Patent 6,881,828

A hybrid fusion protein comprising a first antigenic amino acid sequence fused to a second amino acid sequence substantially homologous to B2M or a fragment thereof.

Description of the Invention

This invention concerns novel hybrid beta-2 microglobulin fusion proteins. It also relates to nucleic acid (DNA and RNA) coding for all or part of such proteins, and to their preparation. Pharmaceutical compositions containing such fusion proteins are useful both as components of prophylactic vaccines and as immuno-therapeutics for the post-exposure treatment of viral infection and malignancy.

Immune responses have two major components, humoral and cell-mediated. The first comprises antibodies, produced by B-lymphocytes, which bind to specific antigens and which serve a number of important functions such as neutralization of viruses, complement fixation and immune complex formation. Antibody production is dependent on helper T cells, which mediate the antigen specific recognition of peptide epitopes displayed by Class II Major Histocompatibility Complex (MHC) molecules on the surface of antigen presenting cells.

The second arm of the immune system involves effector T cells, in particular cytotoxic T lymphocytes (CTLs) which can recognize and kill infected or transformed cells. Such responses are particularly important in the defense against and immunity from viral infections, though they are also involved in host responses against certain tumours. CTL responses are also antigen specific, involving subsets of CD8 positive T cells. Immune recognition is mediated by the T cell receptor which recognizes the peptide antigen displayed on Class I MHC molecules.

Class I MHC molecules are ubiquitously expressed and consist of two polypeptide chains. The first is an alpha chain of about 45 kDa comprising 3 domains. Two of these domains bind to peptides derived from processing endogenously synthesized proteins, such as viral components, and present them to the T cell receptor. These two domains are linked to a single membrane spanning anchor region by a third, immunoglobulin-like domain. The second component is beta-2 microglobulin (B2M), a 100 amino acid protein which can exist free in the serum as well as a part of Class I MHC. The two chains normally associate in the ER, along with peptides produced by the degradation of endogenous proteins to form a ternary complex which is displayed on the cell surface. Although binary complexes lacking peptide can be formed, these are unstable and are normally recycled or degraded (Ljungren et al. Nature 346, 476-(1990).

Crystallographic data reveals that the antigenic peptide binds to a groove between the first two domains of the alpha subunit, the base of which is formed from strands of beta sheet, and the walls from two alpha helices (Biorkman et al. Nature 329, 506-512 (1987)). The nature of the side chains in this region of the alpha chain are critical in determining the peptide selectivity of the molecule, and hence the ability of different Class I MHC alleles to respond to particular epitopes (Bjorkman et al. Nature 329, 512-518 (1987)). Although it is the alpha subunit that binds the peptide, B2M plays an essential role in allowing binding, presumably by stabilising the ternary complex (Vitiello et al. Science 250, 1423-1426 (1990); Rock et al. PNAS 88, 310-304 (1991)). While some free alpha subunit can reach the cell surface in the absence of B2M, and can bind and present peptide, it is considerably less efficient than in the presence of B2M (Bix and Raulet. Exp. Med. 176, 829-834 (1992).

Although the normal route of antigen presentation by Class I MHC involves the degradation of endogenously synthesised proteins, it is possible to achieve antigen presentation by addition of high concentrations of peptide to antigen presenting cells (APC) in vitro. The use of peptides as immunogens has a number of potential advantages, the most significant of which are that it avoids the use of inactivated or attenuated viral particles, and that small peptides can be synthesised chemically, avoiding the requirement for biological production routes. The most efficient presentation is obtained with peptides of from 9-11 residues in length, which corresponds to the length of the groove if they are in an extended conformation.

However, there are major problems associated with this method of eliciting a CTL response. Although such a use of peptides is adequate for demonstrating immune responses such as T cell-mediated killing in vitro, it is not an efficient process, and does not offer a practical route to in vivo immunization. CTL responses may be induced by immunization with certain small peptide fragments but such peptides may elicit poor antibody responses. Further, in an outbred population, individuals of different MHC class I and class II haplotypes will respond to different peptide epitopes; many peptides may be required to prime all the possible haplotypes.

Most of the available evidence also indicates that immunization with whole proteins does not give rise to a CTL response. Such CTL responses that have been induced require the presence of powerful adjuvants or live vector which have not been licensed for use in humans. For example, recombinant HIV gp160 presented in ISCOMs, but not in FCA, FIA or buffered saline, induced HIV-1 envelope-specific CTL activity in BALB/c mice. It is generally felt that approval for the use of ISCOMs in humans is unlikely to be forthcoming. Recombinant vaccinia viruses expressing the V3 region of the HIV envelope gp160 also induce specific CTL responses to the V3 region, but immunization with vaccinia has raised concerns both about safety for immunocompromised patients, and about efficacy. A simpler and more efficient way to induce CTL responses is therefore an important goal, and has been sought actively for a number of years though with only limited success.

It has been shown that the exchange of B2M chains in MHC class I will occur in the presence of free B2M (Hayfil and Strominger, PNAS USA 76(11) 5834-5838 (1979)), and that the binding of exogenous peptides to these molecules occurs upon association and reassociation of B2M light chains (Rock et al., PNAS USA 87 7517-7521 (1990); Kozlowski et al. Nature 349, 74-77 (1991)). The presentation of exogenously added peptide can therefore be made more efficient by the addition of B2M. The use of exogenous B2M in this way to enhance immune responses against peptides in vivo is described in WO-A-91/16924.

However, there are a number of problems with this approach. The use of an admixture, while a convenient tool for in vitro use, is less appropriate for in vivo administration, due to the problems of polypharmacy for product registration.

Further, despite the DNA sequence of B2M being known, it has not been possible to produce recombinant B2M efficiently. B2M for use in the enhancement of immune responses would therefore need to be purified from natural sources such as serum or urine. Not only is this difficult and expensive, but blood products must be screened for a number of contaminants before being acceptable for use in vaccines.

Despite the fact that interactions between the C-terminus of the peptide and the alpha subunit have been shown to be essential for peptide binding and presentation, the present invention has found that peptides fused to the N-terminus of B2M are still capable of binding to the groove in the alpha subunit. This binding is achieved without sacrificing the ability of the ternary complex to bind and trigger the T-cell receptor.

According to the first aspect of the invention there is provided a hybrid fusion protein comprising a first antigenic amino acid sequence fused to a second amino acid sequence substantially homologous with B2M or a fragment thereof.

Preferably the first antigenic sequence is fused to the B2M sequence via a short linker sequence, to span the gap between the peptide and the B2M with minimal disruption to the conformation of the light chain.

The use of B2M fusions offers considerable advantages. The binary complex between the alpha subunit and the B2M fusion is stabilised relative to the corresponding ternary complex, prolonging the ability of the peptide to stimulate the T cell receptor, thereby increasing the effective potency of the peptide. The problems of polypharmacy are avoided, simplifying the development of the compound. This approach also has a number of product registration advantages by minimising the number of proteins present in the vaccine, and may conform more closely to guidelines on quality and safety. Finally, only a single GMP production route is required. These latter considerations are particularly important since multiple epitopes might be required to produce a vaccine effective against the background of MHC diversity. A final advantage is that because the peptide is delivered efficiently to the MHC, the requirement for adjuvants may be obviated.

The expression "substantially homologous", when describing the relationship of an amino acid sequence to a natural protein, means that the amino acid sequence can be identical to the natural protein or can be an effective but truncated or modified form of the natural protein. As a practical matter, though, most analogues will have a high degree of homology with the prototype molecule if biological activity is to be substantially preserved. It will be realised that the nature of changes from the prototype molecule is more important than the amount of them. As guidance, though, at the amino acid level, it may be that at least 60%, 70%, 80%, 90%, 95% or even 99% of the residues will be the same as the prototype molecule; at the nucleic acid level, nucleic acid coding for an analogue may for example hybridize under stringent conditions (such as at approximately 35^oC. to 65^oC. in a salt solution of approximately 0.9 molar) to nucleic acid coding for the prototype molecule, or would do so but for the degeneracy of the genetic code.

Fragments of B2M for use in this invention will retain the ability to augment immune responses to the peptides to which they are fused. Preferably at least 60%, 70%, 80%, 90%, 95% or even 99% of the residues will be retained.

Proteins substantially homologous to B2M include naturally occurring B2M and modified B2M, a variant found in connection with a variety of types of cancer and disorders of the immune system.

The antigenic sequence may correspond to a sequence derived from or associated with an aetiological agent or a tumor. The aetiological agent may be a microorganism such as a virus, bacterium, fungus or parasite. The virus may be: a retrovirus, such as HIV-1, HIV-2, HTLV-I, HTLV-II, HTLV-III, SIV, BIV, LAV, EIAV, CIAV, murine leukaemia virus, Moloney murine leukaemia virus, and feline leukaemia virus; an orthomyxovirus, such as influenza A or B; a paramyxovirus, such as parainfluenza virus, mumps, measles, RSV and Sendai virus; a papovavirus, such as HPV; an arenavirus, such as LCMV of humans or mice; a hepadnavirus, such as Hepatitis B virus; a herpes virus, such as HSV, VZV, CMV, or EBV. HIV is human immunodeficiency virus, HTLV is human T-cell lymphoma leukemia virus, SIV is simian immunodeficiency virus, BIV is bovine immunodeficiency virus, LAV is lymphadenopathy associated virus, EIAV is equine infectious anemia virus, CIAV is chicken infectious anemia virus, RSV is respiratory syncytial virus, HPV is human papilloma virus, LCMV is lymphocytic choriomeningitis virus, HSV is herpes simplex virus, VZV is varicella zoster virus, CMV is cytomegalovirus, and EBV is Epstein Barr virus. The tumor-associated or derived antigen may for example be a proteinaceous human tumor antigen, such as a melanoma-associated antigen, or an epithelial-tumor associated antigen such as from breast or colon carcinoma.

The antigenic sequence may be also derived from a bacterium, such as of the genus Neisseria, Campilobacter, Bordetella, Listeria, Mycobacteria or Leishmania, or a parasite, such as from the genus Trypanosoma, Schizosoma, Plasmodium, especially P. falciparum, or from a fungus, such as from the genus Candida, Aspergillus, Cryptococcus, Histoplasma or Blastomyces.

Preferred antigenic sequences correspond to CTL epitopes from a retrovirus, a paramyxovirus, an arenavirus or a hepadna virus, or a human tumor cell.

Examples include epitopes from:

1) HIV (particularly HIV-1) gp120,

2) HIV (particularly HIV-1) p24

3) VZV gpI, gpII and gpIII

4) LCMV nucleoprotein,

5) Influenza virus nucleoprotein,

6) HPV L1 and L2 proteins,

7) Human papilloma virus E5 and E7

8) Malaria CSP or RESA antigens,

9) Mycobacterium p6,

10) GA 733-2 epithelial tumor-associated antigen,

11) MUC-1 repeat sequence from epithelial tumor-associated antigen,

12) Melanoma MZ2-E antigens

13) Melanoma p97 associated antigen, Particularly preferred antigenic sequences are derived from the third variable domain of an envelope protein of a lentivirus. This region of lentiviruses, known as the V3 loop or GPGR loop is found between amino acids 300 and 330 of gp120 of HIV-1 and in analogous positions of other lentiviruses. The V3 loop is, for HIV-1 at least, the major neutralising epitope of the virus (Putney et al 1986 Science 234, 1392; Rusche et al 1988 Proc. Natl. Acad. Sci. 85, 3198; Palker et al 1988 Proc. Natl. Acad. Sci. 85 1932; and Goudsmit et al., 1988 AIDS 2 157). The antigenic portion of choice may constitute the whole of the V3 loop when derived from different strains. However, multiple copies of a conserved sequence of the V3 loop may be useful in conferring immunity against more than one isolate of a virus (such as HIV-1).

B2M fusion proteins in accordance with the invention can in principle be made by any convenient method including coupling successive amino acid residues together, or by the chemical coupling of two or more oligo- or polypeptide chains or of existing (for example natural) proteins. Although proteins may in principle be synthesised wholly or partly by chemical means, the route of choice will be ribosomal translation, preferably in vivo, of a corresponding nucleic acid sequence.

According to a second aspect of the invention, therefore, there is provided nucleic acid coding for a fusion protein as described above. Both DNA and RNA are within the scope of the invention. DNA may be chemically synthesized and/or recombinant.

Recombinant DNA in accordance with the invention may be in the form of a vector. The vector may for example be a plasmid, cosmid or phage. Vectors will frequently include one or more selectable markers to enable the selection of cells transformed (or transfected: the terms are used interchangeably in this specification) with them and, preferably, to enable selection of cells harboring vectors incorporating heterologous DNA. Appropriate translational initiating and termination signals will generally be present. Additionally, if the vector is intended for expression, sufficient transcriptional regulatory sequences to drive expression will be included. Vectors not including regulatory sequences are useful as cloning vectors. According to a third aspect of the invention there is provided a vector including nucleic acid as described above. It is to be understood that the term "vector" is used in this specification in a functional sense and is not to be construed as necessarily being limited to a single nucleic acid molecule.

Expression vectors in accordance with the invention will usually contain a promoter. The nature of the promoter will depend upon the intended host expression cell. For yeast, PGK is a preferred promoter, but any other suitable promoter may be used if necessary or desirable. Examples include GAPD, GAL1-10, PH05, ADH1, CYC1, Ty delta sequence, PYK and hybrid promoters made from components from more than one promoter (such as those listed). For insect cells, a preferred promoter is the polyhedrin promoter from Autographica californica nuclear polyhedrosis virus (AcNPV). Those skilled in the art will be able to determine other appropriate promoters adapted for expression in these or other cells. Vectors not including promoters may be useful as cloning vectors, rather than expression vectors.

Cloning vectors can be introduced into E. coli or any other suitable hosts which facilitate their manipulation. Expression vectors may be adapted for prokaryotic expression in bacterial cells, such as E. coli.However, for preference vectors are adapted for expression in a microbial eukaryotic cell, such as a yeast (including but not limited to Saccharomyces cerevisiae and Pichia pastoris) or a higher eukaryotic cell such as insect cell lines such as Spodoptera frugiperda SF9, or mammalian cells including Chinese hamster ovary (CHO) cells, mouse myeloma cell lines such as P3X63-Ag8.653, COS cells, HeLa cells, BHK cells, melanoma cell lines such as the Bowes cell line, mouse L cells, human hepatoma cell lines such as Hep G2, mouse fibroblasts and mouse NIH 3T3 cells. Performance of the invention is neither dependent on nor limited to any particular strain of microorganism or cell type: those suitable for use with the invention will be apparent to those skilled in the art, following the teaching of this specification. According to a fourth aspect of the invention there is provided a host cell transfected or transformed with DNA described above.

Despite the DNA sequence of B2M being known, to date it has not been possible to produce recombinant B2M efficiently. Extremely low yields are produced by expression in mammalian cells, and the protein is incorrectly folded following expression in E. coli. Nor are yeasts a universal host for the purpose. Standard techniques for obtaining expression of B2M in perhaps the most widely used yeast for such purposes, namely S. cerevisiae, resulted in very poor yields.

The Pichia pastoris expression system is well known, and has particular advantages in its ease of scalability for large scale production. High level expression has been obtained for a number of proteins in that host, but equally, some have proved difficult to produce. There is no obvious correlation between the properties of a particular polypeptide and its ability to be highly expressed in the Pichia system.

In contrast with the low level expression of B2M in S. cerevisiae, reasonable yields of the fusion proteins of this invention can be obtained in the Pichia system.

Therefore, the invention includes a method of producing fusion proteins of the invention by cultivating a methylotropic yeast harbouring an expression vector comprising DNA encoding the relevant fusion protein, and recovering the expressed fusion protein.

Methylotropic yeast strains include Pichia, in particular P. pastoris, Hansenula, Candida and Torulopsis. Use of Pichia pastoris is presently preferred.

Recombinant DNA encoding the fusion proteins of the invention may be incorporated in a yeast expression vector for expression in methylotropic yeast in accordance with the invention. Such vectors will usually contain a promoter. AOX is a preferred promoter, but any other suitable promoter may be used if necessary or desirable. Examples include GAPD, GAL1-10, PH05, ADH1, PGK, CYC1, Ty delta sequence, PYK and hybrid promoters made from components from more than one promoter (such as those listed).

To obtain secretion of the fusion protein from the yeast cells after expression, the expression vector preferably includes a secretion leader sequence fused to the B2M sequence of the hybrid fusion protein. After secretion, the leader sequence is automatically cleaved from the B2M protein by enzyme(s) produced naturally during the cultivation of the transformed yeast cells. Such secretion techniques are well known. Secretion leaders known to the art include the alpha factor, Pho1, HSA and Suc2. If cleavage is not 100% accurate, the final yield of fusion protein may be contaminated with a fusion of B2M/epitope hybrid with part of the secretion factor sequence, indicating incomplete removal of the secretion factor leader. Although the hybrid protein of the invention may be separated from the contaminant by standard purification methods, for example those based on differing molecular weights, it would be desirable to avoid the difficulty if possible.

It appears the Pho1 leader is in general correctly cleaved from the hybrid fusion proteins of the invention, and it is therefore the presently preferred secretion factor for use in accordance with the invention.

The B2M fusion proteins of the first aspect are useful as vaccines. These might be used prior to exposure as prophylactic agents, or after exposure as immunotherapeutic agents to enhance the clearance of viral infection. Such fusion proteins would be administered by conventional routes, either i.v., i.m. or s.c., though the s.c. route may be preferred.

According to a fifth aspect of the invention, there is provided a pharmaceutical or veterinary formulation comprising a compound of general formula I and a pharmaceutically or veterinarily acceptable carrier. One or more fusion proteins of the first aspect may be present in association with one or more non-toxic pharmaceutically or veterinarily acceptable carriers such as sterile physiological saline or sterile PBS, and/or diluents and/or adjuvants and if desired other active ingredients. Sterility will generally be essential for parenterally administrable vaccines. One or more appropriate adjuvants may also be present. Examples of suitable adjuvants include muramyl peptide compounds such as prototype muramyl dipeptide, aluminium hydroxide and saponin. Coadministration with cytokines may also be considered, eg IFN.gamma. may potentiate the immune response by inducing class I MHC expression.

It may be preferred that, when used as prophylactic vaccines, the B2M agents are used in combination with subunit vaccines designed to induce good neutralising antibody responses. However, this may not be necessary as there is some evidence that CTL responses alone can protect against infection.

According to a sixth aspect of the invention, there is provided a process for the preparation of a pharmaceutical or veterinary formulation in accordance with the fifth aspect, the process comprising admixing a B2M fusion protein of the first aspect and a pharmaceutically or veterinarily acceptable carrier.

The active ingredient may be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the drug can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle.

Particulate antigens produced in accordance with the invention may be useful in the preparation of vaccines, for example immunotherapeutic vaccines, which form a further aspect of the invention.

Claim 1 of 24 Claims

What is claimed is:

1. A hybrid fusion protein comprising a first amino acid sequence which is an antigenic cytolytic T lymphocyte (CTL) epitope, the first sequence being fused through an amino acid linker sequence to a second amino acid sequence which binds to class I major histocompatibility complex (MHC) molecules, said second sequence being selected from the group consisting of a beta-2 microglobulin (B2M) having an amino acid sequence identical to a naturally occurring B2M and B2M having an amino acid sequence that has at least 95% amino acid sequence identity with the amino acid sequence of a naturally occurring B2M, wherein the second sequence has the B2M ability to augment the immune response to the first amino acid sequence.

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