HUMAN ANTIBODIES AND PROTEINS

Abstract

The present invention provides composite proteins, including antibodies, which show reduced immunogenicity. In particular, composite antibodies for use in humans are provided, in particular antibodies which have been modified to remove one or more T-cell epitopes. Methods for generating such proteins are also provided.

Claims

1. A modified antibody or antigen-binding fragment thereof wherein the heavy and light chain variable regions of the modified antibody or antigen-binding fragment are each composed of two or more segments of amino acid sequence from one or more other antibodies or antigen-binding fragments, whereby the segments are neither whole CDRs nor framework regions.

2-36. (canceled)

37. A method for screening composite antibody variable regions comprising: generating a library of genes encoding composite antibody variable regions derived from multiple segments of amino acid sequence of 2 to 31 amino acids long from other antibodies or antigen-binding fragments, wherein the multiple segments are neither whole CDRs nor whole framework regions; screening the composite antibody variable regions to avoid T cell epitopes; and expressing at least a portion of the library and screening the expressed antibody variable regions for binding to one or more antigens of interest.

38. The method of claim 37, wherein the multiple segments of amino acid sequence are derived from human antibodies.

39. The method of claim 37, wherein the expressed antibody variable regions form part of expressed antibodies.

40. The method of claim 37, wherein the expressed antibody variable regions form part of expressed antigen-binding fragments.

41. The method of claim 40, wherein the expressed antigen-binding fragments are selected from Fv's, Fab's, Fab2's, SCA's, single domain antibodies, and multimeric derivatives of each of these.

42. A method for producing copies of a composite antibody variable region of interest comprising: generating a library of genes encoding composite antibody variable regions derived from multiple segments of amino acid sequence of 2 to 31 amino acids long from other antibodies or antigen-binding fragments, wherein the multiple segments are neither whole CDRs nor whole framework regions; screening the composite antibody variable regions to avoid T cell epitopes; expressing at least a portion of the library and screening the expressed antibody variable regions for binding to one or more antigens of interest; identifying an antibody variable region of interest; and producing additional copies of the identified antibody variable region of interest.

43. The method of claim 42, wherein the multiple segments of amino acid sequence are derived from human antibodies.

44. The method of claim 42, wherein the additional copies of the identified antibody variable region of interest form part of produced antibodies.

45. The method of claim 42, wherein the additional copies of the identified antibody variable region of interest form part of produced antigen-binding fragments.

46. The method of claim 45, wherein the produced antigen-binding fragments are selected from Fv's, Fab's, Fab2's, SCA's, single domain antibodies, and multimeric derivatives of each of these.

47. The method of claim 44, wherein the produced antibodies comprise one or more regulatory T cell epitopes which suppress immune reactions.

48. The method of claim 45, wherein the produced antigen-binding fragments comprise one or more regulatory T cell epitopes which suppress immune reactions.

Description

[0088] The following examples should not be considered limiting for the scope of the invention. The figures and tables relate to the examples below and are as follows;

[0089] FIGS. 1/2Sequence of VH (FIG. 1) and VL (FIG. 2) genes used for Composite Human anti-HER2 antibody

[0090] FIG. 3Inhibition of proliferation of human SK-BR-3 cells after 8 days incubation with chimeric 4D5 IgG1/kappa, Composite Human anti-HER2 antibody and epitope avoided anti-HER2 EACHAB with chimeric anti-IgE control (see example 4)

[0091] FIGS. 4/5Sequence of VH (FIG. 4) and VL (FIG. 5) genes used for Composite Human anti-Lewis Y antibody

[0092] FIGS. 6/7Sequence of VH (FIG. 6) and VL (FIG. 7) genes used for Composite Human anti-human IgE antibody

[0093] FIG. 8Sequence of VH and VL genes used for Composite Mouse anti-human TNF antibody including avoidance of human T cell epitopes

[0094] FIG. 9ELISA for binding to human TNF by Composite Mouse and chimeric anti-human TNF antibody

[0095] FIG. 10V region sequences of anti-TNF antibody A2

[0096] FIG. 11Sequences of composite human anti-TNF VH variants

[0097] FIG. 12Sequences of composite human anti-TNF VL variants

[0098] FIGS. 13/14Oligonucleotides for construction of chimeric mouse:human anti-TNF VH (FIG. 13) and VL (FIG. 14)

[0099] FIGS. 15/16Oligonucleotides for construction of primary composite human anti-TNF VII (FIG. 15; corresponding to SEQ ID No. 3 FIG. 11) and VL (FIG. 16; corresponding to SEQ ID No. 4 FIG. 12)

[0100] FIG. 17Oligonucleotides for construction of secondary composite human anti-TNF VH and VL variants

[0101] FIG. 18WEHI-164 protection Assay for composite human anti-TNF antibodies

[0102] FIG. 19Time-Course human T cell assay of lead composite human anti-TNF

[0103] FIG. 20Activity of composite bouganin molecules with inserted human sequence segments

[0104] Tables 1-3CDRs used in Composite Human antibody scFv library comprising 1869 residue-long VH CDR3s (table 1), 778 residue-long VL CDR3s (table 2), and 15310 residue-long VL CDR3s (table 3).

[0105] Table 4Human sequence segments used for primary composite human anti-TNF VH and VL variants

[0106] Table 5Activity of composite human anti-TNF variants

[0107] Table 6Immunogenic peptide sequences of bouganin and replacement human segments in bouganin variants

EXAMPLE 1

Construction of Composite Human Anti-HER2 Antibody

[0108] For creation of a human variable region sequence segment library, amino acid sequences from a range of human immunoglobulins were collected into a single database comprising the in silico human variable region sequence library including heavy (VH) and light (VL) chain variable region sequences. Sources of sequences included the NCBI Igblast database (www.ncbi.nih.gov), Kabat databases (Kabat et al., Sequences of Proteins of Immunological Interest, NIH publication 91-3242, 5.sup.th ed. (1991) (and later updates)), Vbase (www.mrc-cpe.cam.ac.uk/imt.doc), Genbank (Benson et al., Nucl. Acids Res. 25 (1997) p1-6 or via www.bioinf.org.uk/abs) databases. The reference antibody variable region sequences used was a humanised anti-HER2 antibody known as Herceptin (Carter et al., Proc. Nat. Acad. Sci. USA, vol 89 (1992) p4285, U.S. Pat. No. 5,821,337). Segments from the in silico human variable region sequence library were selected for identity to the corresponding amino acids in the Herceptin variable region sequence and combined to produce the composite human VH and VL sequences as shown in FIGS. 1 and 2 respectively.

[0109] Recombinant DNA techniques were performed using methods well known in the art and, as appropriate, supplier instructions for use of enzymes used in these methods. Sources of general methods included Molecular Cloning, A Laboratory Manual, 3.sup.rd edition, vols 1-3, eds. Sambrook and Russel (2001) Cold Spring Harbor Laboratory Press, and Current Protocols in Molecular Biology, ed. Ausubel, John Wiley and Sons. Detailed laboratory methods are also described in example 7 below. Composite human VH and VL sequences corresponding to Herceptin were created using, for each chain, eight synthetic oligonucleotides of 30-60 amino acids in length encoding the entire composite human VH and VL sequences. In parallel, as a control reagent, a chimeric form of the mouse monoclonal antibody 4D5 (Hudziak et al., Mol. Cell. Biol., (March 1989) p1165-1172)), was also created using eight synthetic oligonucleotides per chain. Separate VH and VL oligonucleotides were first phosphorylated, mixed at equal molar ratios, heated to 94oC for 5 min in a thermal cycler followed by cooling to 65oC and incubation at 65oC for 2 min. Incubations were then continued at 45oC for 2 min., 35oC for 2 min., 25oC for 2 min and 4oC for 30 min. Oligonucleotides were then ligated using T4 DNA ligase (Life Technologies, Paisley UK) at 14oC for 18 hours.

[0110] To each of the VH and VL oligonucleotide mixtures, additional oligonucleotides encoding a 5 flanking sequence, including a Kozak sequence, the leader signal peptide sequence and the leader intron, and 3 flanking sequence, including the splice site and intron sequence, were added and annealed as above. The Composite Human V.sub.H and V.sub.K and the 4D5 expression cassettes produced were cloned as HindIII to BamHI fragments into the plasmid vector pUC19 and the entire DNA sequence was confirmed. These were transferred to the expression vectors pSVgpt and pSVhyg which include human IgG1 (V.sub.H) or Kappa (V.sub.K) constant regions respectively and markers for selection in mammalian cells. The DNA sequence was confirmed to be correct for the Composite Human V.sub.H and V.sub.K and 4D5 V.sub.H and V.sub.K in the expression vectors.

[0111] The host cell line for antibody expression was NS0, a non-immunoglobulin producing mouse myeloma, obtained from the European Collection of Animal Cell Cultures, Porton, UK (ECACC No 85110503). The heavy and light chain expression vectors were co-transfected into NS0 cells by electroporation. Colonies expressing the gpt gene were selected in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% foetal bovine serum, 0.8 g/ml mycophenolic acid and 250 g/ml xanthine. Transfected cell clones were screened for production of human antibody by ELISA for human IgG. Cell lines secreting antibody were expanded and the highest producers selected and frozen down in liquid nitrogen. The modified antibodies were purified using Prosep-A (Bioprocessing Ltd, Northumberland, UK). The concentration was determined by ELISA for human IgGK antibody.

[0112] The Composite Human antibody and chimeric 4D5 antibodies were tested for inhibition of proliferation of the HER2+ human breast tumour cell line SK-BR-3 in conjunction with a negative control non-Her-2 binding human IgG1/Kappa antibody exactly as described by Hudziak et al. (ibid). The results (FIG. 3) show that Composite Human antibody and the chimeric 4D5 antibodies have equivalent potency in inhibiting growth of SK-BR-3 cells. FIG. 3 also shows data for an alternative epitope avoided Composite Human antibody produced as below.

[0113] In order to test the epitope avoidance option in the invention, the sequences of the Composite Human heavy and light chain variable regions were analysed for non-self human MHC class II binders using Peptide Threading (www.csd.abdn.ac.uk/gjlk/MHC-thread). This software predicts favourable interactions between amino acid side chains of the peptide and specific binding pockets within the MHC class II binding groove. All overlapping 13 mers from the Composite Human heavy and light chain variable sequences were threaded through a database of MHC class II allotypes and scored based on their fit and interactions with the MHC class II molecules. Peptides predicted to bind MHC class II were 13 mers beginning at residues 16 and 67 in VH, and 9 and 44 in VL. As a result, new segments of the human variable region sequence library were chosen instead of those used in the Composite Human sequences of FIG. 1 in order to introduce the amino acid changes VH 18L-A/69I-G; VL 11L-A, 46L-A. A corresponding epitope avoided Composite Human antibody (EACHAB=Epitope Avoided Composite Human AntiBody) was made by substituting some of the oligonucleotides used to make the antibody corresponding to the sequence in FIG. 1 and the EACHAB was made as in the method described above and tested to show inhibition of SK-BR-5 proliferation equivalent to the standard Composite Human antibody (FIG. 3). This data shows that Composite Human antibodies can be successfully constructed with equal potency to a control chimeric anti-Her-2 antibody and that an EACHAB version of the Composite Human antibody can be generated without loss of potency.

EXAMPLE 2

Immunogenicity of Composite Human Anti-HER2 Antibody

[0114] T cell proliferation assays were carried out to compare the immunogenicity of the Composite Human anti-HER2 antibody, the EACHAB variant and the chimaeric 4D5 antibody (see example 1). These antibodies were prepared from NS0 cells grown in serum-free, animal derived component-free, protein-free medium, HyClone HyQADCF-Mab (Hyclone Cat No: Cat no: SH30349) supplemented with HyQLS1000 Lipid Supplement (Hyclone Cat No: SH30554) and sodium pyruvate (Gibco Cat No: 11360-039). After buffer exchange into 50 mM MES pH6 on a Sephadex G25 (PD10 column), the antibodies were each passed through a cation exchange column (Mono-S 10/10) and eluted with a sodium chloride gradient (0 to 0.5M). The antibody containing fractions were then applied to a Superdex 200 preparative column (XK16/60) run in PBS. Peak fractions were pooled and stored at 4 C. The antibody concentrations were determined by ELISA for human IgG.

[0115] Immunogenicity analysis was performed using PBMCs (peripheral blood mononuclear cells) that were isolated from healthy human donor blood and cryopreserved in liquid nitrogen. Each donor was tissue-typed using an Allset PCR based tissue-typing kit (Dynal, Wirral, UK) and 20 healthy donors were selected according to individual MHC haplotypes. 2 ml bulk cultures containing 410.sup.6 PBMC in AIM V (Invitrogen, Paisley, UK) were incubated in a 24 well tissue-culture plate with test peptides (5 M final concentration) and proliferation was assessed on days 5, 6, 7, and 8 by gently resuspending the bulk cultures and transferring triplicate 100 l samples of PBMC to a U-bottomed 96 well plate. Cultures were harvested onto glass fibre filter mats using a Tomtec Mach III plate harvester (Receptor Technologies, UK) and counts per minute (cpm) values determined by scintillation counting using a Wallac Microbeta TriLux plate reader (using a paralux high efficiency counting protocol). For each test antibody, the stimulation index (SI) was calculated as the ratio of counts per minute (cpm) of the test antibody:cpm of the negative control with SI>2 considered a significant T cell epitope response. The results showed that the chimaeric 4D5 antibody induced significant proliferative responses on at least one of the four days of proliferation tested (SI greater than 2) in five of twenty healthy donors tested (25%), the Composite Human anti-HER2 antibody induced SI>2 in three of twenty donors (15%) whilst the EACHAB anti-HER2 antibody induced SI>2 in none of twenty donors (0%). These results indicated an order of immunogenicity of chimeric 4D5>Composite Human anti-HER2>EACHAB anti-HER2 with the latter showing no evidence of immunogenicity in any donor blood sample tested.

EXAMPLE 3

Construction of Composite Human Anti-Lewis Y Antibody

[0116] A Composite Human antibody specific for sialylated Lewis Y antigen was constructed as described in example 1 using, as the reference antibody variable region sequences, the humanised 3S193 antibody (Scott et al.; Cancer Res., 60 (2000) p3254-3261, U.S. Pat. No. 5,874,060). Segments from the in silico human variable region sequence library were selected for identity to the corresponding amino acids in the humanised 3S193 variable region sequence and combined to produce the Composite Human VH and VL sequences as shown in FIGS. 4 and 5 respectively. In parallel, a reference chimeric anti-Lewis Y antibody was made from the reference V region sequences. Human IgG1 (V.sub.H) and Kappa (V.sub.K) constant regions were used on both the Composite Human anti-Lewis Y antibody and the chimeric reference antibody and antibodies were tested by ELISA against synthetic Lewis Y-HSA conjugate as described in U.S. Pat. No. 5,874,060. The data showed a minimum concentration of 0.1 ug/ml chimeric antibody to give a binding signal in the assay compared to 0.15 ug/ml Composite Human antibody which is consistent with the data of U.S. Pat. No. 5,874,060.

EXAMPLE 4

Construction of Composite Human Anti-IgE Antibody

[0117] A Composite Human Anti-IgE antibody was constructed as described in example 1 using, as the reference antibody variable region sequences, the humanised anti-IgE antibody known as Xolair (Presta et al., J. Immunol., 151(5) (1993) p2623-2632). Segments from the in silico human variable region sequence library were selected for identity to the corresponding amino acids in the Xolair variable region sequence and combined to produce the Composite Human VH and VL sequences as shown in FIGS. 6 and 7 respectively. In parallel, a reference chimeric anti-IgE antibody was made from the reference V region sequences. Human IgG1 (V.sub.H) and Kappa (V.sub.K) constant regions were used on both the Composite Human Anti-IgE antibody and the chimeric reference antibody.

[0118] The specificity of the Fabs was further characterized by surface plasmon resonance (BIAcore 2000, Biacore AB, Uppsala, Sweden). Recombinant human IgE Fab was produced as described by Flicker et al., J. Immunol., 165 (2000) p3849-3859. Test antibodies were purified and immobilized onto flow cells of a CM chip using a NHS/EDC kit (Biacore) to obtain 2010 RU for chimeric anti-IgE and 2029 RU for Composite Human anti-IgE. 10 and 25 nM recombinant human IgE Fab in Hepes-buffered saline (10 mM Hepes, 3.4 mM EDTA, 150 mM NaCl, 0.05% (v/v) surfactant P20, pH 7.4) was passed over the test antibodies at a flow rate of 5 l/min for 10 minutes. The results showed that for both 10 and 25 nM IgE Fab, an equivalent SPR (surface plasmon resonance) curve was detected for the chimeric anti-IgE and the Composite Human anti-IgE antibodies showing that the latter had successfully achieved binding efficiency equivalent to the reference anti-IgE antibody.

EXAMPLE 5

Generation and Screening of Composite Human scFv Libraries

[0119] The strategy for initial construction of the human scFv (single-chain Fv) library was to construct seven consensus human VH and four consensus human VL (kappa) genes as detailed in Knappik et al., J. Mol. Biol., 296 (2000) 57-86 and to clone into these a large number of VH and VL CDR3 segments from databases of human variable regions. This list of CDR3s is shown in table 1 for VH CDR3s, table 2 for VL CDR3s of 8 amino acids and table 3 for VL CDR3s of 10 amino acids. For the master VH and VL construction, 6 overlapping synthetic oligonucleotides encoding VH and VL up to the end of framework 3 were synthesised as detailed by Knappik et al., ibid, and subjected to recursive PCR (Prodromou and Pearl, Protein Engineering, 5 (1992) 827-829). These were ligated into EcoRV digested pZero-1 vector (Invitrogen, Paisley, UK). For addition of CH1 and C kappa, both initially with 4D5 CDR3s (Carter et al, Bio/Technology, 10 (1992) 163-167), the protocol of Knappik et al., ibid, was followed except that the VH-CH1 SapI-EcoRI and VL-C kappa NsiI-SphI fragments were both blunt-end cloned into EcoRV digested pZero-1.

TABLE-US-00001 TABLE1 # Name H3 Length-H3 Subgroup(H) MUC1-1CL DFLSGYLDY 9 I ALL1-1CL VRGSGSFDY 9 III ALL7-1CL DRGGNYFDY 9 III L36CL MYNWNFFDY 9 I 5.M13CL AGLGMIFDY 9 I Au2.1CL RGFNGQLIF 9 I M71CL ALTGDAFDI 9 II VH6.N1CL TKLDWYFDL 9 II E556.XCL RYGGFYFDY 9 II E556.11CL GYSNEGMDV 9 II VH6.A5CL SWDGYSYIY 9 II VH6-EX8CL QMGAEYFQH 9 III E544.2CL DMSLDAFDI 9 II RF-SJ4CL GSVGATLGE 9 II 3.A290CL YGDYHYFDY 9 III A95 GVGSSGWDH 9 III 60P2CL KGSLYYFDY 9 III E553.6CL PNWNDAFDI 9 III E553.16CL RGIPHAFDI 9 III 333CL PPEVESLRS 9 III 1H1CL PPEVESLRS 9 III 126CL PPEVESLRS 9 III 1B11CL PPEVESLRS 9 III 115CL PPEVESLRS 9 III 112CL PPEVESLRS 9 III 2C12CL PPEVESLRS 9 III 2A12CL PPEVESLRS 9 III BUT DLAAARLF? 9 III KOL-based QGTIAGIRH 9 III resh. CAMPATH-9 L2E8CL EDYYYGMDV 9 III s5D4CL DPINWYFDL 9 III ss4CL DRAAGDRDY 9 III P2-57CL HQMYSNSDY 9 I HuHMFG1CL SYDFAWFAY 9 I NEW-based QGTIAGIRH 9 II resh. CAMPATH-9 TR1.10CL VLGIIAADH 9 I L3B2CL DLTGDAFDI 9 I DAW SCGSQYFDY 9 II ss7CL LWNWDAFDI 9 I ss6CL DIMTWGFDY 9 I s5A9CL SNWYWYFDL 9 III NEWM NLIAGCIDV 9 II L2A12CL GGKGGEFDD 9 I B5G10HCL DSGNYRIDY 9 II E553.9CL DPRLDAFDI 9 III SpA1-29CL GYSYPVWGR 9 III AM28CL LVGNSWLDY 9 III BM2CL DL?GLVVEY 9 III CM29CL KVSLSAFDI 9 III B-B10M0CL RGDAMYFDV 9 I HSVCBM8CL DPNPWYFDL 9 III HSVCD53CL DYGDYAFDI 9 III HSVCBG6CL SAHSDAFDM 9 III MICA4CL LEGLGWFDP 9 I 1/11CL RSDYGAIDY 9 III 5/8CL NLGFYHMDV 9 III B6204CL EARGGGGEY 9 III VHCLONE EGWISALNG 9 III 1CL VHCLONE EGEGEYFDY 9 III 32CL MG6-1CL ERTSGDFDF 9 III MG6-3CL NSPGATFES 9 III DaudiCL GNGQKCFDY 9 III IE4CL RGSLQYLDY 9 I IF10CL NNGSYYFDY 9 I hsighvm148CL GSDYSNFAY 9 III E3-MPOCL STHRSAFDV 9 II rev9FdCL EGVHKNFDH 9 III NANUC-1CL LSRAGGFDI 9 III Patient RMPAVAFDY 9 II 14CL 14G1CL RMPAVAFDY 9 II 14G2CL RMRAVAFDY 9 II 14G3CL RMPAVAFDY 9 II A15CL DYGGNPAEL 9 I G15CL GPTCSGGSC 9 I M11CL RKGAAHFDY 9 I RF-DI1CL EEVGGYFQH 9 III AC-18CL DFDGGSFDY 9 III AC-29CL DFDGGSLDY 9 III AC-40CL DFDGGSFDY 9 III TR35CL KVPSHGMDY 9 III TR36CL KVPSHGMDY 9 III TR37CL KVPSHGMDY 9 III TR38CL KVPSHGMDY 9 III L34CL QPLARHFDP 9 III L100CL GPLMRWFDD 9 III WG1CL VAVAGGFDP 9 III RF-ET5CL GVEVAGTAS 9 I RF-ET10CL YYESSAGPP 9 III EW-D1CL EIPRGGSCY 9 III EW-D3CL EIPRGGSCY 9 III KN-D6CL KEKWDSSRC 9 III HH-M2CL GSAAAGTQG 9 III AK-D8CL DFSWAGPHF 9 III BALL-1CL GTHYYDIRV 9 III YJ DGSGSYEGN 9 III K2.2 GGAVAAFDY 9 III E2.5 KPVTGGEDY 9 III MSL5 DYDGAWFAY 9 I Hb-2 WDGRLLVDY 9 III b4CL HKGLRYFDY 9 III b3CL HKGLRYFDY 9 III b2CL HKGLRYFDY 9 III b5CL HKGLRYFDY 9 III b17CL HKGLRYFDY 9 III b19CL HKGLRYFDY 9 III A3-H2CL YRGDTYDYS 9 III A3-M9CL WVGATTSDY 9 III Tmu69CL EDMDYGMDV 9 III Amu1d4-3CL GGRDRYLVY 9 III 1946CL VRVSYGMDV 9 V GN901v1.0 MRKGYAMDY 9 III GN901v1.1 MRKGYAMDY 9 III N901H/KOL MRKGYAMDY 9 III N901H/G36005 MRKGYAMDY 9 III N901H/PLO123 MRKGYAMDY 9 III Patient14CL RMPAVAFDY 9 II 14G1(2)CL RMPAVAFDY 9 II 14G2CL RMRAVAFDY 9 II 14G3CL RMPAVAFDY 9 II CLL-8CL TSIVRGFGP 9 II BA-1FCL DFFRDYFDY 9 I BA-2PCL DFFRDYFDY 9 III L30554.6CL GGTQPFDIR 9 II 15CL SQASGPFDY 9 I CL-GCL GLYQLLFDY 9 III CL-OCL AGGRTSFDP 9 I BA3CL EGNTKAPDY 9 III PSCL NGTSGDFDY 9 II HNK20hu7 YGTSYWFPY 9 I HNK20hu10 YGTSYWFPY 9 I Amu1d4-3CL GGRDRYLVY 9 III Amu1e10-3CL LRYQLLYNY 9 I 1e8-3CL YIAYDAFDI 9 I 1f7-3CL ITPRNAVDI 9 III Agamma41-3CL DGLLAATDY 9 III Agamma8-3CL DRAYLDFWG 9 III Amu10-3CL DKEPAYFDY 9 I Amu2-1CL RGFNGQLIF 9 I Amu40-2CL LSVVVPAAL 9 III Amu70-1CL LADDDPEDF 9 I Tmu69CL EDMDYGMDV 9 III B7-g2B01CL SAGGSAWST 9 III B8-g3C11CL DRSYYGMDV 9 III B8-g3F05CL DKGTRYSDQ 9 III BF1N-g3C12CL WLVEGSFDY 9 III BF1N-g3H05CL GYVGSSLDY 9 III BF1P-g2A11CL WHQLRGPDY 9 III BF2P1-g3D10CL ENSDYYFDY 9 III BF2P1-g3E12CL DGTYGSGVR 9 III BF2P2-g3C10CL GGSMVPFDY 9 III BF2P2-g3D05CL RGWNYYFDS 9 III BF2P2-g3D12CL DAYYYGLDV 9 III BF2P3-g3C10CL DGRYDPIDY 9 III BF2P1-g7B02CL VGSSGWYDY 9 I BF2N1-g1C10CL DLYDYYDEP 9 I BF2N2-g1A11CL DGAAASFDY 9 I BF2N2-g1E01CL VVGADYFDY 9 I BF2N1-g3F03CL DQNWGYFDY 9 III BF2N2-g3B07CL GVLRDAFDI 9 III BF2N2-g3C03CL ASDGYGMDV 9 III BF2N2-g3F07CL GVLRHALDI 9 III BF2N1-g4A03CL GGCGWYKNY 9 III BF2N1-g4B10CL GSNYAKTGY 9 III BF2N1-g4C11CL GKFQLLFDY 9 III BF2N1-g6A07CL ALHGGGMDV 9 III BF2N1-g6F07CL ALHGGGMDV 9 II BF2N2-g6D09CL VYPPDAFDL 9 III mAbRWL1CL PWDYWFFDL 9 II SV-10 DRVAAAGDY 9 III SV-7 DKGTRYSDQ 9 III SV-9 DRVATIPDY 9 III DN6CL ERGITLMDV 9 I DN7CL ERGITLMDV 9 I SC12CL LDWLLPIDY 9 I SC13CL LDWLLPIDY 9 I D11CL DDGDRAFGY 9 III JONCL DPWPAAFDI 9 III DEZCL VRGSWSGDS 9 III BAR RHSSDWYPY 9 III KC13HCL SSPYGALDY 9 III clone15CL GLDQYKTGH 9 II B22CL GAGAAPHDY 9 III P13CL GAGAAPHDY 9 III PSCL NGTSGDFDY 9 II Patient2 ALRPATFDF 9 III

TABLE-US-00002 TABLE2 # Name L3 Length-L3 Class HIV-s8CL QQYADLIT 8 IGG1-KAPPA FOG1-A4CL QQYYSTPT 8 -KAPPA SA-4BCL QQYNTYPT 8 IGG-KAPPA HIV-b5CL QQGNSFPK 8 IGG1-KAPPA HIV-loop8CL QQYGYSLT 8 IGG1-KAPPA Reg-ACL QQFGGSFT 8 KAPPA 9F12FabCL QQSSNTVT 8 KAPPA GP68CL QQYNSLIT 8 IGG1-KAPPA C471CL QQYNNWPT 8 IGM-KAPPA B8807CL LQHNSYPF 8 IGM-KAPPA B122CL QQYNSQYT 8 IGM-KAPPA B6204CL QQYGSLWT 8 IGM-KAPPA IM-9CL QHYNRPWT 8 IGG-KAPPA T48.16-G8CL QQYGSRLT 8 KAPPA 7FCL QHYGTPRT 8 IGG1-KAPPA 1A6CL QQYNNWPT 8 IGM-KAPPA 1.69CL MQATQFPT 8 IGM-KAPPA antiTac HQASTYPL 8 KAPPA WE QQYGRSPR 8 KAPPA D1.1 QQDDLPYT 8 KAPPA K2.2 QNDNLPLT 8 KAPPA E1.1 QQESLPLT 8 KAPPA E2.4 QQDNLPLT 8 KAPPA E2.5 QQESLPCG 8 KAPPA E2.11 QQDSLPLT 8 KAPPA SSaPB QQYGSSRS 8 IGM-KAPPA SEGaBM QQYGSSRT 8 IGM-KAPPA SELcLN QQYCGSLS 8 IGM-KAPPA mAb5.G3CL QQSYSTLT 8 IGM P7CL QLYGSSLT 8 KAPPA PA QQYNNLWT 8 KAPPA CAR QQYNTFFT 8 KAPPA Taykv322CL QQYGSSPT 8 KAPPA Taykv310CL QQYGSSLT 8 KAPPA Taykv320CL QQYGSSLT 8 KAPPA slkv22CL QQYGSSKT 8 KAPPA LES QQYNNWPP 8 KAPPA RF-TMC1CL QHRNNWPP 8 IGM-KAPPA- III slkv4CL QQRSNWPS 8 KAPPA MD3.13CL QQYGSSPT 8 KAPPA VJICL QQYDTIPT 8 KAPPA rsv13LCL QASINTPL 8 IGG1-KAPPA II.2CL MQALQPWT 8 KAPPA I.75CL QQGFSDRS 8 KAPPA II.14CL MQATQFVT 8 KAPPA III.7CL QRCKGMFS 8 KAPPA SPA3-16CL QQYGGSPW 8 KAPPA VLCLONE52CL CRSHWPYT 8 KAPPA 6F5-01CL QQYYSTPP 8 KAPPA 6F7-42CL QQCNTNPP 8 KAPPA 6F8-01CL QQYYSTPP 8 KAPPA 6F9-31CL QQYYSVPP 8 KAPPA D7CL QQYDSLVT 8 IGG1-KAPPA HuVKCL HQYLSSWT 8 KAPPA BC-26CL MQGIHLLT 8 KAPPA VkLaE34CL QHYYGTPH 8 KAPPA FL9-K QQYNTYPT 8 KAPPA HSC7 QEFGDSGT 8 IGG H8C28 QQYGGSPW 8 IGG SEGcPB QQYGSSRT 8 IGM-KAPPA P3CL QQYDSLPT 8 KAPPA A5K3CL QQYGSVFT 8 IGM BZ1K1CL QQYNSYCS 8 IGM BZ2K1CL QQYYSTPL 8 IGM D11K3CL QQYNDWPT 8 IGM D17K2CL MQNIQFPT 8 IGM F21K1CL QQYDNLPP 8 IGM F22K3CL QLLR?LRT 8 IGM SCFV198CL YQYNNGYT 8 KAPPA KC25LCL QQRSNWPT 8 KAPPA ASSYN13CL QQYGTSHT 8 KAPPA BCPBL1CL QQYNHWPS 8 KAPPA BCPBL4CL QQYGSLYT 8 KAPPA BCPBL6CL QQNKDWPL 8 KAPPA BCSYN6CL QQFGTSLT 8 KAPPA ITPBL14CL QQRSNWWT 8 KAPPA ITPBL2CL QQCSNWPT 8 KAPPA SP10CL QQYGSSPT 8 KAPPA

TABLE-US-00003 TABLE3 # Name L3 Length-L3 Class 8E10CL QQYGSSPSIT 10 IGM-KAPPA III-2RCL QKYNSAPPST 10 IGM-KAPPA II-1CL QEYNNWPLWT 10 KAPPA 35G6CL QQYGGSPPWT 10 IGM-KAPPA GF4/1.1CL HEYNGWPPWT 10 IGG3-KAPPA RF-TS5CL QQYNSYSPLT 10 IGM-KAPPA O-81CL MQHTHWSPIT 10 IGM-KAPPA mAb114CL QHYNNWPPWT 10 IGM-KAPPA HIV-B8CL QQSYNTPPWT 10 IGG1-KAPPA HIV-b8CL QQSYNTPPWT 10 IGG1-KAPPA TT117CL QHYGSSPPWT 10 IGG1-KAPPA HIV- QQHNNWPPLT 10 IGG1-KAPPA loop13CL HIV-s3CL QVYGQSPVFT 10 IGG1-KAPPA 1-185-37CL QQYGSSPMYT 10 IGM-KAPPA 1-187-29CL QQYGSSPMYT 10 IGM-KAPPA HIV-s5CL QRFGTSPLYT 10 IGG1-KAPPA HIV-b3CL QQYGDSPLYS 10 IGG1-KAPPA GER QQYDDWPPIT 10 IGG-KAPPA BLICL QQLNSYPPYT 10 IGM-KAPPA 2A4CL QQSYSTPPDT 10 IGG 0-16CL QHYNNWPPSS 10 KAPPA mAb48CL QHYNRLPPWT 10 IGG3-KAPPA 447.8HCL QQYDRSVPLT 10 KAPPA GP13CL QQYYTTPTYT 10 IGG1-KAPPA M37GO37CL QQYYTTPPLT 10 IGG-KAPPA 9500CL QQLYSYPHLT 10 IGM-KAPPA 9702CL CQQYGSSRWT 10 IGG-KAPPA GSD2B5B10CL MQALQTPMST 10 KAPPA MD2F4CL QQRSEWPPLT 10 KAPPA GAN4B.5CL QQYDTSPAWT 10 KAPPA NANUC-2CL QQYGSSQGFT 10 IgG1-kappa SOL10CL MQSIQLPRWT 10 KAPPA AB1/2CL QHYGLSPPIT 10 IGG1-KAPPA AB4CL QEYGSSPPRT 10 IGG1-KAPPA RH-14CL SSYRSSSTRV 10 IGG1-LAMBDA AB1/2CL QHYGLSPPIT 10 IGG1-KAPPA Ab4CL QEYGSSPPRT 10 IGG1-KAPPA L55-81CL QQYYTTLPLT 10 IGM-KAPPA B3 SSYSSTTRVV 10 IGG HUL-mRFCL QQYGSSPQTF 10 IGM-KAPPA 25C1CL FCQYNRYPYT 10 KAPPA LC4aPB LQRSNWGEVT 10 IGM-KAPPA LC4bPB QQRSNWGEVT 10 IGM-KAPPA LC4cPB QQRSNWGEVT 10 IGM-KAPPA mAb3.B6CL QQYGSSPLFT 10 IGM mAb1.C8CL CSYTSSSTLV 10 IGM P9CL QQRSNWPPIT 10 KAPPA 21H9CL QQSYNTLSLT 10 IGG1-KAPPA 19A5CL QHYGNSPPYT 10 IGG1-KAPPA 43F10CL QQSHKTLAWT 10 IGG1-KAPPA FONCL MQGTYWPPYT 10 IGM-KAPPA HuPR1A3 HQYYTYPLFT 10 KAPPA huPR1A3 HQYYTYPLFT 10 KAPPA CLL-412CL QQSYSTPPWT 10 IGG-KAPPA MEV QQSYTNPEVT 10 KAPPA SON QQYGSSPPYT 10 IGM-KAPPA HEWCL QQYGSSPRYT 10 KAPPA HEWCL QQYGSSPRYT 10 KAPPA JH QQFGNSPPL? 10 IGG2-KAPPA HG2B10KCL QQYAGSPPVT 10 IGG-KAPPA CLLCL QQYNNWPPWT 10 IGM-KAPPA slkv12CL QQYNNWPPWT 10 KAPPA bkv6CL QQRSNCSGLT 10 KAPPA slkv11CL QQYNNWPPWT 10 KAPPA slkv13CL QQYNNWPPWT 10 KAPPA bkv7CL QQYNNWPPCT 10 KAPPA bkv22CL QQYNNWPPWT 10 KAPPA bkv35CL QQRSFWPPLT 10 KAPPA MD3.3CL QQRSNWPSIT 10 KAPPA MD3.1CL QQRSNWPPLT 10 KAPPA GA3.6CL QQRTNWPIFT 10 KAPPA M3.5NCL QQRSNWPPGT 10 KAPPA MD3.4CL QQYNNWPPLT 10 KAPPA M3.1NCL QQYNNWPTWT 10 KAPPA GA3.4CL QQRMRWPPLT 10 KAPPA MD3.7CL QQYGSSPKWT 10 KAPPA MD3.9CL QQYGSSPQYT 10 KAPPA GA3.1CL QQYGSSPPYT 10 KAPPA bkv32CL QQYDRSLPRT 10 KAPPA GA3.5CL QQYGNSPLFS 10 KAPPA GA3.8CL QQYGGSPLFS 10 KAPPA E29.1 QQYNNWPTWT 10 IGM-KAPPA KAPPACL R5A3KCL MQALQTLGLT 10 IGM-KAPPA R1C8KCL MQALQTLGLT 10 IGG-KAPPA I.24CL QQSHSAPPYT 10 KAPPA III.12CL QQYGSSPLFT 10 KAPPA III.5CL QQYNDWPPWT 10 KAPPA I.18CL QQYNGNSPLT 10 KAPPA I.67CL QQLNTYPPWT 10 KAPPA III.6CL HKYGGSPPYT 10 KAPPA II.65CL MQDTHWPPWT 10 KAPPA III.14CL QHYGRSPPLT 10 KAPPA 424.F6.24CL QQYGNSPPYT 10 KAPPA T33-5CL QQYGSSPPYT 10 IGM-KAPPA AL-MH QQYFNVPPVT 10 KAPPA AL-Es305 QHYHNLPPTT 10 KAPPA L47CL IQGTHWPQYT 10 IGM-KAPPAAND LAMBDA F29CL QQYGSSRALT 10 IGM-KAPPAAND LAMBDA G28CL QQYYSTPSYT 10 IGM-KAPPAAND LAMBDA G21CL MQALQTLMCS 10 IGM-KAPPAAND LAMBDA VLCLONE QQSYSTPPLT 10 KAPPA 45CL VLCLONE QQSYSTPPIT 10 KAPPA 48CL VLCLONE QQYGGSLPIT 10 KAPPA 56CL C9CL QQYGSSTPLT 10 IGG1-KAPPA ITC88CL QQRSSWPPLT 10 KAPPA AC18CL QQRYSWPPLT 10 KAPPA AC31CL QQRYNWPPLT 10 KAPPA AC32CL QQRSNWPPLT 10 KAPPA AC37CL QQRSSWPPLT 10 KAPPA B20 QQYNNWPPWT 10 IgM-VkIIIa (Humkv328- Jk1)CL B9601(Vg- QQRSNWPPYT 10 IgM-VkIIIa Jk2)CL MF8 QQYNNWPPWT 10 IgM-VkIIIa (Humkv328- Jk1)CL B2 QQYNNWPPWT 10 IgM-VkIIIa (Humkv328- Jk1)CL kappa1CL QQYGSSPPIT 10 IGG2-KAPPA kappa2CL QQYNNWPPIT 10 IGG2-KAPPA kappa3CL QQRSSWPPIT 10 IGG2-KAPPA kappa4CL QQYGSSPRVT 10 IGG2-KAPPA kappa5CL QQYNTNSPIS 10 IGG2-KAPPA kappa7CL QNYGSSPRIT 10 IGG2-KAPPA kappa8CL QQYGSSPPIT 10 IGG2-KAPPA ToP218CL MQSIQLPRFT 10 KAPPA ToP241CL MQSVQLPRFT 10 KAPPA ToP309CL MQSVQLPRFT 10 KAPPA L1236K3CL QQYDKWPPVT 10 KAPPA SOL1CL MQSIQFPRWT 10 KAPPA BC-2CL MQGIHLPPYI 10 KAPPA P3CL NQGTQWLLYT 10 KAPPA P5CL QQYNSYAPYT 10 KAPPA AB1/2CL QHYGLSPPIT 10 IGG-KAPPA AB4CL QEYGSSPPRT 10 IGG-KAPPA MH QQYFNVPPVT 10 KAPPA FL6-K QQLTSYPPWT 10 KAPPA FL2-K QQVNSYPGLT 10 KAPPA FL4-K QQVFSYPGIT 10 KAPPA FL1-K QQYTSLPGIT 10 KAPPA MM4-K QHSYSTLPLT 10 KAPPA MM9-K QQYYNIPYIT 10 KAPPA HSC4 QLYGSSPRVT 10 IGG HSC11 QQYANWPPIT 10 IGG HSC13 QQYNISPRDT 10 IGG HSC23 QQFGSSPLIT 10 IGG HSC35 QQYGDFPRVT 10 IGG REV QQYGDWPPYT 10 KAPPA BLU QQYYTTLSWT 10 KAPPA BK2CL QQYNKWPPLT 10 KAPPA GK6CL MQGTHWLPVT 10 IGG-KAPPA L1236K3CL QQYDKWPPVT 10 KAPPA P1CL QQYDNLPPIH 10 KAPPA H01CL QQLNNYPPFT 10 KAPPA I01CL QQSYSTPPYT 10 KAPPA I10CL QQSYSTPPYS 10 KAPPA I12CL QQSYSTPPYT 10 KAPPA 126TP14KCL QQYNNWLPFT 10 IGG-KAPPA L32CL AAWDDSLTLM 10 IGM-KAPPA

[0120] For insertion of CDR3s, single oligonucleotides encoding each of the CDR3s of table H from the plus strand were synthesised with 12 homologous nucleotides added to each termini for annealing to the consensus VH and VL genes. In addition to these CDR sequences, CDRs from the antibody E25 (see example 4) were included. These primers were extended and secondary primers were added to introduce directly adjacent to the N and C termini of the VH and VL genes (without C regions) 5NotI-3XbaI sites for VH and 5SpeI-3BamHI for VL. Prior to cloning, a further pair of complimentary primers was used to insert the linker sequence (G1y4Ser)3 between VH and VL whilst maintaining XbaI and SpeI sites. Full-sized VH-linker-VL fragments were digested with NotI and BamHI and were cloned into NotI-BamHI digested pBluescript II KS(+/) (Stratagene, Amsterdam, Netherlands).

[0121] Individual Bluescript clones were picked, plasmid DNA was purified and dispensed robotically into 96 well plates as described in WO99/11777. DNAs were then subjected to IVTT including tRNA-biotinyl-lysine and further robotically arrayed onto a streptavidin surface as described in WO99/11777. The immobilised initial scFv library of 10,000 independant clones was then screened by incubation with recombinant human IgE Fab (see example 4). Wells were blocked with PBS/3% BSA at room temperature for 1 hour, washed three times in PBS and treated with 5 ug/ml human IgE Fab in PBS/3% BSA for 1 hour. Wells were then washed a further three times in PBS and treated with 5 ug/ml alkaline phosphatase-labelled chimeric anti-IgE (example 4) in PBS/3% BSA for 1.5 hrs. Wells were further washed five times in PBS and colour developed using the substrates 5-bromo-1-chloro-3-indolyl phosphate and nitro blue tetrazolium (Roche Molecular) for visualization. A strong signal observed at a frequency of 1 of 9600 wells was shown to derive from a VH and VL pair both containing E25 CDR3's.

EXAMPLE 6

Construction of Composite Mouse Anti-TNF Antibody

[0122] A mouse variable region sequence library was created as described in example 1 for the human library using NCBI Igblast, Kabat and Genbank databases. The reference antibody variable region sequences used was a chimeric anti-TNF antibody known as Remicade (Le et al., U.S. Pat. No. 6,277,969) using the variable regions of the mouse cA2 antibody. Segments from the in silico mouse variable region sequence library were selected partly corresponding amino acids in the Remicade variable region but including variations designed to avoid human T cell epitopes in the sequence in the form of non-self human MHC class II binders measured as in example 1. Composite mouse VH and VL sequences compared to sequences used in the chimeric antibody are shown in FIG. 8 indicating differences of 9 and 16 amino acids in VH and VL respectively between the two antibodies as a result of segment selection for epitope avoidance in the Composite Mouse antibody.

[0123] The Composite Mouse and chimeric anti-TNF antibodies were generated as described in example 1. Comparison of purified antibodies for binding to immobilised human TNF in a standard ELISA (described in WO 03/042247A2) showed that the Composite Mouse antibody retained the full binding capacity of the chimeric anti-TNF, antibody (FIG. 9). The immunogenicity of these antibodies was then compared as described in example 2 using 24 HLA-DR typed human blood samples for T cell assays. The results showed that the chimaeric anti-TNF antibody induced significant proliferative responses (SI greater than 2) in nine of the twenty four healthy donors tested (37.5%) compared to the Composite Mouse anti-TNF antibody where none of the twenty four donors (0%) induced SI>2. These results indicated that a Composite Mouse antibody comprising segments of variable region sequence derived totally from mouse V regions with selection of such segments to avoid human T cell epitopes could remove the immunogenicity in human T cell assays displayed by the corresponding chimeric antibody without any epitope avoidance measures.

EXAMPLE 7

Construction of a Composite Human Anti-TNF Antibody

[0124] The reference mouse variable region heavy and light chain sequences of antibody A2 directed against human TNF was obtained from U.S. Pat. No. 5,656,272 (FIG. 10. SEQ. IDs No. 1 and No. 2 respectively). A structural model was made of the mouse reference variable regions and amino-acids critical for CDR conformation were identified based upon their distance from the CDRs (<3 ) and their likely packing close to CDRs. Important, but less critical residues were identified based upon their distance from the CDRs (>3 <6 ) and their likely influence on more critical residues packing closer to the CDRs. A further set of residues were identified based upon their frequency of occurrence in mouse antibody sequences i.e. amino-acids found at a particular location with a frequency of less than 1%.

[0125] Human V region sequence segments that included as many of these residues as possible were selected (table 4) to create full-length VH and VL sequences. Alterations were made to these sequences to include all the identified structurally important residues to create sequences to serve as a template for epitope avoidance and Composite Human Antibody design. A preferred sequence for each composite VH and VL was designed to include important residues from the reference mouse antibody. These variable heavy and light chain amino acid sequences are shown in FIGS. 11 and 12. SEQ IDs No. 3 and No. 4 respectively.

TABLE-US-00004 TABLE4 HumanAntibodyDatabaseDerivationofSequence SegmentsForPrimaryCHABVariants Genbank AccessionNo. Sequencesegment (a)HeavyChain CAA61442 EVQLVESGGGLVQPGCSLKLSC CAD88676 LSCVASGFIFS CAB37182 FSNHWM AAS86088 HWMNWVRQAPGKGLEWVA CAC43592 AEI ABB54411 IRSKS AAL96548 SIN AAK51359 NSA CAA67405 SAT CAB87447 ATHYA AAD30769 HYAESVKGRFTISRD CAC15703 RFTISRDDSKSI AAQ05509 IVYLQM AAT96742 YLQMTDLR AAD20526 LRTEDTGVYYC CAB44788 VYYCSRNY AAO38724 NYYGS AAK14004 GSTY AAD20470 TYDYWGQGT AAB32435 DYWGQGTTVTVSS (b)LightChain CAC06686 DILLTQ AAX57564 LTQSPAILSLSPGERATLSC X72820 LSLSPGERATLSCRASQ AAC15439 QFV AAZ09058 VGSS Z84907 SSI AAL10835 IHWYQQK AAQ21835 QQKPNQSPKLLIK M27751 LLIKYAS AAY16612 YASE AAR89591 ES AAD19534 SM AAV71416 MSG AAZ09098 GIP CAG27043 PSRFSGSGSGTDFTLTINSLE AAQ21937 SLESEDAA AAC41988 ADYYCQQ AAY33370 YYCQQSHS AAD19457 HSWP AAQ55271 WPFTFGQGT AAW69118 TFGQGTNLEIK

[0126] The composite heavy and light chain variable region sequences were scanned for the presence of potential T cell epitopes using a variety of in silico methods (e.g. Propred [http://imtech.res.in/raghava/propred/index.html], Peptide Threading [www.csd.abdn.ac.uk/gjlk/MHC-thread], SYFPEITHI (www.syfpeithi.de), MHCpred (www.jenner.ac.uk/MHCPred/) and compared to homologous human germ-line framework region sequences in conjuction with reference mouse CDRs.

[0127] The following heavy chain variable region variants were made (see FIG. 11):

[0128] SEQ. ID. No. 5 contains the following changes with respect to SEQ. ID. No. 3: T82aN+R83K.

[0129] SEQ. ID. No. 6 contains the following changes with respect to SEQ. ID. No. 3: T82aN+R83K+D82bS

[0130] SEQ. ID. No. 7 contains the following changes with respect to SEQ. ID. No. 3: T82aN+R83K+D82bS+V23A.

[0131] SEQ. ID. No. 8 contains the following changes with respect to SEQ. ID. No. 3: T82aN+R83K+D82bS+V23A+V78A

[0132] The following light chain variable region variants were made (see FIG. 12):

[0133] SEQ. ID. No. 9 contains the following changes with respect to SEQ. ID. No. 4: I10T+N103R.

[0134] SEQ. ID. No. 10 contains the following changes with respect to SEQ. ID. No. 4: I10T+N103R+S80A.

[0135] SEQ. ID. No. 11 contains the following changes with respect to SEQ. ID. No. 4: I10T+N103R+S80A+N41D.

[0136] For construction of a control chimeric antibody, the nucleotide sequences that translate to give SEQ. IDs No. 1 and No. 2 were constructed using a series of overlapping 40 mer synthetic oligonucleotides. The V region sequences were flanked by additional 5 and 3 sequences to facilitate cloning into mammalian expression vectors. The sequences of the oligonucleotides are shown in FIG. 13 and FIG. 14

[0137] Oligonucleotides were purchased from Sigma-Genosys (Poole, UK) and resuspended at a concentration of 100 M. 1 l of each of the heavy chain sense strand oligonucleotides, except the most 5 oligonucleotide, were mixed together and 1.50 (approx. 1 g) of the mix was treated with Polynucleotide Kinase (PNK, Invitrogen, Paisley UK) in a 20 l reaction containing additionally: 2 l 10 PNK buffer, 2 l 10 mM ATP, 14 l H.sub.2O, 0.5 l (5 units) PNK. The reaction was incubated at 37 C. for 30 min and the enzyme inactivated by heating at 70 C. for 20 min. The heavy chain antisense, light chain sense and antisense oligonucleotides were similarly phosphorylated. The 5 oligonucleotide from each set was diluted 1 in 9 with H.sub.2O and 1.5 l added to the appropriate reaction mix. Each reaction was then diluted to 0.5 ml and spin dialyzed in an Amicon microcon YM3 concentrator for 90 min at 8000 rpm until the volume was not more than 44 l.

[0138] The sense and antisense mixes for the heavy chain, and those for the light chain, were combined and made up to 88 l with H.sub.2O. 10 l 10 Ligase Chain Reaction (LCR) buffer and 2 l Pfu ligase (8 units, Stratagene, Cambridge UK) were added to each reaction and incubated as follows in a programmable heating block: 94 C. for 4 min, then 60 C. for 3 min for 1 cycle followed by 20 cycles of 94 C. for 39 sec. then 60 C. for 2 min. Finally the reactions were incubated for 5 min at 60 C. 10 l of each LCR was run through a 1% agarose gel stained with ethidium bromide and compared to 1 Kb ladder markers (Invitrogen). A smear of ligated DNA was observed in each lane, surrounding a faint specific band of approximately 400 bp.

[0139] The heavy and light chain LCRs were amplified via PCR using as primers SEQ. ID. No.s 12 and 22 for the heavy chain and SEQ. ID. No.s 33 and 43 for the light chain. The following were included in each reaction: 5 l LCR, 5 l 10 Expand HiFi buffer (Roche, Lewes UK), 1 l 10 mM NTP mix, 0.25 l each primer (from 100 M stocks), 0.5 l Expand HiFi polymerase (3 units, Roche) and 38 l H.sub.2O. The reactions were cycled as follows: 94 C. 2 min followed by 20 cycles of 94 C. for 30 sec, 60 C. for 30 sec and 72 C. for 30 sec. Finally the reaction was incubated for 5 min at 72 C. The yield and specificity of the reaction was confirmed by agarose gel electrophoresis, as above. Specific, sharp bands at approximately 400 bp were observed for each reaction.

[0140] The reaction products were purified using a Qiagen PCR purification kit and each eluted in 30 l H.sub.2O. The heavy chain product was digested in a standard reaction with restriction enzymes Mlu 1 and Hind III and the light chain product was digested with BssH II and BamH I. The reaction products were again purified using a Qiagen PCR purification kit and each eluted in 30 l H.sub.2O.

[0141] The light chain expression vector pANT08 was based upon a pAT153 backbone and contains in the following order: CMV immediate/early enhancer promoter 590 to +7, a 30 nt 5 UTR derived from a highly expressed mouse antibody light chain RNA, a mouse consensus light chain signal sequence incorporating a BssH II restriction site near the variable region start codon, a short linker (in place of a variable region) to a human composite intron containing 33 nt from the variable region splice site to a BainH I restriction site followed by a fragment of human genomic DNA containing 343 nt of the intron preceding the human constant Kappa (CK) region gene, the CK gene and CK polyA.

[0142] The heavy chain expression vector pANT09 was similar to pANT08 through the promoter region, which is followed by: a 62 nt 5 UTR derived from the heavy chain counterpart of that described above, a mouse heavy chain consensus signal sequence that incorporates a Mlu I restriction site near the variable region start codon, a short linker (in place of a variable region) to the variable region splice site immediately followed by a fragment of human genomic DNA from a Hind III restriction site located in the intron 211 nt upstream of the CH1 gene, to the end of the CH region poly A site. This fragment includes the CH1, hinge, CH2 and CH3 introns and exons of human IgG1. This vector also included a gene for dihydrofolate reductase, controlled by an SV40 promoter and polyA signal, for resistance to methotrexate.

[0143] 2 g each vector was digested with the relevant restriction enzymes in standard reactions in a total volume of 30 l. Each reaction was run through a 1% agarose gel, as above, and the vector specific bands (6.0 Kbp heavy chain and 4.2 Kbp light chain) were excised from the gel and purified using a Qiagen gel extract kit and eluted in 30 l H.sub.2O.

[0144] 1 l each digested vector was ligated to 3 l of the corresponding digested variable gene PCR product using a Ligafast kit (Promega, Southampton UK). 2.5 l each ligation reaction was transformed into sub-cloning efficiency competent XL1-blue (Stratagene), as instructed by the manufacturer, and plated onto LB agar plates containing 100 g/ml ampicillin and incubated overnight at 37 C. Ten bacterial colonies from each ligation were inoculated into 10 ml 2 YT broth containing 100 g/ml ampicillin and grown overnight at 37 C. with shaking. Plasmid was purified from 1.5 ml each overnight culture using a Qiagen plasmid preparation kit and each eluted in 50 l H.sub.2O. The plasmids were sent to a contract sequencing facility and sequenced with a standard CMV promoter primer and clones with the correct V region gene sequence identified.

[0145] For construction of Compsoite Human Antibodies, the nucleotide sequences that translate to give SEQ. IDs No. 3 and No. 4 were constructed using a series of overlapping 40 mer synthetic oligonucleotides. The sequences of the oligonucleotides are shown in FIG. 15 and FIG. 16. The nucleotide sequence that translates to give SEQ. ID. No. 5 was constructed via overlap PCR using oligonucleotide primers SEQ. ID. No.s 94 and 95 (FIG. 17) together with oligonucleotides SEQ. ID. No.s 53 and 63 and the plasmid DNA of the primary Composite Human Antibody heavy chain variant as template. Two PCR reactions were done using as primer pairs either SEQ. ID. No.s 53 and 95, or SEQ. ID. No.s 94 and 63. The following were included in each reaction: 1 l (100 ng) plasmid template, 5 l 10 Expand HiFi buffer (Roche), 1 l 10 mM NTP mix, 0.25 l each primer (from 100 M stocks), 0.5 l Expand HiFi polymerase (3 units, Roche) and 42 l H.sub.2O. The reactions were cycled as follows: 94 C. 2 min followed by 20 cycles of 94 C. for 30 sec, 60 C. for 30 sec and 72 C. for 30 sec. Finally the reaction was incubated for 5 min at 72 C. The entire reactions were electrophoresed through a 1% agarose gel and the specific bands of 295 bp and 126 bp were excised and purified using a Qiagen gel extraction kit. The DNAs were eluted in 30 l H.sub.2O.

[0146] The two purified fragments were joined in a PCR reaction using oligonucleotide primers SEQ. ID. No.s 53 and 63 using PCR conditions as described above, except that the template used was 1l 295 bp product and 1 l 126 bp product, hence the amount of H.sub.2O was reduced to 41 l. The joined PCR product of 396 bp was purified using a Qiagen PCR purification kit and was eluted in 30 l H.sub.2O.

[0147] The nucleotide sequence that translates to give SEQ. ID. No. 6 was constructed via overlap PCR using oligonucleotide primers SEQ. ID. No.s 96 and 97 (FIG. 17) together with oligonucleotides SEQ. ID. No.s 53 and 63 and the plasmid DNA of the primary Composite Human Antibody heavy chain variant as template. Two PCR reactions were done using as primer pairs either SEQ. ID. No.s 53 and 97, or SEQ. ID. No.s 96 and 63. The first stage PCRs were done as described above and yielded fragments of 295 bp and 126 bp. These fragments were purified, joined together and repurified, also as described above.

[0148] The nucleotide sequence that translates to give SEQ. ID. No. 7 was constructed via overlap PCR using oligonucleotide primers SEQ. ID. No.s 98 and 99 (FIG. 17) together with oligonucleotides SEQ. ID. No.s 53 and 63 and the PCR product for SEQ. ID. No. 6 as template. Two PCR reactions were done using as primer pairs either SEQ. ID. No.s 53 and 99, or SEQ. ID. No.s 98 and 63. The first stage PCRs were done as described above and yielded fragments of 98 bp and 318 bp. These fragments were purified, joined together and repurified, also as described above.

[0149] The nucleotide sequence that translates to give SEQ. ID. No. 8 was constructed via overlap PCR using oligonucleotide primers SEQ. ID. No.s 100 and 101 (FIG. 17) together with oligonucleotides SEQ. ID. No.s 53 and 63 and the PCR product for SEQ. ID. No. 7 as template. Two PCR reactions were done using as primer pairs either SEQ. ID. No.s 53 and 101, or SEQ. ID. No.s 100 and 63. The first stage PCRs were done as described above and yielded fragments of 270 bp and 155 bp. These fragments were purified, joined together and repurified, also as described above.

[0150] Each of the above PCR products was digested with Mlu I and Hind III and ligated into similarly digested pANT09. The ligations were transformed and plated, colonies picked, plasmids prepared and sequenced all as described above.

[0151] The nucleotide sequence that translates to give SEQ. ID. No. 9 was constructed via PCR using oligonucleotide primers SEQ. ID. No.s 102 and 103 (FIG. 17) and the plasmid DNA of the primary Composite Human Antibody light chain variant as template. A single PCR reaction was done, as described for the heavy chain variants, that yielded a product of 383 bp. The entire reaction was electrophoresed through a 1% agarose gel and the specific band was excised and purified using a Qiagen gel extraction kit. The DNA was eluted in 30 l H.sub.2O.

[0152] The nucleotide sequence that translates to give SEQ. ID. No. 10 was constructed via overlap PCR using oligonucleotide primers SEQ. ID. No.s 104 and 105 (FIG. 17) together with oligonucleotides SEQ. ID. No.s 74 and 84 and the PCR product for SEQ. ID. No. 9 as template. Two PCR reactions were done using as primer pairs either SEQ. ID. No.s 74 and 105, or SEQ. ID. No.s 104 and 84. The first stage PCRs were done as described above for the heavy chain variants and yielded fragments of 265 bp and 139 bp. These fragments were purified, joined together to create a product of 383 bp and repurified, also as described above for the heavy chain variants.

[0153] The nucleotide sequence that translates to give SEQ. ID. No. 11 was constructed via overlap PCR using oligonucleotide primers SEQ. ID. No.s 106 and 107 (FIG. 17) together with oligonucleotides SEQ. ID. No.s 74 and 84 and the PCR product for SEQ. ID. No. 10 as template. Two PCR reactions were done using as primer pairs either SEQ. ID. No.s 74 and 107, or SEQ. ID. No.s 106 and 84. The first stage PCRs were done as described above for the heavy chain variants and yielded fragments of 148 bp and 256 bp. These fragments were Purified, joined together to create a product of 383 bp and repurified, also as described above for the heavy chain variants.

[0154] Each of the above PCR products was digested with BssH II and BamH I and ligated into similarly digested pANT08. The ligations were transformed and plated, colonies picked, plasmids prepared and sequenced all as described above.

[0155] CHO-K1 cells (ATCC #CCL-61) were propagated in high glucose DMEM containing 10% FCS, L-glutamine, sodium pyruvate and L-proline. Near confluent cultures were harvested for transfection using Lipofectamine 2000 as instructed by the manufacturer (Invitrogen). Transfections were done in 48 well plates seeded with 200 l cells at 310.sup.5 cells/ml using a total of 0.5 g plasmid DNA comprising 0.3 g heavy chain construct and 0.2 g light chain construct.

[0156] Transfections were incubated at 37 C./5% CO.sub.2 for 48 to 72 h before harvesting the supernatants. Antibody expression was quantified by ELISA using: a mouse monoclonal anti-human IgG capture antibody, human IgG1/Kappa standards and HRP conjugated goat anti-human Kappa light chains as detection antibody (all reagents from Sigma).

[0157] All combinations of heavy and light chains were transfected (i.e. 6 heavy chains5 light chains=30 transfections). Antibody expression levels were generally in the range of 0.5 to 2.0 g/ml, however no expression was observed with heavy chain SEQ. ID. No. 8.

[0158] The expressed antibodies were tested for their ability to neutralize the activity of human TNF using TNF-sensitive WEHI-164 cells (Espevik et al., J. Immunol. Methods 1986, 95, 99-105). Cells were plated in 1 g/ml actinomycin D at 510.sup.4 cells per well in 96-well microtiter plates for 3-4 hours. Cells were exposed to 40 pM human TNF and varying concentrations of the chimeric antibody (range 1 ng/ml to 500 ng/ml) to create a standard curve. The various combinations of heavy and light chains were tested at a single concentration point of 25 ng/ml that had previously been determined as the ED.sub.50 of the chimeric antibody. All assays were done in triplicate.

[0159] The mixtures were incubated overnight at 37 C. Cell viability was determined by adding 3-[4,5-dimethyl-thiazol-2-yl]-2, 5-diphenyltetrazoliumbromide dye (MTT) to a final concentration of 0.5 mg/ml, incubating for 4 hours at 37 C., lysing the cells in 0.1M HCl, 0.1% SDS and measuring the optical density at 550 nm wavelength.

[0160] The optical densities from the heavy and light chain combinations were used to calculate the apparent antibody concentrations from the standard curve. The apparent concentration of the chimeric was divided by that of each of the variant combinations to give a fold difference value. Values lower than that for the chimeric indicated that those combinations were more effective at protecting the cells from TNF cytotoxicity, whereas higher values indicated that they were less effective. The values for all combinations are shown in Table 5.

TABLE-US-00005 TABLE 5 Ratio of Activities of Composite Human Antibody Variants compared to Chimeric Antibody SEQ. ID. No. 1 3 5 6 7 8 2 1.00 1.38 1.24 1.20 1.02 ND 4 1.51 2.28 1.28 1.38 1.05 ND 9 1.28 2.14 1.32 1.77 0.95 ND 10 1.31 2.51 1.17 1.63 0.98 ND 11 16.90 15.15 196.49 134.08 105.61 ND

[0161] The following Composite Human Antibody heavy and light chain combinations gave fold differences close to 1.0: SEQ. ID. No.s 5/10, SEQ. ID. No.s 7/4, SEQ. ID. No.s 7/9, SEQ. ID. No.s 7/10. These combinations were selected for further study.

[0162] The expression plasmids carrying the sequences selected above were transfected into NS0 cells (ECACC No. 85110503). The cells were grown in high glucose DMEM containing L-glutamine, sodium pyruvate, 5% ultra low IgG FCS and pen/strep. Cells were harvested during log phase of growth, spun down and resuspended at 510.sup.6 cells/ml in fresh growth media. 750 l cells were mixed with a total of 30 g of each plasmid pair, which had been linearised with Ssp I, in 50 l H.sub.2O. The cell/plasmid mixture was transferred to a 4 mm gap cuvette and electroporated using an Equibio Easyject Plus at 250V, 1500 F, infinite resistance. The electroporate was immediately transferred to 25 ml pre-warmed growth media and plated out in 596 well flat bottomed plates at 100 l/well. The plates were incubated at 37 C./5% CO.sub.2. 48 h post-electroporation, 50 l media containing 300 nM methotrexate was added to each well to give a final concentration of 100 nM. 7 days post-electroporation a further 50 l of media containing 100 nM methotrexate was added to each well.

[0163] Approximately 2 week post-electroporation, the media in some wells began to turn yellow, indicating transfected colony growth. Media from these wells were tested for antibody expression using the anti-human IgG Fc capture/anti-human Ig Kappa light chain HRP conjugate detection ELISA. The test samples were compared to a human IgG1/Kappa standard and antibody expression levels estimated. Colonies expressing useful amounts of antibody were expanded in media containing 200 nM methotrexate.

[0164] Antibodies were purified from 500 ml culture media via protein A affinity chromarography followed by size exclusion chromatography using Sephacryl S200. The purified antibodies were quantified by UV absorbance at 280nm, assuming that OD.sub.2801=1.4 mg/ml.

[0165] Purified chimeric and composite antibodies were tested for activity via the WEHI-164 protection assay described in example 4 above. Each antibody was tested over the full concentration range previously used to create the standard curve (see FIG. 18). Composite Human Antibody 7/10 (i.e. containing SEQ. ID. No.s 7 and 10) was found to be the most active variant and had the same activity as the chimeric antibody. Composite Human Antibodies 7/9 and 5/10 had similar activity that was slightly reduced compared to the chimeric, and Composite Human Antibody 7/4 was the least active.

[0166] Therefore since Composite Human Antibody 7/10 was predicted to have the most favourable MHC class II binding profile and was the most active variant, this was selected for testing in a time course T cell proliferation assay. Human PBMCs were prepared from buffy coats derived from human blood donations via two rounds of Ficoll density centrifugation. The prepared PBMC were resuspended at a density of 310.sup.7 cells/ml in 1 ml aliquots in 90% human AB serum/10% DMSO, and stored under liquid nitrogen. PBMC were tissue typed using a Dynal Allset PCR typing kit.

[0167] The lead Composite Human Antibody was compared to the chimeric antibody in whole protein T cell assays using human PBMC from 20 healthy donors. PBMC from each donor were thawed, washed and resuspended in AIM V serum free lymphocyte growth media. On day 1, 50 g protein was added to 2 ml bulk cultures of 410.sup.6 PBMC in 24 well plates, and triplicate 100 l aliquots were removed and transferred to 96 well plates on days 6 to 9. Each aliquot was pulsed with 75 l media containing 1 Ci tritiated thymidine for 24 h, before harvesting and measuring incorporation of radioactivity. Results were normalised by calculation of the Stimulation Index (SI). Coverage of a wide range of HLA DR allotypes was achieved by selecting donors according to individual MHC haplotypes.

[0168] The results of the time-course assay are shown in FIG. 19 and demonstrated that the chimeric antibody (FIG. 19(a)) elicits a T cell response (SI>=2) on at least one day in 10 of the 20 donors. In contrast, Composite Human Antibody (FIG. 19(b)) failed to elicit a response in any of the donors at any time point. Therefore a non-immunogenic Composite Human Antibody was successfully constructed from segments of human antibodies using a mouse anti-TNF antibody (A2) as reference.

EXAMPLE 8

Construction of a Composite Type I Ribosome Inhibitory Protein

[0169] Composite variants of the plant type I Ribosome Inhibitory Protein (RIP) bouganin (derived from Bougainvillea spectabilis) were generated using methods described in WO2005090579. The location of T cell epitopes in bouganin was tested by analysis of overlapping 15 mer peptides as in WO2005090579 and the peptides of SEQ ID 11-13 in table 6 (corresponding to residues 121-135, 130-144 and 148-162) were identified as epitopes. Bouganin was cloned from leaf tissue from a Bougainvillea spectabilis plant. mRNA was extracted using a polyA Tract System 1000 kit (Promega) from 100 mg tissue as instructed by the manufacturer. cDNA was synthesised from the mRNA template using an AccessQuick RT-PCR system (Promega) with the following primers: ATGTACAACACTGTGTCATTTAAC and TTATTTGGAGCTTTTAAACTTAAGGATACC. The first primer additionally contains an ATG start codon and the second primer additionally contains a TAA stop codon. The PCR product was cloned using a T/A cloning system (pGEM T Easy, Promega) and several clones were sequenced to identify a correct clone orientated with the transcription direction of the T7 promoter contained within the vector.

TABLE-US-00006 TABLE6 ImmunogenicPeptideSequencesofbouganinand ReplacementHumanSequenceSegments SEQIDNo.11: .sup.121AKVDRKDLELGVYKL.sup.135 AAKAD-CAD39157 AKADR-AAH01327 KADRK-XP_372046 AAKSDR-AAH47411 KSDRKD-NP_002678 AAKTD-BAA23704 AKTDR-AAD00450 KTDRK-CAH18368 SEQIDNo.12: .sup.130LGVYKLEFSIEAIHG.sup.144 ELGPQ-BAC04852 LGPQK-NP_056013 GPQKLE-XP_370607 ELGGK-AAI00815 LGGKKL-BAD96533 GGKKLE-AAK68690 ELGNS-BAB14022 LGNSKL-BAD98114 GNSKLE-CAG46875 ELGQAKL-AAF42325 LGQAKLE-AAN63404 ELGQD-CAH71404 LGQDK-BAC04773 QDKLE-NP_004000 SEQIDNo.13: .sup.148NGQEIAKFFLIVIQM.sup.162 GQEQA-CAI95134 QEQAK-AAH55427 EQAKF-NP_079390 GQERA-AAH10634 QERAK-NP_003153 ERAKF-AAH14009

[0170] A series of variants were made containing the human sequence segments identified as shown in table 6. These were constructed using overlap PCR with a high fidelity polymerase (Expand Hi-Fi, Roche). The 5 and 3 primers were as above and the PCR products were cloned into the T/A cloning vector, as above, and correct clones identified that were orientated with the transcription direction of the T7 promoter. Clones were assayed for activity in a coupled transcription and translation reaction that included a control DNA expressing a luciferase gene (Luciferase T7 Control, Promega). Since bouganin is a ribosome inactivating protein, it significantly reduces the levels of translation of the luciferase gene and this reduction is conveniently assayed using a luciferase detection system such as Steady-Glo (Promega). Purified wild type or mutant bouganin plasmids were linearised with Not I and diluted to 10 ng/l. Luciferase T7 Control DNA was diluted to 125 ng/l. 1 l each DNA was mixed with 10 l TnT mix (Promega), 0.25 l Methionine and 0.25 l nuclease free water (supplied in TnT kit). Controls were wt bouganin and Luciferase T7 Control only. Reactions were undertaken in triplicate and incubated for 1 hour at 30 C. 5 l each reaction was transferred to a black walled 96 well luminometer plate and mixed with 45 l water and 50 l Steady-Glo reagent. Luminescence was read in a Wallac Microbeta Trilux luminometer. Activity was expressed as a percentage of the luminescence observed from the Luciferase T7 Control DNA alone.

[0171] FIG. 20 illustrates the activity profile of a number of different variants. This shows that the most active variants are: V123T in peptide 41; V132P/Y133Q in peptide 44; I152Q in peptide 50. A combined mutant was made containing these 4 mutations and re-tested in the activity assay. The activity of this mutant is indicated by COMB in FIG. 20 and retains approximately 75% of the activity of the wt protein.

[0172] Peptides containing the human sequence segments within the active COMB variant corresponding to residues 121-135, 130-144 and 148-162 were synthesised and compared to the corresponding wild type peptides in a time-course T cell assay with human PBMCs from 20 healthy donors as described in example 7. The results showed that peptides containing human sequence segments induced no T cell proliferation in any donor at any time point whilst each of the wild type peptides induced proliferation with SI>2 in >10% of all donors for at least one time point.

EXAMPLE 9

Construction of a Composite Hirudin

[0173] Composite variants of the thrombin inhibitor hirudin (derived from Hirudo medicinalis) were generated using methods described in WO2004113386 using the protein with SEQ ID No 14 in table 7 as wild type. The location of T cell epitopes in hirudin was tested by analysis of overlapping 15 mer peptides as in WO2004113386 and the peptide 27-41 CILGSDGEKNQCVTG was shown to give a significant T cell response with human PBMCs from 20 healthy donors. The human sequence segment KCRH from human melanoma-associated antigen (AAN40505.1) was used to replace the hirudin residues at 26-29 using overlap PCR as in example 8 resulting in a variant hirudin molecule with 28/29IL changed to 28/29RH which retained full activity of the wild type hirudin using assays described in WO2004113386. The modified peptide 27-41 CRHGSDGEKNQCVTG was tested together with the wild type peptide 27-41 CILGSDGEKNQCVTG in T cell assays as in example 8 demonstrating the loss of T cell epitope activity in the modified peptide.

EXAMPLE 10

Construction of Composite Human Anti-IgE Antibody With Tr Epitopes

[0174] VH and VL genes from the Composite Human Anti-IgE antibody of example 4 were cloned according to standard polymerase chain reaction (PCR) methods from Orlandi et al., ibid into separate plasmid vectors as templates for a VL- and VH-specific PCR using oligonucleotide primer pairs. Overlapping complementary sequences were introduced into the PCR products that combined during the subsequent fusion PCR to form the coding sequence either of a 20 amino acid (G.sub.4S.sub.1).sub.4 linker or, alternatively, a 20 amino acid sequence GGSNNLSCLTIPASANNGGS containing a 10 amino acid Tr epitope from the hepatitis C core protein (P19, MacDonald et al., Journal of Infectious Diseases, 185 (2002) p720-727) flanked each side by two asparagines residues and a GGS triplet. This final amplification step was performed with primer pairs for subsequent cleavage with the restriction enzymes EcoRV and BspE1 and cloning into the bluescript KS vector (Stratagene). Dimeric forms of the Composite Human anti-IgE single chain antibodies (scFvs) were then constructed by the method of Mack et al., Proc Natl Acad Sci USA., 92 (1995) p7021-7025. The dimeric VL-linker-VH-VL-linker-VH fragment was subcloned into the EcoR1/Sal1 sites of the expression vector pEF-DHFR (Mack et al., ibid) and transfected into DHFR-deficient Chinese hamster ovary (CHO) cells by electroporation. Selection, gene amplification, and protein production were performed as described by Mach et al., ibid). The dimeric scFv's were purified via the C-terminal histidine tails by affinity chromatography on a nickel-nitrilotriacetic acid (Ni-NTA) column (Qiagen) to give dimeric Fvs designated CHABIgEG4S14 ((G.sub.4S.sub.1).sub.4 linker between VL and VH) and CHABIgEHCVP19 (HCV Tr epitope between VL and VH).

[0175] Dimeric scFvs were subsequently tested in human T cell assays at 50 g/ml exactly as described by Hall et al., Blood 100 (2002) p4529-4536 using PBMCs from 20 healthy donors. The results showed no significant proliferation of T cell for either CHABIgEG4S14 or CHABIgEHCVP19 but showed a significant level of IL-10 production (SI>2) from 4 out of 20 donors stimulated with CHABIgEHCVP19 but not with CHABIgEG4S14 (SI>2 in 0 of 20 donors). This demonstrates the effect of a Tr epitope included within the antibody molecule for the induction of the immunosuppressive cytokine IL-10.