NEUTRALISING ANTIBODY MOLECULES HAVING SPECIFICITY FOR HUMAN IL-17

Abstract

The invention relates to an antibody molecule having specificity for antigenic determinants of IL-17, therapeutic uses of the antibody molecule and methods for producing said antibody molecule.

Claims

1. A neutralising antibody having specificity for human IL-17, wherein the antibody binds the same epitope as an antibody comprising a light chain and a heavy chain, wherein the variable domain of the heavy chain comprises SEQ ID NO:5 for CDR-H1, SEQ ID NO:6 for CDR-H2, and SEQ ID NO:7 for CDR-H3 and wherein the variable domain of the light chain comprises SEQ ID NO:8 for CDRL1, SEQ ID NO:9 for CDR-L2, and SEQ ID NO:10 for CDR-L3.

Description

[0122] The present invention is further described by way of illustration only in the following examples, which refer to the accompanying Figures, in which:

[0123] FIG. 1A) shows the nucleotide and amino acid sequence (SEQ ID NOS:1 and 2, respectively) of the variable domains of the heavy chain, and FIG. 1B) shows the nucleotide and amino acid sequence (SEQ ID NOS:3 and 4, respectively) of the variable domains of the light chain of murine monoclonal antibody IL-17F4.100. In both figures positions 1-57 (nucleotide sequence numbering) are the natural mouse leader sequences associated with these variable regions.

[0124] FIGS. 2A and 2B show the graft design for the IL-17F4.100 heavy (FIG. 2A; SEQ ID NO:11) and light chain (FIG. 2B; SEQ ID NO: 13) sequences. The symbol (I) highlights differences between donor:acceptor:grafted framework sequences. CDR's are single underlined. These are as defined by Kabat, except for CDR-H1 which encompasses both Kabat and Chothia definitions. Double-underlined sequences are donor residues retained in the grafts. Starred (*) residues are common in human sub-group VH3 germline sequences, but not present in this particular germline.

[0125] FIGS. 3A and 3B show the nucleotide and amino acid sequences of the designed genes gH11 (FIG. 3A) and gL3 (FIG. 3B). In both chains the E. coli OmpA leader sequence is shown (bases 1-63 of the nucleotide sequence).

[0126] FIGS. 4A and 4B. Show the amino acid sequence of the antibody Fab fragment of CDP435 (FIG. 4A) light chain and (FIG. 4B) heavy chain.

[0127] FIGS. 5A and 5B. Show the amino acid and nucleotide sequence of the antibody Fab fragment of CDP435. Bases 1-63 and 722-784 represent the E. coli OmpA, leader sequence.

[0128] FIG. 6. Plasmid map of pTTOD (CDP435)

[0129] FIG. 7. A comparison of the effect of CDP435 and IL17F4.100 on human IL-17 induced IL-6 production from Hela cells.

[0130] FIG. 8. The effect of CDP435 on human IL-17 induced IL-6 production from Hela cells.

[0131] FIG. 9. The effect of CDP435 on monkey IL-17 induced IL-6 production from Hela cells.

[0132] FIG. 10. The effect of CDP435 on human IL-17F induced IL-6 production from Hela cells.

[0133] FIG. 11. The effect of CDP435 on mouse IL-17 induced IL-6 production from 3T3-NIH cells.

[0134] FIG. 12. Pharmacokinetics of .sup.125I labelled CDP435 administered subcutaneously in rats FIG. 13. In vivo neutralisation of hIL-17 induced neutrophil accumulation in mice by local administration of CDP435.

[0135] FIG. 14. In vivo neutralisation of hIL-17 induced neutrophil accumulation in mice by subcutaneous administration of CDP435.

[0136] FIG. 15. A diagrammatic representation of the structure of CDP435. n is between 400 and 520.

DNA MANIPULATIONS AND GENERAL METHODS

[0137] E. coli strain INVαF′ (Invitrogen) was used for transformation and routine culture growth. DNA restriction and modification enzymes were obtained from Roche Diagnostics Ltd. and New England Biolabs. Plasmid preparations were performed using Maxi Plasmid purification kits (QIAGEN, catalogue No. 12165). DNA sequencing reactions were performed using the ABI Prism Big Dye terminator sequencing kit (catalogue No. 4304149) and run on an ABI 3100 automated sequencer (Applied Biosystems). Data was analysed using the program AutoAssembler (Applied Biosystems). Oligonucleotides were obtained from OSWEL. The concentration of Fab was determined using Fab assembly ELISA.

In Vitro Neutralisation Assay: Primary Fibroblasts

[0138] Human dermal fibroblasts were grown to 80% confluence in 96 well plates. Antibodies were titrated in half log dilutions from 1 μg/ml and human IL-17 was added to give 25 ng/ml final concentration. The plates containing antibody and human IL-17 were incubated at room temperature for 30 min. Culture medium was removed from fibroblast cultures and 100 μl antibody/IL-17 mix added to the appropriate wells and cultured overnight at 37° C. The amount of IL-8 produced in response to IL-17 was then estimated using the R&D Systems Human IL-8 Duoset Kit DY208.

Example 1: Isolation of IL-17F4.100

[0139] Antibody IL-17F4.100 was obtained using conventional hybridoma techniques. Female BALB/C mice were immunised with recombinant human IL-17 (purchased from R & D systems). Mice received three intra peritoneal immunisations at two weekly intervals of 10 μg IL-17 in 100 μl Freund's adjuvant. Three days prior to performing the fusion the mouse was boosted with 1 g human IL-17 in 100 μl PBS intravenously. The fusion was performed using the method of Galfre et al., 1977, Nature, 266, 550-552 with the mouse myeloma cell line SP2/0 used as the fusion partner. The fusion was screened for antibodies that bound to human IL-17 by ELISA and a number of antibody producing hybridomas were selected from this primary screen one of which was named IL-17F4.100. The hybridoma cells producing IL-17F4.100, were cloned by limiting dilution. The antibody was isotyped and found to be an IgGγ2b with a kappa light chain.

Example 2: Gene Cloning and Expression of the Variable Regions from Murine Monoclonal Antibody IL-17F4.100

PCR Cloning of VH and VL Regions

[0140] Genes for the heavy chain variable domain (VH) and light chain variable domain (VL) of IL-17F4.100 were isolated and sequenced following cloning via reverse transcription PCR.

[0141] The V-region sequences are shown in FIGS. 1A and 1B (starting at base 58) and in SEQ ID NOS:1 to 4.

[0142] The murine V-region genes were sub-cloned into expression vectors containing the human antibody constant region genes (human kappa light chain and gamma-4 heavy chain) and a mouse/human chimeric expressed transiently in CHO cells. Transfections of CHO cells were performed using the lipofectamine procedure according to manufacturer's instructions (InVitrogen, catalogue No. 18324).

Example 3: CDR-Grafting of IL-17F4.100

[0143] A series of humanised VL and VH regions were designed in which the CDR hypervariable regions plus a varying number of framework residues from IL-17F4.100 were grafted onto human V-region acceptor frameworks.

[0144] Three grafted VL regions (gL1-3) were designed and genes were built by oligonucleotide assembly and PCR mutagenesis. A total of 16 grafted VH regions were also constructed (gH1-16). These humanised sequences were sub-cloned into vectors containing human antibody constant region genes, were expressed transiently in CHO cells and their activity in IL-17 binding and neutralisation assays was compared to the chimeric antibody comprising the IL-17F4.100 variable regions and human constant regions.

[0145] The graft most potent at neutralising IL17 was gH11gL3 which contains 1 mouse framework residue in the L chain (Val-2) and 2 mouse framework residues in the H chain (Val-24, Val-78).

[0146] FIGS. 2A and 2B show an alignment between the donor mouse sequence and the acceptor human frameworks. The heavy chain acceptor framework is the human germline sequence VH3 1-3 3.33, with framework 4 coming from this portion of the human JH-region germline JH4. The light chain acceptor framework is the human germline sequence VK1 2-1-(1) 012, with framework 4 coming from this portion of the human JK-region germline JK1. The graft sequences for gH11 and gL3 are given in FIG. 3A (bases 64-420) and FIG. 3B (bases 64-399) respectively (SEQ ID NOS:11-14).

Example 4: Production and Characterisation of CDP435

[0147] CDP435 is a PEGylated antibody fragment according to the present invention in which the antibody component is an antibody Fab fragment constructed from the grafts produced in Example 3. The antibody Fab fragment component of CDP435 was constructed using the genes encoding the selected humanised variable domain graft (gH11gL3) which were sub-cloned into Celltech's E. coli expression vector pTTOD, which contains DNA encoding the human Cγ1 heavy chain CH1 domain and the human C kappa light chain domain (as previously described in WO03/048208). In contrast to WO03/048208 the human heavy chain was truncated in the constant region such that the interchain disulphide cysteine (cys-233 by Kabat numbering system, cys-222 by sequential numbering) is the C-terminal residue. The protein sequence of this CDR-grafted Fab is shown in FIGS. 4A and 4B (Seq ID NOS: 15-18). A map of the pTTOD(CDP435) dicistronic expression vector is shown in FIG. 6 which comprises the construct provided in FIGS. 5A and 5B and SEQ ID NO:19. The construct contains an intergenic sequence, IGS-2, between the light and heavy chain genes (See WO03/048208) and the OmpA leader sequence at the start of both the light and heavy chain genes.

[0148] The pTTOD(CDP435) vector was transformed into the host strain E. coli K12 W3110 and the antibody Fab fragment component of CDP435 produced in E. coli by high cell density cultivation using standard methods. Antibodies were purified using cation exchange followed by anion exchange chromatography using standard methods (Humphreys et al., 2002, Protein Expression and Purification, 26, 309-320).

Production of CDP435

[0149] Two 20 kDa PEG molecules were attached to the purified antibody Fab fragment component of CDP435 using the following method (See also the method provided in International patent application WO2005/003169). The purified antibody Fab fragment produced as described above was reduced to produce 2 thiols per Fab (both interchain cysteines) with 10 mM tris-(2-carboxyethyl)-phosphine (TCEP) for 1 hour at ambient temperature. The reductant was removed by diafiltration into 0.1M phosphate+2 mM EDTA, pH 6.0. The reduced antibody fragment of CDP435 was DiPEGylated on the interchain cysteines with a 3-fold molar excess of 20 kDa PEG-maleimide over Fab, overnight at ambient temperature in order to attach a total of 40 kDa PEG (i.e. 2×20 kDa PEG) to produce CDP435. A diagrammatic representation of CDP435 is shown in FIG. 15.

[0150] After PEGylation the reaction was conditioned for purification of CDP435 by reducing the pH to 4.5 (addition of acetic acid) and reducing the conductivity to 3 mS/cm (addition of water). CDP435 was purified by SP Sepharose HP chromatography in 50 mM acetate pH 4.5. Purified material was concentrated and diafiltered into 50 mM acetate, 125 mM NaCl, pH 5.5, and 0.22 m sterile filtered.

BIAcore Assay

[0151] The assay format used CDP435 captured by anti-human IgG F(ab).sub.2 with a titration of recombinant human IL-17 in the solution phase. BIA (Biamolecular Interaction Analysis) was performed using a BIAcore 3000 (BIAcore AB). Affinipure F(ab′).sub.2 Fragment goat anti-human IgG, F(ab).sub.2 fragment specific (Jackson ImmunoResearch) was immobilised on a CM5 Sensor Chip via amine coupling chemistry to a capture level of =9000 response units (RUs). HBS-EP buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, BIAcore AB) was used as the running buffer with a flow rate of 10 l/min. An injection of CDP435 was made at 10 μl/min in order to obtain around 200Ru of Fab captured by the immobilised anti-human IgG-F(ab).sub.2 to the surface. Human IL-17 was titrated over the captured antibody Fab fragment at various concentrations at a flow rate of 30 l/min. The surface was regenerated by a 2×10 μl injection of 40 mM HCl, followed by a 5 μl injection of 5 mM NaOH at a flow rate of 10 μl/min.

[0152] Background subtraction binding curves were analysed using the BIAevaluation software (version 3.2) following standard procedures. Kinetic parameters were determined from the fitting algorithm. The affinity was measured at human IL-17 concentrations at or below 12.5 nM. The affinity value determined for CDP435 was in the range 133-365 pM with a mean±SD of 223.8±94.5 pM (Table 1).

TABLE-US-00001 TABLE 1 Affinity by BIAcore Replicate k.sub.a (M.sup.−1s.sup.−1) k.sub.d (s.sup.−1) K.sub.d (M) K.sub.d pM 1 1.71E+06 3.23E−04 1.891E−10 189 2 1.35E+06 1.79E−04  1.33E−10 133 3 1.83E+06 4.99E−04  2.72E−10 272 4 2.57E+06 4.11E−04  1.60E−10 160 5 1.62E+06 5.92E−04  3.65E−10 365

[0153] FIG. 7 demonstrates that the neutralisation activity of the antibody Fab fragment of CDP435 is equivalent to that of the murine parental antibody IL-17F4.100 in the Hela cell human IL-17 neuralisation assay (methods as described in Example 5).

Example 5: In Vitro Neutralisation Assays Using CDP435

Hela Cells

[0154] The potency of CDP435 against human recombinant IL-17, monkey recombinant IL-17 and human recombinant IL-17F in Hela cells was tested. Hela cells were obtained from the cell bank at ATCC (ATCC CCL-2). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum, penicillin, gentamycin and glutamine. 1×10.sup.4 cells were plated out into 96 well flat bottomed tissue culture plates. Cells were incubated overnight and washed once in assay buffer. Either human IL-17 (25 ng ml.sup.−1), monkey IL-17 (25 ng ml.sup.−1) or human IL-17F (100 ng ml.sup.−1) was incubated in the presence of a fixed concentration of human TNF-α this mixture was preincubated with CDP435. Cytokine plus antibody was then added to the Hela cells which were incubated overnight. The production of IL-6 in the cell culture supernatant was proportionate to the amount of IL-17/IL-17F added to the cells. Human IL-6 levels were measured by ELISA and quantified by comparison with known standard concentrations of human IL-6.

[0155] The data (FIGS. 8, 9, and 10) indicates that CDP435 potently neutralised both human recombinant IL-17 and monkey recombinant IL-17 but did not inhibit the activity of human recombinant IL-17F. The data from these experiments indicated that CDP435 gave an IC.sub.50 of 158 ng ml.sup.−1±48 against human recombinant IL-17 (25 ng ml.sup.−1) and 147 ng ml.sup.−1±45 against monkey recombinant IL-17 (25 ng ml.sup.−1).

Mouse IL-17 Neutralisation Assay (3T3-NIH Cells)

[0156] The neutralisation potency of CDP435 against mouse recombinant IL-17 was determined. 3T3-NIH cells were obtained from the cell bank at ATCC (ATCC CRL-1658). Cells were grown in DMEM supplemented with 10% calf serum, penicillin, gentamycin and glutamine. The assay buffer used was identical to this buffer with foetal calf serum replacing calf serum. 1×10.sup.4 cells were plated out into 96 well flat bottomed tissue culture plates. Cells were incubated overnight and washed once in assay buffer. Murine IL-17 in the presence of a fixed concentration of human TNF-α was preincubated with CDP435. Cytokine plus CDP435 was then added to the 3T3-NIH cells which were incubated overnight. The production of IL-6 in the cell culture supernatant was proportionate to the amount of mouse IL-17 added to the cells. Mouse IL-6 levels were measured by ELISA and quantified by comparison with known standard concentrations of murine IL-6.

[0157] The data indicates that CDP435 did not inhibit the activity of mouse recombinant IL-17 (FIG. 11).

Example 6: Rat Pharmacokinetic Study with CDP435

[0158] Rats were injected s.c. with .sup.125I labelled CDP435. At various times the animals were bled and the blood counted for radioactivity. The pharmacokinetic trace is shown in FIG. 12. AUC.sub.0-∞=2651% dose*h, t1/2β=52 h, C.sub.max=22.7% dose. The results showed that CDP435 had good pharmacokinetics with a half life of 52 hours.

[0159] CDP435 was labelled with .sup.125I at a specific activity of 0.07 μCi/μg and 77.6 μg antibody administered s.c. in a volume of 100 μl.

In Vivo Neutralisation Assay

[0160] To determine the neutralisation efficacy of CDP435 in vivo, CDP485 was tested in two in vivo models of inflammation.

Intraperitoneal CDP435/Intraperitoneal hIL-17 in Mice

[0161] Male Balb/c mice (18-25 g) were injected intraperitoneally (i.p.) with CDP435 or control Fab′ A33-PEG and then injected i.p. 5 minutes later with hIL-17. After 180 minutes, mice were killed by cervical dislocation and peritoneal lavage performed (3 ml HBSS (Hanks' Balanced Salts)+0.25% BSA, 12 mM HEPES) and neutrophil accumulation quantified by FACS (Neutrophils were identified as those cells expressing CD45 and high levels of GR1 by staining with anti-CD45 CyChrome and anti-GR1 Phycoerythrin antibodies). Neutrophil accumulation in response to 300 ng hIL-17 was significantly reduced with CDP435 at doses of 0.01 and 0.1 mg/kg (FIG. 13).

[0162] In a separate experiment animals were dosed s.c. with 20 mg/kg CDP435 and challenged i.p. with 300 ng hIL-17 24 hours later. After a further 3 hours, peritoneal lavage showed that the CDP435 treatment had blocked neutrophil accumulation (FIG. 14). Thus CDP435 is effective against hIL-17 when given locally with the antigen or administered s.c. at a distant site.

[0163] It will of course be understood that the present invention has been described by way of example only, is in no way meant to be limiting, and that modifications of detail can be made within the scope of the claims hereinafter. Preferred features of each embodiment of the invention are as for each of the other embodiments mutatis mutandis. All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.