The rabbit antibody repertoire, which in the form of polyclonal antibodies has been used in diagnostic applications for decades, would be an attractive source for the generation of therapeutic human antibodies. The humanization of rabbit antibodies, however, has not been reported. Here we use phage display technology to select and humanize antibodies from rabbits that were immunized with human A33 antigen which is a target antigen for the immunotherapy of colon cancer. We first selected rabbit antibodies that bind to a cell surface epitope of human A33 antigen with an affinity in the 1 nM range. For rabbit antibody humanization, we then used a selection strategy that combines grafting of the complementarity determining regions with framework fine tuning. The resulting humanized antibodies were found to retain both high specificity and affinity for human A33 antigen.The growing significance of antibody-based strategies for the treatment of a variety of diseases demands efficient and reliable routes to human or humanized antibodies with high specificity and affinity. Nonhuman antibodies are highly immunogenic in humans thereby limiting their potential use for therapeutic applications, especially when repeated administration is necessary. In order to reduce their immunogenicity, nonhuman antibodies have been humanized using strategies that are based on rational design, in vitro evolution, or a combination of both. An alternative route is the direct generation of human antibodies from transgenic mice containing human immunoglobulin (Ig) 1 loci or by selection from naive or synthetic human antibody libraries displayed on phage (1, 2).Here we exploit a new route to humanized antibodies. We use phage display technology to select and humanize antibodies from rabbits that were immunized with a human antigen. Monoclonal antibodies from multiple species are accessible by screening combinatorial antibody libraries displayed on phage (3). The accessibility of antibody repertoires of multiple species might become substantial in the search for new therapeutic antibodies. In particular the rabbit antibody repertoire, which in the form of polyclonal antibodies has been used in diagnostic applications for decades, would be an attractive source for therapeutic antibodies. The humanization of rabbit antibodies, however, has not been reported.To allow selective antibody targeting, both high specificity of the antibody and restrictive tissue expression of the antigen are required. For cancer therapy, only a few antibody-antigen systems that meet these requirements have been identified (4). The A33 system which has been established in clinical trials is one of them (5-7). Mouse monoclonal antibody A33 (5) detects an antigen that is expressed in normal human colon epithelial cells and by Ͼ95% of human colon cancers. Human A33 antigen (8, 9) is a transmembrane glycoprotein of the Ig superfamily. It consists of 2 extracellular Ig folds, a single transmembrane domain, and a highly polar intracellular tail containing a Spalmitoylation si...

We describe the isolation of a CCR5-specific antibody, ST6, from an antibody phage display library generated from an immune rabbit. ST6 was previously shown to efficiently prevent the surface expression of CCR5 when expressed intracellularly (Steinberger, P., Andris-Widhopf, J., Buhler, B., Torbett, B. E., and Barbas, C. F., III (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 805-810). Because ST6 has therapeutic potential in human immunodeficiency virus, type 1 disease, its humanization was desired to minimize the potential for immunogenicity. ST6 was humanized using a phage display-based approach. Like the parental rabbit clone, the humanized version ST6/34 efficiently prevented the surface expression of CCR5. The conserved linear peptide epitope bound by these antibodies was mapped using phage display. Both ST6 as well as the humanized anti-CCR5 antibody ST6/34 were produced as complete IgG antibodies and shown to bind to cell surface CCR5.

The recombinant expression of immunoglobulin domains, Fabs and scFvs in particular, in Escherichia coli can vary significantly from antibody to antibody. We hypothesized that poor Fab expression is often linked to poor intrinsic stability. To investigate this further, we applied a novel approach for stabilizing a poorly expressing anti-tetanus toxoid human Fab with a predisposition for being misfolded and non-functional. Forty-five residues within the Fab were chosen for saturation mutagenesis based on residue frequency analysis and positional entropy calculations. Using automated screening, we determined the approximate midpoint temperature of thermal denaturation (TM) for over 4000 library members with a maximum theoretical diversity of 855 unique mutations. This dataset led to the identification of 11 residue positions, primarily in the Fv region, which when mutated enhanced Fab stability. By combining these mutations, the TM of the Fab was increased to 92 degrees C. Increases in Fab stability correlated with higher expressed Fab yields and higher levels of properly folded and functional protein. The mutations were selected based on their ability to increase the apparent stability of the Fab and therefore the exact mechanism behind the enhanced expression in E.coli remains undefined. The wild-type and two optimized Fabs were converted to an IgG1 format and expressed in mammalian cells. The optimized IgG1 molecules demonstrated identical gains in thermostability compared to the Fabs; however, the expression levels were unaffected suggesting that the eukaryotic secretion system is capable of correcting potential folding issues prevalent in E.coli. Overall, the results have significant implications for the bacterial expression of functional antibody domains as well as for the production of stable, high affinity therapeutic antibodies in mammalian cells.

mTOR Ser/Thr protein kinase operates in two distinct multi-protein complexes, TORC1 and TORC2, which together regulate growth, metabolism, angiogenesis and survival by integrating nutrient and hormonal environmental signals. The activity of mTOR is frequently up-regulated in human cancer by constitutive mitogen stimuli or oncogenic mutations upstream of TORC1 and TORC2. Rapamycin provides mechanistic rationale and clinical proof of concept for the therapeutic value of targeting mTOR in human cancer, but it also provided insights into how ATP-competitive TORC1/2 inhibitors have the potential to demonstrate superior efficacy. Through rational drug design we have discovered INK128, a potent, selective TORC1/2 inhibitor with excellent drug-like properties. INK128 inhibits mTOR kinase (sub-nanomolar) in an ATP-dependent fashion and demonstrates a high degree of selectivity against closely related kinases as well as against a panel of more than 400 kinases. INK128 inhibits both the phosphorylation of S6 and 4EBP1, the downstream substrates of TORC1, and selectively inhibits AKT phosphorylation at Ser473, the downstream substrate of TORC2, in vitro and in vivo. Interestingly, potent inhibition was also observed in cell lines resistant to rapamycin and pan-PI3K inhibitors. Daily, oral administration of INK128 (alone or in combination) inhibited angiogenesis and tumor growth in multiple xenograft models with predicted dose:exposure PK/PD relationship. We attribute the superior activity of INK128 to the fact that it is a more effective inhibitor of TORC1 relative to rapamycin. We further demonstrated that TORC1/2 inhibitors cause death of murine and human leukemia cells in models of pre-B acute lymphoblastic leukemia. In vivo, oral daily treatment with TORC1/2 inhibitors delayed leukemia onset and augmented the effects of ABL kinase inhibitors. Unexpectedly, these novel TORC1/2 inhibitors had much weaker effects than rapamycin on proliferation and function of normal lymphocytes. These findings establish that transformed lymphocytes are selectively sensitive to active-site TORC1/2 inhibitors and further support the development of such compounds for leukemia therapy in addition to solid tumors. In summary, INK128 is a potent, selective, and orally active TORC1/2 dual inhibitor positioned to enter clinical development. TORC1/2 inhibitors are mechanistically distinct from rapamycin and offer a compelling approach to the treatment of cancer by targeting translational control, cell metabolism, growth and angiogenesis. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B148.

The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.