Proc. Natl. Acad. Sci. USA
`Vol. 88, pp. 2869-2873, April 1991
`Immunology
`
`Humanized antibodies for antiviral therapy
`(herpes simplex virus/computer modeling)
`
`MAN SuNG Co*t, MARGUERITE DESCHAMPS*, RICHARD J. WHITLEY*, AND CARY QUEEN*
`*Protein Design Labs. Inc., 2375 Garcia Avenue. Mountain View, CA 94043; and *Department of Pediatrics. University of Alabama, Birmingham, AL 35294
`
`Communicated by Stanley Falkow, January 14, 1991
`
`Antibody therapy holds great promise for the
`ABSTRACT
`treatment of cancer, autoimmune disorders, and viral infec(cid:173)
`tions. Morine monoclonal antibodies are relatively easy to
`produce but are severely restricted for therapeu~ use by their
`immunogenicity in humans. Production of human monoclonal
`antibodies has been problematic. Humanized antibodies can be
`generated by introducing the six hypervariable regions from
`the heavy and light chains of a murine antibody into a human
`framework sequence and combining it with human constant
`regions. We humanized, with the aid of computer modeling,
`two murine monoclonal antibodies against herpes simplex virus
`gB and gD glycoproteins. The binding, virus neutralization,
`and ceU protection results aU indicate ·that ~ humanized
`antibodies have retained the binding activities and the biolog(cid:173)
`ical properties of the murine monoclonal antibodies.
`
`It was first shown in 1891 that the antibodies induced during
`a viral infection can neutralize the inciting virus (1). For
`certain acute viral infections such as rabies, hyperimmune
`serum from infected patients has been a traditional therapy
`(2). More recently, the development of monoclonal antibody
`technology has allowed generation of specific antibodies
`against various viral antigens (3). Several reports have ap-,
`peared showing that monoclonal antibodies can protect
`against various viral diseases in animal models (4-9). The use
`of monoclonal antibodies thus provides a new approach to
`antiviral therapy.
`The production of murine monoclonal antibodies is rela(cid:173)
`tively straightforward, but problems in the production of
`human monoclonal antibodies have persisted (10). In addition,
`the resulting human antibodies are frequently not of the
`appropriate isotype or do not possess the desired specificity.
`On the other hand, because xenogeneic antibodies are highly
`immunogenic in humans, the potential use of murine mono(cid:173)
`clonal antibodies for human therapy is limited, especially
`when repeated administration is necessary. The immune re(cid:173)
`sponse against a murine monoclonal antibody may potentially
`be reduced by transforming it into a chimeric antibody. Such
`antibodies combine the variable binding domain of a mouse
`antibody with human antibody constant domains (11, 12).
`However, in a study to evaluate the immunogenicity of chi(cid:173)
`meric antibodies, it was found that the anti-variable domain
`response was not attenuated in the chimeric antibody' dem(cid:173)
`onstrating that foreign variable frameworks can be sufficient to
`lead to a strong anti-antibody response (13). Therefore, for
`therapeutic purposes it may be necessary to fully humanize a
`murine monoclonal antibody by reshaping both the variable
`and the constant domains to make them human-like.
`Winter and colleagues (14) first successfully humanized
`both chains of a rat antibody, directed against human lym(cid:173)
`phocytes, by introducing the six hypervariable regions from
`the rat heavy- and light-chain variable regions into human
`variable region framework sequences. Recently, a human-
`
`ized antibody that binds to the human interleukin 2 receptor
`(p55) has also been reported (15). However, generation of
`other fully humanized antibodies has proved unexpectedly
`difficult, because significant loss of binding affinity generally
`resulted from simple grafting of hypervariable regions, prob(cid:173)
`ably due to distortion of the complementarity-determining
`region (CDR) conformation by the human framework.
`Herpes simplex virus (HSY) infections range from asymp(cid:173)
`tomatic to life threatening (Hi). More than 50 HSY polypep(cid:173)
`tides have been identified in HSY-infected cells, including
`seven major cell-surface glycoproteins (17). The specific
`biologic functions ofth~se glycoproteins are not well defined,
`although gB and gD have been shown to be associated with
`cell fusion activity (18, 19). gB and gD express both type(cid:173)
`specific and type-common antigenic determinants. Many of
`the antibodies against gB and gD have shown high neutral(cid:173)
`izing activities in vitro and in vivo (20-24). Oakes and Lausch
`(20) demonstrated that monoclonal antibodies against gB and
`gE suppress replication of HSV-1 in trigeminal ganglia. Dix
`et al. (21) showed that anti-gC and -gD antibodies protect
`mice against acute virus-induced neurological disease. Whit(cid:173)
`ley and colleagues (22-24) produced a panel of murine
`monoclonal antibodies against HSV-1 and showed that sev(cid:173)
`eral of the antibodies protected mice against encephalitis and
`death following ocular inoculation with the virus. Clone Fd79
`(anti-gB) prevented encephalitis even when immunization
`was delayed until 48 hr postinfection. Fd79 and Fdl38-80
`(anti-gD) significantly reduced the severity of epithelial kera(cid:173)
`titis and lowered the frequency of persistent viral infection in
`an outbred mouse model, suggesting potential therapeutic
`uses in humans. Because murine monoclonal antibodies are
`limited by their immunogenicity for human therapy, we chose
`to humanize these two antibodies. In this article, we describe
`the construction of humanized antibodies for Fd79 and
`Fdl38c80. These humanized antibodies retain the binding
`affinities and biological properties of the murine antibodies.
`
`MATERIALS AND METHODS
`Reagents. Vero cells were obtained from American Type
`Culture Collection (CCL 81) and maintained in minimum essen(cid:173)
`tial medium with 10% fetal bovine serum and nonessential amino
`acids. HSV-1 [~05 mutant (F strain)] (25) was a gift of Ed
`Mocarski (Stanford University). All enzymes were obtained
`from New England Biolabs and all chemicals were from Sigma
`unless otherwise specified. Staphylococcal protein A-Sepharose
`CL-4B was from Pharmacia. 125I was from Amersham. Immu(cid:173)
`nostaining reagents were ordered from Tago.
`Synthesis of Variable Domain Genes. The construction of
`variable domain genes for the humanized antibody heavy
`chain and light chain generally follows ref. 15. The nucleotide
`sequences were selected to encode the protein sequences of
`the humanized heavy and light chains, including signal pep(cid:173)
`tides, generally utilizing codons found in the mouse se-
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertisement"
`in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`Abbreviations: HSV, herpes simplex virus; CDR, complementarity(cid:173)
`determining region; pfu, plaque-forming unit(s).
`t'fo whom reprint requests should be addressed.
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`2870
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`Immunology: Co et al.
`
`Proc. Natl. Acad. Sci. USA 88 (1991)
`
`quence. Several degenerate codons were changed to create
`restriction sites or to remove undesirable ones. For each
`variable domain gene, two pairs of overlapping oligonucle(cid:173)
`otides on alternating strands were synthesized (380B DNA
`synthesizer; Applied Biosystems), which encompassed the
`entire coding sequences as well as the signal peptide and the
`splice donor signal. Each oligonucleotide was 110-140 bases
`long with a 15-base overlap. Double-stranded DNA frag(cid:173)
`ments were synthesized with Kienow polymerase, digested
`with restriction enzymes, ligated to the pUC18 vector, and
`sequenced. The two fragments with the correct sequences
`were then ligated into the Xba I sites of expression vectors
`similar to those described in ref. 15.
`Expression and Purification of Humanized Antibodies. For
`each humanized antibody constructed, the heavy-chain and
`light-chain plasmids were linearized at the BamHI sites and
`transfected into Sp2/0 mouse myeloma cells by electropora(cid:173)
`tion. Cells were selected for gpt expression. Clones were
`screened by assaying human antibody production in the
`culture supernatant by ELISA.
`
`Antibodies from the best-producing clones were purified
`by passing tissue culture supernatant over a column of
`staphylococcal protein A-Sepharose CL-4B. The bound an(cid:173)
`tibodies were eluted with 0.2 M glycine·HCI (pH 3.0) and
`neutralized with 1 M Tris·HCI (pH 8.0). The buffer was
`exchanged into phosphate-buffered saline (PBS) by passing
`over a PDlO column (Pharmacia).
`Fluorocytometric Analysis. Vero cells were infected with
`HSV-1 at 3 plaque-forming units (pfu) per cell overnight.
`Cells were trypsinized at 0.5 mg/ml for 1 min, washed
`extensively with PBS, and resuspended in FACS buffer
`(PBS/2% fetal calf serum/0.1% azide) at ,,,,5 x 1<>6 cells per
`ml. One hundred microliters of cell suspension was trans(cid:173)
`ferred to a polystyrene tube and incubated with 100 ng of
`purified antibody on ice for 30 min. The cells were washed
`with F ACS buffer and incubated with fluorescein isothiocy(cid:173)
`anate-labeled goat anti-human antibody on ice for another 30
`min. The cells were washed again and resuspended in
`PBS/1% paraformaldehyde. Cells were analyzed on a FAC(cid:173)
`Scan (Becton Dickinson).
`
`A
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`K
`Fm. 1. Amino acid sequences of the heavy chain (A) and the light chain (B) of the murine and humanized Fd79 antibodies and the heavy
`chain (C) and light chain (D) of the murine and humanized Fd138-80 antibodies. The sequences of the murine antibodies as deduced from the
`cDNA (upper lines) are shown ali~ed with the humanized antibody sequences (lower lines). The humanized Fd79 and Fd138-80 framework
`sequences are derived from Porn and Eu antibodies, respectively. Residues are numbered according to the Kabat system (30). The three CDRs
`in each chain are boxed. Residues in the Porn and Eu framework that have been replaced with murine sequences or consensus human sequences
`are underlined.
`
`107
`105
`G T R L E L K
`GTKVEVK
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`Immunology: Co et al.
`
`Proc. Natl. Acad. Sci. USA 88 (1991)
`
`2871
`
`Affinity Measurements. Binding affinities of the mouse and
`humanized antibodies were determined by competitive bind(cid:173)
`ing. Briefly, Vero cells infected with HSV-1 as described
`above were used as a source of gB and gD antigens. Increas(cid:173)
`ing amounts of competitor antibody (mouse or humanized)
`were added to 1.5 ng of radioiodinated tracer mouse antibody
`(2 µ,Ci/ µ,g; 1 Ci = 37 GBq) and incubated with 4 x HP
`infected Vero cells in 0.2 ml of binding buffer (PBS/2% fetal
`calf serum/0.1 % azide) for 1 hr at 4°C. Cells were washed and
`pelleted, and their radioactivities were measured. The con(cid:173)
`centrations of bound and free tracer antibody were calcu(cid:173)
`lated. The binding affinities were calculated according to the
`methods of Berzofsky and Berkower (26).
`Viral Neutralization Assay. Neutralizing activity of the mu(cid:173)
`rine and humanized antibodies was assayed by a plaque
`reduction method. Briefly, serial dilutions of antibodies were
`mixed with 100 pfu of virus and incubated at 37°C for 1 hr. The
`viruses were then inoculated onto six-well plates with conflu(cid:173)
`ent Vero cells and adsorbed at 37°C for 1 hr. Cells were
`overlaid with 1 % agarose in complete medium and incubated
`for 4 days. Plaques were stained with neutral red. The anti(cid:173)
`body concentration was recorded for 90% plaque reduction.
`In Vitro Protection Assay. Twenty-four-well plates of con(cid:173)
`fluent Vero cells were inoculated with virus at 0.1 pfu per cell
`and allowed to adsorb for 2 hr at 37°C before adding 1 ml of
`antibodies in medium (10, 1, or 0.1 µ,g/ml). At the end of 4
`days, culture medium with antibodies was removed and
`plates were washed and dried by placing overnight in a 37°C
`incubator. To detect viral antigens, each well was incubated
`with 200 µ,l of mouse Fd79 antibody at 0.5 µ,g/ml for 1 hr at
`37°C, washed twice, and incubated with 200 µ,l ofperoxidase(cid:173)
`conjugated goat anti-mouse immunoglobulin (1:300 dilution)
`for 1 hr at 37°C. The plates were washed and then developed
`with the substrate 3-amino-9-ethylcarbazole for 15 min at
`room temperature. The reaction was stopped by rinsing with
`water and air drying.
`Computer Analysis. Sequence analyses and homology
`searches were performed with the MicroGenie sequence
`analysis software (Beckman). The molecular model of the
`variable domains was constructed with the ENCAD program
`(27) and examined with the MIDAS program (28) on an Iris
`4D-120 graphics workstation (Silicon Graphics, Mountain
`View, CA).
`
`RESULTS
`Cloning of Heavy-Chain and Light-Chain cDNA. cDNAs for
`the heavy-chain and light-chain variable domain genes were
`cloned by using anchored polymerase chain reactions (29)
`with 3' primers that hybridized to the constant regions and 5'
`primers that hybridized to the dG tails (details to be published
`elsewhere). The heavy-chain variable domain gene of Fd79
`belongs to mouse heavy-chain subgroup IIIB, and the light
`chain belongs to K-Chain subgroup III. The heavy chain and
`light chain of Fd138-80 belong to the heavy-chain subgroup II
`and K-chain subgroup V, respectively. The translated amino
`acid sequences of the two antibodies are shown in Fig. 1.
`Computer Modeling of Humanized Antibodies. To retain
`high binding affinity in the humanized antibodies, the general
`procedures of Queen et al. (15) were followed. First, a human
`antibody variable region with maximal homology to the
`mouse antibody is selected to provide the framework se(cid:173)
`quence for humanization of the mouse antibody. Normally
`the heavy chain and light chain from the same human
`antibody are chosen so as to reduce the possibility of incom(cid:173)
`patibility in the assembly of the two chains. Based on a
`sequence homology search against the NBRF protein se(cid:173)
`quence data base, the antibody Porn was chosen to provide
`the framework sequences for humanization of Fd79.
`
`The computer program ENCAD (27) was used to construct
`a model of the Fd79 variable region. Inspection of the refined
`model of murine Fd79 revealed two amino acid residues in the
`framework that have significant contacts with the CDR
`residues (Table 1). Lysine in light chain position 49 has
`contacts with three amino acids in CDR2 of the light chain
`(L50Y, L53N, L55E; see Table 1 for explanation of coding
`system) and two amino acids in CDR3 of the heavy chain
`(H99D, HlOOY). Leucine in heavy-chain position 93 shows
`interactions with an amino acid in CDR2 of the heavy chain
`(H35S) and an amino acid in CDR3 of the heavy chain
`(HlOOcF). Hence, L49K and H93L were retained in the
`construction of humanized Fd79, as their replacement with
`human Porn framework residues would be likely to introduce
`distortions into the CDRs. Also, seven other residues in the
`Porn framework (five in the light chain and two in the heavy
`chain) were substituted with consensus human residues
`(identical to the murine Fd79 sequence in six of the choices)
`because of their rare occurrence in other human antibodies.
`The elimination ofunusual amino acids in the framework may
`further reduce immunogenicity. The murine Fd79 sequences
`and the corresponding humanized sequences are shown in
`Fig. 1 A and B. Substituted residues in the Porn framework
`are underlined.
`Similarly, the murine heavy-chain and light-chain se(cid:173)
`quences of Fd138-80 were compared to the NBRF protein
`sequence data base, and the human antibody Eu was selected
`to provide the framework sequence for humanized Fd138-80.
`Inspection of a computer-generated model of Fd138-80 re(cid:173)
`vealed six amino acid residues in the framework that show
`important contacts with CDR residues. The residues and
`their contacting counterparts are listed in Table 1; these
`murine residues were retained in the construction of human(cid:173)
`ized Fd138-80. Two other residues (L87F and H37M) show
`significant contacts with L98F, which is immediately adja(cid:173)
`cent to CDR3, so these two mouse residues were also
`retained. Eight amino acids in the Eu framework (two in the
`light chain and six in the heavy chain) were substituted with
`the murine residues (which are also consistent with the
`human consensus residues) because of their rare occurrence
`in other human antibodies. The murine Fd138-80 sequerices
`and the corresponding humanized sequences are shown in
`Fig. 1 C and D. Substituted residues in the Eu framework are
`underlined.
`Properties of Humanized Antibodies. The humanized Fd79
`and Fd138-80 antibodies were characterized by comparisons
`with the murine and chimeric antibodies. Both humanized
`antibodies bind to Vero cells infected with HSV-1 in a
`fluorocytometric analysis in a manner similar to the chimeric
`
`Fd79
`
`Fd138-80
`
`Table 1. Residues in the framework sequence showing contacts
`with residues in the CDRs
`Amino
`Contacting CDR residues
`acid
`Residue
`L50Y, L53N, L55E, H99D, HlOOY
`U9
`K
`H35S, HlOOcF
`L
`H93
`L34V, L89Q
`H
`L36
`y
`H32H, H341
`H27
`y
`H32H, H53R
`H30
`H63F
`F
`H48
`H63F
`K
`H66
`H63F
`A
`H67
`The amino acid residues are numbered according to the Kabat
`system (30): the first letter (Hor L) stands for the heavy chain or light
`chain, the following number is the residue number, and the last letter
`is the amino acid single-letter code. The CDRs are defined according
`to Kabat. Light chain: CDRl, residues 24-34; CDR2, residues 50-56;
`CDR3, residues 89-97. Heavy chain: CDRl, residues 31-35; CDR2,
`residues 50-65; CDR3, residues 95-102.
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`2872
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`Immunology: Co et al.
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`Proc. Natl. Acad. Sci. USA 88 (1991)
`
`A
`
`B
`
`FIG. 2. Fluorocytometry of HSV-1-infected Vero cells stained with Fd79 (A) and Fd138-80 (B) antibodies. • ·., lsotype-matched control
`antibody; .... ., humanized antibody;--, chimeric antibody.
`
`that polyclonal antibody to glycoprotein D did not prevent the
`spread of virus from cell to cell in culture (31).
`
`DISCUSSION
`The binding, neutralization, and protection results all indi(cid:173)
`cate that the humanized Fd79 and Fdl38-80 antibodies have
`retained the binding activities and the biological properties of
`the murine monoclonal antibodies. The use of murine mono(cid:173)
`clonal antibodies for therapy is hindered by the generation in
`humans of an immune response to the mouse antibodies (32).
`The potential advantages of a humanized antibody are (i) the
`
`120~---------------~
`
`antibodies (Fig. 2), showing that they recognize their respec(cid:173)
`tive viral antigens. Chimeric antibodies (unpublished data)
`rather than the original mouse antibodies were used for this
`analysis so the same second-step staining reagent could be
`used. To more quantitatively assess the binding activity,
`radioiodinated murine antibodies were bound to virally in(cid:173)
`fected cells and Scatchard analysis was performed. The
`affinities of the humanized antibodies were determined by
`competition with the iodinated antibodies. The measure(cid:173)
`ments indicate that there is no significant loss of binding
`affinities in the humanized antibodies. Specifically, there is
`an =2-fold decrease in affinity in humanized Fd79 compared
`to the murine Fd79 (Ku 5.3 x 107 M- 1 vs1.1 x lot' M-1). The
`affinity of humanized Fdl38-80 is comparable to that of the
`murine antibody (K8 , 4.8 x 107 M-1 vs5.2 x 107 M-1). These
`results suggest the general usefulness of computer modeling
`in the design of humanized antibodies.
`Murine Fd79 and Fdl38-80 have been shown to neutralize
`HSV-1 in vitro without complement (22), so the neutralizing
`activities of the humanized antibodies were compared with
`the mouse antibodies. Serial dilutions of equal quantities of
`murine and humanized antibodies were incubated with virus
`for I hr before inoculation onto Vero cells. After 4 days, cells
`were stained with neutral red to visualize plaques. Results
`from these plaque-reduction assays indicated that both hu(cid:173)
`manized Fd79 and Fdl38-80 neutralize virus as efficiently as
`their murine counterparts (Fig. 3). Both humanized and
`murine Fd79 cause a 90% reduction of plaques at an antibody
`concentration of 7 nM (1 µ.g/ml). Similarly, humanized and
`murine Fdl38-80 were able to cause a 90% plaque reduction
`at equivalent levels.
`The antibodies were also investigated for their ability to
`protect cells from viral spread in tissue culture. Vero cells
`were inoculated with virus at 0.1 pfu per cell and allowed to
`adsorb for 2 hr at 37°C before addition of 10, l, or 0.1 µg per
`ml of antibody. After 4 days, antibodies were removed and
`cells were stained with mouse Fd79 antibody for detection of
`viral antigens on infected cells. Results indicated that human(cid:173)
`ized Fd79 at I µg/ml (Fig. 4A) and murine Fd79 (data not
`shown) protected culture cells from viral spread. Cells stained
`with anti-gB antibodies were negative, except isolated single
`cells, which were infected with virus before introduction of
`protective antibodies. However, neither humanized (Fig. 4B)
`nor murine (data not shown) Fdl38-80 was able to protect cells
`against viral spread, despite their ability to neutralize virus
`before inoculation. Fig. 4B shows that total cell lysis and
`staining with anti-gB antibodies were observed even in the
`presence of humanized Fdl38-80 (10 µg/ml). Both gB and gD
`are thought to be associated with cell fusion and virus infec(cid:173)
`tivity (18, 19). However, it is possible that Fd79 blocks both
`the infectivity and cell fusion functions of gB, while Fd138-80
`does not block the fusion epitope of gD, so virus can still
`spread cell to cell. This is not surprising, as it has been reported
`
`A
`
`1po
`
`80
`
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`0
`~
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`~ 60
`<D z
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`
`40
`
`120
`
`B
`
`100
`
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`0
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`
`80
`
`60
`
`40
`
`20
`
`0
`.1
`
`•
`
`100
`10
`1
`Antibody Concentration (nM)
`
`1000
`
`1
`100
`10
`Antibody Concentration (nM)
`
`1000
`
`FIG. 3. Neutralization of HSV-1 by Fd79 (A) and Fd138-80 (B)
`antibodies. e, Mouse; o, humanized .
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`Immunology: Co et al.
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`Proc. Natl. Acad. Sci. USA 88 (1991)
`
`2873
`
`A •• .
`•
`
`Immunostaining of Vero cell monolayers infected with
`Fm. 4.
`HSV-1 in the presence of humanized Fd79 antibodies (1 µg/ml) (A)
`and humanized Fd138-80 antibodies (10 µg/ml) (B).
`
`lack of, or significantly reduced, immune response allowing
`repeated treatment; and (ii) an increased serum half-life,
`reducing the required dose as well as extending the effective
`period. It remains to be evaluated in clinical trials whether
`humanized antibodies will induce an anti-isotypic response
`or, more likely, an anti-idiotypic response, and whether
`humanized antibodies will be superior to chimeric antibodies.
`A humanized antibody (CAMPATH-lH) used to treat two
`patients with non-Hodgkin lymphoma was able to induce
`remission with no anti-globulin response (33).
`The availability of humanized antibodies with specificity
`for HSY gB and gD should provide an opportunity for
`studying the in vivo potency and immunogenicity of human(cid:173)
`ized antibodies in treating viral diseases. The recognition by
`Fd79 and Fd138-80 of type-common epitopes of gB and gD
`(22) expands the therapeutic potential to HSV-2 as well as
`HSV-1. The use of a combination of two or more humanized
`antibodies in therapy could be important to reduce the
`development of antibody-resistant strains. Combination ther(cid:173)
`apy of humanized antibodies with other antiviral agents such
`as acyclovir may provide further opportunities to combat
`diseases when chemotherapeutic agents alone have not been
`effective. The observation that Fd79 and Fd138-80 reduce the
`frequency of viral persistence in a murine ocular model (23)
`suggests that the humanized antibodies, perhaps together
`with other antiviral agents, could reduce episodes of recur(cid:173)
`rent genital infection, an area in which traditional antiviral
`agents have not been effective (34). The effector functions of
`the humanized antibodies remain to be studied. It is antici(cid:173)
`pated that incorporation of the human constant domains may
`
`· enhance effector functions such as antibody-dependent cel(cid:173)
`lular cytotoxicity, leading to greater therapeutic efficiency in
`human patients. The actual efficacy of the antibodies in
`human patients must be evaluated by clinical trials.
`
`We thank Michael Levitt and Phil Payne for helpful discussions in
`designing the humanized antibodies and Barry Selick for the expres(cid:173)
`sion vectors.
`
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`
`5 of 5
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`BI Exhibit 1128
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