Disclosed are bispecific antibodies comprising a first antibody binding specificity which confers the ability of the bispecific antibody to cross the blood-brain barrier, and a second antibody specificity conferring the ability of the bispecific antibody to bind to a .beta.-amyloid epitope. Also disclosed are methods for inhibiting the formation of .beta.-amyloid plaques in the brain of a human, or promoting the disaggregation of a preformed .beta.-amyloid plaque. Such methods recite the administration of a bispecific antibody.

Description of the Invention

SUMMARY OF THE INVENTION

One aspect of the present invention is an antibody which catalyzes hydrolysis of .beta.-amyloid at a predetermined amide linkage. In one embodiment, the antibody preferentially binds a transition state analog which mimics the transition state adopted by .beta.-amyloid during hydrolysis at a predetermined amide linkage and also binds to natural .beta.-amyloid with sufficient affinity to detect using an ELISA. In another embodiment, the antibody preferentially binds a transition state analog which mimics the transition state adopted by .beta.-amyloid during hydrolysis at a predetermined amide linkage, and does not bind natural .beta.-amyloid with sufficient affinity to detect using an ELISA. Antibodies generated are characterized by the amide linkage which they hydrolyze. Specific antibodies include those which catalyze the hydrolysis at the amyloid linkages between residues 39 and 40, 40 and 41, and 41 and 42, of .beta.-amyloid.

Another aspect of the present invention is a vectorized antibody which is characterized by the ability to cross the blood brain barrier and is also characterized by the ability to catalyze the hydrolysis of .beta.-amyloid at a predetermined amide linkage. In one embodiment, the vectorized antibody is a bispecific antibody. Preferably, the vectorized antibody has a first specificity for the transferrin receptor and a second specificity for a transition state adopted by .beta.-amyloid during hydrolysis. Specific vectorized antibodies include those which catalyze the hydrolysis at the amyloid linkages between residues 39 and 40, 40 and 41, and 41 and 42, of .beta.-amyloid.

Another aspect of the present invention is a method for sequestering free .beta.-amyloid in the bloodstream of an animal by intravenously administering antibodies specific for .beta.-amyloid to the animal in an amount sufficient to increase retention of .beta.-amyloid in the circulation. Therapeutic applications of this method include treating patients diagnosed with, or at risk for Alzheimer's disease.

Another aspect of the present invention is a method for sequestering free .beta.-amyloid in the bloodstream of an animal by immunizing an animal with an antigen comprised of an epitope which is present on .beta.-amyloid endogenous to the animal under conditions appropriate for the generation of antibodies which bind endogenous .beta.-amyloid. Therapeutic applications of this method include treating patients diagnosed with, or at risk for Alzheimer's disease.

Another aspect of the present invention is a method for reducing levels of .beta.-amyloid in the brain of an animal by intravenously administering antibodies specific for endogenous .beta.-amyloid to the animal in an amount sufficient to increase retention of .beta.-amyloid in the circulation of the animal. In one embodiment, the antibodies are catalytic antibodies which catalyze hydrolysis of .beta.-amyloid at a predetermined amide linkage. The antibodies may be either monoclonal or polyclonal. In one embodiment, the antibodies specifically recognize epitopes on the C-terminus of .beta.-amyloid.sub.1-43.

Another aspect of the present invention is a method for reducing levels of .beta.-amyloid in the brain of an animal, by immunizing the animal with an antigen comprised of an epitope which is present on endogenous .beta.-amyloid under conditions appropriate for the generation of antibodies which bind endogenous .beta.-amyloid. In one embodiment, the antigen is a transition state analog which mimics the transition state adopted by .beta.-amyloid during hydrolysis at a predetermined amide linkage. In a preferred embodiment, the antigen is comprised of A.beta..sub.10-25. Preferably, the antibodies generated have a higher affinity for the transition state analog than for natural .beta.-amyloid, and catalyze hydrolysis of endogenous .beta.-amyloid.

Similar methods which utilize or generate antibodies which catalyze the hydrolysis of .beta.-amyloid for reducing levels of circulating .beta.-amyloid in an animal, and also for preventing the formation of amyloid plaques in the brain of an animal, are also provided. Also, methods for disaggregating amyloid plaques present in the brain of an animal by utilizing or generating antibodies which catalyze the hydrolysis of .beta.-amyloid are provided.

Another aspect of the present invention is a method for disaggregating amyloid plaques present in the brain of an animal by intravenously administering vectorized bispecific antibodies to the animal in an amount sufficient to cause significant reduction in .beta.-amyloid levels in the brain of the animal. The vectorized bispecific antibodies are competent to transcytose across the blood brain barrier, and have the ability to catalyze hydrolysis of endogenous .beta.-amyloid at a predetermined amide linkage upon binding. Preferably, the vectorized bispecific antibodies specifically bind the transferrin receptor.

Another aspect of the present invention is a method for generating antibodies which catalyze hydrolysis of a protein or polypeptide by immunizing an animal with an antigen comprised of an epitope which has a statine analog which mimics the conformation of a predetermined hydrolysis transition state of the polypeptide, under conditions appropriate for the generation of antibodies to the hydrolysis transition state. This method can be used to generate catalytic antibodies to .beta.-amyloid. A similar method, which utilizes reduced peptide bond analogs to mimic the conformation of a hydrolysis transition state of a polypeptide, is also provided.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to immunologically based methods for controlling levels of .beta.-amyloid in the body of an animal. The invention is based on the finding that antibodies specific for .beta.-amyloid are able to bind .beta.-amyloid in the presence of a physiological level of human serum albumin. The invention is also based on the finding that an animal can tolerate the presence of antibodies specific for .beta.-amyloid in amounts sufficient to sequester .beta.-amyloid in the bloodstream.

One aspect of the present invention relates to a method for sequestering free .beta.-amyloid in the bloodstream of an animal. The soluble and insoluble forms of .beta.-amyloid present within an animal are in dynamic equilibrium. Soluble .beta.-amyloid is thought to translocate between blood and cerebrospinal fluid. Insoluble .beta.-amyloid aggregates deposit from the soluble pool in the brain, as amyloid plaques. Results detailed in the Exemplification section below indicate that intravenous administration of antibodies specific for .beta.-amyloid to an animal impedes the passage of soluble .beta.-amyloid out of the peripheral circulation. This occurs because the .beta.-amyloid specific antibodies, which are restricted to the peripheral circulation, bind to .beta.-amyloid and sequester it in the circulation. Such sequestration is accomplished through intravenous administration of an appropriate amount of antibodies specific for .beta.-amyloid to the animal. The amount of antibody which is sufficient to produce sequestration is dependent upon various factors (e.g., specific characteristics of the antibody to be delivered, the size, metabolism, and overall health of the animal) and are to be determined on a case by case basis.

Administered antibodies can be monoclonal antibodies, a mixture of different monoclonal antibodies, polyclonal antibodies, or any combination therein. In one embodiment, the antibodies bind to the C-terminal region of .beta.-amyloid. Such antibodies specifically bind the less abundant, but more noxious A.beta..sub.1-43 species in the blood as opposed to the smaller and less detrimental A.beta..sub.1-40. In another embodiment, a combination of antibodies having specificity for various regions of .beta.-amyloid are administered. In another embodiment, antibodies which catalyze the hydrolysis of .beta.-amyloid, discussed in more detail below, are administered either alone or in combination with other anti-.beta.-amyloid antibodies.

The animal to which the antibodies are administered is any animal which has circulating soluble .beta.-amyloid. In one embodiment, the animal is a human. The human may be a healthy individual, or alternatively, may be suffering from or at risk for a disease in which elevated .beta.-amyloid levels are thought to play a role, for example a neurodegenerative disease such as Alzheimer's disease.

A related aspect of the present invention is a method for sequestering free .beta.-amyloid in the bloodstream of an animal by stimulating an immune response within the animal to endogenous .beta.-amyloid. The results detailed in the Exemplification below indicate that an animal can tolerate the induction of an immune response which produces antibodies to endogenous .beta.-amyloid, and that the presence of such antibodies will alter the distribution of .beta.-amyloid in the body, in a similar manner as the above described method of administering .beta.-amyloid binding antibodies. The immune response to endogenous .beta.-amyloid is generated by immunizing the animal with one or more antigens comprised of epitopes present on the endogenous .beta.-amyloid. Epitopes present on the inoculated antigens can correspond to epitopes present on any region of the .beta.-amyloid molecule. In a preferred embodiment, epitopes found on the C-terminal region of .beta.-amyloid are used to generate antibodies which specifically bind the A.beta..sub.1-43 species as opposed to the smaller A.beta..sub.1-40. In an alternate embodiment, a combination of different epitopes are administered to generate a variety of antibodies to .beta.-amyloid. A more generalized immune response is generated by immunizing either with a mixture of different small peptide antigens or with the full-length 43 residue .beta.-amyloid peptide. In another embodiment, antigens used for inoculation include transition state analogs of .beta.-amyloid peptides to induce antibodies which have catalytic activity directed towards .beta.-amyloid hydrolysis, described in detail below.

The immunoreactivity of the antigens can be enhanced by a variety of methods, many of which involve coupling the antigen to an immunogenic carrier. In addition, various methods are known and available to one of skill in the art for specifically enhancing the immunogenicity of endogenous molecules or the epitopes contained therein. Various modifications can be made to the .beta.-amyloid antigen(s) described herein to render it more compatible for human use. For example, the peptide(s), can be genetically engineered into appropriate antigenic carriers, or DNA vaccines can be designed.

The above techniques for sequestering .beta.-amyloid in the circulation are also useful for reducing the levels of .beta.-amyloid in the brain. Because the formation of amyloid plaques in the brain is dependent, at least in part, on the levels of free .beta.-amyloid present in the brain, reducing brain .beta.-amyloid levels of an animal will, in turn, reduce the formation of amyloid plaques in the brain. Therefore, the above techniques are useful for preventing the formation of amyloid plaques in the brain of an animal. This is especially applicable to an animal which is considered at risk for the development of amyloid plaques; a risk which may result from a genetic predisposition or from environmental factors. Administration of antibodies, or immunization of the animal to produce endogenous antibodies, to .beta.-amyloid can be of therapeutic benefit to such an animal (e.g., a human who has a family history of Alzheimer's disease, or who is diagnosed with the disease).

Another aspect of the present invention relates to antibodies which are characterized by the ability to catalyze the hydrolysis of .beta.-amyloid at a predetermined amide linkage. Experiments detailed in the Exemplification section demonstrate the generation of different antibodies which have proteolytic activity towards .beta.-amyloid. Such antibodies are generated by immunizing an animal with an antigen which is a transition state analog of the .beta.-amyloid peptide. A transition state analog mimics the transition state that .beta.-amyloid adopts during hydrolysis of a predetermined amide linkage. Transition state analogs useful for generating the catalytic antibodies include, without limitation, statine, phenylalanine statine, phosphonate, phosphonamidate, and reduced peptide bond transition state analogs.

Antibodies generated to epitopes unique to the transition state preferentially bind .beta.-amyloid in the transition state. Binding of these antibodies stabilizes the transition state, which leads to hydrolysis of the corresponding amide bond. The particular amide linkage to be hydrolyzed is chosen based upon the desired cleavage product. For example, cleavage of full length .beta.-amyloid into two peptide fragments which cannot aggregate into amyloid plaques would be of therapeutic use in the methods disclosed herein. Antibodies may be either monoclonal or polyclonal. Several such transition state mimics have been made and used as antigen in the generation of monoclonal antibodies which catalyze the cleavage at the indicated linkage. These antigens and the antibodies generated are listed in Table 8 (see Original Patent) of the Exemplification section below. Antibodies generated to antigens which have transition state mimics incorporated at a specific amide linkage, should bind the natural hydrolysis transition states of these linkages in native .beta.-amyloid, stabilizing the transition state and catalyzing cleavage at that linkage.

At least two different classes of antibodies are generated by the above methods. The first class preferentially binds the transition state analog, and also detectably cross reacts with natural .beta.-amyloid using the ELISA detailed in the Exemplification section, to detect binding. The second class binds the transition state analog, and does not detectably cross react with natural .beta.-amyloid using the ELISA procedure detailed in the Exemplification section to detect binding. Both classes of antibodies have potential value as catalytic antibodies. The respective binding affinities of an anti-transition state antibody is likely to reflect its-activity at catalyzing hydrolysis. It is thought that in order for an antibody to have activity at catalyzing hydrolysis of a protein, it must possess at least a minimal ability to bind the natural (non-transition) state of the protein. Antibodies which retain significant binding for .beta.-amyloid, (that strongly cross react with natural .beta.-amyloid) may be more efficient at catalyzing hydrolysis due to a higher efficiency of binding the .beta.-amyloid. Once bound, these antibodies force the protein into a transition state conformation for hydrolytic cleavage. Alternatively, antibodies which only minimally cross react with natural .beta.-amyloid, although less efficient at binding native .beta.-amyloid, are likely to be more efficient at forcing the bound .beta.-amyloid into the transition state conformation for hydrolytic cleavage. It should be pointed out that failure to detect binding of the anti-transition state antibodies to natural .beta.-amyloid by the ELISA methods presented in the Exemplification herein does not necessarily reflect an inability to bind natural .beta.-amyloid sufficiently to function as a catalytic antibody. More likely, a lack of detection merely reflects the sensitivity limitations of the assay.

Antibodies which have substantial affinity for the predicted cleavage products of the native .beta.-amyloid peptide may be subject to product inhibition and might therefore exhibit low turnover. Such undesirable antibodies can be identified by secondary screening using peptides which contain epitopes of the predicted cleavage products (e.g., via ELISA).

In a preferred embodiment, the antibodies are monoclonal. Monoclonal antibodies are produced by immunizing an animal (e.g., mouse, guinea pig, or rat) with the transition state analog antigen, and subsequently producing hybridomas from the animal, by standard procedures. Hybridomas which produce the desired monoclonal antibodies are identified by screening. One example of a screening method is presented in the Exemplification section which follows. In another embodiment, the antibodies are polyclonal. Polyclonal antibodies are generated by immunizing an animal (e.g., a rabbit, chicken, or goat) with antigen and obtaining sera from the animal. Polyclonal antibodies which have the desired binding specificities can be further purified from the sera by one of skill in the art through the course of routine experimentation.

Catalytic antibodies specific for .beta.-amyloid can alternatively be generated in an individual through the use of anti-idiotype vaccines designed to elicit the production of catalytic antibodies. Such vaccines are described in the disclosure of Raso and Paulus (U.S. patent application Ser. No. 09/102,451, ANTI-IDIOTYPE VACCINES TO ELICIT CATALYTIC ANTIBODIES, filed by Applicants Jun. 22, 1998, currently pending), the contents of which are incorporated herein by reference.

Another aspect of the present invention is the use of statine and reduced peptide bond analogs to elicit catalytic antibodies having proteolytic activity. The Exemplification section below details methods for using statine analogs as antigen in the production of catalytic antibodies, and also lists examples of anti-transition-state antibodies generated using these methods. The "statyl" moiety is derived from naturally evolved protease transition state inhibitors like amastatin, pepstatin, and bestatin. These naturally-occurring statine-based inhibitors have been used to effectively block the activity of aminopeptidases, aspartic proteases and the HIV protease. Synthetic peptides containing a statine residue offer novel features for the induction of catalytic antibodies. The statyl moiety has a tetrahedral bond geometry, its length is extended by two CH.sub.2 units, it has a strategically placed OH group and the structure has no charge. The presence of the additional CH.sub.2 units is expected to elicit a more elongated antibody combining site, and antibodies possessing this extended site will induce extra strain on the peptide substrate, producing an accelerated catalysis. In addition, the --OH group in these statine analogs is thought to better approximate the position and chemistry of the true transition state. Statine-based transition-state analogs should therefore elicit a class of antibodies which is significantly different from those obtained from the more commonly used negatively charged phosphonate analogs.

Reduced peptide bond analogs introduce a tetrahedral configuration, without increasing the distance between amino acid residues. This feature should more closely approximate the true transition state geometry, than previously used analogs. A positively charged secondary amine replaces the amide nitrogen of the natural polypeptide and should elicit a complementary negatively charged side chain at a proximal locus in the antibody combining site. The presence of such ancillary glutamyl or aspartyl groups on the antibody will assist antibody-mediated catalysis of peptide cleavage via acid-base exchange. Reduced peptide bond-based transition-state analogs should therefore elicit a class of antibodies which is significantly different from those obtained from using the more commonly used negatively charged phosphonate analogs. Reduced peptide bond analogs and statine analogs can be used to produce specific transition state analog antigens for a wide variety of proteins or polypeptides. These antigens can in turn be used to generate the respective catalytic antibodies.

Administration of the .beta.-amyloid catalytic antibodies described above can be used in the methods described above for 1) sequestering free .beta.-amyloid in the bloodstream of an animal, 2) reducing levels of .beta.-amyloid in the brain of an animal, and 3) preventing the formation of amyloid plaques in the brain of an animal, to generate the analogous results. Experiments presented in the Exemplification demonstrate that immunization of an animal with a transition state analog results in the generation of an immune response to produce antibodies which recognize the transition state, and which catalyze hydrolysis of the .beta.-amyloid protein. This indicates that the transition state analogs can be used as antigens in these methods to induce the production of antibodies in the animal which recognize and catalyze cleavage of endogenous .beta.-amyloid.

Methods which involve reducing overall levels of .beta.-amyloid in an animal through the proteolytic action of the above described catalytic antibodies are also encompassed by the present invention. The presence of functional catalytic antibodies in the circulation of an animal will reduce the level of intact .beta.-amyloid in the circulation by selective hydrolytic cleavage. Accordingly, the present invention provides a method for reducing levels of circulating .beta.-amyloid in an animal by introducing the above described catalytic antibodies into the animal. Administration of the antibodies to the animal is preferably via intravenous administration. Such antibodies are either monoclonal, mixed monoclonal, polyclonal or any mixture thereof. The origin of the antibody may affect the half-life of the antibody in the animal; antibodies from less related species are more likely to be recognized as foreign by the animal's immune system. Preferably, administered antibodies are derived from a species closely related to the animal, to maximize half-life and minimize adverse reactions by the host. Administration of isolated variable region antibody fragments may produce beneficial results in this regard.

The present invention also provides a method for reducing levels of circulating .beta.-amyloid in an animal by immunizing the animal with a .beta.-amyloid transition state analog to induce endogenous catalytic antibody production. The use and design of such vaccines is described above, and detailed in the Exemplification section below.

The reduction of .beta.-amyloid levels in the circulation of an animal is expected to displace the equilibrium of .beta.-amyloid in the body, and ultimately lead to a reduction in the levels of .beta.-amyloid in the brain of the animal through mass action. In this respect, the present invention provides methods for reducing the levels of .beta.-amyloid in the brain of an animal, by either administering catalytic antibodies to the animal, or by administering a transition state analog to induce endogenous antibody production. It follows that these procedures also have value as methods for preventing the formation of amyloid plaques in the brain of an animal, since the resulting reduction in the levels of .beta.-amyloid in the brain of an animal should prevent the formation of amyloid plaques. These procedures also have value as methods for disaggregating amyloid plaques present in the brain of an animal, since evidence indicates that lower brain .beta.-amyloid levels can lead to the disaggregation of plaques.

Another aspect of the present invention provides a more direct method of altering the distribution of .beta.-amyloid in the brain by actually delivering anti-.beta.-amyloid antibodies to the brain. Methods described above for reducing levels of .beta.-amyloid in the brain and for preventing aggregation of amyloid plaques depend upon exchange between .beta.-amyloid pools in the blood, tissues, cerebrospinal fluid and the brain, the exchange being driven by an antibody-mediated disruption of the equilibrium between these different pools. In contrast, delivery of anti-.beta.-amyloid antibodies to the brain will directly affect .beta.-amyloid aggregation. Evidence presented in the Exemplification section below indicates that the binding of certain anti-.beta.-amyloid antibodies inhibits the initial aggregation of .beta.-amyloid in vitro, and also disaggregates preformed in vitro .beta.-amyloid complexes. Moreover, if insoluble peptide is in equilibrium with a low level of soluble .beta.-amyloid, then an anti-.beta.-amyloid binding antibody could upset this balance and gradually dissolve the precipitate. These observations indicate that the presence of .beta.-amyloid antibodies in the brain will directly inhibit the formation of amyloid plaques and will also disaggregate preformed plaques by disrupting the dynamic equilibrium between soluble .beta.-amyloid and fibrillar .beta.-amyloid deposited as plaques. Furthermore, a highly active catalytic antibody is expected to destroy insoluble .beta.-amyloid plaques by hydrolytically cleaving the constituent aggregated peptides.

One way of delivering antibodies to the brain is by producing vectorized antibodies competent for transcytosis across the blood-brain barrier. Vectorized antibodies are produced by covalently linking an antibody to an agent which promotes delivery from the circulation to a predetermined destination in the body. Examples of vectorized molecules which can traverse the blood-brain barrier are found in the prior art (Bickel et al., Proc. Natl. Acad. Sci. USA 90: 2618-2622 (1993); Broadwell et al., Exp. Neurol. 142: 47-65 (1996)). In these examples, antibodies are linked to another macromolecule, the antibodies being the agent which promotes delivery of the macromolecules. One example of such an agent is an antibody which is directed towards a cell surface component, such as a receptor, which is transported away from the cell surface. Examples of antibodies which confer the ability to trancytose the blood-brain barrier include, without limitation, anti-insulin receptor antibodies, and also anti-transferrin receptors (Saito et al., Proc. Natl. Acad. Sci. USA 92: 10227-31 (1995); Pardridge et al., Pharm. Res. 12: 807-816 (1995); Broadwell et al., Exp. Neurol. 142: 47-65 (1996)). This first antibody is covalently linked to an antibody which binds f-amyloid. Alternatively, coupling the .beta.-amyloid antibodies to ligands which bind these receptors (e.g., insulin, transferrin, or low density lipoprotein) will also produce a vectorized antibody competent for delivery to the brain from the circulation (Descamps et al., Am. J. Physiol. 270: H1149-H1158 (1996); Duffy et al., Brain Res. 420: 32-38 (1987); Dehouck et al., J. Cell Biol. 138: 877-889 (1997)).

A vector moiety can be chemically attached to the anti-.beta.-amyloid antibody to facilitate its delivery into the central nervous system. Alternatively, the moiety can be genetically engineered into the antibody as an integral component. This vector component can be for example, an anti-transferrin receptor antibody or anti-insulin receptor antibody which binds the receptors present on the brain capillary endothelial cells (Bickel et al., Proc. Natl. Acad. Sci. USA 90: 2618-22 (1993); Pardridge et al., J. Pharmacol. Exp. Ther. 259: 66-70 (1991); Saito et al., Proc. Natl. Acad. Sci. USA 92: 10227-31(1995); Friden et al., J. Pharm. Exper. Ther. 278: 1491-1498 (1996)) which make up the blood-brain barrier. The resulting bifunctional antibody will attach to the appropriate receptors on the luminal side of the vessel (Raso et al., J. Biol. Chem. 272: 27623-27628 (1997); Raso et al., J. Biol. Chem. 272: 27618-27622 (1997); Raso, V. Anal. Biochem. 222: 297-304 (1994); Raso et al., Cancer Res. 41: 2073-2078 (1981); Raso et al., Monoclonal antibodies as cell targeted carriers of covalently and non-covalently attached toxins. In Receptor mediated targeting of drugs, vol. 82. G. Gregoriadis, G. Post, J. Senior and A. Trouet, editors. NATO Advanced Studies Inst., New York. 119-138 (1984)). Once bound to the receptor, both components of the bispecific antibody pass across the blood-brain barrier by the process of transcytosis. Anti-.beta.-amyloid antibodies which have entered the brain interact directly with both f-amyloid plaques and the soluble .beta.-amyloid pool. It has been estimated that concentrations of macromolecules in the 10.sup.-8-10.sup.-7 M range can be achieved in the brain using vector-mediated delivery via these brain capillary enriched protein target sites (Maness et al., Life Sciences 55: 1643-1650 (1994); Lerner et al., Science 252: 659-667 (1991)). Importantly, the vector appears safe since animals dosed daily for two weeks with an anti-transferrin receptor antibody displayed no loss of integrity of the blood-brain barrier, using a radioactive sucrose probe (Broadwell et al., Exp. Neurol. 142: 47-65 (1996)).

The Exemplification details the production of vectorized bispecific antibodies which bind .beta.-amyloid. The bispecific antibodies transcytose across the blood brain barrier via a first specificity which binds the transferrin receptor. Use of antibodies which bind the transferrin receptor for delivery of agents across the blood brain barrier is described by Friden et al. in U.S. Pat. Nos. 5,182,107; 5,154,924; 5,833,988; and 5,527,527; the contents of which are incorporated herein by reference.

Results from experiments presented in the Exemplification section which follows indicate that the produced bispecific antibodies retain their separate specificities and are delivered across the blood-brain barrier into the brain parenchyma and brain capillaries of a live animal when administered intravenously.

Alternate methods for the production of bispecific antibodies have been described for genetically engineering bispecific reagents or for producing them intracellularly by fusing the two different hybridoma clones (Holliger et al., Proc. Natl. Acad. Sci. 90: 6444-6448 (1993); Milstein et al., Nature 305: 537 (1983); Mallander et al., J. Biol. Chem. 269: 199-206 (1994)). Vectorized bispecific antibodies produced by these techniques can also be used in the methods of the present invention.

Since the introduction of whole antibodies into the brain might be detrimental if they were to fix complement and promote complement-mediated lysis of neuronal cells, it may be beneficial to produce and utilize smaller vectorized F(ab').sub.2 bispecific reagents. It has been shown that aggregated .beta.-amyloid itself can fix complement in the absence of any antibody and that the resulting inflammation may contribute to the pathology of Alzheimer's disease. The possibility of intracerebral antibody having a similar effect can be greatly reduced by eliminating the Fc region of the antibody. Moreover, since coupling of Fab' halves uses the intrinsic hinge region cysteines, no extraneous substituent linkage groups need be added. Faster or more efficient entry into the brain represents another potential advantage that smaller F(ab').sub.2 or Fv.sub.2 reagents may provide for intracerebral delivery. In addition, the two types of vectorized molecules may have different biodistribution and plasma half-life characteristics (Spiegelberg et al., J. Exp. Med. 121: 323 (1965)).

Depending on their design, anti-.beta.-amyloid bispecific antibodies in the brain can reduce soluble .beta.-amyloid and f-amyloid deposits by three potential mechanisms. An anti-.beta.-amyloid bispecific antibody that tightly binds soluble .beta.-amyloid will not only sequester the peptide but, due to efflux of vectorized molecules from the central nervous system (Kang et al., J. Pharm. Exp. Ther. 269: 344-350 (1994)), may also carry the bound .beta.-amyloid out of the brain, releasing it into the blood stream. Such a clearance mechanism would lead to a continuous cycling of .beta.-amyloid out of the brain. In addition, if the antibodies have catalytic activity, they will directly reduce the levels of harmful .beta.-amyloid by degradation. Since catalytic antibodies exhibit turnover, each antibody can inactivate many .beta.-amyloid molecules. Thus much less vectorized bispecific antibody has to be delivered into the brain to achieve the desired depletion of .beta.-amyloid.

To be effective the anti-.beta.-amyloid sites of a bispecific antibody must be empty before passage out of the blood and into the brain. Therefore the concentration of bispecific antibody in animals must exceed the level of .beta.-amyloid circulating in the blood. Calculations performed based upon known .beta.-amyloid levels (Scheuner et al., Nature Med. 2: 864-870 (1996)) and a medium-range plasma level of bispecific antibody expected in a treated animal indicated 99.9% of the bispecific antibodies that enter the brain will have unoccupied anti-.beta.-amyloid combining sites.

Another way of delivering antibodies to the brain is via direct infusion of anti-.beta.-amyloid antibodies into the brain of an animal. This technique gives these antibodies immediate access to .beta.-amyloid in the brain without having to cross the blood-brain barrier. Direct infusion can be accomplished via direct parenchymal or intracerebroventricular infusion (Knopf et al., J. Immunol. 161: 692-701 (1998)). Briefly, the animal is anesthetized and placed in a stereotaxic frame. A midsagittal incision is made on the scalp to expose the skull and the underlying fascia is scraped away. A hole is drilled to accept a sterilized length of stainless steel hypodermic tubing, which is stereotaxically advanced so that its tip is appropriately located in the brain. A guide cannula is then attached to the skull and sealed. The cannula remains in place for multiple infusions of antibody into the brain. A bolus of a sterile 50 mg/ml solution of a monoclonal anti-.beta.-amyloid can be infused over a 2-8 minute period into an immobilized animal via an injection cannula.

Delivery of catalytic antibodies into the brain of an animal via one of the above described methods, can also be used to disaggregate amyloid plaques present in the brain. The advantage of delivering an .beta.-amyloid-specific catalytic antibody into the brain is two-fold. The .beta.-amyloid peptide is permanently destroyed by such antibodies and, since catalysis is continuous, each antibody inactivates many target .beta.-amyloid molecules in the brain. Thus much less antibody has to be infused into the central nervous system to achieve the desired depletion of .beta.-amyloid.

The amount of antibody to be administered or delivered to the animal should be sufficient to cause a significant reduction in .beta.-amyloid levels in the brain of the animal. The appropriate amount will depend upon various parameters (e.g., the particular antibody used, the size and metabolism of the animal, and the levels of endogenous .beta.-amyloid) and is to be determined on a case by case basis. Such determination is within the means of one of average skill in the art through no more than routine experimentation.

It is expected that additional benefits with respect to lowering brain .beta.-amyloid levels and preventing or disaggregating amyloid plaques can be achieved through utilizing a combination of one or more of the above described approaches.
 

Claim 1 of 10 Claims

1. A therapeutic antibody that specifically binds and cleaves an epitope contained within amino acid positions 10-25 of .beta.-amyloid, wherein said antibody binds soluble .beta.-amyloid and soluble .beta.-amyloid aggregates in a living organism.
 

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