Antibody molecules having specificity for human IL-6

#The present invention relates to antibody molecules having specificity for antigenic determinants of IL-6. The present invention also relates to the therapeutic uses of the antibody molecules and methods for producing the antibody molecules.

#IL-6 is a pleiotropic multi-functional cytokine produced by a variety of cell types. It was originally identified as a B-cell differentiation factor (BSF-2) that induced the final maturation of B-cells into antibody producing cells (Hirano et al., 1986 Nature 324, 73-76). IL-6 has been shown to play a central role in immune regulation, inflammation, heamatopoiesis and oncogenesis. Within the immune system, IL-6 induces B-cell antibody production increasing the amount of polyclonal immunoglobulin. It also induces interleukin-2 (IL-2) receptor expression on T-cells (Nomo et al, 1987, Immunol. letters, 15, 3, 249-253) and promotes IL-2 production in activated T-cells thereby inducing both the growth and the differentiation of cytototoxic T-cells (Okada et al., 1988 J Immunol, 141, 5, 1543-1549). IL-6 is also known to determine the differentiation of monocytes into macrophages (Chomarat P et al., 2000 Nature Immunol., 6, 510-514).

#The function of IL-6 is not restricted to the immune response as it acts in hematopoiesis, thrombopoiesis, osteoclast formation, elicitation of hepatic acute phase response resulting in the elevation of C-reactive protein (CRP) and serum amyloid A (SAA) protein. It is known to be a growth factor for epidermal keratinocytes, renal mesangial cells, myeloma and plasmacytoma cells (Grossman et al., 1989 Prot Natl Acad Sci., 86, (16) 6367-6371; Horii et al., 1989, J Immunol, 143, 12, 3949-3955; Kawano et al., 1988, Nature 332, 6159, 83-85). IL-6 is produced by a wide range of cell types including monocytes/macrophages, fibroblasts, epidermal keratinocytes, vascular endothelial cells, renal messangial cells, glial cells, condrocytes, T and B-cells and some tumour cells (Akira et al, 1990, FASEB J., 4, 11, 2860-2867). Except for tumour cells that constitutively produce IL-6, normal cells do not express IL-6 unless appropriately stimulated.

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

#FIG. 1 shows the graft design for the 240.g1 heavy FIG. 1a; SEQ ID NOS: 2, 5, 6, 7, 11, 19, and 20) and light chain FIG. 1b; SEQ ID NOS: 4, 8, 9, 10, 13, 21, 22, and 23) 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 framework residues retained in the grafts.

#FIG. 2a shows the translated sequence of 240.g1 IgG4 heavy chain, showing intron/exon boundaries. (SEQ ID NOS: 11, 12, 15, 16, and 24).

#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 INVITROGEN. Synthetic genes were built at Entelechon. The concentration of Fab and IgG was determined using assembly ELISA.

#Rats were immunised by subcutaneous injection of recombinant human IL-6 (Peprotech) at three weekly intervals, initially with Freund's Complete Adjuvant and subsequently with Freund's Incomplete Adjuvant. Spleens were harvested one-two weeks after the last immunisation, and single cell suspensions prepared. Immune rat lymphocytes were cultured in the presence of irradiated mouse thymoma EL4 cells and rabbit T cell conditioned media for one week in 96 well microtitre plates. Supernatants were screened for the presence of antibodies specific for human IL-6 in ELISAs. Positives were further screened for the ability to neutralise the biological effects of human IL-6 in a DS1 cell line assay (Bock et al., 1993, Cytokine, 5, 480-489).

#Individual B cells secreting antibody with appropriate binding characteristics were isolated from positive microtitre wells according to the Selected Lymphocyte Antibody Method (Babcook et al., 1996, Proc. Natl. Acad. Sci. USA 93, 7843-7848; WO92/02551), and heavy and light chain variable region genes were cloned via reverse transcription PCR from single rat B cells. Variable regions were expressed in recombinant IgG format to confirm binding, and antibody 132E09 was selected for humanisation and further study. The rat variable region sequence was registered as CA030—00240.