IMMUNOGENIC ANALOGUES OF RANKL

The present invention relates to the field of active specific immune therapy. In particular, the present invention relates to novel immunogenic analogues of RANKL, a protein involved in bone homeostasis. The invention also relates to nucleic acid fragments encoding the novel analogues as well as to vectors and transformed microorganisms and host cells which carry the nucleic acids. The invention also relates to a method of treating or ameliorating disorders characterized by excessive bone resorption. Further, the invention also relates to novel improved adjuvant compositions which are advantageous for formulation of the RANKL derived immunogenic compositions but which are generally applicable as immunogenic adjuvants for the purposes of eliciting immune responses for the purposes of disease prophylaxis or treatment as well as for inducing immune responses in experimental animals and animals used for antibody production.

BACKGROUND OF THE INVENTION

RANKL is a cytokine expressed by osteoblasts and is recognized as a key regulator of osteoclast function and bone resorptive activity (Lacey al. 1998). RANKL (Receptor activator of nuclear factor-kappa B ligand) binds to its receptor (RANK), on the cell surface of osteoclasts, thus stimulating osteoclast differentiation and activation, leading to bone resorption (Burgess et al. 1999). OPG (osteoprotegerin), a natural soluble decoy receptor, neutralises RANKL in vivo and inhibits bone resorption by binding to RANKL. Over-expression of RANKL will disrupt the RANKL-OPG balance and lead to increased bone resorption, as seen in bone diseases such as osteoporosis, rheumatoid arthritis and metastatic bone cancer. Neutralising RANKL can reduce bone destruction in various animal models of these diseases (Kong et al. 1999, Honore et al. 2000).

Recent clinical data show that neutralising RANKL improves bone mineral density in women with postmenopausal osteoporosis and suppressed pathologic bone turnover in patients with metastatic breast cancer (Bekker et al 2004, McClung

et al 2006, Body et al 2006). Immunization capable of inducing immunity against RANKL would inhibit osteoclast activation and subsequent bone loss, by inducing neutralising antibodies against RANKL. Furthermore, such an immunization strategy may be effective at reducing other symptoms related to bone destruction such as bone pain and cartilage damage (Anandarajah and Schwarz, Kostenuik 2005).

Osteoporosis

Osteoporosis which mainly affects postmenopausal women and results from an accelerated rate of bone loss mainly due to the effects of estrogen deficiency. It is estimated that in US the number of patients suffering from osteoporosis and low bone mass in 2010 will be >50 million and that for osteoporosis alone the number will be approximately 12 million (National Osteoporosis Foundation). Comparable numbers are seen for Europe and Australia. The clinical consequences of osteoporosis are fractures, which are associated with considerable morbidity, lengthy hospital admission and correspondingly large economic burden. Thus, osteoporosis in menopausal women is widely recognized as a serious public health issue, and represents an economic and social problem in the light of its consequence in the elderly.

The currently available bone anti-resorptive agents such as estrogen, bisphosphonates, calcitonin, and selective estrogen receptor modulators

(SERMs) are used clinically to decrease bone turnover. Each of these treatments has limitations.

Rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease that affects approximately 1% of the global population. The female-to-male ratio is 2.5 to 1. RA is a costly disease due to its frequent onset in the middle aged, working members of society. Current treatment strategies aim at reduction/elimination of inflammation, maintenance of functional ability, reduction of pain and preventing the disease progression. The limited efficacy and potential severe toxicity related

to the traditional treatment of RA have paved the way for novel therapies such as TNF antagonists and other biological agents.

Several lines of evidence indicate that RANKL/OPG may represent a molecular link between the immune system and bone metabolism. The molecular mechanisms of bone resorption in RA may be described as follows: activated T cells, stromal cells, and synovial fibroblasts express RANKL, which interacts with its specific receptor RANK to promote osteoclast differentiation and activation and to inhibit osteoclast apoptosis, resulting in bone and cartilage damage (and possibly synovial inflammation). It is believed that RANKL/OPG plays a pivotal role in different processes in the pathogenesis of RA involving inflammation, local cartilage and bone damage, and generalised bone loss.

Bone metastasis

The skeleton is a common organ to be affected by malignancy disease, either primary bone lesions, as in multiple myeloma, or lesions secondary to cancer - bone metastasis. It is estimated that more than 1.5 million cancer patients worldwide have skeletal complications. Bone metastatic diseases are common occurrences in patients with various primary malignancies. The prevalence is ranked by an order of 1) breast cancer, 2) prostate cancer, 3) lung cancer, and 4) multiple myeloma.

Metastatic bone disease develops as a result of the many interactions between tumour cells and bone cells. This leads to disruption of normal bone metabolism, with the increased ostoeclast activity seen in most cases. The clinical course of metatastic bone disease in multiple myeloma, breast and prostate cancers is relatively long, with patients experiencing sequential skeletal complications over a period of several years. These include bone pain, fracture, hypercalcemia and spinal cord compression, all of which may profoundly impair a patient's quality of life.

The identification and characterisation of RANKL/OPG/RANK has provided a molecular link between cancer and bone. Breast cancer cells produce PTHrP,

which induces RANKL and inhibit OPG production, thus resulting in an increased RANKL-to-OPG ratio that favours osteolysis. Multiple myeloma cell may express RANKL with the subsequent increased bone loss.

Immunization targeting RANKL

In the present assignee's WO 00/15807 is disclosed a generic strategy for down- regulation of RANKL (also known as OPGL) with an aim to treatment and/or amelioration and/or prophylaxis of conditions characterized by excessive bone resorption. A number of immunization modes are disclosed in the WO 00/15807 as are a number of modified versions of RANKL, where foreign promiscuous T- helper epitopes are introduced in the RANKL amino acid sequence.

SUMMARY OF THE INVENTION

The present inventors have now generated a number of novel RANKL protein variants using a modified human RANKL template, named hRANKL-TB, wherein has further been introduced a promiscuous T-helper lymphocyte epitope. The modifications in hRANKL-TB consists of six point mutations that are introduced in defined regions of the human RANKL wildtype (wt) sequence in order to reduce the biological activity of the human RANKL protein molecules, thereby reducing the potential of the variants for causing adverse effects when administered as a immunogen. The biological activity of hRANKL-TB was studied in a cellular TRAP (tartrate-resistant acid phosphatase) assay (for a model assay, cf. e.g. Clin Chim Acta. 2005 Jun;356(l-2) : 154-63). Here, the variants were tested for their ability to induce differentiation and activation of pre- osteoclast into activated mature TRAP+ osteoclast in vitro. Importantly, from four independent TRAP assays it was concluded that the biological activity of hRANKL-TB as well as the variant hRP1.12 (cf. below) was decreased at least

10-30 fold compared to hRANKL-wt. The sensitivity of the TRAP assay and assay variations did not allow more precise determination of the biological activity.

The RANKL variants were expressed as soluble proteins in Drosophila S2 cells using a constitutive vector. Purification was made using a research process and

characterisation showed that the proteins were structurally similar to native human RANKL wildtype. This was supported by immunological studies in vaccinated rats, where the human RANKL variants all were able to illicit RANKL- specific antibodies that could neutralise native human RANKL protein in a concentration dependent manner. The proteins induced antibodies with similar quality but different quantity. The antibodies were considered functional by being capable of competing with OPG for the binding of human RANKL wild type in a competition ELISA and by inhibiting RANKL-induced differentiation and activation in a cellular osteoclast activation assay in vitro.

Hence, in its broadest aspect, the present invention relates to an immunogenic analogue of a human RANKL polypeptide, said analogue comprising a human RANKL amino acid sequence defined by SEQ ID NO: 1 residues 3-177, which has been modified by

- at least one T-helper lymphocyte epitope (T _H epitope) not naturally present in human RANKL being introduced by means of insertion or substitution in a position corresponding to a flexible loop in the native human RANKL protein or by means of insertion or substitution or addition in a position corresponding to residues 1-23 of SEQ ID NO: 1, and

- in addition to the at least one T _H epitope, at least one point mutation being present in the human RANKL amino acid sequence, wherein said at least one point mutation is one which, when introduced into the amino acid sequence of a biologically active human RANKL polypeptide, produces a RANKL mutant having a significantly lower affinity for OPG than the corresponding non-mutated polypeptide and/or the having a significantly lower ability than the non-mutated RANKL polypeptide to induce differentiation and activation of pre-osteoclast into activated mature TRAP+ osteoclast in vitro. Other related aspects pertain to nucleic acid fragments encoding the immunogenic analogues, vectors comprising such nucleic acid fragments, as well as transformed cells comprising these nucleic acid fragments/vectors. Other related aspect relate to immunogenic compositions comprising the analogues, nucleic acids, vectors or transformed cells.

Another related aspect is a method for a method for down-regulating RANKL in a human being, the method comprising administering an effective amount of an analogue, nucleic acid fragment, vector or transformed cell (or a composition comprising any of these constituents) to a subject in need thereof.

Another main aspect of the invention is based on findings by the present inventors that mixtures of micelle-forming immunogenic adjuvants such as Provax and B5 with non-micelle forming adjuvants such as the alum and calcium adjuvant provide a synergistic adjuvant effect which has as a result that the amount of antigen in an immunogenic composition may be considerably lowered compared to current state of the art immunogenic compositions.

Hence, the other aspect relates to a composition of immunugenic adjuvants comprising a mixture of a micelle forming immunogenic adjuvant, and at least one further non-micelle forming immunogenic adjuvant.

LEGENDS TO THE FIGURES

Fig. 1 : Principle of SOE PCR

Principle of the polymerase chain reaction (PCR) based "gene Splicing by Overlap Extension" (SOE) method used in generation of the RANKL constructs: Fragments from the genes that are to be recombined are generated in separate PCR reactions (Reaction 1 and 2). The primers are designed so that the ends of the products contain complementary sequences. When these PCR products are mixed, denatured, and reannealed (Reaction 3), 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 Reaction 1 & 2, respectively, allows an exponential amplification of the spliced product. In the generation of the present constructs the splicing primers did not only provide the necessary complementary sequences but simultaneously introduced the PADRE epitope coding sequence (SEQ ID NO: 5).

Fig. 2 Flow chart of RANKL variant construction

As all RANKL variants, mutants and wt constructs were made using the same SOE PCR conditions, the details concerning each construct are summarised in table 8. 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 gelpurified and inserted into pET28b+ or the p2ZOp2F vector in the cases were Xbal or Xhol was used as RE. When Ncol was used as restriction enzyme (RE), the fragments were inserted into the hRANKL-TB-pET28b+ or the hRANKL-TB- p2ZOp2F vectors, as the Ncol site is placed 6 aa inside the stalk region.

Fig. 3: Expression levels of single box mutants in HMS174(DE3) in defined media in fermentors.

Figure 4: Immunogenic RANKL variants constructed.

New RANKL variants constructed in the hRANKL-TB template. hRPl.X is hRPl-TB and hRP1.5 to hRP1.14, hRP3.X is hRP3.5 to 3.7, hRP4.X is hRP4.2 to hRP4.4, hRP6.X is hRP6.4 to hRP6.8, hRP7.X is hRP7.2 to hRP7.6.

Fig. 5: Map of the p2475 plasmid

The p2ZOp2F S2 insect cell expression vector carrying the hRP1.12-RA DNA encoding the RANKL variant Protein.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

"RANKL" generally denotes the human RANKL protein, i.e. the protien comprising the amino acid sequence set forth in SEQ ID NO: 2.

A "RANKL polypeptide" is herein intended to denote polypeptides having the amino acid sequence of the above-discussed RANKL protein derived from humans (or truncates thereof sharing a substantial amount of B-cell epitopes

with intact RANKL, i.e. such as the truncate having the amino acid sequence SEQ ID NO: 1).

A "RANKL variant" (also termed a "RANKL analogue") is a RANL 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 RANKL 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 RANKL polypeptide's amino acid sequence. In the present specification and claims, the RANKL variant includes at least one modification in the form of a point mutation which diminishes RANKL's biological activity and at least one foreign T-helper lymphocyte epitope.

A "biologically active human RANKL polypeptide" is a RANKL polypeptide which has retained native RANKL's ability to bind OPG or RANK or has retained native RANKLs' ability to induce differentiation and activation of pre-osteoclast into activated mature TRAP+ osteoclast in vitro.

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 effector functions such as helper activity in the humeral immune response. Likewise, the terms "B-lymphocyte" and "B-cell" will be used interchangeably for antibody-producing lymphocytes.

An "antigen presenting cell" (APC) is a cell which presents epitopes to T-cells. Typical antigen-presenting cells are macrophages, dendritic cells and other phagocytizing and pinocytizing cells. It should be noted that B-cells also functions as APCs by presenting T _H epitopes bound to MCH class II molecules to T _H cells but when generally using the term APC in the present specification and claims it is intended to refer to the above-mentioned phagocytizing and pinocytizing cells.

"Helper T-lymphocytes" or "T _H cells" denotes CD4 positive T-cells, which provide help to B-cells and cytotoxic T-cells via the recognition of T _H epitopes bound to MHC Class II molecules on antigen presenting cells.

The term "cytotoxic T-lymphocyte" (CTL) will be used for CD8 positive T-cells, which require the assistance of T _H cells in order to become activated.

A "specific" immune response is in the present context intended to denote a polyclonal immune response directed predominantly against a molecule or a group of quasi-identical molecules or, alternatively, against cells which present CTL epitopes of the molecule or the group of quasi-identical molecules.

The term "polypeptide" is in the present context intended to mean both short peptides of from 2 to 10 amino acid residues, oligopeptides of from 11 to 100 amino acid residues, and polypeptides 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 comprise prosthetic groups.

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 amino acid sequence or nucleic acid sequence, respectively.

By the term "down-regulation of RANKL protein" is herein meant reduction in the living organism of a mammal, such as a human, of the amount and/or activity of the RANKL protein. The down-regulation can be obtained by means of several mechanisms including removal by scavenger cells (such as macrophages and other phagocytizing cells) or by binding by antibodies which neutralize the biological effect(s) or the RANKL protein.

The expression "effecting presentation ... to the immune system" is intended to denote that the animal's immune system is subjected to an immunogenic

challenge in a controlled manner. 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 immunogen in an immunologically effective manner, whereas the precise mode of achieving this result is of less importance to the inventive idea underlying the present invention.

The term "immunogen" is intended to denote a substance capable of inducing an immune response in a certain animal. It will therefore be understood that an autologous RANKL protein is not normally an immunogen in the autologous host - it is necessary to use either a strong adjuvant and/or to co-present T helper epitopes with the autologous protein in order to mount an immune response against autologous protein and in such a case the "immunogen" is the composition of matter which is capable of breaking autotolerance.

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

The term "vaccine" is used for an immunogenic composition which is capable of inducing an immune response which is either capable of reducing the risk of developing a pathological condition or capable of inducing a therapeutically effective immune response which may aid in the cure of (or at least alleviate the symptoms of) a pathological condition.

When discussing "autotolerance towards RANKL" it is understood that since RANKL is a self-protein in humans, normal individuals do not mount an immune response against RANKL; it cannot be excluded, though, that occasional individuals might be able to produce antibodies against native RANKL, e.g. as part of a autoimmune disorder.

The term "pharmaceutically acceptable" has its usual meaning in the art, i.e. it is used for a substance that can be accepted as part of a medicament for human use when treating the disease in question and thus the term effectively excludes the use of highly toxic substances that would worsen rather than improve the treated subject's condition.

A "foreign T-cell epitope" is a peptide which is able to bind to an MHC molecule and which stimulates T-cells in an animal species. Preferred foreign epitopes are "promiscuous" epitopes, i.e. epitopes, which binds to a substantial fraction of MHC class II molecules in an animal species or population. A term, which is often used interchangeably in the art, is the term "universal T-cell epitopes" for this kind of epitopes. 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 analogue or 2) prepare several analogues wherein each analogue has a different promiscuous epitope inserted. It should be noted 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. It is also important to add that the "foreignness" feature therefore has two aspects: A foreign T _H epitope is 1) presented in the MHC Class II context by the animal in question and 2) the foreign epitope is not derived from the same polypeptide as the target antigen for the immunization - the epitope is thus also foreign to the target antigen.

A "CTL epitope" is a peptide, which is able to bind to an MHC class I molecule.

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 an immune response against the immunogen, vaccination with the immunogen may or may not give rise to an immune response against the immunogen, but the combined vaccination with immunogen and adjuvant induces an 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.

"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.

Description of embodiments of the invention

As mentioned above, a first aspect of the invention relates to an immunogenic analogue of a human RANKL polypeptide, said analogue comprising a human RANKL amino acid sequence defined by SEQ ID NO: 1 residues 3-177, which has been modified by - at least one T-helper lymphocyte epitope (T _H epitope) not naturally present in human RANKL being introduced by means of insertion or substitution in a

position corresponding to a flexible loop in the native human RANKL protein or by means of insertion or substitution or addition in a position corresponding to residues 1-23 of SEQ ID NO: 1, and

- in addition to the at least one T _H epitope, at least one point mutation being present in the human RANKL amino acid sequence, wherein said at least one point mutation is one which, when introduced into the amino acid sequence of a biologically active human RANKL polypeptide, produces a RANKL mutant having a significantly lower affinity for OPG than the corresponding non-mutated polypeptide and/or the having a significantly lower ability than the non-mutated RANKL polypeptide to induce differentiation and activation of pre-osteoclast into activated mature TRAP+ osteoclast in vitro.

Thus, the sequence serving as basis for the analogue is SEQ ID NO: 1, residues 3-177. This sequence is La. the expression product from insect cells transformed with expression vectors encoding SEQ ID NO: 1; it has, as is detailed in the examples below, been found that the two N-terminal amino acids of SEQ ID NO: 1 are processed out of the RANKL analogues/variants of the invention. SEQ ID NO: 1 is a C-terminal subsequence of the complete human RANKL amino acid sequence (SEQ ID NO: 2).

The analogues of the present invention all include the characteristic feature of including at least one point mutation in the SEQ ID NO: 1; the point mutations have been identified in a series of experiments where it has been established that they are less biologically active than native RANKL or corresponding RANKL fragments.

It is preferred that the immunogenic analogue comprises at least 2 point mutations, but more are possible such as at least 3, 4, 5, and at least 6 point mutations. On the other hand, the number of point mutations must be kept at a sufficiently low level so as to ensure that the RANKL analogues obtained are not significantly changed so that they will be incapable of folding up into a 3D conformation which matches that of native RANKL polypeptides (e.g. as evidenced by the analogues' capability of forming trimers or to compete with RANKL with respect to polyclonal and monoclonal antibody binding, e.g. in an

ELISA). As can be seen from example 4 below, one particular RANKL analogue which includes 6 point mutations as well as an in-substitued foreign T-helper epitope, is capable of forming trimeric molecules and of inducing RANKL cross- reacting antibodies.

Hence, the immunogenic analogue is preferably one which comprises at most 10 point mutations, such as at most 9, 8, or at most 7 point mutations.

Preferred analogues of the present invention comprise exactly 6 point mutations.

Point mutations are conveniently substitution mutations and often made with amino acids which do not disturb secondary structure and may even be in the form of conservative substitutions. However, since only structure conservation is an issue (in order to preserve B-cell epitopes) while functionality should be destroyed, not only conservative substitutions are relevant.

The at least one point mutation is conveniently selected from a mutation of a residue corresponding to any one of residues 171, 193, 215, 219, 274, and 301 in SEQ ID NO: 2.

When using the expression "corresponding to" in this context, it is the intention to indicate that amino acids are mutated in a RANKL polypeptide where the amino acids in the vicinity of the mutated amino acid residue are identical to the amino acid residues in the vicinity of the above-indicated amino acid residues in SEQ ID NO: 2 - for example, if the mutated RANKL polypeptide is a fragment of SEQ ID NO: 2, the mutated RANKL polypeptide may be aligned optimally with SEQ ID NO: 2, and a mutated amino acids in the mutated sequence align with any one of residues 171, 193, 215, 219, 274, and 301 in SEQ ID NO: 2, the mutated amino acid is an amino acid residue corresponding to any one of residues 171, 193, 215, 219, 274, and 301 in SEQ ID NO: 2.

Preferred mutations are selected from the group consisting of a mutation corresponding to Ala to Ser in residue 171, Ala to GIy in residue 193, Leu to He in residue 215, He to VaI in residue 219, He to VaI in residue 274, and Asp to GIn

in residue 301. In one particular interesting case, all of these point mutations are present.

The at least one T _H epitope is preferably introduced in SEQ ID NO: 4 without abolishing any of the point mutations discussed above.

The immunogenic analogue preferably comprises the at least one T _H epitope as an insertion or substitution into a RANKL amino acid sequence defined by SEQ ID NO: 107. As apparent from the examples, SEQ ID NO: 107 defines the so- called stalk region in RANKL, and the most promising of the presently disclosed RANKL analogues are those which include a foreign T _H epitope in this region.

The immunogenic analogue is therefore typically selected from analogues comprising an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 120-141 (which are all analogues where a T _H epitope is introduced in the stalk region). Preferred analogues have an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 120- 141, and especially preferred are the analogues having the amino acid sequence SEQ ID NO: 129 or 140 (which only differ with respect to the presence or absence of the N-terminal Arg-Ala residues).

The T _H epitope is herein exemplified by the so-called PADRE (pan DR-binding epitope), but other T _H epitopes are also useful.

WO 00/15807 referred to above includes a thorough discussion of the reasons for including all available knowledge of the epitopes to be inserted, when selecting T _H epitopes for a vaccine: 1) The frequency of responders to the epitope(s) in a population to be immunized, 2) MHC restriction data relative to the epitopes, and 3) frequency in the population of the relevant haplotypes.

There exist a number of naturally occurring "promiscuous" T _H 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 RANKL analogues in the same

vaccine. Further, if such a promiscuous T _H epitopes are also immune dominant {i.e. strong binders to MHC molecules), they are especially suitable.

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, cf. WO 00/15807), diphtheria toxoid, Influenza virus hemagluttinin (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 HLA-DR alleles have been identified and these are all possible T-cell epitopes to be introduced in the RANKL analogues 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. al, 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 al., 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 as exemplified herein be any artificial T-cell epitope which is capable of binding a large proportion of MHC Class II molecues. 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 RANKL 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 A K FVAAWT L KAAA (SEQ ID NO: 6). This, and other epitopes having the same lack of MHC restriction are preferred T-cell epitopes which should be present in the RANKL analogues used in the inventive method.

Although not the main focus of the present invention, it is also possible to utilise some of the other features described in the context of OPGL/RANKL analogues in WO 00/15807: the general description in WO 00/15807 of incorporation in RANKL analogues of targeting moieties, immune stimulating moieties, and presentation-enhancing moieties apply mutatis mutandis to the analogues of the present invention.

It is also a possibility to utilise coupling of the presently disclosed RANKL analogues to larger backbone structures (e.g. polysaccharides, such as dextran, mannan or mannose, or other polymeric backbones) to which multiple polypeptides may be coupled - in some aspects this will allow presentation of multiple epitopes to the immune system, cf. also the discussion of this issue in WO 00/15807.

It will be understood that introduction of a foreign T _H 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 sequence (e.g. insertion of or substition by a complete T-cell epitope) but the important goal to reach is that the RANKL analogue, when processed by an antigen presenting cell (APC), 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 RANKL amino acid sequence 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 fulfill the purpose of the present invention, but merely to make sure that the epitope's amino acid be present in the molecule after processing.

It is preferred that the number or T _H -introducing amino acid insertions, substitutions or additions is at least 2, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 25 insertions, substitutions, additions or deletions. It is furthermore preferred that the number of amino acid insertions, substitutions, additions or is not in excess of 150, such as at most 100, at most 90, at most 80, and at most 70. It is especially preferred that the number of substitutions, insertions, or additions does not exceed 60, and in particular the number should not exceed 50 or even 40. Most preferred is a number of not more than 30. With re-spect to amino acid additions, it should be noted that these, when the resulting construct is in the form of a fusion poly-peptide, is often considerably higher than 150.

The RANKL analogues are prepared according to methods well-known in the art. Since the constitute longer polypeptides, they are normally prepared by means of recombinant gene technology including introduction of a nucleic acid sequence encoding the RANKL 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 subseqeunt purification and optional further modification, e.g. refolding or derivatization.

However, recent advances in this technology has rendered possible the production of full-length polypeptides and proteins by specialized versions of the techniques of solid phase or liquid phase peptide synthese, and therefore it is also within the scope of the present invention to prepare the long constructs by synthetic means.

Compositions of the invention

It will be understood that the present invention aims at providing immunogenic compositions useful for administration to humans in order to cure, alleviate symptoms of or reduce the risk of attaining certain RANKL-associated conditions. Hence, an aspec of the invention is a composition comprising the analogue according to any one of the preceding claims in admixture with at least one pharmaceutically acceptable carrier and/or diluent and/or vehicle and/or excipient and/or immunogenic adjuvant.

The formulation of the RANKL variants 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, intradermal^, subdermally or intramuscularly. Additional formulations which are suitable 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 (as will appear from example 6 herein, the choice of immungenic adjuvant formulation is in this context of great importance). The quantity to be administered also 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 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.

The immune response will normally 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 "Vaccines: New Generationn 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 RANKL is used as the active ingredient in the immunogenic composition. 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 in WO 00/15807, which relate to compounds and agents useful as targeting, immune stimulating and presentation enhancing moieties to include into an immunogenic polypeptide also refer mutatis mutandis to their use in the adjuvant in the present invention.

The application of adjuvants include use of agents such as aluminum hydroxide or phosphate (alum), calcium phosphate, 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 101 ⁰ C 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).

Micelle forming and liposome formulations are also known to confer adjuvant ef- fects, and therefore liposome adjuvants are preferred according to the invention. In this context, the adjuvants known as MF59, B5, and Provax are all interesting possibilities.

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 consists 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. Immunother. 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 Fc receptors on monocytes/macrophages. Especially conjugates between antigen and anti-FcRI have been demonstrated to enhance immunogenicity for the purposes of vaccination.

Other possibilities involve the use of the targeting and immune modulating substances {La. cytokines) mentioned above as candidates for the first and second moieties in the modified versions of RANKL. 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 dipeptide, 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; and latex such as latex beads.

Yet another interesting way of modulating an immune response is to include the RANKL immunogen (optionally together with adjuvants and pharmaceutically acceptable carriers and vehicles) in a "virtual lymph node" (VLN) (a proprietary medical device developed by ImmunoTherapy, Inc., 360 Lexington Avenue, New York, NY 10017-6501). The VLN (a thin tubular device) mimics the structrue 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 Gelber C et al., 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".

As mentioned in Example 6, it has been found that certain combination adjuvants (which are believed to constitute an invention in their own right) provide excellent immunization results with both one exemplary RANKL analogue of the invention but also with other test antigens. Hence, the use of such combination adjuvants which include one micelle-forming adjuvant and at least one non-micelle forming adjuvant is a preferred embodiment of the present invention and all details pertaining to these combination adjuvants apply as especially preferred adjuvant compositions for use with the RANKL analogues of the present invention.

It is expected that the immunogenic compositions of the invention should be administered 1-6 times per year, such as 1, 2, 3, 4, 5, or 6 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 autovaccines according to the invention is not permanent, and therefore the immune system needs to be periodically challenged with the RANKL or modified RANKL polypeptides.

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 _H 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.

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 RANKL). 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 posttranslational processing of the expression product is expected to occur.

Hence, one embodiment of the invention suggests presentation of modified RANKL to the immune system by introducing nucleic acid(s) encoding the RANKL analogues disclosed herein 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 polypeptide, DNA formulated with a targeting protein or polypeptide, DNA formulated with Calcium precipitating agents, DNA coupled to an inert carrier molecule, DNA encapsulated 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 formulation 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. In recent years, good results have been obtained when using electroporation when introducing vaccine vectors into tissue. 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.

Accordingly, the invention also relates to a composition for inducing production of antibodies against RANKL, the composition comprising

a nucleic acid fragment or a vector of the invention (cf. the discussion of vectors below), and

a pharmaceutically and immunologically acceptable vehicle and/or carrier and/or adjuvant as discussed above.

Under normal circumstances, the RANKL 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 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.

It is contemplated that nucleic acid vaccination may be advantageous for priming an immune response, but that it may be followed by boosting with either polypeptide vaccination or live vaccination.

Live vaccines

A third alternative for effecting presentation of RANKL analogues to the immune system is the use of live vaccine technology. In live vaccination, presentation to the immune system is effected by administering a non-pathogenic microorganism which has been transformed with a nucleic acid fragment encoding a modified RANKL or with a vector incorporating 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 or any other suitable pox virus.

Normally, the non-pathogenic microorganism or virus is administered only once, 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.

Use of the method of the invention in disease treatment

The invention also contemplates a method for down-regulating RANKL in a human being, the method comprising administering an effective amount of an analogue of the invention to a subject in need thereof. Alternatively, it is possible to administer an effective amount of nucleic acid fragment or vector of the invention (by emploing nucleic acid vaccination or vaccination with nonpathogenic virus, cf. above). Finally, it is possible to use live vaccination, i.e. administering an effective amount of a transformed cell (non-pathogenic) of the invention.

As will be appreciated from the discussions above, the provision of the method of the invention allows for control of diseases characterized by excessive loss of bone mass. In this context, the disease osteoporosis is the key target for the inventive method but also bone loss associated with complicated bone fractures is a feasible target for treatment/amelioration. Hence, an important embodiment of the method of the invention for down-regulating RANKL activity comprises treating and/or reducing the risk of and/or ameliorating osteoporosis or other conditions characterized by excess bone resorption, the method comprising down-regulating RANKL activity according to the method of the invention to such an extent that the rate of bone resorption is significantly decreased. As indicated above, such conditions include metastasis of cancer to bone tissue, and also rheumatoid arthritis.

In the present context such a significant decrease in bone resorbtion is at least 3% compared to the pathological rate, but higher percentages are contemplated, such as at least 5%, at least 7%, at least 9%, at least 11%, at least 13%, at least 15%, and at least 17%, but even higher percentages are expected, such as at least 20%, or even at least 30%. It is especially preferred that the decrease in bone resorption results in an inversion of the balance between bone formation and bone resorption, i.e. that the rate of bone

formation is brought to exceed the rate of bone resorption. Of course, this imbalance should not be maintained (since it would result in osteopetrosis), but by carefully controlling the number and immunological impact of immunizations of the individual in need thereof it is possible to obtain a balance over time which results in a net conservation of bone mass. Alternatively, if in an individual the method of the invention cannot terminate bone loss, the method of the invention can (optionally in combination with other known methods for reducing bone loss in osteoporosis patients) be used to obtain a significant reduction in bone loss, thereby prolonging the time where sufficient bone mass is present in the individual.

Methods for measuring the rate of bone resorption and bone formation are known in the art. It is by means of biochemical assays possible to gauge the rate of bone resorption by measuring the blood concentration of certain fragments of collagen type I (cf. WO 93/15107 and WO 94/14844). Alterna- tively, the rate of bone loss can be assessed by physical means; representative disclosures in the art of methods for assessing bone mass by non-invasive, physical methods can be found in WO 88/06862, WO 94/12855, WO 95/14431, and WO 97/00643.

Nucleic acid fragments and derivatives; recombinant expression

It will be appreciated from the above disclosure that RANKL analogues can be prepared by means of recombinant gene technology. For this purpose, and of course also for the purpose of nucleic acid immunization, nucleic acid fragments encoding the RANKL analogues are important chemical products. Hence, an important part of the invention pertains to a nucleic acid fragment which encodes RANKL analogue of the invention. 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 invention, 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 reasons preferred that the vector is 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 RANKL analogues 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 RANKL 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 RANKL.

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 cerevisiae), 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, such as a human derived cell. Most preferred are insect cells, e.g. the Drosophila melanogaster cell line (the Schneider 2 (S ₂ ) cell line and vector system available from Invitrogen) for the recombinant production of polypeptides in applicants' lab, and therefore this expression system is particularly preferred, but other insect cell lines such as SF celles are useful.

For the purposes of cloning and/or optimized 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 RANKL analogue or, in the case of non-pathogenic bacteria, as vaccine constituents in a live vaccine.

When producing the RANKL analogue 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 RANKL analogue. Preferably, this stable cell line secretes or carries the RANKL 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 ordinarily carries a replication site, as well as marking se- quences 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 ampicillin 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 prokaryotic microorganism for expression.

Those promoters most commonly used in 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 al., 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 promoter 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 cerevisiase, 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 characteristic 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, phospho- fructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. 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 (Tissue 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 S ₂ available from Invitrogen, 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 fragment 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 Hindlll site toward the BgIl 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 sequences 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.

Adiuvant compositions of the invention

During the work with testing of the immunogenicity of one of the RANKL analogues of the present invention, it was surprisingly discovered that a "mixed adjuvant" comprising the commercially available Provax and Alhydrogel conferred unexpected advantages to the vaccine composition comprising the RANKL analogue. As will appear from Example 6, these advantages appear to be of a more general nature and are not merely related to the precise choice of adjuvants in the mixture or to the precise choice of antigen, as the case often otherwise is.

It has been surprisingly discovered that mixtures of Provax (a micelle forming adjuvant disclosed in US 5,585,103 - it generally incorporates a stabilising detergent, a micelle forming agent and a biodegradable oil) and a non-micelle forming immunogenic adjuvant provides synergy and significantly better

immune responses than any of the adjuvants alone or better than their additive effect. So, in a broad aspect, the present invention relates to a composition of immunugenic adjuvants comprising a mixture of - a micelle forming immunogenic adjuvant, and - at least one further non-micelle forming immunogenic adjuvant.

The composition of immunongenic adjuvant is typically one wherein the micelle forming adjuvant is in the form of an oil-in-water emulsion and typically this emulsion is microfluidized. Examples are Provax (disclosed in US 5,585,103), MF59 (disclosed in US 5,709,879) and B5. Details pertaining to the composition of Provax and B5 can be found in Example 6, cf. the section headed "Adjuvants".

The composition typically has a ratio between the at least one further non- micelle forming immunogenic adjuvant and the micelle forming immunogenic adjuvant is at least 0.01 μg/μl, such as at least 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, and at least 3.0 μg/μl.

On the other hand, the composition typically exhibits a ratio between the at least one further non-micelle forming immunogenic adjuvant and the micelle forming immunogenic adjuvant is at most 50 μg/μl, such as at most 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, and at most 0.5 μg/μl.

Normally, the adjuvant composition is contained in an aquous solution in a concentration of at most 95% v/v, such as at most 85, 75, 65, 55, 45, 35, 25, 20, 15, 10, 9, 8, 7, 6 , 5, 4, 3, 2, and 1% v/v. Such an aqueous solution is normally buffered saline such as phosphate buffered saline.

It is believed that most if not all other immunogenic adjuvants can advantageously be combined with the micelle-forming adjuvant (evidenced by the fact that as diverse adjuvants as alum and CPG both act synergistically with provax) but the non-micelle forming adjuvant is typically selected from a metal ion containing adjuvant and a DNA adjuvant. The metal ion containing adjuvant can e.g. be aluminum hydroxide, aluminium phosphate or calcium phosphate.

The invention consequently comtemplates immunogenic compositions, which comprise comprising an antigen in admixture with an adjuvant composition of the present invention - typically the antigen will be a peptide, polypeptide or protien {i.e. a proteinaceous antigen), but it is also contemplated that other immunogens used in vaccines and immunogenic compositions can be admixed with the inventive compositions. According to the present invention, an especially preferred antigen is an immunogenic RANKL analogue of the invention.

PREAMBLE TO EXAMPLES

In the examples is provided a detailed description of the identification and construction of a new RANKL variant (hRANKL-TB) that contains point mutations within 5 defined areas and is expressed as a soluble product from E.coli HMS174(DE3), as well as of a number of immunogenic variants derived from hRANKL-TB.

The vector system for E.coli expression is the commercially available pET28b+, where expression is controlled by the Lad repressor, and the T7 promoter.

Immunogenic RANKL Variants and Choice of Expression System

Once a point mutated RANKL that fulfilled the requirements was identified, it was used as the template for the construction of immunogenic variants using PADRE (SEQ ID NO: 6) as the inserted epitope.

After testing a large number of RANKL variants in E.coli HMS174(DE3) but obtaining no significant expression, it was decided to change the expression system to insect cells (S2 drosophila cells) using the p2ZOp2F vector as expression vector. Some of the variants were moved from the pET28b+ vector to the p2ZOp2F vector but new variants were also constructed in the p2ZOp2F vector.

-RA RANKL Constructs

N-terminal sequencing combined with Mass-Spectrometri revealed that a large fraction of the S2 expressed variants were lacking the expected first 2 amino acids (RA) with only a small fraction of the purified protein containing the N- terminal RA.

This could be caused by inaccurate cleavage of the signal peptide, and it was therefore decided to construct versions of the most promising RANKL variants without the N-terminal RA.

MATERIALS AND METHODS

Table 1, Raw Materials

Table 2: Composition of main culture medium

Items 1-8 are dissolved in the written order in approximately 50% of the final volume in RO water and mixed thoroughly until fully dissolved. The volume is then adjusted to the final volume (minus the volume added after autoclaving) and transferred to the fermentor and sterilized by autoclaving.

Items 9-11 are mixed and transferred aseptically into the cooled fermentor (37 ⁰ C). pH is adjusted to 7 with H ₃ PO ₄ and NaOH.

Table 3: Composition of pre culture medium

The desired volumes of the stock solutions, items 1-9, are transferred to a 1 I measuring flask. Add RO water to 955 ml . Measure pH and adjust to pH 7.0 if necessary. Stir the solution and transfer it to 4 1000 ml shake flask with 238 ml in each. After autoclaving, the non-autoclavable components, items 10-12, are added aseptically to the shake flasks.

Table 4: Composition of trace salt solution

The components are mixed in approximately 20 % of the final volume. Add acidified (pH 1) RO water to 1000 ml . The solution is stirred until all salts are dissolved. Transfer the solution to a IL blue cap bottle and autoclave it.

Table 5: Composition of Ferro sulfate:

Fermentation Conditions

An Over night culture in LB medium was used to inoculate 250 ml pre culture medium and grown to OD ₆ oo ~ 2-4 at 37 ⁰ C. 50 ml was then used to inoculate 1 defined medium in Infors fermentors, and grown to OD ₆ oo ~ 15-20 at 25 ⁰ C.

Equipment, including fermentors

Table 7 : Infors fermentor system

The InFors Labfors fermentor system consisting of 6 2L fermentors, each with a working volume of 0.5 - 1.6 L, and a Master Controlling Unit connected to a computer installed with software (Iris NT 4.1 for Windows) for data acquisition and -processing.

Beside the fermentors the following equipment were used : Gallenkamp shake incubator

Heraeus Function Line 37 ⁰ C stationary incubator

Sorvall RC5C Plus centrifuge

Biometra PCR machine

ABI Genetic Analyser 310 (DNA sequencing) Electrophoresis equipment (Hybaid)

Methods

SOE PCR

All constructs and mutants were made using SOE PCR technique, cf. Fig. 1.

To construct h RAN KL-TB- pET28b+, we have used hRPmod.Box3wt(pET28) (pl647, cf. SEQ ID NO: 144), which is the codon optimised RANKL containing mutations in 4 of the 5 regions, inserted into the pET28b+ vector, as a template.

First step in the construction of hRANKL-TB-pET28b+ was the setup of two separate PCR reactions using either oligos 1638 and 2356 or 1641 and 2360 as primer pairs (Fig. 1; reaction 1 and reaction 2); for sequence details regarding primers used herein, cf. table 14). We used Expand High fidelity polymerase in all SOE PCR reactions, run with the following 30 touchdown PCR cycles:

1. denaturation temp 94 ⁰ C for 30 sek.

2. annealing temperature 6O ⁰ C (-0,5 ⁰ C pr cycle, for 20 cycles), followed by 10 cycles annealing temp 5O ⁰ C for 30 sek.

3. extension temp, was 68 ⁰ C for 2 min (+ 5 sec pr cycle after the first 10 cycles).

The resulting PCR fragments were 644 bp and 639 bp in size, and were gel purified and used as templates for the second round of SOE PCR, using oligos 1638 and 1641 (Fig. 1; reaction 3). The PCR conditions were the same as the first round PCR, and the resulting 1258 bp large fragment was gel purified and digested with Xbal and Notl. This resulted in three fragments with the following sizes: 585 bp, 361 bp and 312 bp.

pET28b+ vector (p2029) purchased from Novagen was cut with Xbal and Ncol, gel purified and SAP treated (37 ⁰ C 15 min, 65 ⁰ C 20 min). The 585 bp fragment was gel purified and inserted into the above mentioned pET28b+ vector, and ligated overnight in a temperature cycler with cycles comprised of 30 seconds at

1O ⁰ C followed by 30 seconds at 3O ⁰ C according to SP016. The ligation product was transformed into HMS174(DE3) E. coli cells (30 min on ice, 2 min at 42 ⁰ C, 5 min on ice, followed by 1 hour incubation in 500 μl LB at 37 ⁰ C), plated out on kanamycin containing (60 μg/ml) LB Agar plates and incubated at 37 ⁰ C ON. Single colonies from the plates were inoculated into 5 ml LB media, with 60 μg kanamycin/ml, and inkubated at 37 ⁰ C ON shaken at approx 220 rpm. DNA was purified using Qiagen miniprep kits, and the relevant regions were sequenced to identify correct clones. Cf. Fig. 2 for a flow-chart description of the preparation of the RANKL variants.

Table 8: Construction summary

The sequences of all oligos can be found in the sequence listing and in Table 14 infra. The sequences of template plasmids p2075 and pll29 are set forth in SEQ ID NO: 142 and 143, respectively, and as mentioned above, the nucleic acid sequence of template plasmid pl647 is set forth in SEQ ID NO: 144.

EXAMPLE 1

Constructing hRANKL-TB

In the following, all references to amino acid numbering in human RANKL is according to SEQ ID NO: 2 unless otherwise indicated.

A synthetic hRANKL encoding gene containing 5 point mutations (A171S, A193G, N218D, I274V, D301E) was previously purchased and cloned into pET28b+. Expression experiments showed that very low levels of soluble RANKL variant were produced. As 4 of the 5 point mutations had previously been tested as

single point mutations without impairing soluble expression, focus was put on the fifth mutation (N218D) to determine whether this was the mutation resulting in the low expression levels.

Besides the N218 to D mutation, constructs mutating N218 to Q and A217 to S or G have been made in conjunction with the other 4 mutations mentioned above, and these all showed very low expression in rich medium in shake flasks at 37 ⁰ C. These were tested in fermentors (defined medium and 25 ⁰ C) and obtained expression levels below 1 mg soluble RANKL/I.

The template mentioned above is a codon optimised synthetic gene, and the wt hRANKL that has previously been expressed in high amounts (~ 200-400 mg/L) is from a non-codon optimised template. To test whether the codon usage could have an effect on translation, and thereby expression, both the soluble and insoluble fractions of the different fermentations were analysed by Western blotting, and it was found that the overall expression by large is comparable, but that the RANKL is found in the insoluble fractions in the cases where there is low expression of soluble protein. This rules out that impaired translation is the reason for the low expression.

It was then decided to test 5 point mutations (A217S, A193G, A217G, I274V & D301E) as single mutations, to identify whether one of the mutations was causing problems in the expression system. The previous expression analyses were made in E. coli BL21-Star in rich media in shake flasks. It was chosen to test them in E. coli HMS174(DE3) in defined media in fermentors at 25 ⁰ C (cf. the results in Fig. 3). The conclusion of this experiment is that the A217G mutation is the one causing the largest reduction in soluble expression (-30-40 mg/l).

It was then decided to attempt an alternative approach to avoid the A217G (or N218D) mutations by replacing it with 2 separate mutations. The choice fell on a variant where the L215 is mutated to I and 1219 is mutated to V. Hence this template variant includes 6 point mutations (A171S, A193G, L215I, I219V, I274V, D301E) compared with wild-type human RANKL. Further, the template is truncated whereby amino acids 1-139 of SEQ ID NO: 2 are deleted.

The effect of these mutations displayed very encouraging results. Using fermentation conditions as described below, this double mutant combined with the other 4 mutations, expressed ~ 200-500mg/L soluble mutated hRANKL protein. The levels were confirmed both by quantitative ELISA and OPG binding ELISA, and plasmids purified from the samples showing the highest levels of RANKL expression (21 hours post induction) were sequence verified to ensure that it was indeed the mutated version of RANKL that was expressed.

The template was named hRANKL-TB (MR#2875, p2181). This template was subsequently used as template for constructing new immunogenic hRANKL variants. Its coding sequence is set forth in SEQ ID NO: 3 and the encoded, truncated RANKL variant is set forth in SEQ ID NO: 4.

All testing was done in E. coli HMS174(DE3) cells in fermentors using defined media at 25 ⁰ C both pre- and post-induction. 1 ml samples were taken, opened by sonication and analysed for expression of soluble RANKL using quantitative ELISA and/or OPG binding ELISA.

EXAMPLE 2

New immunogenic RANKL Variants

It was decided to construct new RANKL variants (cf. Fig. 4), first in the various loop regions in the TNF-like (structured) domain, and later in the supposedly unstructured stalk region.

"New" RANKL Loop Variants

A large number of new immunogenic RANKL variants have been constructed, addressing new loops in the RANKL protein. The rationale behind these variants is to leave the first and last couple of amino acids in each loop intact, as analysis using modelling software to model protein structures suggested that changing these amino acids tends to displace the β-strands adjacent to the loop.

Loop 4 Construct:

Loop 4 has the sequence RSGEEISIEVSNPSLLDPDQDATYFGAFKVRDID (SEQ ID NO: 85). Based on this sequence, variant hRP 2.3 was constructed where the above-underlined S is substituted by PADRE (underlined) : RSGEEISIEVSNPAKFVAAWTLKAAALLDPDQDATYFGAFKVRDID (SEQ ID NO: 86).

DE Loop Constructs:

The DE loop has the sequence YLQLMVYVJKTSIKIPSSHTLMKGGS (SEQ ID NO:

87), where letters marked in underline, bold and italics were substituted to construct variants hRP 4.3, 4.4 and 4.5. hRP 4.2 was constructed by substituting the underlined TK with PADRE (underlined) :

YLQLMVYVAKFVAAWTLKAAATSIKIPSSHTLMKGGS (SEQ ID NO: 88),

Variant hRP 4.3 was constructed by substituting the KI in italics with PADRE

(italics) :

YLQLMVYVTKTS \AKFVAAWTLKAAAPSSHTUAKGGS (SEQ ID NO: 89), and Variant hRP 4.4 was constructed by substituting the IKI in bold with PADRE

(bold) :

YLQLMVYVTKTSAKFVAAWTLKAAAPSSHTLMKGGS (SEQ ID NO: 90).

EF Loop Constructs:

The EF loop has the amino acid sequence PSSHTLMKGGSTKYWSG/VSEFHFYSINVGGFFK (SEQ ID NO: 91), where letters marked with underline, bold and italics were substituted to arrive at variants hRP3.5, hRP3.6 and hRP3.7. hRP3.5 was constructed by substituting the GN in italics with PADRE (italics) :

PSSHTLMKGGSTKYWSλKFWVWTLK/W*SEFHFYSINVGGFFK (SEQ ID NO: 92), hRP3.6 was constructed by substituting the underlined SGN with PADRE

(underlined) :

PSSHTLMKGGSTKYWAKFVAAWTLKAAASEFHFYSINVGGFFK (SEQ ID NO: 93), and hRP3.7 was constructed by substituting the G in bold with PADRE (bold) :

PSSHTLMKGGSTKYWSAKFVAAWTLKAAANSEFHFYSINVGGFFK (SEQ ID NO: 94).

CD Loop Constructs:

The CD loop has the amino acid sequence VCFRH H ETSG D LATEYLO LMVYVT (SEQ

ID NO: 95), where letters marked with underline are individually substituted to arrive at constructs hRP6.4, hRP6.5, hRP6.6, hRP6.7 and hRP6.8. hRP6.4 was constructed by substituting the underlined A with PADRE (underlined)

VCFRHHETSGDLAKFVAAWTLKAAATEYLOLMVYVT (SEQ ID NO: 96), hRP6.5 was constructed by substituting the underlined L with PADRE

(underlined) :

VCFRHHETSGDAKFVAAWTLKAAAATEYLOLMVYVT (SEQ ID NO: 97), hRP6.6 was constructed by substituting the underlined G with PADRE

(underlined) :

VCFRHHETSAKFVAAWTLKAAADLATEYLOLMVYVT (SEQ ID NO: 98); hRP6.7 was constructed by substituting the underlined S with PADRE

(underlined) :

VCFRHHETAKFVAAWTLKAAAGDLATEYLOLMVYVT (SEQ ID NO: 99), and hRP6.8 was constructed by substituting the underlined D with PADRE

(underlined) :

VCFRHHETSGAKFVAAWTLKAAALATEYLOLMVYVT (SEQ ID NO: 100).

All variants were constructed in the above-discussed hRANKL-TB template, cloned into the pET28b+ vector, and transformed into HMS174(DE3) E.coli cells:

Table 9 : "New" RANKL loop variants

The variants were tested for soluble expression in defined media in shakeflasks at 25 ⁰ C, and anlysed with Western blot and quantitative ELISA. No significant soluble expression was seen, all RANKL protein was present as inclusion bodies.

Further RANKL Variants 7.2-7.6

5 further variants were produced, all placing PADRE in the AA" loop, which is supposed to be the largest and the most flexible loop in the RANKL protein.

AA" loop: MRAEKAMVDGSWLDLAKRSKLEAOPFAHLTINatatosαshkvslSSWYHDRGWAKISN (SEQ ID NO: 101), where amino acids marked with italics, underline, bold and lower case are substituted with PADRE (marked in an identical manner) in constructs hRP7.2, .3, .4, and .6. In hRP7.5, PADRE is inserted in the sequence.

hRP7.2: MRAEKAMVDGSWLDLAKRSKLEAOPFAHLTINATDIPAKFVAAWTLKAAAWYHDRGWAK

ISN (SEQ ID NO: 102) hRP7.3:

MRAEKAMVDGSWLD LAKRSKLEAQPFAHLTINAAKFVAAWTLKAAAGSHKVSLSSWYHD

RGWAKISN (SEQ ID NO: 103) hRP7.4:

MRAEKAMVDGSWLDLAKRSKLEAQPFAHLTINATDIPSGSHAKFVAAWTLKAAAWAK I

SN (SEQ ID NO: 104) hRP7.5:

MRAEKAMVDGSWLDLAKRSKLEAQPFAHLTINATDIPSAKFyAAWTLKAAAGSHKVS LSS WYHDRGWAKISN (SEQ ID NO: 105) hRP7.6:

MRAEKAMVDGSWLDLAKRSKLEAQPFAHLTINakfvaawtlkaaaSSWYHDRGWAKI SN

(SEQ ID NO: 106)

All variants were contructed using an SOE-PCR procedure, in pET28b(+) and transformed into HMS174(DE3).

Table 10: hRP7.2 to hRP7.6 variants

Expression experiments in fermentors, with defined media at 25 ⁰ C, showed low (<5mg/L) or no expression of soluble RANKL protein, when analysed by quantitative ELISA.

After the above mentioned intensive work done to find a RANKL variant with PADRE as epitope, is was concluded that no such variants were found, that were expressed solubly in acceptable amounts.

EXAMPLE 3

Switching Expression System and Vector

As consequence of the sub-optimal results when using E.coli as expression system, it was decided to switch expression system to insect cells.

Expression in insect cells is performed from the p2ZOp2F vector, using the OplE2 promoter and the Bip signal sequence to target the recombinant protein for secretion.

Previous experiments have shown some expression of immunogenic RANKL variants in S2 cells, but these were not produced in the TB template.

All variants that were made in the TB template (see tables 9 and 10) were therefore moved from the pET28b+ vector to the p2ZOp2F vector, in fusion with the Bip signal sequence. The variants were moved using SOE PCR to fuse the Bip signal sequence to RANKL, and inserted into p2ZOp2F with restriction digest and ligation. This means that the codon usage is the same as in the E.coli expression vector.

The previously constructed hRP3.1 and hRPβ. l variants were reconstructed in the hRANKL-TB template, as these variants had been seen to give some expression in S2 cells previously when made in the non-mutated template.

DNA of all variants was made using Qiagen Endotoxin free Maxiprep and used to transfect S2 cells.

All variants are stored at -8O ⁰ C under their respective MR#.

Table 11 : Variants moved to the p2ZOp2F vector

Constructing RANKL Stalk Variants for Expression in S2 Cells

12 variants were constructed where PADRE has been inserted in the N-terminal stalk region of RANKL. This region is expected to be more flexible than the core part of the RANKL molecule.

The following stalk RANKL variants have been constructed in p2ZOp2F.

wtRANKL stalk amino acid sequence: RAEKAMVDGSWLDLAKRSKLEAQ (SEQ ID NO: 107)

amino acid sequence Variant name

RAEKAKFVAAWTLKAAAKRSKLEAQ (SEQ ID NO: 108) hRPl-TB-p2ZOp2F RAKFVAAWTLKAAAKRSKLEAQ (SEQ ID NO: 109) hRP1.4-p2ZOp2F RAEKAKFVAAWTLKAAAKLEAO (SEQ ID NO: 110) hRP1.5-p2ZOp2F RAEKAKFVAAWTLKAAASKLEAQ (SEQ ID NO : 111) hRP1.6-p2ZOp2F

RAEKAMVDGSWLDLAKFVAAWTLKAAAO (SEQ ID NO: 112) hRP1.7-p2ZOp2F RAEKAMAKFVAAWTLKAAAKLEAO (SEQ ID NO: 113) hRP1.8-p2ZOp2F RAEAKFVAAWTLKAAAKRSKLEAO (SEQ ID NO: 114) hRP1.9-p2ZOp2F RAEAKFVAAWTLKAAARSKLEAO (SEQ ID NO: 115) hRP1.10-p2ZOp2F RAEKAMVDGAKFVAAWTLKAAAO (SEQ ID NO: 116) hRPl. ll-p2ZOp2F RAEKAMVDGSAKFVAAWTLKAAAO (SEQ ID NO: 117) hRP1.12-p2Zop2 RAEKAMVDAKFVAAWTLKAAAO (SEQ ID NO: 118) hRP1.13-p2ZOp2F AKFVAAWTLKAAALAKRSKLEAO (SEQ ID NO: 119) hRP1.14-p2ZOp2F

The full length amino acid sequences of these immunogenic variants are set forth in SEQ ID NOs: 120-131.

DNA of all variants was made using Qiagen Endotoxin free Maxiprep and used to transfect S2 cells.

All variants are stored at -8O ⁰ C under their respective MR#.

Table 12: RANKL stalk variants in the p2ZOp2F vector.

Construction of His Tagged Stalk Variants

The stalk variants that were found to be expressed in acceptable amounts, were also constructed in His tagged versions so as to facilitate purification of expressed protein by IMAC (immobilized metal affinity chromatography).

DNA of all variants was made using Qiagen Endotoxin free Maxiprep and used to transfect S2 cells.

All variants are stored at -8O ⁰ C under their respective MR#.

Table 13: RANKL His tagged stalk variants

hRPl.8, hRPl. ll, hRP1.12 and hRP1.13 were made by cutting out the gene using Ncol/Notl digest and inserted into hRANKL-TB-HIS-p2ZOp2F, partially cut with Ncol/Notl. The other constructs were made using SOE-PCR (see table 8).

Constructing hRANKL-TB- I248-p2ZOp2F

To further deactivate RANKL we have constructed a point mutated version of hRANKL-TB where 1248 is mutated to D. This mutation has been reported to decrease activity 8 fold, when compared to wtRANKL. The mutation was made using SOE-PCR in the p2ZOp2F vector, and named hRANKL-TB-I248D-p2ZOp2F, MR#2981, p2354. DNA was made using Qiagen Endotoxin free Maxiprep and used to transfect S2 cells.

Constructing -RA Variants for Expression in S2 Cells

Some heterogenicity in the N-terminal sequence was seen when analysing with N-terminal sequencing and MALDI-TOF. A possible explanation to the heterogenicity could be inaccurate removal of the signal peptide, resulting in 2 versions of the expressed protein, were a small fraction contained the expected full length sequence, but were the largest fraction lacked the first 2 amino acids of the stalk region, namely the RA. To obtain a more homogenous product, versions of the most promising RANKL variants were produced, (hRRP1.5-RA- p2ZOp2F, hRRP1.6-RA-p2ZOp2F, hRRPl. ll-RA-p2ZOp2F, hRRP1.12-RA- p2ZOp2F, hRRP1.13-RA-p2ZOp2F, corresponding to the RANKL variant amino acid sequences set forth in SEQ ID NOs: 133, 134, 139, 140 and 141, respectively.

SEQUENCES

The sequences of truncated and full-length human RANKL are set forth in SEQ ID NOs 1 and 2, respectively. The sequence of the hRANKL-TB encoding gene is set forth in SEQ ID NO: 3:

CGTGCCGAGAAAGCCATGGTGGATGGTAGCTGGCTGGATCTGGCCAAGCGCTCCAAG

TTGGAGGCCCAACCATTTGCCCACCTGACCATTAATAGCACCGATATCCCAAGCGGT AG

TCATAAGGTTTCCTTGTCAAGCTGGTATCATGATCGCGGCTGGGGCAAGATTTCAAA TA TGACCTTCAGCAATGGCAAGTTGATTGTCAATCAAGACGGCTTTTATTATATTTATGCGA ACGTCTGCTTTCGCCACCACGAGACTTCCGGTGATCTTGCAACCGAGTACCTGCAACTT ATGGTCTACGTGACTAAGACCTCCATTAAGATCCCGTCATCCCATACCTTGATGAAGGG TGGGTCGACTAAGTACTGGAGTGGCAATTCCGAGTTTCATTTCTACAGCGTGAATGTGG GTGGCTTTTTTAAGCTTCGGAGCGGCGAGGAGATCTCCATCGAGGTTAGTAATCCCAG CCTGCTGGATCCCGAGCAGGATGCCACCTACTTCGGTGCCTTCAAAGTTCGCGATATTG ATTAA (SEQ ID NO: 3) - the amino acid sequence encoded by this gene is set forth in SEQ ID NO: 4.

The sequence of the PADRE encoding gene is set forth in SEQ ID NO: 5 and the amino acid sequence of PADRE is set forth in SEQ ID NO: 6.

The sequences of the oligos used herein are provided in table 14, and the sequences of the these oligonucleotides are from top to bottom also set forth in SEQ ID NOs: 7-84.

TABLE 14

Name Oligo Sequence

1638 GGTGTCCGGGATCTCGACGCTCTCCC 1641 GCCGGCGAACGTGGCGAGAAAGG 1892 CCTGCGTTATCCCCTGATTCTGTG 1908 TATTATCTGTATTCCAACATCTGCTTT 1909 AAAGCAGATGTTGGAATACAGATAATA 1910 TATTATCTGTATGGCAACATCTGCTTT 1911 AAAGCAGATGTTGCCATACAGATAATA 1912 TATCTGTATGCGCAGATCTGCTTTCGC 1913 GCGAAAGCAGATCTGCGCATACAGATA 2265 GTGCCGGACAACACCCTGGCCTG 2356 GACGTTCGCATAAATATAATAAAAGCCGTCTTGATTGACAATC 2360 CTTTTATTATATTTATGCGAACGTCTGCTTTCGCCACCACGAGACTTCCGG 2388 GCCAAGTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTTGGTATCATGATCGCGGCTGG GGCAAG 2389 AGCTGCGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCTGGGATATCGGTGCTATTAAT GGTCAG 2407 CGTGGCCGCTTGGACCCTGAAGGCCGCAGCTTGGTACCATGATCGTGGCTGGGCGAAAAT TTC 2408 GGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCCGGGATATCCGTCGCATTAATCGTC 2409 GTGGCCGCTTGGACCCTGAAGGCCGCAGCTGGTAGTCATAAAGTTTCGTTGTCAAG 2410 GCGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCATTAATCGTCAGGTGGGCAAACGGC

2411 CGTGGCCGCTTGGACCCTGAAGGCCGCAGCTTGGGCGAAAATTTCAAACATGACCTTC

2412 CTGCGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCATGACTACCGCTCGGGATATCCG TC 2413 GCGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCGCTCGGGATATCCGTCGCATTAATC G 2414 GTGGCCGCTTGGACCCTGAAGGCCGCAGCTTCAAGCTGGTACCATGATCGTGGCTGGGCG 2474 GTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTACCGAGTACCTGCAACTTATGGTC 2475 GGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCAAGATCACCGGAAGTCTCGTGGTGG 2476 GTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTGCAACCGAGTACCTGCAACTTATGG 2477 CCTTCAGGGTCCAAGCGGCCACGAACTTGGCATCACCGGAAGTCTCGTGGTGGCGAAAG 2478 GTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTGATCTTGCAACCGAGTACCTGCAAC 2479 CGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCGGAAGTCTCGTGGTGGCGAAAGCAG 2480 GTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTGGTGATCTTGCAACCGAGTACCTGC 2481 CGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCAGTCTCGTGGTGGCGAAAGCAGACG 2482 CGTGGCCGCTTGGACCCTGAAGGCCGCAGCTCTTGCAACCGAGTACCTGCAACTTATG 2483 GCGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCACCGGAAGTCTCGTGGTGGCGAAAG 2484 CGTGGCCGCTTGGACCCTGAAGGCCGCAGCTCTGGATCCCGAGCAGGATGCCACCTAC

2485 GGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCGCTGGGATTACTAACCTCGATGGAG

2486 CGTGGCCGCTTGGACCCTGAAGGCCGCAGCTCTGCTGGATCCCGAGCAGGATGCCAC

2487 CGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCGGGATTACTAACCTCGATGGAGATC

2488 CGTGGCCGCTTGGACCCTGAAGGCCGCAGCTTCCGAGTTTCATTTCTACAGCGTGAATG

2489 CGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCACTCCAGTACTTAGTCGACCCACCC

2490 GGCCGCTTGGACCCTGAAGGCCGCAGCTACCTCCATTAAGATCCCGTCATCCC

2491 CTGCGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCCACGTAGACCATAAGTTGCAGGT AC

2492 GTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTCCGTCATCCCATACCTTGATGAAGGG

2493 GGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCGGAGGTCTTAGTCACGTAGACCATAAG

2495 GCGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCAATGGAGGTCTTAGTCACGTAGACC

2497 GCGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCCCAGTACTTAGTCGACCCACCCTTC

2498 GTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTAATTCCGAGTTTCATTTCTACAGCG

2509 CCCGAGCGAGAGGCCAACAAAGGCCAC

2510 GTGGCCTTTGTTGGCCTCTCGCTCGGGCGTGCCGAGAAAGCCATGGTGGATGG

2536 GTTGCAGCTTGGACCCTGAAGGCCGCTGCAGCAACCGAGTACCTGCAACTTATGGTCTAC

2537 GCGGCCTTCAGGGTCCAAGCTGCAACGAACTTCGCAGTCTCGTGGTGGCGAAAGCAGACG

2538 GCGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCCGACCCACCCTTCATCAAGGTATGG

2554 GTGACTAAGACCTCCATTAAGGACCCGTCATCCCATACCTTGATGAAG

2555 CTTCATCAAGGTATGGGATGACGGGTCCTTAATGGAGGTCTTAGTCAC

2573 GTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTAAGCGCTCCAAGTTGGAGGCCCAACC

2574 CGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCTTTCTCGGCACGCCCGAGCGAGAGGC C

2575 CGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCACGCCCGAGCGAGAGGCCAACAAAGG CCAC

257 6 GTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTAAGTTGGAGGCCCAACCATTTGCCCA CC

2577 GTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTTCCAAGTTGGAGGCCCAACCATTTG

2578 GTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTCAACCATTTGCCCACCTGACCATTAA TAGC

257 9 CGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCCAGATCCAGCCAGCTACCATCCACC

2580 CGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCCATGGCTTTCTCGGCACGCCCGAGCG

2581 CGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCCTCGGCACGCCCGAGCGAGAGGCCAA C

2582 GTTCGTGGCCGCTTGGACCCTGAAGGCCGCAGCTCGCTCCAAGTTGGAGGCCCAACCATT TG

2583 CGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCACCATCCACCATGGCTTTCTCGGCAC

2584 CGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCGCTACCATCCACCATGGCTTTCTCGG C

2585 CGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCATCCACCATGGCTTTCTCGGCACGCC C

2586 CGGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCCCCGAGCGAGAGGCCAACAAAGGCCA C

2622 GGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCTTGATGTTGATGTTGATGTTGATGTTG

2628 GGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCCTCGGCACGTTGATGTTGATGTTGATG

2630 GGCCTTCAGGGTCCAAGCGGCCACGAACTTGGCTTTCTCGGCACGTTGATGTTGATGTTG

2 681 CAACATCAACATCAACATCAACATCAACGTGCCGAGAAAGCCATGGTGGATGGTAG

2682 CTACCATCCACCATGGCTTTCTCGGCACGTTGATGTTGATGTTGATGTTGATGTTG

2700 GTGGCCTTTGTTGGCCTCTCGCTCGGGGAGAAAGCCAAGTTCGTGGCCGCTTG

2701 GTGGCCTTTGTTGGCCTCTCGCTCGGGGAGAAAGCCATGGTGGATGGTGCCAAG

2702 GTGGCCTTTGTTGGCCTCTCGCTCGGGGAGAAAGCCATGGTGGATGGTAGCG

2703 GTGGCCTTTGTTGGCCTCTCGCTCGGGGAGAAAGCCATGGTGGATGCCAAGTTC

2704 GTGGCCTTTGTTGGCCTCTCGCTCGGGGAGAAAGCCATGGTGGATGGTAGCTG

2705 GTGGCCTTTGTTGGCCTCTCGCTCGGGGAGAAAGCCATGGTGGATGGTAGTTG

901 GGCCTTTTGCTGGCCTTTTGCTC

EXAMPLE 4

Expression in Drososphila cells

Drosophila S2 cells obtained from ATCC were resuscitated and expanded to establish a Drosophila S2 cell bank. Hereafter, the cells were transfected with eight different human RANKL expression vectors and stable polyclonal cell lines were established by adding zeocin to the cells 24 hours post-transfection. The established polyclonal cell lines were evaluated with respect to production of human RANKL variant protein, and the four cell lines expressing hRPl.l l, hRP1.12, hRPl. ll-RA and hRP1.12-RA, respectively, were diluted and seeded in 96-well plates and incubated. Clones from approved 96-well plates were picked and evaluated further in 12-well plates and tissue culture flasks.

Research Cell Bank Stability

Approximately 15 clones expressing each of the four different hRANKL variants, hRPl. ll, hRP1.12, hRPl. ll-RA and hRP1.12-RA, respectively, were selected based on their level of protein expression to enter a stability study. The stability study was designed to evaluate the stability of the clones with respect to expression of hRANKL for approximately 90 days (up to 62 generations). Cells were propagated at 8 x 10 ⁶ cells/ml in shake flasks twice weekly. Samples were taken weekly and saved for later protein analysis (ELISA). After five weeks, the collected samples were evaluated using ELISA and clones with low expression levels were stopped. Thirteen clones expressing hRPl.l l, eleven clones expressing hRP1.12, five clones expressing hRPl .ll-RA and six clones expressing hRP1.12-RA completed the entire study.

In general, cell densities increased from 8 x 10 ⁶ cells/ml to 3-4 x 10 ⁷ cells/ml in 3-4 days for all clones throughout the 90-day study, with few exceptions.

Expression levels varied greatly between clones and in general, few clones expressed high levels of hRANKL (>40 mg/L). Also the number of high expressing clones varied between variants and a higher number of high expressing clones were found between clones expressing hRPl .ll than between clones expressing hRP1.12, hRPl. ll-RA and hRP1.12-RA. Several clones showed stability with respect to expression levels throughout the 90-day study. The highest expressing stable clones were found between clones expressing hRP1.12-RA (clone 166: average 85 mg/L or 1.65 mg/10 ⁶ cell/d and clone 162: average 75 mg/L or 1.2mg/10 ⁶ cells/d. The remaining clones expressing hRP1.12-RA had a much lower expression level of up to 20 mg/L or 0.4 mg/E6 cells as was the case for clones expressing hRP1.12. Several clones expressing hRPl.ll had expression levels of 40-50 mg/L or 0.8-0.9 mg/E6 cells, but only one of these high expressing clones were stable (clone 7). Few clones expressing hRPl. ll-RA expressed well and only one of the clones that completed the stability study was stable (11 mg/L or 0.22 mg/10 ⁶ cells/d).

To further confirm the stability of clone 162 and clone 166, cells from different time points throughout the stability study were analysed with respect to gene copy number at different time points before, during and at the end of the stability study.

The stability study was performed to cover the stability of clones beyond end- point of production. From a single vial of the RCB (4 x 10 ⁷ cells/ml) to a 200 vial MCB (4 x 10 ⁷ cells/ml) it takes eight to nine generations (assuming 93% viability). Accordingly, it takes another eight generations to establish a 200 vial WCB (4 x 10 ⁷ cells/ml) from the MCB. From WCB (or MCB) it takes another nine generations to inoculate a 100 L bioreactor at 15 x 10 ⁶ cells/ml and another 5- 10 generations (depending on the process) to finish a 20-day production run, leaving a margin of at least 25 generations to the maximum number covered here. The stability study was run once in shake flasks when evaluated by specific expression level (each time point analysed in double estimation). However, each time the process is run at larger scale in a bioreactor the stability of the cells is confirmed for the length of the run by both specific RANKL variant protein production and total protein production.

With a stability covering 42 generations (WCB = generation 0), the clone seems suitable for even a very large production size. Inoculation of a 100,000 m ³ production vessel will in theory require 28 generations from resuscitation to inoculation, and with a 20-day production run (5-10 generations) it leaves a margin of at least ten generations.

To confirm that clone 162 and clone 166 were true clones, cells were resuscitated, cultured for a week in T-flasks, diluted and seeded in 96-well plates and incubated. Clones from approved 96-well plates were picked and evaluated further.

It can be concluded that a number of stable clones were established with respect to protein expression for up to 62 generations, including clone 162 and clone 166.

It is likely that it is pure coincidence that the two high expressing clones are expressing hRP1.12-RA {i.e. the expression product has SEQ ID NO: 40) and not hRP1.12 or one of each, since the proteins are so similar and since the remaining clones expressing hRP1.12-RA have expression levels comparable to those of hRP1.12.

Fermentation

A vial of the research cell bank containing 4 x 10 ⁷ cells in 1 ml freeze medium (40% Excell™ 420 + 50% fetal bovine serum, FBS + 10% DMSO) is removed from liquid nitrogen and thawed. The cells are transferred to a T25 flask containing 4 ml 23 ⁰ C medium (Excell™ 420 + 10% FBS). The cells are centrifuged to remove DMSO, followed by resuspension in 5 ml medium (Excell™ 420 + 10% FBS) in a T25 flask and incubated at 23 ⁰ C for 16-24 hours.

The cells are then expanded in three passages through T-flasks. This is followed by a number of passages through shake flasks. At this point FBS is removed as media additive to Excell™ 420 medium in the process. The number of passages in shake flasks is determined by the final cell count /ml that will be used for

inoculating the 1.5L reactor. If a final concentration of 2 x 10 ⁶ cells/ml is used six passages (21-24 days) are needed, while 7 passages (29-36 days) are needed when inoculating to a final concentration of 1.0 x 10 ⁷ cells/ml.

The cell suspension culture from shake flasks is aseptically transferred to the sterilised 2L bioreactor vessel to a final viable cell count of 2-3 x 10 ⁶ cells/ml in 1.5 L working volume. Fresh medium (+ 0.5ml antifoam PD30/ litre) is added to the shake flask culture to make up the 1.5 L.

The 2 I bioreactor is run at the following conditions for three days until the viable cell count is >1.8E7 cells per ml :

pH is controlled at 6.5 ± 0.1 with 5% H3PO4 and 0.5 M KOH.

Dissolved Oxygen Tension (DOT) is kept at 10% with pure oxygen.

Agitation is set to 100 rpm.

The temperature (T) is set to 23 ± I ⁰ C

Air is used as overlay for the fermentation

Daily in process samples are taken to check pH, cell count in the reactor vessel, protein and total protein production, and cell viability.

With this method of cell expansion it takes three days for the cells to expand to the point where perfusion can be started.

When the cell number in the bioreactor exceeds 1.8 x 10 ⁷ viable cells/ ml_, perfusion of fresh medium (Excell™ 420 + 0.5ml antifoam PD30/ litre) is commenced at a rate of 0.5 bioreactor volumes per day for 1 day, followed by 1 reactor volume per day for one more day. In process samples are taken daily from the bioreactor to check pH, cell count (in the reactor vessel and in the perfusion), protein and total protein production, and cell viability.

After the initial two days of perfusion at 0.5 and 1 reactor volume per day, the perfusion rate is set to 1.5 reactor volumes per day, and maintained at this level for the rest of the run. Small upward adjustments in perfusion rate, not

exceeding 10% per day, can be implemented if the glucose concentration in the bioreactor decreases to below 1.5g/l. A bleed will also be implemented as needed when the viable cell count exceeds 70 x 10 ⁶ /ml. The initial bleed rate is set to 0.13 reactor volumes per day, and adjusted daily to maintain a viable cell count of between 50 x 10 ⁶ cells/ml and 80 x 10 ⁶ cells/ml.

The perfusate (~2.3 L) is harvested daily and centrifuged at 4000 rpm for 30 minutes. The centrifuged perfusate is then filtered through a two filter cascade, firstly through a 0.8/0.65 μm pre-filter and then through a 0.45/0.20 μm clarification filter, after which it is stored at -2O ⁰ C. In process samples are taken daily from the bioreactor to check pH, cell count (in the reactor vessel and in the perfusion), protein and total protein production, and cell viability. Processing the clarified perfusate through the first step of the downstream process before freezing would be an alternative to directly freezing the clarified perfusate daily. This would also greatly reduce the volume of liquid to be frozen and stored.

With the above-described perfusion process potentially ten harvests of 15 L can be harvested in 15 days from a 10 L tank. The process is being optimised with regards to inoculation density of cells to minimise number of days. If the expression level is 40 mg/l on average when harvesting 15 L each day for 10 days, the total yield of RANKL variant protein is 6 g.

Materials used in fermentation

Foetal bovine serum, used in cell culture during revival and early preculture is supplied by Life Technologies. It is of New Zealand origin, gamma irradiated and is obtained with a certificate of suitability that meets current EU guidelines. All buffer components and media additions are of USP grade (or Ph Eur). Excell™ 420 media and buffer components are acceptable for GMP production and could be directly transferred to a CMO. Filters used in the processes were selected to be up-scaled and can be used for biopharmaceutical manufacturing.

Results and Evaluation

Process optimisation has to date been performed using the initial stable polyclonal cell line expressing hRP1.12. This process has been applied to the monoclonal lead candidate (clone 166) and been tested in one run. The yields achieved to date for clone 166, 1.12-RA, were on average 80 mg/L for the perfusates from day 3 - day 9 post perfusion initiation. The total protein in the perfusates is below 800 mg/L during the run, leading to RANKL specific productivity being 10-20% of the total protein produced. Cell viability was maintained above 90% for the whole run.

EXAMPLE 5

Purification of RANKL variants

The following description of the optimum purification process relates to purification of one single RANKL variant (SEQ ID NO: 140) obtained from the fermentation described in Example 4, but the purification process is generally applicable for all disclosed variants herein.

Source of Fermentation Supernatant

Fermentations from transformed Drosophila S2 (clone S2pZOp-166hRP1.12-RA) were used in the following . Fermentations were made in a 5 I Braun Biostat fermentor at 4.5 I working volume and 2.5-3.5 x 10 ⁶ cells/ml. The cells were then expanded to 20-30 x 10 ⁶ cells /ml, after which perfusion was initiated at 0.5 RV/d (reactor volumes per day) for one day. The perfusion rate was then increase to 1 RV/d for one more day, before being set to the operating condition of 1.5RV/d total perfusion rate for the rest of the process. The fermentation run lasted 14 days, with 11 days of perfusion of which the first 2 perfusates were discarded.

Purification Process

Three downstream purification experiments were run. The process comprises the following steps: SP-Sepharose FF Cation Exchange (CEX), Source 3OQ Anion Echange (AEX), Blue Sepharose FF affinity column (AC), and a Superdex 200 prep grade size exclusion (SEC). Moreover, two concentration steps before and after SEC were conducted by means of Tangential Flow-Filtration (TFF). Finally, a viral clearance filtration was implemented as last step.

Materials and Media Compositions

Raw materials used in the downstream purification process and media compositions (buffer preparations) were the following :

Raw materials used in protein purifications:

Media Compositions

100 mM Na-Acetate, pH 5.5 buffer (for CEXI Sodium Acetate 8.2 g/L

Acetic Acid 100% Mix sodium acetate with MQ water and adjust pH to 5.5 with acetic acid. Add MQ water to a final volume of IL. Check pH to be 5.5. Filtrate through 0.22 μm Filtertop.

Storage: Room temperature

Expire date: 1 month. Inspect visually regulary and discard if turbid or particulate.

50 mM Na-acetate, pH 5.5 buffer (for CEX^ Sodium Acetate 4.1 g/L

Acetic Acid 100%

Mix sodium acetate with MQ water and adjust pH to 5.5 with acetic acid. Add MQ water to a final volume of IL. Check pH to be 5.5. Filtrate through 0.22 μm Filtertop.

Storage: Room temperature

Expire date: 1 month. Inspect visually regulary and discard if turbid or particulate.

100 mM Tris-HCI, pH 7.0 buffer (for CEXI

Tris 12.1 g/L

Mix Tris with MQ water and adjust pH to 7.0 with 6M HCI or 1OM NaOH. Add MQ water to a final volume of IL. Filtrate through 0.22 μm Filtertop.

Storage: Room temperature Expire date: 1 month. Inspect visually regulary and discard if turbid or particulate.

50 mM Tris-HCI, pH 8.0 buffer (for AEXI Tris 6.1 g/L

Mix Tris with MQ water and adjust pH to 8.0 with 6M HCL. Add MQ water to a final volume of IL. Filtrate through 0.22 μm Filtertop.

Storage : Room temperature

Expire date: 1 month. Inspect visually regulary and discard if turbid or particulate.

50 mM Tris-HCI, 200 mM NaCI, pH 8.0 buffer (for AEXI Tris 6.1 g/L

NaCI 11.7 g/L

Mix Tris with MQ water and adjust pH to 8.0 with 6M HCI . Add MQ water to a final volume of IL. Filtrate through 0.22 μm Filtertop. Storage : Room temperature Expire date : 1 month . Inspect visually regulary and discard if turbid or particulate.

50 mM Tris-HCI, pH 7.5 buffer (for Affinity Chromatography ¹ ) Tris 6.1 g/L

Mix Tris with MQ water and adjust pH to 7.5 with 6M HCL. Add MQ water to a final volume of IL. Filtrate through 0.22 μm Filtertop. Storage : Room temperature

Expire date: 1 month. Inspect visually regulary and discard if turbid or particulate.

50 mM Tris-HCL, 400 mM NaCI, pH 7.5 buffer (for Affinity Chromatography ¹ ) Tris 6.1 g/L

NaCI 23.4 g/L

Mix Tris with MQ water and adjust pH to 7.5 with 6M HCI. Add MQ water to a final volume of IL. Filtrate through 0.22 μm Filtertop.

Storage : Room temperature Expire date: 1 month. Inspect visually regulary and discard if turbid or particulate.

20 mM Tris-HCI, 150 mM NaCI, pH 7.5 (for SEC and Virus filtration) Tris 2.4 g/L

NaCI 8.7 g/L Mix Tris and NaCI with MQ water and adjust pH to 7.5 with 6M HCI . Add MQ

water to a final volume of IL. Filtrate through 0.22 μm Filtertop.

Storage: Room temperature

Expire date: 1 month. Inspect visually regulary and discard if turbid or particulate.

Scale of Purification

*** The size of SP-Sepharose FF was used to apply 5 L (2.5 L fermentation + 2.5 L buffer) of starting material.

The volume recovered after each SP-Sepharose run was 160 ml meaning that 500ml corresponds to 3 combined SP-Sepharose pools.

** Source 30Q intermediate pool was diluted five times before applying onto the Blue-Sepharose column

* Blue-Sepharose intermediate pool was concentrated five times before applying onto a Superdex 200 column.

SP-Sepharose FF cation exchange (CEX)

S2 perfusion batches were processed directly from the fermentation. The supernatants were diluted twice with 100 mM Na-Acetate, pH 5.5 buffer to obtain a final concentration of 50 mM Na-Acetate. The solution was filtrated through the prefilter sartoclean GF (0.8-0.65 μm) followed by a Sartopore 2 filter (0.45-0.2 μm) before loaded onto a SP Sepharose FF CIE column (XK50, bed height; 16 cm, volume 314 ml), equilibrated in 50 mM Na-Acetate, pH 5.5. The solution was kept on ice during load at a flow rate of 120 cm/h. Flow through (FT) was collected in order to detect unbound protein. Bound proteins were eluted with increased pH using 100 mM Tris-HCI, pH 7.0. S2 perfusion batches corresponding to different days of PX107.1 fermentation were processed individually and SP pools derived from each chromatography run were stored at -20 ⁰ C. Once the nine SP Sepharose runs were completed, the SP pools were

thawed, pooled and the pool was divided into three portions which constituted the starting material for the three consistency runs.

Source 3OQ anion exchange (AEX)

Thawed pool from SP-Sepharose was filtrated through a 0.22 μm Filtertop and loaded onto a Source 3OQ column (XK16, bed height 10 cm, volume 20 ml), equilibrated in 50 mM Tris-HCI, pH 8.0. Flow through (FT) was collected in order to detect unbound PX107.1 protein. Bound proteins were eluted with 8 CV of 200 mM NaCI in 50 mM Tris-HCI, pH 8.0.

Blue-Sepharose affinity column (AC)

The pool from Source 3OQ containing the variant in 50 mM Tris-HCI, 200 mM NaCI is diluted five times with 50 mM Tris-HCI pH 7.5 buffer to decrease the NaCI concentration. After dilution, the sample was filtrated through a 0.22 μm Filtertop and loaded onto a Blue Sepharose FF column (XK 26, bed height 20 cm, volume 100 ml), equilibrated in 50 mM Tris-HCI, pH 7.5. Flow through (FT) was collected in order to detect unbound RANKL variant protein. Bound proteins were eluted with 6 CV of 400 mM NaCI in 50 mM Tris-HCI, pH 7.5.

Protein concentration and Superdex 200 prep grade size exclusion (TFF and SEC)

The pool from Blue-Sepharose was applied onto a TFF device in order to concentrate the sample volume. The final volume after TFF must not exceed 10% of the gel filtration CV in order to obtain the expected separation performance of the gel filtration column. The concentrated sample was filtrated through a 0.22 μm Filtertop and applied onto a Superdex 200 column (XK50, bed height 56 cm, volume 1100 ml), equilibrated with 20 mM Tris-HCI, 150 mM NaCI, pH 7.5. The RANKL variant was eluted with 1.4 CV of buffer 20 mM Tris- HCI, 150 mM NaCI, pH 7.5. After SEC, fractions containing the variant were pooled and applied newly onto a TFF device in order to get a protein concentration of 1.2-1.4 mg/ml.

Virus filtration : Planova 2ON filter

The pool from gel filtration run containing 1.2-1.4 mg PX107.1/ml in 20 mM Tris-HCI, 150 mM NaCI, pH 7.5 was filtered through a 0.1 μm pre-filter before virus filtered through a Planova 2ON filter.

Analytical methods

Column chromatography was monitored by optical density and conductivity. Fractions were analysed by SDS-PAGE (Coomasie stained; reduced and non- reduced) and Western blot (using a polyclonal antibody against human RANKL). Intermediate purification pools and final batches were analysed by SDS-PAGE, Western blot, MALDI-TOF , an hOPG ELISA (a receptor based assay used as a quantitative method and also to confirm a correct structure of RANKL variants), and SEC-HPLC whereas N-terminal sequencing analysis was employed only for analyzing final batches.

Procedure and Results

In order to show consistency of the protein purification process, three bioreactors were run in parallel. Material from bioreactor one of these was used for this study. Perfusions were harvested at different times of the fermentation.

The average RANKL variant and total protein concentration in perfusion supernatant material was 44 mg/L and 390 mg/L respectively. Perfusions from 5-11 days exhibited total protein and RANKL variant ratios ranging between 5-8.

One important issue associated with the production of the RANKL variant was protein cleavage at the N-terminal. Purified variant that has been treated with N-glycosidase F exhibit a MALDI-TOF spectrum with two peaks, one major peak with a molecular mass of 19636 Da corresponding to the molecular mass of the full length and other minor peak of 18908 Da which was correlated to the variant being cleaved at the N-terminal leaving a sequence where the first 7 amino acids were missing. This sequence was found as underlying sequence in

the N-terminal sequence analysis confirming the identity of the minor peak visualized in the MALDI-TOF spectrum. It was found that purified batches of variant and perfusion supernatants exhibited the same MALDI-TOF spectrum indicating that cleavage seemed to occur during fermentation.

In view of this, MALDI-TOF MS analysis of the perfusions and purified variant was conducted to assure product quality through the downstream operations.

Cation Exchange Separation (step 1)

The capture step in the purification platform is a SP-Sepharose FF chromatography. Special considerations surround this step due to the above- mentioned cleavage of the variant. The protein is nibbled from one end probably by exoproteases present in the medium. Besides, protein precipitation has been observed in fermentation material after a freeze-thaw cycle leading to difficulties during the filtration process prior to chromatography. In view of this information, a strategy has been laid out to assure the stability of the native structure during downstream processing. Thus, for a production campaign, perfusions were processed daily and handed over for the SP-Sepharose column meaning that the capture step required nine days until all the intermediate SP- Sepharose pools were placed in the freezer.

Material received from upstream was diluted 1/2 with 100 mM acetate buffer pH 5.5 in order to achieve the binding conditions of a low conductivity and pH.

Binding pH was 5.5 and elution was achieved with pH increase in 100 mM Tris- HCI buffer to further avoid the need for sample conditioning after capture.

For each SP-Sepharose run, 5L of diluted perfusion supernatant containing 25 mg PX107.1 per L material (according to hOPG ELISA) was loaded onto a 314 ml SP-Sepharose column meaning that the total amount of variant loaded was approximately 125 mg whereas the amount of variant per ml of chromatographic medium was approximately 0.40-0.45 mg. This step resulted in a considerable minimization of working volume from 5 L to approximately 0.16 L.

In order to verify whether protein integrity was affected at the initial part of the purification process, an aliquot of each SP-Sepharose pool was analyzed by MALDI-TOF MS, SDS-PAGE and Western blotting after a freeze-thaw cycle. MALDI-TOF analysis of SP-Sepharose pools displayed MALDI spectrums equivalent to those obtained from perfusions indicating that new degradation events seemed not to occur during the capture step.

Following this, the SP-Sepharose pools were thawed and combined. The combined pool was divided into three aliquots, each of which constituted the starting material for the next chromatographic step. Thus, each aliquot contained the amount of variant equivalent to process 7.5 L (370 mg variant) of fermentation supernatant. The average yield of variant from the CEX step purification was 60%. Approximately 10-20% of the variant was detected by hOPG ELISA in the CEX flow-through. Some of the variant was recovered during the cleaning of the column but the quantity and nature of this material was not investigated.

Anion Exchange Separation (step 2)

For each run, approximately 450-480 ml containing 200-250 mg of the variant material were then loaded onto a 20 ml Source 3OQ column (15 mg PX107.1 per ml resin material). Binding pH was 8.0 in 50 mM Tris-HCI. Elution was performed by a step elution with 200 mM NaCI in the same buffer. Earlier experiments showed that some remaining variant was eluted from the resin at 300 mM NaCI. In this study, the amount and nature of the material eluted at 300 mM NaCI was investigated. Consistent elution profiles were seen for each of the three purifications runs.

Data obtained from hOPG ELISA and protein assays performed on the Source 3OQ intermediate pools demonstrated that product yields from the AEX step were very similar between the runs with an average overall recovery of 44%. Approximately 65-75% of the variant applied onto the column was recovered in the 200 mM NaCI eluate. No protein was detected by hOPG ELISA in the flow- through pool. Approximately 20% of the variant was found in the 300 mM NaCI

eluate. Source 30Q intermediate pools presented a SEC profile where 95-97% of the peak area corresponded to the molecular mass of trimeric forms of the variant. Material derived from the elution at 300 mM NaCI was estimated to have approximately 40-50% high molecular weight proteins and aggregated variant by SE-HPLC.

Affinity Chromatography Separation (step 3)

After the Source 3OQ purification step, the protein is in buffer 50 mM Tris-HCI, 200 mM NaCI pH 8.0. In order to reach the binding pH and conductivity conditions, the source 3OQ intermediate pool is diluted five times with 50 mM Tris-HCI pH 7.5 buffer. Thus, a volume of approximately 325 ml containing 160 mg of the variant is loaded onto a 100 ml Blue-Sepharose FF column (2 mg of variant per ml of resin). The variant was eluted with 400 mM NaCI in Tris-HCI buffer pH 7.5. Consistent elution profiles were seen for each of the three purifications runs.

Analysis performed on Blue-Sepharose pools demonstrated that the affinity purification step showed consistency in recovery with an average overall recovery of 31%. About 60-80% of the variant loaded into the column was recovered in the 400 mM NaCI eluate. The variant was not detected by hOPG ELISA in the flow-through pool. Analytical SE-HPLC of Blue-Sepharose pools showed a trimer molecular weight dominant profile.

Size exclusion Separation and TFF (steps 4, 5 and 6)

After the Blue-Sepharose step, the sample volume is approximately 200 ml and the protein is in 50 mM Tris-HCI buffer pH 7.5. In order to decrease the size of the gel filtration column, the sample loading volume was reduced five times by means of tangential-flow filtration (TFF). After the concentration step, approximately 40 ml of sample was loaded onto a 1.1 L Superdex 200 prep grade column. Thus, the sample volume is 5% of the total column volume. RANKL variant concentration in the material loaded onto the column was approximately 2.5-3.0 mg/ml. Analytical SEC of this material showed a trimer

molecular weight dominant profile indicating absence of aggregates. The RANKL variant is eluted isocratically with buffer 20 mM Tris-HCI, 150 mM NaCI pH 7.5.

After the gel filtration chromatography, the sample volume was increased approximately 4 times and the RANKL variant concentration was ranging 0.5-0.6 mg/ml. To achieve the desired protein concentration of 1.0 mg/ml, sample volume is reduced approximately 2-2.5 times by means of tangential flow- filtration. Samples after the concentration step were analyzed by hOPG ELISA and Bradford. Product yields after SEC chromatography and concentration steps were similar between the runs with an average overall recovery of 27%. Approximately 80-90% of the concentrated Blue-Sepharose pool loaded onto the column was recovered after chromatography and TFF operation. Analytical SE- HPLC of samples after gel filtration and concentration steps showed a profile where 98% of the peak area corresponded to the molecular weight of the trimeric form of the RANKL variant.

Viral clearance filtration (step 7)

Virus filtration by nanofiltration was included as complementary method for viral clearance. Samples derived from the TFF step were submitted to filtration through a Planova 2ON filter. The three final batches obtained after the virus filtration step were pooled to reference batch RA5607. The recovery of the target protein after the filtration step was investigated : Approximately 80-90% of the concentrated SEC pool was recovered after filtration. Overall recoveries were similar between the runs and the average RANKL variant recovery for the process was 25% with >95% trimeric form of the variant as estimated by SE- HPLC.

Conclusions

• No significant differences in chromatography performance at the capture phase that could be attributed to harvest material derived from the beginning, middle or end of the fermentation were observed.

• Overall, the three purification runs gave consistent yields and column elution profiles.

• The average recovery or the RANKL variant for the process was 25%.

• The RANKL variant from each run demonstrated consistent high purity and product quality.

EXAMPLE 6

Testing adjuvant combinations

Animals

Female C57BL/6 mice, (BALB/c x C57BL/6)F1 mice and female DA rats were used as recipients for vaccinations and were purchased from Harlan Scandinavia (Denmark) or bred at Pharmexa-Epimmune.

Adjuvants

Alhydrogel ^® "85" (2% AI ₂ O ₃ ), Adju-Phos ^® (2% AIPO ₄ ), and Calcium Phosphate Adjuvant (3mg Ca ² VmI) were obtained from Brenntag Biosector (Denmark). ODN1826 20-mer CpG with nuclease-resistant phosphorthioate backbone (TCC ATG ACG TTC CTG ACG TT, SEQ ID NO: 145) was purchased from and made by DNA Technology A/S (Denmark). Provax was prepared according to K. Hariharan et al, Advanced Drug Delivery Reviews 32 (1998) 187-197, except for using 17,000 psi as the internal microfluidization pressure. B5 was microfluidized similar to Provax, but using equimolar concentrations of Squalene & Poloxymer 124, as compared with Squalane & Poloxymer 401 for Provax, respectively. Also, a 25% molar amount of Polysorbate 20 was used for B5, as compared with Polysorbate 80 for Provax.

Antigens

PX107.1 RANKL AutoVac recombinant polypeptide was prepared at Pharmexa A/S (PX107.1 is the RANKL variant obtained from examples 3-5). OVA was purchased from SIGMA, and was purified from endotoxins through Polymyxin B- columns before being used in vaccine formulation. EP-1043, a recombinant poly- HTL-epitope polypeptide antigen was used to evaluate specific cellular responses, and was developed by Pharmexa-Epimmune with the amino acid sequence:

MAEKVYLAWVPAHKGIGGGPGPGQKQITKIQNFRVYYRGPGPGWEFVNTPPLVKLWY QG PGPGYRKILRQRKIDRLIDGPGPGQHLLQLTVWGIKQLQGPGPGGEIYKRWIILGLNKIV R MYGPGPGQGQMVHQAISPRTLNGPGPGIKQFINMWQEVGKAMYGPGPGWAGIKQEFGI PYNPQGPGPGKTAVQMAVFIHNFKRGPGPGSPAIFQSSMTKILEPGPGPGEVNIVTDSQY A LGIIGPGPGHSNWRAMASDFNLPPGPGPGAETFYVDGAANRETKGPGPGGAVVIQDNSDI KVVPGPGPGFRKYTAFTIPSINNE (SEQ ID NO: 146).

Vaccine formulations

Polypeptide antigens were either i) adsorbed to Alhydrogel ^® (700 μg AI ³⁺ /ml in final vaccine), ii) mixed with Provax (33% v/v in final vaccine), B5 (33% v/v in final vaccine) or CpG (500 μg TCC ATG ACG TTC CTG ACG TT/ ml in final vaccine), iii) pre-adsorbed to Alhydrogel ^® (700 μg AI ³⁺ /ml in final vaccine), Adju-Phos ^® (700 μg AI ³⁺ /ml in final vaccine) or Calcium Phosphate Adjuvant (700 μg Ca ² VmI in final vaccine) followed by mixing with either Provax (3.3% or 33% v/v in final vaccine) or B5 (33% v/v in final vaccine), or iv) mixed with CpG (500 μg TCC ATG ACG TTC CTG ACG TT (SEQ ID NO: 145) per ml in final vaccine) followed by mixing with Provax (33% v/v in final vaccine). Polypeptide adsorptions to Alhydrogel ^® , Adju-Phos ^® or Calcium Phosphate Adjuvant were prepared by mixing at room temperature for at least 15 min. Vaccines containing Provax or B5 adjuvants were only vortexed for a few seconds after adding Provax or B5, respectively.

Vaccinations

100 μl of PX107.1 vaccine (1 μg/ml or 10 μg/ml in final vaccine) or OVA vaccine (1 μg/ml in final vaccine) was injected subcutaneously in the back of C57BL/6

mice or DA rats at weeks 0, 2 & 6. 100 μl of EP-1043 vaccine (50 μg/ml in final vaccine) was injected intramuscularly in the quadriceps of (BALB/c x C57BL/6)F1 mice at weeks 0, 2 & 6.

ELISA

Blood was collected biweekly for testing antibody response in prepared sera against vaccine antigens. ELISA was used to measure OD ₅₀ values (i.e. How many times the prepared sera could be diluted to provide 50% of maximal antibody response against coated vaccine antigen). Isotype responses were evaluated by comparing sera OD ₅₀ levels of IgGl (Th2-dependent), IgG2a (ThI- dependent) & IgG2b (Thl-dependent) antibodies restricted against coated vaccine antigen in ELISA.

ELISPOT

CD4 ⁺ T cells were recovered from spleens one week after final vaccination with EP-1043. These effector cells were plated out together with target cells and specific epitope peptides to allow formation, hence IFN-gamma secretion, of

CD4/MHCII-epitope/Target cell synaps. Total spots (i.e. IFN-gamma producing T cells) for 12 specific epitopes were measured.

Results

Synergistic enhancement of humoral response by combining either salt suspensions or CpG with Provax

As depicted in Table I below, Alhydrogel + Provax (No: 3 for mice; No: 15 for rats) provides a stronger humoral response against PX107.1 than Alhydrogel (No: l for mice; No: 13 for rats) or Provax (No: 2 for mice; No: 14 for rats) alone. This effect is observed already after two vaccinations (i.e. week 4), but culminates after third vaccination (i.e. week 8). Similarly, but less pronounced enhancement is observed using Adju-Phos + Provax (No: 5), Calcium Phosphate

Adjuvant + Provax (No:6) or CpG + Provax (No:8), as compared with Provax (No: 2), or CpG (No:7) alone.

Synergistic enhancement of humoral response also seen when combining Alhydrogel with a completely different 3-component oil-in-water adjuvant

As shown in Table I, Alhydrogel + B5 (No: 11) also provides a stronger humoral response against PX107.1 than Alhydrogel (No:9) or B5 (No: 10) alone.

Mainly Th2-dependent IgGl response enhanced by combining salt suspensions with oil-in-water adjuvants

Table II also shows that Alhydrogel + Provax (No: 3), Adju-Phos + Provax (No: 5), Calcium Phosphate Adjuvant + Provax (No:6), as well as Alhydrogel + B5 (No: 11) mainly enhance Th2 responses, as compared with Alhydrogel (No: l & No:9), Provax (No: 2) or B5 (No: 10) alone.

The ratio between salt suspension and oil-in-water adjuvant is crucial for enhanced adjuvanticity

As depicted in Table I, Alhydrogel + Provax ^low (No:4; containing 70 μg Al ³⁺ per 3.3 μl Provax per 100 μl vaccine) does actually ruin the humoral response against PX107.1 as compared with Alhydrogel + Provax (No: 3; containing 70 μg Al ³⁺ per 33 μl Provax per 100 μl vaccine) or even Provax (No: 2; 33 μl Provax per 100 μl vaccine) alone.

Strong humoral response using another antigen in Alhydrogel + Provax

Table I further shows that Alhydrogel + Provax (No: 12) is a potent combination adjuvant for a very low dose of OVA (i.e. 0.1 μg per dose). In fact, the OD50 dilution of 10200 at week 8 corresponds to 1.0 mg/ml of elicited polyclonal IgG against OVA, using a monoclonal mouse-anti-OVA mAb control (SIGMA 6075; data not shown).

Table I. Humoral response in mice and rats using PX107.1 or OVA formulated in various adjuvants

Synergistic enhancement of cellular response by combining Alhydrogel + Provax

As depicted in Table II, Alhydrogel + Provax (No: 3) provides a stronger & broader Helper T-Lymphocyte (HTL) response against a variety of 12 different HTL-epitopes than Alhydrogel (No: l) or Provax (No:2) alone.

Table II. Cellular response in mice using EP-1043 w/wo EP-1033 formulated in various adjuvants

Discussion / Conclusions

Salt suspensions, like Alhydrogel, Adju-Phos, Calcium Phosphate Adjuvant, as well as CpG DNA can enhance adjuvanticity of oil-in-water adjuvants like Provax or a completely different 3-component oil-in-water adjuvant like B5. The main enhancement of the magnitude of the humoral response when combining salt- suspensions with oil-in-water adjuvants is the Th2-dependent antibody production, but there is also a less pronounced enhancement of the ThI- dependent antibody production. In addition, we have found that combined salt- suspension + Provax adjuvants also enhance cellular responses (i.e. HTL), both with respect to magnitude and breadth. Importantly, the molarity ratio between cationic ions and the molarity of oil-in-water particles is crucial in order to enhance, and not ruin, this excel adjuvanticity we show. We have also found that using around 200 μg Al ³⁺ per 33 μl Provax ruins cellular responses (i.e. HTL; data not shown), as well as 70 μg Al ³⁺ per 3.3 μl Provax will ruin the humoral response. We have further performed several boosting-titration experiments for this novel Alhydrogel + Provax combination adjuvant in mice showing that it is optimal to use the week 2 & 6 or week 1 & 6 boosting-interval schedule, as compared with week 2 & 4; week 3 & 6; or week 4 & 8 (data not shown).