Research paper
A dimerized single-chain variable fragment system for the assessment of neutralizing activity of phage display-selected antibody fragments specific for cytomegalovirus

https://doi.org/10.1016/j.jim.2011.11.010Get rights and content

Abstract

Cytomegalovirus (CMV) causes severe sequelae in congenitally infected newborns and may cause life-threatening disease in immuno-deficient patients. Recent findings demonstrate the possibility to alleviate the disease by infusing intravenous immunoglobulin G (IgG) preparations, indicating that antibodies are an effective therapeutic option. Modern molecular methodologies, like phage display, allow for the development of specific antibodies targeting virtually any antigen, including those of CMV. However, such methodologies do not in general result in products that by themselves mediate biological activity. To facilitate a semi-high-throughput approach for functional screening in future efforts to develop efficacious antibodies against CMV, we have integrated two different approaches to circumvent potential bottlenecks in such efforts. Firstly, we explored an approach that permits easy transfer of antibody fragment encoding genes from commonly used phage display vectors into vectors for the production of divalent immunoglobulins. Secondly, we demonstrate that such proteins can be applied in a novel reporter-based neutralization assay to establish a proof-of-concept workflow for the generation of neutralizing antibodies against CMV. We validated our approach by showing that divalent antibodies raised against the antigenic domain (AD)-2 region of gB effectively neutralized three different CMV strains (AD169, Towne and TB40/E), whereas two antibodies against the AD-1 region of gB displayed minor neutralizing capabilities. In conclusion, the methods investigated in this proof-of-concept study enables for a semi-high-throughput workflow in the screening and investigation of biological active antibodies.

Highlights

► Semi-high-throughput strategy for functional screening of CMV-specific scFv. ► Facile cloning and production of divalent immunoglobulins. ► Luciferase-based, automatable neutralization assay. ► Efficient evaluation of anti-viral activity of scFv isolated by phage display.

Introduction

More than half of the global human population is latently infected with human cytomegalovirus (CMV), a prototypical β-herpesvirus. The majority of CMV-infections are inapparent due to a functional immune system, which confines viral replication and provides protection against disease. However, immunodeficient individuals (e.g. AIDS or transplant patients) or infants that have been infected prior to birth are risk groups that suffer substantially from an infection since primary or recurrent infections may lead to life-threatening symptoms or severe disabilities. Considering the severe sequelae, for example sensorineural hearing loss or mental retardation in newborns or retinitis in immuno-compromised individuals, that CMV cause in these risk groups, there is a great need for a vaccine that induce protective immunity against CMV (Stratton et al., 2001). Importantly, in a recent study, Griffiths et al. (2011) were able to show reduced viremia and a shorter duration of therapy with antiviral agents following vaccination with a recombinant viral glycoprotein. Unfortunately, recent infection-studies with the highly homologous rhesus CMV in macaques in vivo have challenged our hope for a truly efficacious protective vaccine (Hansen et al., 2010). Nevertheless, the ability of immuno-competent individuals to control the virus without signs of illness indicates that at least the viral disease can be prevented by an induced immune response. Indeed, several lines of evidence indicate that antibodies are important in this respect (Jonjic et al., 1994, Klenovsek et al., 2007, Nigro and Adler, 2010, Slavuljica et al., 2010) and consequently, passive immunization using highly effective CMV-specific hyperimmune globulin preparations, has shown to be effective for prevention of disease caused by CMV (Nigro et al., 2005). Today, novel molecular tools allow us to understand the nature of protective antibody responses and potential to develop effective defined therapeutic antibody preparations against CMV in vitro.

CMV-specific monoclonal antibodies have been developed using a variety of techniques involving hybridoma technology, cell sorting and molecular engineering approaches (Ohlin et al., 1993, Spaete et al., 1994, Axelsson et al., 2009, Macagno et al., 2010, Pötzsch et al., 2011). With the advent of single cell sorting and in vitro display technologies it is now possible to rapidly generate monoclonal antibody fragments against virtually any antigen. These systems also enables for high-throughput development of recombinant antibody fragments using automated systems for selection and screening (Hallborn and Carlsson, 2002, Turunen et al., 2009, Hust et al., 2011). However, these techniques often involve the development and production of monovalent antibody fragments, a format that is not always suitable for assessment of neutralizing activity against CMV (Lantto et al., 2002b, Barrios et al., 2007). The biological activity of the monovalent antibody format may be poor for instance due to their small size, lack of ability to cross-bind epitopes or faster off-rate reaction kinetics compared to an intact antibody. Additionally, such antibody fragments lack the immunoglobulin Fc region that contributes to effector functions that are vital for biological activity of some CMV-specific antibodies (Spaete et al., 1994).

The combination of phage display technology and high-throughput assay systems is an attractive set of components in a workflow for the generation of specific antibodies against CMV. A limitation in the process (Fig. 1) is to rapidly advance from initial hits, where the antibody fragments are monovalently displayed on phages, to assessment of the biological function of initially identified binders. To address these issues we have investigated tools that are applicable in large scale screening efforts to identify specificities against CMV with appropriate biological activity. The aim is to combine tools facilitating semi-high throughput divalent recombinant antibody production (Moutel et al., 2009) and a rapid screening system to assess biological activity by using a newly developed luciferase-based neutralization test (Reinhard et al., 2011). In this proof-of-concept study, we demonstrate that this approach allows for detection of neutralizing CMV-specific recombinant antibodies. We suggest that this methodology will contribute to future development of antibodies with desired biological function against CMV. It may also provide essential information about functional activity of clones induced in efforts, which despite the associated difficulties, are aimed at developing an effective vaccine against CMV.

Section snippets

Anti-CMV single-chain variable fragments

Recombinant single chain fragment variable (scFv) used in this study have previously been created from transcripts derived from human monoclonal hybridomas or selected from molecular libraries using phage display. The extensively characterized scFv are specific for different epitopes on CMV glycoprotein B: AD-1 (I2-1 and S2-8) (Barrios et al., 2007), AD-2 (AE11F, F3c10/2, D3-3(2)/11 and H3b/31) (Lantto et al., 2002a, Lantto et al., 2002b, Lantto et al., 2003). In addition, a less characterized

Production and characterization of scFv-Fc specific for CMV

Screening the biological activity for a large number of antibodies requires that they are available in a biologically active format. Effective neutralization of human CMV by a number of CMV-specific antibody fragments has previously been shown to be dependent on the presence on dimeric binders (Lantto et al., 2002b, Barrios et al., 2007). To facilitate efficient assessment of biological activity of CMV-specific antibody fragments in future efforts to develop CMV-neutralizing binders, we have

Discussion

Due to their high specificity and stability in vivo, monoclonal antibodies are an important future source of reagents with potential use in protection against viral infection and disease. Recombinant antibody technology has become an important tool for the development of anti-viral antibodies and for the assessment of functional antibody responses against viral disease (Barbas and Burton, 1996). Phage display has been used for the generation of antibody fragments specific for viruses, including

Disclosure statement

This work was supported by grants from BioInvent Int AB (M.O.) and the Deutsche Forschungsgemeinschaft through He2526/7-2 (M.T.). The authors declare no other conflicts of interest.

Role of the funding source

The funding sources have no role in the design of the study, interpretation of the results, in the drafting of the manuscript or in the decision to publish this work.

Acknowledgments

The authors gratefully acknowledge Liselotte Andersson and Mats Mågård for their help with the mass spectrometry experiments and Fredrik Levander and BILS (Bioinformatics Infrastructure for Life Sciences) for performing the analysis of MS data.

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