Daniel G. Yansura

Identification of Heregulin, a Specific Activator of p185erbB2

The proto-oncogene designated erbB2 or HER2 encodes a 185-kilodalton transmembrane tyrosine kinase (p185erbB2), whose overexpression has been correlated with a poor prognosis in several human malignancies. A 45-kilodalton protein heregulin-α (HRG-α) that specifically induced phosphorylation of p185erbB2 was purified from the conditioned medium of a human breast tumor cell line. Several complementary DNA clones encoding related HRGs were identified, all of which are similar to proteins in the epidermal growth factor family. Scatchard analysis of the binding of recombinant HRG to a breast tumor cell line expressing p185erbB2 showed a single high affinity binding site [dissociation constant (Kd) = 105 � 15 picomolar]. Heregulin transcripts were identified in several normal tissues and cancer cell lines. The HRGs may represent the natural ligands for p185erbB2.

Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone

DNA coding for human growth hormone was constructed by using chemically synthesised DNA in conjunction with enzymatically prepared cDNA. This ‘hybrid’ gene was expressed in Escherichia coli under the control of the lac promoter. A polypeptide was produced having the size and immunological properties characteristic of mature human growth hormone.

Expression of full-length immunoglobulins in Escherichia coli: rapid and efficient production of aglycosylated antibodies.

Many research and clinical applications require large quantities of full-length antibodies with long circulating half-lives, and production of these complex multi-subunit proteins has in the past been restricted to eukaryotic hosts. In this report, we demonstrate that efficient secretion of heavy and light chains in a favorable ratio leads to the high-level expression and assembly of full-length IgGs in the Escherichia coli periplasm. The technology described offers a rapid, generally applicable and potentially inexpensive method for the production of full-length therapeutic antibodies, as verified by the expression of several humanized IgGs. One E. coli-derived antibody in particular, anti-tissue factor IgG1, has been thoroughly evaluated and has all of the expected properties of an aglycosylated antibody, including tight binding to antigen and the neonatal receptor. As predicted, the protein lacks binding to C1q and the FcgammaRI receptor, making it an ideal candidate for research purposes and therapeutic indications where effector functions are either not required or are actually detrimental. In addition, a limited chimpanzee study suggests that the E. coli-derived IgG1 retains the long circulating half-life of mammalian cell-derived antibodies.

Amelioration of Type 2 Diabetes by Antibody-Mediated Activation of Fibroblast Growth Factor Receptor 1

Antibody-mediated activation of fibroblast growth factor receptor 1 reverses the diabetic phenotype in mice, likely by affecting brown adipose tissues. Getting at Brown Fat It’s fun to indulge in holiday cheer, if only a holiday miracle allowed one to avoid the often-linked weight gain. At the molecular level, obesity and type 2 diabetes can be linked by the fibroblast growth factor (FGF) family of proteins and their receptors (FGFRs), with some factors showing disease-reversing capabilities. For instance, overweight, diabetic mice treated with FGF21 regain normal metabolism and lose weight, even without spending hours on a treadmill. However, attempts to use this fat-burning factor in humans have not been successful, owing to poor pharmacokinetics as well as concerns over negative effects of modified FGF21 proteins. In this issue, Wu and colleagues describe an antibody-based FGF21 mimic that circumvents these limitations to overcome metabolic disease in mice. The authors reasoned that robust drugs that closely mimic FGF21 function would similarly exert antidiabetic effects. Using phage display technology, Wu et al. identified monoclonal antibodies (R1MAbs) that were specifically targeted tissues that play key roles in diabetes and obesity, including adipose (fat) tissue. In contrast to FGF21, which binds several forms of the FGFR throughout the body, the phage-derived R1MAbs bound only to FGFR1—a receptor present in the pancreas and in brown and white adipose tissues. Diabetic mice with high blood sugar (hyperglycemia) were injected once with either R1MAbs or a control antibody. Within 1 week, blood glucose concentrations in the R1MAb-treated mice were normalized and remained at lower levels compared to placebo-treated mice for more than 1 month without reaching dangerously low blood glucose concentrations (hypoglycemia). The R1MAbs also helped the diabetic mice to lose weight, indicating that this antibody agonist of FGFR1 is a dual-action drug for both diabetes and obesity. Wu et al. also shed light on the mechanism of action of their R1MAbs, showing that they work via FGFR homodimerization in brown adipose tissue. With improved pharmacokinetics over FGF21, in addition to a specific receptor-targeting mechanism, these R1MAbs could enter human clinical trials for diabetes and other obesity-related diseases in the near future. Unfortunately, a miracle drug won’t be available in time for the holidays, so perhaps, this year, opt for the sugar-free egg nog. Clinical use of recombinant fibroblast growth factor 21 (FGF21) for the treatment of type 2 diabetes and other disorders linked to obesity has been proposed; however, its clinical development has been challenging owing to its poor pharmacokinetics. Here, we describe an alternative antidiabetic strategy using agonistic anti-FGFR1 (FGF receptor 1) antibodies (R1MAbs) that mimic the metabolic effects of FGF21. A single injection of R1MAb into obese diabetic mice induced acute and sustained amelioration of hyperglycemia, along with marked improvement in hyperinsulinemia, hyperlipidemia, and hepatosteatosis. R1MAb activated the mitogen-activated protein kinase pathway in adipose tissues, but not in liver, and neither FGF21 nor R1MAb improved glucose clearance in lipoatrophic mice, which suggests that adipose tissues played a central role in the observed metabolic effects. In brown adipose tissues, both FGF21 and R1MAb induced phosphorylation of CREB (cyclic adenosine 5′-monophosphate response element–binding protein), and mRNA expression of PGC-1α (peroxisome proliferator–activated receptor-γ coactivator 1α) and the downstream genes associated with oxidative metabolism. Collectively, we propose FGFR1 in adipose tissues as a major functional receptor for FGF21, as an upstream regulator of PGC-1α, and as a compelling target for antibody-based therapy for type 2 diabetes and other obesity-associated disorders.

Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct half-antibodies

By enabling the simultaneous engagement of two distinct targets, bispecific antibodies broaden the potential utility of antibody-based therapies. However, bispecific-antibody design and production remain challenging, owing to the need to incorporate two distinct heavy and light chain pairs while maintaining natural nonimmunogenic antibody architecture. Here we present a bispecific-antibody production strategy that relies on co-culture of two bacterial strains, each expressing a half-antibody. Using this approach, we produce 28 unique bispecific antibodies. A bispecific antibody against the receptor tyrosine kinases MET and EGFR binds both targets monovalently, inhibits their signaling, and suppresses MET and EGFR-driven cell and tumor growth. Our strategy allows rapid generation of bispecific antibodies from any two existing antibodies and yields milligram to gram quantities of bispecific antibodies sufficient for a wide range of discovery and preclinical applications.

Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent

Significance Therapeutic antibodies have revolutionized the treatment of human disease. Despite these advances, antibody bivalency limits their utility against some targets. Here, we describe the development of a one-armed (monovalent) antibody, onartuzumab, targeting the receptor tyrosine kinase MET. While initial screening of bivalent antibodies produced agonists of MET, engineering them into monovalent antibodies produced antagonists instead. We explain the structural basis of the mechanism of action with the crystal structure of onartuzumab antigen-binding fragment in complex with MET and HGF-β. These discoveries have led to an additional antibody-based therapeutic option and shed light on the underpinnings of HGF/MET signaling. Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coli-derived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF binding and receptor phosphorylation and signaling and has antibody-like pharmacokinetics and antitumor activity. Biochemical data and a crystal structure of a ternary complex of onartuzumab antigen-binding fragment bound to a MET extracellular domain fragment, consisting of the MET Sema domain fused to the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF β-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain (but not β-chain) binding to MET. These data suggest a likely binding site of the HGF α-chain on MET, which when dimerized leads to MET signaling. Onartuzumab, therefore, represents the founding member of a class of therapeutic monovalent antibodies that overcomes limitations of antibody bivalency for targets impacted by antibody crosslinking.

Development of a two-part strategy to identify a therapeutic human bispecific antibody that inhibits IgE receptor signaling

The development of bispecific antibodies as therapeutic agents for human diseases has great clinical potential, but broad application has been hindered by the difficulty of identifying bispecific antibody formats that exhibit favorable pharmacokinetic properties and ease of large-scale manufacturing. Previously, the development of an antibody technology utilizing heavy chain knobs-into-holes mutations and a single common light chain enabled the small-scale generation of human full-length bispecific antibodies. Here we have extended the technology by developing a two-part bispecific antibody discovery strategy that facilitates proof-of-concept studies and clinical candidate antibody generation. Our scheme consists of the efficient small-scale generation of bispecific antibodies lacking a common light chain and the hinge disulfides for proof-of-concept studies coupled with the identification of a common light chain bispecific antibody for large-scale production with high purity and yield. We have applied this technology to generate a bispecific antibody suitable for development as a human therapeutic. This antibody directly inhibits the activation of the high affinity IgE receptor FcϵRI on mast cells and basophils by cross-linking FcϵRI with the inhibitory receptor FcγRIIb, an approach that has strong therapeutic potential for asthma and other allergic diseases. Our approach for producing human bispecific full-length antibodies enables the clinical application of bispecific antibodies to a validated therapeutic pathway in asthma.

Construction of a vector for cloning promoters in Bacillus subtilis

A versatile vector for cloning DNA fragments containing promoter activity in Bacillus subtilis was derived from plasmids pBR322, pUB110 and pC194. Selection is based on chloramphenicol resistance which is dependent upon the introduction of DNA fragments allowing expression of a chloramphenicol acetyl transferase gene. The plasmid contains a second selectable marker, neomycin resistance, and contains functional origins of replication for both B. subtilis and Escherichia coli.

Production of Monoclonal Antibodies in E. coli

The number of monoclonal antibodies approved for use as therapeutic agents by regulatory agencies has increased in the past several years. Monoclonal antibodies are predicted to become an increasingly larger part of biopharmaceutical products, and perhaps dominate the market share by the end of the decade (Walsh 2006). Mammalian expression systems, such as Chinese Hamster Ovary cells (CHO), are currently the preferred system for producing full-length monoclonal antibodies. Fungal systems could become more of a contender for the production of antibodies if titers can be increased (Andersen and Reilly 2004). However, with fungal production systems, there may be concerns about potential non-native mammalian N-linked or O-linked glycosylation that could result in immunogenic responses in humans. Technology developed in recent years (Hamilton et al. 2003) could help to alleviate this concern.

Application of the E. coli trp promoter.

The Escherichia coli tryptophan (trp) promoter has been used extensively for the high level production of proteins on a small and large scale. This regulated promoter is readily available, relatively easy to turn on, and can be used in essentially any E. coli host background. This article gives a detailed use of the trp promoter including the design of expression vectors, subsequent culture conditions for promoter induction, and, finally, a protocol for the most common way of detecting the newly synthesized protein of interest. Its successful use for heterologous protein expression, however, sometimes requires consideration of parameters other than transcription such as translation initiation, translation elongation, and proteolysis. In this respect we offer guidance in getting through these post-transcriptional problems, which can occur with the use of any promoter.