Development of Motavizumab, an Ultra-potent Antibody for the Prevention of Respiratory Syncytial Virus Infection in the Upper and Lower Respiratory Tract

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Abstract

Respiratory syncytial virus (RSV) is the leading cause of viral bronchiolitis and pneumonia in infants and children. Currently, palivizumab is the only approved monoclonal antibody (mAb) for prophylaxis of RSV. However, a small percentage of patients are not protected by palivizumab; in addition, palivizumab does not inhibit RSV replication effectively in the upper respiratory tract. We report here the development and characterization of motavizumab, an ultra-potent, affinity-matured, humanized mAb derived from palivizumab. Several palivizumab variants that enhanced the neutralization of RSV in vitro by up to 44-fold were generated; however, in vivo prophylaxis of cotton rats with these antibodies conferred only about a twofold improvement in potency over palivizumab. This unexpected small increase of in vivo potency was caused by poor serum pharmacokinetics and lung bio-availability that resulted from unexpectedly broad tissue binding. Subsequent analyses revealed that changes at three amino acids arising from the affinity maturation markedly increased the non-specific binding to various tissues. Our results suggested that kon-driven mutations are more likely to initiate non-specific binding events than koff-driven mutations. Reversion of these three residues to the original sequences greatly diminished the tissue binding. The resulting mAb, motavizumab, binds to RSV F protein 70-fold better than palivizumab, and exhibits about a 20-fold improvement in neutralization of RSV in vitro. In cotton rats, at equivalent concentrations, motavizumab reduced pulmonary RSV titers to up to 100-fold lower levels than did palivizumab and, unlike palivizumab, motavizumab very potently inhibited viral replication in the upper respiratory tract. This affinity-enhanced mAb is being investigated in pivotal clinical trials. Importantly, our engineering process offers precious insights into the improvement of other therapeutic mAbs.

Introduction

Respiratory syncytial virus (RSV) poses a great public health threat worldwide. It is highly contagious and can cause repeated infections in both the upper (URT) and lower respiratory tract (LRT). It is the leading cause of viral bronchiolitis and pneumonia in infants and children,1 and can induce severe disease in the LRT in elderly adults and immunocompromised patients. In the United States during 1994–1996, RSV was estimated to cause between 73,400 and 126,300 hospitalizations annually for bronchiolitis and pneumonia among children under 1 year of age.2 From 1990–1999 in the United States, the annual mean number of RSV-associated all-cause deaths for all ages was estimated at 17,358 cases.3 The same study showed that RSV was the most common viral cause of death in children less than 5 years of age, particularly in children under 1 year of age.

RSV is a member of the Pneumovirus genus, of the family Paramyxoviridae. The virus comprises a medium-sized (150–300 nm) envelope with a single negative-strand RNA. The viral genome is transcribed into ten transcripts encoding 11 proteins, including three envelope proteins found on the surface of virions and infected cells, designated F (fusion) protein, G (attachment) protein and SH (small hydrophobic) integral membrane protein.4 The G protein mediates the viral attachment to the host cells, while the F protein plays a role in cell penetration by the virus and promotes spread of the virus from cell to cell through the formation of syncytia. In the late 1980s, a number of neutralizing murine monoclonal antibodies (mAbs) specific for the RSV F glycoprotein were discovered.5 A comparison of the biological and biochemical properties of these neutralizing mAbs led to the identification of three non-overlapping antigenic sites (A, B and C) and one bridge site (AB). The sites A and C, but not B, are generally conserved among clinical virus isolates.

We describe here the development of an ultra-potent anti-RSV mAb, motavizumab (also known as MEDI-524), which is a humanized mAb directed to an epitope in the A antigenic site of the RSV F protein. Motavizumab was derived from affinity maturation of Synagis® (palivizumab; MedImmune, Inc.), a licensed product that is indicated for the prevention of serious LRT disease caused by RSV in children at high-risk, such as infants with bronchopulmonary dysplasia or with a history of premature birth, or children with congenital heart disease. Palivizumab has the ability to neutralize broadly clinical isolates of the RSV A and B subtypes.6 Our attempts to improve the binding kinetics of palivizumab to F protein by improving both the association rate (kon) and dissociation rate (koff) resulted in up to 44-fold improvement in the ability to neutralize RSV in vitro.7 In the current study, several of these affinity-optimized variants have been evaluated for their ability to inhibit prophylactically the replication of RSV in cotton rats (Sigmodon hispidus). Among these, the most potent mAb, A4b4 was further engineered to enhance its efficacy in vivo by improving its pharmacokinetics and removing undesired tissue cross-reactivity. The resulting molecule, motavizumab, has a 34.6 pM avidity for the F protein, differs from palivizumab by only 13 amino acid residues, and reduced RSV titers in the lung and nasal turbinates of cotton rats up to 100-fold better than palivizumab. The enhanced ability of motavizumab to prevent RSV infection in both the URT and LRT may lead to better clinical outcomes and the reduction of RSV-related hospitalizations compared to palivizumab. Motavizumab is being evaluated in multiple human clinical trials.

Section snippets

Prophylaxis of RSV infection in cotton rats by palivizumab variants

Earlier, we reported the generation of many highly potent anti-RSV palivizumab variants.7 A directed evolution approach was applied to improve both koff and kon for F protein binding. In the Fab format, when assayed in the RSV microneutralization assay, these palivizumab variants showed up to a 1500-fold improvement in activity; in the IgG format, the variants showed up to a 44-fold improvement over the parent. Figure 1 shows the amino acid sequences of palivizumab and several improved

Discussion

Currently passive immunization constitutes the only prophylactic treatment for RSV, and palivizumab is the only mAb approved for this approach. Prophylaxis with palivizumab reduced hospitalization from RSV infection in high-risk infants by 55% (10.6% hospitalization in the placebo group versus 4.8% in the palivizumab group).11 Enhancement of the activity of palivizumab may further reduce the hospitalization rate and inhibit RSV replication in the URT more effectively. Thus, a more potent

F protein

The F protein used in this study was the extracellular domain of the F protein (from RSV strain A2) expressed in a baculovirus expression system,26 and purified by an antibody-based affinity column chromatography.5,7

Modifications of palivizumab and its variants, 493L1FR and A4b4

To identify the detrimental residues that affect the behavior of A4b4 in vivo, individual mutations were made at each of the four affinity-matured residues on the VL of A4b4;7 the R29S, F52S, F53K and D55A mutations were generated by the use of the QuickChange XL Site-Directed

Acknowledgements

We thank Joan R. Wicks and Jennifer L. Rojko at Pathology Associates (Frederick, MD), a Charles River Company, for the tissue cross-reactivity study. We thank Kevin Yim at Virion Systems, Inc. (Rockville, Maryland) for performing some of the cotton rat studies.

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    Present address: S. Johnson, MacroGenics, Inc., 1500 East Gude Drive, Rockville, MD 20850, USA.

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