•
`
`RESEARCH
`
`© 1991 Nature Publishing Group http://www.nature.com/naturebiotechnology
`
`RESHAPING A HUMAN MONOCLONAL ANTIBODY TO
`INHIBIT HUMAN RESPIRATORY SYNcmAI. VIRUS
`INFEOION IN VNO
`•2, Patricia Bremner1
`•2, Martin Lambert2 , Geraldine Taylor3,
`Philip R. Temfest1
`Julie M. Furze , Frank J. Carr1 and William J. Harris 1
`•2 ·*
`1Scotgen Limited, 2 Tillydrone Avenue, Aberdeen AB9 2TN, Scotland, UK. 2Department of Molecular and Cell Biology,
`University of Aberdeen, Aberdeen AB9 !AS, Scotland, UK. 3AFRC Institute for Animal Health, Compton, Newbury, Berkshire,
`RG16 ONN , UK. *Corresponding author.
`
`We transferred the complementarity de(cid:173)
`termining regions from a murine mono(cid:173)
`clonal antibody that neutralizes infection
`by respiratory syncytial virus (RSV) to a
`human IgGl monoclonal antibody. The
`resulting reshaped human antibody lost
`affinity for RSV, but an additional alter(cid:173)
`ation to one of the framework regions
`restored binding affinity and specificity.
`This second generation reshaped human
`monoclonal antibody cross-reacted with
`all clinical isolates of RSV tested and both
`prevented disease and cured mice even
`when administered four days after infec(cid:173)
`tion. We expect the antibody will prove
`useful in the management of this major
`childhood disease.
`
`R espiratory syncytial virus (RSV) is the major
`
`cause of acute respiratory illness in young chil(cid:173)
`dren admitted to hospitals, and the community
`practice will treat perhaps five times the num(cid:173)
`ber of hospitalized children. It therefore causes one of the
`major childhood diseases, giving rise to annual epidemics
`of bronchiolitis and pneumonia in children throughout
`the world 1·2
`. While the majority of communit'.y-acquired
`infections resolve themselves in a week to ten days, many
`hospitalized children, especially under six months of age,
`require assisted ventilation. More severe disease may
`result in permanent damage to the lungs leading to
`pulmonary fibrosis . This can leave a child with increased
`susceptibility to chest infections such as chronic bronchitis,
`and indeed about 50% of the more seriously ill children
`go on to show recurrent bouts of wheezing in later years 3
`•
`Efforts to produce an effective vaccine have been un(cid:173)
`successful4 and the major current treatment consists of
`intensive patient management involving the use of oxygen
`
`and possibly intragastric or intravenous feeding. Recently,
`the drug ribavirin has been introduced and has shown
`efficacy5
`. However, the drug has to be administered over
`an 18-hour period by aerosol inhalation. In addition, the
`level of secondary infections following cessation of treat(cid:173)
`ment is significantly higher than in untreated patients.
`Evidence that serum therapy may protect against RSV
`infection comes from a number of sources: ( 1) infants, less
`than one month old, show a low incidence of severe
`bronchiolitis and this has been interpreted as due to
`protecting maternal antibodies6
`; (2) human intravenous
`immunoglobulin (IVIG) prepared from high titer RSV(cid:173)
`immune humans reduces nasal RSV shedding and im(cid:173)
`proves oxygenation7
`; (3) a number of animal studies have
`shown that cotton rats and monkeys are protected against
`9
`infection by passive administration of IVIG8 •
`, and mouse
`monoclonal antibodies both protect against infection 10
`and clear an established RSV infection in mice 11
`• It is
`possible then that a single i~jection of high titer neutral(cid:173)
`izing monoclonal antibody will be more acceptable to the
`clinician to protect and possibly treat the child and pre(cid:173)
`vent virus spread.
`While mouse monoclonal antibodies with high neutral(cid:173)
`izing activity could be used in human therapy, application
`is severely limited by the immune response against mouse
`protein, reduced half-life of mouse antibodies in humans
`and poor recognition of mouse antibody effector domains
`by the human immune system 12·13
`. Two genetic engineer(cid:173)
`ing techniques have been devised in an attempt to reduce
`immunogenicity. Chimaeric antibodies 14 were described
`in which the genetic information encoding the murine
`heavy and light chain variable regions are fixed to genes
`encoding the human heavy and light constant regions.
`The resulting mouse-human hybrid has about 30% of the
`intact immunoglobulin derived from murine sequences.
`In the second approach, only the genetic information for
`the hypervariable complementarity determining regions
`(CDRs) is derived from the murine antibody and trans(cid:173)
`planted in a human monoclonal antibody. With this tech(cid:173)
`nique, only about 3% of the amino acid sequence is of
`murine origin 15
`• Here we have used this CDR grafting
`technique to redesign an anti-RSV mouse monoclonal
`
`266
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`
`antibody as a human IgG l monoclonal antibody and
`demonstrate the ability of this reshaped antibody to pro(cid:173)
`tect and cure mice from RSV infection.
`
`RESULTS
`V region sequence analysis of murine antibody. The
`original murine monoclonal antibody was RSV19
`(lgG2a,K), specific for the fusion (F) protein of RSV. This
`antibody has been shown to neutralize RSV infection in
`vitro and protect mice (see below and E. J. Stott and G. T.,
`in preparation). Cytoplasmic RNA was prepared from
`RSV19 hybridoma cells and cDNA from the immunoglob(cid:173)
`ulin (lg) mRNA using primers specific for lg heavy chain
`variable (VH) regions and lg light chain variable regions
`(VK) (see Experimental Protocol). VH and VK cDNAs
`were then amplified using the polymerase chain reaction
`and cloned into Ml3. Amplified DNAs from two separate
`cDNA preparations were sequenced in both directions
`
`TDLE 1 Binding of anti-RSV antibodies to clinical isolates.
`Extent of Fluorescence•
`
`Isolate Number
`
`HuRSV19VHFNS/VK
`
`Murine RSV19
`
`Sutrsoup A
`818
`V795
`V0040 1
`V00214
`V00764
`V743
`V316
`V369
`Vl249
`V04692
`Vl248
`V01232
`V729
`Su~oupB
`00634
`V4715
`V00463
`V4712
`VOOl65
`V00422
`V837
`V00900
`4677
`4424
`VO I23 1
`
`++++
`+++ +
`++
`+
`++
`++
`++
`+ +++
`+++
`+++
`+
`++
`+
`
`+
`++
`+
`++
`++
`++
`+++
`++
`+++
`++
`+
`
`++++
`+ +++
`++ +
`++
`+++
`++ +
`++
`++++
`+++
`+++
`+
`++
`++
`
`++
`+ ++
`++
`+ +
`++
`++
`+++
`++
`+++
`++
`+
`
`•+, ++, +++ and + +++ refer to relative numbers of fluo(cid:173)
`~esdng cells observed and represent the proponion of cells
`infected.
`
`FIGURE 1 Deduced amino acid sequences of (a) VH and (b) VK of
`murine RSV19 antibody. CDRs are boxed. Amino acids dictated
`by the PCR primers are underlined.
`
`from at least ten independent clones. All VH clones were
`identical as were VK clones except for one unrelated
`sequence, which contained a base pair deletion and con(cid:173)
`sequently would be expected to be non-functional. The
`deduced amino acid sequences following DNA sequenc(cid:173)
`ing of clones is shown in Figure l, with CD Rs defined by
`computer assisted alignment with other VH and VK
`sequences 16
`. The VH sequence is most closely related to
`Kabat subgroup IIC and the VK to Kabat subgroup II.
`The DNA sequence has been lodged with the EMBL data
`library.
`Transplantation of CDR sequences into human frame(cid:173)
`works. The human frameworks chosen to accept the CDR
`sequences were derived from NEWM for VH and REI for
`18 and were within Ml3 based templates with irrel(cid:173)
`VK 17
`•
`evant CDRs as described by Riechmann et al. 19 Synthetic
`oligonucleotides were synthesized containing the VH and
`VK CDRs flanked by short sequences drawn from NEWM
`and REI frameworks respectively and grafted into the
`human frameworks by site-directed mutagenesis20 . The
`resulting constructs (HuRSV19VH/VK) are shown in Fig(cid:173)
`ure 2.
`Expression of reshaped antibody. The reshaped VH
`and VK genes were transferred into vectors pSVgpt
`(conferring resistance to mycophenolic acid) and pSVhyg
`(conferring resistance to hygromycin) respectively21 and
`human IgG 1 and human kappa constant regions added.
`T hese were then cotransfected into YB2/0 rat myeloma
`cells a nd mycophenolic acid resistant clones selected and
`screened for antibody production. Antibody was purified
`by protein A affinity chromatography and analyzed by
`SDS-PAGE under reducing conditions. Two polypeptides
`of approximately 55kD and 25kD, corresponding to the
`heavy and kappa chains, were visible. Such clones secrete
`intact human IgG l with yields of approximately 5 µg/ml/
`106 cells.
`Antigen binding of reshaped antibodies. Figure 3
`shows the binding of HuRSV l 9VH/VK antibody to anti(cid:173)
`gen. Levels of binding were not significantly above back(cid:173)
`ground. Comparison of VH amino acid sequences be(cid:173)
`tween murine RSV19VH (Fig. 1) and HuRSV19VH (Fig.
`2) shows that 3 out of 4 amino acid differences occur
`between amino acids 91-94. The majority of mouse and
`human VHs have arginine at position 94, which is able to
`form a salt bridge with aspartic acid at fosition 101
`contributing to the conformation of CDR3 2 . Since the
`murine RSV19VH lacks Arg94 but has AsplOl we
`thought it possible that in HuRSV19VH an Arg94-
`Aspl01 salt bridge has been imposed thereby altering the
`conformation and antigen interaction of CDR3. Accord(cid:173)
`ingly, a second generation antibody was constructed,
`the murine amino acids
`HuRSV19VHFNS/VK, with
`91-94 FCNS being used to replace the human VH frame(cid:173)
`work amino acids YCAR (Fig. 2). It can be seen from
`Figure 3 that this resulted in the restoration of substantial
`antigen-binding ability. The restored binding affinity was
`lower than that with the original murine monoclonal
`antibody though direct comparison is difficult since dif(cid:173)
`ferent conjugated antibodies were used to detect mouse
`and human immunoglobulins.
`
`(•)
`
`YK
`
`(b)
`
`VK
`
`D I 0 LT 0 SP LS LP YTL GD QA S IS C~
`L 0 E/Q S G T E L E R S C A S V K L S C T A S C
`0 Y 0
`O T L v K T o c N T v L E \v r L o K P c o s r x L L
`F N 1 K lo Y v " HI v " K o R P o o c L E w 1 c ~
`P END DY Q YAP K F Q c lK AT KT AD TS SN T
`1 v IR vs s R r s i c v Po R rs cs cs c Tor TL
`K 1 s R v E A £ o L c v v r c Ir o c s H L P R Tl r c
`I. T F E D T A v v r c N s l w G s D r D
`A y L Q L T s
`~W C 0 GT TV TVS S
`GGTKLE I
`
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`•
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`In vitro biological activity of reshaped antibody. For
`clinical use, an antibody must recognize a high percentage
`of clinical isolates. Table I shows that the human antibody
`HuRSVl9VHFNS/VK recognizes the same broad spec(cid:173)
`trum of isolates as the original murine antibody. Inhibi(cid:173)
`tion of virus induced cell fusion in vitro is a good indicator
`of in vivo protection 10
`• Table 2 shows that the human
`antibody is effective at preventing the formation of giant
`cells associated with RSV-induced cell fusion. Moreover,
`residual giant cells in antibody treated cultures were
`smaller and had fewer nuclei per cell than in untreated
`cultures. The concentration of reshaped antibody which
`produced a 50% reduction in the number of multinucle(cid:173)
`ated giant cells (6.3 µg/ml) was equivalent to that observed
`with the original murine antibody (4.0 µg/ml).
`In vivo biological activity of reshaped antibody.
`BALB/c mice were challenRed intranasally with l 04 pfu of
`A2 strain of human RSV 1 either before or after intrana(cid:173)
`sal or intraperitoneal treatment with HuRSVl9VHFNS/(cid:173)
`VK human antibody. It can be seen from Table 3 that a
`single dose of 25 µg antibody per mouse is extremely
`effective in both prevention and treatment of RSV infec(cid:173)
`tion.
`A comparison of the ability of the original mouse
`antibody and the reshaped human antibody to clear an
`established infection in BALB/c mice, when administered
`intraperitoneally on day 4 of infection, is shown in Figure
`4. In this experiment, mice were infected with 105 pfu of
`virus, which is IO times greater than that used in the
`experiment shown in Table 3. There were no significant
`differences in the ability of the two antibodies to remove
`virus from the lungs of mice. However, with the greater
`challenge dose of virus, approximately 5 mg/kg body
`weight were required to clear the infection.
`
`© 1991 Nature Publishing Group http://www.nature.com/naturebiotechnology
`
`MuRSV19
`
`HuRSV19VHFNS/VK
`
`1.8
`
`1.6
`
`1.4
`
`1.2
`
`1.0
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`A,1>2
`
`0.8
`
`3.1
`
`12.5
`
`50
`
`200
`
`HuRSV19VH/VK
`
`ANTIBOOY,ng
`FIGlll 3 Antigen binding by murine RSV19, HuRSV19VH/VK
`and HuRSV19VHFNS/VK antibodies measured by ELISA.
`
`DISCUSSION
`The potential uses for human monoclonal antibodies in
`diagnosis, prophylaxis and therapy were immediately
`recognized with the first isolation in 1977 of a human cell
`line secreting specific antibod y, but the development of an
`appropriate production technology has been a slow, labo(cid:173)
`26
`rious process23
`. Despite continuing research, a rou(cid:173)
`-
`tinely applicable efficient methodology for isolating hu(cid:173)
`man monoclonals by mammalian cell transformation or
`cell fusion, techniques now so well established for rodent
`monoclonal production, is still not available. As an alter(cid:173)
`native, the use of genetic engineering has allowed the
`partial conversion of rodent antibodies into mouse-hu(cid:173)
`man chimaerics 14 and the use of protein engineering now
`allows the transfer of antigen binding specificity and
`affinity from a rodent antibody into a human immuno(cid:173)
`29
`27
`globulin of any desired subtype 15
`•
`-
`•
`The seminal studies on the use of CDR grafting to
`reshape human antibodies have already demonstrated the
`ability to transfer the properties required for successful
`, inhibition of T-cell proliferation 30
`depletion of T cells 19
`and tumour imaging (M. Verhoeyen, personal communi(cid:173)
`cation). Our data has now shown that it is possible to
`produce a human monoclonal antibody with the compos(cid:173)
`ite of properties necessary for in vivo treatment of an
`infectious disease. The concentrations of human antibody
`
`TAILE 2 Inhibition of RSV induced cell fusion by reshaped
`anti-RSV antibody.
`
`Concentration of
`HuRSVl9VHFNS/VK
`(µ.g/ml)
`
`Number of
`Giant Cells*
`
`Average Number
`of Nuclei per
`Cell
`
`100
`50
`25
`12.5
`6.3
`3.1
`1.6
`0.8
`0.4
`0.2
`0 (virus only)
`0 (no virus)
`
`44
`71
`40
`67
`89
`87
`164
`201
`292
`219
`239,259
`10
`
`4.5
`4.0
`3.8
`N.D.
`N .D.
`N.D.
`N .D.
`N .D.
`N.D.
`N .D.
`14,13.5
`
`*Scored as the number of cells with 2 or more nuclei in 20 fields
`with a 25 x objective microscope lens.
`N.D., not determined.
`
`FIGUIE 2 Amino acid sequences of murine and reshaped VH and
`VK containing RSV19 CDRs. Numbering is according to Kabat et
`al.16
`
`:::".:::;:::::~tt::::: ::::::::: __ ::::::~~'.:::::::::::IT::::::
`--------PG-V-PSOTLS-T--V---T:o::_P-GR- ---- ..._ _ ____ ,.,. V--LV---K-OFS-R-S-V-AA-----F-ND~~~-------
`
`10 1
`
`9..
`
`MuRSV19VH
`
`HuRSV 19VH
`
`HuRSV19VHFNS
`
`HuRSV19VK
`
`HuRSV19VK
`
`268
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`BIOffECHNOLOGY VOL. 9 MARCH 1991
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`

`•
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`© 1991 Nature Publishing Group http://www.nature.com/naturebiotechnology
`
`required to inhibit in vitro virus-induced cell fusion and to
`treat mice in vivo (Tables 2 and 3) are equivalent to those
`needed with the original murine antibody (Fig. 4).
`Structural and computer analysis of antigen antibody
`interactions have clearly demonstrated that individual
`framework residues can be critical for correct interactions
`ofCDRs with antigen. For example, an arginine residue at
`position 94 in VH is thought to interact with the invariant
`aspartic acid residue at position 101 in the CDR22 . It is not
`surprising then that simple transplantation of the CD Rs as
`defined by Kabat from mouse to human often results in
`loss of binding affinity. This occurred with RSVl 9 (Fig. 3)
`and with several other antibodies undergoing reshaping
`
`6
`
`5
`
`~
`
`Q,
`9
`0
`!2
`
`CJ z "
`:::>
`..J
`;/;;
`w
`~ 3
`~ en
`:::>
`a:
`>
`2
`< t.7
`
`1.3
`
`1.9
`
`2.5
`
`3.1
`
`3.7
`
`43
`
`DOSE OF IG ( log,.µg/kgbody welghl )
`
`FIGUIE 4 Effects of monoclonal antibodies on growth of RSV in
`lungs. HuRSV19VHFNS/VK (e) and murine RSV19 (0) given
`intraperitoneally 4 days after intranasal inoculation of mice with
`105 pfu RSV strain A2.
`
`TAILE 3 Prevention and treatment of RSV infection in mice by
`HuRSV19VHFNS/VK antibody.
`
`Antibody Treatment
`
`Route
`
`i.p.
`
`i.n.
`
`i.p.
`
`i.n.
`
`Day*
`
`-I
`
`-1
`
`+4
`
`+4
`
`No antibody
`
`Virus
`Recovered (pfu)
`
`Log pfu/g
`lung*
`
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`0
`1
`0
`1
`0
`2950
`2100
`4400
`4150
`3600
`
`<1.7
`<1.7
`<1.7
`<1.7
`<1.7
`<1.7
`<1.7
`<1.7
`<1.7
`<1.7
`<1.7
`<1.7
`<1.7
`<1.7
`<1.7
`1.7
`<1.7
`1.7
`<1.7
`4.47
`4.32
`4.64
`4.61
`4.55
`
`*-1 refers to administration of HuRSV l 9VHFNS/VK antibody I
`day prior to RSV infection, +4 refers to administration of
`antibod y 4 days post infection.
`*Virus pfu is expressed as the mean virus titre from 100 µI of
`either I 0%, I %, or 0.1 % (w/v) lung homogenates adjusted to pfu
`per gram of lung.
`
`in our laboratories. Ideally, reshaping of antibodies would
`be based upon structural data for each antigen-antibody
`interaction but this is clearly impractical. Our approach is
`to introduce ordered steps of additional alterations to
`achieve the minimal number of changes to the human
`structure necessary to restore binding affinity and speci(cid:173)
`ficity. Comparison of the amino acid sequences of the
`murine and human frameworks (Fig. 2) reveals a number
`of potential sites of framework-CDR interaction, and in
`this case, the interaction between CDR3 and its flanking
`framework residues was considered a significant struc(cid:173)
`tural variation (residues 91-94). An alternative to our
`approach3 0 involves the construction of a consensus hu(cid:173)
`man structure based upon the best homology achievable
`with the original rodent antibody sequence. Which of
`these approaches is more effective at eliminating a human
`immune response remains to be clarified.
`The projected advantages for human therapy of a
`reshaped human antibody compared with a rodent anti(cid:173)
`body are the absence of, or a considerably reduced,
`immune response allowing repeated treatment and an
`increased serum half-life, reducing the dose required and ,
`in the case of prophylactics, extending the period of
`protection provided by a single treatment. The lack of an
`immune response has now been demonstrated with re(cid:173)
`gard to one reshaped antibody constructed upon the same
`human framework used in our studies and antibody was
`still detectable in vivo eight days after administration31
`•
`These data also demonstrate the superiority in half-life
`and reduced immune response of the reshaped product
`compared to a chimaeric antibody 13
`.
`The findings that our reshaped antibody is effective
`against a wide range of clinical isolates of RSV (Table 1)
`and both protects and cures mice (Table 3) raises hope
`that it will be clinically effective, thereby offering an
`additional weapon in the management of this major
`childhood infection.
`
`EXPERIMENTAL PROTOCOL
`Materials. Murine monoclonal antibody hybridoma cell line
`RSV 19 was obtained from AFRC Institute for Animal Health,
`Compton, UK 10. Rat myeloma YB2/0 32
`, obtained from ATTC, is
`a non-lg secreting cell line and was grown in Dulbecco's modified
`Eagle's medium (DMEM) containing 10% fetal calf serum. Vec(cid:173)
`tors M13VHPCR1, Ml3VKPCR1 , pSVgpt and pSVhyg have
`been described in detail21 and were obtamed from G. Winter,
`MRC Laboratory of Molecular Biology, Cambridge, UK. Oligo(cid:173)
`nucleotides were synthesized using an Applied Biosystems 381
`DNA synthesizer. For cDNA synthesis, the primers were
`VH l FOR 5' TGAGGAGACGGTGACCGTGGTCCCTTGGC·
`CCCAG 3' ; VKJFOR 5' GTTAGATCTCCAGCTTGHGTCCC
`3' . For PCR, the additional primers were VHlBACK 5' AGGTS(cid:173)
`MARCTGCAGSAGTCWGG 3' ; VKIBACK 5' GACATTCAGC(cid:173)
`TGACCCAGTCTCCA 3'. For site-directed mutagenesis to
`transplant CDRs, the primers used were: VHCDRl 5' CTGTC(cid:173)
`TCACCCAGTGCATATAGTAGTCGCTGAAGGTGAAGCC
`AGACACGGT 3', VHCDR2 5' CATTGTCACTCTGCCCTG
`GAACTTCGGGGCATATGGAACATCATCATTCTCAGGA
`TCAATCCA 3' , VHCDR3 5' CCCTTGGCCCCAGTGGT(cid:173)
`CAAAGTCACTCCCCCATCTTGCACAATA 3', VKCDRl 5'
`CTGCTGGTACCATTCTAAATAGGTGTTTCCATCAG·
`TATGTACAAGGGTCTGACTAGATCTACAGGTGATG(cid:173)
`GTCA 3', VKCDR2 5' GCTTGGCACACCAGAAAATCGGT(cid:173)
`TGGAAACTCTGTAGATCAGCAG 3 ', VKCDR3 5'
`CCCTTGGCCGAACGTCCGAGGAAGATGTGAACCT·
`TGAAAGCAGT AGT AGGT 3'. For site-directed mutagenesis of
`the human VH framework the oligonucleotide used was 5'
`CTCCCCCATGAA TT ACAGAAATAGACCG 3'.
`Murine variable region DNA sequencing. Cyt~lasmic RNA
`was prepared as described by Favaloro et al. 3 The cDNA
`synthesis reaction consisted of 10-20 µg RNA , 0.4 µM VHlFOR
`or VKIFOR, 250 µMeach of dATP, dCTP, dGTP and dTTP, 50
`mM Tris-HCI, pH 7.5, 75 mM KC!, 10 mM DTT, 3 mM MgCl2
`and 27 units RNase inhibitor (Pharmacia) in a total volume of 50
`µI. Samples were heated at 70°C for 10 min and slowly cooled to
`42°C over a period of 30 min. Then, 100 units MML V reverse
`
`BIOITECHNOLOGY VOL 9 MARCH 1991
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`•
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`
`transcriptase (BRL) was added and incubation at 42°C continued
`for 1 hour. The VH and VK cDNAs were then amplified using
`the PCR as described by Orlandi et al. 21 For PCR amplification of
`VH, DNA/primer mixtures consisted of 5 µl RNA/cDNA hybrid,
`0.5 µM VHlFOR and VHlBACK primers. For PCR amplifica(cid:173)
`tions of VK, DNA/primer mixtures consisted of 5 µI RNA/cDNA
`hybrid, 0.5 µM VHIFOR and VKlBACK primers. To these
`mixtures was added 200 µM each of dA TP, dCTP, dGTP and
`dTTP, 10 mM Tris-HCI, pH 8.3, 50 mM KC!, 1.5 mM MgCl 2 ,
`0.01% (w/v) gelatin, 0.1% (v/v) Tween 20, 0.01% (v/v) NP40 and
`2 units Taq DNA polymerase (United States Biochemicals) in a
`final volume of 50 µI. Samples were subjected to 25 thermal cycles
`of 94°C, I min; 60°C, I min; 72°C, 2 min; ending with 5 min at
`72°C. Amplified VH and VK DNA were purified on low melting
`point agarose gels, and by Elutip-d column chromatography
`(Schleicher and Schuell) and cloned into Ml3. Clones were
`sequenced by the dideoxy method using Sequenase (United
`States Biochemicals).
`Transplantation of CDRs into human frameworks. Oligonu(cid:173)
`cleotide site-directed mutagenesis was based on the method of
`Nakamaya and Eckstein20• To 5 µg of VH or VK single-stranded
`DNA in Ml3 was added a two-fold molar excess of each of the
`three VH or VK phosphorylated oligonucleotides encoding the
`mouse CDR sequences. Primers were annealed to the template by
`heating to 70°C and slowly cooled to 37°C. After site-directed
`mutagenesis, the DNA was transformed into competent E. coli
`TGl cells. Single-stranded DNA was prepared from individual
`plaques and sequenced. If only single or double mutants were
`obtained, then these were subjected to further rounds of muta(cid:173)
`genesis using the appropriate oligonucleotides until the triple
`CDR mutants were obtained.
`The CDR replaced VH and VK genes were cloned in expres(cid:173)
`sion vectors21
`to yield the plasmids termed pHuRSV19VH,
`pHuRSV19VHFNSand pHuRSVl9VK. For pHuRSV19VH and
`pHuRSV19VHFNS, the CDR replaced VH gene together with
`the lg heavy chain promoter, appropriate splice sites and signal
`peptide sequences was excised from Ml3 by digestion with
`Hindlll and BamHI, and cloned into an expression vector
`containing the murine lg heavy chain enhancer, the SV40
`promoter, the gpt gene for selection in mammalian cells and
`genes for replication and selection in E. coli. A human IgG I
`constant region34 was then added as a BamHI fragment. The
`construction of the pHuRSVI 9VK plasmid was essentially the
`same except that the gpt gene was replaced by the hygromycin
`resistance gene and a human kappa chain constant region35 was
`added.
`or
`pHuRSVl9VH
`µg
`expression. Ten
`Antibody
`pHuRSV19VHFNS and 20 µg pHuRSV19VK were linearized by
`digestion with Pvul. The DNAs were mixed together, ethanol
`precipitated and dissolved in 25 µI water. Approximately 107
`YB2/0 cells were grown to semiconftuency, harvested by centrif(cid:173)
`ugation and resuspended in 0.5 ml DMEM together with the
`digested DNA in an electroporation cuvette. After 5 min on ice,
`the cells were given a single pulse of 170V at 960 µF (Gene(cid:173)
`Pulser, Bio-Rad) and left in ice for a further 20 min. The cells
`were then put into 20 ml DMEM plus 10% fetal calf serum and
`allowed to recover for 48 hours. At this time the cells were
`distributed into a 24-well plate and selective medium applied
`(DMEM, 10% fetal calf serum, 0.8 µg/ml mycophenolic acid, 250
`µg/ml xanthine). After 3-4 days, the medium and dead cells were
`removed and replaced with fresh selective medium. Transfected
`clones were visible with the naked eye 8-10 days later.
`The presence of human antibody in the medium of wells
`containing transfected clones was measured by ELISA. Microtiter
`plate wells were coated with goat anti-human lgG (gamma chain
`specific) antibodies (Sera-Lab). After washing with PBST (phos(cid:173)
`phate buffered saline containing 0.02% Tween 20, pH 7.5), 100
`µI of culture medium from the wells containing transfectants was
`added to each microtiter well for I hour at 37°C. The wells were
`then emptied, washed with PBST and either peroxidase-conju(cid:173)
`gated goat anti-human IgG or peroxidase-conjugated goat anti(cid:173)
`human kappa constant region antibodies (Sera-Lab) were added
`and incubated at 37°C for I hour. The wells were then emptied,
`washed with PBST and substrate buffer containing o-phenylene(cid:173)
`diamine added. Reactions were stopped after a few minutes by
`the addition of sulphuric acid and absorbance at 492 nm was
`measured.
`Antigen binding assays. Humanized antibody secreted from
`transfected cell lines and the murine antibody secreted by the
`original hybridoma were purified by protein A affinity chroma(cid:173)
`tography and tested for binding to RSV in an ELISA. Antigen
`consisted of calf kidney (CK) cells infected with the A2 strain of
`
`RSV and treated with 0.5% (v/v) NP40 detergent to yield a cell
`lysate. A control cell lysate was similarly prepared using unin(cid:173)
`fected CK cells. Microtiter plate wells were blocked with PBST
`and humanized or murine anti-RSV antibody applied. After 1
`hour at 37°C wells were washed and biotinylated goat anti-human
`IgG or biotinylated goat anti-mouse IgG antibodies (Sera-Lab)
`added. After a further I hour incubation at 37°C peroxidase(cid:173)
`conjugated streptavidin (Sera-Lab) was added for 20 min at 37°C.
`The wells were washed and peroxidase substrate buffer added.
`Reactions were stopped after a few minutes by the addition of
`sulphuric acid.
`Immunofluorescence analysis of clinical isolates. Twenty-four
`clinical isolates were obtained from children during the winter of
`1983/84 by the Bristol Public Health Laboratory, UK, and repre(cid:173)
`sented both of the major subgroups of RSV. Thirteen isolates were
`serotyped as subgroup A and 11 isolates as subgroup B. HeLa or
`MAI04 cells infected with RSV isolates were grown in tissue
`culture. When the cells showed evidence of cytopathic effect, 20 ml
`0.02% (w/v) disodium EDT A in PBS and 3 ml 0.25% (w/v) trypsin
`in PBS were added and the cell suspension spotted into wells of
`PTFE-coated slides. After 3 hours at 37°C, the slides were dried
`and fixed in 80% acetone. Cells were overlaid with anti-RSV
`antibody for I hour at room temperature. After extensive wash(cid:173)
`ing, either ftuorescein-conjugated rabbit anti-mouse IgG (Nordic
`Laboratories) or ftuorescein-conjugated goat anti-human IgG I
`(Southern Biotechnology, Alabama) were added and incubation
`repeated. After further washing, cells were mounted in glycerol
`and examined under UV light.
`In vitro analysis of inhibition of virus-induced cell fusion.
`The reshaped antibody, HuRSV19VHFNS/VK was tested for
`biological activity in vitro in a fusion inhibition assay. A suspension
`of MA I 04 cells was infected with RSV at 0.1 pfu per cell. After I
`hour at 37°C, 2 ml of cells at 105/ml were distributed to glass
`coverslips in tubes. After a further 24 hours at 37°C, the culture
`medium was replaced by medium containing dilutions of re(cid:173)
`shaped antibody. Twenty-four hours later, coverslip cultures
`were fixed in methanol for 10 min and stained with May
`Grunwald stain (BDH).
`In vivo analysis of efficacy. BALB/c mice (MRC Clinical
`Research Centre, London: category 4, standard) were challenged
`intranasally with 104 pfu of the A2 strain of human RSV 37
`.
`Groups of mice were administered 25 µg of reshaped antibody
`either one day prior to virus infection or 4 days following
`infection. Administration was either by the intranasal (i.n.) or
`intraperitoneal (i.p.) routes. Five days after RSV infection, mice
`were sacrificed and lungs assa~ed for RSV pfu, on secondary CK
`cells as described previous!~ 0
`. In a second experiment, mice
`were inoculated i.n. with 10 pfu of RSV, followed 4 days later
`with various concentrations of reshaped or murine antibody
`administered i. p. and examined on day 5 of infection for the level
`of RSV in the fungs.
`Acknowledgments
`It is a pleasure to acknowledge the advice of the Scotgen
`Scientific Advisory Panel particularly G. Winter, and for gifts of
`vectors from his laboratory in the MRC Laboratory of Molecular
`Biology, Cambridge, UK. We would also like to thank E. J. Stott
`for advice on selection of RSV monoclonal antibodies and 0.
`Caul, Bristol Public Health Laboratory, for clinical isolates.
`Received 12 November 1990; accepted 2 January 1991.
`
`References
`1. Brandt, C. D., Kim, H. W., Orrobio,J. O.,Jeffries, B. C., Wood, S. C.,
`Chanock, R. M., and Parrott, R. H. I 973. Epidemiology of respiratory
`syncytial virus infection in Washington DC III. Composite analysis of
`eleven consecutive yearly epidemics. Am. J. Epidemiol. 98:355-364.
`2. Sims, D. G., Downham, M.A. P. S., McQuillin, J., and Gardner, P. S.
`I 976. Respiratory syncytial virus infection in north-east England. Br.
`Med. J. 2: 1095-1098.
`3. Bruhn, F. W. and Yeager, A.G. 1977. Respiratory syncytial virus in
`early infancy. Am. J. Dis. Child. 131:145-148.
`4. Wright, P. F., Belshe, R. B., Kim, H. W., Van Voris, L. P., and
`Chanock, R. M. 1982. Administration of a highly attenuated live
`respiratory syncytial virus vaccine to adults and children. Infect.
`Immun. 37:397-400.
`5. Conrad, D. A., Christenson,]. C., Waner,]. L., and Marks, M. I. 1987.
`Aerosolized ribavirin treatment of respiratory syncytial virus infection
`in infants hospitalised during an epidemic. Pediatr. Infect. Dis. J.
`6: 152-158.
`6. Ogilvie, M. M., Vathenen, A. S., Radford, M., Codd,]., and Key, S.
`I 981. Maternal antibody and respiratory syncytial virus infection in
`infancy . .J. Med. Virol. 7:263-271.
`7. Hemming, V. G., Rodriguez, W., Kim, H. W., Brandt, C. D., Parrott,
`R.H., Burch, B., Prince, G. A., Baron, P.A., Fink, R.J., and Reaman,
`G. 1987. Intravenous immunoglobulin treatment of respiratory syn-
`
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`cytial virus infections in infants and young children. Anti. Agents and
`Chemotherapy 31: 1882-1886.
`8. Hemming, V. G., Prince, G. A., Horswood, R. L., London, W. T.,
`Murphy, B. R., Walsh, E. E., Fischer, G. W., Weisman, L. E., Baron,
`P.A., and Chanock, R. M. 1985. Studies of passive immunotherapy
`for infections of respiratory syncytial virus in the respiratory tract of a
`primate model. J. Infect. Dis. 152: 1083-1087.
`9. Prince, G. A., Hemming, V. G., Horswood, R. L., and Chanock, R. M.
`1985. Immunoprophylaxis and immunotherapy of respiratory syncy(cid:173)
`tial virus infection in the cotton rat. Virus Res. 3: 193-206.
`10. Taylor, G., Stott, E.J., Bew, M., Femie, B. F., Cote, P.J., Collins,
`A. P., Hughes, M., and Jebbett,]. 1983. Monoclonal antibodies protect
`against respiratory syncytial virus infection in mice. Immunology
`52:137-142.
`11. Stott, E. J. and Taylor, G. 1989. Immune Responses, Virus Infections
`and Disease, p. 85-104. N. J. Dimmock and P. D. Minor (Eds.). I.R.L.
`Press, London.
`12. Benjamin, R. J., Cobbold, S. P., Clark, M. R., and Waldmann, H.
`1986. Tolerance of rat monoclonal antibodies: implications for sero(cid:173)
`therapy. J. Exp. Med. 163: 1539-1544.
`13. Bruggemann, M., Winter, G., Waldmann, H., and Neuberger, M.
`1989. The immunogenicity of chimeric antibodies. J. Exp. Med