Effects of humanization by variable domain resurfacing on the antiviral activity of a single-chain antibody against respiratory syncytial virus

Simon Delagrave1, John Catalan, Charles Sweet, Glenn Drabik, Andrew Henry2, Anthony Rees2,3, Thomas P. Monath and Farshad Guirakhoo4

OraVax Inc., 38 Sidney Street, Cambridge, MA 02139, 1 Hercules Inc.,500 Hercules Road, Mail Stop 8136/216, Wilmington, DE 19808, USA, 2 Oxford Molecular Ltd, The Medawar Centre, Oxford Science Park,Oxford OX4 4GA and 3 School of Biology and Biochemistry, University of Bath, Claverton Down, Bath, Avon BA2 7AY, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
HNK20 is a mouse monoclonal IgA that binds to the F glycoprotein of respiratory syncytial virus (RSV) and neutralizes the virus, both in vitro and in vivo. The single-chain antibody fragment (scFv) derived from HNK20 is equally active and has allowed us to assess rapidly the effect of mutations on affinity and antiviral activity. Humanization by variable domain resurfacing requires that surface residues not normally found in a human Fv be mutated to the expected human amino acid, thereby eliminating potentially immunogenic sites. We describe the construction and characterization of two humanized scFvs, hu7 and hu10, bearing 7 and 10 mutations, respectively. Both molecules show unaltered binding affinities to the RSV antigen (purified F protein) as determined by ELISA and surface plasmon resonance measurements of binding kinetics (Ka {approx} 1x109 M–1). A competition ELISA using captured whole virus confirmed that the binding affinities of the parental scFv and also of hu7 and hu10 scFvs were identical. However, when compared with the original scFv, hu10 scFv was shown to have significantly decreased antiviral activity both in vitro and in a mouse model. Our observations suggest that binding of the scFv to the viral antigen is not sufficient for neutralization. We speculate that neutralization may involve the inhibition or induction of conformational changes in the bound antigen, thereby interfering with the F protein-mediated fusion of virus and cell membranes in the initial steps of infection.

Keywords: antibody/immunotherapy/resurfacing/single-chain Fv


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Humanization by variable domain resurfacing has been proposed as a means of eliminating potential antigenic sites of clinically useful antibodies while preserving binding affinity (Padlan, 1991Go; Roguska et al., 1994Go, 1996Go). This alternative to CDR grafting has been shown experimentally to work well on antibodies recognizing cell-surface ligands (Pedersen et al., 1994Go). Despite the growing importance of humanized recombinant antibodies in the treatment and prevention of viral infections (Co et al., 1991Go; Major et al., 1994Go; Ryu et al., 1996Go; Hamilton et al., 1997Go), there is little information available on the effects of variable domain resurfacing on antiviral activity.

Preservation of the humanized antibody's binding affinity is a primary objective in any humanization attempt. While affinity is clearly an important correlate of in vivo activity for many applications such as anti-tumor immunotherapy (Disis and Cheever, 1997Go), it has also been found that more complex molecular mechanisms than a simple `lock and key' model can underlie antibody activity. For example, the notion that antibodies sterically hinder interactions between viral envelope proteins and cell-surface receptors is not unfounded (Colonno et al., 1989Go), but in many instances it is not supported by experimental evidence. Antibody binding is not sufficient, for instance, to neutralize polio virus (Emini et al., 1983Go; Icenogle et al., 1983Go; Wien et al., 1995Go). In such cases, it has been proposed that antibodies induce conformational changes in the bound viral proteins or prevent conformational changes necessary for infection (Wien et al., 1995Go). In the case of HIV, neutralization by antibody b12 involves inhibition of fusion-uncoating mechanisms while leaving cell attachment unaffected (McInerney et al., 1997Go). Such findings imply that binding affinity is not the only parameter by which the effects of humanization on neutralizing antibodies should be judged.

HNK20 is a mouse monoclonal IgA which efficiently neutralizes respiratory syncytial virus (RSV) and has been investigated in a clinical setting for prevention of RSV infection in premature infants (Weltzin et al., 1994Go; Weltzin, 1996Go). The mechanism by which HNK20 neutralizes virus is not fully understood but it is known that the Fusion protein (F protein) of RSV is recognized specifically and with high affinity (Ka >= 1x109 M–1). It has previously been shown that RSV can be neutralized by a single-chain Fv (scFv) version of HNK20 and that the scFv binds F protein with an affinity similar to that of its parent molecule (Guirakhoo et al., 1996Go). This scFv provides an easily expressed and purified proxy of the monoclonal antibody, allowing us to determine rapidly the effect of humanization substitutions on affinity and neutralization activity. Information gained in these mutagenesis experiments may then be used to construct a fully humanized recombinant immunoglobulin with maximal antiviral activity (Delagrave et al., in preparation).

We present data describing the effects of mutations necessary for humanization by resurfacing on the binding affinity and antiviral activity, in vitro and in an animal model, of a single-chain Fv version of HNK20. As discussed, our observations have implications for the mechanism of RSV neutralization by HNK20 and, more generally, for the humanization of other antiviral antibodies.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Humanization algorithm

The algorithm for humanization of antibody variable domains by resurfacing was followed essentially as described (Roguska et al., 1994Go, 1996Go). Surface residues of HNK20 Fv (defined as having >30% relative solvent accessibility) were found by (i) searching the protein structure database at Brookhaven (PDB) for the subset of antibodies most homologous to the HNK20 Fv, (ii) identifying surface residues in these structures and (iii) aligning the sequences of these antibodies of known structure to the sequence of HNK20 to identify homologous positions.

In order to humanize the surface residues of the murine HNK20 variable domain, a set of highly homologous surface residues from a human target sequence must be selected. The Kabat database (Kabat et al., 1991Go) was searched to identify human VL and VH sequence pairs which are known to be coexpressed in a mature antibody and are most homologous to HNK20 Fv. Sequences of phage-displayed or humanized antibodies were eliminated from the search results.

Mutations in the HNK20 scfv were modeled with the AbM antibody structure modeling program (Oxford Molecular, Oxford, UK). The structures of antibodies HuH52-AA (PDB code 1FGV) and 8F5 (PDB 1BBD) were used to model the framework backbones of VL and VH, respectively. Both sequences were 70–75% identical with HNK20, which is necessary for a good prediction of CDR positions. Canonical structures (Chothia et al., 1992Go) were used to predict the backbone structures of CDRs L1-3 and H2. CDR H1, which did not correspond to a canonical class, was modeled by searching the PDB for protein segments fitting in the framework structure. A PDB search combined with modeling of backbone conformations using CONGEN (Bruccoleri and Karplus, 1987Go) was used to model CDR H3.

DNA manipulations and bacterial strains

The murine and humanized scFv genes were cloned in the pET26b(+) expression vector (Novagen, Madison, WI). Plasmid DNA was propagated in Escherichia coli XL1-Blue (Stratagene, La Jolla, CA) and purified using Qiagen kits (Qiagen, Chatsworth, CA). The E.coli strain BL21 (DE3) (Novagen) was used to express murine and humanized scFv constructs. Restriction and DNA modification enzymes were from New England Biolabs (Beverly, MA). The QuickChange mutganesis kit (Stratagene) was used for resurfacing mutagenesis. The entire open reading frames of all scFv expression vectors were sequenced before expression and purification. Sequencing reactions were carried out using FS sequencing reaction mix and analyzed on a Model 310 Genetic Analyzer (Perkin-Elmer/Applied Biosystems, Foster City, CA).

ScFv purification

Ni2+ or Co2+ Talon resins (Clontech, Palo Alto, CA) were used as described (Guirakhoo et al., 1996Go) or according to manufacturer's instructions to purify all the scFvs described. After purification and dialysis against 1x PBS (phosphate-buffered saline), purified scFv was quantitated by BCA assay (Pierce, Rockford, IL). All purified scFv samples had concentrations of >1 mg/ml. Aliquots were adjusted to a final concentration of 1 mg/ml and quantitated again prior to performing the side-by-side comparisons described here. SDS–PAGE was performed to verify that all samples had similar concentration and purity (data not shown).

ELISA

As described previously (Guirakhoo et al., 1996Go), microtiter plates were coated overnight at 4°C with RSV-infected cell lysate diluted 1:1000 in carbonate–bicarbonate buffer. Plates were blocked, washed and serial dilutions of scFv samples added. Bound scFv was detected by addition of a polyclonal mouse anti-scFv serum followed by an alkaline phosphatase-labeled anti-mouse IgG antibody (Southern Biotechnology, Birmingham, AL). Binding curves were fitted to sigmoidal dose–response curves with variable slopes using Prism 2.0 (GraphPad Software, San Diego, CA).

A whole-virus ELISA was designed to compare the binding characteristics of purified F protein with those of unpurified (presumably native) F protein in the context of the viral envelope. A high-titer suspension of RSV was produced by infecting a nearly confluent monolayer of Vero cells in a T150 flask with a multiplicity of infection of 0.1 and incubating the culture at 37°C in a CO2 incubator for 4 days. Growth medium and cells were recovered from the flask and sonicated for 30 s on ice (setting 5, 50% duty, using a Branson Sonifier 450). The resulting sonicate was centrifuged at 2000 g for 10 min at 4°C and the supernatant was recovered and used as described below.

A microtiter plate was coated with polyclonal anti-RSV serum (Biodesign, Kennebunk, ME), blocked with 2.5% dry milk in PBST (PBS + 0.05% Tween 20) and washed three times with PBST followed by a wash with PBS. RSV 2x106 pfu/ml, diluted in MEM + 10% FBS, was allowed to bind to the plate for 1 h at room temperature. Wells were washed three times with PBS. A solution containing HNK20 at a constant concentration of 250 ng/ml and serially diluted scFv was added to the wells and incubated at room temperature for 1 h. After washing with PBS, an alkaline phosphatase-labeled anti-mouse IgA monoclonal (Southern Biotechnology) was added to the wells for 1 h at room temperature. Following three PBS washes, a colorimetric substrate (p-nitrophenol phosphate, Sigma) was added and optical density at 405 nm was measured with a plate reader.

Binding kinetics and affinity measurements

Surface plasmon resonance technology implemented on a BIAcore instrument (BIAcore, Piscataway, NJ) was used to characterize the binding kinetics of purified scFv. Antibodies were diluted in HEPES-buffered saline (HBS) and injected on CM-5 chips covalently linked to purified RSV F protein as described (Guirakhoo et al., 1996Go).

In vitro neutralization

Microneutralization experiments were performed using a modification of a protocol described by Anderson et al. (1985). In a microtiter plate, dilutions of antibody were mixed with 103 pfu/ml of RSV (strain A2) in MEM at 37°C in a CO2 incubator for 1 h. Vero cells (104/ml) were then added to all wells. Some wells serving as controls contained cells only (0% infection) or virus and cells but no antibody (100% infection). After 4 days of incubation in a CO2 incubator, cell monolayers were fixed in 10% buffered formalin and, from that point, treated as in an ELISA where virus was detected with biotin-labeled anti-RSV (BioDesign) and alkaline phosphatase–streptavidin conjugate (Calbiochem, San Diego, CA). About 20–40 min after addition of a solution containing substrate (p-nitrophenyl phosphate) and 4 mM levamisole (both purchased from Sigma, St Louis, MO), OD405 was measured with an ELISA plate reader. Results are expressed as a percentage of maximum neutralization (= 100 – % infection) and were analyzed with Prism 2.0 (GraphPad Software).

In vivo therapy experiments

In vivo experiments were carried out as described previously (Weltzin et al., 1994Go) with slight modifications: groups of four or five female BALB/c mice, 6–8 weeks old, were infected intranasally (i.n.) on day 0 with 5x105 pfu of RSV (isolated at OraVax from a clinical sample) in a volume of 50 µl. Animals were treated on days 1, 2 and 3 with 77 µl (= 10 µg) i.n. of antibody solution in PBS. Intranasal administration was performed by lightly anesthetizing mice by isoflurane inhalation and pipetting the required volume on to the end of the nose where it was inhaled. On day 4, mice were killed and lung viral titers were determined essentially as described (Weltzin et al., 1994Go), except that plaque assays were enhanced by the use of an immunostaining method. In this assay, Vero cell monolayers infected with virus dilutions were overlayed with 0.75% methylcellulose in MEM containing 10% FBS and, after 3 days in a CO2 incubator, fixed with 10% buffered formalin. The fixed monolayer was blocked with non-fat dry milk (2.5% in PBS containing 0.05% Tween 20). After addition of biotin-labeled goat anti-RSV serum (BioDesign) followed by alkaline phosphatase-labeled streptavidin (Calbiochem), the substrate solution (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium or BCIP/NBT) was added and plaques were counted.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Humanization

Surface residues on the variable domain of HNK20 were identified by aligning its sequence with a set of ten homologous antibody sequences of known three-dimensional structure. Antibodies 1bbd and 1nma (PDB codes), for example, were 69 and 67% identical with HNK20, respectively (alignments not shown). The surface positions of these 10 antibodies were found as described previously (Pedersen et al., 1994Go) and mapped to homologous positions in HNK20, which are predicted to be exposed to solvent to a similar extent.

A human target sequence homologous to HNK20 Fv was identified by searching the Kabat database. By combining information on which positions are surface exposed with an alignment of the most homologous human Fv sequence, surface positions of HNK20 displaying non-human residues were identified. Figure 1AGo shows the human antibody sequence (III-2R) most homologous to surface residues of the HNK20 variable heavy (VH) and variable light (VL) chains. The figure also shows the predicted surface residues of both antibodies. Some sequences were more homologous to HNK20 than III-2R, but these were discounted because they were humanized antibodies themselves or were isolated by phage display. Also, only VL and VH sequences known to be coexpressed in a functional antibody were considered.



View larger version (47K):
[in this window]
[in a new window]
 
Fig. 1. (A) Alignment of mouse antibody HNK20 VL and VH amino acid sequences with human antibody III-2R VL and VH. Dots represent identical amino acids. Conserved surface framework residues are marked by clear boxes, cyan boxes highlight non-conserved surface framework residues and yellow boxes highlight non-conserved surface framework residues which may be involved in antigen binding (`back-mutated' residues). Residues are numbered above each alignment in a sequential scheme. (B) Molecular model of the mu scFv showing the non-conserved surface residues in relation to the CDR [residues numbered as in (A)]. The CDR residues are colored orange, the four back-mutated residues (HN28, HT78, LK3 and LQ108) are shown in yellow and the remaining non-conserved surface residues are in cyan. The two views are of the same molecule rotated 180°.

 
Out of 22 surface residues in the VH chain, 15 (68%) were conserved between human and mouse sequences whereas in the VL chain, 15 of 19 surface residues (79%) were conserved. This leaves seven residues in VH and four residues in VL which could be mutated to make all Fv surface amino acids of HNK20 identical with those of a human Fv. Consideration must be given, however, to potential negative effects on binding affinity which can be caused by mutations near the antigen binding site. Molecular modeling (Figure 1BGo) with AbM identified three residues which were in direct contact with the CDRs: LK3, HN28 and HT78 (amino acids are identified by their chain, H or L, the single-letter amino acid code and their position in the sequence). An additional non-conserved surface residue, LG108, is near the CDRs (within ~5 Å). The mutation needed to introduce a human residue at this position (LG108Q) constitutes a significant change of physico-chemical character which may not be necessary since Gly is observed at position 108 in 33% of human {kappa} chain sequences. Therefore, of the 11 non-conserved surface residues, four (LG108, LK3, HN28 and HT78) will be `back-mutated' (i.e., left unmutated) in the first humanized construct designated `hu7' scFv (Figure 2Go). In an effort to investigate the effects of humanizing further the `mu scFv' (murine scFv) surface, an additional scFv variant bearing 10 changes (all but one, HT78K) was constructed and designated `hu10' scFv, as shown in Figure 2Go. Mutation, HT78K, was previously found to contribute to a decrease in binding affinity of antibody anti-B4 (Roguska et al., 1996Go) and therefore was omitted from the mutations we introduced in the scFv.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2. Schematic diagram of scFv (top) and sequence of mu scFv aligned with sequences of hu7 and hu10 scFv. Unchanged residues are represented by dots and mutations are indicated by the substituting amino acid. The numbers of mutated residues are shown above the mu scFv sequence, as in Figure 1Go.

 
Characterization of binding

A comparison was made between the binding affinities of the hu7 and hu10 scFvs and the affinity of the mu scFv by ELISA (Figure 3Go). An apparent binding affinity (EC50) of 2 nM (overlapping 95% confidence intervals of 1.2– 5.6 nM) was observed for all three molecules, as determined by fitting the data to a sigmoidal dose–response curve (R2 > 0.98 for all three curves). Further characterization of binding kinetics and affinity was performed for all three molecules by surface plasmon resonance measurements of scFv association with purified F protein (Table IGo). The observed Kd values of 0.8 nM for hu7 and hu10 scFvs and of 1.6 nM for mu scFv agree well with the values measured by ELISA.




View larger version (35K):
[in this window]
[in a new window]
 
Fig. 3. (A) ELISA comparison of binding to RSV-infected cell lysate of mu scFv and variants hu7 and hu10. Error bars represent the standard error calculated from triplicate measurements. (B) Competition ELISA in which HNK20 is competing for binding to captured RSV with serially diluted scFv molecules.

 

View this table:
[in this window]
[in a new window]
 
Table I. Binding kinetics and calculated affinity of humanized scFv variants recognizing the RSV F proteina
 
We conducted an experiment to confirm that purified F protein used in ELISA and BIAcore binding assays was in a native conformation and accurately reflected the interactions of antibody and virus. A whole virus competition ELISA was devised to determine whether all scFvs competed similarly with HNK20 for binding to captured RSV. Plates coated with a polyclonal antiserum against RSV were used to capture whole virus. HNK20 was held at a constant concentration in each well and incubated simultaneously with serially diluted aliquots of each scFv (mu, hu7 and hu10). As seen in Figure 3BGo, all three molecules competed equally well with HNK20 for binding to whole virus.

Characterization of antiviral activity

A microneutralization assay (Figure 4Go) shows the relative antiviral activities in vitro of hu7, hu10 and mu scFvs. The data can be fitted with sigmoidal dose–response curves from which EC50 values were determined. The slopes of the curves were roughly equal (ranging from 1.3 to 1.5) and R2 values were good (ranging from 0.96 to 0.97). Because of the range of scFv concentrations used in this assay, the maximum y-value of the hu10 curve was set to 100% and the minimum y-value of the mu curve was set to 20%, in keeping with the values observed with the other curves. The calculated EC50 values are 1, 6 and 20 nM for mu, hu7 and hu10 scFvs, respectively. For each EC50, a 95% confidence interval was also determined: mu 0.8–1.3, hu7 4.4–9 and hu10 15–26 nM. In contrast with affinity measurements reported above, the in vitro potencies of the mu, hu7 and hu10 scFvs are significantly different. Similar results were obtained in two separate experiments.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4. Microneutralization assay comparing the in vitro antiviral activity of mu scFv and humanized variants hu7 and hu10. Each data point is the mean (±SE, error bars) calculated from triplicate measurements obtained in a single experiment. The data were fitted with a variable-slope sigmoidal curve.

 
We determined the ability of the scFv variants to decrease RSV titers in mouse lungs following a 3-day treatment. As shown in Figure 5Go, the mu scFv provided the greatest decrease in RSV infection compared with all other molecules studied. The difference in effectiveness between hu7 and hu10 is statistically significant (p = 0.043 < 0.05) whereas that between mu and hu7 is not (p = 0.087 > 0.05). These results show a clear correlation between in vitro and in vivo antiviral activity; in terms of effectiveness, the mu scFv is slightly superior to hu7 which, in turn, is clearly superior to hu10.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 5. Comparison of viral titers in mice treated with an equal amount (10 µg) of different scFvs or HNK20 (positive control). Titers are reported as log10 of RSV pfu per gram of mouse lung.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have humanized a mouse scFv by variable domain resurfacing while maintaining a high binding affinity. In this process, one of the guiding principles is that the antibody structure should only be minimally modified to avoid problems of decreased affinity often encountered in other approaches such as CDR grafting (Winter and Harris, 1993Go). Of the 11 non-conserved surface residues identified, four were deemed too close to the CDRs to be included in the original set of mutations (LK3, LG108, HN28 and HT78). Three of these substitutions were subsequently shown not to affect adversely antibody binding to antigen. One substitution, HT78K, was previously found to contribute to a decrease in binding affinity of antibody anti-B4 (Roguska et al., 1996Go) and therefore was omitted from the mutations we introduced.

The antiviral activity of humanized clone hu7 was tested in a microneutralization assay as well as in vivo. The in vitro data showed a statistically significant difference in antiviral activity compared with the parental molecule (mu scFv), however, in the mouse model the difference in activity was small and not significant. It seems, therefore, that a conservative resurfacing approach was successful in preserving the biological activity of an antibody while presumably decreasing its immunogenicity. In contrast, adding the three mutations alluded to above proved to be an overly aggressive approach. Antiviral activity of hu10 was significantly decreased compared with hu7 and mu scFv, both in mice and in vitro.

Our experimental findings show that affinity and neutralization are not correlated in this particular antibody–virus system. Through mutagenesis of a few surface residues near the CDRs, we have effectively uncoupled virus binding and neutralization. Trivial explanations such as differing specific activities due to variations in scFv purity are unlikely because the binding affinities are clearly identical among the molecules tested. The possibility of differences in stability was investigated and also shown to be improbable (data not shown). This situation may be interpreted as mirroring the observations made by Blondel et al. (1986), who showed that a neutralization escape mutant of polio virus (Sabin 1-Kcr) was still bound by neutralizing antibody C3 but remained infective. This effect was due to a single amino acid mutation at residue 100 of VP1 from Asn to Lys. In addition, this same antibody is known to neutralize virus without preventing it from attaching to cells (Wien et al., 1995Go). The mechanism underlying this phenomenon is proposed to involve an inhibition or induction of conformational change in the bound viral protein, thereby preventing molecular events necessary for infection (Wien et al., 1995Go). Similarly, McInerney et al. (1997) recently published an analysis of HIV neutralization by antibody b12 where inhibition of cell attachment is clearly shown not to be involved. Since RSV attaches to target cells through its G protein, antibody binding to F protein is unlikely to prevent initial virus–cell interactions, as shown previously (Levine et al., 1987Go). Rather, we hypothesize that fusion of viral and cell membranes is prevented by HNK20 and its derivatives.

It is desirable to improve our understanding of the molecular events underlying our observations, as this could enhance our ability to develop new therapeutic or prophylactic agents against RSV. Although it is possible that some as yet undetermined property of the scFv variants will be shown to correlate with their antiviral activity, we favor the view that our results point to an unsuspected level of complexity in the neutralization mechanism. One possible mechanism would require binding of the antigen by the scFv, followed by an induced conformational change in the antigen. The F protein, after its conformational change, is unable to cause membrane fusion; it is effectively neutralized. In this hypothetical scenario, the mutations in hu10 have somehow decreased the likelihood of this conformational change such that most of the scFv–F protein complex is in a non-neutralized state. Since the binding and conformational change steps are sequential and reversible, the deficit in neutralization can be overcome by increasing scFv concentration as observed in Figure 4Go, yet the apparent affinity of scFv for its ligand would not be affected because neither binding kinetics (BIAcore measurements) nor ELISA binding experiments distinguish between the native and neutralized scFv–F complexes.

To investigate such a mechanism, direct evidence of a conformational change may be obtained in the future by using spectroscopic methods such as X-ray crystallography or NMR on purified scFv and F protein. Alternatively, a panel of conformationally sensitive antibodies against the F protein could be used to compare complexes of F protein with mu or hu10 scFv.

A possible consequence of our observations is that improved neutralization efficiency may be achieved through mutagenesis without increasing affinity and, conversely, that improved affinity may not necessarily translate into a more strongly neutralizing antibody. Certain methods such as affinity-panning provide a means to select for strong antibody–antigen interactions, such as was the case for anti-HIV antibody b12 (Yang et al., 1995Go). However, in order to enhance virus-neutralization potency, a combination of mutagenesis and high-throughput screening of biological activity may prove to be a more valuable approach. The recent elucidation of the neutralization mechanism of HIV by antibody b12 certainly supports this conclusion (McInerney et al., 1997Go).

Finally, by assessing the effects of humanization in an scFv derivative of the full immunoglobulin, we benefit from a convenient and rapid system for mutagenesis and expression without the need for lengthy selection of eukaryotic transfectants expressing sufficient amounts of antibody for complete characterization. This work, therefore, constitutes the initial steps in the humanization of HNK20, a murine IgA. Based on our observations, it would be prudent to humanize the HNK20 Fv by introducing only the first seven mutations (hu7, Figure 2Go). However, one must also note that, in general, neutralization in vivo is a complex phenomenon that relies on several activities of the antibody such as those mediated by the Fc domain. Therefore, small decreases in activity in an scFv may be hidden by the multifaceted biological activity of a full antibody in vivo. In addition, human IgG b12 was recently shown to neutralize HIV by a different mechanism than its Fab fragment (McInerney et al., 1997Go). Such considerations should therefore lead to caution in projecting the results seen in scFv humanization to the case of the whole antibody.


    Acknowledgments
 
Thanks are due to Richard Weltzin for helpful comments and discussions. Thanks also go to the OraVax Biochemistry Department for help in protein purification.


    Notes
 
4 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Anderson,L.J., Hierholzer,J.C., Bingham,P.G. and Stone,Y.O. (1985) J. Clin. Microbiol., 22, 1050–1052.[ISI][Medline]

Blondel,B., Crainic,R., Fichot,O., Dufraisse,G., Candrea,A., Diamond,D., Girard,M. and Horaud,F. (1986) J. Virol., 57, 81–90.[ISI][Medline]

Bruccoleri,R.E. and Karplus,M. (1987) Biopolymers, 26, 137–168.[ISI][Medline]

Chothia,C., Lesk,A.M., Gerardi,E., Tomlinson,I.M., Walter,G., Monks,J.D., Llewelyn,M.B. and Winter,G. (1992) J. Mol. Biol., 227, 799–817.[ISI][Medline]

Co,M.S., Deschamps,M., Whitlery,R.J. and C.Queen (1991) Proc. Natl Acad. Sci. USA, 88, 2869–2873.[Abstract]

Colonno,R.J., Callahan,P.L., Leippe,D.M., Rueckert,R.R. and Tomassini,J.E. (1989) J. Virol., 63, 36–42.[ISI][Medline]

Disis,M.L. and Cheever,M.A. (1997) Adv. Cancer Res., 71, 343–371.[ISI][Medline]

Emini,E.A., Kao,S.Y., Lewis,A.J., Crainic,R. and Wimmer,E. (1983) J. Virol., 46, 466–474.[ISI][Medline]

Guirakhoo,F., Catalan,J., Monath,T.P. and Weltzin,R. (1996) Immunotechnology, 2, 219–228.[ISI][Medline]

Hamilton,A.A., Manuel,D.M., Grundy,J.E., Turner,A.J., King,S.I., Adair,J.R., White,P., Carr,F.J. and Harris,W.J. (1997) J. Infect. Dis., 176, 59–68.[ISI][Medline]

Icenogle,J., Shiwen,H., Duke,G., Gilbert,S., Rueckert,R. and Anderegg,J. (1983) Virology, 127, 412–425.[ISI][Medline]

Kabat,E.A., Wu,T.T., Reid-Miller,M. and Perry,H.M. (1991) Sequences of Immunological Interest. US Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, MD.

Levine,S., Klaiber-Franco,R. and Paradiso,P.R. (1987) J. Gen. Virol., 68, 2521–2524.[Abstract]

Major,J.G., Liou,R.S., Sun,L.K., Yu,L.M., Starnes,S.M., Fung,M.S., Chang,T.W. and Chang,N.T. (1994) Hum. Antibodies Hybridomas, 5, 9–17.[Medline]

McInerney,T.L., Armstrong,S.J. and Dimmock,N.J. (1997) Virology, 223, 313–326.

Padlan, E.A. (1991) Mol. Immunol., 28, 489–498.[ISI][Medline]

Pedersen,J.T., Henry,A.H., Searle,S.J., Guild,B.C., Roguska,M. and Rees,A.R. (1994) J. Mol. Biol., 235, 959–973.[ISI][Medline]

Roguska,M. et al. (1994) Proc. Natl Acad. Sci. USA, 91, 969–973.[Abstract]

Roguska,M.A. et al. (1996) Protein Engng, 9, 895–904.[Abstract]

Ryu,C.J., Padlan,E.A., Jin,B.R., Yoo,O.J. and Hong,H.J. (1996) Hum. Antibodies Hybridomas, 7, 113–122.[Medline]

Weltzin,R. (1996) Drugs Future, 21, 1047–1054.

Weltzin,R., Hsu,S.A., Mittler,E.S., Georgakopoulos,K. and Monath,T.P. (1994) Antimicrob. Agents Chemother., 38, 2785–2791.[Abstract]

Wien,M.W., Filman,D.J., Stura,E.A., Guillot,S., Delpeyroux,F., Carinic,R. and Hogle,J.M. (1995) Nature Struct. Biol., 2, 232–243.[ISI][Medline]

Winter,G. and Harris,W.J. (1993) Trends Pharm. Sci., 14, 139–143.[ISI][Medline]

Yang,W., Green,K., Sweeney,S.P., Briones,A.T., Burton,D.R. and Barbas,C.F.,III (1995) J. Mol. Biol., 254, 392–403.[ISI][Medline]

Received August 11, 1998; revised December 7, 1998; accepted December 28, 1998.