Structure, Function, and Therapeutic Use of IgM Antibodies.

Natural immunoglobulin M (IgM) antibodies are pentameric or hexameric macro-immunoglobulins and have been highly conserved during evolution. IgMs are initially expressed during B cell ontogeny and are the first antibodies secreted following exposure to foreign antigens. The IgM multimer has either 10 (pentamer) or 12 (hexamer) antigen binding domains consisting of paired µ heavy chains with four constant domains, each with a single variable domain, paired with a corresponding light chain. Although the antigen binding affinities of natural IgM antibodies are typically lower than IgG, their polyvalency allows for high avidity binding and efficient engagement of complement to induce complement-dependent cell lysis. The high avidity of IgM antibodies renders them particularly efficient at binding antigens present at low levels, and non-protein antigens, for example, carbohydrates or lipids present on microbial surfaces. Pentameric IgM antibodies also contain a joining (J) chain that stabilizes the pentameric structure and enables binding to several receptors. One such receptor, the polymeric immunoglobulin receptor (pIgR), is responsible for transcytosis from the vasculature to the mucosal surfaces of the lung and gastrointestinal tract. Several naturally occurring IgM antibodies have been explored as therapeutics in clinical trials, and a new class of molecules, engineered IgM antibodies with enhanced binding and/or additional functional properties are being evaluated in humans. Here, we review the considerable progress that has been made regarding the understanding of biology, structure, function, manufacturing, and therapeutic potential of IgM antibodies since their discovery more than 80 years ago.

RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates.

Amber codon suppression for the insertion of non-natural amino acids (nnAAs) is limited by competition with release factor 1 (RF1). Here we describe the genome engineering of a RF1 mutant strain that enhances suppression efficiency during cell-free protein synthesis, without significantly impacting cell growth during biomass production. Specifically, an out membrane protease (OmpT) cleavage site was engineered into the switch loop of RF1, which enables its conditional inactivation during cell lysis. This facilitates extract production without additional processing steps, resulting in a scaleable extract production process. The RF1 mutant extract allows nnAA incorporation at previously intractable sites of an IgG1 and at multiple sites in the same polypeptide chain. Conjugation of cytotoxic agents to these nnAAs, yields homogeneous antibody drug conjugates (ADCs) that can be optimized for conjugation site, drug to antibody ratio (DAR) and linker-warheads designed for efficient tumor killing. This platform provides the means to generate therapeutic ADCs inaccessible by other methods that are efficient in their cytotoxin delivery to tumor with reduced dose-limiting toxicities and thus have the potential for better clinical impact.

DNA binding hairpin polyamides with antifungal activity.

Eight-ring hairpin polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids have been shown to bind with subnanomolar affinity to discrete DNA sites and to modulate a variety of DNA-dependent biological processes. We show here that addition of a second positive charge at the C terminus of an 8-ring hairpin polyamide confers activity against a number of clinically relevant fungal strains in vitro, and activity against Candida albicans in a mouse model. Control experiments indicate that the observed antifungal activity results from a DNA binding mechanism-of-action that does not involve DNA damage or disruption of chromosomal integrity. Hairpin activity is shown to be proportional to yeast DNA content (ploidy). Transcriptional interference is proposed as the likely explanation for fungal cytotoxicity. Experiments with sensitized yeast strains indicate the potential for discrete sites of action rather than global effects.

Engineering toward a bacterial "endoplasmic reticulum" for the rapid expression of immunoglobulin proteins.

Antibodies are well-established as therapeutics, and the preclinical and clinical pipeline of these important biologics is growing rapidly. Consequently, there is considerable interest in technologies to engineer and manufacture them. Mammalian cell culture is commonly used for production because eukaryotic expression systems have evolved complex and efficient chaperone systems for the folding of antibodies. However, given the ease and manipulability of bacteria, antibody discovery efforts often employ bacterial expression systems despite their limitations in generating high titers of functional antibody. Open-Cell Free Synthesis (OCFS) is a coupled transcription-translation system that has the advantages of prokaryotic systems while achieving high titers of antibody expression. Due to the open nature of OCFS, it is easily modified by chemical or protein additives to improve the folding of select proteins. As such, we undertook a protein additive screen to identify chaperone proteins that improve the folding and assembly of trastuzumab in OCFS. From the screen, we identified the disulfide isomerase DsbC and the prolyl isomerase FkpA as important positive effectors of IgG folding. These periplasmic chaperones function synergistically for the folding and assembly of IgG, and, when present in sufficient quantities, gram per liter IgG titers can be produced. This technological advancement allows the rapid development and manufacturing of immunoglobulin proteins and pushes OCFS to the forefront of production technologies for biologics.

In vitro Fab display: a cell-free system for IgG discovery.

Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF.

Cell-free translation of peptides and proteins: from high throughput screening to clinical production.

In the past decade, in vitro transcription/translation technologies have emerged as discovery tools for screening large protein expression libraries, for the selection of engineered polypeptide libraries, and as alternatives to conventional heterologous expression for protein production. Therapeutic proteins and peptides discovered using ribosome-based display methods that link genetic information to the encoded polypeptide generated by cell-free extracts, or purified translation components, are beginning to move forward into human clinical trials. This review details the significant progress in in vitro translation for novel protein and non-natural amino acid containing peptide discovery platforms, as well as advances in the clinical-scale production of therapeutic proteins using cell-free transcription/translation.

Cardiac myosin activation: a potential therapeutic approach for systolic heart failure.

Decreased cardiac contractility is a central feature of systolic heart failure. Existing drugs increase cardiac contractility indirectly through signaling cascades but are limited by their mechanism-related adverse effects. To avoid these limitations, we previously developed omecamtiv mecarbil, a small-molecule, direct activator of cardiac myosin. Here, we show that it binds to the myosin catalytic domain and operates by an allosteric mechanism to increase the transition rate of myosin into the strongly actin-bound force-generating state. Paradoxically, it inhibits adenosine 5'-triphosphate turnover in the absence of actin, which suggests that it stabilizes an actin-bound conformation of myosin. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure.

Discovery of the First Potent and Selective Inhibitor of Centromere-Associated Protein E: GSK923295.

Inhibition of mitotic kinesins represents a novel approach for the discovery of a new generation of anti-mitotic cancer chemotherapeutics. We report here the discovery of the first potent and selective inhibitor of centromere-associated protein E (CENP-E) 3-chloro-N-{(1S)-2-[(N,N-dimethylglycyl)amino]-1-[(4-{8-[(1S)-1-hydroxyethyl]imidazo[1,2-a]pyridin-2-yl}phenyl)methyl]ethyl}-4-[(1-methylethyl)oxy]benzamide (GSK923295; 1), starting from a high-throughput screening hit, 3-chloro-4-isopropoxybenzoic acid 2. Compound 1 has demonstrated broad antitumor activity in vivo and is currently in human clinical trials.