Ig L-chain Shuffling for Affinity Maturation of Phage
Library-derived Human Anti-human MCP-1Antibody Blocking its Chemotactic Activity
Keisuke Yoshinaga1,Miyuki Matsumoto2,Masaharu Torikai2,Kazuki Sugyo1,
Saori Kuroki1,Kentaro Nogami1,Ryo Matsumoto1,Shuhei Hashiguchi1,Yuji Ito1,
Toshihiro Nakashima2and Kazuhisa Sugimura1,*
1Department of Bioengineering,Faculty of Engineering,Kagoshima University,Korimoto1-21-40,
Kagoshima,Kagoshima890-0065;and2The Chemo-Sero-Therapeutic Research Institute,Kyokushi Kikuchi,热电堆
Kumamoto869-1298,Japan
Received November13,2007;accepted January7,2008;published online January23,2008
Monocyte chemotactic protein-1(MCP-1,CC-chemokine ligand2;CCL2)is involved
in the development of various forms of chronic inflammations.Employing the naive
human single-chain Fv displaying phage library,we established seven MCP-1-specific
scFvs.The MC8and MC32clones exhibited blocking activity for the MCP-1-induced
chemotaxis of THP-1cells,in spite of their monovalency.The analysis of V gene
usage showed that all clones bore the identical Vh1gene,IGHV1-24Ã01,with variable
DJ joining sequences,while their Vl usage was relatively varied,suggesting the
preferential contribution of the Vh gene.Based on these findings,to minimize
the deteriorative influences on the MCP-1specificity of MC32,we aimed to achieve
the affinity maturation of MC32using MC32L-chain shuffling library and select
MC32variants.Most MC32variants increased their affinity by reducing the k off value
with no influence of the antigen specificity.MC32variants#22or#56showed»15-fold
higher affinity than MC32,indicating that the L-chain shuffling library is useful if the
Vh is dominantly involved in the determination of the antigen specificity.
Key words:affinity maturation,human antibody,L-chain shuffling,MCP-1,phage
display library.
Abbreviations:AP,alkaline-phosphatase;BSA,bovine serum albumin;CDR,complementary determining
region;FR,framework;HPLC,high performance liquid chromatography;HRP,horseradish peroxidase;
HAS,human serum albumin;IC50,50%inhibitory concentration;IgG,immunoglobulin G;Ig L-chain,
immunoglobulin light chain;IPTG,isopropyl-thio-b-D-galactopyranoside;MCP-1,monocyte chemotactic
protein-1;MIP-1a,macrophage inflammatory protein-1a;PBMC,peripheral blood mononuclear cell;PBS,
phosphate-buffered saline;PCR,polymerase chain reaction;PEG,poly(ethylene glycol);RU,response unit;
scFv,single-chain variable fragment;TU,transforming unit.
Monocyte chemotactic protein-1(MCP-1)is a member of the C-C chemokine family and a potent chemotactic factor for monocytes,T cells,and NK cells(1,2).MCP-1 has been implicated in a wide range of acute and chronic inflammatory processes characterized by monocyte infil-tration,including asthma,glomerulonephritis,rheuma-toid arthritis,atherosclerosis,multiple sclerosis, inflammatory bowel disease and meningitis,as well as in the accumulation of macrophages in the tumour sites (3–6).In these conditions,monocyte infiltration may be a key early event in disease progression.Thus,the inhibition of the chemotactic activity of MCP-1is shown to be a potential intervention point for the therapy of these diseases.In a number of animal models,MCP-1-neutralizing antibodies or biological antagonists showed beneficial activities in reducing the inflammation(7,8).
Recently,antibody medicines are being developed. From the viewpoint of immunogenicity,human antibodies are suitable for use in therapy.Phage-libraries displaying antibody fragments are attractive tools for obtaining human antibodies(9,10).We have established human scFv-displaying phage libraries and isolated several scFvs with inhibitory activities against a target molecule,such as human IL-18,IL-6or Fc"RI a (11–13).
In this study,we attempted to establish human MCP-1-specific human antibodies with inhibitory activity on the chemotactic activity using human scFv-dispalying phage libraries and subsequently to achieve affinity maturation using an IgL-chain shuffling phage library. We isolated seven scFv clones specific to MCP-1and found that two clones,MC8and MC32,showed inhibition in the MCP-1-induced chemotaxis of a human acute monocytic leukaemia cell line,THP-1,in vitro.The analysis of V gene usage showed that all clones bore the identical Vh1gene,IGHV1-24Ã01,with variable DJ joining sequences,while their Vl usage was relatively varied,suggesting a predominant role of the Vh chain in the MCP-1specificity of these clones.
This unique result suggested that the L-chain shuffling of MC32may be an effective strategy for affinity
*To whom correspondence should be addressed.Tel:+81-99-285-8345,Fax:+81-99-258-4706,E-mail:kazu@be.kagoshima-u.ac.jp J.Biochem.143,593–601(2008)包层模
doi:10.1093/jb/mvn009
水
火箭制作方法Vol.143,No.5,2008593ß2008The Japanese Biochemical Society. at Wageningen Universiteit en Research on June 21, 2014/ Downloaded frommaturation,although several other methods have been reported,such as site-directed mutagenesis,error-prone PCR and shuffling of CDRs (14–18).We constructed an MC32-L-chain shuffling library,which was prepared by recombining the MC32Vh chain with a series of k light-chain repertories in a phagemid vector system.Only after the first round of biopanning with a MCP-1-coated plate,every clone exhibited MCP-1-binding specificity.We examined the K d value of these MC32variants and assayed their inhibitory activity on a chemotaxis assay.Seventy-five percent of the variants showed a decreased k off value,resulting in successful affinity maturation in parallel with their IC 50.Thus,this study demonstrated that L-chain shuffling approach could minimize the deteriorative influence on the antigen specificity of a parent clone when Vh domains dominantly contribute for its antigen specificity.
MATERIALS AND METHODS
Human Single-Chain Fv-Displaying Phage Library—Four kinds of human scFv-displaying M13phage libraries were constructed as described previously (9,13).The V genes for g ,m ,k or chains were separately amplified by PCR.The Vh and Vl gene segments were assembled by PCR with a linker DNA encoding (Gly 4Ser)3(scFv linkers)to construct a scFv gene.The scFv genes were recombined to the pCANTAB 5E phagemid vector.The scFv display phage was prepared as described (13).
L-Chain Shuffling Library—An L-chain shuffling library was prepared according to Marks et al .(Fig.1;9,15).The V k genes were amplified from cDNA that had been prepared for scFv-displaing phage library as
described (13).In this PCR,six kinds of back primers for human V k family and five kinds of J k forward primers were employed (9,15).The Vh gene of MC32was assembled with these V k gene segments together with linker DNA encoding (Gly 4Ser)3(scFv linkers)by PCR.Vl shuffling scFv gene fragments were re-amplified by PCR using primers containing restriction sites (Sfi I and Not I).The scFv genes were digested by restriction enzymes (Sfi I and Not I),gel-purified and ligated to the restriction-digested phagemid vector pCANTAB 5E using T4DNA ligase.
The diversity of each library was defined as the number of recombinant single ampicillin-resistant clones obtained from consecutive ligation and transfor-mation of the plasmid into competent cells prior to any amplification process of the genes.These libraries contained 6.4Â106(V k 1),9.8Â106(V k 2), 4.8Â106(V k 3),7.2Â106(V k 4),8.7Â106(V k 5)and 6.4Â106(V k 6)independent clones.
Biopanning—Biopanning was performed as described by Gejima et al .(12).Briefly,5m g/ml of recom
binant human MCP-1(R&D Systems,Inc.,Minneapolis,MN)in 0.1M NaHCO 3(pH 8.6)was incubated at 48C overnight in a 35mm Petri dish (Iwaki Glass,Tokyo).The plates were blocked with 0.5%gelatin,5%skimmed milk or 1%BSA.A library [5Â1011transforming units (TU)]consisted of Vh-V ( -library),or Vh-V k (k -library)was incubated in an MCP-1-coated plastic plate at room temperature for 1h.The plates were washed 10times with PBS containing 0.1%Tween-20(PBST).The bound phages were eluted with 1ml 0.1M glycine-HCl (pH 2.2),immediately neutralized with 0.1vol of 1M-Tris–HCl (pH 9.1),and amplified by infection with log-phase Escherichia coli ,suppressor strain TG1cells (sup E).
mRNA derived PBMC
MC32 Vl shuffling scFv libray
Recombination into pCANTAB5E phagemid vector
Fig.1.Preparation of MC32i -chain shuffling library.The Vh gene was amplified from the scFv gene of MC32by PCR using MC32Vh back and forward primers.The V k genes were amplified from PBMC cDNA using V k -specific primers,asterisk:back primers for human V k family 1to 6.double asterisk:forward primers for human J k family 1to 5(9,15).The MC32Vh gene,the V k gene repertories and linker DNA encoding
(Gly 4Ser)3were assembled by PCR using these primers to produce a heterogeneous population consisting of varying MC32Vh-linker-V k DNA fragments (MC32V k -shuffling genes).This fragment was inserted into the sites of Sfi I and Not I of phagemid vector pCANTAB 5E.Phage clones were recovered as described (11–13).
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To select more specific phage clones,the amplified eluate was incubated with a decreasing amount of MCP-1(3to 1m g)for subsequent rounds.
Soluble scFv—The soluble scFv was prepared by infecting phage clones with E.coli HB2151(a non-suppressor strain)as described(11).These periplasmic scFvs were purified with an anti-E-tag affinity column (GE Healthcare Bio-science Corp,Piscataway,NJ). To eliminate endotoxins,a sample was applied onto Acticlean Etox(Sterogene Bioseparations,Inc.,Carlsbad, CA).Gel permeation chromatography was performed as follows:scFvs were loaded on a10/300GL Superdex75 (GE Healthcare Bio-science Corp.,Piscataway,NJ), which was developed in PBS at a flow rate of0.4ml/ min.The monomer form or the dimer form of scFv was fractionated.
ELISA—ELISA was performed as described(19,20). Briefly,ELISA micro titre plates(Nunc,Denmark)were coated with human MCP-1(80ng/40m l/well,R&D Systems,Inc.,Minneap
olis,MN)or control proteins (80ng/40m l/well)overnight at48C.The plates were blocked with1%BSA in PBS(400m l)at room tempera-ture(RT)for1h.Phage clones[40m l of PEG-precipitated phage(1012TU)]were added to the wells for1h at RT. To detect the bound phage clones,biotinyilated anti-M13 mAb(1/1,000dilution,GE Healthcare Bio-science Corp, Piscataway,NJ)as a primary antibody and alkaline-phosphatase(AP)-conjugated streptavidin(1/1,000dilu-tion,Vector Laboratories,Inc.,Burlingame)were used. Absorbance was measured at405nm in incubation with 50m l of a p-nitrophenyl phosphate/10%diethanol amine solution by use of a microplate reader.The soluble scFv antibody was detected using anti-E-tag mAb(1/1,000 dilution,GE Healthcare Bio-science Corp,Piscataway, NJ)in combination with an AP-conjugated anti-mouse IgG(Jackson Immuno Research,West Grove,PA). Immunoblotting Analysis—The periplasmic fraction of the HB2151culture was subjected to SDS–PAGE(12.5%) and blotted to a PVDF membrane using a semidry electroblotter.After blocking with5%skimmed milk, it was detected by horseradish peroxidase(HRP)-conjugated anti-E-tag mAb using ECL reagents(GE Healthcare Bio-science Corp,Piscataway,NJ)on the image analyzer LAS-1000(Fujifilm,Tokyo).The NEB pre-stained protein marker(New England Biolabs, Beverly,MA)was used as the protein marker.
DNA Sequencing—The nucleotide sequence of the scFv genes was identified using the Dye Termina
tor Cycle Sequencing FS Ready Reaction kit(PE Applied Biosystems,Foster City,CA)with primer pCANTAB5-S1 (50-CAACGTGAAAAAATTATTATTCGC-30)and pCANT AB5-S6(50-GTAAATGAATTTTCTGTATGAGG-30).The amino acid residues of each variable domain were according to Kabat and Wu(21).
Surface Plasmon Resonance Analysis—The BIAcore X100system(BIAcore AB,Uppsala,Sweden)was used. The MCP-1(100m l of50m g/ml of10mM sodium acetate buffer,pH5.0)was immobilized onto a CM5-sensor chip according to the amine-coupling protocol provided by the manufacturer,and unreacted sites were masked with 1M ethanolamine-HCl(pH8.5).The association reaction was initiated by injecting varying concentrations of an scFv solution.The analyte injection was performed at a flow rate of30m l/min.The dissociation reaction was performed washing with PBS.At the end of the cycle,the sensor chip surface was regenerated with a0.1M glycine-HCl buffer(pH2.5).The association(k on,M–1s–1) and dissociation(k off,s–1)constants were calculated using BIAcore system software(BIAcore X100 Evaluation software).In the case of whole IgG antibody, K d was calculated using k on1and k off1values by bivalent analyte model whereas that of scFv was evaluated by 1:1binding model.
Chemotaxis Assay—The migration of cells was assessed in a48-well microchamber(Neuroprobe,Ca
bin John,MD)as previously described(22).Briefly,the lower wells were filled with25.5m l MCP-1at a concentration of 2.2nM with or without varying concentrations of scFvs, while the upper wells were filled with THP-1cells (human acute monocytic leukaemia, 3.75Â105cells/ 50m l/well).The two compartments were separated by polycarbonate filter with8m m pores.After120min incubation at378C in humidified air-5%CO2,the filters were removed.The filter was then wiped to remove non-migrated cells on its upside,fixed in paraformaldehyde and stained with Wright–Giemsa stain(Diff-Quik, Sysmex International Reagents Co.,Ltd.,Hyogo, Japan).The number of migrated monocytes was deter-mined microscopically,and data were calculated as the percentage of the control response induced with MCP-1. Construction of the MC32IgG—IgG form of MC32was constructed as described(11).Briefly,the Vh and Vl genes of MC32were cloned into mammalian cell expression vector pCAG-H with a human IgG1constant region and pCAG-L with human C k,respectively.CHO cells were co-transfected with an equipped molar mixture of these vectors by Lipofectamine(Invitrogen,Carlsbad, CA)according to the manufacturer’s instructions.After 48h of culture in a selective medium,the supernatant was harvested from the culture by centrifuge and filtered through a0.22m m membrane.IgG was purified using a protein A affinity column.The purified MC32-IgG was analysed by SDS–PAGE.
RESULTS
Establishment of MCP-1-Specific scFvs—The scFv phage clones were selected from scFv phage libraries by three rounds of panning with human MCP-1.We screened144clones and found three MCP-1-specific clones from the k-library(2%);on the other hand,we isolated four MCP-1-specific clones(4.4%)from the -library by screening a total of90clones.The clones from the k-library were designated as MC8,MC32or MC62,and those from the -library,as ap20,ap68,apM2 or apM9.
The soluble scFvs were purified from the periplasmic extract of E.coli HB2151on an anti-E tag affinity column.Consisting with the binding specificity of phage clones,all of these scFvs specifically bound to MCP-1but not to unrelated proteins,including MIP-1a(Fig.2). Immunoblotting analysis using HRP-conjugated anti-E-tag mAb gave a single protein band corresponding to the expected molecular weight(Fig.3a).Gel permeation
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chromatography showed that each preparation of scFvs contained significant amounts of a dimer form in addition to a monomer form (Fig.3b).Therefore,the HPLC-purified monomer form was freshly
prepared and analysed to examine the affinity of scFv.
Nucleotide Sequencing of the scFv Gene—The scFv gene sequences of the selected phage clones were examined (Fig.4a and b).The germ-line Vh and Vl gene was assigned by sequence homology based on the database (VBASE)of germ-line V-genes compiled by Tomlinson et al .(Fig.4c;23).This analysis revealed that all of the isolated scFvs were characterized by germline genes IGHV1-24Ã01belonging to the Vh1gene family.D and Jh are relatively variable.Considering that these clones exhibited equal binding specificity to MCP-1,the similarity of CDR2among these clones is noteworthy.On the other hand,it is unlikely that CDR3is preferentially involved in determining the MCP-1specificity in these scFvs.It is also noteworthy that MC8and MC32are different only at one amino acid residue (Q in MC8,while P in MC32at position 114of Vh).In contrast,the Vl gene usage appeared to be variable (IGKV1-39Ã01for two clones,IGLV3-21Ã01for two clones)while the Jl gene was relatively restricted for usage.These results suggested a dominant contribution of the IGHV1-24Ã01heavy-chain gene,particularly,the Vh-CDR2region,rather than of the light-chain gene,to the MCP-1-specific-binding activity.Inhibitory Activity of MCP-1-Specific Human Antibodies on MCP-1-Induced Chemotaxis—To examine the effects of scFvs on the chemotactic activity of MCP-1,we assayed the migration of THP-1cells using 2.2nM MCP-1in the presence or absence of varying co
ncentra-tions of these scFvs.Since scFvs were prepared from the periplasm li ,the contamination of endotoxins may affect the chemotaxis activity of MCP-1.To exclude this possibility,the endotoxin level of the scFv samples was estimated to be below 30ng/m g-scFv.It was shown that,at this level,endotoxins did not affect THP-1chemotaxis by MCP-1(data not shown).
As shown in Fig.5a and b,MC8and MC32scFv clones exhibited the antagonistic activity in a dose-dependent manner.ap20and ap64showed weak inhibitory activity at 3.5m M while apM2and apM9showed no inhibitory activity (Fig.5c).
We constructed the MC32IgG form using MC32scFv DNA as described in M ATERIALS AND M ETHODS section.ELISA showed that MC32IgG preserved its
binding
Fig.2.MCP-1-specific scFv clones.ELISA plates were coated with 80ng/well of MCP-1and various unrelated proteins for 12h.Each scFv was tested for its binding specificity as described in
MATERIALS AND METHODS section.(a):isolates from the k -library;(b):isolates from the
来书网
-library.
Fig.3.Biochemical characterization of scFvs.(a)Immuno-blotting analysis:the periplasmic extract (MC8,MC32,MC62,ap20,ap64,apM2or apM9)of the HB2151cell culture was subjected to SDS–PAGE (12.5%).Immunoblotting was performed with HRP-conjugated anti-E-tag mAb.(b)Gel permeation chro-matography (10/300GL Superdex75)of MC32scFv.
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activity against MCP-1.In the chemotaxis assay,MC32 IgG inhibited the MCP-1-induced THP-1-migration 2.5-fold more strongly than MC32scFv in reference to IC50(Fig.5d).
SPR Analysis of MC32scFv and IgG Form—The affinity of the monomer form of MC32scFv and IgG was analysed.SPR profiles of MC32scFv and IgG form were presented in Fig.6a and b.They showed clear responses in dose–response manners.The K d of MC32IgG was almost similar to that of scFv form (Fig.6c).
喉管Vl Chain Contribution to the Affinity and Anti-MCP-1 Chemotactic Activity of MC32scFv—The analysis of the V gene usage of MCP-1-specific scFvs suggested its preferential contribution of Vh for the commitment of antigen specificity(Fig.4).To avoid a strong influence on the MCP-1-binding specificity but acquire an increase of affinity,L-chain shuffling seemed to be one of the choices for affinity maturation.Therefore,we attempted to evaluate the role of the Vl gene by using an MC32 L-chain shuffling library.In this library,the whole repertoire of V k genes derived from human PBMCs was
Fig. 4.Amino acid sequences and gene usage of MCP-1-specific scFvs.Amino acid sequences of th
e Vh(a)and Vl domains(b):the complementary-determining regions(CDR1–CDR3)and the flanking regions(FR1-4)were deduced according to Kabat and Wu(21).(c)The germ-line gene usage was assigned based on homology to a database(VBASE)of germline V-genes compiled by Tomlinson et al.(23).
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