High-throughput antibody generation and characterization

A phage display library has been constructed containing over 1010 human antibodies, allowing the large-scale generation of antibodies. Over 38,000 recombinant antibodies against 292 antigens were selected, screened and sequenced, and 4,400 resultant unique clones characterized further.

Disregulated Wnt/β-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1-and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1-and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wntdriven cancer.antibody therapeutics | cancer T he Wnt/β-catenin pathway regulates diverse biological processes during development and tissue homeostasis by modulating the protein stability of β-catenin (1-3). In the absence of extracellular Wnt proteins, cytoplasmic β-catenin is associated with the β-catenin destruction complex and degraded by ubiquitinmediated proteolysis. Wnt signals are transduced by two distinct receptors, the serpentine receptor Frizzled (Frz) and the singlespan transmembrane proteins LRP5 or LRP6. Wnt proteins promote the assembly of the Frz-LRP5/6 signaling complex and induce phosphorylation of LRP5 or LRP6. Phosphorylated LRP5 or LRP6 inactivates the β-catenin degradation complex, allowing stabilized β-catenin to enter the nucleus, bind to the TCF transcription factors, and act as a transcriptional coactivator.The extracellular domain of LRP5 or LRP6 contains four YWTD-type β-propeller domains each followed by an EGF-like domain and an LDLR domain. Each propeller contains six YWTD motifs that form a six-bladed β-propeller structure (4). Biochemical studies suggest that Wnt proteins physically interact with both Frz and LRP6 and induce the formation of an Frz-

In the injured liver hepatic stellate cells (HSCs) undergo a dramatic phenotypic transformation known as ''activation'' in which they become myofibroblast-like and express high levels of the tissue inhibitor of metalloproteinase 1 (TIMP-1). HSC activation is accompanied by transactivation of the TIMP-1 promoter. Truncation mutagenesis studies delineated a minimal active promoter consisting of nucleotides ؊102 to ؉60 relative to the major start site for transcription. Removal of an AP-1 site located at nucleotides ؊93 to ؊87 caused almost a complete loss of promoter activity.

BackgroundIn the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44), kinases (EGFR-cytoplasmic domain, CDK2 and 4), proteases (MMP1, CASP2), signal transduction proteins (GRB2, RAF1, HRAS) and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX). Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study.ResultsSeveral protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY), or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx) and maltose binding protein (MBP) were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions.ConclusionsBy analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases proteins may be truncated to minimise molecular weight and the numbers of contiguous hydrophobic amino acids and low complexity regions to aid soluble expression in E. coli.

Retinoic acid, acting through the nuclear retinoic acid receptor β2(RARβ2), stimulates neurite outgrowth from peripheral nervous system tissue that has the capacity to regenerate neurites, namely, embryonic and adult dorsal root ganglia. Similarly, in central nervous system tissue that can regenerate, namely, embryonic mouse spinal cord, retinoic acid also stimulates neurite outgrowth and RARβ2 is upregulated. By contrast, in the adult mouse spinal cord, which cannot regenerate, no such upregulation of RARβ2 by retinoic acid is observed and no neurites are extended in vitro. To test our hypothesis that the upregulation of RARβ2 is crucial to neurite regeneration, we have transduced adult mouse or rat spinal cord in vitro with a minimal equine infectious anaemia virus vector expressing RARβ2. After transduction, prolific neurite outgrowth occurs. Outgrowth does not occur when the cord is transduced with a different isoform of RARβ nor does it occur following treatment with nerve growth factor. These data demonstrate that RARβ2 is involved in neurite outgrowth, at least in vitro, and that this gene may in the future be of some therapeutic use.

Hepatic stellate cells (HSCs)1 represent up to 15% of the resident cells of the liver and play a pivotal role in the cellular pathology underlying hepatic fibrosis (1). In response to liver injury of any etiology, the normally quiescent HSC undergoes a progressive process of trans-differentiation into a proliferating myofibroblast-like activated HSC (1). Through increased secretion of extracellular matrix proteins and the tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2, activated HSCs are responsible for deposition and accumulation of the majority of the excess extracellular matrix in the fibrotic liver (2). Furthermore, activated HSCs can contribute to the fibrogenic process through their ability to secrete and respond to a wide range of cytokines and growth factors (3).Details of the molecular events that regulate HSC activation are beginning to be unraveled, as is the potential for specific members of the AP-1, NF-B, and Kruppel-like transcription factor families to control key profibrogenic features of the activated HSCs (1, 4 -6). Putative AP-1 and NF-B sites are found in the promoters of many genes that are induced upon HSC activation and contribute to the fibrotic process, including TIMP-1 (AP-1), IL-6 (AP-1 and NF-B), and ICAM-1 (NF-B) (4, 5, 7). Since in vivo activation of HSCs can be closely mimicked by culturing HSCs isolated from normal rat liver on plastic and in the presence of serum, it has been possible to investigate the transcriptional control of potential profibrotic genes during HSC activation (1). Investigators including ourselves have previously demonstrated that basal and cytokine/ growth factor-inducible transcription of these genes is dependent on interaction of specific AP-1 and NF-B (Rel) protein dimers with their putative promoter-binding sites (4 -6). These observations indicate that these inducible transcription factors are likely to play a key role in the activation and/or persistence of myofibroblast-like HSCs. Recent studies have identified target genes of NF-B (IL-6 and ICAM-1) and have also indicated that NF-B may protect activated HSCs against apoptosis (5,6,8). Less attention has been directed at studying the role played by AP-1 in HSC activation. Although in vitro studies have shown that activated HSCs express inducible AP-1 DNA-binding activity (4, 9, 10), there is little direct evidence that AP-1 plays a key role in the transcriptional regulation of the activated HSC phenotype. Chen and Davis (11, 12) recently re-

Background: Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression.

Antibody engineering technologies are constantly advancing to improve the clinical effectiveness of monoclonal antibodies (mAbs). Effector functions may be modified by engineering the Fc region, for example to improve or reduce binding to Fc gamma receptors (FcγRs) or complement factors. Other examples for Fc engineering include modification of the half-life of immunoglobulin G (IgG); various studies have shown that half-life can be prolonged by increasing the affinity of Fc for the Fc neonatal receptor (FcRn). Furthermore, engineered pH-dependent antigen binding can be applied to enhance the recycling of IgG via FcRn, enabling binding to additional target molecules. Since bispecific approaches may elicit desired effects on disease targets, a variety of bispecific formats have been developed, including variants that structurally mimic IgG. Finally, antibody-drug conjugates (ADCs) create new opportunities for treatment of certain diseases. Advances in antibody generation, selection of highly cytotoxic molecules and production of stable linkers have paved the way to the development of many ADCs that can be tested in clinical trials. This review covers current antibody engineering strategies for the modification of therapeutic antibodies in the areas of Fc engineering and pH-dependent antigen binding, bispecific antibodies and ADCs.