Characterization of antibodies in single-chain format against the E7 oncoprotein of the Human papillomavirus type 16 and their improvement by mutagenesis

BACKGROUND: Human papillomaviruses (HPV) are the etiological agents of cervical cancer. The viral E7 protein plays a crucial role in viral oncogenesis. Many strategies have been explored to block the E7 oncoprotein activity. The single-chain variable antibody fragments (scFvs) are valuable tools in cancer immunotherapy and can be used as "intracellular antibodies" to knock out specific protein functions. For both in vivo and in vitro employment, the scFv intrinsic solubility and stability are important to achieve long-lasting effects. Here we report the characterization in terms of reactivity, solubility and thermal stability of three anti-HPV16 E7 scFvs. We have also analysed the scFv43 sequence with the aim of improving stability and then activity of the antibody, previously shown to have antiproliferative activity when expressed in HPV16-positive cells. METHODS: The three anti-HPV16 E7 scFv 32, 43 51 were selected from the ETH-2 "phage-display" library. Thermal stability was evaluated with ELISA by determining the residual activity of each purified scFv against the recombinant HPV16 E7, after incubation in the presence of human seroalbumine for different time-intervals at different temperatures. Sequence analysis of the scFvs was performed with BLAST and CLUSTALL programs. The scFv43 aminoacid changes were reverted back to the consensus sequence from the immunoglobuline database by site-directed mutagenesis. ScFv solubility was evaluated with Western blotting by determining their relative amounts in the soluble and insoluble fractions of both prokaryotic and eukaryotic systems. RESULTS: ScFv51 was the most thermally stable scFv considered. Sequence analysis of the most reactive scFv43 has evidenced 2 amino acid changes possibly involved in molecule stability, in the VH and VL CDR3 regions respectively. By mutagenesis, two novel scFv43-derived scFvs were obtained, scFv43 M1 and M2. ScFv43 M2 showed to have improved thermal stability and solubility in comparison with the parental scFv43. CONCLUSION: The characterization of 5 specific anti-HPV16 E7 scFvs shows features important for their activity in vivo. ScFv43 M2 shows higher thermal stability with respect to the parental scFv43, and scFv51 shows high stability and solubility. These properties make the 2 scFvs the best candidates to be tested for anti-E7 activity in vivo

In Vivo Tumor Targeting and Imaging with Engineered Trivalent Antibody Fragments Containing Collagen-Derived Sequences

There is an urgent need to develop new and effective agents for cancer targeting. In this work, a multivalent antibody is characterized in vivo in living animals. The antibody, termed “trimerbody”, comprises a single-chain antibody (scFv) fragment connected to the N-terminal trimerization subdomain of collagen XVIII NC1 by a flexible linker. As indicated by computer graphic modeling, the trimerbody has a tripod-shaped structure with three highly flexible scFv heads radially outward oriented. Trimerbodies are trimeric in solution and exhibited multivalent binding, which provides them with at least a 100-fold increase in functional affinity than the monovalent scFv. Our results also demonstrate the feasibility of producing functional bispecific trimerbodies, which concurrently bind two different ligands. A trimerbody specific for the carcinoembryonic antigen (CEA), a classic tumor-associated antigen, showed efficient tumor targeting after systemic administration in mice bearing CEA-positive tumors. Importantly, a trimerbody that recognizes an angiogenesis-associated laminin epitope, showed excellent tumor localization in several cancer types, including fibrosarcomas and carcinomas. These results illustrate the potential of this new antibody format for imaging and therapeutic applications, and suggest that some laminin epitopes might be universal targets for cancer targeting

Barnase as a New Therapeutic Agent Triggering Apoptosis in Human Cancer Cells

RNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI) presented in the cytoplasm of mammalian cells.In search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC(50)) ranging from 0.2 to 13 microM and to leukemia cell lines with IC(50) values ranging from 2.4 to 82 microM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv) of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC(50) = 1.8 nM) was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3.These results demonstrate that barnase is a potent toxic agent for targeting to cancer cells

Ribosome display: a technology for selecting and evolving proteins from large libraries

The selection and concomitant affinity maturation of proteins to bind to user-defined target molecules have become a key technology in biochemical research, diagnostics, and therapy. One of the most potent selection technologies for such applications is ribosome display. It works entirely in vitro, and this has two important consequences. First, since no transformation of any cells is required, libraries with much greater diversity can be handled than with most other techniques. Second, since a library does not have to be cloned and transformed, it is very convenient to introduce random errors in the library by PCR-based methods and select improved binders. Thus, a true directed evolution, an iteration between randomization and selection over several generations, can be conveniently carried out, e.g., for affinity maturation. Ribosome display has been used successfully for the selection of antibody fragments and other binding proteins, such as Designed Ankyrin Repeat Proteins (DARPins)

Engineering antibodies for stability and efficient folding

Antibody variable domains vary widely in their intrinsic thermodynamic stability. Despite the mutual stabilization of the domains in the scFv fragment, most scFv derived from monoclonal antibodies without further engineering show poor to moderate stability. The situation gets more complex for Fab fragments and full-sized antibodies: while the disulfide-linked C(L)/C(H) heterodimer shows very limited thermodynamic stability, its unfolding kinetics are very slow. The same is true for Fab fragments, which, due to this kinetic stabilization, appear to be more stable than their thermodynamic stability suggests. However, suboptimal variable domains can be engineered for improved stability and folding efficiency while preserving their antigen-binding specificity and affinity, either by a limited number of point mutations or by grafting their antigen specificity to superior variable domain frameworks