Monobody-Mediated Alteration of Lipase Substrate Specificity

Controlling the catalytic properties of enzymes remain an important challenge in chemistry and biotechnology. We have recently established a strategy for altering enzyme specificity in which the addition of proxy monobodies, synthetic binding proteins, modulates the specificity of an otherwise unmodified enzyme. Here, in order to examine its broader applicability, we employed the strategy on <i>Candida rugosa</i> lipase 1 (CRL1), an enzyme with a tunnel-like substrate binding site. We successfully identified proxy monobodies that restricted the substrate specificity of CRL1 toward short-chain fatty acids. The successes with this enzyme system and a β-galactosidase used in the previous work suggest that our strategy can be applied to diverse enzymes with distinct architectures of substrate binding sites

HLA-A2 and HER2/neu expression is high variable on each tumor cell line.

<p>The surface expression of HLA-A2 and HER2/neu for each human tumor cell line was measured by flow cytometry. The representative plots of HLA-A2 versus HER2/neu for each tumor cell line were shown in A-E. In F, a graph of the MFI of one representative experiment from four total was shown. G and H were plots of the MFI of fE75 versus the MFI of HLA-A2 or MFI of HER2/neu respectively. Simple two-dimensional linear regression analysis models do not model the system well. The black lines showed weak correlation of fE75 MFI with HLA-A2 (r² = 0.40) or HER2/neu expression (r² = 0.62).</p

Phage-Display isolation of Fabs Specific for pMHC Molecules.

<p>Fabs specific for either E75/HLA-A2 or ML/HLA-A2 molecules were selected by phage-display technology. Each Fab construct was built from the highly stable 4D5 Fab scaffold containing both a heavy and light chain each with a single variable and constant domain (modeled from PDB ID: 1FVD) (A). The diversity of the Complementary Determining Regions (CDR) for the heavy chain; H1, H2, and H3, and the light chain; L3, was restricted in favor of tyrosine, serine, and other small amino acids. The Cα atoms of the synthetically modified H1, H2, H3, and L3 regions are shown as spheres (B). After three rounds of selection, an ELISA was used to test the specificity of the amplified clones (C). Three clones with three different specificities were identified. The Fab clones fE75 and fML bound E75/HLA-A2 and ML/HLA-A2 respectively with no detectable binding to the opposite pMHC molecule. The Fab clone fE2 showed binding to both E75/HLA-A2 and ML-HLA-A2. Following each Fab clone's specificity determination, the amino acid sequence of each clone was determined (D).</p

T Cell Receptor-Like Recognition of Tumor <em>In Vivo</em> by Synthetic Antibody Fragment

<div><p>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 <em>in vitro</em>. 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.</p> </div

TCR-like Fabs Bind Cognate pMHC with Nanomolar Affinity.

<p>SPR binding response curves of fE75 binding E75/HLA-A2 (A) and ML/HLA-A2 (B) and fML binding E75/HLA-A2 (C) and ML/HLA-A2 (D) are shown. Each Fab was immobilized onto individual flow channels in an NTA-Ni chip. Kinetic data for each Fab binding each pMHC molecule were globally fit to a bimolecular reaction. Green and blue lines designate the start and end respectively of each pMHC injection. Binding curves and curve fits are drawn in black and orange respectively. Each binding curve represents a different concentration of pMHC beginning at 200 nM and decreasing to 1.5 nM in 2 fold dilutions. No binding was observed for either Fab binding non-cognate pMHC molecules up to 400 nM.</p

Fabs bind specifically to endogenously processed and presented levels of pMHC molecules.

<p>Flow cytometric analysis revealed that the Fab tetramer, composed of biotinylated fE75 (A) or fML (B) and streptavidin-Alexa-647, bound to T2 cells incubated with the cognate peptide, but not to control peptides or untreated T2 cells in three separate experiments. In C, a graph of the MFI for several other control peptides against the cognate peptide for each Fab was shown. The staining of HER2/neu positive tumor cell lines by the fE75 and fML tetramers was shown in D and E respectively. The graph of the MFI for one representative experiment from four total was shown in F.</p

TCR-like Fab bind specifically to human tumor cells in HLA-A2 transgenic SCID mice.

<p>Similarly as describe for the SCID mice experiments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043746#pone-0043746-g005" target="_blank">Figure 5</a>), HLA-A2 transgenic SCID mice were injected intravenously with ⁶⁴Cu-DOTA-fE75. Coronal views of the small-animal PET image with coregistered CT images of a scid HLA-A2 transgenic mouse bearing human tumor (MDA-MB-231 and SKOV3) xenographs (A) in flanks at 1 h post-injection. Intensities of PET slices were scaled to the same maximum. Cropped views of only the tumors designated by the white boxes were shown in B respectively. The standardized uptake values (SUV) for ⁶⁴Cu-DOTA-fE75 in the tumors and selected organs over time were shown in C. B  =  bladder, h  =  humeral muscle, k  =  kidneys, l  =  liver, ct  =  control HLA-A2 negative tumor; SKOV3 cell line, pt  =  HLA-A2 positive tumor; MDA-MB-231 cell line.</p

Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

<div><p>We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that <i>in vivo</i> biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 <i>in vivo</i> biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce <i>in vivo</i> biotinylated Fabs in <i>E</i>. <i>coli</i>. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.</p></div

Overview of targets and their success rates.

<p>Overview of the 265 antigen domains included in the study and what protein families they belong to. Family success rate was calculated as percentage of targets that were successful in selections and which also yielded at least one antibody that passed cell-based validation.</p><p>Overview of targets and their success rates.</p

Fab and IgG production.

<p>(A) Comparison of purification yields between different expression vectors using an anti-MBP Fab as an example. The large-scale purification method on the ÄKTA Xpress including a heat denaturation step was used. (B) SDS-PAGE gel showing the anti-MBP Fab produced with various expression vectors and purified in triplicate. (C) IgG production yields with and without the dilution strategy.</p