Detection of amyloid ß oligomers toward early diagnosis of Alzheimer's disease.

Amyloid ß (Aß) peptide accumulation in the brain is considered to be one of the hallmarks of Alzheimer's disease. Here, we compare two analytical techniques for detecting neurotoxic Aß1-42 oligomers - Quartz Crystal Microbalance with Dissipation (QCM-D) and Single Molecule Array (Simoa). Both detection methods exploit a feature of the monoclonal antibody bapineuzumab, which targets N-terminal residues 1-5 of Aß with high affinity and use it as both a capture and detection reagent. Assays developed with the two methods allow us to specifically recognize neurotoxic Aß1-42 oligomers and higher aggregates such as fibrils but discriminate against Aß1-42 monomer species. We find that for detection of Aß1-42 oligomers, Simoa was roughly 500 times more sensitive than the QCM-D technique with limits of detection of 0.22 nM and 125 nM, respectively.

Antibodies specifically targeting a locally misfolded region of tumor associated EGFR.

Epidermal Growth Factor Receptor (EGFR) is involved in stimulating the growth of many human tumors, but the success of therapeutic agents has been limited in part by interference from the EGFR on normal tissues. Previously, we reported an antibody (mab806) against a truncated form of EGFR found commonly in gliomas. Remarkably, it also recognizes full-length EGFR on tumor cells but not on normal cells. However, the mechanism for this activity was unclear. Crystallographic structures for Fab:EGFR(287-302) complexes of mAb806 (and a second, related antibody, mAb175) show that this peptide epitope adopts conformations similar to those found in the wtEGFR. However, in both conformations observed for wtEGFR, tethered and untethered, antibody binding would be prohibited by significant steric clashes with the CR1 domain. Thus, these antibodies must recognize a cryptic epitope in EGFR. Structurally, it appeared that breaking the disulfide bond preceding the epitope might allow the CR1 domain to open up sufficiently for antibody binding. The EGFR(C271A/C283A) mutant not only binds mAb806, but binds with 1:1 stoichiometry, which is significantly greater than wtEGFR binding. Although mAb806 and mAb175 decrease tumor growth in xenografts displaying mutant, overexpressed, or autocrine stimulated EGFR, neither antibody inhibits the in vitro growth of cells expressing wtEGFR. In contrast, mAb806 completely inhibits the ligand-associated stimulation of cells expressing EGFR(C271A/C283A). Clearly, the binding of mAb806 and mAb175 to the wtEGFR requires the epitope to be exposed either during receptor activation, mutation, or overexpression. This mechanism suggests the possibility of generating antibodies to target other wild-type receptors on tumor cells.

Connecting the sequence dots: shedding light on the genesis of antibodies reported to be designed in silico.

Two recent publications out of the same research laboratory report on structure-based in silico design of antibodies against viral targets without sequence disclosure. Cross-referencing the published data to patent databases, we established the sequence identity of said computationally designed antibodies. In both cases, the antibodies align with high sequence identity to previously reported antibodies of the same specificity. This clear underlying sequence relationship, which is far closer than the antibody templates reported to seed the computational design, suggests an alternative origin of the computationally designed antibodies. The lack of both reproducible computational algorithms and of output sequences in the initial publications obscures the relationship to previously reported antibodies, and sows doubt as to the genesis narrative described therein.

Directed evolution to probe protein allostery and integrin I domains of 200,000-fold higher affinity.

Understanding allostery may serve to both elucidate mechanisms of protein regulation and provide a basis for engineering active mutants. Herein we describe directed evolution applied to the integrin alpha(L) inserted domain for studying allostery by using a yeast surface display system. Many hot spots for activation are identified, and some single mutants exhibit remarkable increases of 10,000-fold in affinity for a physiological ligand, intercellular adhesion molecule-1. The location of activating mutations traces out an allosteric interface in the interior of the inserted domain that connects the ligand binding site to the alpha7-helix, which communicates allostery to neighboring domains in intact integrins. The combination of two activating mutations (F265S/F292G) leads to an increase of 200,000-fold in affinity to intercellular adhesion molecule-1. The F265S/F292G mutant is potent in antagonizing lymphocyte function-associated antigen 1-dependent lymphocyte adhesion, aggregation, and transmigration.

Antibody-directed therapy for human hepatocellular carcinoma.

The goals of our research are to develop high-affinity and high-stability antibodies and fragments thereof for targeting tumor-specific antigens in an attempt to develop new therapeutic agents for human hepatocellular carcinoma (HCC). Tumor-associated antigens are excellent targets for drug and gene delivery, and offer the advantage of high cellular specificity. We have explored the use of a monoclonal antibody (Mab) AF-20 raised against a human hepatoma cell line (FOCUS) as a model system. This antibody binds to a 180-kDa homodimeric cell surface glycoprotein with high affinity. The antigen is uniformly expressed in HCC-derived cell lines and human tumors, including those with distant metastasis. There is minimal expression in nontumor tissues, and none detectable in normal liver. Because the AF-20 antigen antibody interactions on the cell surface is rapidly internalized at 37 degrees C, there is an opportunity to deliver cytotoxic payloads to tumor cells. In addition, high-affinity single-chain monoclonal antibody fragments (scFv) have been created using a novel yeast display system. Drug conjugates with AF-20 monoclonal antibodies have been prepared for gene targeting of HCC both in vitro and in vivo using preclinical animal model systems. These studies show that it is possible to generate high-affinity intact scFv antibody fragments that will allow specific tumor targeting of adenoviruses containing suicide genes, chemotherapeutic agents such as methotrexate, and cytotoxic peptides to produce antitumor effects. Therefore, specific antibody targeting of antitumor agents to HCC cells has the potential for therapeutic application in this devastating disease.