WO2004072112A2

WO2004072112A2 - A method for formulating mhc-like molecules for long time storage

Info

Publication number: WO2004072112A2
Application number: PCT/DK2004/000094
Authority: WO
Inventors: Søren Buus; Christina Sylvester-Hvid
Original assignee: Københavns Universitet
Priority date: 2003-02-11
Filing date: 2004-02-11
Publication date: 2004-08-26
Also published as: WO2004072112A3

Abstract

The present invention describes how MHC-like molecules can be formulated in an active form and stored under conditions, which preserve their activity for an extended period of time, while readily allowing the end-user to generate any ligand-MHC-complex of their choice. The present inventors have termed the product of this invention: “formulated MHC molecules”. The utility of such formulated MHC molecules is considerable. One has to keep in mind that an entire arm of the immune system, the T cell immune system, is based upon the recognition of ligands selected and presented by MHC molecules. These ligands constitute the targets of the cellular immune system. They include foreign targets such as vaccine and/or immunotherapy candidates, who can be used to direct immunity against infectious agents and cancers, as well as self-targets involved in the development of autoimmune diseases and transplantation reactions. The availability of formulated MHC molecules affords a non-expert end-user with the enabling technology to identify and validate MHC ligands, and subsequently to generate specific ligand-MHC-complexes for the identification, validation, selection, and induction of specific T cells. The present invention therefore facilitates specific analysis and manipulation of the immune system.

Description

A method for formulating MHC-like molecules for long time storage

Field of invention

The present invention describes how MHC-like molecules, including both MHC class I-like and MHC class II-like molecules, can be formulated in an active "ready to go" form and stored under conditions, which preserve their activity for an extended period of time, while readily allowing the end-user to generate any ligand-MHC-complex of their choice.

The present invention teaches how one can formulate and store MHC-like molecules in a folded conformation and retain their activity over an extended period of time. Therefore, it becomes possible to manufacture and store active MHC-like molecules - and to distribute them thereby enabling non-expert laboratories to generate any ligand-MHC-like complex of their choice. It also facilitates the generation of an inventory of active MHC-like molecules encompassing the more than 1500 different (and still growing number) of known human MHC-like molecules allowing the end-user to handle the enormous complexity of the MHC system easily.

The present invention now enables transportation and storage of MHC-like molecules in a way that allows the generation of a commercial kit, which would allow the final ligand- MHC-like complex formation to be done at the end user. Background of the invention

T lymphocytes are essential in the recognition of foreign antigens and subsequent development of specific immune responses against e.g. invading pathogens. A specific molecular interaction between a T-cell receptor (TCR) and its specific ligand determines activation of T lymphocytes. This ligand consists of antigen-derived peptides presented in complex with tissue (MHC) molecules on antigen presenting cells (APC). The complicated cellular machinery needed to generate these important peptide-MHC target structures has made it difficult to address the peptide specificity of the T cell immune system. Among other things, this would require reliable identification (preferably prediction) and validation (preferably biochemical measurement) of peptide binding to MHC molecules, and/or of peptide-MHC binding to T cell receptors. A convenient source of active MHC class I molecules would be a valuable tool in the structural, biochemical and physiological analysis of peptide-MHC generation and recognition. Such molecules will facilitate the mapping of MHC and T cell specificities and are likely to be useful for T cell mediated immune manipulations.

It has proven very difficult indeed to produce active MHC molecules, the word active meaning the ability to efficiently bind appropriate ligands. Natural MHC complexes are by their very nature pre-saturated with high-affinity binding ligands. They are therefore unable to bind offered ligand with any degree of efficiency and they will never give rise to pure homogeneous preparations of ligand-MHC complexes. In fact, due to the essential requirement for ligands during the assembly of MHC molecules, it is difficult to envision an in vivo strategy, which would lead to the production of large amounts of ligand-free, natural MHC molecules. A way to remove high-affinity ligands would be to unfold, i.e. denature the in vivo generated MHC complexes in vitro and isolate the receptor component under denaturing conditions. Although such a strategy would be highly efficient in removing any high-affinity ligand, it does create a seemingly insurmountable problem namely that denatured MHC molecules refold very inefficiently.

The present inventors have recently reported a possible solution to this conundrum by generating large amounts of recombinant MHC class I heavy chain molecules in bacteria, extracting the recombinant MHC class I product into denaturing buffers and isolating the active moieties. To their surprise, the present inventors found that MHC class I heavy chain and light chain (β2m) molecules with pre-formed disulphide bonds could fold with high efficiency and become active [WO 00/15665]. However, this work by Pedersen, L. O. et al., and further described in WO 00/15665 only concerns a method for producing active MHC class I molecules by isolating heavy chains under non reducing conditions from crude bacteria lysate. These molecules can be stored at least for three months in the presence of a strong denaturing agent (e.g. 8 M Urea) - however this work does not refer to any formulation approach for long time storage active MHC molecules in a hydrophilic buffer, under non-denaturing conditions - which impairs the handling process of the molecules prior analysis of the molecules.

The present inventors later identified methods to purify the active subspecies to homogeneity under denaturing conditions [DK PA 2002 00766]. The present inventors also found that the unfolded heavy chain molecules could be stored for three months in denaturing buffers at -20°C, in some cases the storage buffer included 50% glycerol, [WO 00/15665], and still be able to fold and become active upon removal of the denaturant. Upon renaturation, however, the present inventors were only able to keep these heavy chain molecules active for a brief period of time, the half-life of the MHC molecules was only 48 hr at 4°C, [WO 00/15665].

Several methods have been used to generate active antigen binding MHC molecules (e.g. Garboczi et al., and US 6,153,408). However none of the methods describe how to formulate these molecules for long time storage in a hydrophilic buffer under non- denaturing conditions.

WO 02/0726319 describes a method for production of MHC class I molecules conjugated to different tags. This work only concerns production of conjugated molecules for diagnostic purposes not a method for long-time storage.

Yet others have investigated the thermal stability of both empty - and peptide filled MHC class I molecules (Fahnestock et al.). This again is not related to formulation of active MHC class I molecules for long-time storage under non-denaturing conditions. Production methods for increasing antigen-binding capacity of MHC molecules have been published (US 5,364,762), but these methods do not deal with ways of formulating MHC molecules while retaining their peptide binding capacity during storage.

In other words, long-term storage only appeared possible for the denatured form. From the point of view of protein stability, denaturing storage is problematic since denatured proteins are very susceptible to modifications such as those induced by the denaturant (e.g. urea-induced carbamylation), by disulphide shuffling, and/or by proteolytic cleavage.

From a practical point of view, denaturing storage is also a hassle since the high concentrations of denaturant used lead to crystal formation complicating liquid handling. This handling problem is further worsened by the requirement for fairly concentrated MHC heavy chain stock preparation and the resulting low volume aliquots needed for tests. Thus, a storage enigma exists, namely that only denaturing storage conditions are feasible, however, the stability and handling of denatured proteins are not satisfying.

Finally, it should be noted that the refolding protocol is most critical for the activation of the denatured MHC molecules, and therefore is a possible source of experimental error. Thus, there is an unmet need for folded and active MHC molecules that are formulated in a way that allows long-term storage under non-denaturing conditions. This will improve the stability and purity of MHC preparation and it will minimise the technical expertise required of the end-user to use these molecules. The important implication is that it will facilitate the standardisation of strategies and methods to address both ligand-MHC complex formation as well as the corresponding T cell recognition. Detailed description of the invention

The present invention describes how MHC-like molecules, including both MHC class I-like and MHC class II-like molecules, can be formulated in an active form and stored under conditions, which preserve their activity for an extended period of time, while readily allowing the end-user to generate any ligand-MHC-complex of their choice.

In one of its broadest aspect, the present invention relates to a method for formulating a renaturated, active and stable MHC class I-like molecule comprising diluting a denatured heavy chain of said MHC class I-like molecule which is reduced, partially oxidised and/or fully oxidised, into a hydrophilic buffer with an admixture of a co-factor facilitating the formation of the renatured, active and stable MHC class I-like molecule, whereby said MHC class I-like molecule can be long-term stored.

In another broad aspect, the present invention relates to a method for formulating a renaturated, active and stable MHC class I-like molecule comprising diluting a denatured heavy chain of said MHC class I-like molecule which is reduced, partially oxidised and/or fully oxidised, into a hydrophilic buffer with an admixture of a co-factor facilitating the formation of the renaturated, active and stable MHC class I-like molecule, but in absence of high-affinity binding ligand, whereby said MHC class I-like molecule can be long-term stored.

In essence, the inventive concept described herein relates to a method for formulating a renaturated, active and stable empty MHC class I-like molecule comprising diluting a denatured heavy chain of said MHC class I-like molecule, which is reduced, partially oxidised and/or fully oxidised, into a hydrophilic buffer with an admixture of a co-factor facilitating the formation of the renaturated, active and stable MHC class I-like molecule, but in absence of any ligand, whereby said MHC class I-like molecule can be long-term stored.

In the broadest sense, a MHC class I-like molecule is any polypeptide encoded within the MHC gene complex referred to as classical MHC molecules and non classical MHC molecules including the MHC class I-like molecules encoded in the CD1 locus, respectively, and further relates to any recombinant molecule including engineered and truncated molecules, with the proviso that MHC class II molecules are excluded.

The MHC class I complex contains a heavy chain, the α-chain, associated non-covalently with a smaller light chain, the β₂-microglobulin (β₂m) and a peptide placed in a peptide binding groove made up by the heavy chain.

In the present application, one aspect relates to MHC class I-like molecules, however it should be clear to a person skilled in the art that any of these experimental conditions and data are similar for CD1, since the structure and assembly of a CD1 presenting complex is analogously to MHC class I, especially the dependency of β₂m and ligand. Thus, the subsequent paragraphs describes experimental conditions for MHC class I.

In the broadest sense, co-factor relates to any factor capable of facilitating the formation of an active and stable MHC class I-like molecule, such as chaperones or any other compound capable of said formation. It is understood that an active MHC class I-like molecule is any MHC class I-like molecule, which will fold into an MHC class I-like complex in the presence of the relevant co-factor in an entirely ligand-dependent manner, and thus a stable MHC class I-like molecule is a molecule which retains these characteristics as they emerge.

In a preferred embodiment of the method of the present invention, the co-factor is selected form the group consisting of chaperones, Adaptor Protein 1 (API), AP2, AP3, AP4, Tap, tapasin, calreticulin, calnexin, β₂m and Erp57. It should be understood that the co- factor relates to both natural molecules and recombinant molecules including engineered and truncated molecules.

A co-factor interacts with the heavy chain of the MHC class I-like molecule with a certain affinity. The K_D (equilibrium dissociation constant) for e.g. β₂m to MHC class I heavy chain is only « 1 nM. Therefore, in a presently preferred embodiment of the present invention the concentration of the co-factor is at least 1 nM, such as but not limited to at least 10 nM, at least 100 nM, at least 1 μM, at least 5 μM, at least 10 μM, at least 20 μM, at least 25 μM, at least 50 μM, at least 100 μM, at least 200 μM, or at least 300 μM, but at the most. 7.500 mM co-factor.

In a special embodiment the said hydrophilic buffer, further comprises low-affinity binding ligands.

It should be noted that the present invention preferably relates to hydrophilic buffers without contaminating peptides, however, this does not exclude the addition of low affinity or non-binding protein to the hydrophilic buffer so that simply adding peptides, polypeptides or proteins such as, but not limited, to bovine serum albumin, skimmed milk protein or foetal calf serum to the buffer. Such simple addition of low affinity or non- binding compounds are still within the scope of the present invention.

The term "low-affinity" binding ligand relates to any ligand binding to an empty MHC-like molecule with a K_D greater than 500 nM. In contrast, the term "high- affinity" binding ligands relates to any ligand binding to the empty MHC-like molecule" with a K_D less than 500 nM.

The term "admixture" relates to the presence of any co-factor in the hydrophilic buffer. It is optional, whether this c co-factor is present in the hydrophilic buffer when the MHC class I-like heavy chains is diluted into the buffer and/or added subsequently.

In the present context, the term "active" relates to MHC class I-like molecules, which will refold into a ligand-MHC class I-like complex in the presence of relevant co-factor in a ligand dependent manner. Thus, such active MCH class I-like molecules are, contrary to a natural produced MHC complexes presaturated with peptide, all capable of binding a ligand.

The concentration of active MHC class I-like molecules can be measured by e.g. a quantitative ELISA method [DK PA 2002 00768]. In brief, a specific amount of MHC class I-like molecules is diluted into a buffer containing a high-affinity binding ligand. The reagents are incubates at 18°C until steady state and the exact amount of generated ligand-MHC class I-like complexes is measured by the quantitative ELISA method. OD values obtained by the colourmetric reaction are converted into nM ligand-MHC class I-like complex by including a standard curve. The exact amount of MHC class I-like complexes is determined by using non-linear regression. The output from the non-linear regression Bmax is an approximation of the concentration of active MHC class I-like molecules. Thus, to describe their stability at least 2 measurements of the concentration of MHC class I-like complexes over a time interval are required.

The term "stable" relates to MHC-like molecules, which retain their activity for at least 1 week after their generation, at least 2 weeks after their generation, at least 3 weeks after their generation, at least 1 month after their generation, at least 12 months after their generation, at least 2 years after their generation, at least 5 years after their generation, at least 10 years after their generation, such as 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or 15 years, but at the most 20 years after their generation. The term "stability" can further be defined as the ability to resist change e.g. determinably by measuring the concentration of active MHC-like molecules before and after storage, and be expressed as the concentration of active molecules after storage in percentage of the concentration of active molecules before storage. A person skilled in the art would thus recognise the term "stable", as being activity of the MHC-like molecules, which has an activity close to the starting point of said activity, such as at least 100% retained of the starting point activity, at least 99% retained of the starting point activity, at least 98% retained of the starting point activity, at least 97% retained of the starting point activity, at least 97% retained of the starting point activity, at least 96% retained of the starting point activity, at least 95% retained of the starting point activity, at least 90% retained of the starting point activity , at least 85 % retained of the starting point activity, at least 80% retained of the starting point activity, at least 75% retained of the starting point activity, at least 60% retained of the starting point activity, at least 55 % retained of the starting point activity, at least 50% retained of the starting point activity, at least 45% retained of the starting point activity or still has at least 40% retained of the starting point activity.

By "starting point" is meant the time that the molecules are formulated.

The term "long-term storage" relates to storage of MHC-like molecules for periods extending at least 1 week after their generation, at least 2 weeks after their generation, at least 3 weeks after their generation, at least 1 month after their generation, at least 2 months after their generation, at least 12 months after their generation, at least 2 years after their generation, at least 10 years after their generation, such as at least 2 weeks, at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, at least 15 years, but at the most or 20 years after their generation.

The term "empty" MHC-like molecule relates to a MHC-like molecule, which is essentially devoid of ligand, but which in presence of a appropriate ligand will generate a ligand-MHC- like complex. By the term "essentially devoid" is meant that at least 50% of the MHC-like molecules are devoid of peptides.

The term "ligand" refers to any compound, which will bind to the MHC-like molecule and generate a ligand-MHC-like complex. Such ligands could be high-affinity binding peptides and/or high-affinity binding glycolipids able to bind to the ligand-binding domain of the MHC-like molecule.

In the present context, the term "renaturated" protein relates to a previously unfolded (i.e. denatured) protein, which has been subjected to a refolding treatment and thereby regained some, or all, of the native structure and/or function.

In the present context, the term "renaturated empty" MHC class I-like molecule relates to any MHC class I-like heavy chain in complex with a co-factor in a hydrophilic solution in absence of a high-affinity binding ligand, which will de novo fold into a ligand-MHC class I- like complex in the presence of said high-affinity binding ligand, soluble in said hydrophilic solution.

A simple test to detect if a renaturated MHC class I-like molecule has been generated is by use of an ELISA technique such as [DK PA 2002 00768].

In the present context, the term "denatured MHC-like molecule" relates to any unfolded MHC-like polypeptide, in which the molecular structure of the MHC-like molecule has been modified by a denaturing agent such as any chaeotrophic ligand like urea, or guanidine hydrochloride, heat, acid, alkali or ultraviolet radiation.

"Reduced MHC-like heavy chain" should be understood as any MHC-like polypeptide chain devoid of disulphide bridges which due to the molecular structure of the MHC-like heavy chain has been modified by a reducing agent such as 2-mercaptoethanol (β- Mercaptoethanol), Dithiothreitol (DTT), Tri-(2-carboxyethyl)phosphine reductant (TCEP), reductase enzyme or any other reducing agent.

"Partially oxidised MHC-like heavy chain" should be understood as any MHC-like polypeptide chain containing at least one-disulphide bridge. As understood by a person skilled in the art a disulphide bridge is a formation of a covalent disulphide bond between two cystein residues, which can contribute to the stability of protein tertiary structure. The "S-S" bond covalently cross-links two regions of the structure that may be distant in sequence, but nearby in the folded state. Further, a "fully oxidised MHC-like heavy chain" contains at least two disulphide bridges. Furthermore, a "fully oxidised MHC class II-like heavy chain" is a molecule where the maximum number of possible disulfide bonds have been formed.

By way of example, MHC class I heavy chains contains at least four cysteines, therefore a MHC class I-like heavy chain can generate at least ten different isomers, consisting of partially oxidised heavy chains or totally reduced or oxidised heavy chains. It is expected that only one of these preoxidised isomers can renaturate into a ligand-MHC class I-like complex in an entirely ligand co-factor dependant manner. This particular isomer contains at least one correct disulphide bridge. Due to limitations in purification steps of preoxidised heavy chains of the present state of the art, some MHC class I-like heavy chains preparations contain less than 100% active isomers which will bind a ligand in the presence of a co-factor. It has previously been shown that preoxidised MHC class I-like heavy chain isomers can be stored for more than 3 months in a denaturing agent such as, 8 M urea at -20 °C, and retain equal activity [WO 00/15665]

In another presently preferred embodiment, the denatured MHC class I-like heavy chains are incubated in the hydrophilic buffer according to the present invention at least 0 sec at 0°C and is substantially active and stable. The amount of generated empty MHC class I- like molecules depends on the affinity, K_D, of co-factor to the heavy chain of the MHC class I-like molecule, the temperature and the incubation time. Thus, it is evident for a skilled person that there is an expected proportionality between time and temperature. The present invention thus relates to even the absolute minimum of required incubation time for generating an MHC class I molecule, such as but not limited to 1/100.000 sec, such as 1/10.000 sec, such as 1/1000 sec. and such as 1/100 sec. Further, it is an object of preferred embodiments of the present invention to allow sufficient time for a generation of at least one empty MHC class I-like molecule at any temperature selected, such as but not limited to an formulation time at 0°C of 0 sec, 60 sec, 5 min, 10 min, 60 min, 2 hours and 5 hours or any time limit above.

In another embodiment of the present invention, the storage further increases the amount of active MHC-like heavy chain isomers as measured by the quantitative ELISA method described above. In yet another preferred embodiment, the amount of active isomers is increased at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and at least 100% by long term storage.

A further embodiment of the present invention relates to a hydrophilic buffer further comprising any cryoprotectant suitable for storage of protein. Said cryoprotectant stabilises and protects the protein activity at 0°C to -150°C, which is the normal range of protein storage. A person skilled in the art would easily recognise examples of such cryoprotectants such as, but limited to, glycerol, polyethylene-glycol, lactose, sucrose and polyvinyl-pyrrolidone or a solution containing a mixture of two or more of the above.

In a presently preferred embodiment of the invention, the amount of cryoprotectant in the hydrophilic buffer is 50% vol./vol. However, any amount of cryoprotectant suitable for stabilising and protecting the MHC-like molecules could be applied, such as but not limited to 99% vol. /vol., 90% vol./vol., 80% vol./vol., 70% vol./vol., 65% vol. /vol., 60% vol./vol., 59% vol./vol., 58% vol./vol., 57% vol./vol., 56% vol./vol., 55% volJvol., 54% vol./vol., 53% vol./vol., 52% vol./vol., 51% volJvol. or 49% vol./vol., 48% vol./vol., 47% vol./vol., 46% vol./vol., 45% vol./vol., 44% vol./vol., 43% vol./vol., 42% vol./vol., 41% vol./vol., 40% vol./vol., 35% vol./vol., 30% vol./vol., 25% vol./vol., 20% vol./vol., 15% vol./vol., 14% vol./vol., 13% vol./vol., 12% vol./vol., 11% vol./vol., 10% vol./voL, 9% vol./vol., 8% vol./vol., 7% vol./vol., 6% vol./vol., 5% vol./vol., 4% vol./vol., 3% vol./vol., 2% vol./vol.. 1% vol./vol., or 0.5% volJvol.

In a presently most preferred embodiment of the present invention, the cryoprotectant is glycerol.

It should be noted that the amount of cryoprotectant present in the hydrophilic buffer, especially in the final solution at the end user, should not exceed an amount wherein the empty MHC-like molecule is unable to renaturate into a MHC-like complex in the presence of a high-affinity binding ligand, thus the present invention relates to a concentration of cryoprotectant wherein said empty MHC-like molecule, when suitably diluted, is capable of renaturating into a MHC-like complex in the presence of a high-affinity binding ligand.

It is an object of preferred embodiments of the present invention to obtain a final cryoprotectant concentration prior to de novo folding of MHC-like complexes, which is below 50% vol./vol., below 40% vol./vol., below 30% vol./vol., below 20% vol./vol., below 15% vol./vol., below 14% vol./vol., below 13% vol./vol., below 12% vol./vol., below 11% vol./vol., below 10% vol./vol., below 9% vol./vol., below 8% vol./vol., below 7% vol./vol., below 6% vol./vol., below 5% vol./vol., below 4% vol./vol., below 3% vol./vol., below 2% vol./vol., below 1% vol./vol., below 0.5% vol./vol., below 0.1% vol./vol., or below 0.01% vol./vol..

MHC class I

As described above, the examples presented in the present application relate to studies based on MHC class I molecules and thus a presently preferred embodiment of the invention relates to a method for the generation of a renaturated, active and stable MHC class I molecule comprising diluting a denatured MHC class I heavy chain which is reduced, partially oxidised and/or fully oxidised into a hydrophilic buffer with an admixture of a co- factor facilitating the formation of the renaturated, active and stable MHC class I molecule, but in absence of high-affinity binding ligand, whereby said MHC class I molecule can be long-term stored.

In a presently more preferred embodiment of the present invention, the MHC class I-like molecule is a MHC class I molecule and the MHC class I ligand is a peptide.

In a presently preferred context, the wording co-factor relates to any factor capable of facilitating the formation of an active and stable MHC class I molecule. It is understood that an active MHC class I molecule is any MHC class I molecule which will fold into a peptide-MHC class I complex in the presence of co-factor in an entirely peptide-dependent manner, and thus a stable MHC class I molecule is a molecule which retains these characteristics as they emerge.

In a presently preferred embodiment of the present invention relating to MHC class I molecules, the co-factor is selected form the group consisting of chaperones, tapasin, calreticulin, calnexin, β₂m and Erp57. In a most preferred embodiment, the co-factor is β₂m. It should be understood that the co-factor relates to both natural molecules and/or recombinant molecules including engineered and truncated molecules.

As described above, the concentration of β₂m should be at least 1 nM, such as but not limited to at least at least 10 nM, at least 100 nM, at least 1 μM, at least 5 μM, at least 10 μM, at least 20 μM, at least 25 μM, at least 50 μM, at least 100 μM, at least 200 μM, or at least 300 μM.

In the present context β₂m (beta-2 microglobulin) or light chain relates to any polypeptide chain encoded within the β₂m locus containing one immunoglobulin-like domain which is able to interact with a MHC class I molecule. As described above, such β₂m obviously relates to both natural β₂m molecules and recombinant β₂m molecules including engineered truncated molecules and/or mutated β₂m molecules.

In a presently preferred context, an "MHC class I heavy chain" relates to a polypeptide chain encoded by any allele located in locus Mhc-I, or equivalent polypeptide chains encoded recombinantly, or fragments thereof. Further, since any Vertebrae studied to date possess the major histocompatibility gene complex, the invention relates to any MHC heavy chain MHC encoded by any Vertebrae. For purposes of the present invention, Vertebrae include but are not limited to the order Rodentia, such as mice; order Logomorpha, such as rabbits; more particularly the order Carnivora, including Felines (cats) and Canines (dogs); even more particularly the order Artiodactyla, Bovines (cows) and Suines (pigs); and the order Perissodactyla, including Equines (horses); and most particularly the order Primates, Ceboids and Simoids (monkeys) and Anthropoids (humans and apes). In the most preferred embodiment the Vertebraeare humans.

In a preferred embodiment of the present invention, the long-term storage further increases the amount of active MHC class I-like heavy chain isomers.

In a presently preferred context, the term "MHC class I molecule" relates to any MHC class I molecule in complex with β₂m in absence of a high-affinity binding peptide in a hydrophilic buffer, which will de novo fold into a peptide-MHC class I complex in the presence of a peptide.

The term "empty" MHC class I molecule relates to an MHC class I molecule, which is essentially devoid of peptide, but which in presence of a appropriate ligand will generate a peptide-MHC class I complex.

In another presently preferred aspect the present invention relates to a renaturated MHC class I-like molecule comprising denatured MHC class I-like heavy chain, which is reduced, partially oxidised and/or fully oxidised, diluted into a hydrophilic buffer with an admixture of a co-factor, but in absence of high affinity binding ligand. It is possible to obtain at least 90% saturation of MHC class I like molecules during storage, such as at least 91% saturation, such as at least 92% saturation, such as at least 93% saturation, such as at least 94% saturation, such as at least 95% saturation, such as at least 96% saturation, such as at least 97% saturation, such as at least 98% saturation, 5 such as at least 99% saturation, or such as at least 100% saturation. By subsequently dilution the receptor and ligand one 100 fold reduces the saturation is reduced to about 10% leaving approximately 90% of the molecules available for ligand binding.

As described above the activity of denatured MHC class I-like heavy chain crucial relies on 10 the presence of a co-factor during storage in a hydrophilic buffer. Prefolded MHC class I- like heavy chains might further increase the stability during storage with the admixture of a low affinity binding ligand in a high concentration. The ligand would interact with the ligand-binding site and facilitate the formation of an active conformation of the molecule during storage. 15

Formulating MHC class I heavy chains with a low-affinity binding ligand leading at least 90% saturation of the heavy chains would stabilise the ligand binding site. Upon usage End-user can dilute the formulated molecules e.g. a 100 fold and add any ligand of interest. Lowering the concentration of low-affinity binding ligand by e.g. a 100 fold will 20 decrease the saturation of the receptor to about 10%. A least 90% of the heavy chains will be available for ligand binding (Buus, S., et al. 1995).

In another presently preferred aspect of the present invention relates to a renaturated MHC class I-like molecule comprising denatured MHC class I-like heavy chain, which is 25 reduced, partially oxidised and/or fully oxidised, diluted into a hydrophilic buffer with an admixture of a co-factor and a low-affinity binding ligand.

In a preferred embodiment the low affinity binding ligand will occupy at least 100%, at least 99%, at least 98%, at least 97 %, at least 96%, at least 95%, at least 99%, at least

30 98%, at least 97 %, at least 96%, at least 95%, at least 94%, at least 93%, at least 92 %, at least 91%, at least 90%, at least 89%, at least 88%, at least 87 %, at least 86%, at least 85%, at least 84%, at least 83%, at least 82 %, at least 81%, at least 80%, at least 79%, at least 78%, at least 77 %, at least 76%, at least 75%, at least 74%, at least 73%, at least 72 %, at least 71%, at least 70%, at least 69%, at least 68%, at least 67 %, at

35 least 66%, at least 65%, at least 64%, at least 63%, at least 62 %, at least 61%, at least 60%, at least 59%, at least 58%, at least 57 %, at least 56%, at least 55%, at least 54%, at least 53%, at least 52%, at least 51%, at least 50% during storage of formulated MHC class I like heavy chains.

40 Yet another presently preferred aspect of the present invention relates to a renaturated empty MHC class I-like molecule comprising denatured MHC class I-like heavy chain, which is reduced, partially oxidised and/or fully oxidised, diluted into a hydrophilic buffer with an admixture of a co-factor, but in absence of any binding ligand. In a presently special preferred embodiment, said renaturated MHC class I-like molecule is a MHC class I molecule.

In addition to the storage and transportation of the MHC class I-like molecules described in the present invention, the molecules can be easily modified to generate empty oligomers (dimers, tetramers) of MHC class I-like conjugates to stain specific T cells. Oligomer forms of recombinant MHC class I complexes are e.g. known to activate cytotoxic T cells and represent a new golden standard in analysis of complex T cell populations. The efficient generation of well-defined MHC class I-like complexes may generate new approaches for manipulation of immune responses.

Exemplified, enumeration of specific T cells for quantitative and qualitative characterisation of T cell populations, by Mark Davis and co-workers in 1996, demonstrated that recombinant, pure peptide-MHC class I complexes could be generated with an added biotinylation signal attached to the C-terminal of the heavy chain. After an enzymatic biotinylation process these complexes could be tetramerized with Phycoerythin labelled Streptavidin. These multimerized, labelled peptide-MHC class I complexes could subsequently be used to label T cells in a peptide-specific, MHC class I-restricted manner. The stability of the T cell receptor for peptide-MHC class I complexes is generally thought to be too low to effect a stable biochemical binding. However, after tetramerization, the avidity of the multimerized complexes is sufficient to effect biochemical binding. Thus, such tetramers can be used to count by fluorescence activated cell sorter (FACS) analysis of the number of T cells in any given cell suspension. It has subsequently been shown that older methods for counting specific T cells, limiting dilution analysis, is grossly incorrect ^" and underestimate the number of specific T cells. Thus, it has become imperative both from a scientific point of view, but also from a publication point of view, to do "tetramer" analysis. The "Davis method" has two major problems: it is difficult to produce large amounts of pure MHC class I molecules, and the biotinylation process is expensive, cumbersome, and in particular, it requires extended incubation at 37°C. Many peptides do not remain associated to the MHC class I for the time of this latter incubation, and this may explain the variable result even within the same laboratory. The present patent application discloses a method whereby MHC class I-like complexes can be generated in a fast and efficient process leading to a minimum post-complex formation clean up. It is envisioned that the MHC class I-like molecules produced according to the present method will be amenable to transport and storage in a way that allows the generation of a commercial kit, which would further allow the final ligand-MHC class I-like complex production to be done in the any non-expert laboratory using any relevant peptide of the end-users choice. Finally, many better ways of tagging the MHC class I-like than the enzymatic biotinylation process can be envisioned.

Thus, it is an object of preferred embodiments of the present invention to provide a MHC class I-like molecule comprising a biotinylation site. It is obvious to a skilled person that any biotinylation site within said MHC class I-like molecule could be biotinylated. One specially preferred embodiment of the present invention relates to any MHC class I- like molecule according to the present invention which is biotinylated. Further, another specially preferred embodiment of the present invention relates to a MHC-tetramer comprising said biotinylated MHC class I-like molecules.

Additionally, it is envisaged that it may be possible in a similar manner to generate MHC class I-like molecule wherein a further reagent is added as a marker making the kit suitable for diagnostic purposes. In a more specially preferred embodiment of the invention such marker could be selected from the group consisting of flourochromes, enzymes, chemiluminescense, and radioactive markers.

CDl like molecules

Several other MHC class I-like genes have been discovered that reside on chromosomes other than those carrying the MHC. One example is CDl. Humans possess five CDl genes (CDla, CDlb, CDlc, CDld and CDle), while mice possess two, termed CDldl and CDld2. CDl molecules have a similar structure to MHC class I molecules and are dependent on non-covalent interaction with a cofactor in order to refold similar to MHC.

As described above, the resemblance between MHC class I and CDl related molecules is striking. Although the present invention is exemplified with reference to MHC class I proteins, it is possible in a similar manner to generate all proteins consisting of at least one heavy chain containing at least one disulphide bond in its native state such as but not limited to loci, HLA-E, F, G in human and H2-Q, H2-T and H2-M in mice.

In the present context, a CDl heavy chain relates to a polypeptide chain encoding non- classical MHC-like heavy chain encoded within the CD-I gene locus or equivalent polypeptide chains encoded recombinantly, or fragments thereof. Further, since not only human but also mouse and guinea-pig possess the CDl genes the invention relates to any CDl protein encoded in any of human, guinea-pig or mouse CDl locus or any other Vertebrae possessing the CDl genes.

Obviously, it is clear for a person skilled within the art that the present invention is easily adapted to comprise any protein within the CDl family. Thus a further embodiment of the present invention relates to a method for the generation of a renaturated CDl molecule comprising diluting a denatured heavy chain of said CDl molecule which is reduced, partially oxidised and/or fully oxidised into a hydrophilic buffer with an admixture of a co- factor facilitating the formation of an active and stable CDl molecule, but in absence of high-affinity binding glycolipids, whereby said CDl molecule can be long-term stored.

Like for MHC class I heavy chains the CDl heavy chains are translated into the endoplasmatic recticulum and rapidly thereafter associate with chaperones like calnexin and calreticulin, which facilitates the refolding and generation of correct disulphide bridges prior to ligand binding in the CDl binding groove. The second event of the CDl assembly in the endoplasmatic recticulum involves the non- covalent association of CDl heavy chains with β₂m. All five of the human CDl isoforms associates with β₂m, but they do so with varying affinity e.g. CDlb- β₂m interactions are particularly strong.

In relation to the aspect of the invention concerning CDl molecules, the co-factor is selected form the group consisting of Tap, tapasin, calreticulin, calnexin, β₂m, and Erp57. However, new studies show that CDl proteins interact with protein complexes at the cytoplasmatic face of the plasma membrane, allowing the various CDl isoforms to be sorted from one another and delivered to sub-compartments of the endosomal network. Thus, API, AP2, AP3 and AP4 or any other adaptor-protein-related factor known to interact with CDl is also included in the present invention.

One presently particularly preferred embodiment of the present invention relates to a method for the generation of a renaturated CDlb molecule comprising diluting a denatured heavy chain of said CDlb molecule which is reduced, partially oxidised and/or fully oxidised into a hydrophilic buffer with an admixture of a β₂m facilitating the formation of an active and stable CDlb molecule, but in absence of high-affinity binding glycolipid, whereby said CDlb molecule can be long-term stored.

MHC class II

The present invention is exemplified with reference to MHC class I proteins, but it is possible in a similar manner to generate MHC class II molecules.

The detailed structure of MHC class II molecules has been solved at the X-ray crystallography level. MHC class II has an overall structure similar to MHC class I although the domain distribution is slightly different. The class II molecule contains an α-chain non- covalently associated with a β-chain and a peptide bound in a peptide-binding groove made up by the two heavy chains.

In the broadest sense a MHC class Il-like molecule is any polypeptide encoded within the Mhc-II gene complex referred to as classical MHC class II molecules and non classical MHC class II molecules respectively, and further relates to any recombinant molecule including engineered and truncated molecules.

Another broad embodiment of the present invention relates to a method for the generation of a renaturated active and stable MHC class Il-like molecule comprising diluting denatured heavy chains of said MHC class Il-like molecule which are reduced, partially oxidised and/or fully oxidised, into a hydrophilic buffer, whereby said active and stable MHC class Il-like molecule can be long-term stored.

A further broad embodiment of the present invention relates to a method for the generation of a renaturated active and stable MHC class Il-like molecule comprising diluting denatured heavy chains of said MHC class Il-like molecule which are reduced, partially oxidised and/or fully oxidised, into a hydrophilic buffer in absence of high-affinity binding peptides, whereby said active and stable MHC class Il-like molecule can be long- term stored.

One particularly preferred embodiment of the invention thus relates to a method for the generation of a renaturated empty MHC class Il-like molecule comprising diluting denatured heavy chains of said MHC class Il-like molecule which are reduced, partially oxidised and/or fully oxidised into a hydrophilic buffer in absence of any peptides, whereby said active and stable MHC class Il-like molecule can be long-term stored.

By "a MHC class Il-like molecule" is meant a protein, which comprises a complex of at least one MHC class II α-chain and/ or at least one MHC class II β-chain. The MHC class II chains may be natural, recombinant, and additionally truncated in order to make the complex soluble in aqueous solution. In a preferred embodiment, the MHC class II molecule comprises an /β dimer.

By "an empty" MHC class Il-like molecule" is meant a class II like molecule which comprises an α/β dimer but which is essentially devoid of ligand. By the term "essentially devoid" is meant that at least 50% of the MHC class Il-like molecules are devoid of peptides.

In a preferred embodiment of the present invention, the denatured active and stable MHC class Il-like heavy chains is formulated in the hydrophilic buffer at least 0 sec. at 0°C and retains equal activity after at least 2 weeks as described above. The interpretation of the time and temperature is described above for MHC class I but also relates to MHC class II.

The concentration of active MHC class Il-like molecules can be measured by e.g. a quantitative ELISA method [DK PA 2002 00768].

In the present context, the term empty MHC class Il-like molecule relates to any empty MHC class Il-like heavy chain in complex in absence of a high-affinity binding ligand, such as but not limited to peptides, in a hydrophilic buffer, which will de novo fold into an MHC class Il-like complex in the presence of such high-affinity binding ligands.

In the present context, the term "renaturated MHC class Il-like molecule " relates to any MHC class Il-like heavy chain in a hydrophilic solution in absence of said high-affinity ligand, which will generate a MHC class Il-like complex in the presence of a high-affinity binding ligand soluble in said hydrophilic solution.

A simple test to detect if a renaturated MHC class Il-like molecule has been generated is by use of an ELISA technique, as described above.

In another presently preferred embodiment, the denatured MHC class Il-like heavy chains is formulated in the hydrophilic buffer according to the present invention at least 0 sec at 0°C and is substantially active and stable. The amount of generated empty MHC class Il- like molecules depends on the affinity, K_D, of the α and β chain of the MHC class Il-like molecule, the temperature and the incubation time. Thus, it is clear for a skilled person that there is an expected proportionality between time and temperature. Thus it is an object of preferred embodiments of the present invention to allow at least sufficient time for a generation of at least one empty MHC class Il-like molecule at any temperature selected, such as but not limited to an formulation at 0°C of 0 sec, 60 sec, 5 min, 10 min, 60 min, 2 hours and 5 hours or any time limit above.

In a preferred embodiment of the present invention, the long-term storage further increases the amount of active heavy chain isomers of the MHC class II.

MHC class Il-like β chains contains at least 2 cysteines, and the MHC class Il-like α chain contains at least 4 cysteines, therefore a MHC class Il-like α heavy chain can generate at least ten different isomers, consisting of partially oxidised heavy chains or totally reduced or oxidised heavy chain. It is expected that only one of these preoxidised isomers can renaturate into a ligand-MHC class Il-like complex in an entirely ligand dependant manner. This particular isomer contains at least one correct disulphide bridge. Due to limitations in purification steps of preoxidised heavy chains some MHC class Il-like heavy chains preparations contains less than 100% active isomers which will bind a ligand.

A further embodiment of the present invention relates to a hydrophilic buffer for the purpose of generating empty MHC class Il-like molecules further comprising any cryoprotectant suitable for storage of protein. Said cryoprotectant stabilises and protects the protein activity at (0 °C to -150 °C), as described above.

Another presently preferred aspect of the present invention relates to a renaturated MHC class Il-like molecule comprising denatured MHC class Il-like heavy chain, which is reduced, partially oxidised and/or fully oxidised, diluted into a hydrophilic buffer with an admixture of a co-factor, but in absence of high affinity binding ligand, thus generating an active and stable MHC class Il-like molecule.

Yet another presently preferred aspect of the present invention relates to a renaturated empty MHC class Il-like molecule comprising denatured MHC class Il-like heavy chain, which is reduced, partially oxidised and/or fully oxidised, diluted into a hydrophilic buffer with an admixture of a co-factor, but in absence of any binding ligand.

In a presently special preferred embodiment said renaturated MHC class Il-like molecule is a MHC class II molecule.

In addition to the storage and transportation of the active and stable MHC class Il-like molecules described in the present invention, the MHC class II molecules can be easily modified to generate empty oligomers (dimers, tetramers) MHC class Il-like conjugates to stain specific T cells. Oligomer forms of recombinant MHC class II complexes are e.g. known to activate T helper cells and could represent a new standard in analysis of T cell populations. The efficient generation of well-defined MHC class Il-like complexes may generate new approaches for manipulation of immune responses.

Also, as described for MHC class I, biotinylation is possible for any MHC class Il-like molecule, thus it is an object of preferred embodiments of the present invention to provide a MHC class Il-like molecule comprising a biotinylation site. It is obvious to a skilled person that any biotinylation site within said MHC class Il-like molecule could be biotinylated.

One preferred embodiment of the present invention relates to any active and stable MHC class Il-like molecule according to the present invention, which is biotinylated. Further, another preferred embodiment of the present invention relates to an MHC class Il-like tetramer comprising said biotinylated MHC class Il-like molecules.

Kits of the present invention

One way to obtain specific knowledge of the structure and function of ligand-MHC complexes, including MHC class I, MHC class II and CDl complexes, is e.g. generation of MHC class I-like molecules and/ or of MHC class Il-like molecules, where one only has to add relevant ligand in order to generate complexes for analysis. This enables large-scale production of pure ligand-MHC complexes for crystal analysis or for the determination of MHC binding specificity.

Another important aspect of the invention is a kit comprising a MHC molecule allowing the recipient to produce any ligand-MHC-like complex simply by adding a peptide of interest. The MHC molecules will preferably be produced by the method according to the invention.

Multimeric MHC/peptide complexes have a high avidity for specific T cell receptors (TCRs), and their use as detection reagents has revolutionised our ability to understand T cell responses to foreign, allo- and auto-antigens. The involvement of the MHC/ T cell pathway in immune responses to a wide range of diseases demonstrates the broad applicability of recombinant MHC-peptide complexes in many different fields of immunological research today.

In one embodiment, the kit comprises an oligomer of empty MHC-like molecules, such as two, three, four or more.

Cancer research, viral immunity, transplantation immunology, autoimmunity and vaccine development are just some of the areas in which MHC-peptide complexes have begun to impact our understanding of T cell responses. E.g. peptide-MHC complexes can also be used in staining tissue sections, which can render specific understanding of the spatial and temporal relationships of T cell immune responses, e.g. the homing of T cells to infected tissue region, furthermore, MHC tetramer allows simple and rapid determination of the frequency of alloreactive T cells following transplantation. In a specific embodiment, the kit comprises a further reagent added as a marker making the kit suitable for diagnostic purposes. The marker is preferably selected from the group consisting of flourochromes, enzymes, chemiluminescense, and radioactive markers.

In a preferred embodiment of the present invention, the kit contains a molecule for quantitative analysis of ligand binding.

A presently preferred embodiment of the invention relates to a kit comprising a molecule for analysing ligand binding affinities by use of a quantitative method and optionally a radioactive biochemical binding assay, wherein the quantitative method is selected from the group consisting of ELISA, RIA, fluorescense correlation spectroscopy, fluorescence polarization, and flourometry.

It is further an object of preferred embodiments of the present invention to provide a kit comprising a molecule according to any of the preceding claims that can be long-term stored for use in a structure and functional analysis of said molecules.

In one embodiment, the kit comprises reagents that will allow the end-user to determine the binding of any peptide of his/her choice using detection systems such as enzyme linked immuno sorbent assay (ELISA), radio immuno assay (RIA), or others known to the person skilled in the art.

The present inventors have termed the any of the products obtainable by the present invention for commercial purposes: "ready-to-go" MHC-like molecules. The utility of such "ready-to-go" MHC-like molecules is considerable.

One such utility relates to a renaturated, active, stable and optionally empty MHC-like molecule obtainable by the method presented in the present application, further comprising a ligand for diagnostic purposes and use of such a renaturated, active, stable and optionally empty MHC-like molecule obtainable by the method according of the present invention for diagnostic purposes.

Legends to figures

Figure 1

The upper frames (1A & B) illustrate how ligand-MHC-like complexes are generated using prior art of exploiting preoxidised denatured MHC heavy chains; 1A: these steps are performed by the manufacturer, IB: these steps are performed by the End-user. The lower frames (2A & B) illustrate how preoxidised denatured MHC heavy chains are used to generate ligand-MHC-like complexes according to the present invention. 2A: Formulation of MHC molecules by the manufacturer, 2B: this step is performed by the End-user. Figure 2

Formulation of Ready to Go MHC class I molecule with graded concentrations of β₂m: Denatured MHC class I heavy chains were diluted into a buffer containing graded concentrations of β₂m. Subsequent to formulation, the molecules were stored at -20 °C in 50% vol/voi glycerol. The activity, measured by the quantitative ELISA, was increasing with increasing concentrations of β₂m present during storage. (•) : A*1101, (■): A*0301 in urea no β₂m present. Figure 3

Preoxidised denatured MHC class I heavy chains stored in less than 8 M urea showed impaired binding activity. Preoxidied denatured A*1101 and A0301 were diluted into a buffer containing decreasing concentrations of Urea. The activity, measured by the quantitative ELISA, was ineffective compared to formulated class I molecules. (•): A*1101 in urea no β₂m present, (■): A*0301 in urea no β₂m present, (O): A*1101 stored with 5 μM β₂m present, (D): A*0301 stored with 5 μM β₂m present.

Figure 4a

A*0301 molecules retain peptide binding activity for more than 2 months. Four peptides were offered to A*0301 molecules. The binding activity of the molecule remained intact for an extended period of more than 2 months. . Peptides:

(0): ALNFPGSQK; (■): ATIGTAMYK; (Δ) : IVTDFSVK; (X): KTGGPIYKR.

Figure 4b

A*1101 molecules retains peptide binding activity for more than 2 months. Four peptides were offered to A*1101 molecules. The binding activity of the molecule remained intact for an extended period of more than 2 months. Peptides: (0): ALNFPGSQK; (■): ATIGTAMYK; (Δ): IVTDFSVK; (X): KTGGPIYKR.

Figure 5

5 A A*0301 molecules retains peptide binding activity formulated with (♦) low-binding peptide or (•) non-binding peptide or (A) no peptide for more than 8 months, when stored at -80 °C.

5B

A*1101 molecules retains peptide binding activity formulated with (•) low-binding peptide or (A) non-binding peptide or (♦) no peptide for more than 8 months, when stored at -80 °C.

Figure 6

A*1101 and A*0301 heavy chains were formulated with or without glycerol in the presence of 5 μM β₂m. The Ready to go molecules were stored for 2 months at -20 °C before analysis. The presence of glycerol increased the amount of de novo folded A*0301 complexes with 10% (from 3.4 nM to 3.7 nM) while glycerol increased the amount of folded A*1101 molecules with up to 34% (from 1.8 nM to 2.5 nM). (•): A*0301 + β₂m + glycerol; (O): A*0301 + β₂m no glycerol; (■): A*1101 + β₂m + glycerol; (□): A*1101 + β2m no glycerol. Figure 7

Two different MHC class I heavy chains; A*0301(#), A*1101 (■) respectively were formulated by diluting the MHC molecules into a hydrophilic buffer/50% glycerol with increasing concentrations of bovine serum albumin without β₂m. When no BSA was present (A*0301 :O; (A*1101:D) only » 25% was able active and refolded into a peptide-MHC class I complex. With the admixture of 0.25% BSA 100% of the heavy chains retained activity for at least 1 year.

Figure 8.

A*0201 heavy chain was diluted into a buffer containing D2m and graded concentration of a high affinity binding ligand. Recombinant Tapasin was added either as dissolved in PBS or in a denatured form in 8 M urea. The amount of de novo folded A*0201 class I molecule was determined by Prism GraphPad® (Bmax) and the peptide binding affinity, KD including a 95% (Table 2). The peptide binding curves are plotted as amount of de novo folded complex as a function of amount of peptide offered: (A): Admixture of Tapasin in Urea, (O): Admixture of Tapasin in PBS, (▼): without Tapasin.

Examples

Reagents and peptides

The pan-specific mouse anti-HLA class I antibody, W6/32 (from ATCC # HB-95), was purified from ascites by protein A affinity chromatography. The polyclonal rabbit anti- human β₂m-HRP (Dako #P174) and the EnVision+™, Peroxidase, Rabbit enhancing reagent (Dako #K4003) were both from Dako (Copenhagen). 96 well Maxisorp ™ ELISA plates were from Nunc, (Roskilde, Denmark). Pluronic Lutrol F-68 (BASF, Denmark).

Peptides were synthesised by Schafer-N, (Copenhagen, Denmark), and analysed by high performance liquid chromatography and mass spectrometry. All other reagents were purchased from Sigma-Aldrich, Denmark or Merck, Denmark.

Expression and purification of recombinant HLA molecules

The E.coli strain XA90 was transformed with the vector pHNl containing a HLA-A*0201 (1- 275) gene under T7 polymerase control. The gene encoding the HLA-A*1101 (1-276) was PCR amplified from a cDNA library from the EBV transformed cell line, KHAGNI (IHW 9248), inserted into pET28 (Novagen, Madison, Wisconsin), codon optimised for E.coli expression using QuickChange (Stratagene, La Jolla, California), verified by sequencing (ABI 310, Perkin Elmer), and finally transformed into E.coli strain BL21(DE3) (Novagen). Protein expression and purification were performed as follows. Briefly, inclusion bodies containing the recombinant HLA protein product were extracted into 8M Urea, 20 mM Tris, and pH 8 under non-reducing conditions. They were subsequently purified under denaturing conditions by ion-exchange chromatography, hydrophobic interaction chromatography and gel filtration chromatography. Active fractions were identified, pooled, concentrated by diafiltration (Amicon YM10) and stored in 8M Urea, 20 mM Tris, pH 8 at 20°C until use. Human β₂-microglobu!in was obtained from the urine of ureamic patients and purified by chromatofocusing, or generated recombinantly.

5

Formulation of empty class I molecules

Preoxidised denatured MHC class I heavy chains in 8 M urea were diluted into PBS containing graded concentrations of β₂m (Figure 1C) to a final concentration of 150 nM active molecules. Heavy chains and β₂m were mixed on ice for 60 min and subsequently 10 50% volJvol. glycerol was added and samples were stored at -20 °C. After different time periods at -20 °C, the molecules were analysed for peptide binding activity.

Analysis of peptide binding activity of class I molecules by quantitative ELISA

Empty MHC class I molecules were analysis as described in [Sylvester-Hvid, C, et al., 15 2002]. In brief, ninety-six well MaxiSorp ELISA plates (Nunc) were coated and left overnight at 4°C with W6/32, using 50 μl/ well at 5 μg/ml in 100 mM carbonate buffer, pH9.6. Residual binding sites were blocked with 320 μl/well 1% w/v bovine serum albumin in PBS (1% BSA/PBS). To remove unbound W6/32 and blocking reagent, the plate was washed twice using 600 μl/well of 0.05% Tween-20 (Sigma) in PBS at room temperature 20 using an automated plate-washer (Skanwasher300; Skatron, Norway). On ice, empty class I molecules were diluted 50-fold into a 50 mM Tris-maleat buffer, pH6.6, containing 100 nM human β2tτι, excess peptide and 0.3 mg/ml Lutrol-F68.

To allow complex formation, the reaction mixtures were incubated at 18°C for 48 h. Just

25 prior to the ELISA analysis, the reaction volume was diluted 10 times into 2%SMP/PBS at 4° C, and 50 μl/well with PBS/0.05% Tween-20 were transferred in duplicate to a W6/32 coated plate. The plate was incubated for 2 h at 4°C, and then washed 6 times with 600 μl/well with PBS/0.05% Tween 20 at room temperature. To detect the bound complexes, the plate was incubated for 1 h at 4°C with 50 μl/well of horseradish peroxidase (HRP)-

30 conjugated, polyclonal rabbit antihuman β₂m antibody (DAKO p0174) diluted 1 : 2500 into 2% SMP-PBS, and then washed 6 X 600 μl/well with PBS/0.05% Tween-20 at room temperature. To enhance detection, the plate was subsequently incubated for 30 min at room temperature with a dextran polymer conjugated with goat antirabbit IgG and HRP (DAKO EnVision^®+ Peroxidase, Rabbit K4003) diluted 1 : 15 in 2% SMP-PBS containing 1%

35 normal mouse serum, and then washed 6 X 600 μl/well with PBS/0.05% Tween-20 at room temperature. Finally, the ELISA was developed with 3,3 ^' 5,5 ^' -tetramethylbenzidine hydrogenperoxide (TMB-one, Kem-En-Tech, Copenhagen, Denmark) for 30 min at room temperature, and the colorimetric reaction was read at 450 nm using a Victor² Multilabel ELISA counter (Perkin Elmer, Copenhagen, Denmark). The activity (%) was determined as

40 data analysis output Bmax/amount of offered Ready to go molecules * 100. Results

Operationally, the activity analysis was conducted in two steps. First, MHC class I molecules were formulated in a hydrophilic buffer with admixture of a graded concentrations of β₂m. The reagents were incubated at 0 °C for 60 min and glycerol was ; added to a final concentration of 50% volJvol. The MHC molecules were stored at -20 °C for at least 24 h and op to 75 days. Secondly the molecules generated in the first step were analysed in the quantitative ELISA Step. This step was able to measure the concentration of MHC complexes and thereby determine the activity of said "Ready-to-go" MHC class I molecules.

Example 1

Formulation of "Ready-to-go" MHC class I molecules in a hydrophilic buffer.

Three different denatured molecules, A*0101, A*030i and A*1101 molecules, were formulated with increasing amount of β₂m to generate MHC class I molecules. The molecules were subsequently stored in 50% glycerol for 10 days and diluted 50-fold to a final concentration of 1% glycerol into a buffer containing 100 nM β₂m and 10 μM high- affinity binding peptide. The reagents were incubated for 48 hr at 18 °C until steady state was achieved. The amount of de novo folded A*0101, A*0301 and A*1101 class I complexes was analysed in a quantitative ELISA assay. Like shown in Figure 2, a β₂J-Q dependent activity could be observed with as little as between 30 nM and 100 nM nM ϋ m present during storage. In fact, the ELISA signal was proportional to the amount of β₂m present in the storage buffer for more than fore decades of β₂m present. The reason for the increased signal for A*1101 compared to e.g. A*0301 and A*0101 is probably due to difference to the concentration of active empty molecules.

Example 2

Less than 8 M Urea will induce inactive MHC molecules when β₂m is absent.

In addition the consequences of lowering the concentration of the denaturing agent urea, from the denatured MHC heavy chains during storage, were analysed. A fixed concentration of A*0301 and A*1101 was diluted into a buffer containing graded concentrations of urea without β₂m. The heavy chains were incubated for 60 min at 0 °C and stored in 50% vol/vol glycerol for 4 days until analysed. Less than 8 M urea present during storage did induce inactive MHC heavy chains (Figure 3). Not even at the highest concentration of urea present (4 M) did the heavy chain fold into a peptide-MHC class I complex or at least the amount was too low to be detected in the ELISA. In comparison, heavy chains stored in the presence of 5 μM β₂m during storage retained 55% and 95% for A*0301 and A*1101, respectively, as seen in Figure 3. The presence of β₂m did indeed rescue the heavy chains from aggregation. Example 3

MHC class I molecules retain 100% activity for more than 2 month of storage.

Formulated molecules (A*0301, A*1101) were diluted 50-fold into a buffer to a final concentration of 3 nM active molecules and 100 nM β₂m. Graded concentrations of peptide were offered and the folding mix was incubated at 18 °C for 48 for the de novo folding of peptide-MHC complex had reached a steady state. The amount of active molecules was determined as the Bmax from the non-linear regression of Bmax/amount of offered Ready to go molecules * 100. Four different peptides (binding to A*0301 and A*1101 for peptide identity Table 1), were offered to A*0301 and A*1101 molecules which had been stored for various times periods. Both MHC class I molecules retained 100% activity for more than two months (Figure 4a, 4b). In addition, the determined binding affinity (K_D) for the peptides was identical, further proving that the "Ready to go molecules" retained their peptide bindings properties during long-time storage. Taken together, we have shown a novel approach for formulation "Ready to go " MHC molecules, which can be stored for an extended time and maintain the peptide binding activity.

Table 1

Identification of peptides used in the activity studies of A*1101 and A*0301 class I molecules.

Example 4

Formulation of "Ready-to-go" MHC class I molecules in a hydrophilic buffer with the admixture of a low binding peptide.

Two different MHC class I heavy chains were formulated like previously described in example 1. Denatured recombinant A*0301 and A*1101 were formulated in PBS/ β₂m [5 μM] and mixed with either 100 μM low binding peptide or 100 μM non-binding peptide or no peptide. Ready to go preparations were stored at -80 °C, 4 °C, 18 °C and 37 °C for various times periods prior to activity analysis. Activity of the Ready to Go MHC class I molecules were examined like described previously.

Formulated A*0301 was able to retain full activity when stored for more than 9 weeks at 4 °C, in contrast to storage at 18 °C, which decreased the activity to just 50% after 3 three weeks (data not shown). When stored at 37°C the formulated A*0301 Ready to go lost all activity after just 3 weeks (data not shown). Furthermore, when stored at -80 °C all three formulations of A*0301 molecules retained full activity for more than 8 months (Figure 5A).

The activity of the three formulations of A*1101 were examined like described above. Ready to go molecules retained activity for more than 9 weeks when stored at 4°C and 18 °C in the presence of either a non-binding peptide, no peptide of a low binding peptide. (data not shown). When stored at -80°C the three formulations of A*1101 molecules retained a 100% activity for more than 8 months (Figure 5B). These observations demonstrate -80°C as the optimal storage temperature for formulated Ready to Go MHC class I molecules - however, some MHC class I alleles retain activity for a long period of time event when stored at 18°C (A*1101). This might be due to differences in the optimal concentration of β₂m during the storage for A*1101 and A*0301. We demonstrate that for each MHC class I molecule, the optimal β₂m concentration should be examined before formulation.

Example 5

Formulation of "Ready-to-go" MHC class I molecules in a hydrophilic buffer with the admixture of a cryoprotectant glycerol.

Two different MHC class I heavy chains were formulated like previously described in example 1. Denatured recombinant A*0301 and A*1101 were formulated in PBS containing 5 μM of β₂m with or without the admixture of glycerol to a final concentration of 50%. Activity of the Ready to Go MHC class I molecules was examined like described previously after storage in a two months at -20°C. The presence of glycerol increased the amount of de novo folded A*0301 complexes with 10% (from 3.4 nM to 3.7 nM) while glycerol increased the amount of folded A*1101 molecules with up to 34% (from 1.8 nM to 2.5 nM). We demonstrate that the presence of glycerol during storage of formulated molecules is crucial in order to protect the MHC molecules most efficiently.

Example 6

Two different MHC class I heavy chains; A*0301 and A*1101, respectively were formulated by diluting the MHC molecules into a hydrophilic buffer/50% glycerol with increasing concentrations of bovine serum albumin, without β₂m. The molecules were stored up to one year at -20 °C and subsequently analyzed for peptide binding activity like previously described. When no BSA was present only 26% of the MHC molecules were active with respect to bind peptide. In contrast when adding just 0.03% BSA during the storage, 70-80% of the heavy chains were rescued (Figure 7). Adding 0.25% of BSA was adequate to rescue 100% of the heavy chains. By increasing the BSA concentration further only increased the background noise (folding efficiency above 100%.) This was confirmed in subsequently analysis - where increasing BSA was added to a refolding assay of MHC molecules. When BSA was present in concentration above 0.25% BSA was able to facilitate the de novo folding of MHC molecules when no peptide was offered (data not shown).

We demonstrate that BSA might possess the ability the act as a functional co-factor able to rescue the MHC heavy chains during the storage in a hydrophilic buffer.

The admixture of both BSA and β₂m might further increase the activity and stability of the MHC molecules under non denaturing conditions - It is highly likely that different MHC class I alleles display different requirement for BSA and β₂m during storage. Therefore we recommend that each MHC molecule is analyzed for optimal storage conditions.

Example 7:

Tapasin facilitates the formation of peptide-MHC complexes.

Tapasin was generated recombinant like described for β₂m and either eluted in 8 M Uread or diluted into PBS.

Recombinant A*0201 heavy chains was diluted into PBS containing saturation amount of β₂m and high affinity binding peptide with or without the chaperone Tapasin. After 48 hours of incubation the amount of de novo folded MHC class I molecule (approximately Bmax) as well as the peptide binding affinity were determined. As shown in Figure 8, Bmax was significantly higher (95% confidential interval: 4.06 - 4.79 nM) for the refolding of A*0201 molecule in the presence of TAPASIN pre diluted in 8 m Urea compared to the reaction with A*0201 molecule without Tapasin (95% confidential interval: 3,12 - 3,65 nM). Tapasin was added pre diluted in PBS. 3.7 nM A*0201 class I complexes was de novo folded -more than when leaving out Tapasin. Clearly Tapasin have a significant impact on the formation of class I complexes, however further analysis will have to clarify whether Tapasin should be added under denatured or refolded conditions. Tapasin is known to interact with the MHC class I molecules keeping it in an active peptide binding conformation in the ER, while the heavy chain is waiting for the appropriate peptide.

Thus, Tapasin can protect class I heavy chain during formulation in a hydrophilic buffer for long time storage, associating with the unfolded part of the MHC class I heavy chain and reduce aggregation. Table 2

References

DK PA 2002 00766

DK PA 2002 00768

WO 00/15665

WO 02/072631

US 5,364,762

US 6,153,408

Buus, S., Pedersen, L. O. and Stryhn, A. "The analysis of peptide binding to MHC and of MHC specificity". Immunological recognition of peptides in medicine and biology 1995: 61- 77 Fahnestock ML et al., Thermal Stability Comparision of Purified Empty and Peptide-Filled Forms of a Class I MHC Molecule. Science 1992, 258: 1658-1662.

Garboczi DN et al., HLA-A2-peptide complexes: Refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proceedings of the National Academy of Science USA, 1992, 89: 3429-33

Pedersen, L.0., Nissen, M. H., Hansen, N .V., Nielsen, L. L., Lauemøller , S. L.L., Blicher, T., Nansen, A., Sylvester-Hvid, C, Thomsen A.R. and Buus, S. "Efficient assembly of recombinant major histocompatibility complex class I molecules with preformed disulphide bonds". Eur. J. Immunol. 2001, 31: 2986-2996.

Sylvester-Hvid, C, Kristensen, N.S., Blicher, T., Ferre, H., Lauemøller, S.L., Wolf, Z.A., Lamberth, K., Nissen, M.H., Pedersen L.0. and Buus S. "Establishment of a quantitative ELISA capable of determining peptide-MHC class I interaction". Tissue antigens. 2002, 59:251-258.

Claims

1. A method for formulating a renaturated, active and stable MHC class I-like molecule comprising diluting a denatured heavy chain of said MHC class I-like molecule which is reduced, partially oxidised or fully oxidised, in a hydrophilic buffer with an admixture of a co-factor to facilitate the formation of the renatured, active and stable MHC class I-like molecule, whereby said MHC class I-like molecule can be long-term stored.

2. A method according to claim 1, wherein the hydrophilic buffer is in absence of high- affinity binding ligand.

3. A method according to claim 1 and/or 2, wherein the hydrophilic buffer further comprises a low-affinity-binding ligand.

4. A method for formulating a renaturated, active, stable and empty MHC class I-like molecule comprising diluting a denatured heavy chain of said MHC class I-like molecule which is reduced, partially oxidised and/or fully oxidised, in a hydrophilic buffer with an admixture of a co-factor to facilitate the formation of the renaturated, active, stable and empty MHC class I-like molecule, but in absence of any ligand, whereby said MHC class I- like molecule can be long-term stored.

5. A method according to any of the preceding claims, wherein the co-factor is selected from the group consisting of a chaperone, API, AP2, AP3, AP4, Tap, tapasin, calreticulin, calnexin, β₂m and Erp57.

6. A method according to any of the preceding claims, wherein the denatured MHC class I- like heavy chain is formulated in the hydrophilic buffer at least 0 sec. at 0°C whereby it becomes active and substantially retains said activity for at least 1 week.

7. A method according to any of the preceding claims, wherein the long-term storage further increases the amount of active heavy chain isomers.

8. A method according to any of the preceding claims, wherein the hydrophilic buffer further comprises glycerol.

9. A method according to any of the preceding claims, wherein the empty MHC class I-like molecule is capable of refolding into a ligand-MHC class I-like complex in the presence of a high-affinity binding ligand.

10. A method according to any of the preceding claims, wherein the MHC class I-like molecule is CDl.

11. A method according to any of the preceding claims, wherein the MHC class I-like molecule is MHC class I.

12. A method according to any of the preceding claims, wherein the co-factor is β₂m.

13. A method according to any of the preceding claims, wherein the concentration of β₂m 5 is at least 1 nM.

14. A formulated renaturated, active and stable MHC class I-like molecule obtainable by the method according to any of claims 1-13.

10 15. A formulated renaturated, active, stable and empty MHC class I-like molecule obtainable by the method according to any of claims 1-13.

16. A formulated renaturated, active, stable and empty MHC class I-like molecule according to claim 15, wherein the MHC class I-like molecule is a MHC class I molecule.

15

17. A formulated MHC class I-like molecule according to any one of claim 15 or claim 16, which is biotinylated.

18. A formulated MHC-tetramer comprising a MHC class I-like molecule according to claim 20 17.

19. A method for formulating a renaturated active and stable MHC class Il-like molecule comprising diluting denatured heavy chains of said MHC class Il-like molecule which are reduced, partially oxidised and/or fully oxidised, into a hydrophilic buffer, whereby said

25 MHC class Il-like molecule can be long-term stored.

20. A method for formulating a renaturated active and stable MHC class Il-like molecule comprising diluting denatured heavy chains of said MHC class Il-like molecule which are reduced, partially oxidised and/or fully oxidised, into a hydrophilic buffer in absence of

30 high-affinity binding peptides, whereby said MHC class Il-like molecule can be long-term stored.

21. A method according to any of claims 19 or 20, wherein the hydrophilic buffer further comprises low-affinity-binding peptides.

35

22. A method for formulating a renaturated active, stable and empty MHC class Il-like molecule comprising diluting denatured heavy chains of said MHC class Il-like molecule which are reduced, partially oxidised and/or fully oxidised, into a hydrophilic buffer in absence of any peptides, whereby said MHC class Il-like molecule can be long-term stored.

40

23. A method according to any of claims 19-22, wherein the denatured MHC class II heavy chains is formulated in the hydrophilic buffer at least 0 sec at 0°C whereby it becomes active and substantially retains said activity for at least 1 week.

24. A method according to any of claims 19-23, wherein the long-term storage further increases the amount of active heavy chain isomer.

25. A method according to any of claims 19-24, wherein the hydrophilic buffer further 5 comprises glycerol.

26. A method according to any of claims 19-25, wherein the empty MHC class II molecule is capable of renaturating into a MHC class II complex in the presence of a high-affinity binding peptide.

10

27. A formulated renaturated MHC class Il-like molecule comprising denatured MHC class II heavy chain which is reduced, partially oxidised and/or fully oxidised and contained in a hydrophilic buffer in absence of high-affinity binding peptides whereby said MHC class Il- like molecule can be long-term stored.

15

28. A formulated renaturated empty MHC class Il-like molecule comprising denatured MHC class II heavy chain which is reduced, partially oxidised and/or fully oxidised and contained in a hydrophilic buffer in absence of any binding peptides whereby said MHC class Il-like molecule can be long-term stored.

20

29. A formulated molecule according to claim 28 which is biotinylated.

30. A formulated MHC-tetramer comprising a molecule according to claim 29.

25 31. A kit comprising a formulated molecule according to any of the preceding claims that can be long-term stored for use in a structure and functional analysis of said molecules.

32 A kit according to claim 31, wherein said kit is selected from the group consisting of ELISA, RIA, fluorescence correlation spectroscopy, fluorescence polarisation, and 30 flourometry.

33. A kit according to any of claims 31-32, which comprises an oligomer of the molecule, such as two, three, four or more molecules.

35 34. A kit according to any of claims 31-33, wherein a further reagent is added as a marker making the kit suitable for diagnostic purposes.

35. A kit according to claim 34, wherein the marker is selected from the group consisting of flourochromes, enzymes, chemiluminescense, and radioactive markers.

40

36. A formulated renaturated, active, stable and optionally empty MHC-like molecule obtainable by the method according to any of claims 1-13, further comprising a ligand for diagnostic purposes.

37. Use of a formulated renaturated, active, stable and optionally empty MHC-like molecule obtainable by the method according to any of claims 1-13, for diagnostic purposes.