C. Ronald Geyer

Recombinant Antibodies and In Vitro Selection Technologies

Over the past decade, the accumulation of detailed knowledge of antibody structure and function has enabled antibody phage display to emerge as a powerful in vitro alternative to hybridoma methods for creating antibodies. Many antibodies produced using phage display technology have unique properties that are not obtainable using traditional hybridoma technologies. In phage display, selections are performed under controlled, in vitro conditions that are tailored to suit demands of the antigen and the sequence encoding the antibody is immediately available. These features obviate many of the limitations of hybridoma methodology, and because the entire process relies on scalable molecular biology techniques, phage display is also suitable for high-throughput applications. Thus, antibody phage display technology is well suited for genome-scale biotechnology and therapeutic applications. This review describes the antibody phage display technology and highlights examples of antibodies with unique properties that cannot easily be obtained by other technologies.

Loss of imprinting of the insulin-like growth factor II (IGF2) gene in esophageal normal and adenocarcinoma tissues

To evaluate loss of imprinting (LOI) and expression of the IGF2 gene in matched esophageal normal and adenocarcinoma tissues, we studied a prospective cohort of 77 patients who underwent esophageal resection between 1998 and 2003. IGF2 imprinting status was determined by reverse transcription-polymerase chain reaction (PCR) following ApaI digestion, and quantitative PCR was used to evaluate IGF2 expression, which was correlated with clinicopathologic findings, disease-free and overall survival. In total, 32% (14/44) of informative tissues showed loss of IGF2 imprinting, with a strong correlation between the tumor and normal esophageal epithelia (Kappa = 0.89, P < 0.01). Normal epithelia with LOI had increased expression of IGF2 [median: 2.91, 95% confidence interval (CI): 0.93-5.06] compared with imprinted normal epithelia (median: 1.13, 95% CI: 0.85-1.39) (P = 0.03). In contrast, tumors with LOI had significantly reduced IGF2 expression (median: 1.87, 95% CI: 0.53-5.21) compared with normally imprinted tumors (median: 6.79, 95% CI: 3.39-15.89) (P = 0.016). Patients below the age of 65 years with normally imprinted tumors had significantly reduced 5 year disease-free survival (DFS) (24%) compared with patients whose tumors had LOI for IGF2 (55%) (P = 0.03). Cox regression analysis showed that IGF2 overexpression was associated with significantly reduced disease-free survival (P = 0.04). We conclude that in a subgroup of younger patients, loss of IGF2 imprinting was associated with improved outcome following esophageal resection. Expression of IGF2 in esophageal adenocarcinoma and normal esophageal epithelia depended on imprinting status and tissue type, suggesting novel molecular regulatory mechanisms in esophageal tumorigenesis.

RIZ1 is potential CML tumor suppressor that is down-regulated during disease progression

BackgroundRIZ1 expression and activity are reduced in many cancers. In AML cell lines and patient material, RIZ1 expression is reduced relative to normal bone marrow. In chronic myelogenous leukemia (CML), blastic transformation is associated with loss of heterozygosity in the region where RIZ1 is located. RIZ1 is a PR domain methyltransferase that methylates histone H3 lysine 9, a modification important for transcriptional repression. In CML blast crisis cell lines RIZ1 represses insulin-like growth factor-1 expression and autocrine signaling. Together these observations suggest that RIZ1 may have a role in the chronic phase to blast crisis transition in CML.ResultsIn CML patient material, we observed that RIZ1 expression was decreased during progression from chronic phase to blast crisis. RIZ1 was expressed in mature myeloid and CD34+ cells demonstrating that decreased RIZ1 expression in blast crisis is not due to an increased immature cell population. Expression of RIZ1 CML blast crisis cell lines decreased proliferation, increased apoptosis, and enhanced differentiation.ConclusionRIZ1 is a candidate tumor suppressor gene whose expression is decreased in blast crisis. Loss of RIZ1 activity results in decreased apoptosis and differentiation and enhanced proliferation. Together these results suggest that loss of RIZ1 expression will lead to an increase in myeloid blast cell population resulting in CML progression.

Synthetic Modular Antibody Construction by Using the SpyTag/SpyCatcher Protein‐Ligase System

Efforts to engineer recombinant antibodies for specific diagnostic and therapy applications are time consuming and expensive, as each new recombinant antibody needs to be optimized for expression, stability, bio‐distribution, and pharmacokinetics. We have developed a new way to construct recombinant antibody‐like “devices” by using a bottom‐up approach to build them from well‐behaved discrete recombinant antibody domains or “parts”. Studies on antibody structure and function have identified antibody constant and variable domains with specific functions that can be expressed in isolation. We used the SpyTag/SpyCatcher protein ligase to join these parts together, thereby creating devices with desired properties based on summed properties of parts and in configurations that cannot be obtained by using genetic engineering. This strategy will create optimized recombinant antibody devices at reduced costs and with shortened development times.

Near infrared fluorescence imaging of EGFR expression in vivo using IRDye800CW-nimotuzumab

Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) monoclonal antibody that is approved in many countries for the treatment of EGFR-positive cancers. Near infrared (NIR) fluorescent dye-labeled antibodies represent an attractive class of image-guided surgical probes because of their high specificity, tumor uptake, and low dissociation from tumor cells that express the antigen. In this study, we developed a NIR fluorescent dye-labeled nimotuzumab immunoconjugate, IRDye800CW-nimotuzumab, and evaluated in vitro binding with EGFR-positive cells, in vivo tumor uptake by NIR fluorescent imaging, and ex vivo biodistribution. There was no difference in binding between nimotuzumab and IRDye800CW-nimotuzumab to EGFR-positive cells. In mice bearing EGFR-positive xenografts, IRDye800CW-nimotuzumab uptake peaked at 4 days post injection and slowly decreased thereafter with high levels of accumulation still observed at 28 days post injection. In EGFR-positive xenografts, IRDye800CW-nimotuzumab showed more than 2-fold higher uptake in tumors compared to IRDye800CW-cetuximab. In addition, liver uptake of IRDye800CW-nimotuzumab was two-fold lower than cetuximab. The lower liver uptake of IRDye800CW-nimotuzumab could have implications on the selected dose for clinical trials of the immunoconjugate. In summary, this study shows that nimotuzumab is a good candidate for NIR fluorescent imaging and image-guided surgery.

A Single-Framework Synthetic Antibody Library Containing a Combination of Canonical and Variable Complementarity-Determining Regions

Synthetic antibody libraries have been used to generate antibodies with favorable biophysical and pharmacological properties. Here, we describe the design, construction, and validation of a phage‐displayed antigen‐binding fragment (Fab) library built on a modified trastuzumab framework with four fixed and two diversified complementarity‐determining regions (CDRs). CDRs L1, L2, H1, and H2 were fixed to preserve the most commonly observed “canonical” CDR conformation preferred by the modified trastuzumab Fab framework. The library diversity was engineered within CDRs L3 and H3 by use of custom‐designed trinucleotide phosphoramidite mixes and biased towards human antibody CDR sequences. The library contained ≈7.6 billion unique Fabs, and >95 % of the library correctly encoded both diversified CDR sequences. We used this library to conduct selections against the human epidermal growth factor receptor‐3 extracellular domain (HER3‐ECD) and compared the CDR diversity of the naïve library and the anti‐HER3 selection pool by use of next‐generation sequencing. The most commonly observed CDR combination isolated, named Her3‐3, was overexpressed and purified in Fab and immunoglobulin G (IgG) formats. Fab HER3‐3 bound to HER3‐ECD with a KD value of 2.14 nm and recognized cell‐surface HER3. Although HER3‐3 IgG bound to cell‐surface HER3, it did not inhibit the proliferation of HER3‐positive cells. Near‐infrared imaging showed that Fab HER3‐3 selectively accumulated in a murine HER3‐postive xenograft, thus providing a lead for the development of HER3 imaging probes.

Allosteric Lariat Peptide Inhibitors of Abl Kinase

Going against tradition: although most kinase inhibitors are ATP competitive, lariat peptides inhibit Abl kinase activity in an ATP-uncompetitive manner. Further, lariat peptides discriminated Src family kinases, and recognize the allosteric region that lies adjacent to the ATP binding pocket in the Abl kinase catalytic cleft.

EPHB6 augments both development and drug sensitivity of triple-negative breast cancer tumours

Triple-negative breast cancer (TNBC) tumours that lack expression of oestrogen, and progesterone receptors, and do not overexpress the HER2 receptor represent the most aggressive breast cancer subtype, which is characterised by the resistance to therapy in frequently relapsing tumours and a high rate of patient mortality. This is likely due to the resistance of slowly proliferating tumour-initiating cells (TICs), and understanding molecular mechanisms that control TICs behaviour is crucial for the development of effective therapeutic approaches. Here, we present our novel findings, indicating that an intrinsically catalytically inactive member of the Eph group of receptor tyrosine kinases, EPHB6, partially suppresses the epithelial–mesenchymal transition in TNBC cells, while also promoting expansion of TICs. Our work reveals that EPHB6 interacts with the GRB2 adapter protein and that its effect on enhancing cell proliferation is mediated by the activation of the RAS-ERK pathway, which allows it to elevate the expression of the TIC-related transcription factor, OCT4. Consistent with this, suppression of either ERK or OCT4 activities blocks EPHB6-induced pro-proliferative responses. In line with its ability to trigger propagation of TICs, EPHB6 accelerates tumour growth, potentiates tumour initiation and increases TIC populations in xenograft models of TNBC. Remarkably, EPHB6 also suppresses tumour drug resistance to DNA-damaging therapy, probably by forcing TICs into a more proliferative, drug-sensitive state. In agreement, patients with higher EPHB6 expression in their tumours have a better chance for recurrence-free survival. These observations describe an entirely new mechanism that governs TNBC and suggest that it may be beneficial to enhance EPHB6 action concurrent with applying a conventional DNA-damaging treatment, as it would decrease drug resistance and improve tumour elimination.

Evaluation of antibody fragment properties for near-infrared fluorescence imaging of HER3-positive cancer xenografts

In vivo imaging is influenced by the half-life, tissue penetration, biodistribution, and affinity of the imaging probe. Immunoglobulin G (IgG) is composed of discrete domains with known functions, providing a template for engineering antibody fragments with desired imaging properties. Here, we engineered antibody-based imaging probes, consisting of different combinations of antibody domains, labeled them with the near-infrared fluorescent dye IRDye800CW, and evaluated their in vivo imaging properties. Antibody-based imaging probes were based on an anti-HER3 antigen binding fragment (Fab) isolated using phage display. Methods: We constructed six anti-HER3 antibody-based imaging probes: a single chain variable fragment (scFv), Fab, diabody, scFv-CH3, scFv-Fc, and IgG. IRDye800CW-labeled, antibody-based probes were injected into nude mice bearing FaDu xenografts and their distribution to the xenograft, liver, and kidneys was evaluated. Results: These imaging probes bound to recombinant HER3 and to the HER3-positive cell line, FaDu. Small antibody fragments with molecular weight <60 kDa (scFv, diabody, and Fab) accumulated rapidly in the xenograft (maximum accumulation between 2-4 h post injection (hpi)) and cleared primarily through the kidneys. scFv-CH3 (80 kDa) had fast clearance and peaked in the xenograft between 2-3 hpi and cleared from xenograft in a rate comparable to Fab and diabody. IgG and scFv-Fc persisted in the xenografts for up to 72 hpi and distributed mainly to the xenograft and liver. The highest xenograft fluorescence signals were observed with IgG and scFv-Fc imaging probes and persisted for 2-3 days. Conclusion: These results highlight the utility of using antibody fragments to optimize clearance, tumor labeling, and biodistribution properties for developing anti-HER3 probes for image-guided surgery or PET imaging.

Synthetic antigen-binding fragments (Fabs) against S . mutans and S . sobrinus inhibit caries formation

Streptococcus mutans and Streptococcus sobrinus are the main causative agents of human dental caries. Current strategies for treating caries are costly and do not completely eradicate them completely. Passive immunization using nonhuman antibodies against Streptococcal surface antigens has shown success in human trials, however they often invoke immune reactions. We used phage display to generate human antigen-binding fragments (Fabs) against S. mutans and S. sobrinus. These Fabs were readily expressed in E. coli and bound to the surface S. mutans and S. sobrinus. Fabs inhibited sucrose-induced S. mutans and S. sobrinus biofilm formation in vitro and a combination of S. mutans and S. sobrinus Fabs prevented dental caries formation in a rat caries model. These results demonstrated that S. mutans and S. sobrinus Fabs could be used in passive immunization strategies to prevent dental caries. In the future, this strategy may be applied towards a caries therapy, whereby Fabs are topically applied to the tooth surface.