3 Cbl-induced signaling was investigated by comparing the molecular requirements for 70Z/3 Cbl- and TCR-induced phospholipase C gamma 1 (PLC gamma 1) activation. 70Z/3 Cbl-induced PLC gamma 1 tyrosine phosphorylation required, in addition to the PLC gamma 1 N-terminal SH2 domain, the C-terminal SH2 and SH3 domains that were dispensable for TCR-induced phosphorylation.

Phosphorylation and O-linked glycosylation of Elf-1 leads to its translocation to the nucleus and binding to the promoter of the TCR zeta-chain.

Elf-1, a member of the E 26-specific transcription factor family with a predicted molecular mass of 68 kDa, is involved in the transcriptional regulation of several hematopoietic cell genes. We demonstrate that Elf-1 exists primarily as a 98-kDa form in the nucleus and as an 80-kDa form in the cytoplasm. Phosphorylation and O-linked glycosylation contribute to the increased posttranslational molecular mass of Elf-1.

Membrane raft-dependent regulation of phospholipase Cgamma-1 activation in T lymphocytes.

Numerous signaling molecules associate with lipid rafts, either constitutively or after engagement of surface receptors. One such molecule, phospholipase Cgamma-1 (PLCgamma1), translocates from the cytosol to lipid rafts during T-cell receptor (TCR) signaling. To investigate the role played by lipid rafts in the activation of this molecule in T cells, an influenza virus hemagglutinin A (HA)-tagged PLCgamma1 was ectopically expressed in Jurkat T cells and targeted to these microdomains by the addition of a dual-acylation signal.

Differential effects of Cbl and 70Z/3 Cbl on T cell receptor-induced phospholipase Cgamma-1 activity.

We demonstrate that the differential effects Cbl and oncogenic 70Z/3 Cbl have on Ca(2+)/Ras-sensitive NF-AT reporters is partially due to their opposing ability to regulate phospholipase Cgamma1 (PLCgamma1) activation as demonstrated by analysis of the activation of an NF-AT reporter construct and PLCgamma1-mediated inositol phospholipid (PI) hydrolysis. Cbl over-expression resulted in reduced T cell receptor-induced PI hydrolysis, in the absence of any effect on PLCgamma1 tyrosine phosphorylation. In contrast, expression of 70Z/3 Cbl led to an increase in basal and OKT3-induced PLCgamma1 phosphorylation and PI hydrolysis.

Functional independence and interdependence of the Src homology domains of phospholipase C-gamma1 in B-cell receptor signal transduction.

B-cell receptor (BCR)-induced activation of phospholipase C-gamma1 (PLCgamma1) and PLCgamma2 is crucial for B-cell function. While several signaling molecules have been implicated in PLCgamma activation, the mechanism coupling PLCgamma to the BCR remains undefined. The role of PLCgamma1 SH2 and SH3 domains at different steps of BCR-induced PLCgamma1 activation was examined by reconstitution in a PLCgamma-negative B-cell line.

Sequences surrounding the Src-homology 3 domain of phospholipase Cgamma-1 increase the domain's association with Cbl.

SH3 domains are protein modules that interact with proline-rich polypeptide fragments. Cbl is an adapter-like protein known to interact with several SH3 domains, including the PLCgamma1 SH3 domain and the Grb2 amino terminal SH3 domain. Here we explore whether sequences surrounding the PLCgamma1 SH3 domain core sequence (aa.

The amino-terminal Src homology 2 domain of phospholipase C gamma 1 is essential for TCR-induced tyrosine phosphorylation of phospholipase C gamma 1.

TCR engagement activates phospholipase C gamma 1 (PLC gamma 1) via a tyrosine phosphorylation-dependent mechanism. PLC gamma 1 contains a pair of Src homology 2 (SH2) domains whose function is that of promoting protein interactions by binding phosphorylated tyrosine and adjacent amino acids. The role of the PLC gamma 1 SH2 domains in PLC gamma 1 phosphorylation was explored by mutational analysis of an epitope-tagged protein transiently expressed in Jurkat T cells.

Elf-1 regulates basal expression from the T cell antigen receptor zeta-chain gene promoter.

In mature T cells, limited synthesis of the TCR-zeta subunit is primarily responsible for regulating surface expression of TCRs. Transcription of zeta is directed by a complex promoter that includes two potential binding sites for the Ets family of transcription factors at -52 (zEBS1) and -135 (zEBS2). Mutation of these two sites results in a marked reduction of transcription from this promoter.

Cbl-mediated regulation of T cell receptor-induced AP1 activation. Implications for activation via the Ras signaling pathway.

The functional role of Cbl in regulating T cell receptor (TCR)-mediated signal transduction pathways is unknown. This study uses Cbl overexpression in conjunction with a Ras-sensitive AP1 reporter construct to examine its role in regulating TCR-mediated activation of the Ras pathway. Cbl overexpression in Jurkat T cells inhibited AP1 activity after TCR ligation.

HIV Tat represses transcription through Sp1-like elements in the basal promoter.

MHC class I genes are potently repressed by HIV Tat, which transactivates the HIV LTR. Tat represses class I transcription by binding to complexes associated with a novel promoter element, consisting of Sp1-like DNA binding sites. Transcription by other Sp1-dependent promoters, such as MDR1 and the minimal SV40 promoters, is also repressed by Tat, whereas the human beta-actin promoter is neither activated by Sp1 nor repressed by Tat.

Transcriptional regulation of the T cell antigen receptor zeta subunit: identification of a tissue-restricted promoter.

The cell surface expression of T cell antigen receptors (TCR) is regulated in part by the limiting synthesis of the zeta subunit. Utilizing fragments from the 5' region of the human zeta gene, two discrete regions that promote transcription were characterized. Both of these elements are located within 125 bases of the most 3' site of transcription initiation.

T cell antigen receptor-eta subunit. Low levels of expression and limited cross-species conservation.

The eta-subunit of the TCR is derived from an alternative splice product of the TCR-zeta gene. The eta-subunit has been extensively characterized in murine T cells, where up to 10% of TCR bear zeta-eta heterodimers rather than zeta-homodimers. In contrast to the significant levels of eta found in murine T cells, in human PBL an eta-like region is expressed spliced to upstream exons of the zeta-gene at no more than 0.