Title:  Synthetic immunogenic but non-amyloidogenic peptides homologous to amyloid .beta. for induction of an immune response to amyloid .beta. and amyloid deposits

United States Patent:  6,713,450

Issued:  March 30, 2004

Inventors:  Frangione; Blas (New York, NY); Wisniewski; Thomas (Staten Island, NY); Sigurdsson; Einar M. (New York, NY)

Assignee:  New York University (New York, NY)

Appl. No.:  861847

Filed:  May 22, 2001

Abstract

The present invention relates to synthetic immunogenic but non-amyloidogenic peptides homologous to amyloid .beta. which can be used alone or conjugated to an immunostimulatory molecule in an immunizing composition for inducing an immune response to amyloid .beta. peptides and amyloid deposits.

SUMMARY OF THE INVENTION

The present invention provides a synthetic immunogenic but non-amyloidogenic peptide homologous to amyloid .beta. which can be used for induction of an immune response to amyloid .beta. peptides and amyloid deposits and would overcome or avoid the complications and problems encountered in the prior art.

The synthetic immunogenic but non-amyloidogenic peptide homologous to amyloid .beta. includes the first thirty amino acid residues of A.beta.1-42 (SEQ ID NO:1), where zero, one or two of residues 17-21 are substituted with Lys, Asp, or Glu, and preferably includes an N-terminal and/or C-terminal segment of 4-10 Lys or Asp residues.

The present invention also provides a conjugate in which the peptide is cross-linked to an immunostimulatory polymer molecule.

Another aspect of the present invention is directed to an immunizing composition/vaccine which contains an immunizing effective amount of the synthetic non-amyloidogenic but immunogenic peptide homologous to amyloid .beta., or a conjugate thereof.

A further aspect of the present invention is directed to a method for immunotherapy to induce an immune response to amyloid .beta. peptides and amyloid deposits.

A still further aspect of the invention is directed to molecules which include the antigen-binding portion of an antibody raised against the synthetic non-amyloidogenic but immunogenic peptide according to the present invention. Also provided are pharmaceutical compositions containing this peptide-binding molecule and a method for reducing the formation of amyloid fibrils and deposits.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have designed synthetic non-amyloidogenic peptides homologous to amyloid .beta. (A.beta.) which have not only a reduced ability to adopt a .beta.-sheet conformation as an antigenic source but also would have a much lower risk of leading to any toxic effects in humans. By using these synthetic non-amyloidogenic peptides, or conjugates thereof, in an immunizing composition, the present invention provides a means for rendering A.beta. peptides and amyloid deposits as targets for the immune system. An important object of the present invention is therefore to provide a method for immunization which minimizes the toxicity associated with injected A.beta. peptides while maximizing the immune response to A.beta. peptides and amyloid deposits.

The synthetic non-amyloidogenic but immunogenic peptides homologous to A.beta. according to the present invention are designed to have reduced fibrillogenic potential while maintaining the two major immunogenic sites of A.beta. peptides, which are residues 1-11 and 22-28 of A.beta.1-42 based on the antigenic index of Jameson et al. (1988) and results/observations obtained in the laboratory of the present inventors. Accordingly, the present inventors have based the design of the synthetic non-amyloidogenic peptide on the first thirty amino acid residues (SEQ ID NO:1) of A.beta.1-42, where one or two of the hydrophobic residues at positions 17-21 of SEQ ID NO:1 are substituted with charged residues Lys, Asp, or Glu. The first thirty residues of A.beta. lack the hydrophobic C-terminus of A.beta.1-42 but retains the two immunogenic sites corresponding to residues 1-11 and 22-28 of SEQ ID NO:1.

By modifying one or two residues at positions 17-21 of A.beta.1-30 (SEQ ID NO:1) with Lys, Asp, or Glu, which are hydrophilic residues that have a low probability of adopting .beta.-sheet conformation, the fibrillogenic potential of the peptide is greatly reduced. SEQ ID NOs: 12 and 13 are examples of such modified A.beta.1-30. Furthermore, the presence of a series of Lys or Asp residues at the N-terminus and/or C-terminus of the synthetic peptide of the present invention would further enhance immunogenicity (Werdelin, 1981) and reduce the propensity of the synthetic peptide to adopt a .beta.-sheet conformation and form amyloid fibrils/deposits. The coupling of lysine residues to A.beta. peptides of 4 to 8 residues in length has recently been proposed by Pallitto et al. (1999) in the design of anti-.beta.-sheet peptides or A.beta. fibrillogenesis inhibitors, but the use of Pallitto's peptides as immunogens has never been proposed. Polycationic amino acids have been previously used to enhance protein transport into cells by endocytosis/phagocytosis processes (Martinez-Fong et al., 1994; Wang et al., 1989; Shen et al., 1985; Peterson et al., 1984; Deierkauf et al., 1977; DiNicola et al., 2000). Buschle et al., (1997) reported that polycationic amino acids enhanced uptake of peptides by antigen presently cells, thereby initiating an immune response. They also reported that, whereas peptide uptake mediated by polylysine appears to be due to an at least transient permeabilization of cell membranes, peptide delivery in the presence of polyarginine may rely on endocytic processes.

The synthetic immunogenic but non-amyloidogenic peptide homologous to A.beta. according to the present invention, which is not considered to be a peptide inhibitor of A.beta. fibrillogenesis, is represented by the formula

(A)_m -(N-Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ -C)_n -(B)_p

wherein:

m is 0, 4, 5, 6, 7, 8, 9, or 10;

p is 0, 4, 5, 6, 7, 8, 9, or 10;

A is Lys or Asp;

B is Lys or Asp;

n is 1 or 2;

N is residues 1-16 of SEQ ID NO:1;

C is residues 22-30 of SEQ ID NO:1;

Xaa₁ Xaa₂, Xaa₃, Xaa₄, and Xaa₅ are Leu, Val, Phe, Phe, and Ala, respectively, in which zero, one or two of residues Xaa₁, Xaa₂ Xaa₃, Xaa₄, and Xaa₅ is substituted with Lys, Asp, or Glu; and

when zero residues are substituted, then either or both of m or p is not zero.

The amino acid sequences of the peptide represented by the above formula are presented and identified as SEQ ID NOs:2-5.

The basic thirty amino acid sequence (A.beta.1-30) in which zero, one or two of residues 17-21 are substituted is represented in the above formula by N-Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ -C (SEQ ID NO:15). This thirty amino acid residue segment can be repeated (n is 2) in the synthetic peptide according to the present invention. Preferably, a polylysine or polyaspartate segment of 4 to 10 residues is present at the N-terminus and/or the C-terminus of the peptide. When no residues are substituted in residues 17-21 of A.beta.1-30, the peptide has a polylysine or polyaspartate segment of 4 to 10 residues at the N-terminus and/or C-terminus. If a polylysine or polyaspartate segment is not present at the C-terminus, then the C-terminus is preferably amidated, as exemplified by SEQ ID NO:6 as a preferred embodiment. SEQ ID NO:11 is an embodiment of an unsubstituted A.beta.1-30 peptide with a polylysine or polyaspartate segment of 4 to 10 residues at the C-terminus.

Furthermore, when m is 0, the N-terminal polylysine or polyaspartate segment of 4 to 10 residues is absent, and it is then preferred that either the C-terminus of the peptide be amidated to reduce the possibility that the C-terminal charge of the peptide would reduce the immunogenicity of the residue 22-28 region of A.beta. or that a polylysine or polyaspartate segment of 4 to 10 residue be present at the C-terminus. Another preferred embodiment of the peptide according to the present invention is as follows:

when m is not zero, p is zero;

when p is not zero, m is zero; and

Xaa₁, Xaa₂, Xaa₃, Xaa₄, and Xaa₅ are Leu, Val, Phe, Phe, and Ala, respectively, in which one or two residues Xaa₁, Xaa₂, Xaa₃, Xaa₄, and Xaa₅ is substituted with Lys, Asp, or Glu (SEQ ID NOs:2-5).

Those of skill in the art will also appreciate that peptidomimetics of the synthetic peptide of the present invention, where the peptide bonds are replaced with non-peptide bonds, can also be used.

As is well-known in the art, the reduced fibrillogenic potential for the synthetic peptides according to the present invention can be readily determined by measuring the .beta.-sheet conformation of the peptides using conventional techniques such as circular dichroism spectra, FT-IR, and electron microscopy of peptide suspensions.

It is also well-known that immunogens must be presented in conjunction with major histocompatibility (MHC) class II antigens to evoke an efficient antibody response. The MHC class II antigens produced by antigen-presenting cells (APCs) bind to T cell epitopes present in the immunogen in a sequence specific manner. This MHC class II-immunogen complex is recognized by CD4⁺ lymphocytes (T_h cells), which cause the proliferation of specific B cells capable of recognizing a B cell epitope from the presented immunogen and the production of B cell epitope-specific antibodies by such B cells. An additional approach to further increase immunogenicity of the synthetic peptides of the present invention is to form a conjugate with an immunostimulatory polymer molecule such as mannan (polymer of mannose), glucan (polymer of .beta.1-2 glucose), tripalmitoyl-S-glycerine cysteine, and peptides which are currently approved for use in vaccines in humans. Such peptides approved for use in vaccines provide strong T helper cell (T_h) epitopes from potent immunogens such as tetanus toxin, pertussis toxin, the measles virus F protein, and the hepatitis B virus surface antigen (HBsAg). The T_h epitopes selected to be conjugated to the synthetic peptide are preferably capable of eliciting T helper cell responses in large numbers of individuals expressing diverse MHC haplotypes. These epitopes function in many different individuals of a heterogeneous population and are considered to be promiscuous T_h epitopes. Promiscuous T_h epitopes provide an advantage of eliciting potent antibody responses in most members of genetically diverse population groups.

Moreover, the T helper cell epitopes conjugated/cross-linked to the synthetic peptide of the present invention are also advantageously selected not only for a capacity to cause immune responses in most members of a given population, but also for a capacity to cause memory/recall responses. When the mammal is human, the vast majority of human subjects/patients receiving immunotherapy with the synthetic peptide of the present invention will most likely already have been immunized with the pediatric vaccines (i.e., measles+mumps+rubella and diphtheria+pertussis+tetanus vaccines) and, possibly, the hepatitis B virus vaccine. These patients have therefore been previously exposed to at least one of the T_h epitopes present in pediatric vaccines. Prior exposure to a T_h epitope through immunization with the standard vaccines should establish T_h cell clones which can immediately proliferate upon administration of the synthetic peptide (i.e., a recall response), thereby stimulating rapid B cell responses to A.beta. peptides and amyloid deposits.

While the T_h epitopes that may be used in the conjugate with the synthetic peptide of the invention are promiscuous, they are not universal. This characteristic means that the T_h epitopes are reactive in a large segment of an outbred population expressing different MHC antigens (reactive in 50 to 90% of the population), but not in all members of that population. To provide a comprehensive, approaching universal, immune reactivity for the synthetic non-amyloidogenic peptide according to the present invention, a mixture of conjugates with different T_h epitopes cross-linked to a synthetic peptide can be prepared. For example, a combination of four conjugates with promiscuous T_h epitopes from tetanus and pertussis toxins, measles virus F protein and HBsAg may be more effective.

The T_h epitopes in the immunostimulatory peptide cross-linked to the synthetic non-amyloidogenic peptide according to the present invention include hepatitis B surface antigen T helper cell epitopes, pertussis toxin T helper cell epitopes, tetanus toxin T helper cell epitopes, measles virus F protein T helper cell epitope, Chlamydia trachomitis major outer membrane protein T helper cell epitopes, diphtheria toxin T helper cell epitopes, Plasmodium falciparum circumsporozoite T helper cell epitopes, Schistosoma mansoni triose phosphate isomerase T helper cell epitopes, Escherichia coli TraT T helper cell epitopes and are disclosed in U.S. Pat. No. 5,843,446, the entire disclosure of which is incorporated herein by reference.

It will be appreciated by those of skill in the art that the term "synthetic" as used with the peptide of the present invention means that it is either chemically synthesized or is produced in an organism only when the host organism is genetically transformed from its native state to produce the peptide. The synthetic peptides of the present invention can be made by synthetic chemical methods which are well known to the ordinary skilled artisan. Accordingly, the synthetic peptides can be synthesized using the automated Merrifield techniques of solid phase synthesis with either t-Boc or F-moc chemistry on Peptide Synthesizers such as an Applied Biosystems Peptide Synthesizer.

Alternatively, longer peptides can be synthesized by well-known recombinant DNA techniques. Any standard manual on DNA technology provides detailed protocols to produce the synthetic peptides of the invention. To construct a nucleotide sequence encoding a synthetic peptide of the present invention, the amino acid sequence is reverse transcribed into a nucleic acid sequence, and preferably using optimized codon usage for the organism in which the peptide will be expressed. Next, a synthetic gene is made, typically by synthesizing overlapping oligonucleotides which encode the peptide and any regulatory elements, if necessary. The synthetic gene is inserted in a suitable cloning vector and recombinant clones are obtained and characterized. The synthetic peptide of the present invention is then expressed under suitable conditions appropriate for the selected expression system and host, and the desired peptide is purified and characterized by standard methods.

An immunostimulatory peptide that can be cross-linked to the synthetic non-amyloidogenic peptide of the invention is also obtainable from the invasin protein of a Yersinia species. The invasins of the pathogenic bacteria Yersinia spp. are outer membrane proteins which mediate entry of the bacteria into mammalian cells (Isberg et al., 1990). Invasion of cultured mammalian cells by the bacterium was demonstrated to require interaction between the Yersinia invasin molecule and several species of the .beta.1 family of integrins present on the cultured cells (Tran Van Nhieu et al., 1991) Since T lymphocytes are rich in .beta.1 integrins (especially activated immune or memory T cells) the effects of invasin on human T cell have been investigated (Brett et al., 1993). It is thought that integrins facilitate the migration of immune T cells out of the blood vessels and through connective tissues to sites of antigenic challenge through their interaction with extracellular matrix proteins including fibronectin, laminin and collagen. The carboxy-terminus of the invasin molecule was found to be co-stimulatory for naive human CD4₊ T in the presence of the non-specific mitogen, anti-CD3 antibody, causing marked proliferation and expression of cytokines. The specific invasin domain which interacts with the .beta.1 integrins to cause this stimulation also was identified (Brett et al., 1993). Because of the demonstrated T cell co-stimulatory properties associated with this domain, it can be cross-linked to the synthetic peptide of the present invention to enhance immunogenicity.

Many of the outer membrane proteins of Gram-negative bacteria are both lipid-modified and very immunogenic. Because of the apparent correlation between covalent lipid linkage and immunogenicity, tripalmitoyl-S-glycerine cysteine (Pam₃ Cys), a lipid common to bacterial membrane proteins, can be coupled to the synthetic peptides in a conjugate to also enhance immunogenicity.

Immunogenicity can further be significantly improved if the synthetic peptides are co-administered with adjuvants. Adjuvants enhance the immunogenicity of an antigen but are not necessarily immunogenic themselves. Adjuvants may act by retaining the antigen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system. Adjuvants can also attract cells of the immune system to an antigen depot and stimulate such cells to elicit immune responses.

Immunostimulatory agents or adjuvants have been used for many years to improve the host immune responses, e.g. to vaccines. Intrinsic adjuvants, such as lipopolysaccharides, normally are the components of the killed or attenuated bacteria used as vaccines. Extrinsic adjuvants are immunomodulators which are typically non-covalently linked to antigens and are formulated to enhance the host immune responses. Thus, adjuvants have been identified that enhance the immune response to antigens delivered parenterally. Some of these adjuvants are toxic, however, and can cause undesirable side-effects, making them unsuitable for use in humans and many animals. Indeed, only aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established and a HBsAg vaccine has been adjuvanted with alum as well.

A wide range of extrinsic adjuvants can provoke potent immune responses to antigens. These include saponins complexed to membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, killed mycobacteria in mineral oil, Freund's complete adjuvant, bacterial products, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A, and liposomes. To efficiently induce humoral immune responses (HIR) and cell-mediated-immunity (CMI), immunogens are emulsified in adjuvants. Many adjuvants are toxic, inducing granulomas, acute and chronic inflammations (Freund's complete adjuvant, FCA), cytolysis (saponins and Pluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPS and MDP). Although FCA is an excellent adjuvant and widely used in research, it is not licensed for use in human or veterinary vaccines because of its toxicity.

U.S. Pat. No. 4,855,283 teaches glycolipid analogues including N-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each of which is substituted in the sugar residue by an amino acid, as immuno-modulators or adjuvants. U.S. Pat. No. 4,258,029 teaches that octadecyl tyrosine hydrochloride (OTH) functions as an adjuvant when complexed with tetanus toxoid and formalin inactivated type I, II and III poliomyelitis virus vaccine. Also, Nixon-George et al., 1990, reported that octadecyl esters of aromatic amino acids complexed with a recombinant hepatitis B surface antigen enhanced the host immune responses against hepatitis B virus.

The addition of exogenous adjuvant/emulsion formulations which maximize immune responses to A.beta. peptides and amyloid deposits are preferred. The adjuvants and carriers that are suitable are those: (1) which have been successfully used in Phase I human trials; (2) based upon their lack of reactogenicity in preclinical safety studies, have potential for approval for use in humans; or (3) have been approved for use in food and companion animals. Some of the adjuvants that are currently undergoing clinical tests are reported in Aguado et al., (1999).

Immunotherapy regimens which produce maximal immune responses following the administration of the fewest number of doses, ideally only one dose, are highly desirable. This result can be approached through entrapment of immunogen in microparticles. For example, the absorbable suture material poly(lactide-co-glycolide) co-polymer can be fashioned into microparticles containing immunogen. Following oral or parenteral administration, microparticle hydrolysis in vivo produces the non-toxic byproducts, lactic and glycolic acids, and releases immunogen largely unaltered by the entrapment process. The rate of microparticle degradation and the release of entrapped immunogen can be controlled by several parameters, which include (1) the ratio of polymers used in particle formation (particles with higher co-glycolide concentrations degrade more rapidly); (2) particle size, (smaller particles degrade more rapidly than larger ones); and, (3) entrapment efficiency, (particles with higher concentrations of entrapped antigen degrade more rapidly than particle with lower loads). Microparticle formulations can also provide primary and subsequent booster immunizations in a single administration by mixing immunogen entrapped microparticles with different release rates. Single dose formulations capable of releasing antigen ranging from less than one week to greater than six months can be readily achieved. Moreover, delivery of the synthetic peptide according to the present invention entrapped in microparticles can also provide improved efficacy when the microparticulate immunogen is mixed with an exogenous adjuvant/emulsion formulations.

The efficacy of the synthetic peptides can be established and analyzed by injecting an animal, e.g., mice or rats, with the synthetic peptide formulated in alum and then following the immune response to amyloid .beta. peptides.

Another aspect of the present invention provides an immunizing composition which includes an immunizing effective amount of one or more of the synthetic peptides of the invention, or conjugates thereof, and a pharmaceutically acceptable carrier, excipient, diluent, or auxiliary agent, including adjuvants. Accordingly, the synthetic peptides, or conjugates thereof, can be formulated as an immunizing composition using adjuvants, pharmaceutically-acceptable carriers, excipients, diluents, auxiliary agents or other ingredients routinely provided in immunizing compositions. Such formulations are readily determined by one of ordinary skill in the art and include formulations for immediate release and for sustained release, e.g., microencapsulation. The present immunizing compositions can be administered by any convenient route including subcutaneous, oral, intramuscular, or other parenteral or internal route. Similarly the vaccines can be administered as a single dose or divided into multiple doses for administration. Immunization schedules are readily determined by the ordinary skilled artisan. For example, the adjuvants or emulsifiers that can be used in this invention include alum, incomplete Freund's adjuvant, liposyn, saponin, squalene, L121, emulsigen and ISA720. In preferred embodiments, the adjuvants/emulsifiers are alum, incomplete Freund's adjuvant, a combination of liposyn and saponin, a combination of squalene and L121 or a combination of emulsigen and saponin.

The immunizing compositions of the present invention contain an immunoeffective amount of one or more of the synthetic peptides or conjugates thereof and a pharmaceutically acceptable carrier. Such compositions in dosage unit form can contain about 0.5 .mu.g to about 1 mg of each peptide or conjugate per kg body weight. When delivered in multiple doses, the dosage unit form is conveniently divided into the appropriate amounts per dosage.

Immunizing compositions which contain cocktails of two or more of the synthetic peptides, or conjugates thereof, of the present invention enhance immunoefficacy in a broader population and thus provide a better immune response to amyloid .beta. peptides and amyloid deposits. Other immunostimulatory synthetic peptide immunogens are arrived at through modification into lipopeptides so as to provide built-in adjuvanticity for potent vaccines. The immune response to synthetic peptide immunogens of the present invention can be improved by delivery through entrapment in or on biodegradable microparticles of the type described by O'Hagan et al (1991). The immunogens can be encapsulated with or without adjuvant, including covalently attached lipid moiety such as Pam₃ Cys, and such microparticles can be administered with an immunostimulatory adjuvant such as Freund's Incomplete Adjuvant or alum. The microparticles function to potentiate immune responses to an immunogen and to provide time-controlled release for sustained or periodic responses. for oral administration, and for topical administration (O'Hagan et al., 1991).

A further aspect of the present invention is a method for immunization with the synthetic peptide or conjugate thereof of the present invention. This method according to the present invention involves administering to a mammal, in need thereof, preferably human, an immunizing composition containing the synthetic peptide(s) or conjugates thereof. Efficacy will be tested first in transgenic mouse models of AD such as the mouse model used in Schenk et al. (1999) or other publicly or commercially available AD transgenic mouse model.

Yet another aspect of the present invention provides for antibodies raised against the immunogenic peptides of the present invention and molecules which includes the antigen-binding portion of such antibodies.

It should be understood that when the term "antibodies" is used with respect to the antibody embodiments of the present invention, this is intended to include intact antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs), as well as proteolytic fragments thereof such as the Fab or F(ab')₂ fragments. Furthermore, the DNA encoding the variable region of the antibody can be inserted into other antibodies to produce chimeric antibodies (see, for example, U.S. Pat. No. 4,816,567) or into T-cell receptors to produce T-cells with the same broad specificity (see Eshhar, et al., (1990) and Gross et al., (1989)). Single chain antibodies can also be produced and used. Single chain antibodies can be single chain composite polypeptides having antigen binding capabilities and comprising a pair of amino acid sequences homologous or analogous to the variable regions of an immunoglobulin light and heavy chain (linked V_H -V_L or single chain F_v). Both V_H and V_L may copy natural monoclonal antibody sequences or one or both of the chains may comprise a CDR-FR construct of the type described in U.S. Pat. No. 5,091,513 (the entire content of which is hereby incorporated herein by reference). The separate polypeptides analogous to the variable regions of the light and heavy chains are held together by a polypeptide linker. Methods of production of such single chain antibodies, particularly where the DNA encoding the polypeptide structures of the V_H and V_L chains are known, may be accomplished in accordance with the methods described, for example, in U.S. Pat. Nos. 4,946,778, 5,091,513 and 5,096,815, the entire contents of each of which are hereby incorporated herein by reference.

An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term "epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody. Epitopes or "antigenic determinants" usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.

Monoclonal antibodies (mAbs) are a substantially homogeneous population of antibodies to specific antigens. MAbs may be obtained by methods known to those skilled in the art. See, for example Kohler et al., (1975); U.S. Pat. No. 4,376,110; Harlow et al., (1988); and Colligan et al., (1993), the entire contents of which references are incorporated entirely herein by reference. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and any subclass thereof. The hybridoma producing the mAbs of this invention may be cultivated in vitro or in vivo. High titers of mAbs can be obtained by in vivo production where cells from the individual hybridomas are injected intraperitoneally into pristane-primed Balb/c mice to produce ascites fluid containing high concentrations of the desired mAbs. MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.

Chimeric antibodies are molecules, the different portions of which are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. Chimeric antibodies are primarily used to reduce immunogenicity during application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric or humanized mAbs are used. Chimeric and humanized antibodies and methods for their production are well-known in the art, such as Cabilly et al., 1984; Morrison et al., 1984; Boulianne et al., 1984; Cabilly et al., 1984; Neuberger et al., 1985; Taniguchi et al., 1985; Morrison et al., 1986; Neuberger et al., 1986; Kudo et al., 1986; Morrison et al., 1986; Sahagan et al., 1986; Robinson et al., 1987; Liu et al., 1987; Sun et al., 1987; Better et al., 1988; and Harlow et al., 1988. These references are hereby incorporated herein by reference.

A "molecule which includes the antigen-binding portion of an antibody," is intended to include not only intact immunoglobulin molecules of any isotype and generated by any animal cell line or microorganism, but also the antigen-binding reactive fraction thereof, including, but not limited to, the Fab fragment, the Fab' fragment, the F(ab')₂ fragment, the variable portion of the heavy and/or light chains thereof, and chimeric or single-chain antibodies incorporating such reactive fraction, as well as any other type of molecule or cell in which such antibody reactive fraction has been physically inserted, such as a chimeric T-cell receptor or a T-cell having such a receptor, or molecules developed to deliver therapeutic moieties by means of a portion of the molecule containing such a reactive fraction. Such molecules may be provided by any known technique, including, but not limited to, enzymatic cleavage, peptide synthesis or recombinant techniques.

The present invention also provides a pharmaceutical composition containing a molecule which includes the antigen-binding portion of an antibody raised against a peptide of the present invention, and a pharmaceutically acceptable, carrier, diluent, excipient or auxiliary agent. The formulation of pharmaceutical compositions, which formulation is conventionally used in a highly skilled art and which compositions are suitable for its intended use as a therapeutic for reducing the formulation of amyloid fibrils and deposits, can be developed with only routine experimentation by those of skill in the art.

According to the present invention, the molecule which includes the antigen-binding portion of an antibody raised against the immunogenic peptides of the present invention can be administered to a subject in need thereof to reduce the formation of amyloid fibrils and deposits. The site of administration, the dosage, and the schedule of administration are determined according to well-established procedures used by those of skill in the art.

Claim 1 of 30 Claims

What is claimed is:

1. An isolated peptide comprising the amino acid sequence

(Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Glu Asp Val Gly Ser Asn Lys Gly Ala)_n (SEQ ID NO:15)

wherein n is 1 or 2; and

Xaa₁, Xaa₂, Xaa₃, Xaa₄, and Xaa₅ are Leu, Val, Phe, Phe, and Ala, respectively, in which one or two of residues Xaa₁, Xaa₂, Xaa₃, Xaa₄, and Xaa₅ is substituted with Lys, Asp, or Glu.

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