Viral receptor blockage by multivalent recombinant antibody fusion proteins: inhibiting human rhinovirus (HRV) infection with CFY196

Fang Fang* and Mang Yu

NexBio, Inc., 6650 Lusk Boulevard, Suite B102, San Diego, CA 92121, USA


    Abstract
 Top
 Abstract
 Introduction
 The common cold and...
 Blocking HRV receptor by...
 Receptor blockage by multivalent...
 References
 
Viral receptor blockage by monoclonal antibodies (mAbs) is a common strategy to inhibit viral infection; however, no drug candidate acting by this mechanism has reached the market so far. Analysis of the experimental and clinical data on a receptor-blocking mAb, mAb 1A6, against human rhinovirus (HRV) major receptor intercellular adhesion molecule 1 (ICAM-1) reveals that insufficient avidity of the mAb is probably the key factor to blame for its unsatisfactory clinical efficacy. In this article, we have summarized the recently published work from Perlan Therapeutics, Inc. and others that involves creation of multivalent Fab fusion proteins against the HRV major receptor ICAM-1. The multivalent Fab fusion proteins have exhibited much improved avidities over conventional mAbs, as well as superior HRV inhibition in vitro. This work has led to a promising drug candidate, CFY196, for prevention and treatment of HRV infections. Multivalent recombinant Fab fusion proteins may herald a new generation of potent antiviral receptor blockers and other therapeutic molecules.

Keywords: monoclonal antibodies, multivalent receptor blockers, antiviral


    Introduction
 Top
 Abstract
 Introduction
 The common cold and...
 Blocking HRV receptor by...
 Receptor blockage by multivalent...
 References
 
One of the proven methods to prevent and inhibit viral infections is to block host cell receptors that are used by viruses to gain cell entry. Receptor blockage can be achieved by various strategies, one of which is using monoclonal antibodies (mAbs) that bind to specific epitopes on the receptor molecules. A plethora of in vitro studies have reported effective viral inhibition by receptor-blocking mAbs. However, so far these studies have not yielded any approved drug on the market. In some cases, such as human rhinovirus (HRV) infection, efficacies of the mAbs in humans were less than satisfactory. Careful evaluation of the strategy reveals that insufficient avidities, or functional affinities, of the receptor-blocking antibodies are often the factors that limit the therapeutic efficacy of the mAbs. This point is clearly demonstrated in the case of HRV receptor-blocking antibodies.

High avidity binding is afforded by multivalency, in which multi-subunit molecular complexes simultaneously bind to more than one identical target molecules that are present on a solid or semi-solid surface, such as cell membrane. Kinetically, multivalency prolongs binding by decreasing the rate of dissociation (koff). The same mechanism is well adapted by viral particles, which have multiple receptor binding sites for attachment to host cell receptors. To compete effectively against viruses for sustained receptor binding, viral receptor blockers need to exhibit superior binding avidities; otherwise viruses would eventually gain an upper hand in the competition.

The avidity of a single molecule can be enhanced exponentially by a complex that contains two, three or more identical molecular subunits. Applying this mechanism to the design of receptor blockers against HRV major receptor intercellular adhesion molecule 1 (ICAM-1), Perlan Therapeutics, a San Diego-based biotech company, has created multivalent recombinant antibody Fab fusion proteins that have shown significantly higher avidities and in vitro functional improvements over conventional mAbs.1 In this article, we will briefly summarize the history of developing HRV receptor-blocking antibodies and discuss the scientific rationale and experimental findings of a new drug candidate, CFY196. In addition, the technology for generating high avidity binding molecules like CFY196 will have much broader applications; some potential applicable areas for the technology will also be discussed.


    The common cold and HRV
 Top
 Abstract
 Introduction
 The common cold and...
 Blocking HRV receptor by...
 Receptor blockage by multivalent...
 References
 
The common cold is the most prevalent disease affecting humans. An average individual experiences about 200 colds in a lifetime, totalling over 5 years of suffering with colds. Social and economic impacts of the common cold are enormous, with more than $40 billion in economic loss each year in the USA alone.2 Viruses causing colds, in particular HRV, are responsible for many complications, including ear infections, asthma attacks and exacerbation of sinusitis, bronchitis, chronic obstructive pulmonary disease, etc. Over-the-counter cold remedies only manage cold symptoms, and do not prevent the common cold or cold complications. Therefore, effective prophylaxis against the common cold is highly desirable, especially for people who have high risk of developing common cold complications.

Approximately 50% of colds on an annual basis are caused by human rhinoviruses (HRV);3,4 in the autumn, HRV can cause over 80% of colds.5 HRV includes over 100 distinct serotypes with a large genetic diversity,6 which renders vaccine development unfeasible. HRV infects the human upper respiratory tract; the attachment and entry of HRV is mediated by specific interactions between HRV virions and receptors on the surface of respiratory epithelial cells. Based on their selection of receptors, HRVs are grouped into two families (except for HRV-87): the major group and the minor group.6 The major group contains at least 91 serotypes, all of which exclusively bind to host cell receptor ICAM-1.79 The minor group contains about 10 serotypes, and their cellular receptor is either the low-density lipoprotein receptor (LDLR) or 2-macroglobulin receptor/LDLR-related protein (2MR/LRP), or both.10


    Blocking HRV receptor by monoclonal antibody
 Top
 Abstract
 Introduction
 The common cold and...
 Blocking HRV receptor by...
 Receptor blockage by multivalent...
 References
 
Since binding to major receptor ICAM-1 is the critical step in the infectious cycle of the majority of HRV serotypes, blocking HRV’s access to ICAM-1 will prevent infection. This concept is primarily supported by the work of Colonno over a decade ago.11 The group led by Colonno generated a monoclonal antibody against ICAM-1, referred to as mAb 1A6 or RRMA. mAb 1A6 prevented HRV infection both in tissue culture and in chimpanzees.11 In addition, intranasal administration of the antibody in chimpanzees for up to 1 year did not produce detectable local or systemic toxicity.11 Furthermore, Hayden et al.12 demonstrated in a human challenge trial that short-term intranasal administration of mAb 1A6 at high dosage delayed the onset of HRV-induced colds in human volunteers by up to 2 days, and reduced the severity of symptoms by up to 50%. However, the antibody treatment failed to reduce the incidence of the common cold in the trial, although it did delay the onset of the disease.12 This result indicated that receptor blockage by mAb 1A6 was partial and unsustainable, although it was unclear whether the lack of sustained effect was the result of incomplete blockade, receptor turnover or dissociation of antibody from receptors.

Casasnovas & Springer13 later provided further evidence that anti-ICAM-1 mAbs do not have real kinetic advantage over HRV virions to provide sustained receptor blockage against HRV. By comparing the ICAM-1 binding dissociation kinetics of three anti-ICAM-1 mAbs and HRV virions, they showed that even the best mAb (mAb RR1/1, Kd = 38 nM) would dissociate from ICAM-1 at essentially the same rate (koff) as HRV virion dissociation from ICAM-1; i.e. the half-life of the mAb–ICAM-1 binding complex is almost identical to that of the HRV–ICAM-1 complex (t = 0.69/koff). Under such conditions, HRV particles can eventually gain access to the receptor and infect cells. These findings suggest that the failure of mAb 1A6 to prevent HRV infection in human trial was, at least in part, due to its insufficient avidity as an HRV receptor blocker.


    Receptor blockage by multivalent antibody fusion proteins
 Top
 Abstract
 Introduction
 The common cold and...
 Blocking HRV receptor by...
 Receptor blockage by multivalent...
 References
 
Theoretically, the problem of insufficient avidity can be overcome by multivalent recombinant antibodies that exhibit higher avidity than conventional mAbs. Previously, a variety of multivalent antibody fusion proteins have been engineered by fusing scFv fragments of antibodies with polymerization domains, such as streptavidin,14 yeast GCN4 leucine zipper domain15 or the tetramerization domain of human p53 protein.15 Although some of the prior multivalent scFv fusion proteins indeed exhibited improved avidities, it was reasoned that multivalent Fab fusion proteins would be superior, since antibody Fab fragments are generally more stable and have higher binding affinities than scFv fragments. The method developed at Perlan Therapeutics involves fusing Fab with a linker sequence and a multimerization domain derived from human coiled-coil sequences. Depending on the design of coiled-coil sequences, bivalent, trivalent and tetravalent Fab fusion proteins have been made. Such fusion molecules can be expressed and assembled in Escherichia coli, and purified in soluble homogeneous forms at good yields.1 Using this method, Perlan Therapeutics has generated a tetravalent Fab fusion protein, CFY196, against the HRV major receptor ICAM-1.1

CFY196 is composed of an Fab fragment of a humanized version of mAb 1A616 fused with a linker derived from human immunoglobulin D hinge and a tetramerization domain derived from the coiled-coil sequence of human transcription factor ATF{alpha}. CFY196 is expressed and purified as a homogenous molecular complex that is consistent with tetravalent Fab as determined by size-exclusion chromatography, analytical ultracentrifugation and dynamic light scattering.1 Based on the kinetic parameters measured by BIAcore analysis at 1650 response units of immobilized ICAM-1, the tetravalent CFY196 exhibited an almost two orders of magnitude reduction in koff compared with its bivalent counterpart. Such kinetic improvement also directly leads to functional superiorities of CFY196. In in vitro assays using three HRV serotypes (HRV-14, -16 and -39), CFY196 consistently and significantly outpaced its bivalent, trivalent counterparts, and the best commercial anti-ICAM-1 mAbs, in preventing HRV infection as measured by reduction of cytopathic effects and HRV viral titres. CFY196 was 13 times as effective as mAb RR1/1 in preventing HRV-induced cell death in HeLa cells.1

Hayden’s group has also independently performed tests on CFY196. They reported that 50% effective concentrations (EC50s) of CFY196 against three major group HRV serotypes were in the range of 15.9–105 ng/mL on HeLa-I and human embryonic lung fibroblast WI-38 cells.17 No inhibitory effect was observed for one minor serotype, which uses a different cell receptor. Yield reduction assays on HeLa-I monolayers showed that CFY196 inhibited the replication of HRV-39 in a concentration-dependent manner and, at low viral inocula, completely prevented HRV replication when added up to 2 h after infection at 1 µg/mL. It was much more active than an mAb against ICAM-1. Single cycle of replication experiments with the addition of CFY196 at 10 µg/mL at different time intervals against high inoculum (multiplicity of infection = 2) showed that the inhibitory effect occurred only when CFY196 was added before or at the same time as the virus. When viral absorption was carried out at 4°C and subsequent incubation at 34°C, it was found that CFY196 had to be present during the absorption period to have an inhibitory effect. When CFY196 at 10 µg/mL was exposed to cells and then removed at different time intervals before HRV-39 inoculation, single cycle studies found that an inhibitory effect was detectable up to 4 h prior to virus infection. In contrast, a capsid binder (pleconaril) and a 3C protease inhibitor (ruprintrivir) were not inhibitory under such conditions.17

In summary, CFY196 has demonstrated superior avidity and in vitro potency against HRV infection over conventional mAbs. Such findings indicate that testing of human efficacy of CFY196 is warranted; especially since mAb 1A6 has already exhibited positive effects in a human trial. Further preclinical and clinical development of CFY196 is warranted to fully evaluate its potential as a prophylactic and therapeutic agent for the HRV-induced common cold.

The technology used to generate CFY196 can also be applied to other areas where high avidity binding is desired. Additional multivalent receptor blockers may be developed to inhibit binding of not only viruses, but also various bioactive ligands such as growth factors, hormones, cytokines, etc. On the other hand, making multivalent receptor agonists can be yet another exciting application of the technology, as it may yield some highly desirable therapeutic effects, such as killing cancer cells by multivalent cross-linking and subsequent activation of receptors that induce apoptosis. As a novel multivalent recombinant fusion protein, CFY196 may herald a new generation of potent therapeutic molecules.


    Footnotes
 
* Corresponding author. Tel: +1-858-452-2631; Fax: +1-858-452-2534; E-mail: ffang{at}nexbio.com Back


    References
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 Abstract
 Introduction
 The common cold and...
 Blocking HRV receptor by...
 Receptor blockage by multivalent...
 References
 
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