Construction and characterization of a humanized single chain Fv antibody fragment against the main immunogenic region of the acetylcholine receptor

Journal of Neuroimmunology 94 Ž1999. 182–195

Construction and characterization of a humanized single chain Fv antibody fragment against the main immunogenic region of the acetylcholine receptor Danai Papanastasiou, Avgi Mamalaki ) , Elias Eliopoulos, Konstantinos Poulas, Christos Liolitsas, Socrates J. Tzartos 1 Department of Biochemistry, Hellenic Pasteur Institute, 127 Vas. Sofias AÕenue, 115 21 Athens, Greece Received 29 July 1998; revised 23 October 1998; accepted 16 November 1998

Abstract The single chain Fv fragment of mAb198 ŽscFv198. directed against the main immunogenic region ŽMIR. of the nicotinic acetylcholine receptor ŽAChR., can efficiently protect the AChR in muscle cell cultures against the destructive activity of human myasthenic autoantibodies. Humanization of the scFv198 antibody fragment should prove useful for therapeutic application by reducing its immunogenicity. Framework sequences from human immunoglobulins homologous to the rat scFv198 sequences were selected and a totally synthetic humanized scFv198 antibody fragment was constructed in vitro. Humanized VH and VL domains were synthesized using two overlapping sets of 225 bases long oligonucleotides overlap extension and polymerase chain reaction ŽPCR., then assembled into a full-length gene by overlap extension of single-stranded DNA ŽssDNA. fragments and PCR. The initial humanized antibody fragment had a very low affinity for the AChR. Molecular modeling was then performed and four residues from the framework regions ŽFR. of the humanized VH domain were selected to be replaced by the corresponding amino acid from the rat sequence. Three mutants were constructed by overlap extension, using PCR. The humanized variant containing replacements at VH residues 27, 29, 30 and 71 showed very good recovery of AChR binding activity; its binding affinities for Torpedo or human AChR Ž K D : 8.5 or 323 nM, respectively. being only four times lower than those of the parental scFv198 Ž K D : 2 or 80 nM, respectively.. This variant was able to protect the human AChR against the binding of anti-MIR mAb and anti-a autoantibodies from a myasthenic patient. It was also able to protect AChR against antigenic modulation induced by the anti-MIR mAb198. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Acetylcholine receptor; Myasthenia gravis; Main immunogenic region; Single chain Fv; Humanization

1. Introduction Myasthenia gravis ŽMG. is an autoimmune disease characterized by fatigability and weakness of skeletal muscles. The interaction of autoantibodies with acetylcholine receptors ŽAChR. leads to the destruction of AChRs at the neuromuscular junction ŽLindstrom et al., 1988.. AntiAChR antibodies are detected in approximately 90% of MG patients, but are essentially absent in healthy humans. The AChR of the neuromuscular junction is an integral membrane glycoprotein ŽMW ; 290,000 Da., consisting of five homologous subunits in the stoichiometry a 2 bgd or a 2 b´d, which form the cation channel ŽChangeux, 1990; Unwin, 1993.. AChR loss is caused mainly by comple) 1

Corresponding author. Telefax: q30-1-6457831. Also corresponding author.

ment-mediated destruction of the postsynaptic membrane and cross-linking of the membrane-bound AChR by bivalent antibodies, resulting in an increased rate of internalization and degradation Žantigenic modulation. ŽHeinemann et al., 1977; Tzartos et al., 1987; Loutrari et al., 1992.. The majority of anti-AChR antibodies in rats immunized with intact AChR and, possibly, in MG patients, are directed against an extracellular region of the AChR a-subunit, named the main immunogenic region ŽMIR. ŽTzartos and Lindstrom, 1980; Tzartos et al., 1998.. The ability of anti-MIR monoclonal antibodies ŽmAbs. to cause AChR loss by antigenic modulation has been demonstrated using muscle cell cultures ŽConti-Tronconi et al., 1981; Tzartos et al., 1985. and animal models ŽTzartos et al., 1987; Loutrari et al., 1992.. In contrast, univalent Fab fragments of anti-MIR mAbs do not bind complement and do not cross-link AChRs and thus do not cause antigenic modula-

0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 8 . 0 0 2 4 9 - 5

D. Papanastasiou et al.r Journal of Neuroimmunology 94 (1999) 182–195

tion, but can protect the AChR in muscle cultures from the modulating activity of intact antibodies and human MG sera ŽSophianos and Tzartos, 1989.. It has also been shown that rats can be efficiently protected against the pathogenic activity of anti-MIR mAbs by the use of high affinity Fab fragments derived from an anti-MIR mAb ŽD. Papanastasiou, K. Poulas, A. Kokla, S.J. Tzartos, unpublished data.. Univalent fragments of anti-MIR mAbs are therefore expected to be useful therapeutic agents in humans. The anti-MIR mAb198 was derived from rats immunized with intact AChR isolated from human muscle ŽTzartos et al., 1983.. The single chain Fv ŽscFv. fragment of mAb198, expressed in Escherichia coli in a soluble form, retains all the functional characteristics of the proteolytic Fab fragment ŽMamalaki et al., 1993. and most interestingly, efficiently protects the AChR in human muscle cell cultures against degradation caused by myasthenic antibodies ŽMamalaki et al., 1993.. This in vitro protective effect of scFv198 makes it a good candidate for a therapeutic approach. However, the potential use of rodent antibodies in the clinic has the drawback that they are potentially immunogenic in man ŽAdair, 1992., making long-term repeated administration difficult. One approach of overcoming this problem is to construct a humanized antibody by transferring the complementarity determining regions ŽCDR. of scFv198 into a human antibody ŽRiechmann et al., 1988.. However, structural analysis of antibody variable regions has demonstrated that several residues in the framework regions ŽFR. are important in determining the structure of the CDR loops and in producing proper interdomain contacts ŽChothia et al., 1985; Foote and Winter, 1992; Singer et al., 1993; Padlan, 1994.. Thus, in order to preserve the binding affinity, the majority of CDR-grafted antibodies require additional amino acid changes in the FRs. In addition, the number of changes to the human FRs should be minimal to avoid introducing an immunogenic antibody into human patients. The aim of this work was to produce a recombinant antibody fragment of potentially reduced immunogenicity and with functional properties identical to those of the rat scFv198. The initial version of the humanized scFv198 Žh-scFv198. did not show significant binding to the AChR, while a variant with four substitutions in the heavy chain, selected by computer modeling of the variable regions of rat and humanized Fv198, retained very significant AChR binding affinity and protective ability of the rat scFv198.

2. Materials and methods 2.1. Antibodies and cell lines The hybridoma cell line producing mAb198 ŽIgG2a. was derived from spleen cells from a Lewis rat immunized with intact AChR isolated from human muscle ŽTzartos et al., 1983.. mAb155, which binds to the cytoplasmic region

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a 373–380 of the AChR, was produced from rats immunized with denatured Torpedo AChR ŽTzartos et al., 1986.. Myc-9E10.2 is a mouse hybridoma cell line ŽATCC CRL 1729. that secretes mAb9E10, directed against a c-myc epitope ŽEvan et al., 1985. and used for the immunodetection and purification of recombinant c-myc-tagged antibody fragments ŽWard et al., 1989.. MG5234 is a serum from a myasthenic patient, selected for its high anti-a subunit antibody content, while MG2853 was selected for its low anti-a subunit antibody content, determined as described previously ŽLoutrari et al., 1997. by comparing the binding of MG antibodies to the human asubunitrTorpedo b, g, d-subunit hybrid AChR and to the human TE671 AChR. The AChR sources used for assays were detergent extracts of membranes from Torpedo marmorata electric organ prepared according to Lindstrom et al. Ž1981. or TE671 cells ŽStratton et al., 1989., as described previously ŽLuther et al., 1989.. 2.2. Selection of human FR sequences and construction of the synthetic humanized scFÕ198 antibody fragment For the humanization of scFv198, human antibody sequences were obtained from the Kabat database ŽKabat et al., 1987. and those heavy and light chain sequences showing the highest degree of homology with the FR sequences of scFv198 selected. A 798-bp long sequence, covering the entire humanized scFv198 Žh-scFv198. antibody fragment, was synthesized using the polymerase chain reaction ŽPCR.. The sequences of the oligonucleotides used are shown in Fig. 2. In separate PCRs, the VH and VL domains were constructed by overlap extension and PCR. In each reaction, the templates used to generate the double-stranded antibody fragments were two long Ž225 bases. internal single-chain oligonucleotides ŽHUM1–HUM2 or HUM3–HUM4., containing 50 base complementary sequences in their 3X ends, plus two short outer PCR primers ŽHUM5–HUM6 or HUM11–HUM8. that hybridized at the two ends of the target sequences ŽFig. 3A.. The internal oligonucleotides could not be purified prior to use, as this mixture included a small proportion of the full length sequence ŽCiccarelli et al., 1991.. Three to 4 mg of the internal oligonucleotides were subjected to three rounds of extension Ždenaturation at 948C for 1 min, annealing at 608C or 558C for 2 min for the VH or VL fragments, respectively, and extension at 728C for 5 min., then 50 pmol of the two short outer primers were added. PCR was carried out for 30 cycles Ždenaturation at 948C for 1 min, annealing at 608C or 558C for 1 min for the VH or VL fragments, respectively, and extension at 728C for 2 min.. Five microliters of this PCR-amplified mixture were used in a subsequent 30 cycle PCR Ždenaturation at 948C for 1 min, annealing at 708C or 658C for 1 min for the VH or VL fragments, respectively, and extension at 728C for 2 min.. The two synthetic fragments were purified in a 2% low melting temperature

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agarose gel prepared with TAE buffer ŽTris acetate 0.04 M, EDTA 0.001 M., extracted from the gel using a Geneclean kit ŽBIO 101. according to the manufacturer’s protocol and then digested with SmaI restriction endonuclease and ligated into the pBluescript II KS vector ŽStratagene, CA. for sequence analysis ŽSequenase Version 2.0, United States Biochemical.. The full-length humanized scFv198 antibody fragment was generated by three-step PCR ŽFig. 3B.. Firstly, overlapping regions between the two synthetic genes, VH and VL, were constructed using the HUM5–HUM10 and HUM9–HUM12 primers, respectively, the HUM9 and HUM10 primers containing 60 complementary nucleotide residues. HUM9 is the forward primer for the VL fragment and HUM10 is the reverse primer for the VH fragment ŽFig. 2.. PCR was carried out using 100 pg of plasmid DNA Ždenaturation at 948C for 1 min, annealing at 608C for 1 min and extension at 728C for 1 min.. Single-stranded DNA ŽssDNA. was prepared from these double-stranded products by asymmetric PCR Ždenaturation at 958C for 30 s, annealing at 608C for 30 s, and extension at 728C for 1 min., then the two ssDNA fragments were assembled by overlap extension and PCR. Three extended cycles were performed, then the outer primers ŽHUM5 and HUM8. were added for a further 30 rounds Ždenaturation at 948C for 1 min, annealing at 658C for 1 min, and extension at 728C for 1.5 min.. The PCR product was purified on an agarose gel as described above, then digested with SfiI and NotI restriction endonucleases and ligated into the phagemid pHEN1 in frame between the pelB leader sequence and the c-myc tag peptide sequence ŽHoogenboom et al., 1991.. All DNA manipulations were carried out as described in Maniatis et al. Ž1982.. 2.3. Expression and purification of humanized scFÕ The initial humanized h-scFv198 and its mutants were inserted into the phagemid pHEN1 and the resulting construct electroporated into the E. coli HB2151 strain ŽHoogenboom et al., 1991.. Positive clones, detected by PCR screening ŽGussow and Clackson, 1989., were sequenced ŽSequenase Version 2.0, United States Biochemical.. Bacterial clones were grown to a density of 0.5–0.9 OD at 600 nm in 2=TY medium containing 100 mgrml of ampicillin and 0.1% glucose for approximately 3 h at 378C, then induced with 1 mM IPTG for a further 16 h at 258C ŽHoogenboom et al., 1991.. Supernatants of bacteria cultures were analyzed by 15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis ŽSDS-PAGE. under reducing conditions ŽLaemmli, 1970., followed by transfer to Hybond C nitrocellulose membrane ŽAmersham, Life Science. for immunoblotting. The h-scFv198 fragment was detected using mAb9E10, directed against the c-myc peptide fused in the C-terminus of h-scFv198 and horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin

ŽDako.. Bacterial culture supernatants containing the hscFv198 fragment were filtered Ž0.2 mm pore size., concentrated 10 times by ultrafiltration ŽAmicon. and passed over an affinity column ŽmAb9E10-protein A. ŽEMBO, 1991; Mamalaki et al., 1993.. The affinity column was prepared by binding of mAb9E10 to Sepharose protein A column ŽPIERCE.. mAb9E10 was covalently coupled to protein A using 20 mM dimethylpimelidate ŽEMBO, 1991.. The bound h-scFv198 antibody fragments were eluted in 0.2 M glycine–HCl ŽpH 2.8.. Fractions were immediately neutralized with 1 M Tris–HCl ŽpH 7.4., supplemented with 0.1 mgrml BSA Žfinal concentration. and dialysed extensively against PBS containing 0.2 mM EDTA. Samples were analyzed on a 12% SDS-PAGE under reducing conditions. The protein concentration was estimated by comparing the intensity of the stained band of the purified protein with that of a known concentration of a protein of similar molecular weight Žcarbonic anhydrase.. The binding activity of h-scFv198 was tested by radioimmunoassay ŽRIA. using Torpedo AChR ŽLindstrom et al., 1981.. 2.4. Molecular modeling The molecular models of the variable domains of rat scFv198 and humanized h-scFv198 were obtained using the XABgen Žversion X-1.0. program ŽMandal et al., 1996. Žhttp:rrabgen.cvm.tamu.edurXABGENrXABgen.html. and analyzed using the O program ŽJones et al., 1991.. The X-ray crystallography-solved structures were obtained from the data bank incorporated in the XABgen program ŽKABS.. The 15-residue linker ŽGly4 Ser. 3 , which connects the VH C-terminal and VL N-terminal in scFvs, was not built into the model, as no regular secondary structure could be predicted and there was no tendency to interfere with domain folding ŽHuston et al., 1988.. All calculations and model buildings were performed on a Silicon Graphics Elan Indigo ŽIrix 5.3. work station. 2.5. Site-directed mutagenesis Mutations in the h-scFv198 antibody were generated by the overlapping PCR method ŽHo et al., 1989.. Following digestion with SfiI or NotI restriction endonucleases, the plasmid pHEN1Žh-scFv198. was used as the PCR template to generate the three mutants, h-scFv-M1 ŽVH71 Leu ™ Arg., h-scFv-M3 ŽVH27 Gly ™ Phe, VH29 Phe ™ Leu and VH30 Ser ™ Thr. and h-scFv-M4, containing all four replacements. The primers used for mutagenesis were the HUM14 Ž5X-GCTGTCTTTGGTTTCTCCCTCACTAGCTTTAGTGTA-3X . and its complementary primer HUM15 Ž5X-TACACTAAAGCTAGTGAGGGAGAAACCAAAGACAGC-3X . for h-scFv-M3 and h-scFv-M4, the primer HUM16 Ž5X-GTCACCATATCTAGAGACACGTCC-3X . and its complementary primer HUM17 Ž5X-GGACGTGTC TCTAGATATGGTGAC-3X . for h-scFv-M1. Each muta-

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tion was generated by two steps of PCR. Firstly, in separate PCRs, the primers, HUM5–HUM15 and HUM14–HUM8, were used to construct two fragments containing the h-scFv-M3 mutation Ždenaturation at 948C for 1 min, annealing at 608C for 1 min and extension at 728C for 1.5 or 3 min for HUM5–HUM15 and HUM14– HUM8, respectively.. The two PCR products were isolated on an agarose gel, as described above, and extracted by electroelution. Then the two purified products were assembled by overlap extension and a second PCR. Eight extended cycles were performed, then the outer primers ŽHUM5 and HUM8. were added for a further 26 cycles Ždenaturation at 948C for 1 min and annealing-extension at 658C for 2.5 min.. The same conditions were used for the construction of h-scFv-M1 mutant. For the construction of the h-scFv-M4 mutant, the h-scFv-M1 mutant was used as template. The PCR products were assembled, as described previously, by adding the primers HUM5–HUM15 Ždenaturation at 948C for 1 min, annealing at 608C for 1 min and extension at 728C for 1.5 min. and HUM14–HUM8 Ždenaturation at 948C for 1 min, annealing at 488C for 1 min and extension at 728C for 3 min. and the final PCR products were purified on an agarose gel and electroeluted before ligation into phagemid pHEN1.

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immunoprecipitated AChR was measured on a g-counter. After subtraction of background values, the percentage residual binding activity of mAb198 in the presence of h-scFv-M4 or h-scFv198 was calculated from the difference between the radioactivity precipitated in the presence or absence of h-scFv-M4 or h-scFv198, respectively. 2.8. Antibody competition experiments for human AChR The ability of h-scFv-M4 to inhibit the binding to the human AChR of either mAbs or antibodies from MG

2.6. Affinity measurements The dissociation constant Ž K D . of h-scFv-M4 for Torpedo AChR was determined by Scatchard plot analysis ŽScatchard, 1949; Tzartos et al., 1981.. A constant amount of purified h-scFv-M4 was incubated with increasing concentrations of 125 I-a-Bgt-labeled Torpedo AChR, then the antigen–antibody complexes were immunoprecipitated using anti-myc mAb9E10 and rabbit anti-rat immunoglobulins. For human AChR, K D was calculated by RIA at the point at which 50% of the antigen was bound to antibody. Since, at this point, the concentration of free antigen is equal to the concentration of antigen in the antigen–antibody complex, according to the equation K D s wAbx free wAChRx freerwAChRx bound , the K D is equal to the concentration of the free antibody. 2.7. Antibody competition experiments using solubilized Torpedo AChR Competition experiments were performed as described previously ŽMamalaki et al., 1993. with minor modifications. An amount of 20 fmol of 125 I-a-Bgt-labeled Torpedo AChR was incubated with various amounts of h-scFv-M4 or h-scFv198. After 2 h at 48C, 20 fmol of mAb198 were added and incubation continued for a further 2 h at 48C. Ten microliters of rabbit anti-rat immunoglobulin Žwhich was found unable to precipitate the scFv fragments. were then added to the mixture and incubation continued for another hour at 48C. After centrifugation and washing, the

Fig. 1. Amino acid sequences of the cloned scFv198 heavy ŽA. and light ŽB. variable regions. The one-letter amino acid code is used. Amino acid positions are numbered according to Kabat et al. Ž1987. and the CDRs determined according to hypervariable sequence definition ŽKabat et al., 1987. and structural definition ŽChothia et al., 1989. are indicated by a line below or above the sequence respectively. h-scFv198 denotes humanized heavy and light variable regions constructed by grafting the CDRs from scFv198 into the HIG1 and DAUDI framework regions. The dashes indicate residues that are identical to the corresponding rat residues. The amino acid sequences of the human variable regions HIG1 and DAUDI are also aligned.

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patients was tested in competition radioimmunoassay experiments. An amount of 38 fmol of 125 I-a-Bgt labeled human AChR was incubated with various amounts of h-scFv-M4 or scFv198. After 2 h at 48C, an amount of mAb198 sufficient, in the absence of competing fragments, to precipitate approximately 60% of the AChR was added,

and incubation continued for a further 2 h at 48C. mAb155, which recognizes a cytoplasmic epitope of human AChR, was used as a negative control. Human AChR was immunoprecipitated and measured as described for Torpedo AChR and the residual binding activity of the mAbs calculated as described previously.

Fig. 2. Nucleotide sequence of humanized h-scFv198. Sequences of the long internal oligonucleotides used in PCR are shown in italics, while the short outer primers are shown by horizontal arrows. SfiI and NotI restriction endonuclease sites, used for cloning into the pHEN1 vector, are shown by vertical arrows. The unique sites for restriction endonucleases, BamH1, HinfI, Bst NI, TthIII 1, SmaI and DraII, introduced are also indicated by vertical arrows. The CDRs are shown in boxes while the nucleotide sequence of the linker is shown in a box marked with dashed lines.

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Competition experiments were also performed using MG sera. An amount of 14 fmol of human AChR labeled with 125 I-a-Bgt was preincubated with various amounts of h-scFv-M4 or scFv198 for 2 h at 48C, then 10 ml of MG serum Žcontaining 10 fmol of anti-AChR antibody, sufficient, in the absence of competing fragments, to precipitate approximately 70% of the 125 I-a-Bgt AChR. were added. After incubation for 2 h at 48C, sheep anti-human immunoglobulin antiserum, treated with normal rat serum before use in order to avoid cross-precipitation of the rat scFv198, was added and incubation continued for 1 h at 48C. After centrifugation and washes, the precipitated radioactivity was counted. Background precipitation was determined using normal human serum instead of MG serum and subtracted from the values obtained.

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Fab198 and AM mA b corresponds to antigenic modulation in the presence of mAb198 only.

2.9. In Õitro protection of AChR against antigenic modulation The protective effect of h-scFv-M4 against AChR loss induced by mAb198 due to antigenic modulation ŽAM. was determined in vitro using TE671 cells as previously described ŽSophianos and Tzartos, 1989; Mamalaki et al., 1993. with minor modifications. TE671 cells were transferred 7 days after confluence to 96-microwell plates. In each microwell were added 50 ml, containing 5 = 10 5 TE671 cells in DMEM supplemented with 10% FCS, plus 10 6 Url benzylpenicillin sodium salt, 0.1 grl streptomycin sulfate and 40 mgrml cycloheximide at 378C. One hour after plating, 20 ml containing various amounts of h-scFv-M4 or Fab198 were added and incubated for 1 h at 378C. Subsequently, 10 ml of fresh medium containing 50 fmol mAb198 were added and incubated for additional 4 h at 378C. To label surface AChR, 10 ml of fresh medium containing 40 nM 125 I-a-Bgt were added and incubated for another 3 h. Subsequently, cells were washed five times with medium and cell bound radioactivity was released by 2% SDS and counted on a g-counter. Background radioactivity was determined by adding to the 125 I-a-Bgt an 100-fold excess of unlabeled a-Bgt and was subtracted from values obtained Ž Dcpm .. The percent of surface AChR loss by antigenic modulation was calculated from the percent reduction in cell bound 125 I-a-Bgt as follows: AM s 100Ž1 y Dcpm AChR ab rDcpm AChR no – ab .. Where Dcpm AChR ab is the Dcpm released from cultures treated with mAb198 with or without protecting antibody fragment and Dcpm AChR no – ab is the Dcpm released from cultures without antibody or treated only with protecting antibody fragment. Protection of AChR against mAb198 was calculated as the percentage of reduction in the modulating effect of mAb198 from the equation: percentage of protection s 100 w 1 y AM protected rAM m A b x . W here AM protected corresponds to antigenic modulation induced by mAb198 in the presence of protecting h-scFv-M4 or

Fig. 3. ŽA. Strategy used for the construction of the humanized VH and VL regions. VH and VL antibody regions were constructed in separate PCR. For each reaction, two long internal oligonucleotides ŽHUM1– HUM2 or HUM3–HUM4. complementary by 50 bases were used to construct the target sequence. The external primers ŽHUM5–HUM6 or HUM11–HUM8. were then added to the reaction and the full length DNA product amplified. The VL synthetic gene encodes the VL fragment and the 15-residue linker. ŽB. Strategy used for the construction of the full-length humanized h-scFv198 antibody fragment. In the first PCR, overlapping regions were constructed between VH and VL synthetic genes, using complementary primers HUM9 or HUM10 Ž60 bases. and HUM5 or HUM12, respectively. ssDNA was prepared in a second PCR. In the final PCR, the two ssDNA fragments were assembled by their own complementarity and the full length dsDNA amplified using the external primers ŽHUM5–HUM8.. The PCR product was digested with SfiI and NotI and cloned into the pHEN1 phagemid vector.

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3. Results

3.1. Selection of human V region framework sequences To retain the binding affinity and specificity of the rodent antibody in the humanized antibody, human VH and VL regions with maximum sequence homology to the respective rat variable regions were selected from the Kabat data base ŽKabat et al., 1987. for use as templates for CDR grafting. Thus, the human HIG1 VH region ŽKudo et al., 1985., a member of subgroup II, and the human DAUDI VK region, ŽKlobeck et al., 1984., a member of subgroup I, were chosen as heavy and light chain templates, respectively, into which the CDRs of the rat scFv198 were grafted. The extent of amino acid identity in the FR regions of the human sequences and scFv198 was 62.2% for the VH region and 77.2% for VL region ŽFig. 1..

3.2. Construction and analysis of the initial humanized scFÕ antibody fragment The variable domain genes of the humanized heavy and light chains were generated by whole gene synthesis. The nucleotide sequences selected to encode the protein sequences of the humanized heavy and light chains also included the linker sequence ŽVaughan et al., 1996.. Several codons were changed either to create restriction sites or to remove undesirable ones without affecting the coded protein sequence. The specific restriction endonuclease sites Ž BamHI, HinfI, BstNI, TthIII 1, SmaI and DraII., introduced on both sides of the CDRs, were absent in the vector ŽFig. 2.. This construct will be useful for future humanization attempts. Two long internal oligonucleotides and two short outer PCR primers were used for each variable domain ŽFig. 3.. VH- and VL- coding segments were inserted into the pBluescript II KS vector for sequence verification. All VH clones tested had deletions in different sites in their se-

Fig. 4. ŽA. A ribbon diagram of rat Fv198 model. Residues relevant to this work are shown as a ball and stick representation. Residues VH27, VH29 and VH30 belong to the H1 structural loop and are involved in the formation of the antigen binding pocket. ŽB, C. Diagram of parts of the FR3, H1-CDR1 and H2-CDR2 loops showing the relative position of residue VH71 and residues VH27, VH29, VH30, VH51, VH52 and VH53 in rat ŽB. and humanized ŽC. Fv. Diagrams generated using the program, MOLSCRIPT ŽKraoulis, 1991..

D. Papanastasiou et al.r Journal of Neuroimmunology 94 (1999) 182–195 Table 1 Residue substitutions in humanized scFv198 antibody fragments and their effect on the binding activity for Torpedo AChR compared with that of rat scFv198 Constructs scFv198 h-scFv198 h-scFv-M1 h-scFv-M3 h-scFv-M4

Residue substitution

Binding activity for Torpedo AChR Ž%.

VH and VL: straight CDR swap VH: L71R VH: G27F, F29L, S30T VH: L71R, G27F, F29L, S30T

100 2 8 F2 63

quence, as a result of PCR errors. Two clones were selected and reconstructed by replacing their SacII restriction fragments Ž329 bp. in order to obtain the correct VH gene. To obtain the full length h-scFv198 antibody fragment, overlapping regions were first constructed between the VH and VL synthetic genes. Their sequences were then converted into ssDNA and the two ssDNAs assembled by extended PCR. The synthetic gene for the h-scFv198 antibody fragment was ligated into the phagemid pHEN1 and used to transform E. coli HB2151. Soluble-expressed protein in the culture supernatant was detected by Western blot analysis Ždata not shown.; following purification by a cross-linked protein A-mAb9E10 affinity column, the material migrated as a single band on SDS-PAGE under reducing conditions Ždata not shown., with a mobility consistent with the expected size of ; 30 kDa. The expression level of h-scFv198 was low, estimated at 8.6 mgrl of bacteria culture. When tested by RIA, the binding activity of the purified h-scFv198 antibody fragment to Torpedo AChR was very low, never exceeding approxi-

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mately 2% of that of the parental scFv198, despite the high excess of the amounts used Ž0.2 mg. per test tube. Its binding to human AChR was questionable since the precipitated counts per minute did not exceed twice those of the negative control. 3.3. Molecular modeling The computer program, XABgen, was used to construct models of the rat Fv198 and humanized h-Fv198 molecules. Using the O program ŽJones et al., 1991., the rat and humanized Fv models were superimposed and compared. Residues with a possible adverse effect on antigen binding pocket or with differences in side chain properties were identified. In particular, amino acids in the humanized framework region showing changes in size, charge, hydrophobicity or potential to form hydrogen bonds, compared with the parental model, were further examined. No amino acid residues in the VL region were selected for replacement, while four amino acid residues Žpositions 27, 29, 30 and 71. in the rat VH region were identified as having significant effects on CDR conformation ŽFig. 4A.. The substitution of Phe by Gly ŽVH27. in h-Fv198 seems to affect the hydrophobicity of the molecule and the lack of the bulky Phe side chain appears to alter the binding site ŽFig. 4B, C.. The charged, but flexible side chain of Arg VH71 in the rat Fv198 stabilizes the conformation of the CDR1 and CDR2 loops ŽFig. 4B., providing polar interactions between the amino groups of the Arg and the main chain carbonyl groups of residues 29, 32, 51, 52 and 53. In h-scFv198 VH71, Arg is replaced by the shorter and hydrophobic residue, Leu, eliminating favorable polar in-

Fig. 5. Detection of h-scFv-M4 antibody fragment ŽA. by SDS-PAGE and ŽB. by Western blot analysis. Lane 1: bacterial culture supernatant containing the h-scFv-M4 fragment; Lanes 2, 3: affinity-purified h-scFv-M4 fragment from culture supernatant, plus BSA added to avoid antibody absorption on the tube surface.

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Table 2 K D of rat scFv198 and humanized h-scFv-M4 for AChR

scFv198 h-scFv-M4

K D for Torpedo AChR ŽnM.

K D for human AChR ŽnM.

2 8.5

80 323

teractions with the main chain carbonyls ŽFig. 4C. and creating an empty surface cavity that is partially hydrophobic due to the Leu side chain. The substitution of Leu by Phe ŽVH29. and Thr by Ser ŽVH30. in h-Fv198 does not significantly change the hydrophobic character or modify

side chain properties. Residues VH27, VH29 and VH30 in FR1 are part of the H1 structural loop ŽChothia et al., 1989. and, therefore, are probably directly involved in the antigen binding pocket and have also been reported to interact with the CDR1 and CDR2 loops ŽCo et al., 1994; Corti et al., 1994.. 3.4. Construction and binding actiÕities of mutants of the humanized scFÕ fragment Modifications to the humanized scFv198 were made in order to obtain an antibody fragment with a binding activity comparable to that of rat scFv198. Those FR residues

Fig. 6. Inhibition of mAb binding to Torpedo ŽA. or human ŽB. AChR by affinity-purified recombinant antibody fragments. 100% precipitation represents antigen binding by the mAb in the presence of 0.3 mg BSA instead of test fragment. ŽA. Precipitation of Torpedo AChR by the anti-MIR mAb198 in the presence of 0.06 mg of h-scFv198 or 0.09 mg Ža. or 0.3 mg Žb. of mutant h-scFv-M4. ŽB. Precipitation of human AChR by anti-MIR mAb198 or mAb155 Žwhich binds to the cytoplasmic region of the AChR. in the presence of different amounts of humanized mutant h-scFv-M4 and rat scFv198 antibody fragments.

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in h-scFv198, predicted, by molecular modeling, as interfering with the conformation of the CDRs, were replaced by the corresponding residues from rat scFv198. Three variants of h-scFv198 were prepared by site-directed mutagenesis using two subsequent PCR amplifications, namely, h-scFv-M1 ŽVH71 Leu ™ Arg., h-scFv-M3 ŽVH27 Gly ™ Phe, VH29 Phe ™ Leu and VH30 Ser ™ Thr. and h-scFvM4, containing all four replacements ŽTable 1.. These variants were expressed by the same procedure used for h-scFv198. Supernatants from bacterial clones containing similar amounts of the different variants were first assayed for binding to Torpedo AChR and their binding activity compared with that of an equal amount of h-scFv198 Žthe protein concentration of the variants being determined by Western blot analysis.. The binding activity of h-scFv-M1 for Torpedo AChR was four times higher than that of the original h-scFv198, while that of h-scFv-M3 was no better than that of h-scFv198. h-scFv-M4 showed the greatest improvement in binding activity, with a value 30 times greater than that of h-scFv198 ŽTable 1., and was selected for further analysis. Purification of h-scFv-M4 was carried out by affinity chromatography using cross-linked mAb9E10-protein A column. The purified h-scFv-M4 antibody fragment was then analyzed by SDS-PAGE electrophoresis under reducing conditions and immunoblotting ŽFig. 5.. The yield of purified h-scFv-M4 was approximately 45 mgrl of bacteria culture. To assess the binding affinity of h-scFv-M4 for the antigen, the dissociation constants Ž K D . for Torpedo and human AChR were calculated and the affinity of h-scFv-M4

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for both antigens was found to be only four times lower than that of the parental scFv198 ŽTable 2.. 3.5. Specificity of h-scFÕ-M4 and ability to protect the AChR against binding of MG antibodies The specificity of h-scFv-M4 for Torpedo and human AChR was examined by competition experiments using the parental mAb198. Torpedo AChR labeled with 125 I-a-Bgt was preincubated with an excess of protecting h-scFv-M4 or h-scFv198, then mAb198 was added and the antigen bound to the intact mAb selectively precipitated. h-scFv-M4 was able to protect Torpedo AChR almost completely Ž86–90%. against the binding of the intact anti-MIR mAb198, while h-scFv198 inhibited the binding of only a small percentage of the intact mAb198 Ž23%. ŽFig. 6A.. The ability of h-scFv-M4 to protect human AChR against the binding of mAb198 was then tested and compared with the protecting activity of scFv198. The amounts of h-scFv-M4 used inhibited the binding of mAb198 by up to 66%, while the same amount of parental scFv198 inhibited mAb198 binding by up to 83%. As expected, human AChR was not protected against the binding of mAb155, which recognizes a cytoplasmic epitope on the AChR ŽFig. 6B.. The ability of h-scFv-M4 and scFv198 to protect the AChR against the binding of MG antibodies was then investigated. When an MG serum in which the majority of antibodies were directed against the a-subunit ŽMG5234.

Fig. 7. Inhibition of MG serum binding to human AChR by antibody fragments. Binding of human AChR by an anti-a MG serum Ž86% of the anti-AChR autoantibodies being directed against the a subunit. or a non-a MG serum Ž7.9% of the anti-AChR autoantibodies being directed against the a-subunit. in the presence of h-scFv-M4 or scFv198 antibody fragments. 100% binding represents AChR precipitation by MG serum in the presence of 0.3 mg BSA instead of test fragment.

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Fig. 8. Protection of TE671 AChR by h-scFv-M4 against antigenic modulation induced by intact anti-MIR mAb198. TE671 cells were incubated with h-scFv-M4 or Fab198 before incubation with mAb198. Incubation with plain medium or with plain mAb198 gave the 0% or 100% antigenic modulation, respectively.

was used, h-scFv-M4 and scFv198 gave about 34% and 72% protection, respectively; the difference probably being due to the higher affinity of scFv198 for human AChR. In contrast, using an MG serum ŽMG2853. containing a low proportion of anti-a-subunit antibodies, essentially no protection was seen ŽFig. 7.. 3.6. Protection of TE671 AChR by h-scFÕ-M4 against antigenic modulation caused by mAb198 The ability of the recombinant h-scFv-M4 antibody fragment to protect AChR from antigenic modulation caused by the anti-MIR mAb 198 was also examined. TE671 cells were preincubated with h-scFv-M4 or Fab198 and then intact mAb198 was added and incubated for 7 h. Surface AChR were labeled by 125 I-a-Bgt and the cell bound radioactivity was measured. The amounts of h-scFvM4 used were able to protect against AChR loss by up to 50%, whereas Fab198 was able to essentially elicit complete protection, apparently due to much higher affinity Ž K D of Fab198 for human AChR was approximately 22 nM. ŽFig. 8..

4. Discussion Because of the high specificity of mAbs for a wide variety of cellular targets, antibody therapy with mAbs is considered as a promising treatment of various diseases, such as cancer and autoimmune diseases. However, this promise has not yet been widely achieved because rodent antibodies used in clinical trials have been shown to be immunogenic ŽSchroff et al., 1985; Shawler et al., 1985.. A partial solution to this problem is the use of humanized

antibodies. Generally, in designing humanized antibodies, human FRs are selected, based on the identification of those human variable regions that are most homologous to the original rodent mAb. Although the CDR grafting technique has been described as resulting in the retention of antibody specificity ŽRiechmann et al., 1988., in most cases, a number of murine FR amino acids, in addition to the CDRs, have to be transferred in order to retain a functional antigen binding site ŽChothia et al., 1985; Foote and Winter, 1992; Singer et al., 1993; Padlan, 1994.. These FR residues are believed to affect the conformation of the CDRs. However, the number of changes in the human FRs should be minimum in order to retain low immunogenicity in human patients. Some humanized antibodies have already been used in clinical trials. The humanized antibody CAMPATH-1H, used to treat patients with non-Hodgkin lymphoma, was able to induce remission in the absence of any anti-immunoglobulin response ŽHale et al., 1988.. The results of the treatment of patients with refractory rheumatoid arthritis and recalcitrant plaque psoriasis with the humanized antibody, Orthoclone OKTcdr4a, were encouraging in terms of safety and tolerability, and again, no human antimurine antibodies were detected ŽBachelez et al., 1998; Moreland et al., 1998.. The recombinant scFv198 fragment can protect AChR against loss mediated by the intact parental mAb198 or MG antibodies in human muscle cell cultures ŽMamalaki et al., 1993. and is therefore a potentially useful tool in the clinical treatment of MG. In this article, we describe the humanization of scFv198 in an attempt to enhance its clinical potential by reducing its immunogenicity. To achieve this end, human FR amino acid sequences were chosen to be as similar as possible to the original rat sequence in order to reduce any deformation of the rat

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CDRs. Since the conformations of the VH and VL domains in the binding site are independent of each other or only dependent on a few residues, VH and VL FRs were chosen from two different antibodies. The gene coding for the humanized antibody fragment, h-scFv198, was synthesized entirely by PCR using long oligonucleotides Ž225 bases in length., the VH and VL synthetic genes being created separately and their dsDNAs converted into ssDNA before assembly by overlap extension and PCR. The synthetic gene was then expressed in E. coli and the recombinant protein produced was translocated to the periplasm, where disulfide bond formation occurred and a soluble, biologically active conformation was apparently obtained. The poor level of expression seen for soluble h-scFv198 is probably due to the specific antibody framework regions ŽJung and Pluckthun, 1997.. RIA assays with soluble Torpedo AChR demonstrated that h-scFv198 had very low binding activity for AChR. Molecular modeling was then used to identify framework region amino acids present in the original rat antibody but different in the humanized antibody that might interact with the CDRs, then these amino acids were substituted into the human FR, along with the CDRs. According to the models obtained, residue VH71 shows strong interactions with residues VH29 and VH53, stabilizing the conformation of the CDR1 and CDR2 loops. The importance of residue VH71 in antibody conformation has already been reported ŽTramontano et al., 1990; Carter et al., 1992; Sato et al., 1994; Xiang et al., 1995.. When this residue is replaced, it seems that significant conformation distortions occur in the CDR1 and CDR2 loops. Moreover, by substitution of residues VH27, VH29, and VH30, the rat CDR1 was essentially extended to include the whole H1 structural loop. The crucial role of molecular modeling in the humanization of scFv198 is illustrated by the hscFv-M4 variant produced, which binds Torpedo AChR 30 times more efficiently than the simple CDR loop swap h-scFv198 variant. Furthermore, the affinity of h-scFv-M4 for either Torpedo or human AChR was only four times lower than that of the rat scFv198. Finally, antigenic modulation studies have shown that this humanized antibody fragment was capable of protecting half of the cell surface AChR from the modulating effect of intact mAb, whereas the rat scFv198 was capable of protecting cell surface AChR against mAb198 nearly completely Ž80%. ŽMamalaki et al., 1993.. These results suggest that further efforts should be performed towards the design and construction of a new mutant with further improved affinity.

5. Conclusion We present the humanization and subsequent modification of an scFv antibody fragment Žh-scFv-M4. constructed entirely by PCR, which was able to protect the AChR from the binding of anti-MIR mAbs and anti-a MG

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antibodies. However, for this molecule to be suitable for therapeutic approaches, its AChR binding affinity must be further improved. It is possible that a panel of several different protective molecules may have to be used to obtain near-complete protection of all patients against their full spectrum of antibodies. The Fab fragment of mAb198 has been crystallized and the determination of its 3D structure is in progress ŽVatzaki et al., 1993; K. Poulas, K.R. Acharya, E. Eliopoulos, N. Oikonomakos and S.J. Tzartos, unpublished data.. The data obtained from the X-ray crystallographic studies might then identify FR residues in the rat antibody that influence antigen binding. Subsequently, these residues will be used to replace the corresponding residues in the humanized antibody in order to obtain a yet higher affinity, possibly capable of in vivo protection of the human AChR.

Acknowledgements We thank Dr. G. Winter for kindly providing us with pHEN1 vector, A. Kokla and N. Trakas for excellent technical assistance and T. Barkas for valuable suggestions. This work was supported by grants from the Greek General Secretariat of Research and Technology ŽPENED 567., the Association Francaise contre les Myopathies and the contract CHRXCT94-0547 of the HCM program of CEC.

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