Patent Application
20060222624
The present invention relates to the field of therapeutic immunotherapy, and in particular to the field of active immunotherapy targeted at down-regulating autologous (“self”) proteins and other weakly immunogenic antigens. The invention thus provides novel and improved immunogenic, detoxified variants of tumour necrosis alpha (TNFα) as well as the necessary tools for the preparation of such variants. The invention further relates to methods of immunotherapy as well as compositions useful in such methods.
Use of active immunotherapy (“vaccination”) as a means of curing or alleviating disease has received growing attention over the last 2 decades. Notably, the use of active immunotherapy as a means for breaking tolerance to autologous proteins that are somehow related to a pathological (or otherwise undesired) physiologic condition has been known since the late seventies where the first experiments with anti-fertility vaccines where reported.
Vaccines against autologous antigens have traditionally been prepared by “immunogenizing” the relevant self-protein, e.g. by chemical coupling (“conjugation”) to a large foreign and immunogenic carrier protein (cf. U.S. Pat. No. 4,161,519) or by preparation of fusion constructs between the autologous protein and the foreign carrier protein (cf. WO 86/07383). In such constructs, the carrier part of the immunogenic molecule is responsible for the provision epitopes for T-helper lymphocytes (“TH epitopes”) that render possible the breaking of autotolerance.
Later research has proven that although such strategies may indeed provide for the breaking of tolerance against autologous proteins, a number of problems are encountered. Most important is the fact that the immune response that is induced over time will be dominated by the antibodies directed against the carrier portion of the immunogen whereas the reactivity against the autologous protein often declines, an effect that is particularly pronounced when the carrier has previously served as an immunogen—this phenomenon is known as carrier suppression (cf. e.g. Kallyaperumal et al. 1995., Eur. J. Immunol 25, 3375-3380). However, when using therapeutic vaccination it is usually necessary to re-immunize several times per year and to maintain this treatment for a number of years and this also results in a situation where the immune response against the carrier portion will be increasingly dominant on the expense of the immune response against the autologous molecule.
Further problems involved when using hapten-carrier technology for breaking autotolerance is the negative steric effects exerted by carrier on the autologous protein part in such constructs: The number of accessible B-cell epitopes that resemble the conformational patterns seen in the native autologous protein is often reduced due to simple shielding or masking of epitopes or due to conformational changes induced in the self-part of the immunogen. Finally, it is very often difficult to characterize a hapten-carrier molecule in sufficient detail.
WO 95/05849 provided for a refinement of the above-mentioned hapten-carrier strategies. It was demonstrated that self-proteins wherein is in-substituted as little as one single foreign TH epitope are capable of breaking tolerance towards the autologous protein. Focus was put on the preservation of tertiary structure of the autologous protein in order to ensure that a maximum number of autologous B-cell epitopes would be preserved in the immunogen in spite of the introduction of the foreign TH element. This strategy has generally proven extremely successful inasmuch as the antibodies induced are broad-spectred as well as of high affinity and that the immune response has an earlier onset and a higher titre than that seen when immunizing with a traditional carrier construct.
WO 00/20027 provided for an expansion of the above principle. It was found that introduction of single TH epitopes in the coding sequence for self-proteins could induce cytotoxic T-lymphocytes (CTLs) that react specifically with cells expressing the self-protein. The technology of WO 00/20027 also provided for combined therapy, where both antibodies and CTLs are induced—in these embodiments, the immunogens would still be required to preserve a substantial fraction of B-cell epitopes.
Tumour necrosis factor (TNF, TNFα, cachectin, TNFSF2) is a potent paracrine and endocrine mediator of inflammatory and immune functions. TNFα is cytotoxic for many cells especially In combination with gamma-interferon. TNFα was initially identified in 1975 and demonstrated to initiate tumour necrosis and regression. The anti-cancer effect has later been investigated in detail, but the treatment has not been a success as cancer therapy, although there are still cancer trials using TNFα running. TNFα was later discovered as the cause of cachexia and it was discovered that TNFα exerts its function through a receptor-mediated process. Two different TNFα receptors (TNFR55 and TNFR75) have been identified that mediate cytotoxic and inflammatory effects of TNFα. TNFα induces and perpetuates inflammatory processes during chronic inflammatory diseases like rheumatoid arthritis (RA) and is suspected to have a critical role in allergies and psoriasis. Blocking of the TNFα signal by soluble receptors, receptor-specific inhibitors, down-regulation of TNFα production or monoclonal anti-TNFα antibodies are attractive therapy forms to adverse the biological effects of TNFα up-regulation and signalling.
It is evident from the results obtained from treatment with soluble TNFα receptors and monoclonal anti-TNFα antibodies that anti-TNFα therapy is a success in several diseases, like RA and Crohn's disease. The anti-TNFα treatment is both considered safe and effective.
To date, two TNFα antagonists, Remicade (Infliximab, Centocor/Johnson&Johnson) and Enbrel (Etanercept, Immunex) have been approved for clinical use.
Remicade is a chimeric mouse-human monoclonal IgG1 antibody directed against soluble and cell associated TNFα. Remicade blocks the binding of TNF with its endogenous cell surface TNFα receptor. The Food and Drug Administration (FDA) approved Remicade in October 1998 for use in moderate to severe or fistulizing Crohn's Disease refractory to conventional therapies. The indication was extended to include adjunctive use with methotrexate in rheumatoid arthritis refractory to methotrexate therapy alone and in July 2002 maintenance therapy in Crohn's disease.
Enbrel is a recombinant protein consisting of the extracellular portion of the human TNFα receptor fused to the Fc portion of human IgG1. Enbrel inhibits TNFα activity by serving as a decoy TNFα receptor. FDA approved Enbrel for use in rheumatoid arthritis in November 1998. More than 350.000 patients have been treated with these TNFα antagonists. Review of clinical efficacy and safety information of these agents are performed continuously and although infections and other immune-related adverse events remain a major concern for TNFα antagonists, recent safety evaluation of post-marketing experience performed by the FDA and the Committee for Proprietary Medicinal Products (CPMP) states that anti-TNFα therapies have a favourable risk-benefit balance although labelling changes, including changes on serious infections have been required.
Compared with the established anti-TNFα therapies, the presently suggested TNFα immuno-therapy has the advantages of microgram amount vaccinations and less frequent injections to keep a high anti-TNFα in vivo titre compared with large infusions of monoclonal antibodies. The positive consequences are a lower risk for side effects and less expensive therapy. It is also believed that a natural polyclonal antibody response will act as a more efficient down-regulator of TNFα than other anti-TNFα therapies.
TNFα is translated as a 233 amino acid precursor protein and secreted as a trimeric type II transmembrane protein, which is cleaved by specific metalloproteases to a trimeric soluble protein where each identical monomeric subunit consists of 157 amino acids (the amino acid sequence of which is set forth in SEQ ID NO: 10, residues 2-158). Human TNFα is non-glycosylated while murine TNFα has a single N-glycosylation site. The TNFα monomer has a molecular weight of 17 kDa while the trimer has a theoretical MW of 52 kDa, although a cross-linked trimer moves as 43 kDa In SDS-PAGE. TNFα contains two cysteines that stabilize the structure by forming an intramolecular disulphide bridge. Both the N and C-terminus of TNFα are important for the activity. Especially the C-terminus is sensitive as deletion of three, two and even one amino acid drastically decreases the solubility and bioactivity. The important amino acid is Leu157, which forms a stabilizing salt bridge between two monomers in the trimer. On the other hand deletion of the first eight amino acids increases the activity with a factor 1.5-5 while deletion of the first nine amino acids restores the full-length activity. TNFα is a well-studied protein and many of the intra- and inter-molecular interactions leading to trimer formation and receptor binding have been identified.
Hence, in nature, human TNFα (SEQ ID NO: 10, residues 2-158) exists as both a dimer and a trimer, but the molecule is in both cases very suitable as a candidate target for the present invention.
WO 95/05849 and WO 98/46642 both disclose vaccine technology that is suitable for down-regulating the activity of TNFα (tumour necrosis factor a), a cytokine involved in the pathology of several diseases such as type I diabetes, rheumatoid arthritis, and inflammatory bowel disease. Both disclosures teach preservation of the tertiary structure of monomer TNFα when this molecule confronts the immune system. Also, WO 03/042244 (not yet published) discloses a number of generic and specific TNFα variants.
Even though the above-referenced technologies have provided for very promising results, there are several factors that may come into play when assessing the viability of a vaccine approach in combating a disease. One of these factors is the expression level of the immunogenic protein.
For instance, in order for a nucleic acid vaccine to be functional, the cells transfected in vivo with a construct encoding an “immunogenized” autologous protein must be able to express the immunogen in sufficient amounts so as to induce a suitable immune response. Also, polypeptide based vaccines require that the immunogenic protein can be produced in satisfactory amounts in an industrial fermentation process. However, it is often observed that even slight changes in the amino acid sequence of a known protein can have dramatic effects on the amounts of protein that can be recovered.
Further, the stability of genetically modified protein sequences may also be less than optimal (both in terms of shelf-life and in terms of stability in vivo).
Also, when, as is the case for TNFα, the self-protein that it is desired to down-regulate is a heteropolymer or homopolymer it is not necessarily so that a variant of a monomeric unit of this protein will be capable of inducing antibodies that are sufficiently specific for the conformation native to the polymeric protein.
Finally, TNFα is a toxic substance, and unfortunately it has been observed that the optimum folded immunogenic variants of TNFα preserve the native toxicity of TNFα because these variants are capable of forming biologically active trimers (or fold op as biologically active monomers that mimic the trimer structure).
It is an object of the invention to provide for improved immunogenic and detoxified analogues of human TNFα as well as to provide for improved methods for inducing humeral immunity against this protein. Further, it is an object of the invention to provide improved methods for culturing soluble TNFα variants as well as variants of other proteins. Finally, it is also objects of the invention to provide for other means and measures that are useful when preparing or utilising the improved immunogens.
When producing large-scale amounts of recombinant protein in bacterial host cells, it is often desired that the expression product becomes available as inclusion bodies inside the bacteria. The reasons for this are several: For example the expression yields are normally considerably higher when the protein is expressed as insoluble inclusion bodies, and the purification of the protein is also facilitated because the desired expression product is easily and conveniently separated from soluble protein from the bacterial fermentation.
When expressing a recombinant protein as insoluble inclusion bodies, it is, however, often necessary to subject the expression product to various protein refolding processes in order to obtain it in a biologically active form, but this is normally acceptable even though such a step leads to a certain loss of total recombinant protein that is never folded into the correct biologically active form.
However, when producing recombinant immunogenic variants of non-immunogenic self-proteins such as TNFα it is necessary to introduce TH epitopes and thereby the primary structure of the protein product becomes altered when compared to the native self-protein. The present inventors have experienced that even the slightest of changes renders the traditional approach of inclusion body expression followed by refolding impractical: The yields of protein after refolding that has preserved a satisfactory fraction of B-cell epitopes compared to the native self-protein are very often low, and this problem increases with the complexity of the protein in question.
It has now been found that designing and effecting expression of protein constructs that are produced as soluble protein from bacteria is a superior way of preparing immunogenic variants of self-proteins—even though subsequent purification steps become more complicated because other soluble proteins have to be removed, the final purified and correctly folded product is obtained in significantly higher yields than when compared to the traditional approach outlined above. And, very importantly, the purified proteins obtained from this type of expression exhibit a hitherto unprecedented ability to preserve B-cell epitopes of the native self-protein from which they are derived.
In brief, according to the present invention, soluble expression of variant proteins is an excellent selection criterion when initially selecting for immunogenic variants of a self-protein that are suitable for vaccination purposes.
In order to obtain the goal of soluble protein expression of such immunogenized self-proteins (and other proteins where changes have been introduced in the primary sequence), a number of parameters can be varied—multimeric proteins that are difficult to assemble can be produced by stabilising their structure both on the monomeric level but also by preparing monomeric mimics of the TNFα multimer, and also simple monomeric proteins can be stabilised according to the teachings set forth herein.
Another important factor is the fermentation conditions—findings in the present inventors' lab have e.g. indicated that fermentation of bacteria at lower temperatures than those normally used for obtaining high level expression greatly facilitate the production of soluble forms of the variant proteins.
The present inventors have previously found that preparation of “monomerized” or stabilised forms TNFα may provide for immunogenic molecules having a high stability, superior immunogenicity and desirable production characteristics. The present invention focuses on improvements to these concepts, where a number of specific detoxifying mutations have been introduced in the variants so as to make these more patient compilable while preserving the immunogenicity.
Apart from the detoxifying mutation, the TNFα variants of the present invention include a number of variations in the TNFα monomer structure that are sufficiently non-destructive so as to allow correct folding of the TNFα monomers while at the same time Introducing at least one MHC Class II binding amino acid sequence—these variations are already disclosed in detail in WO 03/042244. It has e.g. been found that insertion of a foreign TH epitope can be made in one particular loop structure in native TNFα without this having a negative impact on the expression characteristics of the protein or on the monomer's capability of forming a functional TNFα dimer or trimer.
Hence, a one aspect of the invention relates to a detoxified, immunogenic analogue of human TNFα, wherein the analogue includes at least one foreign MHC Class II binding amino acid sequence and further has the characteristic of being
wherein toxicity is reduced or abolished by introduction of at least one point mutation selected from the group consisting of Y87S, D143N, and A145R, the amino acid numbering beginning with the N-terminal V in human TNFα.
In general, it has been found that all of the best suited immunogenic analogues of the invention are those that are soluble proteins already at the stage when they are produced and isolated in soluble form from their recombinant host cells.
The invention further provides for nucleic acid fragments (such as DNA fragments) encoding such immunogenic analogues and also to vectors including such DNA fragments.
The invention also provides for transformed cells useful for preparing the analogues.
The invention further provides for immunogenic compositions comprising the analogous or the vectors of the invention.
Also provided by the invention are methods of treatment, where multimeric proteins are down-regulated and to treatment of specific diseases related to the particular multimeric proteins.
Definitions
In the following, a number of terms used in the present specification and claims will be defined and explained in detail in order to clarify the metes and bounds of the invention.
The terms “T-lymphocyte” and “T-cell” will be used interchangeably for lymphocytes of thymic origin that are responsible for various cell mediated immune responses as well as for helper activity in the humeral immune response. Likewise, the terms “B-lymphocyte” and “B-cell” will be used interchangeably for antibody-producing lymphocytes.
A “polymeric protein” is herein defined as a protein that includes at least two polypeptide chains that are not joined end-to-end via a peptide bond (the term “multimeric protein” is used interchangeably therewith). Hence, polymeric proteins may be polymers consisting of several polypeptides that are kept together in polymeric form by means of disulfide bonds and/or non-covalent binding. Also included within the term are processed pre-proteins and pro-proteins that after processing include at least two free C-termini and at least two free N-termini. Finally, included within the term is also temporarily existing complexes between at least two polypeptides that may form up an unstable but yet biologically active molecular entity that has a distinct 3-dimensional structure.
“An immunogenic analogue” (or an “immunogenized” analogue or variant) is herein meant to designate a single polypeptide that includes substantial parts of the sequence information found in a complete polymeric protein. That is, the analogue protein of the invention includes one polypeptide chain whereas a polymeric protein includes at least 2 polypeptide chains. It should be noted that the analogue may be a variation of the polymers monomeric subunit structure, but in that case, the immunogenic analogue is capable of forming polymeric protein complexes that resemble the native polymer.
A “monomerized” analogue or variant of a polymeric protein is in the present context a single polypeptide that includes, in covalently linked form via a peptide bond, at least 2 polypeptide chains found in a polymeric protein in nature, where these 2 polypeptide chains are not linked via a peptide bond.
“A substantial fragment” of a monomeric unit of a multimeric protein is intended to mean a part of a monomeric polypeptide that constitutes at least enough of the monomeric polypeptide so as to form a domain that folds up in substantially the same 3D conformation as can be found in the multimeric protein.
A “TNFα polypeptide” is herein intended to denote polypeptides having the amino acid sequence of TNFα proteins derived from humans and other mammals. Also unglycosylated forms of TNFα, which are prepared in prokaryotic systems, are included within the boundaries of the term as are forms having varying glycosylation patterns due to the use of e.g. yeasts or other non-mammalian eukaryotic expression systems. It should, however, be noted that when using the term “a TNFα polypeptide” it is intended that the polypeptide in question is normally non-immunogenic when presented to the animal to be treated. In other words, the TNFα polypeptide is a self-protein or is a xeno-analogue of such a self-protein, which will not normally give rise to an immune response against TNFα of the animal in question.
A “TNFα analogue” is a TNFα polypeptide which has been either subjected to changes in its primary structure and/or that is associated with elements from other molecular species. Such a change can e.g. be in the form of fusion of a TNFα 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 TNFα polypeptide's amino acid sequence. Also encompassed by the term are derivatized TNFα molecules, cf. the discussion below of modifications of TNFα.
It will be understood, that TNFα analogues also include monomeric variants that contains substantial parts of complete TNFα multimeric proteins.
When using the abbreviation “TNFα” herein, this is intended as references to the amino acid sequences of mature, wild-type TNFα (also denoted “TNFαm” and “TNFαwt” herein), respectively. Mature human TNFα is denoted hTNFα, hTNFαm or hTNFαwt, and murine mature TNFα are denoted mTNFα, mTNFαm, or mTNFαwt. In cases where a DNA construct includes information encoding a leader sequence or other material, this will normally be dear from the context.
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 TNFα amino acid sequence or nucleic acid sequence, respectively.
A “detoxifying mutation” is in the present context defined as a mutation (e.g. a point mutation) in the TNFα amino acid sequence that renders the resulting molecule significantly less toxic in a relevant animal model (or in the autologous host from where the TNFα amino acid sequence is derived). It will be understood, however, that the detoxifying mutation should not be one that interferes significantly with the correct folding of the TNFα molecule, since it is desired to preserve B-cell epitopes.
The term “animal” is in the present context in general intended to denote an animal species (preferably mammalian), such as Homo sapiens, Canis domesticus, etc. and not just one single animal. However, the term also denotes a population of such an animal species, since it is important that the individuals immunized according to the method of the invention all harbour substantially the same TNFα allowing for immunization of the animals with the same immunogen(s). If, for instance, genetic variants of TNFα exist in different human populations it may be necessary to use different immunogens in these different populations in order to be able to break the autotolerance towards TNFα in each population. It will be clear to the skilled person that an animal in the present context is a living being which has an immune system. It is preferred that the animal is a vertebrate, such as a mammal.
By the term “down-regulation” is herein meant reduction in the living organism of the biological activity of TNFα (e.g. by interference with the interaction between the TNFα protein and biologically important binding partners for this molecule). The down-regulation can be obtained by means of several mechanisms: Of these, simple interference with the active site in the multimeric protein by antibody binding is the most simple. However, it is also within the scope of the present invention that the antibody binding results in removal of the multimeric protein by scavenger cells (such as macrophages and other phagocytic cells).
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 accomplished 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 amellorate ongoing disease) or nucleic acid “pharmaccine” vaccination. The important result to achieve is that immune competent cells in the animal are confronted with the antigen in an immunologically effective manner, whereas the precise mode of achieving this result is of less importance to the inventive idea underlying the present invention.
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.
When using the expression that the TNFα has been “modified” is herein meant a chemical modification of the polypeptide, which constitutes the backbone of the self-protein. Such a modification can e.g. be derivatization (e.g. alkylation, acylation, esterification etc.) of certain amino acid residues in the amino acid sequence, but as will be appreciated from the disclosure below, the preferred modifications comprise changes of (or additions to) the primary structure of the amino acid sequence.
When discussing “autotolerance towards TNFα” it is understood that since TNFα is a self-protein in the population to be vaccinated, normal individuals in the population do not mount an immune response against it; it cannot be excluded, though, that occasional individuals in an animal population might be able to produce antibodies against the native TNFα, e.g. as part of an autoimmune disorder. At any rate, an animal species will normally only be autotolerant towards its own TNFα, but it cannot be excluded that analogues derived from other animal species or from a population having a different phenotype would also be tolerated by said animal.
A “foreign T-cell epitope” (or: “foreign T-lymphocyte epitope”) is a peptide which is able to bind to an MHC molecule and which stimulates T-cells in an animal species—an alternate term is therefore. Preferred foreign T-cell epitopes in the invention are “promiscuous” (or “universal” or “broad-range”) epitopes, i.e. epitopes that bind to a substantial fraction of a particular class of MHC molecules in an animal species or population. Only a very limited number of such promiscuous T-cell epitopes are known, and they will be discussed in detail below. It should be noted that in order for the immunogens which are used according to the present invention to be effective in as large a fraction of an animal population as possible, it may be necessary to 1) insert several foreign T-cell epitopes in the same analogue or 2) prepare several analogues wherein each analogue has a different promiscuous epitope inserted. It should be noted also that the concept of foreign T-cell epitopes also encompasses use of cryptic T-cell epitopes, i.e. epitopes that 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 TH epitope) is a foreign T cell epitope that 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.
An “MHC Class II binding amino acid sequence that is heterologous to a multimeric protein” is therefore an MHC Class II binding peptide that does not exist in TNFα. Such a peptide will, if it is also truly foreign to the animal species harbouring the multimeric protein, be a foreign TH epitope.
A “functional part” of a (bio)molecule is in the present context intended to mean the part of the molecule, which is responsible for at least one of the biochemical or physiological effects exerted by the molecule. It is well-known in the art that many enzymes and other effector molecules have an active site, which is responsible for the effects exerted by the molecule in question. Other parts of the molecule may serve a stabilizing or solubility enhancing purpose and can therefore be left out if these purposes are not of relevance in the context of a certain embodiment of the present invention. However, according to the present invention, it is preferred to utilise as much of the polymeric molecule as possible, because the increased stability has in fact been demonstrated when using the monomers described herein.
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 combination of 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. These issues will be discussed in detail below.
“Stimulation of the immune system” means that a substance or composition of matter exhibits a general, non-specific immunostimulatory effect. A number of adjuvants and putative adjuvants (such as certain cytokines) share the ability to stimulate the immune system. The result of using an immunostimulating agent is an increased “alertness” of the immune system meaning that simultaneous or subsequent immunization with an immunogen induces a significantly more effective immune response compared to isolated use of the immunogen.
Characteristics of the Detoxified Immunogenic TNFα Analogues of the Invention
It is advantageous if the immunogenic analogue according to the invention displays, in the substantial fragments, a substantial fraction of B-cell epitopes found in native, biologically active TNFα. A substantial fraction of B-cell epitopes is herein intended to mean a fraction of B-cell epitopes that antigenically characterises TNFα versus other proteins. It is preferred that the substantial fragments display essentially all B-cell epitopes found in the corresponding TNFα monomers when being part of the polymeric protein—of course, introduction of minor changes in the monomer TNFα sequence may be necessary. For instance, as explained above, an amino acid sequence derived from a monomeric unit is modified by means of amino acid insertion, substitution, deletion or addition so as to reduce toxicity of the TNFα analogue as compared to the native protein and/or so as to introduce the MHC Class II binding amino acid sequence, if it is undesired of irrelevant to have that sequence positioned in a linker.
An especially preferred embodiment provides for an immunogenic TNFα analogue of the invention, wherein each of the substantial fractions comprises essentially the complete amino acid sequence of each monomeric TNFα unit, either as a continuous sequence or as a sequence including inserts. That is, only insignificant parts of the monomeric TNFα unit's sequence are left out of the analogue, e.g. in cases where such a sequence does not contribute to tertiary structure of the monomeric unit or quaternary structure of TNFα. However, this embodiment allows for substitution or insertion of the monomer, as long as the 3D structure of the multimeric TNFα protein is maintained. Hence, it is especially advantageous if the immunogenic TNFα analogue is one, wherein amino acid sequences of all monomeric units of TNFα are represented in the analogue, and it is particularly advantageous if the analogue includes the complete amino acid sequences of (all) the monomers constituting TNFα, either as unbroken sequences or as sequences including inserts.
As will appear, it is therefore preferred that the 3-dimensional structure of the complete native, biologically active TNFα is essentially preserved in the analogue.
Demonstration of preservation of a substantial fraction of B-cell epitopes or even the 3-dimensional structure of TNFα that is subjected to modification as described herein can be achieved in several ways. One is simply to prepare a polyclonal antiserum directed against the native TNFα (e.g. an antiserum prepared in a rabbit) and thereafter use this antiserum as a test reagent (e.g. in a competitive ELISA) against the modified proteins, which are produced. Modified versions (analogues) which react to the same extent with the antiserum as does the native molecule must be regarded as having the same 3D structure as the native molecule whereas analogues exhibiting a limited (but still significant and specific) reactivity with such an antiserum are regarded as having maintained a substantial fraction of the original B-cell epitopes.
Alternatively, a selection of monoclonal antibodies reactive with distinct epitopes on the native TNFα can be prepared and used as a test panel. This approach has the advantage of allowing 1) an epitope mapping of TNFα and 2) a mapping of the epitopes, which are maintained in the analogues prepared.
Of course, a third approach is to compare the resolved 3D structure of native TNFα with the resolved three-dimensional structure of the analogues prepared. Three-dimensional structure can be resolved by the aid of X-ray diffraction studies and NMR-spectroscopy. Further information relating to the tertiary structure can to some extent be obtained from circular dichroism studies which have the advantage of merely requiring the polypeptide in pure form (whereas X-ray diffraction requires the provision of crystallized polypeptide and NMR requires the provision of isotopic variants of the polypeptide) in order to provide useful information about the tertiary structure of a given molecule. However, ultimately X-ray diffraction and/or NMR are necessary to obtain conclusive data since circular dichroism can only provide indirect evidence of correct 3-dimensional structure via information of secondary structure elements.
The immunogenic TNFα analogue of the invention may include a peptide linker that includes or contributes to the presence in the analogue of at least one MHC Class II binding amino acid sequence that is heterologous to the multimeric protein. This is particularly useful in those cases where it is undesired to alter the amino acid sequence corresponding to monomeric units in TNFα. Alternatively, the peptide linker may be free of and not contributing to the presence of an MHC Class II binding amino acid sequence in the animal species from where the TNFα protein is derived; this can conveniently be done in cases where it is necessary to utilise a very short linker or where it, as in the present invention, is essential to detoxify a potentially toxic analogue by e.g. introducing the MHC Class II binding element in an active site.
It is preferred that the MHC Class II binding amino acid sequence binds a majority of MHC Class II molecules from the animal species from where the multimeric protein has been derived, i.e. that the MHC Class II binding amino acid sequence is universal or promiscuous.
It is of course important that this sequence serves its purpose as a T cell epitope in the species for which the immunogen is intended to serve as a vaccine constituent. There exists a number of naturally occurring “promiscuous” T-cell epitopes which are active in a large proportion of individuals of an animal species or an animal population and these are preferably introduced in the vaccine, thereby reducing the need for a very large number of different analogues in the same vaccine. Hence, the at least one MHC Class II binding amino acid sequence is preferably selected from a natural T-cell epitope and an artificial MHC-II binding peptide sequence. Especially preferred sequences are a natural T-cell epitope is selected from a Tetanus toxoid epitope such as P2 (SEQ ID NO: 2) or P30 (SEQ ID NO: 4), a diphtheria toxoid epitope, an influenza virus hemagluttinin epitope, and a P. falciparum CS epitope.
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 analogues used according to the present invention. Cf. also the epitopes discussed in the following references, which are hereby all incorporated by reference herein: WO 98/23635 (Frazer IH et al., assigned to The University of Queensland); Southwood S et. al, 1998, J. Immunol. 160: 3363-3373; Sinigaglia F et al., 1988, Nature 336: 778-780; Chicz R M et al., 1993, J. Exp. Med 178: 27-47; Hammer J et al., 1993, Cell 74: 197-203; and Falk 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 that share common motifs with these.
Alternatively, the epitope can be any artificial T-cell epitope, which is capable of binding a large proportion of MHC Class II molecules. In this context the pan DR epitope peptides (“PADRE”) described in WO 95/07707 and in the corresponding paper Alexander J et al., 1994, Immunity 1: 751-761 (both disclosures are incorporated by reference herein) are interesting candidates for epitopes to be used according to the present invention. It should be noted that the most effective PADRE peptides disclosed in these papers carry D-amino acids in the C- and N-termini in order to improve stability when administered. However, the present invention primarily aims at incorporating the relevant epitopes as part of the analogue, 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 AKFVAAWTLKAAA (SEQ ID NO: 6 and 8) or an immunologically effective subsequence thereof. This, and other epitopes having the same lack of MHC restriction are preferred T-cell epitopes, which should be present in the analogues used in the inventive method. Such super-promiscuous epitopes will allow for the simplest embodiments of the invention wherein only one single modified TNFα is presented to the vaccinated animal's immune system.
Preferred embodiments of the invention include modification by introducing at least one foreign immunodominant TH epitope. It will be understood that the question of immune dominance of a TH epitope depends on the animal species in question. As used herein, the term “immunodominance” simply refers to epitopes which in the vaccinated individual gives rise to a significant immune response, but it is a well-known fact that a TH epitope which is immunodominant in one individual is not necessarily immunodominant in another individual of the same species, even though it may be capable of binding MHC-II molecules in the latter individual.
As mentioned above, the introduction of a foreign T-cell epitope can be accomplished by introduction of at least one amino acid insertion, addition, deletion, or substitution. Of course, the normal situation will be the introduction of more than one change in the amino acid sequence (e.g. insertion of or substitution by a complete T-cell epitope) but the important goal to reach is that the analogue, when processed by an antigen presenting cell (APC), will give rise to such a T-cell epitope being presented in context of an MCH Class II molecule on the surface of the APC. Thus, if the amino acid sequence of the monomeric unit in appropriate positions comprises a number of amino acid residues which can also be found in a foreign TH epitope then the introduction of a foreign TH 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 TH epitope by insertion or substitution.
According to the present invention, the analogue may also form part of larger molecule wherein it is coupled to at least one functional moiety, the presence of which does not interfere negatively to a significant degree with the antibody-accessibility of the analogue. The nature of such moieties (which may be fused to the analogue) can be to target the modified molecule to an antigen presenting cell (APC) or a B-lymphocyte, to stimulate the immune system, and to optimise presentation of the analogue to the immune system.
Targeting moieties are conveniently selected from the group consisting of a substantially specific binding partner for a B-lymphocyte specific surface antigen or for an APC specific surface antigen, such as a hapten or a carbohydrate for which there is a receptor on the B-lymphocyte or the APC. The immunostimulating moieties may be selected from the group consisting of a cytokine, a hormone, and a heat-shock protein. The presentation-optimising moiety may be selected from the group consisting of a lipid group, such as a palmitoyl group, a myristyl group, a farnesyl group, a geranyl-geranyl group, a GPI-anchor, and an N-acyl diglyceride group.
A suitable cytokine is, or is an effective part of any of, interferon γ (IFN-g), Flt3L, interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15), and granulocyte-macrophage colony stimulating factor (GM-CSF).
A preferred heat-shock protein is, or is an effective part of any of, HSP70, HSP90, HSC70, GRP94, and calreticulin (CRT).
It is preferred that the number of amino acid insertions, deletions, 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, 21, and 25 insertions, substitutions, additions or deletions. It is furthermore preferred that the number of amino acid insertions, substitutions, additions or deletions 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, deletions, 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 respect to amino acid additions, it should be noted that these, when the resulting construct is in the form of a fusion polypeptide, is often considerably higher than 150.
Preferred embodiments of the invention include modification by introducing at least one foreign immunodominant TH epitope (=“foreign MHC Class II binding amino acid sequence”). It will be understood that the question of immune dominance of a TH epitope depends on the animal species in question. As used herein, the term “immunodominance” simply refers to epitopes which in the vaccinated individual gives rise to a significant immune response, but it is a well-known fact that a TH epitope which is immunodominant in one individual is not necessarily immunodominant in another individual of the same species, even though it may be capable of binding MHC-II molecules in the latter individual.
Another important point is the issue of MHC restriction of TH epitopes. In general, naturally occurring TH epitopes are MHC restricted, i.e. a certain peptide constituting a TH epitope will only bind effectively to a subset of MHC Class II molecules. This in turn has the effect that in most cases the use of one specific TH epitope will result in a vaccine component which is effective in a fraction of the population only, and depending on the size of that fraction, it can be necessary to include more TH epitopes in the same molecule, or alternatively prepare a multi-component vaccine wherein the components are variants which are distinguished from each other by the nature of the TH epitope introduced.
If the MHC restriction of the T-cells used is completely unknown (for instance in a situation where the vaccinated animal has a poorly defined MHC composition), the fraction of the animal population covered by a specific vaccine composition can be determined by means of the following formula:
where pi is the frequency in the population of responders to the ith foreign T-cell epitope present in the vaccine composition, and n is the total number of foreign T-cell epitopes in the vaccine composition. Thus, a vaccine composition containing 3 foreign T-cell epitopes having response frequencies in the population of 0.8, 0.7, and 0.6, respectively, would give
1−0.2×0.3×0.4=0.976
i.e. 97.6 percent of the population will statistically mount an MHC-II mediated response to the vaccine.
The above formula does not apply in situations where a more or less precise MHC restriction pattern of the peptides used is known. If, for instance a certain peptide only binds the human MHC-II molecules encoded by HLA-DR alleles DR1, DR3, DR5, and DR7, then the use of this peptide together with another peptide which binds the remaining MHC-II molecules encoded by HLA-DR alleles will accomplish 100% coverage in the population in question. Likewise, if the second peptide only binds DR3 and DR5, the addition of this peptide will not increase the coverage at all. If one bases the calculation of population response purely on MHC restriction of T-cell epitopes in the vaccine, the fraction of the population covered by a specific vaccine composition can be determined by means of the following formula:
wherein φj is the sum of frequencies in the population of allelic haplotypes encoding MHC molecules which bind any one of the T-cell epitopes in the vaccine and which belong to the jth of the 3 known HLA loci (DP, DR and DQ); in practice, it is first determined which MHC molecules will recognize each T-cell epitope in the vaccine and thereafter these MHC molecules are listed by type (DP, DR and DQ)—then, the individual frequencies of the different listed allelic haplotypes are summed for each type, thereby yielding φ1, φ2, and φ3.
It may occur that the value pi in formula I exceeds the corresponding theoretical value ni:
wherein vj is the sum of frequencies in the population of allelic haplotypes encoding MHC molecules which bind the ith T-cell epitope in the vaccine and which belong to the jth of the 3 known HLA loci (DP, DR and DQ). This means that in 1−ni of the population there is a frequency of responders of fresidential
where the term 1−fresidential
Therefore, when selecting T-cell epitopes to be introduced in the analogue of the invention, it is important to include all knowledge of the epitopes which is available: 1) The frequency of responders in the population to each epitope, 2) MHC restriction data, and 3) frequency in the population of the relevant haplotypes.
It should be noted that preferred analogues of the invention comprise modifications which result in a polypeptide that includes stretches having a sequence identity of at least 70% with the corresponding monomeric units of TNFα or with subsequences thereof of at least 10 amino acids in length. Higher sequence identities are preferred, e.g. at least 75% or even at least 80% or 85%. The sequence identity for proteins and nucleic acids can be calculated as (Nref−Ndif)·100/Nref, wherein Ndif is the total number of non-identical residues In the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (Ndif=2 and Nref=8).
Finally, In order to conclusively verify that a TNFα analogue of the invention is indeed effective as an immunogen, various tests may be performed in order to provide the necessary confirmation. In this context, reference is also made to the discussion of identification of useful IL5 analogues in WO 00/65058—this disclosure may be used for verification of the usefulness of an analogue (IL5 derived or not) subject to the present inventive technology.
A preferred TNFα analogue is selected from the group consisting of 1) two or three complete TNFα monomers joined end-to-end by a peptide linker, wherein at least one peptide linker includes at least one MHC Class II binding amino acid sequence, and 2) two or three complete TNFα monomers joined end-to-end by an inert peptide linker, wherein at least one of the monomers include at least one foreign MHC Class II binding amino acid sequence or wherein at least one foreign MHC Class II binding amino acids sequence is fused to the N- or C-terminal monomer, optionally via an inert linker.
Particularly interesting are immunogenic TNFα molecules with high stability, since it has earlier been found by the inventors that monomeric TNFα constructs tend to be relatively unstable, cf., however, the discussion below.
Previously, a gene encoding the 3 TNFα subunits linked together by epitopes and/or inert peptide linkers has been produced. The goal was to generate variant TNFα molecules with a conformation as close to the native TNFα trimer as possible. The variants were designed to efficiently elicit neutralizing antibodies against wtTNFα. The most suitable TNFα variants were found to be soluble and stable proteins in the absence of detergents or other kinds of additives that could disrupt the protein conformation.
The following discussion focuses on the preferred modifications of TNF that do not relate to the detoxification per se.
By expressing the three monomers linked together as one single polypeptide chain using linkers and TH epitopes, it is intended to prepare TNFα variants that are more stable than previous variant TNFα immunogens. This allows preservation of the TNFα structure, by introduction of the necessary TH epitopes outside of stabilizing hydrogen bonds, salt bridges or disulfide bridges.
From the X-ray crystal structure of TNFα it is seen that the first 5 residues of the N terminal are too flexible to allow a structure determination. The C-terminus, however, is located close to the middle of the monomer interface and is less flexible. The distance between the C alpha atoms of Arg-6 and Leu-157 is 10 Å, which is the distance of 3-4 amino acid residues. Therefore it seems to be possible to link the monomeric subunits directly together, but since the C-terminals are located at a delicate site, it is advantageous to use flexible linkers, e.g. glycine linkers, for this connection.
Five variants have until now been designed utilising the “monomerized trimer” approach. The control TNF_T0 (TNFα Trimer number 0, SEQ ID NO: 22 in WO 03/042244) consists of the three monomers directly linked together by 2 separate glycine linkers (GlyGlyGly). TNF_T0 is designed so as to be as stable as the wild type trimeric protein. Of course, other inert flexible linkers known in the art of protein chemistry may be used instead of the above-mentioned glycine linkers, the important feature being that the flexible linker does not interfere adversely with the monomerized protein's capability of folding into a 3D structure that is similar to the 3D structure of physiologically active wtTNFα.
The TNF_T0 construct is expressed as a soluble protein in E. coli, and it has been used to prepare the exemplary construct TNF_T4 (cf. WO 03/042244), which is a variant wherein the PADRE MHC Class II binding peptide (SEQ ID NO: 6) is introduced. In this construct, the ratio between monomeric units and foreign epitopes are thus 1 epitope per 3 monomers, instead of 1 epitope per monomer as is the case in prior art variants that relied on immunogenized monomeric proteins—this is also the case for SEQ ID NO: 55 in WO 03/042244). This fact provides a potentially positive influence on the trimer stability. An offspring from this approach is the TNF13 C2 variant (cf. WO 03/042244), which is a TNFα monomer with a PADRE epitope in the same position as in TNF_T4.
In parallel, the tetanus toxoid P2 and P30 epitopes (SEQ ID NOs: 2 and 4, respectively), have been used in the TNF_T1 and TNF_T2 variants (cf. WO 03/042244), containing one epitope in each linker region, and also in TNF_T3 (cf. WO 03/042244) that contains one C-terminal epitope and one in the second linker region. Proteins are mostly folded from the N-terminal toward the C-terminal. The idea underlying TNF_T3 is that when the first two N-terminal domains fold up they will function as internal chaperones for the third domain (monomer), which is enclosed by epitopes.
It has been disclosed in WO 03/042244 that in addition to the technology described in detail above, where polymeric proteins are “monomerized”, TNFα (and possibly many other multimeric proteins) allows for the production of monomers that 1) include at least one stabilising mutation and/or 2) include at least one non-TNFα derived MHC Class II binding amino acid sequence, where these TNFα monomer variants are capable of folding correctly into a tertiary structure that subsequently allows for the formation of dimeric and trimeric TNFα proteins having a correct quaternary structure (as evidenced by these having receptor binding activity). Hence, in these constructs it has been possible to prepare variants of monomeric TNFα that does not necessarily need to be produced as monomerized trimers because the changes introduced in the monomer sequences introduce so limited disruption of the monomer's tertiary structure that a di- or trimer can be formed. In accordance with the ideas underlying the present invention, it has further been found that all such variants are expressible as soluble proteins from bacterial cells.
Hence, it has proven possible to prepare immunogenic TNFα variants according to the following strategies that can be combined and which may further be combined with the already discussed “monomerization approach” of the invention (since these particular modifications all are non-destructive by nature):
The Flexible Loop Strategy
It has been discovered by the present inventors that insertion of the PADRE epitope (SEQ ID NO: 6) into loop 3 in position Gly108-Ala109 is a promising approach to prepare TNFα variants with a structure closely resembling the native TNFα molecule. It has been deduced from the TNFα crystal structure that a TH epitope inserted directly into this position will not have any neighbouring amino acid residues in close proximity to interact with. Studies with TNF34 (cf. WO 03/042244), the first PADRE construct made according to this approach, has shown that approximately 5% of the expressed protein TNF34 was soluble in E. coli and 95% of the TNF34 was expressed as inclusion bodies when the bacterial host cells were grown at 37° C. but after an adaptation of the fermentation process where the fermentation temperature is 25° C., the yields of soluble protein from the fermentation is close to 100%. Hence, optimisation of growth conditions increases the yield of soluble protein.
A number of other constructs have been prepared (TNF35-TNF39, cf. WO 03/042244), where all of these solely rely on introduction of PADRE in the flexible loop 3. It is further contemplated according to the present invention that introduction of a foreign epitope can be made in other parts of loop 3 or just outside loop 3. Especially insertions are considered to be interesting, and insertion of a foreign epitope such as PADRE or P2 or P30 may advantageously be made after any one of amino acids 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, and 118 in human TNFα. However, also substitutions in the same region of TNFα are considered advantageous, so substitutions that involve the same range of amino acids (i.e. substitution of one single or several consecutive amino acids 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 in human TNFα).
Stability Enhancing Mutations
Introduction of TH epitopes in the flexible loop 3 could potentially destabilize the structure of the TNFα variant. However, this potential destabilization can be counteracted by stabilization of the structure through introduction of cysteines that will form a disulfide bridge. A cystine pair in two different positions have until now been introduced in variants TNF34-A and TNF34-B (cf. WO 03/042244). Also, the flexible N-terminal (the first 8 amino acids) that is known to reduce the strength of the receptor interaction can be deleted in parallel, hence the variant TNF34-C (cf. WO 03/042244). The disulfide bridge is introduced in the monomer for stabilization of the epitope insertion site together with the naturally occurring disulfide bridge (Cys-67 Cys-101). This strategy is believed to also stabilise both a TNFα monomer as such and a monomerized di- or trimer.
Other Constructs
Several different strategies have been employed in the design of variants that will be soluble expression products. TNFX1.1-2 (cf. WO 03/042244) are based on insertions of PADRE in the first loop of TNFα, where the insertion site is located at an intron position. In TNFX2.1 (cf. WO 03/042244) an artificial “stalk” region is created containing an insertion of PADRE.
Mutations of TNFα have revealed that large hydrophobic amino acid substitutions, pointing into the trimer interface, stabilize the trimer structure. TNFX3.1 and TNFX3.2 (cf. WO 03/042244) are proposals to stabilize the existing TNF34 variant. TNFX4.1 (cf. WO 03/042244) uses di-glycine linkers to diminish structural constrains from the PADRE peptide on the overall TNF34 structure. TNFX5.1 (cf. WO 03/042244) employs, as an insertion point, a loop structure found in the TNF family member BlyS. TNFX6.1-2, TNF7.1-2 and TNFX8.1 (cf. WO 03/042244) are further variants. TNFX9.1 and TNFX9.2 (cf. WO 03/042244) are TNF34 variants that utilize identical overlapping TNFα sequences of 4-6 amino acids both pre and post the epitope. Finally, two variants (SEQ ID NOs: 46 and 47 in WO 03/042244) are P2/P30 double variants in the same location as for the PADRE peptide in TNF34.
Further, from the crystal structure of TNFα it is observed that one stabilizing salt bridge is present within the TNFα monomer between the residues Lys-98 and Glu-116. The definition of a salt-bridge is an electrostatic interaction between side chain oxygens in Asp or Glu and positive charged atom side chain nitrogens in Arg, Lys or His with an interatomic distance less than 7.0 Ångstrom. By site directed substitution mutations of Lys-98 with Arg or His at this position in combination with substitutions of Glu 116 with Asp, an improvement of the stability for this salt bridge and thereby the stability of the trimer molecule could be attained. It is also possible to exchange these salt bridges with disulphide bridges, in a manner described above.
It has been observed that murine TNFα is considerably more stable than the human TNFα regarding to solubility and proteolysis. Improvement of TNFα variants includes making site directed mutants so as to mimic murine TNFα crystal structure to obtain more proteolytically stable TNFα product.
From the x-ray structures of human and murine TNFα it is seen that the centre of the trimer (in the middle of the three TNFα monomers) is held together due to hydrophobic forces, whereas the top and the bottom of the trimer is connected due to natural occurring salt bridges. Therefore, by screening these salt bridges for stronger connections, the stability of the TNFα trimer would also be improved.
In summary, the following specific TNFα variants have until now been prepared:
The numbers used are from the N-terminal V In SEQ ID NO: 10 (that is, from amino acid no. 2 in SEQ ID NO: 10). Preceding the N-terminal Valine is in some sequences a Methionine used for translation start.
All of the above-discussed variants of TNFα that are disclosed in WO 03/042244 are detoxified according to the present invention.
A number of point mutations are known in the art to detoxify TNFα or at least reduce toxicity to a large extent. These point mutations will, if necessary, be introduced into the variants of the present invention. Especially preferred mutations are substitutions corresponding to mature TNFα of Tyr-87 with a Ser, of Asp-143 with Asn, and of Ala-145 with Arg. Further, all effective mutations mentioned in Loetscher, H., Stueber, D., Banner, D., Mackay, F. and Lesslauer, W. 1993 JBC 268 (35) 26350-7, are also interesting embodiments in the detoxifying embodiments of the present invention. These point mutations may be used with any one of the specific constructs disclosed in WO 03/042244.
The most preferred protein constructs of the invention are thus those represented by any one of SEQ ID NOs: 12, 13, 14, 16, 17, and 18, as well as any amino acid sequence derived thereof that only include conservative amino acid changes.
At any rate, it is an important embodiment that all of these TNFα variants discussed above are expressible as soluble proteins from bacterial cells such as E. coli.
The preferred vector is pET28b+ when the goal is expression from E. coli, p2Zop2F (SEQ ID NO: 60 in WO 03/042244) is the vector used for insect cell expression, and pHP1 (or its commercially available “twin” pCI) is the vector used for expression in mammalian cells.
General Therapies Provided by the Invention
The invention provides for methods whereby it becomes possible to down-regulate TNFα in a very advantageous manner.
In general, there is provided a method for down-regulating TNFα in an autologous host, the method comprising effecting presentation to the animal's immune system of an immunogenically effective amount of at least one immunogenic TNFα analogue of the invention. It is preferred that the autologous host is a mammal, most preferably a human being.
The method can be put into practice in a number of ways, of which administration of a protein vaccine is one choice, but also a nucleic acid vaccination strategy or a live vaccination strategy are of great interest.
Protein/Polypeptide Vaccination and Formulation
When effecting presentation of the analogues to an animal's immune system by means of administration thereof to the animal, the formulation of the polypeptide follows the principles generally acknowledged in the art.
Preparation of vaccines which contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorporated herein by reference. Typically, such vaccines are prepared as injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients, which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants, which enhance the effectiveness of the vaccines; cf. the detailed discussion of adjuvants below.
The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously, intracutaneously, or intramuscularly. Additional formulations which are 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. The quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to mount an immune response, and the degree of protection desired. Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 0.1 μg to 2,000 μg (even though higher amounts in the 1-10 mg range are contemplated), such as in the range from about 0.5 μg to 1,000 μg, preferably in the range from 1 μg to 500 μg and especially in the range from about 10 μg to 100 μg. Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.
The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the age of the person to be vaccinated and the formulation of the antigen.
Some of the analogues of the vaccine are sufficiently immunogenic in a vaccine, but for some of the others the immune response will be enhanced if the vaccine further comprises an adjuvant substance.
Various methods of achieving adjuvant effect for the vaccine are known. General principles and methods are detailed in “The Theory and Practical Application of Adjuvants” 1995, Duncan E. S. Stewart-Tull (ed.), John Wiley & Sons Ltd, ISBN 0-471-95170-6, and also in “Vaccines: New Generation immunological Adjuvants”, 1995, Gregoriadis G et al. (eds.), Plenum Press, New York, ISBN 0-306-45283-9, both of which are hereby incorporated by reference herein.
It is especially preferred to use an adjuvant, which can be demonstrated to facilitate breaking of the autotolerance to autoantigens. 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 (IS-COM matrix); a particle; DDA; aluminium adjuvants; DNA adjuvants; γ-inulin; and an encapsulating adjuvant. In general it should be noted that the disclosures above which relate to compounds and agents useful as first, second and third moieties in the analogues also refer mutatis mutandis to their use in the adjuvant of a vaccine of the invention.
The application of adjuvants include use of agents such as aluminium hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in buffered saline, admixture with synthetic polymers of sugars (e.g. Carbopol®) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70° to 101° 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).
Liposome formulations are also known to confer adjuvant effects, and therefore liposome adjuvants are preferred according to the invention.
Also immunostimulating complex matrix type (ISCOM® matrix) adjuvants are preferred choices according to the invention, especially since it has been shown that this type of adjuvants are capable of up-regulating MHC Class II expression by APCs. An ISCOM® matrix 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 I G and Mitchell G F, 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 Fcy receptors on monocytes/macrophages. Especially conjugates between antigen and anti-FcyRI have been demonstrated to enhance immunogenicity for the purposes of vaccination.
Other possibilities involve the use of the targeting and immune modulating substances (i.a. cytokines) mentioned in the claims as moieties for the protein constructs. 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 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, N.Y. 10017-6501). The VLN (a thin tubular device) mimics the structure and function of a lymph node. Insertion of a VLN under the skin creates a site of sterile inflammation with an upsurge of cytokines and chemokines. T- and B-cells as well as APCs rapidly respond to the danger signals, home to the inflamed site and accumulate Inside the porous matrix of the VLN. It has been shown that the necessary antigen dose required to mount an immune response to an antigen is reduced when using the VLN and that immune protection conferred by vaccination using a VLN surpassed conventional immunization using Ribi as an adjuvant. The technology is i.a. described briefly in 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, Oct. 12th-15th 1998, Seascape Resort, Aptos, Calif.”.
It is expected that the vaccine should be administered at least once a year, such as at least 1, 2, 3, 4, 5, 6, and 12 times a year. More specifically, 1-12 times per year is expected, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times a year to an individual in need thereof. It has previously been shown that the memory immunity induced by the use of the preferred auto-vaccines according to the invention Is not permanent, and therefore the immune system needs to be periodically challenged with the analogues.
Due to genetic variation, different individuals may react with immune responses of varying strength to the same polypeptide. Therefore, the vaccine according to the invention may comprise several different polypeptides in order to increase the immune response, cf. also the discussion above concerning the choice of foreign T-cell epitope introductions. The vaccine may comprise two or more polypeptides, where all of the polypeptides are as defined above.
The vaccine may consequently comprise 3-20 different analogues, such as 3-10 analogues. However, normally the number of analogues will be sought kept to a minimum such as 1 or 2 analogues.
Nucleic Acid Vaccination
As a very important 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 proteins). 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 posttranslation processing of the expression product is expected to occur; this is especially important in the case of autovaccination, since, as mentioned above, a significant fraction of the original B-cell epitopes of the polymer should be preserved in the modified molecule, and since B-cell epitopes in principle can be constituted by parts of any (bio)molecule (e.g. carbohydrate, lipid, protein etc.). Therefore, native glycosylation and lipidation patterns of the immunogen may very well be of importance for the overall immunogenicity and this is expected to be ensured by having the host producing the immunogen.
It should be noted that the enhanced expression levels observed with some of the presently disclosed analogues is very important for efficacy of DNA vaccination, since the in vivo expression level is one of the determining factors in the immunogenic efficacy of a DNA vaccine
Hence, a preferred embodiment of the invention comprises effecting presentation of the analogue of the invention to the immune system by introducing nucleic acid(s) encoding the analogue 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 a polymer, e.g. in PLGA (cf. the microencapsulation technology described in WO 98/31398) or in chitin or chitosan, and DNA formulated with an adjuvant. In this context it is noted that practically all considerations pertaining to the use of adjuvants in traditional vaccine 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 intraarterlally. Furthermore, it is well known in the art that nucleic acid vaccines can be administered by use of a so-called gene gun, and hence also this and equivalent modes of administration are regarded as part of the present invention. Finally, also the use of a VLN in the administration of nucleic acids has been reported to yield good results, and therefore this particular mode of administration is particularly preferred.
Furthermore, the nucleic acid(s) used as an immunization agent can contain regions encoding the moieties specified in the claims, e.g. in the form of the immunomodulating substances described above such as the cytokines discussed as useful adjuvants. A preferred version of this embodiment encompasses having the coding region for the analogue and the coding region for the immunomodulator in different reading frames or at least under the control of different promoters. Thereby it is avoided that the analogue or epitope is produced as a fusion partner to the immunomodulator. Alternatively, two distinct nucleotide fragments can be used, but this is less preferred because of the advantage of ensured co-expression when having both coding regions included in the same molecule.
Accordingly, the invention also relates to a composition for inducing production of antibodies against TNFα, the composition comprising
a nucleic acid fragment or a vector of the invention (cf. the discussion of nucleic acids and vectors below), and
a pharmaceutically and immunologically acceptable vehicle and/or carrier and/or adjuvant as discussed above.
Under normal circumstances, the nucleic acid is introduced in the form of a vector wherein expression is under control of a viral promoter. For more detailed discussions of vectors and DNA fragments according to the invention, cf. the discussion below. Also, detailed disclosures relating to the formulation and use of nucleic acid vaccines are available, cf. Donnelly J J et al, 1997, Annu. Rev. Immunol. 15: 617-648 and Donnelly J J et al., 1997, Life Sciences 60: 163-172. Both of these references are incorporated by reference herein.
Live Vaccines
A third alternative for effecting presentation of the analogues of the invention to the immune system is the use of live vaccine technology. In live vaccination, presentation to the immune system is effected by administering, to the animal, a non-pathogenic microorganism that has been transformed with a nucleic acid fragment encoding an analogue of the invention 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 P D, 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 to the animal, but in certain cases it may be necessary to administer the microorganism more than once in a lifetime in order to maintain protective immunity. It Is even contemplated that immunization schemes as those detailed above for polypeptide vaccination will be useful when using live or virus vaccines.
Alternatively, live or virus vaccination is combined with previous or subsequent polypeptide and/or nucleic acid vaccination. For instance, it is possible to effect primary immunization with a live or virus vaccine followed by subsequent booster immunizations using the polypeptide or nucleic acid approach.
The microorganism or virus can be transformed with nucleic acid(s) containing regions encoding the moieties mentioned above, e.g. in the form of the immunomodulating substances described above such as the cytokines discussed as useful adjuvants. A preferred version of this embodiment encompasses having the coding region for the analogue and the coding region for the immunomodulator in different reading frames or at least under the control of different promoters. Thereby it is avoided that the analogue or epitopes are produced as fusion partners to the immunomodulator. Alternatively, two distinct nucleotide fragments can be used as transforming agents. Of course, having the adjuvating moieties in the same reading frame can provide, as an expression product, an analogue of the invention, and such an embodiment is especially preferred according to the present invention.
Combination Treatment
One especially preferred mode of carrying out the invention involves the use of nucleic acid vaccination as the first (primary) immunization, followed by secondary (booster) immunizations with a polypeptide based vaccine or a live vaccines as described above.
Use of the Method of the Invention in Disease Treatment
The diseases/conditions that are relevant are rheumatoid arthritis, juvenile chronic arthritis, spondylarthropathies, polymyositis, dermatomyositis, vasculitis, psoriasis (plaque) and psoriatic arthritis, Mb. Crohn, chronic obstructive pulmonary disorder, myelodysplastic syndrome, uveitis in rheumatoid arthritis, acute pulmonary dysfunction, asthma (both acute and chronic), Wegener's granulomatosis, irritable bowel disease, temporomandibular disorder (painful jaw joint), stomatitisosteoporosis, and cancer cachexia as well as other inflammatory diseases and other conditions generally appreciated in the art to be linked to the adverse effects of TNFα. It is therefore possible to treat or ameliorate symptoms that are associated with any of these diseases by employing the method of the invention for down-regulating activity of a multimeric protein.
Compositions of the Invention
The invention also pertains to compositions useful in exercising the method of the invention. Hence, the invention also relates to an immunogenic composition comprising an immunogenically effective amount of an analogue defined above, said composition further comprising a pharmaceutically and immunologically acceptable diluent and/or vehicle and/or carrier and/or excipient and optionally an adjuvant. In other words, this part of the invention concerns formulations of analogues, essentially as described hereinabove. The choice of adjuvants, carriers, and vehicles is accordingly in line with what has been discussed above when referring to formulation of the analogues for peptide vaccination.
The analogues are generally prepared according to methods well known in the art. Longer polypeptides are normally prepared by means of recombinant gene technology including introduction of a nucleic acid sequence encoding the analogue into a suitable vector, transformation of a suitable host cell with the vector, expression of the nucleic acid sequence (by culturing the host cell under appropriate conditions), recovery of the expression product from the host cells or their culture supernatant, and subsequent purification and optional further modification, e.g. refolding or derivatization. Details pertaining to the necessary tools are found below under the heading “Nucleic acid fragments and vectors of the invention” but also in the examples. In this section is also described the preferred method of recombinant preparation of the analogues, i.e. low-temperature fermentation of E. coli in order to obtain soluble TNFα variants.
Recent advances in peptide synthesis technology has rendered possible the production of full-length polypeptides and proteins by these means, and therefore it is also within the scope of the present invention to prepare the long constructs prepared by means of the well-known techniques of solid- or liquid-phase peptide synthesis.
Nucleic Acid Fragments and Vectors of the Invention
It will be appreciated from the above disclosure that modified polypeptides can be prepared by means of recombinant gene technology but also by means of chemical synthesis or semi-synthesis; the latter two options are especially relevant when the modification consists of or comprises coupling to protein carriers (such as KLH, diphtheria toxoid, tetanus toxoid, and BSA) and non-protelnaceous molecules such as carbohydrate polymers and of course also when the modification comprises addition of side chains or side groups to an polymer-derived peptide chain. These embodiments, are, as will be understood from the above, not the preferred ones.
For the purpose of recombinant gene technology, and of course also for the purpose of nucleic acid immunization, nucleic acid fragments encoding the analogues are important chemical products (as are their complementary sequences). Hence, an important part of the invention pertains to a nucleic acid fragment which encodes an analogue as described herein, i.e. a polymer derived artificial polymer polypeptide as described in detail above. The nucleic acid fragments of the invention are either DNA or RNA fragments.
Most preferred DNA fragment of the invention comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences encoding any one of SEQ ID NOs: 12, 13, 14, 16, 17 and 18 or a nucleic acid sequence complementary to any of these.
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, phage, cosmids, mini-chromosomes, or virus, but also naked DNA, which is only expressed transiently in certain cells, is an important vector (and may be useful in DNA vaccination). 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 periplasm) 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 not incapable of being integrated in the host cell genome; typically, naked DNA or non-integrating viral vectors are used, the choices of which are well-known to the person skilled in the art.
The vectors of the invention are used to transform host cells to produce the modified TNFα polypeptide of the invention. Such transformed cells, which are also part of the invention, can be cultured cells or cell lines used for propagation of the nucleic acid fragments and vectors of the invention, or used for recombinant production of the modified TNFα 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 TNFα.
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. Most preferred are cells derived from a human being, cf. the discussion of cell lines and vectors below. Recent results have shown great promise in the use of a commercially available Drosophila melanogaster cell line (the Schneider 2 (S2) cell line and vector system available from Invitrogen) for the recombinant production of TNFα analogues of the invention, and therefore this expression system is particularly preferred, and therefore this type of system is also a preferred embodiment of the invention in general.
For the purposes of cloning and/or optimised expression it is preferred that the transformed cell is capable of replicating the nucleic acid fragment of the invention. Cells expressing the nucleic fragment are preferred useful embodiments of the invention; they can be used for small-scale or large-scale preparation of the analogue or, in the case of non-pathogenic bacteria, as vaccine constituents in a live vaccine.
When producing the analogues 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, since both of these options facilitate subsequent purification of the expression product.
When an effective producer cell has been identified it is preferred, on the basis thereof, to establish a stable cell line which carries the vector of the invention and which expresses the nucleic acid fragment encoding the modified TNFα. Preferably, this stable cell line secretes or carries the TNFα analogue of the invention, thereby facilitating purification thereof.
In general, plasmid vectors containing replicon and control sequences that 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 sequences, which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (see, e.g., Bolivar et al., 1977). The pBR322 plasmid contains genes for 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 that can be used by the prokaryotic microorganism for expression.
Those promoters most commonly used in prokaryotic recombinant DNA construction include the B-lactamase (penicillinase) and lactose promoter systems (Chang et al., 1978; Itakura et al., 1977; Goeddel et al., 1979) and a tryptophan (trp) promoter system (Goeddel et al., 1979; EP-A-0 036 776). 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.
According to the present invention, it is preferred that the preparation of the TNFα analogues in prokaryotic cells results in the provision of soluble proteins, cf. Example 9, and it is especially preferred that the host cell used for production is an E. coli cell.
Soluble TNFα variants have now been shown by the present inventors to be relatively easy and convenient to prepare in E. coli at lowered temperatures. Where conventional methods for fermentation of E. coli normally utilises temperatures in the range around 37° C., it has been found by the present inventors that increased yields of soluble TNFα variants can be obtained by fermenting at temperatures below 32° C.—even though the total yield of recombinant protein is lower than after fermentation at around 37° C., the yield of the most suitable variants Is considerably higher, and no refolding of insoluble, denatured protein is necessary.
In brief, a typical fermentation process of the invention involves the steps of inoculation of a fermentor with a transformed bacterium, subsequent fermentation to obtain a sufficient amount of biomass followed by induction of recombinant expression, and finally, harvest of the recombinant protein. It is not mandatory that inducible systems are used, but it is more convenient.
Hence, an important aspect of the invention relates to recombinant production in bacteria, notably E. coli, of the TNFα variants of the present invention, where at least the phase after induction of recombinant TNFα analogue production is performed at a temperature of less than 32° C. (that is, in case an inducible system is used). Example 9 has, however, demonstrated the highest yields of soluble recombinant TNFα analogues when all fermentation (both before and after induction) is performed at such low temperatures.
It is preferred that the lowered temperature in either of the two-phases of fermentation (before and after induction) is below 30° C., such as below 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20° C. It is preferred that the temperature is in the range between 20 and 30° C., more preferably in the range between 22 and 28° C., and it is most preferred that the temperature is about 25° C. As is shown in Example 9, yields of close to 100% soluble TNFα analogues have been obtained when fermenting at this temperature.
It should be noted that the inventors believe that the low temperature conditions for production of soluble TNFα variants of the present invention are generally applicable for recombinant production of soluble variants of immunogenic proteins—even though the total protein yields obtained by such fermentation are lower than what is achieved by e.g. fermentation at 37° C., the final yield of protein having a useful 3D structure is considerably higher when using the low temperature conditions, and, importantly, subsequent time and resource consuming refolding procedures can be avoided. Or, in brief, the yield of the desired conformation of the variant protein is higher.
Hence, the present inventors also suggest that the strategy of using the above-specified low-temperature fermentation conditions is a generally applicable way of producing useful immunogenic variants of proteins.
In addition to prokaryotes, eukaryotic microbes, such as yeast cultures may also be used, and here the promoter should be capable of driving expression. Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al., 1980). This plasmid already contains the trpl gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan for example ATCC No. 44076 or PEP4-1 (Jones, 1977). The presence of the trpl lesion as a 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, phosphofructokinase, 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 i.a. Protein Sciences, 1000 Research Parkway, Meriden, Conn. 06450, U.S.A. and from Invitrogen), and MDCK cell lines. In the present invention, an especially preferred cell line the insect cell line S2, 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) or cytomegalovirus (CMV). 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 HindIII site toward the BglI 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.
Preparation of TNFα Variants
A synthetic DNA sequence “SMTNFWT3” (SEQ ID NO: 9) encoding the wild type human TNFα monomer polypeptide (SEQ ID NO: 10) was delivered as a ligation product from Entelechon GmbH. The DNA sequence of the human hTNFα was optimised for expression in E. coli according to the Codon Usage Database by exclusion of all codons with a frequency in E. coli of less than 10%. Further, the sequence was designed to include a 5′ NcoI restriction site for subsequent cloning steps.
The SMTNFWT3 ligation product was introduced into the pCR 4 TOPO Blunt vector and E. coli DH10B cells were transformed. Plasmid DNA from 10 of the resulting SMTNFWT3TOPO clones was purified and five clones containing the expected fragment (when analysed by Restriction Enzyme (RE) digest) were selected.
The NcoI/EcoRI DNA fragments from the five potentially correct SMTNFWT3TOPO clones were isolated and transferred to the pET28b(+) vector and sequence determined. Insertions, deletions or substitutions were identified in four clones whereas one clone appeared to be correct. The correct construct—SMTNFWT3pET28 was subsequently used as template for the generation of all single TNFα variants.
TNF34 Construction
The PanDR epitope amino acid sequence (SEQ ID NOs: 6 and 8) was manually “reverse-translated” to a DNA sequence (SEQ ID NO: 7) optimised for expression in E. coli, see below, and inserted in loop 3 of TNFα by SOE PCR.
The resulting construct (a DNA sequence encoding SEQ ID NO: 19 of WO 03/042244) was placed In the pET28b+ vector to generate TNF34-pET28b+.
Monomerized Trimer Construction
The monomerized trimer constructs are based on 3 TNFα encoding regions, separated by either a tri-glycine linker and/or an epitope-encoding region.
The TNFα gene was synthesized as three separate entities. The three fragments were assembled by SOE PCR, and the assembled gene (SEQ ID NO: 21 in WO 03/042244) was cloned into pCR2.1-TOPO. After sequence verification, a correct clone was isolated. The hTNFT—0 gene (SEQ ID NO: 21 In WO 03/042244 encoding TNFα-GlyGlyGly-TNFα-GlyGlyGly-TNFα, SEQ ID NO: 22 in WO 03/042244, i.e. 3 copies of SEQ ID NO: 17 separated by two tri-glycine linkers) was then transferred to pET28b+ to generate hTNFT—0-pET28b+. A correct clone was isolated, sequence verified and transformed into E. coli lines BL21-STAR, BL21-GOLD and HMS174.
hTNFT—0-pET28b+ was used as template to generate the following four monomerized trimer variants: hTNFT—1, hTNFT—2, hTNFT—3 and hTNFT—4 (SEQ ID NOs: 49, 51, 57, and 59 in WO 03/042244) by SOE PCR. A further variant (SEQ ID NO: 53 In WO 03/042244) can be made in a similar way.
hTNFT—1, hTNFT—2 and hTNFT—3 are variants including tetanus toxoid epitopes P2 and P30 (SEQ ID NOs: 2 and 4, respectively) that need to be assembled by two rounds of SOE PCR. hTNFT—4 is a variant with a PADRE (SEQ ID NO: 6) insert and can be assembled by a single round of SOE PCR. A further variant (SEQ ID NO: 55 in WO 03/042244) can be made in a similar way.
hTNFT—4 was constructed by the above-mentioned methods, and a correct clone of hTNFT—4-pET28b+ was found in TOP 10 cells and the construct was transferred to BL21-STAR and HMS174 cells.
To generate hTNFT—1, hTNFT—2 and hTNFT—3 the epitopes were inserted by SOE PCR in very small fragments of the trimer, which were inserted into hTNFT—0-pET28b+ by RE cutting and ligation.
Stabilising TNF34 Mutants
To further stabilise the TNF34-pET28b+ variant described above, variants containing the introduction of an extra disulfide bridge as well as a deletion mutant were constructed. 3 different variants were constructed:
TNF34-A-pET28b+ contains the substitutions Q67C and A111C, TNF34-B-pET28b+ contains A96C and I118C, and TNF34-C-pET28b+ that contains a deletion of the 8 most N-terminal amino acids—the amino acid sequences of the expression products are set forth in SEQ ID NOs: 20, 30, and 31 in WO 03/042244.
All 3 constructs were made using SOE PCR, and were cloned in BL21-STAR, BL21-GOLD and HMS174, followed by sequence verification.
Flexible Loop Variants
In order to find a variant that might exhibit improved characteristics compared to the TNF34-pET28b+ variant, constructs were made where the PADRE insert (SEQ ID NO: 6) is moved around in flexible loop 3 of the TNFα molecule.
All of these: TNF35-pET28b+, TNF36-pET28b+, TNF37-pET28b+, TNF38-pET28b+, TNF39-pET-28b+, and a variant with PADRE placed in the C terminus of the molecule; TNFC2-pET28b+, were made with SOE PCR technique and were cloned in BL21-STAR, BL21-GOLD and HMS174, followed by sequence verification. The amino acid sequences of the expression products are set forth in SEQ ID NOs: 23, 24, 24, 26, 27 and 28 in WO 03/042244.
To also evaluate the possibility of using insect cells as expression system, TNFWT, TNF34, TNF35, TNF36, TNF37, TNF38, TNF39 and TNFC2 were transferred into the p2Zop2f vector (cf.
Other Constructs
A large number of further TNFα variants have been prepared, all termed TNFX, cf. above. The DNA encoding these variants has being made by SOE PCR, and cloned directly into pET28b+.
The correct TNFX clones have been transformed into BL21-STAR and HMS174, and subsequently sequence verified.
Periplasmic Expression
The LTB leader sequence has been added directly upstream of SEQ ID NO: 16 of WO 03/042244 in TNF34-pET28b+, to target the expression to the periplasmic space.
Mammalian Expression
To test for expression in mammalian cells, SEQ ID NO: 16 of WO 03/042244 and the DNA encoding TNF34 have been transferred to the pHP1 vector, which is a variant of the commercially available pCI vector (Promega Corporation). pHP1 includes a kanamycin resistance gene as marker instead of the AmpR gene of pCI.
Optimised Production of Soluble Protein from E. coli
Introduction
As the initial expression of TNFα and variants thereof only resulted in very limited amounts of soluable protein, when expressed from E. coli cells, the task was to improve the expression levels and the solubility of the TNFα variants significantly to ensure a fast and convenient in expression system.
Approach
Initial experiments were made in shake flasks and the experience gained was used when working with fermentors. 3 fermentors were run at a time, and samples were taken out during the fermentation for analysis of cell growth, TNFα production and degradation levels.
Objective
The objective of the study was to define the optimal growth conditions for expression of immunogenic TNFα variants when expressed in HMS174 E. coli cells in defined growth media.
Materials and Methods
Raw Materials
Starting Materials for fermentation with a defined minimal medium:
Media and Buffer Compositions
Preparation of Main Culture Medium
Items 1-8 are dissolved in the written order in approximately 50% of the final volume in deionized 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° C.). pH is ted to 7.
Preparation of Pre Culture Medium
The desired volumes of the stock solutions, items 1-9, are transferred to a 1 l 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.
Preparation 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 1 l blue cap bottle and autoclave it.
Equipment
Fermentors
The InFors Labfors fermentor system consisting of 6 2 L 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.
Methods
The initial experiments to evaluate the percentage of soluble protein expressed was done in 250 ml shake flasks, in a incubator shaken at approx 200 rpm/min in rich media w/60 μ/ml kanamycin.
TNFα protein expression was evaluated with Western blots and Coommassie stained SDS PAGE, in both HMS174 and BL21 Star strains. Both total and soluble TNFα expression was evaluated, by lysing the cells, and removing a sample from the supernatant before and after a centrifugation step (20.000×g for 10 minutes). The percentage of soluble TNFα was estimated by “eyeballing” the Western Blot.
Different temperature combinations were tested: mostly, biomass was produced at 37° C. followed by induction, and expression temperatures of 25° C. (37/25) or 37° C. (37/37) was tested.
TNF37(A145R) (SEQ ID NO: 13) was chosen as the model variant for expression and was then tested with defined media, in shake flasks, using the 37/25° C. combination.
Creating a Research Cell Bank
A Research cell bank (RCB) of TNF37(A145R) was established in Yeast Media (YEM) with 60 μ/ml kanamycin, by inoculating a pre-warmed 37° C. 1 l baffled shake flask containing 225 ml YEM, with 25 ml from a 250 ml ON culture in YEM with 60 μg/ml kanamycin, and growing at 37° at 200 rpm for 3½ hours. Identical glycerol freeze stocks were made by mixing 60 ml exponentially growing cell culture with 140 ml 86% glycerol, and aliquotting in 1 ml aliquots. These aliquots are stored at −80° C. and a single aliquot is used for pre-culture prior to fermentation.
To determine the quality of the RCB, 3 pre-culture flasks containing 250 ml pre-culture media w/60μg/ml kanamycin were inoculated with an RCB aliquot each and OD600 was followed.
The cultures behaved identically until 11 hours after inoculation where they reached an OD600 of ˜3.2. It was decided to inoculate 1 l fermentors with 50 ml pre-culture that have been grown for 11 hours under the above-mentioned conditions, in all the following experiments.
Optimising Fermentor Conditions
To determine the optimal fermentation conditions, samples were taken from growing cultures in Inforss fermentors, and analysed for OD600, by spectrophotometry, TNF expression levels were determined using a quantitative TNF specific ELISA, and TNF degradation was determined by Western Blotting.
Fermentations were tested at different temperature conditions, 25°/25°, 25°/25°/16°, 37°/25°and 37°/37°.
OD600 and TNF expression was tested on a large number of samples, and few representative samples with regard to TNF expression were further analysed by Western Blot to determine degradation.
Schematic Presentation
General Process Parameters:
Specific Process Paramaters
The IPTG concentration was 1 mM and the temperature at induction was lowered from 37° C. to 25° C. except from the 37/37° C. and the 25/25° C. processes. The total fermentation time was between 14 and 18 hours for the 37/37° C. process and up to 61 hours for the 25/25° C. and the 37/25° C. processes, including propagation, induction and protein production. The total fermentation time depends on the growth of the culture. OD600start in the fermentor was between 0.1-0.3 (2-6 in the pre culture) as calculated from the OD in the inoculation culture. Induction, 37/25° C. process, was performed at OD600=20±1-2 or nine to eleven hours after inoculation. Protein production took place for 20-24 hours. Induction at the 25/25° C. process was performed at OD600=10±1-2 or 22-24 hours after inoculation. Protein production took place for 24-36 hours.
TNFα Variant Expression
The TNF expression levels were tested by taking out 2×1 ml samples and storing at −20° C. The samples were then thawed on ice and sonicated 3×30 sec, with at least 30 sec on ice in between to prevent heating of the samples. The samples were centrifuged at 20.000×g for 10 min and the supernatant was tested for TNFα content using the TNFα quantitative ELISA.
A general overview of the process can be seen in the flow chart of
Results and Discussion
The experiments carried out in this study, showed that expression of soluble TNF significantly increased when expressed at 25° C. both in shake flasks and in fermentors.
Shake Flask Experiments
100 ml Rich medium was inoculated with 5 ml ON culture, and grown at 37° C. When the culture reached an OD600 the culture was Induced with 1 mM IPTG. At the same time, the cultures grown at 37° C. were either kept at 37° C. or moved to 25° C., were they were grown over night. The following day, the cells were harvested, lysed and assayed for total and soluble expression of TNFα variant.
The results showed that the temperature shift from 37° C. to 25° C. at the point of induction, had a significant positive effect on the amount of soluble TNF expressed, so it was decided to keep this temperature shift for the following experiments.
Selected Variants
All variants were tested as described above, and TNF34, TNF34A, TNF37, TNFX5.1, TNFX2.1 and TNF_T2 were selected as expressing in high enough levels for further investigation.
Where the detoxified versions of the above-mentioned constructs were available, expression of these variants were tested and the results showed comparable expression levels to the non-detoxified versions.
Expression in Fermentors
To test TNFα variant expression in defined media, we made an initial experiment in shake flasks, with the same conditions as described above (37°/25°). The over night Induced culture showed satisfactory levels of TNFα variant expression, and we therefore decided to test the expression in fermentors to optimise the amounts of TNFα variant expressed, and also to try an minimise the degradation of the variants that still is seen in the expressed variants.
Cell Growth
The Research cell bank was made as described above, and we used this pre-culture to inoculate 1 l fermentors with 50 ml of pre-culture at OD600 ˜3-4. The fermentors were preheated to the chosen temperature (37° C. or 25° C.) to minimise the temperature shock. In the case of 37° C. fermentors, the cells continued exponential growth without any lag-phase. When the 37° C. pre-culture was inoculated into a 25° C. fermentor, the cells had a lag-phase of approx. 17 hours, before commencing exponential growth at a slower rate. This is to be expected as a consequence of the temperature shock the cells are subjected to in this case.
The same lag-phase was seen when we shifted the 37° C. culture to 25° C. immediately before induction. An attempt to gradually shift the temperature from 37° C. to 25° C. over an hour (2° pr 10 min) did not reduce the lag-phase.
TNFα Variant Expression Levels
The expression levels were determined via the TNFα specific quantitative ELISA.
The highest levels seen are in the 25/25° C. process, were levels of approx 250 mg/l are obtained. These experiments are still single verifications, and the time between 15 h and 35 h after induction still remains to be analysed.
The 37/37° C. process only gives very limited amounts of soluble TNFα variant approx 15 mg/l and the 37/25° C. process seems to peak at around 100 mg/l
Conclusions
Based on results from a total of more than 100 fermentations, 2 upstream processes (from a total of 6) for the production of soluble TNFα variant proteins have been chosen. The fermentations were performed in a defined minimal medium (see description below). The chosen fermentation processes were based on temperature studies where 4 different temperature combinations were tested. The tested TNFα variant was TNF37-145 in the E. coli strain HMS174.
The optimal results have been obtained using 25° both pre- and post-induction. The optimal harvest time remains to be determined.
Selection assays
A direct receptor ELISA together with a polyclonal ELISA and a cytotoxicity assay with KD-4 and Wehi cells are used as first line assays to screen and follow purification. Antibodies produced against TNFα variants are used to inhibit wtTNFα binding in both the receptor and the cytotoxic assay, to measure the antibody quality.
Purification Procedures
In this example, recombinant production and subsequent purification of one of the TNFα variants (TNF37) is described in detail. However, the purification procedure is the preferred one according to the present invention and will also be applicable (with small adjustments relevant for each variant) for other TNFα variants of the present invention.
However, at present the inventors are working on an improved purification scheme that is especially suited for large-scale fermentation of TNFα variants. Basically, the same individual steps described below are used, but the order is reversed so as to finish the purification with a hydroxyapatite chromatographic step after SP-sepharose and Q-sepharose chromatographic steps.
An E. coli strain BL21 STAR/TNF37 colony from a LB-kanamycin plate (60 mg kanamycin/L LB media containing 1.5% Agar) is resuspended In 5 ml LB-media (60 mg kanamycin/L LB) and grown over night (16 hours) at 37° C. while shaking 220 RPM in a New Braunswick shaker.
2×2 ml of this culture is transferred to 2×1 L LB (60 mg kanamycin/L) in 2 L baffled shake flasks and the cells are allowed to grow in a New Braunswick shaker at 220 RPM to OD436=0.6-0.8. This step has been performed at the exemplary temperatures 37° C. and 25° C., but the temperature may be optimised for each culture.
1 ml 1 M IPTG is added to each flask and the cells are allowed to grow for 16-20 hours. Before induction, the temperature is adjusted to 25° C. if this is not already the fermentation temperature.
The cells are harvested in centrifuge tubes (500 ml) by centrifugation at 5000 RPM for 15 min using an SLA-3000 head in a Sorvall centrifuge.
The cells are transferred to one 500 ml pre-weight centrifuge tube using 0.9% NaCl and harvest cells by centrifugation as before.
The supernatant is discharged and the tube is weighed to determine the cell weight (should be 7-11 grams).
200 ml 50 mM Na2HPO4, pH=7.0 is added (if cells are re-suspended they should be used directly, otherwise it is possible to freeze).
Cell Disruption, Centrifugation, and Filtration
A mechanical disruption of the cells offer several advantages over enzymatic disruption in terms of efficiency, reliability and the ability to choose any buffer necessary in the following steps of the purification. The APV-1000 is kept cool during the operation by adding ice water to the sample-chamber before use and pas ice water through the machine between the two passages of sample. Centrifugation and filtration serves to remove any particles or aggregates from solution prior to chromatographic separation of the proteins. The cell disruption and HA-chromatography should be done the same day as this might minimize the apparent protease activity as a consequence of the separation from these in the chromatographic step. The procedure for disruption, centrifugation and filtration is as follows:
The carefully re-suspended cell material is transferred from to the cell-disrupter (APV-1000). The cell-suspension is careful passed 2× through the disrupter (cooling on ice after each passage and passing ice water through the APV-1000 in between the passages) using 700 bars of backpressure (the solution ought to be clear at this point).
The disrupted cells are transferred to a 500 ml centrifuge tube and the cells are spun for 45 min at 10000 RPM in a Sorvall centrifuge using the SLA-3000 head.
The extract (approx 225 ml) is passed through a 0.22 μm filter.
Hydroxyapatite(HA) Chromatography
Hydroxyapatite Bio-Gel HTP Gel (BIO-RAD; catalogue # 130-0420) is a crystalline form of calcium phosphate having proven itself as a unique tool in the separation of proteins such as monoclonal antibodies and other proteins otherwise not separable by other methods. However, in our experience the flow properties of the material are somewhat critical in that sense that a flow higher than 2 ml/min raises the pressure to an unacceptable high level. Also the material has collapsed several times when attempt has been made to regenerate with sodium hydroxide as recommended by the manufacturer.
Buffers and Column
Stock for buffer A+B: 1 M Na2HPO4×2H2O pH=7.0 (pH adjusted to 7 with HCl). Buffers A+B made from dilutions of stock.
Buffer A: 50 mM Na2HPO4×2H2O, pH=7.0
Buffer B: 0.3 M Na2HPO4×2H2O, pH=7.0
Column packed to approximately 50-60 ml with hydroxyapatite Bio-Gel HTP Gel (BIO-RAD; catalogue # 130-0420) using a suspension in Buffer A and a XK 26/40 (Amersham Biosciences) column.
Chromatography Program
Purge system 20 ml at a flow of 30 ml/min.
Equilibration: 4 CV of Buffer A at a flow of 2 ml/min
Load sample through pump (inlet F on the BioCad) (approx 225+5-10 ml if the sample in the tubing is needed) at a flow of 2 ml/min.
Wash column with 1.5 CV Buffer A at a flow of 2 m/min.
Elution: Elute protein with a gradient of 4 CV from 0% to 100% Buffer B at a flow of 2 ml/min.
Clean column with 2 CV Buffer B at a flow of 2 ml/min.
Re-equilibration with 4 CV Buffer A at a flow of 2 ml/min.
Select fractions, pool, and dialyse ON at 4° C. against 15× volume 20 mM Tris-HCl, 0.075 M NaCl, pH 8.0.
Selecting TNF37-Containing Fractions After HA Chromatography
The HA chromatography elution fraction profile basically consist of a “run through” fraction and one eluted peak that can be separated into several peaks. The TNF37-containing fractions has to be selected on the basis of a Coommassie stained gel of the entire peak since a peak containing TNF37 is not directly identifiable. However, as a consequence of subsequent purification steps the selection of fractions at this point is less critical and it is possible to remove contaminants later in the procedure. Thus, a less conservative selection of fractions ensures maximum yield of variant.
Initially the “run through” was checked with “dot blots” for any TNF37. This gave a positive result that In theory should indicate that a significant part of the variant did not bind to the column. However, when the “run through” is subjected to the very efficient SP-sepharose cation Exchange Chromatography (cf. next step) and the fractions are analysed with Coommassie stained gels they do not contain any detectable TNF37-variant indicating some false positive reaction in the “dot blot” or a fraction of the variant that binds completely different to the SP-sepharose.
SP-Sepharose Cation Exchange Chromatography
SP-sepharose is a basic cation exchange step selected as consequence of the rather high, calculated pI of 9.4 of the variant compared to the wtTNFα pI of 7.8. This increase in pI is a consequence of the 2 lysines introduced via the PADRE epitope. This chromatography is very efficient and fast for the TNF37 variant and is expected to be useful for a large number of other loop variants of TNFα.
The sample applied should have a lower conductivity than 8 mS/cm and pH should be at least 7.7 before continuing with SP-sepharose chromatography since variations from this in our experience has made the binding properties of the protein different from time to time.
Buffers and Column
Stocks to buffers A+B: 1 M Tris-HCl. pH=8.0.
Buffer A: 20 mM Tris-HCl, 0.075 M NaCl pH=8.0.
Buffer B: 20 mM Tris-HCl, 1 M NaCl, pH=8.0.
Column packed to approximately 60 ml with SP-sepharose FF (Amersham Biosciences; catalogue # 17-0729-01) using a suspension in Buffer A and a XK 26/40 (Amersham Biosciences) column.
Chromatography Program
Purge system 20 ml at a flow of 30 ml/min
Equilibration: 4 CV of Buffer A at a flow of 4 ml/min.
Load sample through pump (inlet F on the BioCad) (Sample+10 ml if the sample in the tubing is needed) at a flow of 4 ml/min.
Wash column with 1.5 CV Buffer A at a flow of 4 m/min.
Elution: Elute protein with a gradient of 4 CV from 0% to 100Buffer B at a flow of 4 ml/min.
Clean column with 2 CV Buffer B at a flow of 4 ml/min.
Re-equilibration with 4 CV Buffer A at a flow of 4 m/min.
Select fractions, pool, and dialyse ON at 40° C. against 15× volume 20 mM Tris-HCl, 0.075 M NaCl, pH 8.0.
Selecting TNF37 Containing Fractions after SP Sepharose Chromatography
The profile basically consists of a “run through” fraction and several protein containing peaks. However two peaks contains the variant with some contaminants. It is at this point important not to include to many fractions on the right side of peak two since this in our experience includes to many contaminants that are not easily removed in subsequent chromatographic steps.
Q-Sepharose Anion Exchange Chromatography
Q-sepharose is a basic anion exchange step selected for removing a major contaminant protein that with high reproducibility follows the purification of TNF37 including the HA-chromatography and SP-sepharose. The TNF37 variant itself does not bind to the column but the major unknown contaminant does. It is, however, possible to select fractions in a conservative fashion already in the SP-sepharose step in that way avoiding the contaminant. However, this compromises the yield of TNF37 variant compared to when the Q-sepharose is used in the procedure and since also other minor contaminants are removed in this step, it is preferred to include it in the total procedure. In conclusion the Q-sepharose step is important in the purification of variant 37 and offers an even better end product with a high yield.
Buffers and Column
Stocks to buffers A+B: 1 M Tris-HCl. pH=8.0.
Buffer A: 20 mM Tris-HCl, 0.075 M NaCl, pH=8.0.
Buffer B: 20 mM Tris-HCl, 1 M NaCl, pH=8.0.
Column packed to approximately 50-60 ml with Q-sepharose FF (Amersham Biosciences; catalogue # 17-0510-01) using a suspension in Buffer A and a XK 26/40 (Amersham Biosciences) column.
Chromatography Program
Purge system 20 ml at a flow of 30 ml/min.
Equilibration: 4 CV of Buffer A at a flow of 4 ml/min
Load sample through pump (inlet F on the BioCad) (Sample+10 ml if the sample in the tubing is needed) at a flow of 2 m/min.
Wash column with 3 CV Buffer A at a flow of 4 ml/min.
Elution: Elute remaining protein with 2 CV 100% Buffer B at a flow of 4 ml/min.
Re-equilibration with 4 CV Buffer A at a flow of 4 ml/min.
Select fractions, pool and apply directly on SP-sepharose column.
The elution profile basically consists of a “Run through” fraction and several protein containing peaks. The “Run through” fraction can sometimes be divided into several purely resolved peaks which all contains the TNF37 variant and therefore all are pooled. This heterogeneity of the TNF37 is probably solved when the problem with the apparent proteolytic degradation is solved.
Immunisation Studies
Materials:
Saline (0,9% NaCl in sterile water, Fresenius Kabi Norge AS, Norway)
Complete Freund's Adjuvant (Sigma, F-5881, 39H8926)
Incomplete Freund's Adjuvant (Sigma, F-5506, 60K8937)
Alhydrogel 2% [10 mg Al/ml](Brenntag Bilosector, Batch 96 (3176))
Adjuphos[5 mg Al/ml] (Brenntag Biosector, Batch 2 (8937))
Wild type human TNF (Invitrogen cat.no: 10062-024).
KYM-1D4: Provided by A. Meager (A. Meager, J. Immunol. Methods 1991, 144:141-143)
WEHI 164 clone 13: Provided by T. Espevik (T. Espevik and J. Nissen-Myer, J. Immunol. Methods 1986, 95:99-105)
Tetrazolium salt (MTS, CellTiter 96 Aqueous one solution; Promega, G3581)
Rotating bar (Rotamix, Heto, Denmark)
Vortex (OLE DICH instrumentmakers ApS, Denmark)
Choice of Formulation/Adjuvant
The purified TNFα variant proteins (in 20 mM Tris-HCl, 0.075 M NaCl, pH 8.0) are diluted to 0.5 mg/ml with saline (0.9% NaCl), batched (375 μg/vial) and stored at −20° C. until used for immunizations.
For each TNFα variant, Immunizations are made with two adjuvants: 1) Complete Freund's Adjuvant (CFA, for the primary immunization) and Incomplete Freund's Adjuvant (IFA, for boost immunizations) and 2) Alhydrogel or Adjuphos (state-of-the-art Aluminum hydroxide and aluminum phosphate adjuvants, respectively)—these are used for both prime and boost injections.
Before primary immunization, a decision on the choice of either Alhydrogel or Adjuphos as adjuvant for the TNF variant is made. The adjuvant with the best ability to adsorb the TNFα variant is chosen for further use in the immunization experiment. Two aliquots of the TNFα variant are mixed with an equal volume of Alhydrogel and Adjuphos in two vials. The vials are gently mixed at room temperature for 30 minutes on a rotating bar. Vials are then centrifuged at 13000 g for 15 minutes and supernatant is tested for the soluble TNFα variant content on a gradient (4-12%) SDS gel. The adjuvant/varlant aliquot containing the least free variant (i.e. where more variant has bound to aluminum-particles) is then selected as the best adjuvant.
Preparation of Antigen/Adjuvant Emulgate
CFA/IFA emulgates are prepared through the following procedure:
Vials with TNFα variant [0.5 mg/ml] is thawed, transferred to a 10 ml sterile vial and mixed with an equal volume of CFA or IFA. The vial is then mixed further on a vortex at 3300 rpm for 30 minutes at 20° C.
Alhydrogel/Adjuphos emulgates are prepared through the following procedure:
Alhydrogel/Adjuphos are diluted to 1.4 mg Al/ml with saline. Vials with TNFα variant [0.5 mg/ml] is thawed, transferred to a 10 ml sterile vial and mixed with an equal volume of Alhydrogel [1.4 mg Al/ml] or Adjuphos [1.4 mg Al/ml]. The vial is then mixed further on a rotating bar for 30 minutes at 20° C.
Choice of Animal Model
Six-eight weeks old Balb/Ca female mice are repetitively immunized with TNFα variants. Blood samples are collected at different intervals and isolated sera are investigated for anti-wtTNFα antibody titers. Mice are ordered from Taconic Farms, Inc. Acquires M&B A/S, Denmark. Mice are housed at the animal facility of Pharmexa for one week before initiation of experiment.
Immunization Scheme and Dosage
Groups of 10+10 mice are immunized with each TNFα variant in CFA/IFA and Alhydrogel/Adjuphos respectively. 20+20 mice are used for immunization with wild type TNFα.
At the first immunization, 50 μg of protein in adjuvant will be injected subcutaneously. All mice will receive additional booster immunizations subcutaneously with 25 μg of protein in adjuvant 2, 6 and 10 weeks after the first immunization.
Blood samples will be collected immediately before the first immunization and 1 week after each boost immunization.
Assays Employed
Cytotoxicity bioassay using WEHI 164 clone 13- or KYM-1D4-cells: This assay is used to determine the toxicity of TNFα variants of the invention. Cells are cultured for 48 hours in the presence of titrated amounts of TNFα variants and cell death is determined by addition of Tetrazolium salt (MTS), which is bioreduced into a colored formazan product by living cells. Cytotoxicity of TNFα variants are compared to that of human wild type TNFα.
Cytotoxicity-inhibition bioassay using WEHI 164 clone 13- or KYM-1D4-cells: This assay is used to investigate the ability of anti-sera raised In TNFα immunized mice to neutralize the cytotoxic effect of wild type TNFα. Cells are cultured for 48 hours with titrated amounts of anti-sera and a constant concentration of wild type human TNFα, which is sufficient to induce cell death in 50% of cells in the absence of anti-sera. Cell death is determined by MTS, as described above. Neutralization-ability of sera from TNFα variant-immunized mice are compared to sera obtained from mice immunized with human wild type TNFα.
In Vitro Studies
Cytotoxicity bioassay using WEHI 164 clone 13- or KYM-1D4-cells: Cytotoxicity-inhibition bioassay using WEHI 164 clone 13- or KYM-1D4-cells.
Criteria for Choice of Best Immunogenic Constructs
TNFα variants should display minimal cytotoxicity. Immunization of mice with TNFα variants should generate anti-sera with better or equal ability to neutralize human wild type TNFα-mediated cytotoxidty in WEHI- or KYM-1D4 cells as sera obtained from human wild type TNFα-immunized mice.
The invention will now by further described by the following numbered paragraphs:
1. An immunogenic analogue of a human TNFα protein, wherein said analogue comprises
a) two or three complete TNFα monomers joined end-to-end by a peptide linker, wherein at least one peptide linker includes at least one MHC Class II binding amino acid sequence, or
b) two or three complete TNFα monomers joined end-to-end by an inert peptide linker, wherein at least one of the monomers include at least one foreign MHC Class II binding amino acid sequence or wherein at least one foreign MHC Class II binding amino acids sequence is fused to the N- or C-terminal monomer, optionally via an inert linker, or
c) a human TNFα monomer or an analogue defined in a or b, wherein has been inserted or insubstituted at least one foreign MHC Class II binding amino acid sequence into flexible loop 3, or
d) a human TNFα monomer or an analogue defined in a or b, wherein has been introduced at least one disulfide bridge that stabilises the TNFα monomer 3D structure, or
e) a human TNFα monomer or an analogue defined in a or b, wherein any one of amino acids 1, 2, 3, 4, 5, 6, 7, 8, and 9 in the amino terminus have been deleted, or
f) a human TNFα monomer or an analogue defined in a or b, wherein is inserted or in-substituted at least one foreign MHC Class II binding amino acid sequence into loop 1 in an intron position, or
g) a human TNFα monomer or an analogue defined in a or b, wherein at least one foreign MHC Class II binding amino acid sequence is introduced as part of an artificial stalk region in the N-terminus of human TNFα, or
h) a human TNFα monomer or an analogue defined in a or b, wherein at least one foreign MHC Class II binding amino acid sequence is introduced so as to stabilize the monomer structure by increasing the hydrophobicity of the trimeric interaction interface, or
i) a human TNFα monomer or an analogue defined in a or b, wherein at least one foreign MHC Class II binding amino acid sequence flanked by glycine residues is inserted or in-substituted in the TNFα amino acid sequence, or
j) a human TNFα monomer or an analogue defined in a or b, wherein at least one foreign MHC Class II binding amino acid sequence is inserted or in-substituted in the D-E loop, or
k) a human TNFα monomer or an analogue defined in a or b, wherein at least one foreign MHC Class II binding amino acid sequence is inserted or in-substituted between two identical subsequences of human TNFα, or
l) a human TNFα monomer or an analogue defined in a or b, wherein at least one salt bridge in human TNFα has been strengthened or substituted with a disulphide bridge, or
m) a human TNFα monomer or an analogue defined in a or b, wherein solubility or stability towards proteolysis is enhanced by introducing mutations that mimic murine TNFα crystalline structure,
wherein potential toxicity is reduced or abolished by introduction of at least one point mutation selected from the group consisting of Y87S, D143N or A145R, the amino acid numbering setting out from the N-terminal valine in human TNFα.
2. The immunogenic analogue according to paragraph 1 wherein the MHC Class II binding amino acid sequence binds a majority of MHC Class II molecules from the animal species from where the multimeric protein has been derived.
3. The immunogenic analogue according to paragraph 2, wherein the at least one MHC Class II binding amino acid sequence is selected from a natural T-cell epitope and an artificial MHC-II binding peptide sequence.
4. The immunogenic analogue according to paragraph 3, wherein the natural T-cell epitope is selected from a Tetanus toxoid epitope such as P2 or P30, a diphtheria toxoid epitope, an influenza virus hemagluttinin epitope, and a P. falciparum CS epitope.
5. An immunogenic analogue according to paragraph 1, wherein the amino acid sequence of the analogue is selected from the group consisting of SEQ ID NO: 12, 13, 14, 16, 17, and 18, and any amino acid sequence that only include conservative amino acid changes thereof.
6. An immunogenic analogue according to paragraph 1, which can be expressed as a soluble protein from bacterial cells.
7. A nucleic acid fragment that encodes an immunogenic analogue according to paragraph 1, or a nucleic acid fragment complementary thereto.
8. The nucleic acid fragment according to paragraph 7 that is a DNA fragment.
9. The nucleic acid fragment according to paragraph 7 which comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences that encode any one of SEQ ID NOs: 12, 13, 14, 16, 17, and 18, or a nucleic acid sequence complementary thereto.
10. A method for down-regulating autologous TNFα in a host animal, the method comprising effecting presentation to the animal's immune system of an immunogenically effective amount of at least one immunogenic analogue according to paragraph 1.
11. The method according to paragraph 10, wherein the autologous host is a mammal, such as a human being.
12. The method according to paragraph 10, wherein presentation is effected by administering the immunogenic analogue according to paragraph 1 to the autologous host, optionally in admixture with an adjuvant.
13. The method according to paragraph 12, wherein the adjuvant is 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 (an ISCOM matrix); a particle; DDA; aluminium adjuvants; DNA adjuvants; Γ-inulin; and an encapsulating adjuvant.
14. The method according to paragraph 10, wherein an immunogenically effective amount of analogue is administered to the animal via a route selected from the parenteral route such as the intradermal, the subdermal, and the intramuscular routes; the peritoneal route; the oral route; the buccal route; the sublinqual route; the epidural route; the spinal route; the anal route; and the intracranial route.
15. The method according to paragraph 14, wherein the effective amount is between 0.5 μg and 2,000 μg.
16. The method according to paragraph 14, which includes at least one administration per year, such as at least 2, at least 3, at least 4, at least 6, and at least 12 administrations per year.
17. The method according to paragraph 10, wherein presentation of the analogue to the immune system is effected by introducing nucleic acid(s) encoding the analogue into the animal's cells and thereby obtaining in vivo expression by the cells of the nucleic acid(s) introduced.
18. The method according to paragraph 17, wherein the nucleic acid(s) introduced is/are selected from 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 such as the adjuvants defined in paragraph 13.
19. The method according to paragraph 17, wherein the nucleic acids are administered intraarterially, intraveneously, or by the routes defined in paragraph 14.
20. The method according to paragraph 17, which includes at least one administration of the nucleic acids per year, such as at least 2, at least 3, at least 4, at least 6, and at least 12 administrations per year.
21. The method according to paragraph 10, wherein presentation to the immune system is effected by administering a non-pathogenic microorganism or virus which is carrying a nucleic acid fragment which encodes and expresses the analogue.
22. The method according to paragraph 21, wherein the virus is a non-virulent pox virus such as a vaccinia virus.
23. The method according to paragraph 22, wherein the microorganism is a bacterium.
24. The method according to paragraph 21, wherein the non-pathogenic microorganism or virus is administered one single time to the animal.
25. A composition for inducing production of antibodies against a multimeric protein, the composition comprising
an immunogenic analogue according to paragraph 1, and
a pharmaceutically and immunologically acceptable carrier and/or vehicle and/or adjuvant.
26. A composition for inducing production of antibodies against a multimeric protein, the composition comprising
a nucleic acid fragment according to paragraph 7, and
a pharmaceutically and immunologically acceptable carrier and/or vehicle and/or adjuvant.
27. The composition according to paragraph 25, wherein the analogue is formulated as defined in paragraph 13.
28. A method for the preparation of the analogue according to paragraph 1, the method comprising culturing a host cell transformed with the nucleic acid fragment according to paragraph 7 under conditions that facilitate expression thereof and subsequently recovering the analogue as a protein expression product from the culture.
29. The method according to paragraph 28, wherein the host cell is a bacterial host cell.
30. The method according to paragraph 29, wherein the analogue is a soluble expression product.
31. The method according to paragraph 28, where the host cell is cultured at a temperature of less than 32° C. during a substantial period at which the expression product is produced by the host cell.
32. The method according to paragraph 31, wherein the temperature is about 25° C.
33. The method according to paragraph 31 wherein the temperature is kept substantially constant during the complete period of culturing of the host cell.
34. The method according to paragraph 32 wherein the temperature is kept substantially constant during the complete period of culturing of the host cell.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2003 00701 | May 2003 | DK | national |
This application is a continuation-in-part of International Patent Application PCT/DK2004/000329 filed May 6, 2004 and published as WO 2004/099244 which published on Nov. 18, 2004 and claimed priority from Danish Patent Application PA 2003 00701 filed May 9, 2003 and from U.S. Patent Application Ser. No. 60/469,491, filed May 9, 2003. Each of the above referenced applications, and each document cited in this text (“application cited documents”) and each document cited or referenced in each of the application cited documents, and any manufacturer's specifications or instructions for any products mentioned in this text and in any document incorporated into this text, are hereby incorporated herein by reference; and, technology in each of the documents incorporated herein by reference can be used in the practice of this invention. It is noted that in this disclosure, terms such as “comprises”, “comprised”, “comprising”, “contains”, “containing” and the like can have the meaning attributed to them in U.S. Patent law; e.g., they can mean “includes”, “included”, “including” and the like. Terms such as “consisting essentially of” and “consists essentially of” have the meaning attributed to them in U.S. Patent law, e.g., they allow for the inclusion of additional ingredients or steps that do not detract from the novel or basic characteristics of the invention, i.e., they exclude additional unrecited ingredients or steps that detract from novel or basic characteristics of the invention, and they exclude ingredients or steps of the prior art, such as documents in the art that are cited herein or are incorporated by reference herein, especially as it is a goal of this document to define embodiments that are patentable, e.g., novel, nonobvious, inventive, over the prior art, e.g., over documents cited herein or incorporated by reference herein. And, the terms “consists of” and “consisting of” have the meaning ascribed to them in U.S. Patent law; namely, that these terms are closed ended.
| Number | Date | Country | |
|---|---|---|---|
| 60469491 | May 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/DK04/00329 | May 2004 | US |
| Child | 11270276 | Nov 2005 | US |
