The present invention is related to the fields of molecular biology, virology, immunology and medicine. The invention provides a composition comprising an ordered and repetitive antigen or antigenic determinant array. The invention also provides a process for producing an antigen or antigenic determinant in an ordered and repetitive array. The ordered and repetitive antigen or antigenic determinant is useful in the production of vaccines for the treatment of infectious diseases, the treatment of allergies and as a pharmaccine to prevent or cure cancer and to efficiently induce self-specific immune responses, in particular antibody responses.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention provides compositions, which comprises highly ordered and repetitive antigen or antigenic determinant arrays, as well as the processes for their production and their uses. Thus, the compositions of the invention are useful for the production of vaccines for the prevention of infectious diseases, the treatment of allergies and cancers, and to efficiently induce self-specific immune responses, in particular antibody responses.

In a first aspect, the present invention provides a novel composition comprising, or alternatively consisting of, (A) a non-natural molecular scaffold and (B) an antigen or antigenic determinant. The non-natural molecular scaffold comprises, or alternatively consists of, (i) a core particle selected from the group consisting of (1) a core particle of non-natural origin and (2) a core particle of natural origin; and (ii) an organizer comprising at least one first attachment site, wherein said organizer is connected to said core particle by at least one covalent bond. The antigen or antigenic determinant is a self antigen or a fragment thereof and has at least one second attachment site which is selected from the group consisting of (i) an attachment site not naturally occurring with said antigen or antigenic determinant; and (ii) an attachment site naturally occurring with said antigen or antigenic determinant. The invention provides for an ordered and repetitive self antigen array through an association of the second attachment site to the first attachment site by way of at least one non-peptide bond. Thus, the self antigen or self antigenic determinant and the non-natural molecular scaffold are brought together through this association of the first and the second attachment site to form an ordered and repetitive antigen array.

In a second aspect, the present invention provides a novel composition comprising, or alternatively consisting of, (A) a non-natural molecular scaffold and (B) an antigen or antigenic determinant. The non-natural molecular scaffold comprises, or alternatively consists of, (i) a core particle and (ii) an organizer comprising at least one first attachment site, wherein said core particle is a virus-like particle comprising recombinant proteins, or fragments thereof, of a bacteriophage, and wherein said organizer is connected to said core particle by at least one covalent bond. The antigen or antigenic determinant has at least one second attachment site which is selected from the group consisting of (i) an attachment site not naturally occurring with said antigen or antigenic determinant; and (ii) an attachment site naturally occurring with said antigen or antigenic determinant. The invention provides for an ordered and repetitive antigen array through an association of the second attachment site to the first attachment site by way of at least one non-peptide bond.

In a third aspect, the present invention provides a novel composition comprising, or alternatively consisting of, (A) a non-natural molecular scaffold and (B) an antigen or antigenic determinant. The non-natural molecular scaffold comprises, or alternatively consists of, (i) a core particle selected from the group consisting of (1) a core particle of non-natural origin and (2) a core particle of natural origin; and (ii) an organizer comprising at least one first attachment site, wherein said organizer is connected to said core particle by at least one covalent bond. The antigen or antigenic determinant is an amyloid beta peptide (A.beta..sub.1-42) or a fragment thereof, and has at least one second attachment site which is selected from the group consisting of (i) an attachment site not naturally occurring with said antigen or antigenic determinant; and (ii) an attachment site naturally occurring with said antigen or antigenic determinant. The invention provides for an ordered and repetitive antigen array through an association of the second attachment site to the first attachment site by way of at least one non-peptide bond.

In a fourth aspect, the present invention provides a novel composition comprising, or alternatively consisting of, (A) a non-natural molecular scaffold and (B) an antigen or antigenic determinant. The non-natural molecular scaffold comprises, or alternatively consists of, (i) a core particle selected from the group consisting of (1) a core particle of non-natural origin and (2) a core particle of natural origin; and (ii) an organizer comprising at least one first attachment site, wherein said organizer is connected to said core particle by at least one covalent bond. The antigen or antigenic determinant is an anti-idiotypic antibody or an anti-idiotypic antibody fragment and has at least one second attachment site which is selected from the group consisting of (i) an attachment site not naturally occurring with said antigen or antigenic determinant; and (ii) an attachment site naturally occurring with said antigen or antigenic determinant. The invention provides for an ordered and repetitive antigen array through an association of the second attachment site to the first attachment site by way of at least one non-peptide bond.

Further aspects as well as preferred embodiments and advantages of the present invention will become apparent in the following as well as, in particular, in the light of the detailed description, the examples and the accompanying claims.

In a preferred embodiment of the present invention, the core particle is a virus-like particle comprising recombinant proteins of a RNA-phage, preferably selected from the group consisting of a) bacteriophage Q.beta.; b) bacteriophage R17; c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g) bacteriophage M11; h) bacteriophage Mx1; i) bacteriophage NL95; k) bacteriophage f2; and 1) bacteriophage PP7. Most preferred are bacteriophage Q.beta. and bacteriophage fr.

In another preferred embodiment of the invention, the recombinant proteins of the RNA-phages comprise wild type coat proteins.

In further preferred embodiment of the invention, the recombinant proteins of the RNA-phages comprise mutant coat proteins.

In yet another embodiment, the core particle comprises, or alternatively consists of, one or more different Hepatitis core (capsid) proteins (HBcAgs). In a related embodiment, one or more cysteine residues of these HBcAgs are either deleted or substituted with another amino acid residue (e.g., a serine residue). In a specific embodiment, the cysteine residues of the HBcAg used to prepare compositions of the invention which correspond to amino acid residues 48 and 107 in SEQ ID NO:134 are either deleted or substituted with another amino acid residue (e.g., a serine residue).

Further, the HBcAg variants used to prepare compositions of the invention will generally be variants which retain the ability to associate with other HBcAgs to form dimeric or multimeric structures that present ordered and repetitive antigen or antigenic determinant arrays.

In another embodiment, the non-natural molecular scaffold comprises, or alternatively consists of, pili or pilus-like structures that have been either produced from pilin proteins or harvested from bacteria. When pili or pilus-like structures are used to prepare compositions of the invention, they may be formed from products of pilin genes which are naturally resident in the bacterial cells but have been modified by genetically engineered (e.g., by homologous recombination) or pilin genes which have been introduced into these cells.

In a related embodiment, the core particle comprises, or alternatively consists of, pili or pilus-like structures that have been either prepared from pilin proteins or harvested from bacteria. These core particles may be formed from products of pilin genes naturally resident in the bacterial cells.

In a particular embodiment, the organizer may comprise at least one first attachment site. The first and the second attachment sites are particularly important elements of compositions of the invention. In various embodiments of the invention, the first and/or the second attachment site may be an antigen and an antibody or antibody fragment thereto; biotin and avidin; strepavidin and biotin; a receptor and its ligand; a ligand-binding protein and its ligand; interacting leucine zipper polypeptides; an amino group and a chemical group reactive thereto; a carboxyl group and a chemical group reactive thereto; a sulfhydryl group and a chemical group reactive thereto; or a combination thereof.

In a further preferred embodiment, the composition further comprises an amino acid linker. Preferably the amino acid linker comprises, or alternatively consists of, the second attachment site. The second attachment site mediates a directed and ordered association and binding, respectively, of the antigen to the core particle. An important function of the amino acid linker is to further ensure proper display and accessibility of the second attachment site, and thus to facilitate the binding of the antigen to the core particle, in particular by way of chemical cross-linking. Another important property of the amino acid linker is to further ensure optimal accessibility and, in particular, reactivity of the second attachment site. These properties of the amino acid linker are of even more importance for protein antigens.

In another preferred embodiment, the amino acid linker is selected from the group consisting of (a) CGG; (b) N-terminal gamma 1-linker; (c) N-terminal gamma 3-linker; (d) Ig hinge regions; (e) N-terminal glycine linkers; (f) (G).sub.kC (G).sub.n with n=0-12 and k=0-5; (g) N-terminal glycine-serine linkers; (h) (G).sub.kC(G).sub.m(S)1(GGGGS).sub.n with n=0-3, k=0-5, m=0-10, 1=0-2 (SEQ ID NO:424); (i) GGC; (k) GGC-NH2; (l) C-terminal gamma 1-linker; (m) C-terminal gamma 3-linker; (n) C-terminal glycine linkers; (o) (G).sub.nC(G).sub.k with n=0-12 and k=0-5; (p) C-terminal glycine-serine linkers; (q) (G).sub.m(S).sub.l(GGGGS).sub.n(G).sub.oC(G).sub.k with n=0-3, k=0-5, m=0-10, 1=0-2, and o=0-8 (SEQ ID NO:425).

An important property of glycine and glycine serine linkers is their flexibility, in particular their structural flexibility, allowing a wide range of conformations and disfavoring folding into structures precluding accessibility of the second attachment site. As glycine and glycine serine linkers contain either no or a limited amount of side chain residues, they have limited tendency for engagement into extensive interactions with the antigen, thus, further ensuring accessibility of the second attachment site. Serine residues within the glycine serine linkers confer improved solubility properties to these linkers. Accordingly, the insertion of one or two amino acids either in tandem or isolation, and in particular of polar or charged amino acid residues, in the glycine or glycine serine amino acid linker, is also encompassed by the teaching of the invention.

In a further preferred embodiment, the amino acid linker is either GGC-NH2, GGC-NMe, GGC-N(Me)2, GGC-NHET or GGC-N(Et)2, in which the C-terminus of the cysteine residue of GGC is amidated. These amino acid linkers are preferred in particular for peptide antigens, and in particular for embodiments, in which the antigen or antigenic determinant with said second attachment site comprises A.beta. peptides or fragments theerof. Particular preferred is GGC-NH2. In another embodiment, the amino acid linker is an Immunoglobulin (Ig) hinge region. Fragments of Ig hinge regions are also within the scope of the invention, as well as Ig hinge regions modified with glycine residues. Preferably, the Ig hinge regions contain only one cysteine residue. It is to be understood, that the single cysteine residue of the Ig hinge region amino acid linker can be located at several positions within the linker sequence, and a man skilled in the art would know how to select them with the guidance of the teachings of this invention.

In one embodiment, the invention provides the coupling of almost any antigen of choice to the surface of a virus, bacterial pilus, structure formed from bacterial pilin, bacteriophage, virus-like particle or viral capsid particle. By bringing an antigen into a quasi-crystalline `virus-ike` structure, the invention exploits the strong antiviral immune reaction of a host for the production of a highly efficient immune response, i.e., a vaccination, against the displayed antigen.

In yet another embodiment, the antigen may be selected from the group consisting of: (1) a protein suited to induce an immune response against cancer cells; (2) a protein suited to induce an immune response against infectious diseases; (3) a protein suited to induce an immune response against allergens; (4) a protein suited to induce an improved response against self-antigens; and (5) a protein suited to induce an immune response in farm animals or pets. In another embodiment, the first attachment site and/or the second attachment site are selected from the group comprising: (1) a genetically engineered lysine residue and (2) a genetically engineered cysteine residue, two residues that may be chemically linked together.

In a yet further preferred embodiment, first attachment site comprises or is an amino group and said second attachment site comprises or is a sulfhydryl group. Preferably, the first attachment site comprises or is a lysine residue and said second attachment site comprises or is a cysteine residue.

The invention also includes embodiments where the organizer particle has only a single first attachment site and the antigen or antigenic determinant has only a single second attachment site. Thus, when an ordered and repetitive antigen array is prepared using such embodiments, each organizer will be bound to a single antigen or antigenic determinant.

In a further aspect, the invention provides compositions comprising, or alternatively consisting of, (a) a non-natural molecular scaffold comprising (i) a core particle selected from the group consisting of a core particle of non-natural origin and a core particle of natural origin, and (ii) an organizer comprising at least one first attachment site, wherein the core particle comprises, or alternatively consists of, a virus-like particle, a bacterial pilus, a pilus-like structure, or a modified HBcAg, or fragment thereof, and wherein the organizer is connected to the core particle by at least one covalent bond, and (b) an antigen or antigenic determinant with at least one second attachment site, the second attachment site being selected from the group consisting of (i) an attachment site not naturally occurring with the antigen or antigenic determinant and (ii) an attachment site naturally occurring with the antigen or antigenic determinant, wherein the second attachment site is capable of association through at least one non-peptide bond to the first attachment site, and wherein the antigen or antigenic determinant and the scaffold interact through the association to form an ordered and repetitive antigen array.

Other embodiments of the invention include processes for the production of compositions of the invention and a methods of medical treatment using vaccine compositions described herein.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed.

In a still further aspect, the present invention provides a composition comprising a bacteriophage Q.beta. coat protein attached by a covalent bond to phospholipase A.sub.2 protein, or a fragment thereof. In a preferred embodiment, the phospholipase A.sub.2 protein, or a fragment thereof, and the bacteriophage Q.beta. coat protein interact through the covalent bond to form an ordered and repetitive antigen array. In another preferred embodiment, the covalent bond is not a peptide bond. In another preferred embodiment, the phospholipase A.sub.2 protein includes an amino acid selected from the group consisting of the amino acid sequence of SEQ ID NO:168, the amino acid sequence of SEQ ID NO:169, the amino acid sequence of SEQ ID NO:170, the amino acid sequence of SEQ ID NO:171, the amino acid sequence of SEQ ID NO:172, the amino acid sequence of SEQ ID NO:173, the amino acid sequence of SEQ ID NO:174, and the amino acid sequence of SEQ ID NO:175.

The present invention also provides a method of making the composition comprising combining the bacteriophage Q.beta. coat protein and the phospholipase A.sub.2 protein, wherein the bacteriophage Q.beta. coat protein and the phospholipase A.sub.2 protein interact to form an antigen array.

In another aspect, the present invention also provides a composition comprising a non-natural molecular scaffold comprising a bacteriophage Q.beta. coat protein, and an organizer comprising at least one first attachment site, wherein the organizer is connected to the bacteriophage Q.beta. coat protein by at least one covalent bond; and phospholipase A.sub.2 protein, or a fragment thereof, or a variant thereof with at least one second attachment site, the second attachment site being selected from the group consisting of: an attachment site not naturally occurring with the a phospholipase A.sub.2 protein, or a fragment thereof; and an attachment site naturally occurring with the a phospholipase A.sub.2 protein, or a fragment thereof, wherein the second attachment site associates through at least one non-peptide bond to the first attachment site, and wherein the antigen or antigenic determinant and the scaffold interact through the association to form an ordered and repetitive antigen array. In a preferred embodiment, the phospholipase A.sub.2 protein includes an amino acid selected from the group consisting of the amino acid sequence of SEQ ID NO:168, the amino acid sequence of SEQ ID NO:169, the amino acid sequence of SEQ ID NO:170, the amino acid sequence of SEQ ID NO:171, the amino acid sequence of SEQ ID NO:172, the amino acid sequence of SEQ ID NO:173, the amino acid sequence of SEQ ID NO:174, and the amino acid sequence of SEQ ID NO:175.

The present invention also provides a method of making the composition comprising combining the bacteriophage Q.beta. coat protein and the phospholipase A.sub.2 protein, wherein the bacteriophage Q.beta. coat protein and the phospholipase A.sub.2 protein interact to form an antigen array. Preferably, the antigen array is ordered and/or repetitive.

The present invention also provides a pharmaceutical composition comprising a phospholipase A.sub.2 protein, and a pharmaceutically acceptable carrier. The present invention also provides a vaccine composition comprising a phospholipase A.sub.2 protein. In a preferred embodiment, the vaccine composition of claim 31, further comprising at least one adjuvant.

The present invention also provides a method of treating an allergy to bee venom, comprising administering the pharmaceutical composition or the vaccine composition to a subject. As a result of such administration the subject exhibits a decreased immune response to the venom.

The invention also relates to a vaccine for the prevention of prion-mediated diseases by induction of anti-lymphotoxin.beta., anti-lymphotoxin.alpha.: or anti-lymphotoxin.beta.-receptor antibodies. The vaccine contains protein carries foreign to the immunized human or animal coupled to lymphotoxin.beta. or fragments thereof, lymphotoxinc.alpha. or fragments thereof or the lymphotoxin.beta. receptor or fragments thereof. The vaccine is injected in humans or animals in order to induce antibodies specific for endogenous lymphotoxin.beta., lymphotoxin.beta. or lymphotoxin.beta. receptor. These induced anti-lymphotoxin.beta., lymphotoxin.alpha. or anti-tymphotoxin.beta. receptor antibodies reduce or eliminate the pool of follicular dendritic cells present in lymphoid organs. Since prion-replication in lymphoid organs and transport to the central nervous system are impaired in the absence of follicular dendritic cells, this treatment inhibits progression of prion-mediated disease. In addition, blocking lymphotoxin.beta. is beneficial for patients with autoimmune diseases such as diabetes type I.
 

Claim 1 of 52 Claims

1. A composition comprising: (a) a non-natural molecular scaffold comprising: (i) a core particle that is a virus-like particle of an RNA bacteriophage; and (ii) an organizer comprising at least one first attachment site, wherein said organizer is connected to said core particle by at least one covalent bond; (b) an antigen or antigenic determinant with at least one second attachment site, wherein said antigen or antigenic determinant is amyloid beta peptide (A.beta..sub.1-42) or a fragment thereof, and wherein said second attachment site is selected from the group consisting of: (i) an attachment site not naturally occurring with said antigen or antigenic determinant; and (ii) an attachment site naturally occurring with said antigen or antigenic determinant, wherein said second attachment site associates with said first attachment site through at least one non-peptide bond; and wherein said antigen or antigenic determinant and said non-natural molecular scaffold interact through said association to form an ordered and repetitive antigen array.

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