The present invention provides a recombinant immunogenic obtained by tandem multimerization of a B-cell epitope bearing fragment of A.beta.42, within the active loop site of a carrier (display site), preferably bacterial thioredoxin (Yrx). Polypeptides bearing multiple copies of A.beta.42 fragments, preferably with an interposed amino acid linker, were constructed and injected into mice in combination with an adjuvant. Elicited antibodies were found to selectively bind to fibrillar and/or oligomers A.beta. within neuritic AD plaques.

Description of the Invention

FIELD OF THE INVENTION

The present invention relates to immunogenic constructs comprising a fragment of A.beta.42 and a carrier characterized in that said fragment is positioned within the active loop site (display site) of the carrier, method of production and uses of the same.

BACKGROUND OF THE INVENTION

Amyloidogenic diseases such as Alzheimer's disease (AD) have been recognized as the major cause of dementia in elderly people. The decline of cognitive abilities in AD is associated with histopathological changes in the brain, the most relevant being the formation of amyloid plaques and neurofibrillary tangles.

While amyloid plaques contain many proteins, they have as their main constituent the amyloid-.beta. (A.beta.) peptide. The formation of the A.beta. peptide, and thereby A.beta. amyloid plaques, arises from aberrant processing of the amyloid precursor protein (APP).

Currently, several pharmacological approaches have being developed to slow or reverse the progression of AD. While several approaches are directed to inhibit the metabolic generation of the A.beta. peptide, others are directed to prevent the aggregation of the A.beta. amyloid in the brain of AD affected patients.

However, the most promising approaches are directed to increase the brain clearance of A.beta. plaques through the administration of either antigens able to generate an immune response against A.beta. (active immunization) or antibodies directed against A.beta. (passive immunization).

Antigens or immunogens are usually macromolecules that contain distinct antigenic sites or "epitopes" that are recognized and interact with the various components of the immune system. They usually comprise a small molecule or "hapten", such as short peptide, coupled to a suitable carrier. Carriers typically are proteins of higher molecular weight that are able to cause an immunological response when administered in vivo.

In an immune response, antibodies are produced and secreted by the B-lymphocytes in conjunction with the T-helper (TH) cells. In the majority of hapten-carrier systems, the B cells produce antibodies that are specific for both the hapten and the carrier. In these cases, the T lymphocytes will have specific binding domains on the carrier, but will not recognize the hapten alone. In a kind of synergism, the B and T cells cooperate to induce a hapten-specific antibody response.

Therefore, in constructing an effective antigen, the selection of the proper carrier and the proper hapten is crucial to guarantee a robust and selective immunogenic response. The safety of the antigen is also of crucial importance. For example, the administration to AD patients of the promising AN-1792 vaccine constituted by pre-aggregated A.beta.42 and the immune adjuvant QS-21 led to severe meningoencephalitis in about 6% of the treated subjects. Both central activation of cytotoxic T cells and autoimmune reactions were proposed as potential mechanisms of toxicity. An immunological response against endogenous monomeric A.beta. may be harmful since non-aggregated A.beta. species have a physiological role in neuronal activity.

Thus, it is of great importance the proper selection of both the hapten and the carrier to guarantee antibody selectivity towards the harmful A.beta. species and to prevent autoimmune toxicity.

WO2005058940 proposes conjugating peptide immunogen comprising A.beta. peptide or a fragment thereof to a protein/polypeptide carrier.

The immunogenic constructs are produced by a chemical method comprising derivatizing functional groups of amino acid residues of the carrier wherein any unconjugated, derivatized functional groups of the amino acid residues are inactivated via capping to block them from reacting with other molecules. Such a method results in immunogens wherein the A.beta. fragment is bound to the amino acid side chains of the carrier. While in WO2005058940 several different carriers and haptens have been proposed their in vivo histopathological efficacy has not been shown.

Kim, H. D. et al in Biochem. Biophys, Res. Commun. Volume 336, pages 84-92 propose an anti-A.beta. DNA vaccine, composed of unscaffolded 11-fold repeats of A.beta.1-6.

Such construct yielded antibodies that indiscriminately recognized monomeric, oligomeric and fibrillar A.beta.42 species.

In general, selective targeting of immunogens against the different assembly states of A.beta.42 (monomers, oligomers or fibrils) has not been achieved so far.

In view of the above considerations there is still a need to develop a safe and effective immunogenic construct which may be used in therapeuthic vaccination compositions to prevent the aggregation of A.beta. amyloid in the brain of patients affected by AD or other amyloidogenic deseases such as Down Syndrome.

The present invention provides a recombinant immunogenic construct characterized in that the A.beta. fragments is positioned within the active loop site (display site) of the carrier rather than bound to the ends of the carrier. Said peptide is obtained by tandem multimerization of a B-cell epitope bearing fragment of A.beta.42, within the active loop site (display site) of a carrier, preferably thioredoxin (Trx).

The immunogens of the present invention were found to elicit antibodies recognizing neurotoxic oligomeric species of the A.beta. amyloid which recently have been indicated as the most proximate causative agents of amyloidogenic diseases.

This capability has been associated with the construction of the immunogen featuring the A.beta. amyloid within the carrier. Such configuration to some extent permits the right folding of the immunogenic protein and more effectively presents it to the immune system. When the immunogen bears more than one A.beta. amyloid fragment, and in particular specific numbers of said fragments, the resemblance of the immunogen to the A.beta. amyloid oligomers, is believed to further improve its efficacy as well as to increase the selectivity.

A linker between the carrier and the fragments further helps in preserving the peptide epitope assembly state.

SUMMARY OF THE INVENTION

The present invention provides an immunogenic construct (also indicated hereinafter as immunogen) comprising a fragment bearing the immunodominant B-cell epitope of A.beta.42 and a carrier characterized in that said fragment is positioned within the active loop site (display site) of the carrier. The carrier is preferably thioredoxin whereas the A.beta. fragment is advantageously a N-terminal fragment of less than 30 amino acid residues, preferably less than 20 amino acid, more preferably is A.beta.1-15.

Even more preferably the immunogenic construct bears more than one fragment, preferably 2 to 16, most preferably 4 fragments.

The present invention also provides a method to construct said immunogen, the method comprising a linker assisted tandem multimerization of a B-cell epitope bearing a fragment of A.beta.42 within the display of the carrier, preferably a N-terminal fragment of less than 30 amino acid residues.

In another aspect the present invention provides a composition comprising said immunogen for active vaccination against amyloidogenic diseases.

In a further aspect the present invention provides the use of said immunogen to develop antibodies, preferably monoclonal antibodies, to be used as passive vaccine against amyloidogenic diseases.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides an immunogenic construct (or immunogen) comprising a carrier bearing at least one A.beta.42 fragment. Said fragment is positioned within a surface exposed region (active loop site or display site) of the carrier which stabilizes it conformationally.

The exact size and chemical homogeneity of the construct is routinely determined by both gel electrophoresis and mass spectrometry.

The structure of the construct may be determined by analytical techniques; however nuclear magnetic resonance (NMR) is preferably employed.

The carrier is preferably thioredoxin (Trx). Trx is particularly suitable for its small size (109 amino acids), peptide display capacity, and ability to act as a non-toxic immunoenhancer capable of stimulating murine T-cell proliferation. However other carriers may be used.

The A.beta. amyloid fragment is a N-terminal end, advantageously a N-terminal fragment having less than 30 amino acid residues, preferably less than 20 amino acid, and more preferably selected from the group consisting of A.beta.1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-15 reported in Table 1 (see Original Patent), according to the one-letter code for amino acids. Preferably, the A.beta. amyloid fragment is A.beta.1-15.

Advantageously the immunogenic construct of the invention bears more than one fragment, preferably from 2 to 16, more preferably 4 fragments.

In a preferred embodiment the fragments are bound to the carrier throughout a linker to prevent the formation of junctional epitopes. Said linker is a short amino acid sequence, preferably a linker constituted of 1 to 5 amino acid residues, more preferably Glycine-Glycine-Proline (Gly-Gly-Pro). However other linkers may be used, such as Glycine-Proline-Glycine-Proline-Glycine (Gly-Pro-Gly-Pro-Gly) (SEQ ID NO: 1), or Serine-Glycine-Serine-Glycine (Ser-Gly-Ser-Gly) (SEQ ID NO: 2).

The preferred immunogen construct consists of thioredoxin linked, optionally through a suitable linker, to four A.beta.1-15 fragments, indicated hereinafter as Trx(A.beta.1-15).sub.4.

The method to construct said immunogen is a cloning method that comprises amplifying the carrier in a suitable bacterium, inserting the carrier in a suitable vector, said vector comprising a T7 promoter for the protein expression throughout the pET system; preparing an A.beta. fragment DNA insert; restricting and ligating the carrier-vector and the A.beta. fragment DNA insert.

Preferably the A.beta. fragment DNA insert comprises an amino acid linker.

Whenever multimers are prepared an excess of A.beta. fragment DNA insert is employed.

The preferred immunogenic construct of the present invention, upon injection once-a-month for 4 months in transgenic mice in which a brain .beta.-amyloid pathology had been induced, appears to reduce the number and the size of A.beta. plaques in hippocampus and cerebral cortex. Moreover the preferred immunogenic construct of the invention was found to elicit antibodies which recognize determined species of A.beta.42.

Said antibodies upon intra-hippocampal injection are capable of clearing A.beta.42-positive plaques in hippocampus and cortex of the transgenic mice, said clearing effect being particularly evident for oligomeric A.beta. species. Said antibodies were also found to strongly improve A.beta.-associated astrogliosis (Example 2).

Accordingly, the immunogenic constructs of the present invention may form compositions for use as both active and passive vaccine against amyloidogenic diseases.

For active vaccination, a pharmaceutical composition comprising the immunogenic construct of the invention is advantageously administered in combination with an adjuvant.

The selection of an adjuvant and/or carrier depends on the stability of the vaccine containing the adjuvant, the route of administration, the dosing schedule, the efficacy of the adjuvant for the species being vaccinated, and, in humans, a pharmaceutically acceptable adjuvant is one that has been approved or is approvable for human administration by pertinent regulatory bodies. For example, Complete Freund's adjuvant is not suitable for human administration. Suitable adjuvants include 3 De-O-acylated monophosphoryl lipid A (MPL), muramyl-di-peptide and saponins such as QS21 and Quil A.

A preferred class of adjuvants is aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate. Further adjuvants include cytokines, such as interleukins (IL-1, IL-2, and IL-12), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF).

An adjuvant can be administered with the immunogen as a single composition, or can be administered before, concurrent with or after administration of the immunogen. Optionally, two or more different adjuvants can be used simultaneously.

Immunogen and adjuvant can be packaged and supplied either in the same vial or in separate vials and mixed before use.

The pharmaceutical compositions comprising the immunogenic construct of the invention may also include a variety of other pharmaceutically acceptable components. See Remington's Pharmaceutical Science (15.sup.th Ed., Mack Publishing Company, Easton, Pa., 1980).

The preferred pharmaceutical form depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.

The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.

For the parenteral administration, the immunogenic construct of the invention can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier which can be a sterile liquid such as water oils, saline, glycerol, or ethanol.

Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in the compositions.

The compositions of the invention may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.

The immunogenic construct of the invention may be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained release of the active ingredient.

Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal formulations.

For passive vaccination, the composition is injected into a mammal, such as a Guinea pig or other animal species and the resulting antibodies are purified and subsequently injected into humans.

Preferably the antibodies are monoclonal and are produced by immunizing a mammal with the Trx(A.beta.1-15).sub.4 immunogenic construct. Said antibodies are used for the prevention and treatment of amyloidogenic diseases, in particular Alzheimer's disease.
 

Claim 1 of 30 Claims

1. An immunogenic construct comprising a carrier, said carrier bearing at least one fragment of A.beta.42 within an active loop site of the carrier.

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