WO2007054090A1

WO2007054090A1 - Therapeutic vaccines targeting hmgb1

Info

Publication number: WO2007054090A1
Application number: PCT/DK2006/000614
Authority: WO
Inventors: Tomas Bratt; Florence Dal Degan; Katja Bergholdt Nielsen; Bjørn VOLDBORG; Jens Holmberg
Original assignee: Affitech AS
Current assignee: Affitech AS
Priority date: 2005-11-09
Filing date: 2006-11-08
Publication date: 2007-05-18
Anticipated expiration: 2008-05-09
Also published as: CA2628546A1; WO2007054090A8; AU2006312847A1; EP1957534A1

Abstract

A method is disclosed for inducing an immune response against autologous high mobility group box 1 (HMGB1) in the autologous host. The method relies on specific active immunotherapy utilising variants of HMGB1 and targeting conditions such as cancer, Alzheimer's disease, atherosclerosis, and inflammatory disturbances. Typical modes of the invention utilises modified HMGB1 polypeptides that comprise a substantial fragment of at least one of the A and B boxes of HMGB1. Also provided are variant HMGB1 polypeptides and nucleic acid fragments encoding these as well as vectors and transformed host cells.

Description

THERAPEUTIC VACCINES TARGETING HMGBl

FIELD OF THE INVENTION

The present invention relates to the field of active specific immunotherapy. In particular, the present invention provides for novel immunotherapeutic agents and methods that target the protein antigen HMGBl. The invention further provides for various tools and methods that are practical in the provision of the immunotherapeutic agents and methods.

BACKGROUND OF THE INVENTION

The HMGBl protein

HMGBl (high mobility group box 1), also known as HMG-I and amphoterin, is a well-known 215 amino acid non-histone chromosomal protein which has recently been demonstrated to exert cytokine-like function. HMGBl has two DNA-binding domains (boxes) that associate with DNA without sequence specificity, but with higher binding to distorted or bent DNA and the protein thereby facilitates assembly of site-specific DNA binding proteins within the chromatin. The two boxes are not in direct interaction.

HMGBl is normally imported into the nucleus upon acetylation and is recycled between the nucleus and cytosol depending on the acetylation status of the protein. HMGBl associates with exocytotic vesicles when present in the cytosol and is subsequently exported through an IL-1-like mechanism. Almost the whole of HMGBl is rich in positively charged lysines, while the C-terminal thirty amino acids are constituted of charged aspartic and glutamic acids. More than 50% of the amino acids in HMGBl are charged at physiological pH. The pi of HMGBl increases from 5.6 to 10.5 when the acidic C-terminal is removed.

Human HMGBl has 95% sequence identity with HMGBl across all mammalian species wherein the protein has been sequenced and only differs at two amino acids from murine HMGBl. The two non-conserved amino acids are C-terminal aspartic and glutamic acids that are conservatively replaced with glutamic and aspartic acids, respectively.

The acidic tail stabilizes the tail-less HMGBl AB to identical thermal stability as wt (Knapp, S., Muller, S., Digilio, G., Bonaldi, T., Bianchi, M. E., and Musco, G. (2004) Biochemistry 43, 11992-11997), while all residues in the linker regions are unstructured and flexible. Full- length HMGBl is expressed in very. low yields in E. coli, the most popular explanation being that the full-length variant is toxic in E. CoIi. Another possible explanation is that the acidic tail with 30 consecutive acidic amino acid leads to shortage of tRNA for aspartic and glutamic acid and thereby slows down the expression rate (Lee, K. B., Brooks, D. J., and Thomas, J. O. (1998) Gene 225, 97-105). Structural studies have demonstrated the importance of the primary structure for the formation of the arrow-like α-helical box structure (Read, C. M., Cary, P. D., Crane-Robinson, C, Driscoll, P. C, and Norman, D. G. (1993) Nucleic Acids Res. 21, 3427-3436), although the initial amino acids (PKDP) of the B box are very flexible (Weir, H. M., Kraulis, P. J., Hill, C. S., Raine_ΛA. R., Laue, E. D., and Thomas, J. O. (1993) EMBO J. 12, 1311-1319). HMGBl is acetylated and potentially phosphorylated intracellular^, but it is not known if these modifications have any influence on the cytokine function.

Endogenous properties

Apart from the nuclear functions HMGBl is secreted and has cytokine-like extracellular functions. Most of the cell dependent effects that have been ascribed to HMGBl are through interactions with RAGE (Receptor of Advanced Glycation Endproducts) that elicit intracellular chemotactic and/or proinflammatory signals.

HMGBl is released into the circulation during the late phase of inflammation and during tumour metastasis. One of the pro-inflammatory roles of HMGBl is the induction of monocyte migration through the endothelium but it also induces secretion of several proinflammatory cytokines. HMGBl is mainly produced and actively released from stimulated monocytes and macrophages, in a so far unknown IL-I like mechanism. HMGBl is also passively released from necrotic, but not from apoptotic cells. HMGBl has also been reported as a developmentally regulated protein abundantly expressed in the embryonic brain, where it is associated with the leading edges of migrating neuronal cells. HMGBl can induce chemotaxis in tumor, stem and immune cells. The chemotactic properties are regulated by both soluble and membrane-associated HMGBl that bind and signal through RAGE. The B box and C- terminal linker of HMGBl has been reported to be involved in RAGE binding. The proinflammatory effect, which is toxic at high concentrations, is partly distinct from RAGE binding. It has been suggested that HMGBl also binds TLR2 and/or TLR4. HMGBl also associates with negatively charged carboxylated or sulphated glycosides e.g. heparan sulphate that can be present on cell membranes or in the extracellular matrix. It has also been demonstrated that HMGBl has anti-microbial effects and can be part of the innate immune response, although the exact mechanisms have not been determined and the effect could be more indirect (Zetterstrom, C. K., Bergman, T., Rynnel-Dagoo, B., Erlandsson, H. H., Soder, O., Andersson, U., and Boman, H. G. (2002) Pediatr.Res. 52, 148-154). Recently, it has been suggested that HMGBl is directly involved in the mismatch repair of DNA damage (Yuan, F., Gu, L, Guo, S., Wang, C, and Li, G. M. (2004) J.Biol.Chem. 279, 20935- 20940).

Exogenous properties

Both HMGBl and the B box also exhibit adjuvant properties as they induce maturation and activation of dendritic cells (APCs) and enhance antibody responses (Rovere-Querini, P., Capobianco, A., Scaffidi, P., Valentinis, B., Catalanotti, F., Giazzon, M., Dumitriu, I. E., Muller, S., Iannacone, M., Traversari, C, Bianchi, M. E., and Manfredi, A. A. (2004) EMBO Rep. 5, 825-830), and the use of HMGBl as an adjuvant has been sought patented (WO 03/026691).

It is not known whether this means that HMGBl produced by tumor cells will trigger an antitumor immune response or that anti-HMGBl will attenuate anti-self responses in autoimmunity. Another interesting property of HMGBl is that it can be used as transfection agent if mixed with plasmid material. HMGBl acts by DNA binding and promote supercoiling of the plasmid (Mistry, A. R., Falciola, L, Monaco, L., Tagliabue, R., Acerbis, G., Knight, A., Harbottle, R. P., Soria, M., Bianchi, M. E., Coutelle, C, and Hart, S. L (1997) Biotechniques 22, 718-729), and it could also be speculated that HMGBl enhances the cellular uptake of associated plasmids by receptor binding or binding to negatively charged carbohydrates on the cell membrane.

Role in sepsis

HMGBl is a late essential downstream mediator of endotoxin lethality in sepsis and although other cytokine antagonists have not been effective in treatment of sepsis, the delayed release of HMGBl and the successful anti-HMGBl antibody treatment in murine sepsis models shows some promise for treatment of sepsis if targeting HMGBl.

Role in arthritis

HMGBl is abundantly expressed in synovium and intra-articular fluid of RA patients as well as in experimental arthritis and is involved in the mechanism that sustains chronic inflammation. Intra-articular injections of HMGBl induce synovitis while anti-HMGBl antibodies protect mice during experimental inflammatory conditions. The HMGBl A box has an antagonistic function, inhibiting the effects of HMGBl and/or the B box, as efficiently as anti-HMGBl antibodies. Inhibition of HMGBl by antibodies is not only preventive but also therapeutic.

HMGBl release induces several cytokines e.g. IL-I, TNF and interferon^ and this induction is amplified by IL-2 in combination with IL-12 or IL-I. Interestingly, in IL-IR-/- knockout mice HMGBl does not induce arthritis (Pullerits, R., Jonsson, I. M., Verdrengh, M., Bokarewa, M., Andersson, U., Erlandsson-Harris, H., and Tarkowski, A. (2003), Arthritis Rheum. 48, 1693- 1700). Protection against arthritis requires a quite high amount of anti-HMGBl antibodies (2 mg/mouse) in murine arthritis models, while 0,5 mg/mice does not protect. Both the antagonistic A-box and anti-B-box polyclonal antibodies inhibit CIA in mice and rats, analogous to or better than anti-TNF antibodies (Kokkola, R., Li, J., Sundberg, E., Aveberger, A. C, Palmblad, K., Yang, H., Tracey, K. J., Andersson, U., and Harris, H. E. (2003) Arthritis Rheum. 48, 2052-2058).

Role in cancer

Both HMGBl and RAGE are overexpressed in many cancer forms. HMGBl accelerate tumor growth in murine models and is upregulated in 63% of metastatic prostate cancer cases (Kuniyasu, H., Chihara, Y., Kondo, H., Ohmori, H., and Ukai, R. (2003) Oncol. Rep. 10, 1863-1868). Administration of A box reduces formation of lung metastasis. RAGE is a multifactorial receptor that is also activated by glycated proteins, which is present in diabetic patients, calgranulin, and A-beta in Alzheimer patients. HMGBl is an anti-apoptotic RAGE ligand and sRAGE and anti-HMGBl antibodies suppress growth of implanted tumors (Taguchi, A., Blood, D. C, del Toro, G., Canet, A., Lee, D. C, Qu, W., Tanji, N., Lu, Y., LaIIa, E., Fu, C, Hofmann, M. A., Kislinger, T., Ingram, M., Lu, A., Tanaka, H., Hori, O., Ogawa, S., Stern, D. M., and Schmidt, A. M. (2000) Nature 405, 354-360).

Alzheimer's disease

An amyloidogenic sequence has been identified in the first alpha helix of the A box (Kallijarvi, J., Haltia, M., and Baumann, M. H. (2001) Biochemistry 40, 10032-10037). HMGBl is upregulated in the brain around dying neurons and is associated with A-beta plaques (Takata, K., Kitamura, Y., Kakimura, J., Shibagaki, K., Tsuchiya, D., Taniguchi, T., Smith, M. A., Perry, G., and Shimohama, S. (2003) Biochem.Biophys.Res.Commun. 301, 699-703). HMGBl has also a potent proinflammatory role within the CNS (O'Connor, K. A., Hansen, M. K., Rachal, P. C, Deak, M. M., Biedenkapp, J. C, Milligan, E. D., Johnson, J. D., Wang, H., Maier, S. F., Tracey, K. J., and Watkins, L. R. (2003) Cytokine 24, 254-265). Injected HMGBl inhibits A-beta clearance and enhance neurotoxicity, thus making HMGBl a potential target in Alzheimer's disease (Takata, K., Kitamura, Y., Tsuchiya, D., Kawasaki, T., Taniguchi, T., and Shimohama, S. (2004) J.Neurosci.Res. 78, 880-891). HMGBl is, however, also found at the leading edge of neurons, where it activates plasmin (through binding to tPA), which degrades both HMGBl and amyloid plaques. Therefore, more investigations are needed to establish the true endogenous role of HMGBl in plaque formation and removal (Kranenburg, O., Bouma, B., Kroon-Batenburg, L. M.,. Reijerkerk, A., Wu, Y. P., Voest, E. E., and Gebbink, M. F. (2002) Curr.Biol. 12, 1833-1839).

Atherosclerosis

Expression of HMGBl is also upregulated during atherosclerosis, where RAGE is highly activated (Kalinina, N., Agrotis, A., Antropova, Y., DiVitto, G., Kanellakis, P., Kostolias, G., Ilyinskaya, O., Tararak, E., and Bobik, A. (2004) Arterioscler.Thromb.Vasc.Biol.) and HMGBl is released by damage to or necrosis of endothelial cells (Degryse, B., Bonaldi, T., Scaffidi, P., Muller, S., Resnati, M., Sanvito, F., Arrigoni, G., and Bianchi, M. E. (2001) J.Cell Biol. 152, 1197-1206).

OBJECT OF THE INVENTION

It is an object of the invention to provide novel immunotherapeutics that target the protein HMGBl so as to enable treatment via active specific immunotherapy of diseases where down- regulation of HMGBl will provide for beneficial therapeutic effects.

SUMMARY OF THE INVENTION

The present inventors have concluded that HMGBl is a potential target in many different chronic and acute inflammatory disturbances, such as autoimmune diseases e.g. arthritis, but also as target in diseases as disparate as cancer, Alzheimer's disease and atherosclerosis. As sepsis and treatment thereof is acute, within a day after disease onset, this is not a primary indication for HMGBl targeting active immunotherapy, whereas an anti-HMGBl vaccine could be used preventively e.g. before major operations that are known to cause sepsis.

HMGBl is a comparatively new therapeutic target and no specific HMGBl antagonists have as of yet been examined in clinical trials. However, several agents with a general antiinflammatory activity are known to down-regulate HMGBl. Ethyl pyruvate is an agent that is in early clinical trials, but also corticosteroids have been reported to have an effect on HMGBl regulation. A more direct approach through stimulation of acetylcholine receptors on macrophages downregulates HMGBl release. The acetylcholine receptor is also activated by nicotine. This can potentially explain the beneficial effect that smoking has on the incidence on ulcerative colitis (Wang, H., Liao, H., Ochani, M., Justiniani, M., Lin, X., Yang, L, Al Abed, Y., Wang, H., Metz, C, Miller, E. J., Tracey, K. J., and Ulloa, L. (2004) Nat.Med.). However, none of these approaches directly and specifically target HMGBl. In contrast, the present invention suggests targeting of HMGBl by means of active specific immune therapy. It is believed that this approach is novel, i.e. the present invention for the first time proposes and enables down-regulation of HMGBl by immunizing actively against this protein. Any general active immunization method available in the art where is used an immunogen which induces an effective immune response against HMGBl can be used. Hence, traditional vaccination approaches using an immunogen including an epitope of HMGBl or being capable of providing such an immunogen in vivo (typically DNA vaccines and viral vaccines function this way) are useful agents according to the present invention. So, in its most general scope the present invention relates to a method for inducing humoral immunity against HMGBl by administering an effective dose of an immunogen capable of effecting presentation of at least one HMGBl B-cell epitope to the immune system.

In a general aspect, the present invention relates to a method for inducing an immune response against autologous high mobility group box 1 (HMGBl) in a mammal, including a human being, the method comprising effecting uptake and processing by antigen presenting cells (APCs) in the subject of at least one modified HMGBl polypeptide, said at least one modified HMGBl polypeptide comprising

- a substantial fraction of the B-cell epitopes from the autologous HMGBl, and

- at least one T helper epitope (TH epitope) which is heterologous to the mammal and the HMGBl protein, thereby inducing an antibody response that targets the autologous HMGBl.

In another aspect, the present invention relates to a modified HMGBl polypeptide that is capable of inducing an immune response against autologous HMGBl in a human subject, comprising a substantial fraction of the B-cell epitopes and optionally CTL epitopes from the B box of HMGBl, and at least one non-human T helper epitope (T_H epitope). Also part of the invention is an immunogenic composition which comprises, as an effective immunogenic agent this modified human HMGBl in admixture with a pharmaceutically and immunologically acceptable carrier or vehicle, and optionally an adjuvant.

The invention further relates to a nucleic acid fragment which encodes a modified HMGBl polypeptide of the invention, and also a vector carrying this nucleic acid fragment is a part of the invention. Similarly, a transformed cell carrying this nucleic acid fragment or vector is also a part of the invention.

Furhter, the invention also relates to a composition for inducing production of antibodies a- gainst HMGBl, the composition comprising

- a nucleic acid fragment or a vector of the present invention, - and a pharmaceutically and immunologically acceptable diluent and/or vehicle and/or adjuvant.

Another part of the invention is a stable cell line which carries the vector of the invention and which expresses the nucleic acid fragment of the invention, and which optionally secretes or carries the modified HMGBl of the invention on its surface. A method for preparing the cell line is also part of the invention, said method comprising transforming a host cell with the nucleic acid fragment of the invention or with the vector invention.

LEGENDS TO THE FIGURE

Fig. 1: Tertiary structure of HMGBl B box. The tertiary structure of Chinese hamster HMGBl B box (aa 92-171) was determined by 2D ¹H NMR (Read, C. M. Gary, P. D., Crane-Robinson, C, Driscoll, P. C. and Norman D. G. (1993) Nucleic Acids Res. 21, 3427-3436). The all alpha-helical fold (dark color) is in the form of an L- or arrow-shape. The primary sequence of the Chinese hamster HMGBl B box is identical with the human sequence. The 3D structure of the HMGBl A box has also been determined and is highly homologous to the B box structure (Weir, H. M., Kraulis, P. J., Hill, C. S. Raine, A. R. C, Laue, E. D. and Thomas J. O. (1993) EMBO J. 12, 1311-1219).

Fig. 2: The HMGBl amino acid sequence.

The A box domain consists of amino acid 9-81. The B box consists of amino acid 91-165. The acidic C-terminal is defined by amino acid 186-215. A heparin binding site is found at amino acid 5-12. An amyloidgenic sequence is found at amino acid 13-27. Six alpha helices are found in the protein at amino acid positions 16-30, 39-50 and 53-77 in the A box and at amino acid positions 100-116, 123-136 and 140-162 in the B box. There are two potential N- glycosylation sites, at positions 37 and 134, which are presumably not glycosylated as the protein is synthesized in the cytosol and not in the ER. Polylysines at positions 28-30 and 180, 182-185 are frequently acetylated.

Fig. 3: Reduced SDS-PAGE and western blot of the purified HMGBl variants.

Lane 1 molecule weight standard, lane 2 HMG-AB4, lane 3 HMG-AB8, lane 4 HMG-ABlO, lane

5 HMG-AB13 and lane 6 HMG-AB3.1.

Fig. 4: Reduced and non-reduced SDS-PAGE of HMG-AB8 variants. Reduced (left) and unreduced (right) purified HMG-AB8 at 1 mg/ml, 0.5mg/ml and 0.25 mg/ml. Fig. 5: RP-HPLC chromatogram of purified HMGBl.

Main peak purity is 90%. The sample is purified variant HMG-AB3.1 (Sample 02017).

Fig. 6: SE-HPLC chromatogram of purified HMGBl (Sample variant HMG-AB8 D01650).

Fig. 7: Anti-HMGBl response in HMG-ABlO-vaccinated C57BL/6 mice and DA rats. WT-HMGBl (lug/ml) was coated on Maxisorp plates over night, blocked with 3% fish gelatin, and anti-HMGBl response in sera (1/1000 for mice, 1/100 for rats) was detected by HRP- conjugated anti-mouse Ig or anti-rat Ig, respectively.

Left figure: mice (n=5 males per group) were vaccinated at day 0 (100 μg HMG-ABlO), day 14 (50 μg HMG-ABlO) and day 42 (50 μg HMG-ABlO), using ISA51 (A), Adjuphos (B) or Alhydrogel (C). PBS alone (D) was used in control mice.

Right figure: rats (n=4 males or females per group) were vaccinated at day 0 (100 μg ABlO), day 15 (50 μg ABlO), day 41 (50 μg ABlO), and day 70 (50 μg ABlO), using ISA51 (A- females, D-males), Adjuphos (B-females, E-males) or Alhydrogel (C-females, F-males). Rat control groups (i.e. KLH emulsified in ISA51, Adjuphos or Alhydrogel; and PBS alone) showed no anti-HMGBl response (data not shown).

Fig. 8: Anti-HMGBl IgG isotype response in HMG-AB10-vaccinated rats. WT-HMGBl (1 μg/ml) was coated on Maxisorp plates o.n., blocked with 3% fish gelatin, and HMGBl-specific total Ig-, IgGl- and IgG2b responses in sera were detected by HRP- conjugated anti-rat Ig, anti-rat IgGl and anti-rat IgG2b, respectively. Pooled sera from HMG- ABIO-Alhydrogel (A), HMG-ABIO-Adjuphos (B) and HMG-AB10-ISA51 (C) were investigated.

Fig. 9: Transfer of Pristane-induced arthritis (PIA) in in-house DA rats. Five DA females were injected i.d. at the base of the tail with 500 μl of Pristane, and spleen (SPL) and inguinal lymph nodes (LN) were collected at arthritis onset (day 14 post-injection). Pristane-primed spleen and lymph node cells were stimulated in vitro for 48h in presence of Concanavalin A (1.5 μg/ml). 37 million viable cells were then transferred i.p. to naϊve DA recipients. Clinical arthritis score (scale from 0-60), and index body weight (grams) were examined.

Fig. 10: HMGBl-induced TNF-α production in RAW264.7 cells.

RAW264.7 cells (180 000 / 96-well) were stimulated with native or trypsinated WT-HMGBl, HMG-AB4, HMG-ABlO and Lysozyme (negative control) for 4h at 37⁰C in presence or absence of Polymyxin B (50ug/ml). Supernatants were then collected to measure TNF-α production in ELISA. Fig. 11: Neutralization of HMGBl-induced TNF-α production from RAW264.7 cells. RAW264.7 cells (180 000 / 96-well) were stimulated with WT-HMGBl (i.e. OD50 concentrations) for 4h at 37⁰C in media with Polymyxin B (50 μg/ml) in presence of rabbit anti-HMGBl PAb (ABD-012 from JENA; 1 mg/ml), rabbit anti-HMGBl PAb (07-584 from Upstate, 1 mg/ml), human recombinant RAGE (1145-RG from R&D Systems, 1 mg/ml) or PBS. Supernatants were measured for TNF-α in ELISA.

Fig. 12: HMGBl-induced TNF-α production from RAW264.7 cells are reduced by any sera. RAW264.7 cells (180 000 / 96-well) were stimulated with WT-HMGBl (i.e. OD50 concentration) for 4h at 37⁰C in media with Polymyxin B (50 μg/ml) in presence of pooled sera (1:100) from vaccinated mice at day 28 after 1^st vaccination (A28m-AB10/ISA51;

B28m-AB10/Adjuphos; C28m-AB10/Alhydrogel; or X28m-PBS), pooled sera (1:100) from rats at day 41 after 1^st vaccination (A41r-AB10/Alhydrogel; B41r-AB10/Adjuphos; C41r- AB10/ISA51; D41r-KLH/Alhydrogel; E41r-KLH/Adjuphos; F41r-KLH/ISA51; or X41r-PBS). Lysozyme (LZ) served as negative control, WT-HMGBl (WTa-3ug/ml; WTb-1.5 μg/ml) served as positive controls. Additional sera controls include WT-HMGBl diluted in 10% FCS media (10% FCS) or in 10% sera from PBS-treated rats day 41 after 1^st injection (10% X41r). Supernatants were measured for TNF-α in ELISA.

Fig. 13: HMGBl Sandwich ELISA.

Mouse anti-HMGBl MAb (abl2029 from ABCAM, 3 μg/ml) was coated on Maxisorp plates o.n., blocked with 3% fish gelatin, incubated for 2h with HMGBl (human recombinant His- tagged from SIGMA; in-house WT- and ABlO-HMGBl; or post-translationally modified calf HMGBl from WAKO). Biotinylated mouse anti-HMGBl MAb (MAB1690 from R&D Systems, 1:250) was used as detector Ab or alternatively the monoclonals were substituted with each other.

Fig. 14: HMGBl Sandwich ELISA to quantify HMGBl-variants from lysate.

Left figure; mouse anti-HMGBl MAb (abl2029 from ABCAM, 3 μg/ml) was coated on Maxisorp plates o.n., blocked with 3% fish gelatin, incubated for 2h with cell lysates containing HMGBl variants (1:2 dilution, dil. 5x each step until 1:31250), and biotinylated mouse anti-HMGBl MAb (MAB1690 from R&D Systems, 1:250) was used as detector Ab. Right figure; mouse anti-HMGBl MAb (MAB1690 from R&D Systems, 8 μg/ml) was coated on Maxisorp plates o.n., blocked with 3% fish gelatin, incubated for 2h with cell lysates containing HMGBl variants (1:2 dilution, diluted 5x each step until 1:31250), and biotinylated mouse anti-HMGBl MAb (abl2029 from ABCAM, 1:250) was used as detector Ab. Fig. 15: Expression of HMGBl variants in cell lysate.

The expression levels of all the variants that have been constructed were analysed by coomassie stained reduced SDS-PAGE. Every lane show a specific HMGBl variant. The variants based on AB box templates have a size that correspond to about 30 kDa and the variants based on the B box correspond to about 15 kDa. Arrows indicate the bands representing the AB box and B box variants.

Fig. 16: Clinical arthritis scores in female rats immunized with HMG-ABlO. A: The difference in clinical arthritis score between female rats immunised with HMG-ABlO in Alhydrogel or controls immunized with KLH in Alhydrogel. B: Summary results of three groups of female rats that were immunised with HMG-ABlO in either Alhydrogel, Adjuphos or ISA-51 compared with female rats immunized with KLH with the same adjuvants.

Fig. 17: HMGBl templates and variants according to the present invention. The amino acid sequences of the templates and variants in this figure are also set forth in the sequence listing in SEQ ID NOs: 1-24 and SEQ ID NOs: 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77. The A and B box are shown in bold and the inserted epitope in italics. The amino acids that are underlined constitute the alpha helical regions in the A and B box.

Fig. 18: Immunoprecipitation of antisera raised against HMG-ABlO with wild-type HMGBl. The figure shows a classical precipitation assay where there is only a visible precipitation line between wtHMGBl and antisera from rats and mice immunized with HMG-ABlO, whereas there are no visible precipitation line between wtHMGBl and antisera from rats and mice immunized with controls.

DETAILED DISCLOSURE OF THE INVENTION

In the following, a number of terms used in the present specification and claims will be defined and explained in detail in order to clarify the metes and bounds of the invention.

The term "immunogen" in the present context refers to an agent (a substance or a composition of matter) that induces a specific immune response against the immunogen in a host which has been immunized therewith. It will be understood that certain molecules (e.g. tra- ditional small haptens or self-proteins that are tolerated in the autologous host) are incapable of inducing a specific immune response and therefore cannot be termed "immunogens" in that particular setting. However, some self-proteins are, when formulated in very strong immunologic adjuvants, capable of inducing a specific immune response in spite of the normally tolerant state of the immunized animal. In such a context, the "immunogen" is therefore the composition of matter (self-protein with adjuvant) and not just a single molecule.

The terms "T-lymphocyte" and "T-cell" will be used interchangeably for lymphocytes of thymic origin which are responsible for various cell mediated immune responses as well as for helper activity in the humeral immune response. Likewise, the terms "B-lymphocyte" and "B-cell" will be used interchangeably for antibody-producing lymphocytes.

An "HMGBl polypeptide" is herein intended to denote polypeptides having the amino acid se- quence of the above-discussed HMGBl proteins derived from humans and other mammals (or truncates thereof sharing a substantial amount of B-cell epitopes from at least the B box of human HMGBl). Also forms of HMGBl polypeptides having varying degrees of glycosylation (because they are produced in cells having varying capabilities with respect to effecting glycosylation of proteins). It should, however, be noted that when using the term "an HMGBl polypeptide" it is intended that the polypeptide in question is normally non- immunogenic when presented to the animal to be treated. In other words, the HMGBl polypeptide is a self-protein or is a xeno-analogue of such a self-protein which will not normally give rise to an immune response against HMGBl of the animal in question.

An "HMGBl analogue" or a "modified HMGBl polypeptide" or an "HMGBl variant" is an HMGBl polypeptide which has been subjected to changes in its primary structure. Such a change can e.g. be in the form of fusion of an HMGBl polypeptide to a suitable fusion partner {i.e. a change in primary structure exclusively involving C- and/or N-terminal additions of amino acid residues) and/or it can be in the form of insertions and/or deletions and/or substitutions in the HMGBl polypeptide's amino acid sequence. Also encompassed by the term are derivatized HMGBl molecules, cf. the discussion below of modifications of HMGBl.

It should be noted that the use as a vaccine in a human of a mammal analogue of human HMGBl could be imagined to produce the desired immunity against HMGBl in humans. Such use of a xeno-analogue for immunization is also considered to be a "HMGBl analogue" as defined above.

When using the abbreviation "HMGBl" herein, this is intended as a reference to the amino acid sequence of wildtype HMGBl (also denoted "HMGBl" and "HMGBl-wt" and "wtHMGBl" herein). In cases where a DNA construct includes information encoding a leader sequence or other material, this will normally be clear from the context. The term "polypeptide" is in the present context intended to mean molecules comprising polyamino acids covalently linked via peptide bonds, and the term encompasses both short peptides of from 2 to 10 amino acid residues, oligopeptides of from 11 to 100 amino acid residues, and poly-peptides of more than 100 amino acid residues. Furthermore, the term is also intended to include proteins, i.e. functional biomolecules comprising at least one polypeptide; when comprising at least two polypeptides, these may form complexes, be covalently linked, or may be non-covalently linked. The polypeptide(s) in a protein can be glycosylated and/or lipidated and/or acetylated and/or phosphorylated and/or comprise prosthetic groups. Thus the term includes enzymes, antibodies, antigens, transcription factors, binding proteins e.g. DNA binding proteins, or protein domains or fragments of proteins or any other amino acid based material.

The term "polyamino acid" denotes a molecule consitituted by at least 3 covalently linked amino acid residues.

The term "subsequence" means any consecutive stretch of at least 3 amino acids or, when relevant, of at least 3 nucleotides, derived directly from a naturally occurring HMGBl amino acid sequence or nucleic acid sequence, respectively.

The term "animal" is in the present context in general intended to denote an animal species (preferably mammalian), such as Homo sapiens, Cam^'s domesticus, etc. and not just one single animal. However, the term also denotes a population of such an animal species, since it is important that the individuals immunized according to the method of the invention all harbour substantially the same HMGBl allowing for immunization of the animals with the same immunogen(s). If, for instance, genetic variants of HMGBl exist in different human populations it may be necessary to use different immunogens in these different populations in order to be able to break the autotolerance towards HMGBl in each population. It will be clear to the skilled person that an animal in the present context is a living being which has an immune system. It is preferred that the animal is a vertebrate, such as a mammal.

By the term "in vivo down-regulation of HMGBl activity" is herein meant reduction in the living organism of the number of interactions between HMGBl and its natural binding partners and ligands (or between HMGBl and other possible biologically important binding partners for this molecule). The down-regulation can be obtained by means of several mechanisms: Of these, simple interference with the active site in HMGBl by antibody binding or by antibody binding to HMGBl and removal by Fc receptor carrying cells is the most simple. However, it is also within the scope of the present invention that the antibody binding results in removal of HMGBl-carrying cells by secondary immunological mechanisms (such as complement activation and killing of cells by IMK cells. Also within the scope of the present invention is the killing of HMGBl expressing cells by cytotoxic T lymphocytes.

The expression "effecting uptake and processing by antigen presenting cells ... thereby inducing an antibody response" is intended to denote that the animal's immune system is subjected to an immunogenic challenge in a controlled manner which ensures that the essential mechanisms in a humoral immune response are triggered {i.e. antigen uptake and processing followed by recognition of presented T-helper eptitopes from the antigen by T- helper cells and stimulation of B-cells by which also present the same T-helper epitopes). As will appear from the disclosure below, such challenge of the immune system can be effected in a number of ways of which the most important are vaccination with polypeptide containing "pharmaccines" {i.e. a vaccine which is administered to treat or ameliorate ongoing disease) or nucleic acid "pharmaccine" vaccination. The important result to achieve is that immune competent cells in the animal are confronted with the antigen in an immunologically effective manner, whereas the precise mode of achieving this result is of less importance to the in- ventive idea underlying the present invention.

The term "immunogenically effective amount" has its usual meaning in the art of immunology, i.e. an amount of an immunogen which is capable of inducing an immune response which significantly engages molecules which share immunological features with the immunogen.

When using the expression that the HMGBl has been "modified" is herein meant a chemical modification of the polypeptide which constitutes the backbone of HMGBl. Such a modification can e.g. be derivatization {e.g. alkylation, acylation, esterification etc.) of certain amino acid residues in the HMGBl sequence, but as will be appreciated from the disclosure below, the preferred modifications comprise changes of (or additions to) the primary structure of the HMGBl amino acid sequence.

When discussing "autotolerance towards. HMGBl" it is understood that since HMGBl is a self- protein in the population to be vaccinated, normal individuals in the population do not mount an immune response against HMGBl; it cannot be excluded, though, that occasional individuals in an animal population might be able to produce antibodies against native HMGBl, e.g. as part of an autoimmune disorder. At any rate, an animal will normally only be autotolerant towards its own HMGBl, but it cannot be excluded that HMGBl analogues derived from other animal species or from a population having a different HMGBl phenotype would also be tolerated by said animal. A "foreign T-cell epitope" (or: "foreign T-lymphocyte epitope") is a peptide which is able to bind to an MHC molecule and which stimulates T-cells in an animal species. Preferred foreign T-cell epitopes in the invention are "promiscuous" (also known as "universal") epitopes, i.e. epitopes which bind to a substantial fraction of a particular class of MHC molecules in an ani- mal species or population. Only a very limited number of such promiscuous T-cell epitopes are known, and they will be discussed in detail below. It should be noted that in order for the immunogens which are used according to the present invention to be effective in as large a fraction of an animal population as possible, it may be necessary to 1) insert several foreign T-cell epitopes in the same HMGBl analogue or 2) prepare several HMGBl analogues wherein each analogue has a different promiscuous epitope inserted. It should be noted also that the concept of foreign T-cell epitopes also encompasses use of cryptic T-cell epitopes, i.e. epitopes which are derived from a self-protein and which only exerts immunogenic behaviour when existing in isolated form without being part of the self-protein in question.

A "foreign T helper lymphocyte epitope" (a foreign T_H epitope) is a foreign T cell epitope which binds an MHC Class II molecule and can be presented on the surface of an antigen presenting cell (APC) bound to the MHC Class II molecule.

A "functional part" of a (bio)molecule is in the present context intended to mean the part of the molecule which is responsible for at least one of the biochemical or physiological effects exerted by the molecule. It is well-known in the art that many enzymes and other effector molecules have an active site which is responsible for the effects exerted by the molecule in question. Other parts of the molecule may serve a stabilizing or solubility enhancing purpose and can therefore be left out if these purposes are not of relevance in the context of a certain embodiment of the present invention. For instance it is possible to use certain cytokines as a modifying moiety in HMGBl (cf. the detailed discussion below), and in such a case, the issue of stability may be irrelevant since the coupling to HMGBl provides the stability necessary.

The term "adjuvant" has its usual meaning in the art of vaccine technology, i.e. a substance or a composition of matter which is 1) not in itself capable of mounting a specific immune response against the immunogen of the vaccine, but which is 2) nevertheless capable of enhancing the immune response against the immunogen. Or, in other words, vaccination with the adjuvant alone does not provide a specific immune response against the immunogen, vaccination with the immunogen may or may not give rise to a specific immune response against the immunogen, but the combination of vaccination with immunogen and adjuvant induces a specific immune response against the immunogen which is stronger than that induced by the immunogen alone. "Targeting" of a molecule is in the present context intended to denote the situation where a molecule upon introduction in the animal will appear preferentially in certain tissue(s) or will be preferentially associated with certain cells or cell types. The effect can be accomplished in a number of ways including formulation of the molecule in composition facilitating targeting or by introduction in the molecule of groups which facilitates targeting. These issues will be discussed in detail below.

"Stimulation of the immune system" means that a substance or composition of matter exhibits a general, non-specific immunostimulatory effect. A number of adjuvants and putative adjuvants (such as certain cytokines) share the ability to stimulate the immune system. The result of using an immunostimulating agent is an increased "alertness" of the immune system meaning that simultaneous or subsequent immunization with an immunogen induces a significantly more effective immune response compared to isolated use of the immunogen.

"Productive binding" means binding of a peptide to the MHC molecule (Class I or II) so as to be able to stimulate T-cells that engage a cell that present a peptide bound to the MHC rπole- cule. For instance, a peptide bound to an MHC Class II molecule on the surface of an APC is said to be productively bound if this APC will stimulate a T_H cell that binds to the presented peptide-MHC Class II complex and, accordingly, a peptide bound to an MHC Class I molecule on the surface of a cell is said to be productively bound if activated CTLs that recognize the peptides are capable of exerting a cytotoxic effect on the cell.

Preferred modes of anti-HMGBl immunization

The present invention entails active immunization against HMGBl. More precisely, the invention in a preferred embodiment pertains to a method for inducing an immune response against autologous high mobility group box 1 (HMGBl) in a mammal, including a human being, the method comprising effecting uptake and processing by antigen presenting cells (APCs) in the subject of at least one modified HMGBl polypeptide, said at least one modified HMGBl polypeptide comprising

- a substantial fraction of the B-cell epitopes from the autologous HMGBl, and

This active immunization approach is preferably used to treat, ameliorate or reduce the risk of attracting a condition selected from the group consisting of cancer, Alzheimer's disease, atherosclerosis, and an inflammatory disturbance, including a chronic or acute condition such as an autoimmune disease, e.g. arthritis. The HMGBl polypeptide being the immunogen which ultimately confronts the immune system is in the preferred embodiment of the invention thus a modified molecule wherein at least one change is present in the HMGBl polypeptide amino acid sequence, since the chances of obtaining the all-important breaking of autotolerance towards HMGBl is greatly facilitated that way. It should be noted that this does not exclude the possibility of using such a modified HMGBl in formulations which further facilitate the breaking of autotolerance against HMGBl, e.g. formulations containing certain adjuvants discussed in detail below. It should be further noted that the invention also contemplates using unmodified HMGBl or fragments thereof formulated (e.g. with strong adjuvants) in such a way that tolerance against HMGBl is broken, cf. the description above of the most general scope of the invention.

The following formula describes the preferred HMGBl constructs generally covered by the invention:

(MOD₁)_sl(E_el)_nl(MOD₂)_s2(E_e2)n2....(MOD_x)_sx(Eeχ)n>c (D

-where E_ei-E_exare x B-cell epitope containing subsequences of an HMGBl polypeptide, which independently are identical or non-identical and which may contain or not contain foreign side groups, x is an integer ≥ 3, nl-nx are x integers > 0 (at least one is > 1), MOD₁-MOD_x are x modifications introduced between the preserved B-cell epitopes, and Si-S_x are x integers > 0 (at least one is > 1 if no side groups are introduced in the E_ex sequences). Thus, given the general functional restraints on the immunogenicity of the constructs, the invention allows for all kinds of permutations of the original sequence of the HMGBl polypeptide, and all kinds of modifications therein. Thus, included in the invention are modified HMGBl polypeptides obtained by omission of parts of the sequence of the HMGBl polypeptide, which e.g. exhibit adverse effects in vivo and thus could give rise to undesired immunological reactions.

In one embodiment, side groups (in the form of foreign T-cell epitopes or the herein- discussed first, second and third moieties) are covalently or non-covalently introduced. This is intended to mean that stretches of amino acid residues derived from HMGBl are derivatized without altering the primary amino acid sequence, or at least without introducing changes in the peptide bonds between the individual amino acids in the chain.

An alternative embodiment utilises amino acid substitution and/or deletion and/or insertion and/or addition (which may be effected by recombinant means or by means of peptide synthesis; modifications which involves longer stretches of amino acids can give rise to fusion polypeptides). One version of this embodiment is the technique described in WO 95/05849, which discloses a method for immunizing against self-proteins by immunising with analogues of the self-proteins wherein a number of amino acid sequence(s) has been substituted with a corresponding number of amino acid sequence(s), which each comprise a foreign immunodominant T-cell epitope, while at the same time maintaining the overall 3 dimensional structure of the self-protein in the analogue. For the purposes of the present invention, it is however sufficient if the modification (be it an amino acid insertion, addition, deletion or sub- stitution) gives rise to a foreign T-cell epitope and at the same time preserves a substantial number of the B-cell epitopes in HMGBl. However, in order to obtain maximum efficacy of the immune response induced, it is advantageous that the 3-dimenstional structure of at least the A box and/or the B box of HMGBl is maintained in the modified molecule. This means that it is advantageous if modifications in the HMGBl structure are made in a non- destructive way, e.g. in flexible loops or termini.

One embodiment of the invention utilises multiple presentations of B-lymphocyte epitopes of the HMGBl polypeptide (i.e. formula I wherein at least one B-cell epitope is present in two positions). This effect can be achieved in various ways, e.g. by simply preparing fusion polypeptides comprising the structure (modified HMGBl polypeptide)™, where m is an integer > 2 and then introduce the modifications discussed herein in at least one of the HMGBl sequences. Or, it is possible to couple multiple HMGBl peptides or polypeptides to a carrier backbone, whereby multiple presentations of identical HMGBl B-cell epitopes are achieved. It is hence preferred that the modifications introduced includes at least one duplication of a B- lymphocyte epitope and/or the introduction of a hapten. These embodiments including multi- pie presentations of selected epitopes are especially preferred in situations where merely minor parts of the HMGBl polypeptide are useful as constituents in a vaccine agent.

In the following will be provided a description of the characteristic features of preferred immunogens that ultimately confronts the immune system when utilising the method of the present invention.

It has been shown (in Dalum I et al., 1996, J. Immunol. 157: 4796-4804) that potentially self-reactive B-lymphocytes recognizing self-proteins are physiologically present in normal individuals. However, in order for these B-lymphocytes to be induced to actually produce antibodies reactive with the relevant self-proteins, assistance is needed from cytokine producing T-helper lymphocytes (T_H-cells or T_H-lymphocytes). Normally this help is not provided be- cause T-lymphocytes in general do not recognize T-cell epitopes derived from self-proteins when presented by antigen presenting cells (APCs). However, by providing an element of "foreignness" in a self-protein (i.e. by introducing an immunologically significant modification), T-cells recognizing the foreign element are activated upon recognizing the foreign epitope on an APC (such as, initially, a mononuclear cell). Polyclonal B-lymphocytes (which are also specialised APCs) capable of recognising self-epitopes on the modified self-protein also internalise the antigen and subsequently presents the foreign T-cell epitope(s) thereof, and the activated T-lymphocytes subsequently provide cytokine help to these self-reactive polyclonal B-lymphocytes. Since the antibodies produced by these polyclonal B-lymphocytes are reactive with different epitopes on the modified polypeptide, including those which are also present in the native polypeptide, an antibody cross-reactive with the non-modified self-pro- tein is induced. In conclusion, the T-lymphocytes can be led to act as if the population of polyclonal B-lymphocytes have recognised an entirely foreign antigen, whereas in fact only the inserted epitope(s) is/are foreign to the host. In this way, antibodies capable of cross-reacting with non-modified self-antigens are induced.

Several other ways of modifying a peptide self-antigen in order to obtain improved breaking of autotolerance (in addition to the inclusion of foreign T helper epitopes) are known in the art. Hence, according to the invention, the modification can include (optionally in combination with any other relevant modifications of HMGBl discussed herein) that

- at least one first moiety is introduced which effects targeting of the modified molecule to an antigen presenting cell (APC), and/or - at least one second moiety is introduced which stimulates the immune system, and/or

- at least one third moiety is introduced which optimises presentation of the modified HMGBl polypeptide to the immune system.

However, all these modifications should be carried out while ensuring that a substantial fraction of the original B-lymphocyte epitopes in the relevant parts of HMGBl will ultimately confront the immune system, since the B-lymphocyte recognition of the native molecule is thereby enhanced.

It is therefore preferred that the modified HMGBl polypeptide comprises a substantial fragment of at least one of the A and B boxes. In particular, it is preferred that the modified HMGBl polypeptide comprises at least a complete A box or a complete B box.

A "substantial fragment" is in this context meant to denote a fragment (or a mutated version) of a complete A box or complete B box which preserves the 3D structural features of the complete box. Hence, the term allows for non-destructive deletions or substitution of amino acids, notably for conservative substitutions, but the immunologic profile of a substantial fragment and the unmodified A or B box will be the same. This can readily be tested by determining cross-reactivity of a fragment of an A or B box with a polyclonal antiserum raised against the relevant complete A or B box. In the event the reactivity with the polyclonal serum cannot be distinguished from the reactivity between the complete box and the same serum, then the fragment is truly a "substantial fragment" The modified HMGBl polypeptide may comprise substantial fragments of both the A and B boxes, in particular it may comprise complete A and B boxes.

The at least one T_H epitope which is introduced in the preferred modified HMGBl may be introduced into any one of the linker regions of HMGBl. It is preferred that the modified HMGBl polypeptide is provided by introduction of the T_H epitope in the region of HMGBl corresponding to the linker between the A and B boxes or corresponding to the linker, region C-terminally to the B box.

Especially preferred modified HMGBl polypeptides are those, wherein the foreign T_H epitope is introduced - as an insertion in one or both of the above-specified linker regions; or

- as a substitution in one or both the above-specified linker regions, where said substitution may include deletion of any one or all amino acids in said linker regions.

Hence, the modified HMGBl polypeptide may be selected from the group consisting of

- complete A and B boxes separated by an amino acid sequence including the T-helper epitope,

- a complete B box and an N-terminal amino acid sequence including the T-helper epitope and no substantial fragments of the A box, and

- a complete B box and a C-terminal amino acid sequence including the T-helper epitope and no substantial fragments of the A box, wherein the modified HMGBl polypeptide may include amino acids from the linker regions (meaning that it is inessential whether or not amino acids from the linker regions are present).

As mentioned above, the introduction of a foreign T-cell epitope can be accomplished by introduction of at least one amino acid insertion, addition, deletion, or substitution. Of course, the normal situation will be the introduction of more than one change in the amino acid se- quence (e.g. insertion of or substitution by a complete T-cell epitope) but the important goal to reach is that the analogue, when processed by an antigen presenting cell (APC) such as a macrophage or a dendritic cell, will give rise to such a foreign immunodominant T-cell epitope being presented in context of an MCH Class II molecule on the surface of the APC. Thus, if the amino acid sequence of the HMGBl polypeptide in appropriate positions comprises a number of amino acid residues which can also be found in a foreign T_H epitope then the introduction of a foreign T_H epitope can be accomplished by providing the remaining amino acids of the foreign epitope by means of amino acid insertion, addition, deletion and substitution. In other words, it is not necessary to introduce a complete T_H epitope by insertion or substitution in order to fulfil the purpose of the present invention. The number of amino acid insertions, deletions, substitutions or additions is typically at least 2, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 25 insertions, substitutions, additions or deletions. The number of amino acid insertions, substitutions, additions or deletions does normally not exceed 150, so that at most 100, at most 90, at most 80, and at most 70 changes are introduced. The number of substitutions, insertions, deletions, or additions does in some embodiments not exceed 60, and in particular the number does not exceed 50 or even 40 in these embodiment. In certain embodiments, the number is not more than 30. With respect to amino acid additions, it should be noted that these, when the resulting construct is in the form of a fusion polypeptide, is often considerably higher than 150.

Some embodiments of the invention include modification by introducing at least one foreign immunodominant T-cell epitope. It will be understood that the question of immune dominance of a T-cell epitope depends on the animal species in question. As used herein, the term "immunodominance" simply refers to epitopes which in the vaccinated individual/population gives rise to a significant immune response, but it is a well-known fact that a T-cell epitope which is immunodominant in one individual/population is not necessarily immunodominant in another individual of the same species, even though it may be capable of binding MHC-II molecules in the latter individual. Hence, for the purposes of the present invention, an immune dominant T-cell epitope is a T-cell epitope which will be effective in providing T-cell help when present in an antigen. Typically, immune dominant T-cell epitopes has as an inherent feature that they will substantially always be presented bound to an MHC Class II molecule, irrespective of the polypeptide wherein they appear.

Another important point is the issue of MHC restriction of T-cell epitopes. In general, naturally occurring T-cell epitopes are MHC restricted, i.e. a certain peptides constituting a T-cell epitope will only bind effectively to a subset of MHC Class II molecules. This in turn has the effect that in most cases the use of one specific T-cell epitope will result in a vaccine component which is only effective in a fraction of the population, and depending on the size of that fraction, it can be necessary to include more T-cell epitopes in the same molecule, or alternatively prepare a multi-component vaccine wherein the components are variants of the HMGBl polypeptide which are distinguished from each other by the nature of the T-cell epitope introduced.

If the MHC restriction of the T-cells used is completely unknown (for instance in a situation where the vaccinated animal has a poorly defined MHC composition), the fraction of the population covered by a specific vaccine composition can be determined by means of the fol- lowing formula

-where p, is the frequency in the population of responders to the /^th foreign T-cell epitope present in the vaccine composition, and n is the total number of foreign T-cell epitopes in the vaccine composition. Thus, a vaccine composition containing 3 foreign T-cell epitopes having response frequencies in the population of 0.8, 0.7, and 0.6, respectively, would give

1 - 0.2 x 0.3 x 0.4 = 0.976

-i.e. 97.6 percent of the population will statistically mount an MHC-II mediated response to the vaccine.

The above formula does not apply in situations where a more or less precise MHC restriction pattern of the peptides used is known. If, for instance a certain peptide only binds the human MHC-II molecules encoded by HLA-DR alleles DRl, DR3, DR5, and DR7, then the use of this peptide together with another peptide which binds the remaining MHC-II molecules encoded by HLA-DR alleles will accomplish 100% coverage in the population in question. Likewise, if the second peptide only binds DR3 and DR5, the addition of this peptide will not increase the coverage at all. If one bases the calculation of population response purely on MHC restriction of T-cell epitopes in the vaccine, the fraction of the population covered by a specific vaccine composition can be determined by means of the following formula:

-wherein ψ_j is the sum of frequencies in the population of allelic haplotypes encoding MHC molecules which bind any one of the T-cell epitopes in the vaccine and which belong to the/^h of the 3 known HLA loci (DP, DR and DQ); in practice, it is first determined which MHC molecules will recognize each T-cell epitope in the vaccine and thereafter these are listed by type (DP, DR and DQ) - then, the individual frequencies of the different listed allelic haplotypes are summed for each type, thereby yielding φ_lf φ_2l and φ₃.

It may occur that the value p,- in formula II exceeds the corresponding theoretical value π,:

-wherein U₇- is the sum of. frequencies in the population of allelic haplotype encoding MHC molecules which bind the /^th T-cell epitope in the vaccine and which belong to the/^h of the 3 known HLA loci (DP, DR and DQ). This means that in 1-π,- of the population is a frequency of responders of f_resi__uau ⁼ (Pr^πi)ld~^πi)- Therefore, formula III can be adjusted so as to yield formula V:

w

-where the term 1-ή-_esi_duai-_/ is set to zero if negative. It should be noted that formula V re- quires that all epitopes have been haplotype mapped against identical sets of haplotypes.

Therefore, when selecting T-cell epitopes to be introduced in the HMGBl analogue, it is important to include all knowledge of the epitopes which is available: 1) The frequency of responders in the population to each epitope, 2) MHC restriction data, and 3) frequency in the population of the relevant haplotypes.

There exists a number of naturally occurring "promiscuous" T-cell epitopes which are active in a large proportion of individuals of an animal species or an animal population and these are preferably introduced in the vaccine thereby reducing the need for a very large number of different analogues in the same vaccine.

The promiscuous epitope can according to the invention be a naturally occurring human T-cell epitope such as epitopes from tetanus toxoid (e.g. the P2 and P30 epitopes exemplified herein, cf. SEQ ID NOs: 26 and 27, respectively), diphtheria toxoid, Influenza virus hemaglu- ttinin (HA), and P. falciparum CS antigen.

Over the years a number of other promiscuous T-cell epitopes have been identified. Especially peptides capable of binding a large proportion of HLA-DR molecules encoded by the different HL-A-DR alleles have been identified and these are all possible T-cell epitopes to be introduced in the analogues used according to the present invention. Cf. also the epitopes discussed in the following references which are hereby all incorporated by reference herein: WO 98/23635 (Frazer IH et al., assigned to The University of Queensland); Southwood S et ai, 1998, J. Immunol. 160: 3363-3373; Sinigaglia F et al., 1988, Nature 336: 778-780; Chicz RM et al., 1993, J. Exp. Med 178: 27-47; Hammer J et a/., 1993, Cell 74: 197-203; and FaIk K et al., 1994, Immunogenetics 39: 230-242. The latter reference also deals with HLA-DQ and -DP ligands. All epitopes listed in these 5 references are relevant as candidate natural epitopes to be used in the present invention, as are epitopes which share common motifs with these.

Alternatively, the epitope can be any artificial T-cell epitope which is capable of binding a large proportion of MHC Class II molecules. In this context the pan DR epitope peptides ("PADRE") described in WO 95/07707 and in the corresponding paper Alexander J et al., 1994, Immunity 1: 751-761 (both disclosures are incorporated by reference herein) are interesting candidates for epitopes to be used according to the present invention. It should be noted that the most effective PADRE peptides disclosed in these papers carry D-amino acids in the C- and N-termini in order to improve stability when administered. However, the present invention primarily aims at incorporating the relevant epitopes as part of the modified HMGBl polypeptide, which should then subsequently be broken down enzymatically inside the lysosomal compartment of APCs to allow subsequent presentation in the context of an MHC-II molecule and therefore it is not expedient to incorporate D-amino acids in the epitopes used in the present invention.

One especially preferred PADRE peptide is the one having the amino acid sequence SEQ ID NO: 25 (AKFVAAWTLKAAA) or an immunologically effective subsequence thereof. This and other epitopes having the same lack of MHC restriction are preferred T-cell epitopes which should be present in the analogues used in the inventive method. Such super-promiscuous epitopes will allow for the simplest embodiments of the invention wherein only one single modified HMGBl polypeptide is presented to the vaccinated animal's immune system.

As mentioned above, the modification of the HMGBl polypeptide can also include the introduction of a first moiety which targets the modified HMGBl polypeptide to an APC or a B- lymphocyte. For instance, the first moiety can be a specific binding partner for a B- lymphocyte specific surface antigen or for an APC specific surface antigen. Many such specific surface antigens are known in the art. For instance, the moiety can be a carbohydrate for which there is a receptor on the B-lymphocyte or the APC (e.g. mannan or mannose). Alternatively, the second moiety can be a hapten. Also an antibody fragment which specifically recognizes a surface molecule on APCs or lymphocytes can be used as a first moiety (the surface molecule can e.g. be an FCγ receptor of macrophages and monocytes, such as FCγRI or, alternatively any other specific surface marker such as CD40 or CTLA-4). It should be noted that all these exemplary targeting molecules can be used as part of an adjuvant also, cf. below.

As an alternative or supplement to targeting the modified HMGBl polypeptide to a certain cell type in order to achieve an enhanced immune response, it is possible to increase the level of responsiveness of the immune system by including the above-mentioned second moiety which stimulates the immune system. Typical examples of such second moieties are cytokines, and heat-shock proteins or molecular chaperones, as well as effective parts thereof.

Suitable cytokines to be used according to the invention are those which will normally also function as adjuvants in a vaccine composition, i.e. for instance interferon γ (IFN-γ), interle- ukin 1 (IL-I), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 12 (IL- 12), interleukin 13 (IL-13), interleukin 15 (IL-15), and granulocyte-macrophage colony stimulating factor (GM-CSF); alternatively, the functional part of the cytokine molecule may suffice as the second moiety. With respect to the use of such cytokines as adjuvant substances, cf. the discussion below.

According to the invention, suitable heat-shock proteins or molecular chaperones used as the second moiety can, be HSP70 (heat shock protein 70), HSP90 (heat shock protein 90), HSC70 (heat shock cognate protein 70), GRP94 (also known as gp96, cf. Wearsch PA et al. 1998, Biochemistry 37: 5709-19), and CRT (calreticulin).

Alternatively, the second moiety can be a toxin, such as listeriolycin (LLO), lipid A and heat- labile enterotoxin. Also, a number of mycobacterial derivatives such as MDP (muramyl dipep- tide), CFA (complete Freund's adjuvant) and the trehalose diesters TDM and TDE are interesting possibilities.

Also the possibility of introducing a third moiety which enhances the presentation of the modified HMGBl polypeptide to the immune system is an important embodiment of the invention. The art has shown several examples of this principle. For instance, it is known that the palmitoyl lipidation anchor in the Borrelia burgdorferi protein OspA can be utilised so as to provide self-adjuvating polypeptides (cf. e.g. WO 96/40718) - it seems that the lipidated proteins form up micelle-like structures with a core consisting of the lipidation anchor parts of the polypeptides and the remaining parts of the molecule protruding there from, resulting in multiple presentations of the antigenic determinants. Hence, the use of this and related approaches using different lipidation anchors (e.g. a myristyl group, a myristyl group, a farnesyl group, a geranyl-geranyl group, a GPI-anchor, and an N-acyl diglyceride group) are preferred embodiments of the invention, especially since the provision of such a lipidation anchor in a recombinant^ produced protein is fairly straightforward and merely requires use of e.g. a naturally occurring signal sequence as a fusion partner for the modified HMGBl polypeptide. Another possibility is use of the C3d fragment of complement factor C3 or C3 itself (cf. Dempsey et al., 1996, Science 271, 348-350 and Lou & Kohler, 1998, Nature Biotechnology 16, 458-462).

Another attractive way of presenting multiple copies of epitopic regions is the technology dis- closed in WO 001 '32227 ^', where antigens are presented in ordered, repetitive patterns, thereby giving rise to T-cell independent immunogens that resemble virus capsids. In the context of the present invention, the technology of WO 00/32227 is regarded as application of a specialized adjuvant. The disclosure of WO 00/32227 is hereby incorporated by reference herein. An alternative embodiment of the invention which also results in the preferred pre- sentation of multiple {e.g. at least 2) copies of the important epitopic regions of the HMGBl polypeptide to the immune system is the covalent coupling of polyamino acids selected from the HMGBl polypeptide, the subsequence thereof, or the analogues thereof to certain molecules and, when necessary, together with foreign T_H epitopes or one of the first, second or third moieties discussed above. For instance, polymers can be used, e.g. polyhydroxypoly- mers, notably carbohydrates such as dextran, cf. e.g. Lees A et al., 1994, Vaccine 12: 1160-

1166; Lees A et al., 1990, J Immunol. 145: 3594-3600, but also mannose and mannan are . useful alternatives. Integral membrane proteins from e.g. E. coll and other bacteria are also useful conjugation partners. The traditional carrier molecules such as keyhole limpet hemo- cyanin (KLH), tetanus toxoid, diphtheria toxoid, and bovine serum albumin (BSA) are also preferred and useful conjugation partners.

Alternatively, dimeric and multimeric variants which include at least two copies of a substantially complete or complete B box are also contemplated. These variants may include multiple repeats of complete B boxes and one or more foreign T_H epitopes.

In particular, the invention includes embodiments where the modified HMGBl polypeptide includes duplication of at least a substantial fragment of the B box, such as at least 3, at least 4 at least 5, at least 6, at least 7, at least 8, and at least 9 substantial fragments of the B boxes. These substantial fragments are in some embodiments identical.

In certain embodiments, the modified HMGBl polypeptide includes at least 2 complete B boxes, such as at least 3, at least 4 at least 5, at least 6, at least 7, at least 8, and at least 9 complete B boxes.

The substantial fragments of the B boxes or the complete B boses may be part of a fusion polypeptide, wherein the substantial fragments or complete B boxes are optionally separated by the at least one foreign T helper epitope.

Considerations underlying chosen areas of introducing modifications in HMGBl polypeptides are a) preservation of known and predicted B-cell epitopes in the A and/or B boxes, and b) preservation of 3D structure of the A and/or B boxes.

Since the invention involve immunization against of human HMGBl, it is consequently preferred that the HMGBl polypeptide discussed above is a human HMGBl polypeptide - however, any discussions below of human HMGBl could be used for HMGBl from other species (which, however, are known to be highly homologous to the human sequence). It will then be understood that teachings relating to changes in the human sequence should be - transposed to the relevant sequence in the relevant animal: From the sequence listing it appears where the boundaries for the mature HMGBl peptide sequence can be found, and it will be understood that any specific sequence data referred to in the human sequence should take offset in the corresponding sequences in the various mammalian HMGBl sequences.

Another embodiment of the present invention is the presentation of the HMGBl analogues which do not include any subsequence of HMGBl that binds productively to MHC class II molecules initiating a T-cell response.

The rationale behind such a strategy for design of the immunogen that engages the immune system to induce an anti-HMGBl immune response is the following: It has been noted that when immunizing with autologous proteins formulated in an adjuvant which is sufficiently strong to break the body's tolerance towards the autologous protein, there is a danger that in some vaccinated individuals the immune response induced cannot be discontinued simply by discontinuing the immunisation. This is because the induced immune response in such individuals is most likely driven by a native T_H epitope of the autologous protein, and this has the adverse effect that the vaccinated individual's own protein will be able to function as an immunizing agent in its own right: An autoimmune condition has thus been established.

The preferred methods including use of foreign T_H epitopes have to the best of the inventors' knowledge never been observed to produce this effect, because the anti-self immune response is driven by a foreign T_H epitope, and it has been repeatedly demonstrated by the inventors that the induced immune response invoked by the preferred technology indeed declines after discontinuation of immunizations. However, in theory it could happen in a few individuals that the immune response would also be driven by an autologous T_H epitope of the relevant self-protein one immunises against) - this is especially relevant when considering self-proteins that are relatively abundant, whereas other therapeutically relevant self- proteins are only present locally or in so low amounts in the body, that a "self-immunization effect" is not a possibility; however, for HMGBl, this effect cannot be excluded.

One very simple way of avoiding this self-immunisation is hence to altogether avoid inclusion in the immunogen of peptide sequences that could serve as T_H epitopes (and since peptides shorter than about 9 amino acids cannot serve as T_H epitopes, the use of shorter fragments is one simple and feasible approach). Therefore, this embodiment of the invention also serves to ensure that the immunogen does not include peptide sequences of the target HMGBl that could serve as "self-stimulating T_H epitopes" including sequences that merely contain conservative substitutions in a sequence of the target protein that might otherwise function as a T_H epitope.

Preferred embodiments of the immune system presentation of the analogues of HMGBl involve the use of a chimeric peptide comprising at least one HMGBl derived peptide, which does not bind productively to MHC class II molecules, and at least one foreign T-helper epitope. Moreover, it is preferred that the HMGBl derived peptide harbours a B-cell epitope. It is especially advantageous if the immunogenic analogue is one, wherein the amino acid sequences comprising one or more B-cell epitopes are represented either as a continuous se- quence or as a sequence including inserts, wherein the inserts comprise foreign T-helper epitopes.

Again, such an embodiment is most preferred when the suitable B-cell epitope carrying regions of HMGBl are constituted by short peptide stretches that in no way would be able to bind productively to an MHC Class II molecule. The selected B-cell epitope or -epitopes of HMGBl should therefore comprise at most 9 consecutive amino acids of hHMGBl. Shorter peptides are preferred, such as those having at most 8, 7, 6, 5, 4, or 3 consecutive amino acids from the hHMGBl amino acid sequence.

It is preferred that the analogue comprises at least one subsequence of HMGBl so that each such at least one subsequence independently consists of amino acid stretches from HMGBl selected from the group consisting of 9 consecutive amino acids, 8 consecutive amino acids, 7 consecutive amino acids, 6 consecutive amino acids, 5 consecutive amino acids, 4 consecutive amino acids, and 3 consecutive amino acids.

In the most preferred embodiments, the modified HMGBl polypeptide comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 5-24, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, and an amino acid sequence set forth in Table 1 herein. The polypeptide may be encoded by a nucleic acid sequence selected from any one SEQ ID NOs: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, and 76.

Polypeptide and protein vaccination

In one embodiment of the therapeutic method of the invention, unmodified or modified HMGBl polypeptide is formulated together with a pharmaceutically and immunologically acceptable carrier and/or vehicle and, optionally an adjuvant and can subsequently be administered to the patient in need thereof.

When effecting presentation of the HMGBl polypeptide or the modified HMGBl polypeptide to an animal's (in this case to a human's) immune system by means of administration of an immunogenically effective amount thereof, the formulation of the polypeptide follows the principles generally acknowledged in the art. Preparation of vaccines which contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorporated herein by reference. Typically, such vaccines are prepared as injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines; cf. the detailed discussion of adjuvants below.

The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously, intracutaneously, or intramuscularly. Additional formulations which are suit- able for other modes of administration include suppositories and, in some cases, oral, buccal, sublinqual, intraperitoneal, intravaginal, anal, epidural, spinal, and intracranial formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredient, preferably 25-70%. For oral formulations, cholera toxin is an interesting formulation partner (and also a possible conjugation partner).

The polypeptides may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to mount an immune response, and the degree of protection desired. Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 0.1 μg to 2,000 μg (even though higher amounts in the 1-10 mg range are contemplated), such as in the range from about 0.5 μg to 2,000 μg or 0.5 μg to 1,000 μg, preferably in the range from 1 μg to 500 μg and especially in the range from about 10 μg to 100 μg. Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.

The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the age of the person to be vaccinated and the formulation of the antigen.

Some of the polypeptides of the vaccine are sufficiently immunogenic in a vaccine, but for some of the others the immune response will be enhanced if the vaccine further comprises an adjuvant substance.

Various methods of achieving adjuvant effect for the vaccine are known. General principles and methods are detailed in "The Theory and Practical Application of Adjuvants", 1995, Duncan E.S. Stewart-Tull (ed.), John Wiley & Sons Ltd, ISBN 0-471-95170-6, and also in "Vac- cines: New Generation Immunological Adjuvants", 1995, Gregoriadis G et al. (eds.), Plenum Press, New York, ISBN 0-306-45283-9, both of which are hereby incorporated by reference herein.

It is especially preferred to use an adjuvant which can be demonstrated to facilitate breaking of the autotolerance to autoantigens; in fact, this is essential in cases where unmodified HMGBl is used as the active ingredient in the autovaccine. Non-limiting examples of suitable adjuvants are selected from the group consisting of an immune targeting adjuvant; an immune modulating adjuvant such as a toxin, a cytokine, and a mycobacterial derivative; an oil formulation; a polymer; a micelle forming adjuvant; a saponin; an immunostimulating complex matrix (ISCOM matrix); a particle; DDA; aluminium adjuvants; DNA adjuvants; γ-inulin; and an encapsulating adjuvant. In general it should be noted that the disclosures above which relate to compounds and agents useful as first, second and third moieties in the analogues also refer mutatis mutandis to their use in the adjuvant of a vaccine of the invention.

The application of adjuvants include use of agents such as aluminium hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in buffered saline, admixture with synthetic polymers of sugars (e.g. Carbopol®) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70° to 101⁰C for 30 second to 2 minute periods respectively and also aggregation by means of cross-linking agents are possible. Aggregation by reactivation with pepsin treated antibodies (Fab fragments) to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed. Admixture with oils such as squalene and IFA is also preferred.

According to the invention DDA (dimethyldioctadecylammonium bromide) is an interesting candidate for an adjuvant as is DNA and γ-inulin, but also Freund's complete and incomplete adjuvants as well as quillaja saponins such as QuilA and QS21 are interesting as is RIBI. Further possibilities are monophosphoryl lipid A (MPL), the above mentioned C3 and C3d, and muramyl dipeptide (MDP).

Liposome formulations are also known to confer adjuvant effects, and therefore liposome adjuvants are preferred according to the invention.

Also immunostimulating complex matrix type (ISCOM® matrix) adjuvants are preferred choices according to the invention, especially since it has been shown that this type of adjuvants are capable of up-regulating MHC Class II expression by APCs. An ISCOM® matrix con- sists of (optionally fractionated) saponins (triterpenoids) from Quillaja saponaria, cholesterol, and phospholipid. When admixed with the immunogenic protein, the resulting particulate formulation is what is known as an ISCOM particle where the saponin constitutes 60-70% w/w, the cholesterol and phospholipid 10-15% w/w, and the protein 10-15% w/w. Details relating to composition and use of immunostimulating complexes can e.g. be found in the above- mentioned text-books dealing with adjuvants, but also Morein B et al., 1995, Clin. Immuno- ther. 3: 461-475 as well as Barr IG and Mitchell GF, 1996, Immunol, and Cell Biol. 74: 8-25 (both incorporated by reference herein) provide useful instructions for the preparation of complete immunostimulating complexes.

Another highly interesting (and thus, preferred) possibility of achieving adjuvant effect is to employ the technique described in Gosselin et al., 1992 (which is hereby incorporated by reference herein). In brief, the presentation of a relevant antigen such as an antigen of the present invention can be enhanced by conjugating the antigen to antibodies (or antigen binding antibody fragments) against the Fey receptors on monocytes/macrophages. Especially conjugates between antigen and anti-FcyRI have been demonstrated to enhance immuno- genicity for the purposes of vaccination. Other possibilities involve the use of the targeting and immune modulating substances (/.a. cytokines) mentioned above as candidates for the first and second moieties in the modified versions of HMGBl. In this connection, also synthetic inducers of cytokines like poly I:C are possibilities.

Suitable mycobacterial derivatives are selected from the group consisting of muramyl dipep- tide, complete Freund's adjuvant, RIBI, and a diester of trehalose such as TDM and TDE.

Suitable immune targeting adjuvants are selected from the group consisting of CD40 ligand and CD40 antibodies or specifically binding fragments thereof (cf. the discussion above), mannose, a Fab fragment, and CTLA-4.

Suitable polymer adjuvants are selected from the group consisting of a carbohydrate such as dextran, PEG, starch, mannan, and mannose; a plastic polymer such as; and latex such as latex beads.

Yet another interesting way of modulating an immune response is to include the HMGBl im- munogen (optionally together with adjuvants and pharmaceutically acceptable carriers and vehicles) in a "virtual lymph node" (VLN) (a proprietary medical device developed by Immu- noTherapy, Inc., 360 Lexington Avenue, New York, NY 10017-6501). The VLN (a thin tubular device) mimics the structure and function of a lymph node. Insertion of a VLN under the skin creates a site of sterile inflammation with an upsurge of cytokines and chemokines. T- and B- cells as well as APCs rapidly respond to the danger signals, home to the inflamed site and accumulate inside the porous matrix of the VLN. It has been shown that the necessary antigen dose required to mount an immune response to an antigen is reduced when using the VLN and that immune protection conferred by vaccination using a VLN surpassed conventional immunization using Ribi as an adjuvant. The technology is i.a. described briefly in GeI- ber C et a/., 1998, "Elicitation of Robust Cellular and Humoral Immune Responses to Small Amounts of Immunogens Using a Novel Medical Device Designated the Virtual Lymph Node", in: "From the Laboratory to the Clinic, Book of Abstracts, October 12^th - 15^th 1998, Seascape Resort, Aptos, California".

Microparticle formulation of vaccines has been shown in many cases to increase the immuno- genicity of protein antigens and is therefore another preferred embodiment of the invention. Microparticles are made either as co-formulations of antigen with a polymer, a lipid, a carbohydrate or other molecules suitable for making the particles or the microparticles can be homogeneous particles consisting of only the antigen itself. Examples of polymer based microparticles are PLGA and PVP based particles (Gupta RK et al., 1998) where the polymer and the antigen are condensed into a solid particle. Lipid based particles can be made as micelles of the lipid (so-called liposomes) entrapping the antigen within the micelle (Pietrobon PJ, 1995). Carbohydrate based particles, are typically made of a suitable degradable carbohydrate such as starch or chitosan. The carbohydrate and the antigen are mixed and condensed into particles in a process similar to the one used for polymer particles (Kas HS et a/., 1997).

Particles consisting only of the antigen can be made by various spraying, crystallization and freeze-drying techniques. Especially suited for the purposes of the present invention is the super critical fluid technology that is used to make very uniform particles of controlled size (York P, 1999 & Shekunov B ef al., 1999).

It is expected that the vaccine should be administered at least once a year, such as at least 1, 2, 3, 4, 5, 6, and 12 times a year. More specifically, 1-12 times per year is expected, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times a year to an individual in need thereof. It has previously been shown that the memory immunity induced by the use of the preferred auto- vaccines according to the invention is not permanent, and therefore the immune system needs to be periodically challenged with the analogues.

Due to genetic variation, different individuals may react with immune responses of varying strength to the same polypeptide. Therefore, the vaccine according to the invention may comprise several different polypeptides in order to increase the immune response, cf. also the discussion above concerning the choice of foreign T-cell epitope introductions. The vaccine may comprise two or more polypeptides, where all of the polypeptides are as defined above.

The vaccine may consequently comprise 3-20 different modified or unmodified polypeptides, such as 3-10 different polypeptides. However, normally the number of polypeptides will be sought kept to a minimum such as 1 or 2 polypeptides.

As an alternative to the HMGBl analogues of the invention, it is also possible to immunize by using anti-idiotypic antibodies or even mimotopes. The technologies for preparing anti-idio- typic antibodies that mimic an HMGBl epitope are known in the art, but one especially inter- esting version involves use of autologous anti-idiotypic antibodies, which are reactive with an anti-HMGBl antibody and which are modified by introduction of a foreign T helper epitope as generally described herein. Mimotopes can be isolated from libraries of random peptides that are screened in phage display against antibodies that bind HMGBl specifically. Nucleic acid vaccination

As an alternative to classic administration of a peptide-based vaccine, the technology of nucleic acid vaccination (also known as "nucleic acid immunisation", "genetic immunisation", and "gene immunisation") offers a number of attractive features.

First, in contrast to the traditional vaccine approach, nucleic acid vaccination does not require resource consuming large-scale production of the immunogenic agent (e.g. in the form of industrial scale fermentation of microorganisms producing modified HMGBl). Furthermore, there is no need to device purification and refolding schemes for the immunogen. And finally, since nucleic acid vaccination relies on the biochemical apparatus of the vaccinated individual in order to produce the expression product of the nucleic acid introduced, the optimum post- translational processing of the expression product is expected to occur; this is especially important in the case of autovaccination, since, as mentioned above, a significant fraction of the original HMGBl B-cell epitopes should be preserved in the modified molecule, and since B-cell epitopes in principle can be constituted by parts of any (bio)molecule (e.g. carbohydrate, lipid, protein etc.). Therefore, native glycosylation and lipidation patterns of the immunogen may very well be of importance for the overall immunogenicity and this is expected to be ensured by having the host producing the immunogen.

Hence, a preferred embodiment of the invention comprises effecting presentation of modified HMGBl to the immune system by introducing nucleic acid(s) encoding the modified HMGBl into the animal's cells and thereby obtaining in vivo expression by the cells of the nucleic acid(s) introduced.

In this embodiment, the introduced nucleic acid is preferably DNA which can be in the form of naked DNA, DNA formulated with charged or uncharged lipids, DNA formulated in liposomes, DNA included in a viral vector, DNA formulated with a transfection-facilitating protein or poly- peptide, DNA formulated with a targeting protein or polypeptide, DNA formulated with Calcium precipitating agents, DNA coupled to an inert carrier molecule, DNA encapsulated in a polymer, e.g. in PLGA (cf. the microencapsulation technology described in WO 98/31398) or in chitin or chitosan, and DNA formulated with an adjuvant. In this context it is noted that practically all considerations pertaining to the use of adjuvants in traditional vaccine formula- tion apply for the formulation of DNA vaccines. Hence, all disclosures herein which relate to use of adjuvants in the context of polypeptide based vaccines apply mutatis mutandis to their use in nucleic acid vaccination technology.

As for routes of administration and administration schemes of polypeptide based vaccines which have been detailed above, these are also applicable for the nucleic acid vaccines of the invention and all discussions above pertaining to routes of administration and administration schemes for polypeptides apply mutatis mutandis to nucleic acids. To this should be added that nucleic acid vaccines can suitably be administered intraveneously and intraarterially. Furthermore, it is well-known in the art that nucleic acid vaccines can be administered by use of a so-called gene gun, and hence also this and equivalent modes of administration are regarded as part of the present invention. Finally, also the use of a VLN in the administration of nucleic acids has been reported to yield good results, and therefore this particular mode of administration is particularly preferred.

Furthermore, the nucleic acid(s) used as an immunization agent can contain regions encoding the above-discussed 1^st, 2^nd and/or 3^rd moieties, e.g. in the form of the immunomodulating substances described above such as the cytokines discussed as useful adjuvants. A preferred version of this embodiment encompasses having the coding region for the analogue and the coding region for the immunomodulator in different reading frames or at least under the control of different promoters. Thereby it is avoided that the analogue or epitope is produced as a fusion partner to the immunomodulator. Alternatively, two distinct nucleotide fragments can be used, but this is less preferred because of the advantage of ensured co-expression when having both coding regions included in the same molecule.

Under normal circumstances, the HMGBl variant-encoding nucleic acid is introduced in the form of a vector wherein expression is under control of a viral promoter. For more detailed discussions of vectors and DNA fragments according to the invention, cf. the discussion below. Also, detailed disclosures relating to the formulation and use of nucleic acid vaccines are available, cf. Donnelly JJ et al, 1997, Annu. Rev. Immunol. 15: 617-648 and Donnelly JJ et al., 1997, Life Sciences 60: 163-172. Both of these references are incorporated by reference herein.

Live and viral vaccines

A third alternative for effecting presentation of modified HMGBl to the immune system is the use of live vaccine technology. In live vaccination, presentation to the immune system is effected by administering, to the animal, a non-pathogenic microorganism which has been transformed with a nucleic acid fragment encoding a modified HMGBl or with a vector incor- porating such a nucleic acid fragment. The non-pathogenic microorganism can be any suitable attenuated bacterial strain (attenuated by means of passaging or by means of removal of pathogenic expression products by recombinant DNA technology), e.g. Mycobacterium bovis BCG., non-pathogenic Streptococcus spp., E. coli, Salmonella spp., Vibrio cholerae, Shigella, etc. Reviews dealing with preparation of state-of-the-art live vaccines can e.g. be found in Saliou P, 1995, Rev. Prat. 45: 1492-1496 and Walker PD, 1992, Vaccine 10: 977-990, both incorporated by reference herein. For details about the nucleic acid fragments and vectors used in such live vaccines, cf. the discussion below.

As an alternative to bacterial live vaccines, the nucleic acid fragment of the invention discussed below can be incorporated in a non-virulent viral vaccine vector such as a vaccinia strain (e.g. in a modified vaccinia Ankara, MVA) or any other suitable pox virus.

Normally, the non-pathogenic microorganism or virus is administered only once to the animal, but in certain cases it may be necessary to administer the microorganism more than once in a lifetime in order to maintain protective immunity. It is even contemplated that immunization schemes as those detailed above for polypeptide vaccination will be useful when using live or virus vaccines.

Alternatively, live or virus vaccination is combined with previous or subsequent polypeptide and/or nucleic acid vaccination. For instance, it is possible to effect primary immunization with a live or virus vaccine followed by subsequent booster immunizations using the polypeptide or nucleic acid approach.

The microorganism or virus can be transformed with nucleic acid(s) containing regions encoding the 1^st, 2^nd and/or 3^rd moieties, e.g. in the form of the immunomodulating substances described above such as the cytokines discussed as useful adjuvants. A preferred version of this embodiment encompasses having the coding region for the analogue and the coding region for the immunomodulator in different reading frames or at least under the control of dif- ferent promoters. Thereby it is avoided that the analogue or epitopes are produced as fusion partners to the immunomodulator. Alternatively, two distinct nucleotide fragments can be used as transforming agents. Of course, having the 1^st and/or 2^nd and/or 3^rd moieties in the same reading frame can provide as an expression product, an analogue of the invention, and such an embodiment is especially preferred according to the present invention.

Peptides, polypeptides, and compositions of the invention

As will be apparent from the above, the present invention is based on the concept of immunising individuals against the HMGBl antigen. The preferred way of obtaining such an immunization is to use modified versions of HMGBl, thereby providing molecules which have not previously been disclosed in the art.

It is believed that the modified HMGBl molecules discussed herein are inventive in their own right, and therefore an important part of the invention pertains to an HMGBl analogue which is derived from an animal HMGBl wherein is introduced a modification which has as a result that immunization of the animal with the analogue induces production of antibodies cross- reacting with the unmodified HMGBl polypeptide. Preferably, the nature of the modification conforms with the types of modifications described above when discussing various embodiments of the method of the invention when using modified HMGBl. Hence, any disclosure presented herein pertaining to modified HMGBl molecules are relevant for the purpose of describing the HMGBl analogues of the invention, and any such disclosures apply mutatis mutandis to the description of these analogues.

It should be noted that preferred modified HMGBl molecules comprise modifications which results in a polypeptide having a sequence identity of at least 70% with HMGBl or with a subsequence thereof of at least 10 amino acids in length. Higher sequence identities are preferred, e.g. at least 75% or even at least 80% or 85%. The sequence identity for proteins and nucleic acids can be calculated as {N_ref - N_di_f)-100/N_ref, wherein Λ/_d/f is the total number of non-identical residues in the two sequences when aligned and wherein N_ref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (N_dif=2 and /V_ref=8). It is especially preferred that the sequence identity relative to the A and B boxes is very high, typically above 90% (above 91, 92, 93, 94, 95, 96, 97 and 98 percent) and as mentioned above, up to 100%.

The invention also pertains to compositions useful in exercising the method of the invention. Hence, the invention also relates to an immunogenic composition comprising an immunogeni- cally effective amount of an HMGBl polypeptide which is a self-protein in an animal or a subsequence of such an HMGBl polypeptide, said HMGBl polypeptide or subsequence being formulated together with an immunologically acceptable adjuvant so as to break the animal's autotolerance towards the HMGBl polypeptide, the composition further comprising a pharmaceutically and immunologically acceptable vehicle and/or carrier. In other words, this part of the invention pertains to the formulations of naturally occurring HMGBl polypep- tides/subsequences which have been described in connection with embodiments of the method of the invention.

The invention also relates to an immunogenic composition comprising an immunologically effective amount of an HMGBl analogue defined above, said composition further comprising a pharmaceutically and immunologically acceptable diluent and/or vehicle and/or carrier and/or excipient and optionally an adjuvant. In other words, this part of the invention concerns formulations of modified HMGBl, essentially as described hereinabove. The choice of adjuvants, carriers, and vehicles is accordingly in line with what has been discussed above when referring to formulation of modified and unmodified HMGBl for use in the inventive method for the immunizing against autologous HMGBl. The polypeptides are prepared according to methods well-known in the art. Longer polypeptides are normally prepared by means of recombinant gene technology including introduction of a nucleic acid sequence encoding the HMGBl analogue into a suitable vector, transformation of a suitable host cell with the vector, expression of the nucleic acid sequence, recovery of the expression product from the host cells or their culture supernatant, and subsequent purification and optional further modification, e.g. refolding or derivatization.

Shorter peptides are preferably prepared by means of the well-known techniques of solid- or liquid-phase peptide synthesis. However, recent advances in this technology has rendered possible the production of full-length polypeptides and proteins by these means, and there- fore it is also within the scope of the present invention to prepare the long constructs by synthetic means.

Nucleic acid fragments and vectors of the invention

It will be appreciated from the above disclosure that modified HMGBl polypeptides can be prepared by means of recombinant gene technology but also by means of chemical synthesis or semisynthesis; the latter two options are especially relevant when the modification consists in coupling to protein carriers (such as KLH, diphtheria toxoid, tetanus toxoid, and BSA) and non-proteinaceous molecules such as carbohydrate polymers and of course also when the modification comprises addition of side chains or side groups to an HMGBl polypeptide-derived peptide chain.

For the purpose of recombinant gene technology, and of course also for the purpose of nucleic acid immunization, nucleic acid fragments encoding modified HMGBl are important chemical products. Hence, an important part of the invention pertains to a nucleic acid fragment which encodes an HMGBl analogue, i.e. an HMGBl derived polypeptide which either comprises the natural HMGBl sequence to which has been added or inserted a fusion partner or, preferably an HMGBl derived polypeptide wherein has been introduced a foreign T-cell epitope by means of insertion and/or addition, preferably by means of substitution and/or deletion. The nucleic acid fragments of the invention are either DNA or RNA fragments.

The nucleic acid fragments of the invention will normally be inserted in suitable vectors to form cloning or expression vectors carrying the nucleic acid fragments of the invention; such novel vectors are also part of the invention. Details concerning the construction of these vectors of the invention will be discussed in context of transformed cells and microorganisms below. The vectors can, depending on purpose and type of application, be in the form of plasmids, phages, cosmids, mini-chromosomes, or virus, but also naked DNA which is only expressed transiently in certain cells is an important vector. Preferred cloning and expression vectors of the invention are capable of autonomous replication, thereby enabling high copy- numbers for the purposes of high-level expression or high-level replication for subsequent cloning.

The general outline of a vector of the invention comprises the following features in the 5'-→-3' direction and in operable linkage: a promoter for driving expression of the nucleic acid fragment of the invention, optionally a nucleic acid sequence encoding a leader peptide enabling secretion (to the extracellular phase or, where applicable, into the periplasma) of or integration into the membrane of the polypeptide fragment, the nucleic acid fragment of the inven- tion, and optionally a nucleic acid sequence encoding a terminator. When operating with expression vectors in producer strains or cell-lines it is for the purposes of genetic stability of the transformed cell preferred that the vector when introduced into a host cell is integrated in the host cell genome. In contrast, when working with vectors to be used for effecting in vivo expression in an animal {i.e. when using the vector in DNA vaccination) it is for security rea- sons preferred that the vector is not incapable of being integrated in the host cell genome; typically, naked DNA or non-integrating viral vectors are used, the choices of which are well- known to the person skilled in the art.

The vectors of the invention are used to transform host cells to produce the modified HMGBl polypeptide of the invention. Such transformed cells, which are also part of the invention, can be cultured cells or cell lines used for propagation of the nucleic acid fragments and vectors of the invention, or used for recombinant production of the modified HMGBl polypeptides of the invention. Alternatively, the transformed cells can be suitable live vaccine strains wherein the nucleic acid fragment (one single or multiple copies) have been inserted so as to effect secretion or integration into the bacterial membrane or cell-wall of the modified HMGBl.

Preferred transformed cells of the invention are microorganisms such as bacteria (such as the species Escherichia [e.g. E. coli], Bacillus [e.g. Bacillus subtilis], Salmonella, or Mycobacterium [preferably non-pathogenic, e.g. M. bovis BCG]), yeasts (such as Saccharomyces cere- visiae), and protozoans. Alternatively, the transformed cells are derived from a multicellular organism such as a fungus, an insect cell, a plant cell, or a mammalian cell. Most preferred are cells derived from a human being, cf. the discussion of cell lines and vectors below. Recent results have shown great promise in the use of a commercially available Drosophila melanogaster cell line (the Schneider 2 (S₂)cell line and vector system available from Invitro- gen) for the recombinant production of HMGBl analogues of the invention, and therefore this expression system is particularly preferred. Also the spodoptera cells (SF cells) SF9 and SF21 are preferred. For the purposes of cloning and/or optimised expression it is preferred that the transformed cell is capable of replicating the nucleic acid fragment of the invention. Cells expressing the nucleic fragment are preferred useful embodiments of the invention; they can be used for small-scale or large-scale preparation of the modified HMGBl or, in the case of non-patho- genie bacteria, as vaccine constituents in a live vaccine.

When producing the modified HMGBl of the invention by means of transformed cells, it is convenient, although far from essential, that the expression product is either exported out into the culture medium or carried on the surface of the transformed cell.

When an effective producer cell has been identified it is preferred, on the basis thereof, to establish a stable cell line which carries the vector of the invention and which expresses the nucleic acid fragment encoding the modified HMGBl. Preferably, this stable cell line secretes or carries the HMGBl analogue of the invention, thereby facilitating purification thereof.

In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with the hosts. The vector ordi- narily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (see, e.g., Bolivar et al., 1977). The pBR322 plasmid contains genes for ampiciliin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the pro- karyotic microorganism for expression.

Those promoters most commonly used in prokaryotic recombinant DNA construction include the B-lactamase (penicillinase) and lactose promoter systems (Chang et al., 1978; Itakura et al., 1977; Goeddel et al., 1979) and a tryptophan (trp) promoter system (Goeddel et a/., 1979; EP-A-O 036 776). While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plasmid vectors (Siebwenlist et al., 1980). Certain genes from prokaryotes may be expressed efficiently in E. coli from their own promoter sequences, precluding the need for addition of another pro- moter by artificial means.

In addition to prokaryotes, eukaryotic microbes, such as yeast cultures may also be used, and here the promoter should be capable of driving expression. Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al., 1980). This plasmid already contains the trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan for example ATCC No. 44076 or PEP4-1 (Jones, 1977). The presence of the trpl lesion as a charac- teristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzman et al., 1980) or other glycolytic enzymes (Hess et al., 1968; Holland et al., 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and gluco- kinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.

Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing a yeast-compatible promoter, origin of replication and termination sequences is suitable.

In addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate in culture (tissue culture) has become a routine procedure in recent years (Tis- sue Culture, 1973). Examples of such useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7 293, Spodoptera frugiperda (SF) cells (commercially available as complete expression systems from La. Protein Sciences, 1000 Research Parkway, Meriden, CT 06450, U.S.A. and from Invitrogen), and MDCK cell lines. In the present invention, an especially preferred cell line is S₂ available from Invitro- gen, PO Box 2312, 9704 CH Groningen, The Netherlands.

Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences. For use in mammalian cells, the control functions on the expression vectors are often provided by viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a frag- ment which also contains the SV40 viral origin of replication (Fiers et al., 1978). Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the Hindlϊl site toward the BgII site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequen- ces are compatible with the host cell systems.

An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

Accordingly, since the nucleic acids of the invention may be immunogens in their own right, the invention also relates to a composition for inducing production of antibodies against

HMGBl, the composition comprising

- a nucleic acid fragment or a vector of the invention (cf. the discussion of vectors above), and - a pharmaceutically and immunologically acceptable vehicle and/or carrier and/or adjuvant as discussed above.

Identification of useful HMGBl analogues

It will be clear to the skilled person that not all variants or modifications of native HMGBl will have the ability to elicit antibodies in an animal which are cross-reactive with the native form. It is, however, not difficult to set up an effective standard screen for modified HMGBl molecules which fulfil the minimum requirements for immunological reactivity discussed herein. Hence, another part of the invention concerns a method for the identification of a modified HMGBl polypeptide which is capable of inducing antibodies against unmodified HMGBl in an animal species where the unmodified HMGBl polypeptide is a self-protein, the method comprising preparing, by means of peptide synthesis or by molecular biological means, a set of mutually distinct modified HMGBl polypeptides wherein amino acids have been added to, inserted in, deleted from, or substituted into the amino acid sequence of an HMGBl polypeptide of the animal species thereby giving rise to amino acid sequences in the set which comprise T-cell epitopes which are foreign to the animal species, or preparing a set of nucleic acid fragments encoding the set of mutually distinct modified HMGBl polypeptides, testing members of the set for their ability to induce production of antibodies by the animal species against the unmodified HMGBl, and identifying and optionally isolating the member(s) of the set which significantly induces antibody production against unmodified HMGBl in the animal species, or identifying and optionally isolating the polypeptide expression products encoded by members of the set of nucleic acid fragments which significantly induces antibody production against unmodified HMGBl polypeptide. in the animal species.

In this context, the "set of mutually distinct modified HMGBl polypeptides" is a collection of non-identical modified HMGBl polypeptides which have e.g. been selected on the basis of the criteria discussed above {e.g. in combination with studies of circular dichroism, NMR spectra, and/or HMGBl-ray diffraction patterns). The set may consist of only a few members but it is contemplated that the set may contain several hundred members. Likewise, the set of nucleic acid fragments is a collection of non-identical nucleic acid fragments, each encoding a modified HMGBl polypeptide selected in the same manner.

The test of members of the set can ultimately be performed in vivo, but a number of in vitro tests can be applied which narrow down the number of modified molecules which will serve the purpose of the invention.

Since the goal of introducing the foreign T-cell epitopes is to support the B-cell response by T-cell help, a prerequisite is that T-cell proliferation is induced by the modified HMGBl. T-cell proliferation can be tested by standardized proliferation assays in vitro. In short, a sample enriched for T-cells is obtained from a subject and subsequently kept in culture. The cultured T-cells are contacted with APCs of the subject which have previously taken up the modified molecule and processed it to present its T-cell epitopes. The proliferation of T-cells is monitored and compared to a suitable control (e.g. T-cells in culture contacted with APCs which have processed intact, native HMGBl). Alternatively, proliferation can be measured by determining the concentration of relevant cytokines released by the T-cells in response to their recognition of foreign T-cells.

Having rendered highly probable that at least one modified HMGBl of the set is capable of inducing antibody production against HMGBl, it is possible to prepare an immunogenic com- position comprising at least one modified HMGBl polypeptide which is capable of inducing antibodies against unmodified HMGBl in an animal species where the unmodified HMGBl polypeptide is a self-protein, the method comprising admixing the member(s) of the set which significantly induces production of antibodies in the animal species which are reactive with HMGBl with a pharmaceutically and immunologically acceptable carrier and/or vehicle and/or diluent and/or excipient, optionally in combination with at least one pharmaceutically and immunologically acceptable adjuvant.

Likewise, it is also possible to prepare an immunogenic composition which as an immunogen contains a nucleic acid fragment encoding an immunogenic HMGBl analogue, cf. the discus- sion of nucleic acid vaccination above.

The above aspects of the invention are conveniently carried out by initially preparing a number of mutually distinct nucleic acid sequences or vectors of the invention, inserting these into appropriate expression vectors, transforming suitable host cells with the vectors, and expressing the nucleic acid sequences of the invention. These steps can be followed by isola- tion of the expression products. It is preferred that the nucleic acid sequences and/or vectors are prepared by methods comprising exercise of a molecular amplification technique such as PCR or by means of nucleic acid synthesis.

EXAMPLE 1

Molecule Design

The full-length HMGBl protein has previously been reported to be toxic for host cells during expression. It has been shown that peptides shorter than one of the DNA binding boxes are not expressed in either E. coli or cell-free systems (Bianchi, M. E., Falciola, L., Ferrari, S., and Lilley, D. M. (1992) EMBO J. 11, 1055-1063). The biological function of HMGBl has been assigned to the B box and the A box has been described as antagonist to HMGBl and B box function. Therefore, the B box is the shortest domain used as template for expression. Both the A and B boxes aggregate when expressed with C-terminal linkers and have a tendency to form dimers during purification, probably through the unpaired cysteines. The dinners are, however, not affected by reduction, indicating that a more complex dimerisation event than a reducable cysteine bridge has occurred or that the cysteines are not at all involved in the dimer formation.

Two different templates are presently being examined, the full-length protein without the acidic C-terminal and the HMGBl B box with parts of the N and C-terminal linker regions (HMGB-AB and B-box, respectively). The DNA binding boxes are highly conserved with low probability for successful epitope insertions. So, as a first generation the linker regions between the two boxes and between the B box and the acidic C-terminal will, respectively, be used for insertions, cf. Fig. 1, 2 and 17. A strategy that is pursued in parallel is to produce synthetic immunogenic variants of the B box. This approach utilizes a synthetic B box as a control and could potentially provide fast results on the feasibility of a HMGBl immunization approach and help to evaluate any safety concerns that potentially could arise in connection with inhibition of HMGBl functions.

In Fig. 2 and its accompanying legend is provided an overview of the protein motifs found in HMGBl. Among HMGBl mutants reported an RlOG mutation of HMGBl was expressed in similar amounts to the wild-type molecule, while other mutations resulted in lower yields. No obvious insertion site outside the linker regions can be identified from alignment with related proteins. There exist related proteins with two amino acids longer β-turn between the first two alpha-helices compared with HMGBl, but none of the related proteins have a β-turn long enough to allow epitope insertions or substitutions (Falciola, L, Murchie, A. I., Lilley, D. M., and Bianchi, M. (1994) Nucleic Acids Res. 22, 285-292).

In the following table is shown the different HMGBl constructs contemplated so far:

TABLE 1

HMGBl Last aa beFirst aa after Amino acids deleted by Total amino acid length

Constructs fore epitope epitope insert using PADRE as epitope

HMGBl-ABl 85 88 KK 194

HMGB1-AB2 83 84 - 196

HMGB1-AB3 86 90 KKF 193

HMGB1-AB4 85 90 KKKF 192

HMGB1-AB5 85 91 KKKFKD 190

HMGB1-AB6 83 91 ETKKKFKD 188

HMGB1-AB7 169 172 AA 194

HMGB1-AB8 169 179 AAKKGVVKA and C-. 185 terminal KK

HMGB1-AB9 163 182 AKGKPDAAKKGVVKAEKS 178

HMGBl-ABlO 163 179 AKGKPDAAKKGVVKA 179 and C-terminal KK

HMGBl-ABIl 163 181 AKGKPDAAKKGVVKAEK 177 and C-terminal KK

HMGB1-AB12 160 181 AYRAKGKPDAAKKGVVKA 174

EK and C-terminal KK

HMGB1-AB13 160 179 AYRAKGKPDAAKKGVVKA 176 and C-terminal KK

HMGB1-AB14 160 172 AYRAKGKPDAA 185 HMGBl Last aa beFirst aa after Amino acids deleted by Total amino acid length

Constructs fore epitope epitope insert using PADRE as epitope

HMGBl-Bl 85 88 KK 113

HMGB1-B2 83 84 - 115

HMGB1-B3 86 90 KKF 112

HMGB1-B4 85 90 KKKF 111

HMGB1-B5 85 91 KKKFKD 109

HMGB1-B6 83 91 ETKKKFKD 107

HMGB1-B7 169 172 AA 113

HMGB1-B8 169 179 AAKKGVVKA and C- 104 terminal KK

HMGB1-B9 163 182 AKGKPDAAKKGVVKAEKS 97

HMGBl-BlO 163 179 AKGKPDAAKKGVVKA 98 and C-terminal KK

HMGBl-BIl 163 181 AKGKPDAAKKGVVKAEK 96 and C-terminal KK

HMGB1-B12 160 181 AYRAKGKPDAAKKGVVKA 93

EK and C-terminal KK

HMGB1-B13 160 179 AYRAKGKPDAAKKGVVKA 95 and C-terminal KK

HMGB1-B14 160 172 AYRAKGKPDAA 104

The numbers used are from the N-terminal MGKGDP— of the HMGBl sequence where M is amino acid 1 and E from the C-terminal — DDDE is number 215. HMGBl-AB variants end at amino acid 183. HMGBl-B variants start with glycine in position 83 as GET— after an inserted methionine as translation start. HMGBl-B variants can also be produced without either the N- or C-terminal linker region, corresponding to amino acids 83-88 or amino acids 166-183.

EXAMPLE 2

Molecular biology and fermentation

HMGBl is potentially ideal for E. coli expression as no glycosylation or cysteine bridge are needed. It is, however, possible that HMGBl is toxic to E. coli through binding to DNA, therefore only short induction times have been used (Bianchi, M. E. (1991) Gene 104, 271- 275). A suitable alternative expression system is induced expression in S2 cells, cf. below.

Construction of different templates comprising various domains of HMGBl were performed to investigate how well these are expressed in E. coli. After selection of templates, a first generation of HMGBl variants, using the PADRE epitope (SEQ ID NO: 25) were constructed for expression and characterisation. If it should turn out that an E. coli system is unsuitable for HMGBl expression, the variants can be moved to a vector for a eukaryotic expression system.

General construction technique

All constructs were generated from an E. coli codon optimised synthetic HMGBl Wt encoding DNA template.

The generation of the variants from the synthetic HMGBl template was done by introducing epitopes using SOE PCR technique.

Truncated wt constructs

Four different wt templates were tested for expression. 1 template containing the wt sequence except the acidic tail HMG-AB, and 3 B-box templates containing only the B-box with or without N- & C-terminal linker regions HMG-B

4Four families of variants

Four different "families" of variants were constructed:

1. Variants containing both the A & B box, with the PD epitope in the linker region between the A and the B box, HMG-AB-I to HMG-AB-6.

2. Variants containing both the A & B box, with the PD epitope C-terminally to the B box, HMG-AB-7 to HMG-AB-14

3. Variants containing only the B box, with the PD epitope N-terminally of the B box, HMG- Bsh + HMG-B4 to HMG-B-6

4. Variants containing only the B box, with the PD epitope C-terminally of the B box, HMG- BlO and HMG-B-Il Moving HMGB-I variants to pPX4

An expression vector has been constructed that uses the pTrc promoter (pHP8/pPX4). To be able to transfer the HMGB-I from the pET28b+ vector to the pHP8 vector (SR-106.1-042- 01), an Notl site has been inserted in the MCS of the pHP8 vector, thereby creating the pPX4 vector.

All HMGB-I variants and wt encoding genes were inserted into the Ncol/Notl sites of the pPX4 vector. Where the Ncol site was not present in the pET28b+ construct, the upstream Xbal site was used instead of the Ncol. This results in constructs that contain the upstream region of pET28b+, but not the optimised Shine-Delgano sequence of pHP8.

Raw Materials

Synthetic Gene

The synthetic gene encoding HMGBl was purchased as a sequence-verified clone in pCR- Script-Amp from GENEART GmbH, BioPark Josef- Engert-Strasse 9, D-93053 Regensburg in Germany.

The HMGBl synthetic gene has the sequence set forth in SEQ ID NO: 80):

cccatgggtaaaggcgatccgaaaaaaccgcgtggcaaaatgagcagctacgcgtttttcgtgcagacctgccgcgaagaacata aaaagaaacatccggatgcgagcgtgaactttagcgaattcagcaaaaaatgcagcgaacgctggaaaaccatgagcgcgaaag aaaaaggcaaattcgaagatatggcgaaagcggataaagcgcgctacgaacgcgaaatgaaaacctatattccgccgaaaggcg aaaccaaaaagaaattcaaagatccgaacgcgccgaaacgtccgccgagcgcgttttttctgttttgcagcgaatatcgcccgaaaa tcaaaggcgaacatccgggcctgagcatcggcgatgtggcgaaaaaactgggcgaaatgtggaacaacaccgcggcggatgata aacagccgtacgaaaaaaaagcggcgaaactgaaagaaaaatacgaaaaagatatcgcggcgtatcgcgcgaaaggtaaaccg gatgccgcgaaaaaaggcgtggtgaaagcggaaaaatcaaaaaagaaaaaagaggaagaagaagatgaagaggatgaaga agatgaagaagaagaggaagatgaagaggacgaagatgaagaggaagatgacgatgacgaataagcggccgcc

And the synthetic DNA encoding the Padre epitope:

gccaagttcgtggccgcttggaccctgaaggccgcagct (SEQ ID NO: 78)

Synthetic oligonucleotides had the following sequences:

Oligo Name Oligo Sequence Purpose

2540 CTTTAAGAAGGAGATATACCATGGGTAAAGGCGATCCG Constructing HMG-AB-pET28b+ Oligo Name Oligo Sequence Purpose

SEQ ID NO: 81 AAAAAACCGCGTGGC ^"

2541 GTGGTGGTGCTCGAGTGCGGCCGCTTACTTTTTTGATTr Constructing HMG-AB-pET28b+ SEQ ID NO: 82 TTCCGCTTTCACCACGC

2542 CCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATAT constructing HMG-B¹ SEQ ID NO: 83 ACCATGTTCAAAGATCCGAACGCGCCGAAACG

2543 GAAGGAGATATACCATGGGCGAAACCAAAAAGAAATTC constructing HMG-'B SEQ ID NO: 84 AAAGATCCGAACGCGCCG

2544 GGTGGTGGTGCTCGAGTGCGGCCGCTTATTTCGCGCGA constructing HMG-¹B SEQ ID NO: 85 TACGCCGCGATATC

2559 CGTGGCCGCTTGGACCCTGAAGGCCGCAGCTAAATTCA Constructing HMG-B-I SEQ ID NO: 86 AAGATCCGAACGCGCCGAAACG

2560 GCGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCGG Constructing HMG-B-I SEQ ID NO: 87 TTTCGCCCATGGTATATCTCCTTC

2561 CAAGTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTG Constructing HMG-B-2 SEQ ID NO: 88 AAACCAAAAAGAAATTCAAAGATCCG

2562 CCTTCAGGGTCCAAGCGGCCACGAACTTGGCGCCCATG Constructing HMG-B-2 SEQ ID NO: 89 GTATATCTCCTTCTTAAAGTTAAAC

2563 GTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTAAAG Constructing HMG-B-4 SEQ ID NO: 90 ATCCGAACGCGCCGAAACGTCCGCCG

2564 GCGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCCTT Constructing HMG-B-4 SEQ ID NO: 91 TTTGGTTTCGCCCATGGTATATCTCC

2565 GTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTCCGA Constructing HMG-B-5 SEQ ID NO: 92 ACGCGCCGAAACGTCCGCCGAGCGCG

2566 GTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTAAAA Constructing HMG-B-7 SEQ ID NO: 93 AAGGCGTGGTGAAAGCGGAAAAATC

2567 GCGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCATC Constructing HMG-B-7 SEQ ID NO: 94 CGGTTTACCTTTCGCGCGATACGCC

2568 GTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTGAAA Constructing HMG-B-8 SEQ ID NO: 95 AATCATAAGCGGCCGCACTCGAGCACCACCACCACC

2569 GTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTAAAA Constructing HMG-B-9 SEQ ID NO: 96 AGTAAGCGGCCGCACTCGAGCACCACCACC

2570 GCGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCGC Constructing HMG-B-9 SEQ ID NO: 97 GATACGCCGCGATATC 1 1 I I ICG

2571 GTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTTCAT Constructing HMG-B-Il SEQ ID NO: 98 AAGCGGCCGCACTCGAGCACCACCACCACC

2572 GCGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCCG Constructing HMG-B-12 SEQ ID NO: 99 CGATATC I I I I I CGTA I I I I I CTTTCAG

2588 CGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCGGTT Constructing hHMG-AB-X SEQ ID NO: 100 TCGCCTTTCGGCGGAATATAGG

2589 CCTTCAGGGTCCAAGCGGCCACGAACTTGGCGCCTTTC Constructing hHMG-AB-X SEQ ID NO: 101 GGCGGAATATAGGTTTTCATTTC

2590 GGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCCTTTT Constructing hHMG-AB-X SEQ ID NO: 102 TGGTTTCGCCTTTCGGCGGAATATAG

2591 GGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCTTTGG Constructing hHMG-AB-X SEQ ID NO: 103 TTTCGCCTTTCGGCGGAATATAGG Oligo Name Oligo Sequence Purpose

2592 GGGGATCTAGATCGGGGTACCAAAGTCACCATGGGTAA Constructing hHMG-AB-X

SEQ ID NO: 104 AGGCGATCCGAAAAAACCG

2594 GGTGGTGGTGGTGCTCGAGTGCGGCCGCTTAATACGCC Constructing HMG-Bsh-pET28b+

SEQ ID NO: 105 GCGATATCTTTTTCG

Truncated templates

The truncated templates were made using PCR to generate the necessary restriction sites (Ncol/Xbal & Notl) 5' and 3' of the coding region. The PCR fragments were made using Expand High fidelity polymerase run with the following 30 touchdown PCR cycles :

1. Denaturation temp 94°C for 30 sek.

2. Annealing temperature 6O⁰C (-0,5⁰C pr cycle, for 20 cycles), followed by 10 cycles annealing temp 50⁰C for 30 sek.

3. Extension temp, was 68°C for 2 min (+ 5 sec pr cycle after the first 10 cycles).

The resulting PCR fragment was gel purified and treated with Taq polymerase, 15 min at

72⁰C to add A-overhangs. The fragments were then inserted into pCR2.1-TOPO according to the manufacturers protocol, transformed into DHlOB E.coli cells using electroporation, followed by 1 hour incubation in 500μl LB at 37°C), plated out on kanamycin containing (60μg/ml) LB Agar plates and incubated at 37°C ON. Single colonies from the plates were inoculated into 5 ml LB media, with 60 μg kanamycin/ml, and incubated at 37⁰C ON shaken at approx 220 rpm. DNA was purified using Qiagen miniprep kits, and the relevant regions were sequenced to identify correct clones.

DNA from a sequence verified clone was cut with either Ncol/Notl (HMG-AB & HMG-'B) or Xbal/Notl (HMG-B & HMG-B') at 37 ⁰C for 1 hour, gel purified and inserted into pET28b+, cut with the same enzymes using the following procedure.

pET28b+ vector (p2029) purchased from Novagen was cut with Notl and Ncol, gel purified and treated with SAP, in SAP buffer, 37⁰C for 15 min followed by inactivation at 65° for 20 min.

The gel purified HGMBl fragment from the cut pCR2.1-TOPO construct was inserted into the above-mentioned pET28b+ vector, and ligated overnight in a temperature cycler with cycle comprised of 30 seconds at 10⁰C followed by 30 seconds at 30⁰C according to SP016. The ligation product was transformed into HMS174(DE3) E.coli cells (30⁰C on ice, 2' at 42°C, 5' on ice, followed by 1 hour incubation in 500μl LB at 37°C), plated out on kanamycin containing (60 μg/ml) LB Agar plates and incubated at 37⁰C ON. Single colonies from the plates were inoculated into 5 ml LB media, with 60 μg kanamycin/ml, and incubated at 37°C ON shaken at approx 220 rpm. DNA was purified using Qiagen miniprep kits, and the relevant regions were sequenced to identify correct clones.

The oligo combinations used and the fragment sizes are summarised in the following table, where the fragments written in bold are cloned into pET28b+.

All constructs were made using the SOE (.splicing by overlap extension) PCR technique.

(Fragments from the genes that are to be recombined are generated in 2 separate PCR reactions. The primers are designed so that the ends of the products contain complementary sequences. When these PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3' ends overlap and act as primers for each other.

Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are 'spliced' together. Addition of 5' and 3' oligo primers from the 2 first reactions, respectively, allows an exponential amplification of the spliced product. In the generation of the constructs, the splicing primers did not only provide the necessary complementary sequences but simultaneously introduced the PADRE epitope.

To construct HMG-ABl-pET28b+, HMG-AB-pET28b+ (p2345) has been used, which is a truncated synthetic HMGB-I gene inserted in pET28b+, as a template. HMG-AB-pET28b+ was made from the synthetic gene HMGB-I (HMGB-1-pPCRscript) ■

First step in the construction of HMG-AB-l-pET28b+ was two separate PCR reactions, using either oligos 1638 and 2588 or 1641 and 2559 as primer pairs. Expand High fidelity polymerase was used in all SOE PCR reactions, run with the following 30 touchdown PCR cycles :

1. Denaturation temp 94°C for 30 sek.

2. Annealing temperature 60⁰C (-0,5⁰C pr cycle, for 20 cycles), followed by 10 cycles. annealing temp 5O⁰C for 30 sek.

The resulting PCR fragments were 690 bp and 636 bp in size, and were gel purified and used as templates for the second round of SOE PCR, using oligos 1638 and 1641. The PCR conditions were the same as the first round PCR, and the resulting 1300 bp large fragment was gel purified and digested with Ncol and Notl. This resulted in three fragments with the following sizes: 588 bp, 400 bp and 312 bp.

pET28b+ vector (p2029) purchased from Novagen was cut with Notl and Ncol, gel purified and SAP treated. The 588 bp fragment was gel purified and inserted into the above mentioned pET28b+ vector, and ligated overnight in a temperature cycler with cycle comprised of 30 seconds at 10°C followed by 30 seconds at 30⁰C according to SP016. The ligation product was transformed into HMS174(DE3) E.coli cells (30⁰C on ice, 2' at 42°C, 5' on ice, followed by 1 hour incubation in 500μl LB at 37°C), plated out on kanamycin containing (60 μg/ml) LB Agar plates and incubated at 37⁰C ON. Single colonies from the plates were inoculated into 5 ml LB media, with 60 μg kanamycin/ml, and incubated at 37⁰C ON shaken at approx 220 rpm. DNA was purified using Qiagen miniprep kits, and the relevant regions were sequenced to identify correct clones.

As all HMGB-I AutoVac variants and wt constructs were made using the same SOE PCR conditions, the details concerning each construct are summarised in the tables below. The relevant oligos are listed with respect to the template used, and the resulting fragment sizes. The size of the fragments resulting from the restriction digests are listed, with the fragment written in bold being the fragment that is gel purified and inserted into pET28b+ vector.

Construction of HMGBl-pPX4 vectors

We have constructed a new vector (pPX4) that uses the pTrc promoter instead of the T7 promoter found in the pET vector. The induction of expression has been shown to be identical to that of the pET system (Lad, induced with IPTG).

DNA of all HMGBl-pET28b+ constructs and pPX4 p2428 were cut with either Ncol or Xbal and Notl following the procedure described in section 3.3.1.

The HMGBl containing fragments (»550-600 bp for the HMG-AB type constructs and « 250- 300 bp for the HMG-B type constructs) were gel purified and ligated into pPX4 p2428, a derivative of pHP8, with a Notl site inserted.

The equipment used for the construction work are summarized here:

Protein expression of various HMGBl domains has initially been examined in E. coli. Conflicting reports has been published concerning the efficiency of HMGBl expression in E. coli, and it was the initial task to examine if E. coli is a suitable system for HMGBl variants of the invention.

Two HMGBl wt templates have been investigated (HMG-AB and -B box) to construct different variants with PADRE (SEQ ID NO: 25) as the T-helper epitope introduced. Other T- helper epitopes may be used, such as the P2 and P30 epitopes (SEQ ID NOs: 26 and 27).

15 variants were made with HMGBl-AB-pET28b+ as template, and 6 variants with only the HMGBl-B box part of the gene as template.

All these variants have been prepared in the pET28+ system, using the T7 promoter in HMS174(DE3) E. coli cells with IPTG induction.

Table

HMGBl-pET28b+ constructs

* Denotes a wt template

EXAMPLE 3

Fermentation and screening of HMGBl variants

The fermentation and screening of HMGBl constructs presently cover 12 fermentations of various variants for purification purposes and 2 screening experiments in shake flasks.

As the ELISA procedure is not fully developed, quantification of the amount of HMGBl protein in the samples were estimated from the standard used in the SDS-PAGE analysis. A "+" in the results table above (titled "HMGBl-pET28b+ constructs") indicates that HMGBl protein is present in an amount that can be used for purification. A "-" indicates that the variant did not appear as an expression product or only at very low levels.

Fermentations in tanks: The variants HMG-B5, HMG-AB4, HMG-AB3.1, HMG-AB8, HMG-B4, and HMG-AB13 were produced for purification. They all demonstrated high expression levels as well as reasonable high yields in the purification process. Based on the results from the analysis the fermentations should be harvested after 4 hours as the strongest bands on the coomassie stained gels appears after 2 - 4 hours whereafter the expression seems to decline.

The fermentation process parameters were as follows: Batch fermentation in defined medium (except from the 2 experiments in rich medium) with initial glucose concentration at 30 g/L (3%). pH = 7.0, biomass accumulation temperature = 37°C, induction point = OD_60O = 15 - 30, IPTG concentration = 1 mM, protein expression temperature = 37⁰C and expression time = 4 - 7 hours. At induction, additional medium containing glucose was added to a final glucose concentration at 20 g/L (2 %).

Screening of pET HMGBl variants and new pPX4 HMGBl variants:

The expression was also analysed by a preliminary sandwich ELISA procedure. Here it was necessary to use different setups, in order to detect all the different HMGBl variants, with both polyclonal and monoclonal anti-HMGBl. One set-up included coating over night with 3 μg/ml mice anti-HMGBl monoclonal (Abl2029 from Abeam) in 0.1 M Na₂CO₃, pH 9.5. Blocking with 3% fish gelatine in 20 mM Na-phosphate, pH 7.2 and 0,15 M NaCI (ELISA Buffer) and incubated with samples and standards for 2h all diluted in ELISA buffer. After Ih incubation with mouse biotinylated anti-HMGBl monoclonal (Mab 1690 from R&D Systems) and subsequent HRP-labelled streptavidin and developed with OPD (o-phenylenediamine dihydrochloride), the signal was measured at 490 nm on an EIx 808 ELISA reader (Bio-Tek Instruments) and compared with a standard curve. After every incubation step the plates were washed extensively in ELISA buffer with high salt (0.5M NaCI) and 1% Triton X-IOO. (See also the explanatory text for Figs. 13 and 14)

Here, the different variants present very different responses, see Fig. 13 although SDS-PAGE with subsequent coomassie staining do not detect any major differences in expression levels. It is noticeable that the three groups of variants (AB variants with the PADRE epitope between the boxes, AB variants with the epitope after the B box and the short B box variants) have different antibody reactivity in the ELISA. It is thus obvious that the ELISA is unsuitable for comparing the expression of different variants, but could be used to compare expression of the same HMGBl variant for optimisation of expression and purification for that variant.

Screening of some of the new pPX4 variants, also using rich medium, in shake flasks has been performed. Results from the screening:

Initial results indicated that all variants expressed with the pET system could also be expressed with the pPX4, except for HMG-B5-pPX4.

EXAMPLE 4

Protein purification and characterisation

The amphoteric nature of HMGBl is probably the main reason for the multitude of interactions that has been ascribed to HMGBl. Several reports indicate that HMGBl interact with both itself and several different substances. For example, HMGBl interacts with several transcription factors, DNA, sulphated glycosylated structures e.g. heparan sulphate and receptors like syndecan, RAGE and TLR2 and TLR4, in many cases through binding to negatively charged glycans or glycolipids.

Hence, the high pi and specific binding properties of HMGBl can conveniently be used for purification. As a consequence it is not initially expected that the variants will be produced including purification tags, even though this possibility is not excluded. The stability of purified HMGBl is probably low (according to the literature) and covalent dimers have been reported. HMGBl stability/solubility will be investigated and it can be envisioned that cysteines potentially responsible for dimerisation and aggregation could be mutated. One purification alternative is described by Weir, H. M., Kraulis, P. J., Hill, C. S., Raine, A. R., Laue, E. D., and Thomas, J. O. (1993) EMBO J. 12, 1311-1319. The disrupted E. coli cells are applied on a S-Sepharose and eluted at high salt, after ammonium sulphate addition the soluble material is applied on a Phenyl Sepharose and the eluted material is reported as "pure" product. Potential contamination of the product with endotoxin can be removed by a polymyxin B column (Pierce) or Prosep RemTox (Millipore).

Five variants (HMG-AB3.1, HMG-AB4, HMG-AB8, HMG-ABlO and HMG-AB13) and WtHMGBl have been purified from expression in minimal media to a purity of 75-90%. Typical purification yields are about 40-80 mg variant from cell pellets representing ~700 ml cell suspension; the purified proteins are depicted in Fig. 3.

For all the AB variants, the same purification scheme was followed. After cell disruption and filtration the filtrate was applied to a Heparin Sepharose column and eluted with a NaCI gradient. HMGBl containing fractions were pooled and changed to a phosphate buffer without salt. The sample was further purified and concentrated by a 5 ml SP-Sepharose column and eluted with a salt gradient. Pefabloc is used to inhibit protease degradation in the start material.

MALDI-TOF analysis indicates that the N-terminal methionine on purified HMGBl is cleaved. Reduced SDS-PAGE demonstrates a single band of the variants, while non-reduced conditions reveal two bands, see Fig. 4.

It has not been possible to separate the two bands enough to examine if they constitute the +/- methionine HMGBl or if it is due to other modifications. MALDI-TOF of reduced and non- reduced variants together with trypsin peptide map needs to be performed to reveal the nature of the two bands. The CD spectra for most variants have high homology to the spectrum of wtHMGBl.

wtHMGBl with and without the C-terminal acidic tail has been expressed in S2 cells. This material will be used as reference material in the different assays.

The short B box variants have lower expression levels than the AB variants. The B box variants cannot be purified with the same purification process as the AB variant. A first purification step using Phosphocellulose or other cation exchange matrices has replaced the Heparin step.

Initial experiments indicate that there are endotoxins present in purified proteins, part of this contamination could come from the buffers and a purification with endotoxin free buffers will be performed and the product analysed for endotoxin to evaluate if an extra endotoxin removal step is required. Potential contamination of the product with endotoxin can be removed by a polymyxin B column (Pierce) or Prosep RemTox (Millipore).

The variant HMG-ABlO has been mixed with the Adjuphos® and Alhydrogel® adjuvants. The variant binds both adjuvants.

Development of RP-HPLC method for HMGBl variants:

Samples were analysed by reverse phase HPLC (Jupiter C4 column 50x2 mm, Phenomex) using a linear gradient from 10% solvent B (0,1% TFA in 95% acetonitrile/5% H₂O) to 64% in solvent A (0,1% TFA in H2O) at a flow rate of 0,2 ml/minute for 59 minutes at 4OC. Elution was monitored at 220 nm.

The method is used for purity measurements during purification process development and for stability analysis. In the future the RP-HPLC method will also be used for quantification purposes. For that purpose a well-characterised reference is needed. In Fig. 5 is shown a chromatogram for purified AB3.1 variant indicating a purity of approximately 90%.

Development of SE-HPLC method for HMGBl variants

Samples were analysed on a Tosoh Biosep- TSK super SW 2000 using running buffer 50 mM Na-phosphate pH 7.0, 0.5 M NaCI, 10% Acetonitrile (0,2 ml/min). Proteins were detected at 220 nm.

The SE-HPLC method will be used for dimer/ aggregates detection during purification process development and stability studies. In Fig. 6 is shown a SEC chromatogram of purified HMG- AB3.1. Further development of the procedure is needed for better separation as the main peak exhibit tailing.

EXAMPLE 5

Immunological assays and animal models

It is advantageous that HMGBl is homologous in the experimental species and in man. Only one series of HMGBl variants need to be constructed because of the absolute sequence identity of HMGBl in rodents and man, except for the two amino acid difference in the C- terminal, which is not used to construct the HMGBl variants. The variants can be tested in inflammatory models such as C57BI mice with collagen induced arthritis (Williams, R. O., Williams, D. G. and Maini, R. N. (1992) J. Immunol. Methods 147, 93-100), although C57BI mice are not as responsive to HMGBl induced arthritis as other strains (Pullerits, R., Jonsson, I. M., Verdrengh, M., Bokarewa, M., Andersson, U., Erlandsson-Harris, H., and Tarkowski, A. (2003) Arthritis Rheum. 48, 1693-1700) and in DA rats.

It is known that PADRE is a poor T-helper epitope in several rodent strains and therefore it is important to select a strain that has strong PADRE reactivity for the selection experiments. Apart from measuring disease scores e.g. number of arthritic animals, number of swollen joints and paw thickness, antisera will be collected and analysed for direct anti-HMGBl titer in ELISA and neutralising titer in the macrophage-TNF release assay combined with the TNF cell-based toxicity assay or mTIMF ELISA. To broaden the potential indication for HMGBl immunization, it could also be tested in tumor models. This could be done in a metastasis model that has been established using B16-F1 mouse melanoma cells (Huttunen, H. J., Fages, C, Kuja-Panula, J., Ridley, A. J., and Rauvala, H. (2002) Cancer Res. 62, 4805-4811).

HMG-ABlO variant triggers anti-HMGBl IgG response in rats and mice

A direct ELISA for measuring the humoral response against HMGBl in rodents upon vaccination with the HMGBl-ABlO variant has been established. The animals were immunized at week 0, week 2, week 6 and week 10 and for the rats week 14.

Measurements of anti-HMGBl antibodies were done by a direct ELISA. 1 microgram/ml

HMGBl was coated on microtiter plates over night at +4C and blocked with 3% fish gelatin for 2 hours. The sera were diluted in 10 fold steps starting at 100 times dilution and anti- HMGBl reactivity were detected with either HRP-labelled anti-mouse Ig or anti rat Ig, respectively. After OPD development the signal was measured at 490 nm.

C57BL/6 mice as well as DA rats were primed with 100 microgram ABlO followed by boosters with 50 μg emulsified in either Alhydrogel, Adjuphos or ISA51 elicit significant anti-HMGBl humoral responses after two or three vaccinations, respectively (Fig. 7). Female rats have a more consistent high titre compared with male rats. For vaccinated DA rats, we have further shown that the anti-HMGBl humoral response is of Th2 type (Fig. 8), since elicited antibodies are exclusively of IgGl isotype, which could be beneficial for treatment of arthritis that is a Thl-driven disease.

PIA-transfer A study in HMG-AB10-vaccinated DA rats was initiated and PIA-transfer was chosen as an arthritis model (Fig, 9) since the therapeutic phase of arthritis then can be investigated directly, without interfering with priming of arthritogenic T cells. The PIA-transfer model is detailed in P. Olofsson et al., Nat Genet. 2003 Jan;33(l):25-32.

Cell assay for evaluating neutralizing efficiency of anti-HMGBl sera

A cell assay, in which RAW264.7 cells (murine macrophage cell line) are subjected to HMGBl, leading to TNF-α production has been established.

The murine macrophage-like cell line RAW 264.7 was purchased from American Type Culture collection and cultured in RPMI-1640 media (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% pencillin/streptomycin. Before HMGBl addition the cells were changed to medium without FBS and 1-10 microgram HMGBl were added to the cells with or without concomitantly added purified antibodies from immunised rats and mice. TNF-α release to the media after 3 hours was measured by mTNF-α ELISA (MTAOO, R&D Systems).

In this assay, wtHMGBl as well as HMG-ABlO and -AB4 variants are shown to similarly stimulate TNF-α production in a concentration-dependent way. In addition, trypsinated HMG- ABlO or -AB4 does not induce TNF-α production, which verifies the assay not to be endotoxin-dependent (Fig. 10). A minor practical drawback of this assay is that it requires approximately 3 μg/ml of HMGBl to reach OD₅₀ for TNF-α production, which means that high amounts of antibodies are required for neutralization. So far, we have demonstrated that a polyclonal anti-HMGBl antibody preparation (ABD-012 from JENA) could be used to significantly reduce the TNF-α production (Fig. 11). However, sera from non-vaccinated (i.e. PBS-treated) mice and rats reduced the secretion of TNF-α from stimulated RAW264.7 cells (Fig. 12), and IgG from HMG-AB10-vaccinated animals is currently purified for this assay.

HMGBl Sandwich ELISA

A sandwich ELISA for HMGBl has been established.

The expression was also analysed by sandwich ELISA. Different setups using polyclonal and monoclonal anti-HMGBl were used, including coating with 3 microgram/ml mice anti-HMGBl monoclonal (Abl2029 from Abeam). Blocking with 3% fish gelatin and incubated with samples and standards for 2h. After incubation with detecting mouse biotinylated anti- HMGBl monoclonal (Mab 1690 from R&D Systems) and HRP-labelled streptavidin and subsequent OPD incubation, the signal was measured at 490 nm and compared with a standard curve. After every incubation step the plates were washed extensively. (See also the text relating to Figs. 13 and 14 and the description of the same assay in Example 3)

This assay has higher reactivity for human recombinant HMGBl (SIGMA), since the in-house produced wtHMGBl and post-translationally modified HMGBl purified from calf thymus (WAKO) requires 100 times higher concentration to reach OD₅₀ (Fig. 14). We are in the process to optimising a sandwich ELISA for WT-HMGBl that can be used to quantify HMGBl- variants (Fig. 13), as well as being used for neutralization assays, cf above "Cell assay for evaluating neutralizing efficiency of anti-HMGBl sera".

Claims

1. A method for inducing an immune response against autologous high mobility group box 1 (HMGBl) in a mammal, including a human being, the method comprising effecting uptake and processing by antigen presenting cells (APCs) in the subject of at least one modified HMGBl polypeptide, said at least one modified HMGBl polypeptide comprising

- a substantial fraction of the B-cell epitopes from the autologous HMGBl, and

- at least one T helper epitope (T_H epitope) which is heterologous to the mammal and the HMGBl protein, thereby inducing an antibody response that targets the autologous HMGBl.

2. The method according to claim 1, for use in treatment or prophylaxis of a condition selected from the group consisting of cancer, Alzheimer's disease, atherosclerosis, and inflammatory disturbances, including chronic and acute conditions such as autoimmune diseases, e.g. arthritis.

3. The method according to claim 1 or 2, wherein the modified HMGBl polypeptide comprises a substantial fragment of at least one of the A and B boxes.

4. The method according to claim 3, wherein the modified HMGBl polypeptide comprises at least a complete A or a complete B box.

5. The method according to claim 3, wherein the modified HMGBl polypeptide comprises substantial fragments of both the A and B boxes.

6. The method according to claim 5, whererin the modified HMGBl polypeptide comprises complete A and B boxes.

7. The method according to any one of the preceding claims, wherein the at least one T_H epitope is introduced in any one of the linker regions of HMGBl.

8. The method according to any one of the preceding claims, wherein the modified HMGBl polypeptide is provided by introduction of the T_H epitope in the region of HMGBl corresponding to the linker between the A and B boxes or corresponding to the linker region C-terminally to the B box.

9. The method according to claim 8, wherein the foreign T_H epitope is introduced

- as an insertion in one or both of the specified linker regions; or - as a substitution in one or both the specified linker regions, where said substitution may include deletion of any one or all amino acids in said linker regions.

10. The method according to claim 9, wherein the modified HMGBl polypeptide is selected from the group consisting of - complete A and B boxes separated by an amino acid sequence including the T-helper epitope,

- a complete B box and a C-terminal amino acid sequence including the T-helper epitope and no substantial fragments of the A box, wherein the modified HMGBl polypeptide may include amino acids from the linker regions.

11. The method according to any one of the preceding claims, wherein the at least one foreign T_H epitope is immunodominant and/or wherein the at least one foreign T_H epitope is promiscuous.

12. The method according to any one of the preceding claims, wherein foreign T_H epitope(s) is/are selected from a natural T_H epitope and an artificial MHC-II binding peptide sequence

13. The method according to claim 12, wherein the natural T-cell epitope is selected from a Tetanus toxoid epitope, a diphtheria toxoid epitope, an influenza virus hemagluttinin epitope, and a P. falciparum CS epitope, and wherein the artificial MHC-II binding peptide sequence is a pan DR binding peptide such as the one having the amino acid sequence set forth in SEQ ID NO: 25.

14. The method according to any one of the preceding claims, wherein the modified HMGBl polypeptide has an amino acid sequence selected from any one of SEQ ID NOs: 5-24, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, and an amino acid sequence set forth in Table 1 herein.

15. The method according to any one of the preceding claims, wherein the modified HMGBl polypeptide is encoded by a nucleic acid sequence selected from any one SEQ ID NOs: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, and 76.

16. The method according to any one of the preceding claims, wherein the modified HMGBl polypeptide further comprises

- at least one first moiety effecting targeting of the modified HMGBl polypeptide to an antigen presenting cell (APC), and/or

- at least one second moiety stimulating the immune system, and/or

- at least one third moiety optimising presentation of the modified HMGBl to the immune system.

17. The method according to any one of the preceding claims, wherein the modified HMGBl , polypeptide includes duplication of at least one B-cell epitope of the autologous HMGBl.

18. The method according to claim 17, wherein the modified HMGBl polypeptide includes duplication of at least a substantial fragment of the B box, such as at least 3, at least 4 at least 5, at least 6, at least 7, at least 8, and at least 9 substantial fragments of the B boxes.

19. The method according to claim 18, wherein the substantial fragments are identical.

20. The method according to claim 18 or 19, wherein the modified HMGBl polypeptide includes at least 2 complete B boxes, such as at least 3, at least 4 at least 5, at least 6, at least 7, at least 8, and at least 9 complete B boxes.

21. The method according to any one of claims 18-20, wherein the substantial fragments or complete B boxes are part of a fusion polypeptide, wherein the substantial fragments or complete B boxes are optionally separated by the at least one foreign T helper epitope.

22. The method according to any one the preceding claims, wherein non-HMGBl derived components such as foreign T_H epitopes or first, second and third moieties as defined in claim 14 are present in the form of - side groups attached covalently or non-covalently to suitable chemical groups in the amino acid sequence of the autologous HMGBl or a subsequence thereof, and/or

- fusion partners to the amino acid sequence derived from the autologous HMGBl.

23. The method according to claim 22, wherein

- the first moiety is a substantially specific binding partner for an APC specific surface antigen such as a carbohydrate for which there is a receptor on the APC, e.g. mannan or mannose, or wherein the first moiety is a hapten,

- the second moiety is a cytokine selected from interferon y (IFN-γ), Flt3L, interleukin 1 (IL- 1), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15), and granulocyte-macrophage colony stimulating factor (GM-CSF), or an effective part thereof; a heat-shock protein selected from heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), heat shock cognate 70 (HSC70), glucose-regulated protein 94 (GRP94), and calreticulin (CRT), or an effective part thereof; or a hormone,

- the third moiety is a lipid such as a palmitoyl group, a myristyl group, a farnesyl group, a geranyl-geranyl group, a GPI-anchor, and an N-acyl diglyceride group.

24. The method according to claim any one the preceding claims, wherein the modified HMGBl polypeptide substantially preserves the 3-dimensional structure of the B box and, where applicable, the A box.

25. The method according to any one of the preceding claims, wherein the APC is a dendritic cell or a macrophage.

26. The method according to any one of the preceding claims, comprising administering an immunogenically effective amount of the at least one modified HMGBl polypeptide.

27. The method according to claim 26, wherein said modified HMGBl is formulated together with a pharmaceutically and immunologically acceptable carrier and/or vehicle and, optionally an adjuvant.

28. The method according to claim 27, wherein the adjuvant is selected from the group con- sisting of an immune targeting adjuvant; an immune modulating adjuvant such as a toxin, a cytokine, and a mycobacterial derivative; an oil formulation; a polymer; a micelle forming adjuvant; a saponin; an immunostimulating complex matrix (ISCOM matrix); a particle; DDA; aluminium adjuvants; DNA adjuvants; γ-inulin; and an encapsulating adjuvant.

29. The method according to claim 28, wherein the cytokine is as defined as in claim 23, or an effective part thereof, wherein the toxin is selected from the group consisting of listerioly- cin (LLO), Lipid A (MPL, L180.5/RalLPS), and heat-labile enterotoxin, wherein the mycobacterial derivative is selected from the group consisting of muramyl dipeptide, complete Freund's adjuvant, RIBI, and a diester of trehalose such as TDM and TDE, wherein the immune targeting adjuvant is selected from the group consisting of CD40 ligand, CD40 antibo- dies or specifically binding fragments thereof, mannose, a Fab fragment, and CTLA-4, wherein the oil formulation comprises squalene or incomplete Freund's adjuvant, wherein the polymer is selected from the group consisting of a carbohydrate such as dextran, PEG, starch, mannan, and mannose; a plastic polymer; and latex such as latex beads, wherein the saponin is Quillaja saponaria saponin, Quil A, and QS21, and wherein the particle comprises latex or dextran.

30. The method according to any one of claims 26-29, which includes administration via a route selected from the oral route and the parenteral route such as the intracutaneous, the subcutaneous, the peritoneal, the buccal, the sublinqual, the epidural, the spinal, the anal, and the intracranial routes.

31. The method according to any of claims 26-30, which includes at least one administration a year, such as at least 2, 3, 4, 5, 6, and 12 administrations a year.

32. The method according to any one of claims 1-25, comprising administering a non-pathogenic microorganism or virus which is carrying a nucleic acid fragment encoding and expressing the at least one modified HMGBl polypeptide, such as a nucleic acid fragment comprising any one SEQ ID NOs: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, and 76.

33. The method according to claim 32, wherein the non-pathogenic microorganism or virus is administered once to the animal.

34. The method according to any one of claims 1-25, comprising administering, to the animal, at least one nucleic acid fragment which encodes and can express the at least one modified HMGBl polypeptide, such as a nucleic acid fragment comprising any one SEQ ID NOs: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, and 76.

35. The method according to claim 34, wherein the at least one nucleic acid fragment is selected from naked DNA, DNA formulated with charged or uncharged lipids, DNA formulated in liposomes, emulsified DNA, DNA included in a viral vector, DNA formulated with a transfec- tion-facilitating protein or polypeptide, DNA formulated with a targeting protein or polypeptide, DNA formulated with a targeting carbohydrate, DNA formulated with Calcium precipitating agents, DNA coupled to an inert carrier molecule, and DNA formulated with an adjuvant.

36. The method according to claim 35, wherein the adjuvant is selected from the group con- sisting of the adjuvants defined in claim 28 or 29.

37. The method according to any one of claims 34-36, wherein the mode of administration is as defined in claim 30 or 31.

38. A modified human HMGBl polypeptide that is capable of inducing an immune response against autologous HMGBl in a human subject, comprising a substantial fraction of the B-cell epitopes and optionally CTL epitopes from the B box of HMGBl, and at least one non-human T helper epitope (T_H epitope).

39. The modified human HMGBl polypeptide according to claim 38, which is as defined in any one of claims 1, and 3-24.

40. An immunogenic composition which comprises, as an effective immunogenic agent the modified human HMGBl according to claim 38 or 39 in admixture with a pharmaceutically and immunologically acceptable carrier or vehicle, and optionally an adjuvant.

41. A nucleic acid fragment which encodes a modified HMGBl polypeptide according to claim 38 or 39.

42. A vector carrying the nucleic acid fragment according to claim 41.

43. The vector according to claim 42 being capable of autonomous replication.

44. The vector according to claim 42 or 43 being selected from the group consisting of a plasmid, a phage, a cosmid, a mini-chromosome, and a virus.

45. The vector according to any one of claims 42-44, comprising, in the 5'→3' direction and in operable linkage, a promoter for driving expression of the nucleic acid fragment according to claim 41, optionally a nucleic acid sequence encoding a leader peptide enabling secretion of or integration into the membrane of the polypeptide fragment, the nucleic acid fragment according to claim 41, and optionally a nucleic acid sequence encoding a terminator.

46. The vector according to any one of claims 42-45 which, when introduced into a host cell, is integrated in the host cell genome or is not capable of being integrated in the host cell genome.

47. A transformed cell carrying the vector of any one of claims 42-46.

48. A composition for inducing production of antibodies against HMGBl, the composition comprising

- a nucleic acid fragment according to claim 41 or a vector according to any one of claims 42-46, and a pharmaceutically and immunologically acceptable diluent and/or vehicle and/or adjuvant.

49. A stable cell line which carries the vector according to any one of claims 42-46 and which expresses the nucleic acid fragment according to claim 41, and which optionally secretes or carries the modified HMGBl according to claim 38 or 39 on its surface.

50. A method for the preparation of the cell line according to claim 49, the method comprising transforming a host cell with the nucleic acid fragment according to claim 41 or with the vector according to any one of claims 42-46.