A major difficulty in treating cancer is the inability to differentiate between normal and tumor cells. The immune system differentiates tumor from normal cells by T cell receptor (TCR) binding of tumor-associated peptides bound to Major Histocompatibility Complex (pMHC) molecules. The peptides, derived from the tumor-specific proteins, are presented by MHC proteins, which then serve as cancer markers. The TCR is a difficult protein to use as a recombinant protein because of production issues and has poor affinity for pMHC; therefore, it is not a good choice for use as a tumor identifier outside of the immune system. We constructed a synthetic antibody-fragment (Fab) library in the phage-display format and isolated antibody-fragments that bind pMHC with high affinity and specificity. One Fab, fE75, recognizes our model cancer marker, the Human Epidermal growth factor Receptor 2 (HER2/neu) peptide, E75, bound to the MHC called Human Leukocyte Antigen-A2 (HLA-A2), with nanomolar affinity. The fE75 bound selectively to E75/HLA-A2 positive cancer cell lines in vitro. The fE75 Fab conjugated with 64Cu selectively accumulated in E75/HLA-A2 positive tumors and not in E75/HLA-A2 negative tumors in an HLA-A2 transgenic mouse as probed using positron emission tomography/computed tomography (PET/CT) imaging. Considering that hundreds to thousands of different peptides bound to HLA-A2 are present on the surface of each cell, the fact that fE75 arrives at the tumor at all shows extraordinary specificity. These antibody fragments have great potential for diagnosis and targeted drug delivery in cancer.
The γ-secretase complex, composed of presenilin, anterior-pharynxdefective 1, nicastrin, and presenilin enhancer 2, catalyzes the intramembranous processing of a wide variety of type I membrane proteins, including amyloid precursor protein (APP) and Notch. Earlier studies have revealed that nicastrin, a type I membrane-anchored glycoprotein, plays a role in γ-secretase assembly and trafficking and has been proposed to bind substrates. To gain more insights regarding nicastrin structure and function, we generated a conformationspecific synthetic antibody and used it as a molecular probe to map functional domains within nicastrin ectodomain. The antibody bound to a conformational epitope within a nicastrin segment encompassing residues 245-630 and inhibited the processing of APP and Notch substrates in in vitro γ-secretase activity assays, suggesting that a functional domain pertinent to γ-secretase activity resides within this region. Epitope mapping and database searches revealed the presence of a structured segment, located downstream of the previously identified DAP domain (DYIGS and peptidase; residues 261-502), that is homologous to a tetratricopeptide repeat (TPR) domain commonly involved in peptide recognition. Mutagenesis analyses within the predicted TPR-like domain showed that disruption of the signature helical structure resulted in the loss of γ-secretase activity but not the assembly of the γ-secretase and that Leu571 within the TPR-like domain plays an important role in mediating substrate binding. Taken together, these studies offer provocative insights pertaining to the structural basis for nicastrin function as a "substrate receptor" within the γ-secretase complex.A lzheimer's disease (AD), a progressive neurodegenerative disease, is the most prevalent cause of dementia in humans. The principal neuropathological hallmark of AD is the presence of senile plaques composed of dystrophic neurites surrounding extracellular aggregates of Aβ peptides (1). Aβ peptides are liberated from amyloid precursor proteins (APP) by the concerted action of β-site APP cleaving enzyme 1 and γ-secretase (2, 3). γ-Secretase is a macromolecular complex consisting of presenilin 1 or presenilin 2 (PS1 or PS2), anterior-pharynx-defective 1 (APH-1), nicastrin (NCT), and presenilin enhancer 2 (PEN-2) that catalyzes intramembranous proteolysis of several membrane-tethered substrates (4). Evidence has emerged to reveal the functions of each subunit: PS is the catalytic subunit (5); APH-1 serves as a scaffold for the complex assembly (6); NCT seems to be responsible for substrate binding (7); and PEN-2 promotes endoproteolytic cleavage of PS and "activation" of the enzyme complex (5, 8). Although γ-secretase cleaves a multiplicity of substrates at heterogeneous sites within individual transmembrane domains, the molecular mechanism(s) underlying substrate recognition and processing remain elusive.NCT, a 709-aa type I transmembrane glycoprotein with a large, heavily glycosylated ectodomain (ECD), was first identified as a PS-inter...
We show the efficacy of a therapeutic strategy that combines the potency of a DNA-binding photosensitizer, UV A Sens, with the tumor-targeting potential of receptor-mediated endocytosis. The photosensitizer is an iodinated bibenzimidazole, which, when bound in the minor groove of DNA and excited by UV A irradiation, induces cytotoxic lesions attributed to a radical species resulting from photodehalogenation. Although reminiscent of photochemotherapy using psoralens and UV A irradiation, an established treatment modality in dermatology particularly for the treatment of psoriasis and cutaneous T-cell lymphoma, a critical difference is the extreme photopotency of the iodinated bibenzimidazole, f1,000-fold that of psoralens. This feature prompted consideration of combination with the specificity of receptor-mediated targeting. Using two in vitro model systems, we show the UV A cytotoxicity of iodo ligand/protein conjugates, implying binding of the conjugate to cell receptors, internalization, and degradation of the conjugate-receptor complex, with release and translocation of the ligand to nuclear DNA. For ligand-transferrin conjugates, phototoxicity was inhibited by coincubation with excess native transferrin. Receptor-mediated UV A -induced cytotoxicity was also shown with the iodo ligand conjugate of an anti-human epidermal growth factor receptor monoclonal antibody, exemplifying the potential application of the strategy to other cancer-specific targets to thus improve the specificity of phototherapy of superficial lesions and for extracorporeal treatments.
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