,.
`
`'
`
`Protein Engineering vol.6 no.8 pp.971-980, 1993
`
`Humani7.ation of a mouse anti-human lgE antibody: a potential
`therapeutic for IgE-mediated allergies
`
`Frank Kolblnger1.3, J� Saldanha2, Norman Hardmanl
`and Mary M.Bendigl.'
`
`1CIBA-<iEIGY AG, CH-4002, IWcl, Switzerland and 2Mcdical Rescarcb
`Council Collabonuivc Centre, 1-3 Burtonbole Lane, Mill Hill,
`London NW7 !AD, UK
`3Pr=t address: F.mwicklungslabor filr TmrnnnmSS11ys, Obcrc Hardtstrassc 18,
`Po&fach 1050, 780'.l Frul>urg, Germany
`4To whom correspondence should be addressed
`
`Mouse mAb TES-C21(C21) recognires an epitope on human
`lgE and, therefore, has potential as a therapeutic agent in
`patients with lgE-mediated allergies such as hay fever, food
`and drug allergies and extrinsic asthma. The clinical
`antibodies is limited, however, due to
`usefulness of mouse
`their immunogenkity in humans. Mouse C21 andbody was
`humanized by complementarity determining region (CDR)
`grafting with the aim of developing an effective and safe
`therapeutic for the treatment of IgE-mediated allergies. The
`CDR-grafted, or reshaped human, C21 variable �ns were
`carefully designed using a speclally constructed molecular
`model of the mouse C21 variable regions. A key step in the
`design of reshaped human variable regions Is the selection
`of the human framework regions (FRs) to serve a.s the
`backbones of the reshaped human variable regjom. Two
`approaches to the selection of human FRs were tested: (i)
`selection from human consensus sequences and (10 selection
`from individual human anbDodles. The reshaped human and
`mouse C21 antibodies were tested and compared using a
`r to measure the kinetics of binding to human lgE.
`biosenso
`Surprisingly, a few of the reshaped human C21 antibodies
`exhibited patterns of binding and affinities that were
`essen tially identical to those of mouse C21 antibody.
`Key words: antibody/biosensor/CDR grafting/human IgE/
`molecular modelling
`
`Introduction
`Mouse mAb TES-C21 (C21) was isolated from mice inununized
`with polyclonal IgE purified from human serum (Davis et al.,
`human IgE circulating
`1993). Mouse mAb C21 binds to secreted
`in plasma and to the membrane-anchored IgE present on the
`surface of IgE-expressing B cells, but does not interact with IgE
`when it is bound to its low- or high-affinity receptors (F�Rl and
`F�RII respectively) on mast cells, basophils and other cells.
`Mouse mAb C2 l , therefore, does not induce mast cells and
`basophils to release histamine and other mediators that cause
`allergic symptoms. Antibodies that recognize this very defined
`region of human IgE may be useful and safe for clearing
`circulating lgE from the blood and for specifically targeting IgE­
`secreting B cells, but not other cells bearing IgE. These
`antibodies, therefore, may have therapeutic applications in the
`treatment of IgE-mediated allergies (Chang et al., 1990).
`The development and use of mouse mAbs as therapeutic agents
`have been hindered by the human anti-mouse antibody response
`
`© Oxford U niverxity Press
`
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`(HAMA) which reduces the half-life and, therefore, the efficacy
`of the mouse antibody in patients (see review by Adair et al.,
`1990). In addition, there are risks of adverse side-effects
`associated with repeated administrations of highly immunogenic
`foreign protein to patients. Many of the problems associated with
`the use of mouse mAbs as therapeutic agents could be overcome
`with the use of human mAbs. It has proven technically difficult,
`however, to isolate the latter . In addition, it would be particularly
`difficult to isolate high-affinity human antibodies recognizing self­
`antigens such as human IgE. In order to make nxiuse monoclonal
`antibodies more acceptable as therapeutic agents, a variety of
`approaches have been developed for rendering mouse mAbS less
`immunogenic in humans by making them resemble human anti­
`bodies. Most approaches focus on replacing parts of the mouse
`antibody with parts of human antibodies (see review by Presta,
`1992). The most complete method for 'humanization' consists
`of taking only the complementarity determining regions (CDRs)
`from the mouse antibody variable regions and grafting these
`mouse CDRs into human variable regions (Jones et al., 1986).
`The CDR-grafted variable regions, or reshaped human variable
`regions, are then joined to human constant regions to create a
`reshaped human antibody. This study describes the succes.sful
`humanization of mouse C21 antibody by CDR grafting.
`
`Materials and methom
`Molecular TnO<h,lling of the mouse C21 variable regions
`of the variable regions of mouse mAb C21
`The DNA sequences
`were provided by Tanox Biosystems Inc. (Houston, TX). A 3-D
`model of the variable regions was built based on protein sequences
`derived from the DNA sequences. The model was developed on
`a Silicon Graphics IRIS 40 workstation using the molecular
`modelling package QUANTA (Polygen Corporation, Waltham,
`MA). The light chain variable region (VJ was modelled on the
`structure of the mouse anti-lysozyme antlbody HyHEL-10 as
`solved by X-ray crystallography (Padlan et al., 1989). The heavy
`chain variable region (V w was modelled on the structure of the
`mouse anti-lysozyme antibody HyHEL-5 (Sheriff et al., 1987).
`The VL and VH regions of mouse C2i antibody have 79 and
`80% identity, respectively, to mouse HyHEL-10 and HyHEL-5
`antibodies. Identical residues in the framework regions (FRs)
`were retained and non-identical residues were substiwted using
`QUANTA. CORI, CDR2 and CDR3 of the VL region and
`CDR l and CDR2 of the VH region from mouse C2 l antibody
`well to the canonical forms postulated by Chothia
`corresponded
`et al. (1989). Minor variations from the canonical sequences
`were
`seen, however, at residue 33 in CDRl of the VL region and
`residue 55 in CDR2 of the V H region. The main chain torsion
`angles of these loops were the same as those of the original
`antibody structures (HyHEL-10 for CORI, CDR2 and CDR3
`of the VL region; HyHEL-5 for CDRl and CDR2 of the VH
`region). Because there are no canonical structures for CDR3s
`of V H regions, CDR3 of the V H region of mouse C21 antibody
`was modelled on a loop selected from 91 high-resolution protein
`structures in the Brookhaven Databank (Bernstein et al., 1977).
`
`EXHIBIT 1194 Ian A. Wilson, D.Phil.
`4/21/18 Planet Oepos - Tricia Rosate, RDR. CRR, CSR No. 10891
`
`971
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`F.Kolblnger et al.
`
`Thirty candidate loops were extracted using the algorithm of Jones
`and Thirup (1986) as implemented in QUANTA. The best loops
`were selected by eye. The loops were anchored on three amino
`acid residues in the FRs on either side of the CDR3 in the mouse
`C21 VH region. The CDR3 in the mouse C21 VH region was
`modelled on residues 87 - I 06 of the Bence-Jones protein RHE
`(Furey et al., 1983). This region of RHE corresponds
`approximately to the CDR3 of a VL region. The model was
`subjected to steepest descents and conjugate gradients energy
`minimization using the CHARMM potential (Brooks et al.,
`1983), as implemented in QUANTA, to relieve unfavourable
`atomic contacts and to optimize van der Waals and electrostatic
`interactions.
`Construction of the reshaped human C21 VL and VH regions
`The first versions of reshaped human C2 l V L and V H regions
`(Ll and Hl) were constructed by gene synthesis using six
`overlapping synthetic DNA oligonucleotides for each construction
`(Table I, panel A; Table II, panel A). In each case, the six
`5'-phosphorylated and PAGE-purified oligonucleotides (Genosys
`Biotechnologies, Houston, TX) were assembled using a PCR­
`based protocol. Aliquots of each oligonucleotide (5 pmol) were
`annealed and extended in a 100 111 reaction containing 10 mM
`Tris-HCI (pH 8.3), 1.5 mM MgC12, 50 mM KCI, 10 mM {3-
`mercaptoethanol, 0.05% (w/v) Tween-20, 0.05% NP-40, 200
`11M dNTPs and 5 U Vent DNA polymerase (New England
`Biolabs, Beverly, MA). Following one cycle at 95°C for 1 min,
`50°C for 2 min and 72 °C for 4 min in a Techne PHC-2 tempera­
`ture cycler, 50 pmol of oligonucleotide primers, designed to
`hybridize at the 5'- and 3'-ends of the full-length DNA fragment,
`
`Table I. Oligonuclrrtides used for the collSIIUction of the reshaped human
`C2 J V L regions
`
`Panel A. Oligonuclcotides forthe synthesis of vcrnon LI of reshaped hu1T1M1 C2 I V._ region
`Oligo I: C21LI
`5' -TCAAGA>.AGC
`GCTGCTGCTG
`
`TTGCCGCCAC CATGGAGACC CCCGCCCAGC TGCJ'G'l'TCCT
`TGGCTG
`CCCG ACACCACCGG CGACATCCTG CTGACCCAGA GCCCC
`
`Oh&o 2: C2 J L2
`5' -GATGTTCGTG
`CCGATGCTCT GGCTGG
`CCCT GCAGCl'CAGG GTGGCCCTCT
`CGCCGGGGCT CAGG CTCAGG GTGCCGGOCIC TCTGGGTC
`AG CAGGA
`
`Oligo 3: C2 I LI
`5' -CAGAGCATCG GCACCAACAT CCA=AC
`CTGATCMGT ACGCC
`CCCCAGGCTG
`
`CAGCAGAAGC CCGGCCAGGC
`
`were added (C21-5', and C21-L3' or C21-H3', Tables I and II,
`panels A). Then the full-length DNA fragment was amplified
`in 20 cycles at 95°C for 1 min, 60°C for 2 min and 72°C for
`2 min. Next the reaction was chloroform-extracted. The DNA
`was ethanol-precipitated, digested with HindIII and BamHI, and
`fragments of the correct size purified from an agarose gel. The
`Hindill -BamHI DNA fragments were cloned into a pBluescript
`KS+ vector (Stratagene, La Jolla, CA) and sequenced using
`Sequenase (United States Biochemical Corporation, Cleveland,
`OH). Point mutations and/or deletions within the DNA sequence
`were corrected by exchanging DNA restriction enzyme fragments
`between different clones and/or using PCR-based mutagenesis
`methods (Kammann et al., 1989). HindIII -BamHl fragments
`exhibiting the correct DNA sequences were then subcloned into
`
`Table ll. Oligonucleotidc.s used for the construction of the reshaped human
`C2 J V H regions
`
`l'llDel A. Oligonucl coodcs for 1he synthcsi• of vcnion Hl of rcVlapcd
`rqwn.
`
`human C21 v.
`
`Ollgo J: C21HI
`5'-TGAAGAMGC TTGCCGCCAC CATGGA=
`TG'l'TCTGCCT
`ACCTGGAGGG
`CCTGGCCGTG GCCCCCGGCG CCCACAGCCA GGTGCAGCTC CTGCAGA
`
`Ollgo 2: C21H2
`5' -CA.GCCAGT
`AC ATGCTGAAGG
`CG=cc GGGCTTCTTC
`
`CAG CTCACCTrCA
`TGTAGCCGCT GGCC'M'G
`ACCTCGG
`CGC CGCTCTGCAC
`CAGCTGCACC TGG
`
`Oligo 3: C2IH3
`5' -CACCTT CAGC ATGTACTGGC TGGAGTGGGT G.V.GCAGAGG CCCGGCCACG
`GCCTGG AGTC GGTGG GCGAG ATCAGCCCCG GCACC'l'TCAC CACCAACTAC AACGA
`
`OJi&o 4. C21H4
`5'-GTC=<:TC
`TCTCGGCGGT
`
`GTCAGGCTGC TCAGCl'CCAT GTAGGCGGTC TTGG TGCI'GG
`GAAGGTGGCC TTGGC=A ACTrCTCG'l'T GTAGTTGGTG GTGAAGG
`
`OIJ&o 5: C2JH5
`5' -AGCAGCCTG>. CCAGCGAGGA CACCGCCGTC TACTACTGCG CCAGGTTCAG
`GGCAGCAACT ACGACTACTI'
`CCACTTCAGC
`CGA
`
`Oli&O 6: C2IH6
`TCTA.GAA.CTC ACCTGAGCTC ACGGTCACCA GGGTGCCCTG
`5' -TTTGGATCCT
`GCCCCAGTAG TCGAAGTAGT CGTAGTTGCT
`GCC
`
`5' Primer. C21·5'
`5' -TGAAGAMGC
`TTGCCGCCAC C
`3' Primer: C21-HJ'
`5' -T'M'GGATCCT TCTAGA.ACTC
`
`ACC
`
`for the iubseqllelll conllruetion of vervons H3, Hay J. and Hay3
`Paod B. Oligom.cleotidcs
`of ll1c reshaped buman C2 I V • rq:ioo.
`
`Ol1go4: C21L4
`5' -AGGGTGMGT CGGTG
`TCGCTGG
`GCTGATGCTC
`
`CCGCT GCCGCTGCCG CTGAACCTGC TGGGGA TGCC
`CGT A=ATCAG
`CAGCCTG
`
`Primer H/R38K·A40R-L (tnlroduocs R38K and A40R inlO HI. complementary strand)
`5' -� Gq;TCACCCA CTCCAGCC
`
`Olieo S: C21LS
`CTTC A.CCCTG ACCATCAGCA GGCTGCAGCC
`S'�CGGCACCGA
`GCCATGTACT ACTGCCAGCA GAGCGACAGC TGGC
`
`CGAGGACTTC
`
`Oligo 6: C2 J L.6
`5' -TTI'GGATCCT TCTAGAATAC TCACGT'l'TGA
`CCGAAGG TGG TGGG CCAGCT GTCGCTCTGC
`
`TCTCCAC
`TC
`
`CTT GGTCCC=
`
`S' Primer: C21·S'
`5' -TCMGA>.AGC TTCCCGCCAC C
`
`C21·LI"
`J' Prima:
`5' -'l"I'TGGA TCCT TCT ACM TAC TCAC
`
`Pand B. Olipuclcorides for the subsequent <XWl.5t1UCtion of vrnions L2 and LI of ll1c
`reshaped human C2 I V L re&ion
`Prima !JD60S..L (introduces D60S inlO LI, oomplam:ntary strand)
`5'-CTGAA
`ccrc:t kGGGG ATGCC GCTCATGCTC
`Primer U!XiOS·SL (introduces D60S tnlO LI, oodlnz smnd)
`5' -cccitACAGGT �CAG CGGCA
`Pn ma UE I� VJL (mtroduccs EID and V3 L inlO LI , a>mplementary stnnd)
`GCAi;G AT!;TCGCCGG TC
`5 ' -GGTC>.
`EID and V3L tnlO LI. codui£ s<rand)
`Primer UEl�V3L-SL (Ultroduces
`5' --GAi;ATqiTGC
`TCACCCAGAG CCCCGGC
`
`Primcr H/R3&K-A40R-SL (in1roch.icco R38K and A40R into HI, codtnK Slnnd)
`CACG GCCTGGAGT
`5'�
`CCCGGC
`Primer H/R66K-L (lntroducc R66K lnlO HI , oompleme:ltary stntnd)
`GA AC.-.-1\.-1-u;TI' GTJ\G
`5'-GAACGTGGCC >;TGGCCTT
`Pnmcr H/R66K-SL (lntroduccs R66K into HI , coding strand)
`5' -CAAGG CCAi;G GCCAC CTTCA CCGCCG
`AC
`Pnmcr H/R83T-L (inlroductJ R83T tnlO H I , complcmcnwy strand)
`5'�TC=cTJ;;
`l:J'CAGGCTGC TCAGCTCCAT G
`R83T into HI i""'· coding stmid)
`Primer HIR83T·S (mlroduocs
`5' -CAGCCTCAJ;Qi AGCGAGGACA C
`Primer HayFR2 (FR2 changes f mm HI lO Hay I • complemenwy suand)
`5' -CCA:J:CCACTC CA� CCGGGCC
`Primer HayFR2·L (FR2 changes from H3 !O Hay3, complcmentary wand)
`cq;
`5' -CC>.I:CCACl'C
`CA� c:rs;TGG
`C�CT
`Primer HayFR2-S (FR2 changes from HI 10 Hay! and H3 lO HayJ, coding strand)
`5 ' -CAliAG!;CTGG AGTGGll TGGG CGAGA TC
`Pnmcr HayFR3 (FR3 c1wi,es from HI IO Hay! and HJ IO Hay3. complcmcnwy 51rand)
`5' � TGGTGTCGGC
`Primer HayFRJ·S (FR3 changcs from HI to Hayland H3 io HayJ. codin' stru>d)
`!;CACCGCCTA C
`5' -ACCAGqiCCA
`
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`HlllJUUl.lzation of an antl-lgE antibody
`
`vectors designed to express human x light chains or human )'-1
`heavy chains in mammalian cells (Maeda et al., 1991).
`Additional versions of reshaped human C21 V L regions (I2
`and L3) were generated from version L1 by oligonucleotide­
`directed PCR mutagenesis. The PCR primers used to create
`versions L2 and L3 from version Ll are listed in Table I, panel
`B. Similarly, additional versions of reshaped human C21 VH
`regions (H3, Hay l and Hay3) were generated from version HI
`by oligonucleotide-directed PCR mutagenesis. The PCR primers
`used to create the new versions of reshaped human C21 VH
`regions are
`listed
`in Table II, panel B. The resulting
`Hindill. -Baml-Il fragments were cloned, sequenced and
`subcloned into the expression vectors as de&'ribed previously.
`
`Expression of reshaped human C21 antibodies in cos cells
`The cos cells were co-transfected by ele.ctroporation with the
`plasmid DNAs designed to express the reshaped human C2 l light
`and heavy chains (Maeda et al., 1991). After a 10 min recovery
`period, the cells were plated out in 10 ml Dulbecco's minimal
`essential medium containing 5% "Y globulin-free, heat-inactivated
`fetal calf serum. After 72 h incubation, the medium was collected
`and centrifuged to remove cells and cellular debris. The
`supernatant was filtered through a 0.45 µm membrane and
`analysed by ELISA for assembled antitxxly with human x light
`chains and human "Y heavy chains.
`
`Analysis of the mouse, chimeric and reshaped lwman C21
`/nzeraction Analysis (BIA)
`antibodies try Biospecific
`A biosensor-based analytical system (Pharmacia BIAcore) was
`used to analyse the kinetics of interaction between the C2 l
`antitxxlies and their antigen, human IgE. Mouse C21 antitxxly
`(TES-C21), chimeric C21 antibody (TESC-2) and mouse-human
`chimeric IgE antibody (SE44, Sun et al., 1991) were provided
`by Tanox Biosystems Inc. As capture antitxxlies, - 11,000
`resonance units (RU) (l l ng/mm2) of polyclonal rabbit anti­
`mouse IgG 1 (Pharmacia Biosensor AB, Freiburg, Germany) or
`rabbit anti-human IgG (kindly donated by Dr U.Roder, Pharmacia
`Biosensor AB) were immobilized to the CM5 senSor chip surface
`via their amino groups (Jonsson et al., 1991). For each C21 test
`antitxxly, four experimental cycles were performed. Each cycle
`consisted of binding a constant amount of test C2 l antibody to
`the respective capture antitxxly followed by the interaction of
`this test C2 l antitxxly with fixed concentrations of human IgE.
`The assays were carried out at 25°C. Test C21 antitxxly was
`diluted in HBS (10 mM HEPES, 3.4 mM EDTA, 150 mM
`NaCl, 0.05% BIAsurfactant, pH 7.4) to a final concentration of
`5 - 10 µg/ml and bound
`to catching antibody to obtain
`1300-2200 RU ( l.3-2.2 ng/mm2) of bound test antitxxly.
`Human lgE, at concentrations of 3.125, 6.25, 12.5 and 25 nM,
`was passed over the bound test C2 l antitxxly at a flow rate of
`5 JLl/rnin for 9 min. An aliquot of 4 µI of 40 mM HCl was used
`to remove antitxxly-antigen complexes and to prepare the
`of the reshaped hwnan C21 antibodies
`Protein A purification
`surface for the next cycle. The surface plasmon resonance (SPR)
`Reshaped human C21 antitxxlies were purified from the cos cell
`signals were measured and illustrated as a sensorgram. The
`supematants by affinity chromatography on I ml immobilized
`rates of association for the antitxxly -antigen interactions were
`protein A (Prosep A, Bioprocessing Ltd, Durham, UK) packed
`calculated using computer programs implemented in the BIAcore
`into HR 515 FPLC columns (Pharmacia, Uppsala, Sweden). The
`system.
`columns were run at constant flow rates of 2 ml/min on an FPLC
`For the detennination of the rates of dissociation, a similar
`system (Pharmacia). Eluted protein was detected in a flow cell
`protocol was used. Test C21 antitxxlies were first bound to the
`(UV absorbency at 280 nm). The columns were prepared by
`sensor chip surface via the immobilized capture antitxxlies.
`washing in 10 column volumes of PBS (20 mM sodium
`Human lgE (25 nM) was allowed to bind to the C2 l test annbody.
`phosphate, 150 mM NaCl, pH 8.0), pre-elution with 10 column
`Then HBS buffer was passed over the sensor chip surface at a
`volumes of 100 mM sodium citrate buffer (pH 3.0) and re­
`constant flow rate of 5 µl/min and the decrease in resonance signal
`equilibration with 10 column volumes of PBS (pH 8.0). The cos
`monitored over a period of 15-25 min . The sensor chip surface
`cell supematants (20-50 ml) were clarified by filtration through
`was later regenerated with 4 µ1 40 mM HO. Because the
`a 0.45 µ.m membrane and then loaded directly onto the column
`dissociation of antitxxly-antigen complexes is a first order
`with a peristaltic pump. The column was washed with PBS
`reaction, the linear parts of the sensorgrams were used to calculate
`(pH 8.0) until the UV absorbency returned to baseline. Bovine
`the rates of dissociation using computer programs implemented
`IgG was then eluted by washing with 100 mM sodium citrate
`in the BIAcore system.
`buffer (pH 5.0) until the baseline returned to zero. Finally,
`The specificities of mouse, chimeric and reshaped human C21
`reshaped human antitxxlies were eluted with 100 mM sodium
`antitxxlies for human IgE were also tested using the Pb.arm.acia
`citrate buffer (pH 3.0). The pH was adjusted immediately to
`BIAcore machine. The test C21 antibodies were bound to
`pH 7.0 with 1 M Tris. The neutralized eluates containing the
`immobilized capture antitxxlies on the sensor chip surface as
`reshaped human antitxxlies were concentrated in a Centricon-10
`descnbed previously. Human Igs with x light chains and a variety
`microconcentrator (Amicon, Stonehouse, UK) and the buffer
`of heavy chains (lgM, IgD; Serotec, Oxford, UK) (lg?\l , IgA2;
`changed to PBS (pH 7.2). Purity of the reshaped human anti­
`Calbiochem, Nottingham, UK) (lgG4, IgG3, IgG2, IgG I; Sigma)
`bodies was analysed by SOS-PAGE and Coomassie blue
`(lgE; Tanox Biosystems Inc.) were passed over the surface at
`staining (Laernmli, 1970). Protein concentration was detennined
`concentrations of 5 µglml. The SPR signals were measured and
`by UV absorption at 280 nm and by ELISA.
`illustrated as sensorgrarns.
`
`ELISA for human )'Ix antitxxly
`Microtiter 96-well plates were coated with goat anti-human IgG
`(Fe specific) (Dianova). After washing, the plates were blocked
`with 1 % bovine serum albumin in PBS (pH 7.2) plus 0.05%
`Tween. Sample and sample dilutions were added and, after
`incubation and washing, bound human IgG/x antitxxly was
`detected using affinity-purified goat anti-human x light chain
`polyclonal antibody conjugated with horseradish peroxidase
`(Sigma, Poole, UK). A purified recombinant human antibody
`(hwnan IgG l / x) of known concentration was used as a standard.
`
`Results
`Mokcular model of the srructure of the nwuse C21 variabk
`regions
`To design reshaped human variable regions that recreate as
`closely as possible the antigen binding site in the original mouse
`antitxxly, it would be useful to know the strucrure of the mouse
`lg variable regions (Verboeyen et al., 1988). In most cases,
`however, the structure of the mouse antitxxly to be humanized
`
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`F.Kolbinger et al.
`
`A
`
`Fig. 1. A view of the molecular model of the variable regions of mouse C21 antibody. (A) The Ca trace of the variable regions with the FRs in yellow, the
`CDRs in the VL region in blue, the CDRs in the VH region in green, residues of special interest in the FRs of the VL region in purple, and residues of
`special interest in the FRs of the VH region in red. (B) A line drawing of (A) with the residues of special intereSt labelled.
`
`has not, as yet, been determined. In these cases, a molecular
`model of the mouse antibody can be constructed as a guide to
`the design of the reshaped human variable regions (Kettleborough
`
`et al. . 1991). In preparation for the design of the reshaped human
`C21 variable regions, a molecular model of the VL and VH
`regions of mouse C2 l antibody was built. Details of the
`
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`..
`
`ffqmeninrtkm of an anti-lgE antfbody
`
`CDRl
`CD�
`P'R2
`FRl
`5
`4
`3
`2
`1
`12345678901234567890123 45678901234 567890123456789 0123456
`* ***
`*********
`*
`DILLTQSPAILSVSPGERVSFSC RASQSIGTNIH WYQQRTDGSPRLLIK YASESIS
`
`C21
`
`SGIII EIVLTQSPGTLSLSPGERATLSC
`
`WYQQKPGQAPRLLIY
`
`EIVLTQSPGTLSLSPGERATLSC
`
`WYQQKPGQAPRLLI§
`
`D-L-------------------- RASQSIGTNIH --------------K YASESIS
`
`D-L-------------------- ----------- ------------- -K -------
`
`--------- ----- --------- ----------- --------------K -------
`
`P'Jl4
`CDR3
`FR3
`10
`9
`8
`7
`6
`78901234567890123456789012345678 90123456 78901234567
`*******
`*
`*
`GIPSRFSGSGSGTEFTLNINSVESEDIADYYC QQSDSWPTT FGGGTKLEIK
`
`Ll
`
`L2
`
`L3
`
`CH
`
`SGIII GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC
`
`FGQGTKVE Ill
`
`llP'
`
`GIPDRFSGSGSGTDFTLTISRLEPEDFAMYYC
`
`FGQGTKVEIK
`
`Ll
`
`Ll
`
`L3
`
`---&------ --------------------- - QQSDSWPTT ----------
`
`-------------------------------- --------- ----------
`
`---&---------------------------- --------- ----------
`
`Fl&· 2. Comparisons of chc amino acid scquence.s of mowc and reshaped bwnan C2! VL regions. C21 shows the FRs and CDRs of the mouse C21 VL
`region. The amioo acid residues that arc part of !he canonical sequences for the CDR loop stnx:turcs arc !Illlrtcd with an asterisk ( Oiotbi.a er al. • 1989). The
`numbering i! according to Kabat et al. (1987). SGID shows the FRs of the consemus sequence for human x VL regions of subgroup ill (Kabat et al., 1987).
`KAF shows the FR5 from the VL region of human KAF annbody (Newkirl: er al., 1988). The residue8 in the FRs of KAF that differ from those in the
`conseruus sequeoce arc WKlcrlined. LI, L2 and L3 arc the versions of reshaped human C2 l V L region. The residues in the FRs !hat differ from the KAF
`scqucnce arc shown in bold.
`
`comtruction of the model are descnbed in Materials and methods.
`A view of the model highlighting the amino acid residues that
`were of particular interest is shown in Figure l.
`
`Design of the reshaped human C21 variable regions
`The design of the reshaped human C21 VL and VH regions was
`based on either the consensus sequences for certain subgroups
`of human V L and V H regions (Kabat et al., 1987) or the
`sequences from individual human antibodies. The amino acid
`sequences of the mouse C2 l V L and V H regions were most
`similar to the consensus sequence for human X V L subgroup ill
`(69% identity within the FRs) and for human VH subgroup I
`(70% identity within the FRs). In the first step of the design
`process, the mouse C21 CDRs were linked to the FRs
`· these
`human consensus sequences. The preliminary designs were
`examined and certain amino acid residues in the human FRs were
`identified as possible key residues in determining binding to
`antigen. For example, the amino acid residues that were part of
`the canonical structures for CDR loop formation, as proposed
`by Chothia et al. (1989), were highlighted (see residues marked
`with an asterisk in Figures 2 and 3). Residues that were potentially
`involved in VL -VH packing, as descnbed by Chothia et al.
`
`( 1985), were examined. The rare occurrence of certain amino
`acids at specific positions was noted. With this information, and
`with reference to the model of the mouse C21 variable regions,
`decisions were made as to whether or not certain amino acid
`residues in the selected human FRs should be replaced with the
`amino acid residues that occurred at those positions in the mouse
`C21 variable regions.
`For the design of the first version of reshaped human C2 l V L
`region (Ll), changes in the human FRs were made at positions
`l , 3, 49 and 60 (numbering according to Kabat et al., 1987)
`(Figure 2). The amino acids at positions land 3 were considered
`important because the model showed that the N-terminus of the
`mouse C21 light chain lay between CDRl and CDR 3 of the VL
`region. Therefore, the N-terminus either could be directly
`involved in antigen binding or could alter the conformation of
`the CDRs. The amino acid at position 49 is lysine and is located
`between two amino acids that are part of the canonical structure
`for CDR2 of the VL region. It is in the binding pocket created
`by CDR2 of the V L region and may form an interaction with
`the glutamic acid at position 53 in CDR2 of the V L region. The
`serine at position 60 in mouse C2 l V L region was located in the
`model at the edge of the binding site and could be influencing
`
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`P'Rl
`
`CDRl
`
`FR2
`6
`5
`4
`3
`2
`1
`123456789012345678901234567890 12345 67890123456789 012A3456789012345
`****
`***** ** *
`QVQLQQSGAELMKP GASVKISCKTTGYTFS KYWLE WVKQRPGHGLEWVG EISPGTFTTNYNEKFKA
`
`CDR:.Z
`
`F. Kolblnger et al.
`
`Cll
`
`SGI
`
`Bl
`
`B3
`
`BAY
`
`�
`QVQLVQSGAEVKKPGXSVXVSCKASGYTFS
`
`WVRQAPGXGLEWVG
`
`Q--------------A--K----------- MYWLE --K-R--H------ EISPGTFTTNYNEKFKA
`-------H------ -----------------
`
`Q--------------A--K-----------
`
`QVQLVQSGAEVKKPG�SVKVSCKASGYTFT WVRQAPGQBLEWMG
`
`Bayl ------------------------ -----5 KYWLE --lt-R--------- EISPGTFTTNYNEKFKA
`
`Bay3 -----------------------------$
`
`-------------- -----------------
`
`P'R3
`
`CDR3
`11
`10
`7
`8
`9
`67890123456789012ABC345678901234 567890ABCDEF12 34567890123
`*
`*
`FSHFSGSNYDYFDY WGQGTSLTVSS
`KATFTADTSSNTAYLQLSGLTSEDSAVYFCAR
`
`P'R4
`
`RVT XTXDXSXNTAYMELSSLRSEDTAVYYCAR
`
`WGQGTLVTVSS
`
`Jt.A- F-A-T-T----------T-----------FSHFSGSNYDYFDY -----------
`
`-A-F-A-T-T------------------------------------ -----------
`
`RVTITBDISASTAYMELSSLRSEDTAVYYCAR
`
`WGQGTLVTVSS
`
`Cll
`
`SGI
`
`Bl
`
`BJ
`
`BAY
`
`Bayl ltA -F-A--------------T-----------FSHFSGSNYDYFDY -----------
`
`-------------- -- ---------
`Bay3 -A-F-A--------------------------
`
`f\!. 3. Comparisons of the amino acid sequences of the mouse and reshaped human C21 VH regions. C21 shows the FRs and CDRs of the mouse C21 VH
`region. The amino acid residues that are pan of the canonical sequcnccs for the CDR loop wucrurcs are marked with an asterisk (Chochia et a/., 1989). The
`numbering is according to Kabat et al. (1987). SGI shows the FRs of the consensus sequence for human VH regions of subgroup I (Kabat �1 al., 1987) HAY
`shows the FRs from the VH region of human HAY antibody �imonian et al., 1987). The rc5idues in the FRs of HAY that differ from those in the
`con.scnsus sequence arc underlined. HI and H3 are the venrions of reshaped human C2 I V H region that were designed based on the consensus scqucncc. The
`residues in the FRs that differ from the con.scnsus scqucnce are shown in bold. Hay I and Hay3 arc the versions of reshaped human C2 I V H region that were
`designed based on the HAY scqucncc. The residues in the FRs that differ from the HAY SCG uence are shown in bold.
`
`antigen binding. Two further versions of reshaped human C2 l
`VL regions (L2 and L3) were made (Figure 2). Version L2
`contained only the changes at positions l, 3 and 49. Version L3
`contained only the changes at positions 49 and 60.
`For the design of the first version of reshaped human C2 l V H
`region (Hl), changes in the human FRs were made at positions
`38, 40, 66, 67 and 83 (shown in oold in Figure 3). In the model,
`the arginine at position 40 and the lysine at position 66 formed
`salt bridges with the glutamic acid at position 85 and the aspartic
`acid at position 86, respectively. For this reason, the arginine
`(position 40) and the lysine (position 66) were used in version
`HI of reshaped human C21 VH region. At positions 38 and 83,
`lysine and threonine were used in version H l of reshaped human
`C2 l V H region so as not to create a too highly positively
`charged area that could disrupt the overall structure. In the model,
`the alanine at position 67 appeared to be packed under the CDR2
`loop of the VH region. It was decided, therefore, to use alanine
`
`instead of valine because a size change at this position might
`disrupt the conformation of the CDR2 loop. One further version
`of reshaped human C2 l V H region was designed based on the
`human subgroup I consensus sequence (H3 in Figure 3). Version
`H3 contained only the change at position 67.
`There was no single preferred amino acid at positions I, 16,
`19, 43, 69, 71, 73 and 75 in the FRs of the consensus sequence
`for human subgroup I V H regions (as indicated by an E/Q or
`an X in the SGI sequence in Figure 3). In the design of versions
`H l and H3 of reshaped hwnan C2 l V H regions, the amino acids
`that occurred at these positions in the mouse C2 l V H region
`were used because there were examples of human subgroup I
`V H regions that had those amino acids at the same positions.
`The one exception was position 75 where threonine was selected
`because it is the most frequent amino acid at this position in
`human subgroup I VH regions. Because the amino acids used
`at these eight positions were observed in examples of human V H
`
`976
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`on 20 April 2018
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`6 of 10
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`

`

`regions, they were considered to resemble human sequences.
`To determine how similar the first versions of reshaped human
`C2 l variable regions were to variable regions present in individuaJ
`human antibodies, the amino acid sequences of the first versions
`of reshaped human C21 VL and VH regions (L l and Hl) were
`compared with human variable regions in a database derived from
`the Leeds Database (OWL Composite Protein Sequence
`Database, version 15, University of Leeds, UK).
`The first version of reshaped human C2 l V L region (L 1)
`showed the geatest similarity (80 % identity) to the V L region in
`human KAF anobody (hybridoma EV1-15'CL) (Newkirk er al.,
`1988). Within the FRs, KAF and the first version of reshaped
`human C21 VL region (LI) differed by only four amino acids.
`These four amino acid positions (I, 3, 49 and 60) were the same
`four positions in the human FRs that had been highlighted during
`the design of the first version of reshaped human C2 l V L
`region. Thus, in the design of the reshaped human C21 VL
`region, there would be no significant difference if FRs derived
`from a human x V L consensus sequence or those from an
`indiviclual human x VL sequence were used. Therefore, there
`was no reason to design additional versions of reshaped human
`C21 VL regions based on human FRs from individual human
`antibodies.
`The first version of reshaped human C21 VH region (Hl)
`showed greatest similarity (68% identity) to the VH region of
`human HAY antibody (hybridoma 8ElO'CL) (Dersimonian
`et al., 1987). Within the FRs, HAY and the first version of
`reshaped human C21 VH region (Hl ) differed by 13 amino
`acids. Th