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"Robinson, Matthew K."
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Combining Anti-ERBB3 Antibodies Specific for Domain I and Domain III Enhances the Anti-Tumor Activity over the Individual Monoclonal Antibodies
Inappropriate signaling through the epidermal growth factor receptor family (EGFR1/ERBB1, ERBB2/HER2, ERBB3/HER3, and ERBB4/HER4) of receptor tyrosine kinases leads to unregulated activation of multiple downstream signaling pathways that are linked to cancer formation and progression. In particular, ERBB3 plays a critical role in linking ERBB signaling to the phosphoinositide 3-kinase and Akt signaling pathway and increased levels of ERBB3-dependent signaling is also increasingly recognized as a mechanism for acquired resistance to ERBB-targeted therapies.
We had previously reported the isolation of a panel of anti-ERBB3 single-chain Fv antibodies through use of phage-display technology. In the current study scFv specific for domain I (F4) and domain III (A5) were converted into human IgG1 formats and analyzed for efficacy.
Treatment of cells with an oligoclonal mixture of the A5/F4 IgGs appeared more effective at blocking both ligand-induced and ligand-independent signaling through ERBB3 than either single IgG alone. This correlated with improved ability to inhibit the cell growth both as a single agent and in combination with other ERBB-targeted therapies. Treatment of NCI-N87 tumor xenografts with the A5/F4 oligoclonal led to a statistically significant decrease in tumor growth rate that was further enhanced in combination with trastuzumab.
These results suggest that an oligoclonal antibody mixture may be a more effective approach to downregulate ERBB3-dependent signaling.
Journal Article
Diversification and shared features of tumor‐binding antibody repertoires in tumor, sentinel lymph node and blood of three patients with breast cancer
Objectives B cell‐mediated immunity can be associated with favorable clinical outcomes in cancer patients. However, the mechanism and features of anti‐tumor immune responses are not well understood. In particular, how B cells expressing tumor‐specific antibodies are distributed (in the tumor vs. the circulation) has not been well defined. Methods We performed an in‐depth analysis of B cell antibody repertoires derived from the tumor, sentinel lymph nodes and peripheral blood of three treatment‐naïve patients with breast cancer. We integrated transcriptional analysis, next‐generation sequencing of immunoglobulin heavy‐chain gene rearrangements and phage display to define B cell responses and clonal architecture in the tumor microenvironment, and to identify antibodies against autologous tumor tissues. Results B cell clonal lineage mapping across sequencing libraries generated from the tumor, sentinel lymph node and blood revealed that some expanded B cell clones overlap between tumor and lymph node, and fewer clones overlapped with the peripheral blood. Notably, tumor‐associated or tumor‐binding clones recovered in the phage panning and tracked back through the tissues harboured extensive somatic hypermutations in both the tumor and the lymph node. Conclusions These findings suggest an evolving humoral immune response that targets the tumor and the possibility of monitoring B cell clones of interest in blood after identifying them in tumor or lymph node samples. Studying the dynamics and specificity of B cell responses may provide insights into the characteristics of successful anti‐tumor immunity, provide a means for monitoring therapy and yield novel targets for personalised therapies. This study combined antibody phage display and specificity analysis with immune repertoire and transcriptional profiling to provide an in‐depth analysis of B cell clonal networks and specificities in three treatment‐naïve patients with breast cancer. Phage panning and binding studies against autologous tumors allowed us to identify tumor‐binding antibodies and tracked back through the tissues harboured extensive somatic hypermutations in both the tumor and the lymph node.
Journal Article
Is This Not the Τέκτων?
The term τέκτων in Mark 6:3 has received various interpretations, with “carpenter” and “woodworker” being the most prominent. However, a brief survey of the use of τέκτων in the LXX, Graeco-Roman, and early Jewish literature reveals that this term is more ambiguous than scholars have often argued and requires contextual clues for precise translation. Because of this, one must look to the socio-historical reality of a common Nazarene τέκτων in order to determine the best translation of τέκτων in Mark 6:3. Historical evidence, including recent archeological findings, indicates that a Nazarene τέκτων would have likely been expected to carry out a plethora of tasks in the vicinity of building and crafting. Thus, this article argues that the best translation for τέκτων in Mark 6:3 is “builder-craftsman.”
Journal Article
Compounds identified by virtual docking to a tetrameric EGFR extracellular domain can modulate Grb2 internalization
Background
Overexpression or mutation of the epidermal growth factor receptor (EGFR) potently enhances the growth of many solid tumors. Tumor cells frequently display resistance to mechanistically-distinct EGFR-directed therapeutic agents, making it valuable to develop therapeutics that work by additional mechanisms. Current EGFR-targeting therapeutics include antibodies targeting the extracellular domains, and small molecules inhibiting the intracellular kinase domain. Recent studies have identified a novel prone extracellular tetrameric EGFR configuration, which we identify as a potential target for drug discovery.
Methods
Our focus is on the prone EGFR tetramer, which contains a novel protein-protein interface involving extracellular domain III. This EGFR tetramer is computationally targeted for stabilization by small molecule ligand binding. This study performed virtual screening of a Life Chemicals, Inc. small molecule library of 345,232 drug-like compounds against a molecular dynamics simulation of protein-protein interfaces distinct to the novel tetramer. One hundred nine chemically diverse candidate molecules were selected and evaluated using a cell-based high-content imaging screen that directly assessed induced internalization of the EGFR effector protein Grb2. Positive hits were further evaluated for influence on phosphorylation of EGFR and its effector ERK1/2.
Results
Fourteen hit compounds affected internalization of Grb2, an adaptor responsive to EGFR activation. Most hits had limited effect on cell viability, and minimally influenced EGFR and ERK1/2 phosphorylation. Docked hit compound poses generally include Arg270 or neighboring residues, which are also involved in binding the effective therapeutic cetuximab, guiding further chemical optimization.
Conclusions
These data suggest that the EGFR tetrameric configuration offers a novel cancer drug target.
Journal Article
Impact of expression system on the function of the C6.5 diabody PET radiotracer
The ability of engineered antibodies to rapidly and selectively target tumors that express their target antigen makes them well suited for use as radioimaging tracers. The combination of molecular size and bivalent nature makes diabody molecules a particularly promising structure for use as radiotracers for diagnostic imaging. Previous data have demonstrated that the anti-HER2 C6.5 diabody (C6.5db) is an effective radiotracer in preclinical models of HER2-positive cancer. The aim of this study was to evaluate the impact on radiotracer performance, associated with expressing the C6.5db in the
Pichia pastoris
(P-C6.5db) system as compared to
Escherichia coli
(E. C6.5db). Glycosylation of P-C6.5db led to faster blood clearance and lower overall tumor uptake than seen with
E. coli
-produced C6.5db. However, P-C6.5db achieved high tumor/background ratios that are critical for effective imaging. Dosimetry measurements determined in this study for both
124
I-P-C6.5db and
124
I-E-C6.5db suggest that they are equivalent to other radiotracers currently being administered to patients.
Journal Article
Isolation of anti-MISIIR scFv molecules from a phage display library by cell sorter biopanning
While cell surface antigens represent the most common targets for antibody-based cancer therapy, isolation of new antibodies specific for these targets from single-chain Fv phage display libraries has been hindered by limitations associated with traditional selection techniques. Solid phase panning is often associated with conformational changes to the target protein due to its immobilization on plastic tubes that can limit the ability of the isolated scFv to bind to conformational epitopes and solution panning methods require the use of secondary tags that often mask desired sequences and create unintended epitopes. Commonly utilized cell-based panning methods typically yield a panel of single-chain Fv (scFv) molecules that are specific for numerous cell surface antigens, often obscuring the desired clones. Here, we describe a novel cell sorter-based system to isolate single-chain Fv molecules specific for defined antigen targets expressed on stably-transformed mammalian cells. We employed these methods to isolate promising scFv clones that bind specifically to the Müllerian inhibiting substance type II receptor, a cell surface ovarian cancer antigen that has proven to be a difficult target for selection strategies.
Journal Article
Quantitation of small-animal (124)I activity distributions using a clinical PET/CT scanner
Time-dependent PET imaging can be an important tool in the assessment of radiotracer performance in murine models. We have performed a quantitative analysis of PET images of (124)I, acquired on a clinical PET system using a small-animal phantom. We then compared the recovered activity concentrations with the known activity concentration in the phantom spheres. The recovery coefficients found from the phantom data were applied to in vivo (124)I anti-HER2/neu C6.5 diabody PET data and compared with necropsy biodistribution data from the same tumor-bearing immunodeficient mouse.
The small-animal phantom consisted of a 4 x 8 cm water-filled acrylic cylinder with hollow spheres filled with water ranging in volume from 0.0625 to 1.0 mL and activity concentration of 27 +/- 2 kBq/mL. The background activity concentrations varied from 0 to 0.05 to 0.10 of the spheres. Data were acquired at 0, 5, and 10 cm from the scanner longitudinal axis. Recovery coefficients were theoretically calculated for spheres of different volume, background-to-target concentrations, and distance from the scanner's longitudinal axis. The theoretic recovery coefficients were applied to the maximum sphere activity concentration measured from the PET images, thus obtaining a recovered activity concentration to be compared with the known activity concentration of the spheres.
The mean recovered activity concentration for the phantom spheres was 25 +/- 2 kBq/mL. The (124)I diabody PET image of a mouse with a tumor xenograft was then analyzed using the techniques described. The tumor percentage injected dose per gram estimated from the murine PET image (4.8 +/- 0.4) compared well with those obtained from necropsy studies (5.1).
This study indicates the feasibility of performing quantitative imaging on murine (124)I antibody fragment PET images using a large-bore clinical scanner, which enables high-throughput studies to evaluate the performance of PET tracers in a timely and cost-effective manner by imaging multiple animals simultaneously. Tracers deemed promising by this screening method can then be further evaluated using traditional necropsy studies. Our group is currently conducting time-dependent (124)I diabody PET and necropsy comparative studies with larger numbers of mice.
Journal Article
Isolation of scFvs to In Vitro Produced Extracellular Domains of EGFR Family Members
The members of the epidermal growth factor receptor (EGFR) family are over expressed in a variety of malignancies and are frequently linked to aggressive disease and a poor prognosis. Although clinically effective monoclonal antibodies (MAbs) have been developed to target HER2 and EGFR, the remaining two family members, HER3 and HER4, have not been the subject of significant efforts. In this paper, we have taken the initial steps required to generate antibodies with potential clinically utility that target the members of the EGFR family. The genes for the extracellular domains (ECDs) of all four members of the EGFR family were cloned and used to stably transfect 293 (HEK) cells. Milligram quantities of each ECD were produced and characterized. The HER3, HER4, and EGFR ECDs were then employed as targets for the selection of antibodies from naïve human scFv (single-chain Fv) phage display libraries. Six unique scFv clones were isolated that bound specifically to HER3, 13 unique clones were isolated with specificity for HER4 and 52 unique anti-EGFR clones were isolated. These scFvs provide a valuable and potentially clinically relevant panel of agents to target the members of the EGFR family.
Journal Article
Combining Anti-ERBB3 Antibodies Specific for Domain I and Domain III Enhances the Anti-Tumor Activity over the Individual Monoclonal Antibodies
Inappropriate signaling through the epidermal growth factor receptor family (EGFR1/ERBB1, ERBB2/HER2, ERBB3/HER3, and ERBB4/HER4) of receptor tyrosine kinases leads to unregulated activation of multiple downstream signaling pathways that are linked to cancer formation and progression. In particular, ERBB3 plays a critical role in linking ERBB signaling to the phosphoinositide 3-kinase and Akt signaling pathway and increased levels of ERBB3-dependent signaling is also increasingly recognized as a mechanism for acquired resistance to ERBB-targeted therapies. We had previously reported the isolation of a panel of anti-ERBB3 single-chain Fv antibodies through use of phage-display technology. In the current study scFv specific for domain I (F4) and domain III (A5) were converted into human IgG1 formats and analyzed for efficacy. Treatment of cells with an oligoclonal mixture of the A5/F4 IgGs appeared more effective at blocking both ligand-induced and ligand-independent signaling through ERBB3 than either single IgG alone. This correlated with improved ability to inhibit the cell growth both as a single agent and in combination with other ERBB-targeted therapies. Treatment of NCI-N87 tumor xenografts with the A5/F4 oligoclonal led to a statistically significant decrease in tumor growth rate that was further enhanced in combination with trastuzumab. These results suggest that an oligoclonal antibody mixture may be a more effective approach to downregulate ERBB3-dependent signaling.
Journal Article