A recombinant or isolated humanized or chimeric antibody, or antigen binding fragment thereof, that specifically binds to a neoepitope of human amyloid a peptide, a pharmaceutical composition comprising the antibody and its use in the preparation of a medicament for the treatment of amyloidosis

206641/2 A recombinant or isolated humanized or chimeric antibody, or antigen-bindingfragment thereof, that specifically binds to a neoepitope of human amyloid Apeptide, a pharmaceutical composition comprising the antibody and its use inpreparation of a medicament for treatment of amyloidosis

TECHNICAL FIELD

The invention resides in the technical fields of immunology and medicine.

BACKGROUND OF THE INVENTION

Amyloidosis is a general term that describes a number of diseases characterized by theexistence of pathological forms of amyloid proteins, often involving extracellulardeposition of protein fibrils, which form numerous "amyloid deposits" or "amyloidplaques," which may occur in local sites or systematically. These deposits or plaques arecomposed primarily of a naturally occurring soluble protein or peptide, assembled intoextensive Insoluble deposits 10-100 micro m in diameter in a variety of tissue sites. Thedeposits are composed of generally lateral aggregates of fibrils that are approximately 1 Ο-Ι 5 nm in diameter. Amyloid fibrils produce a characteristic apple green birefringence inpolarized light, when stained with Congo Red dye. Generally, the fibrillar composition ofthese deposits is an Identifying characteristic for the various forms of amyloid disease.

The peptides or proteins forming the plaque deposits are often produced from a largerprecursor protein. More specifically, the pathogenesis of amyloid aggregates such as fibrildeposits generally involves proteolytic cleavage of an "abnormal" precursor protein intofragments that aggregate into anti-parallel beta pleated sheets. The fibrillar composition ofthese deposits is an Identifying characteristic for the various forms of amyloid disease. Forexample, intracerebral and cerebrovascular deposits composed primarily of fibrils of betaamyloid peptide (beta -AP) are characteristic of Alzheimer's disease (both familial andsporadic forms), islet amyloid protein peptide (IAPP; amylin) is characteristic of the fibrilsin pancreatic islet cell amyloid deposits associated with type II diabetes, and beta 2-microglobulin is a major component of amyloid deposits which form as a consequence oflong term hemodialysis treatment. More recently, prion-associated diseases, such asCreutzfeld- Jacob disease, have also been recognized as amyloid diseases.

In general, primary amyloidoses of the disease are characterized by the presence of"amyloid light chain-type" (AL-type) protein fibrils, so named for the homology of the N- 1 206641/2 terminal region of the AL fibrils to the variable fragment of immunoglobulin light chain(kappa or lambda). Malle et al. (1998), Scand. J. Immunol., 48:557-561 describes acollection of polyclonal antibodies raised against synthetic peptides corresponding tolimited regions of serum amyloid Ai (SAAi). US 2007/0178504 describes methods ofusing biomarkers in determining the risk of lung cancer. These biomarkers include awide variety of antigens and antibodies. WO 2005/025516 describes methods forproducing antibodies that are specific to conformational epitopes on amyloidogenicpeptides, including Αβ fragments, IAPP, and transthyretin.

The various forms of disease have been divided into classes, mostly on the basis of whetherthe amyloidosis is associated with an underlying systematic illness. Thus, certain disordersare considered to be primary amyloidoses, in which there is no evidence for preexisting orcoexisting disease. In secondary or reactive (AA type) amyloidosis characterized by thepresence deposition of amyloid protein A (AA) fibrils, there is an underlying or associatedchronic inflammatory or infectious disease state.

Heredofamilial amyloidoses may have associated neuropathic, renal, or cardiovasculardeposits of the ATTR transthyretin type. Other heredofamilial amyloidoses include othersyndromes and may have different amyloid components (e.g., familial Mediterranean feverwhich is characterized by AA fibrils). Other forms of amyloidosis include local forms,characterized by focal, often tumor-like deposits that occur in isolated organs. Otheramyloidoses are associated with aging, and are commonly characterized by plaqueformation in the heart or brain. Also common are amyloid deposits associated with longterm hemodialysis. These and other forms of amyloid disease are summarized in Table 1(Tan, S. ¥. and Pepys, Histopathology 25:403-414, 1994; Harrison's Handbook of InternalMedicine, 13th Ed., isselbacher, K.J., et al, eds, McGraw-Hill, San Francisco, 1995) and aredescribed in U.S. Patent Nos. 6,<875,434, 6,890,535, 6,913,745, 6,923,964, and 6,936,246. 2 WO 2009/086539 PCT/US2008/088493 T Classification ABLE 1 of Amyloid Diseases Amyloid Protein/ Peptide Protein Precursor Protein Variants Clinical AA Serum Amyloid AProtein (ApoSSA) Reactive (secondary)Amyloidosis: Familial Mediterraneanfever Familial amyloidnephropathy with urticariaand deafness (Muckle-Wells syndrome) AA Serum amyloid A protein (ApoSSA) Reactive systemicamyloidosis associatedwith systemicinflammatory diseases AL Monoclonalimmunoglobulin lightchains (kappa, lambda) Ak, A, (e.g.,AkIII) Idiopathic (primary)Amyloidosis: myeloma ormacro globulinemia-associated; systemicamyloidosis associatedwith immunocytedyscrasia; monoclonalgammopathy; occultdyscrasia; local nodularamyloidosis associatedwith chronic inflammatorydiseases AH IgG(l(yl)) Αγί Heavy chain amyloidosisassociated with severalimmunocyte dyscrasias ATTR Transthyretin (TTR) At least 30known pointmutations Familial amyloidpolyneuropathy(e.g., Met 30, Portuguese) ATTR Transthyretin (TTR) e.g., Met 111 Familial amyloid cardiomyopathy (Danish) 3 WO 2009/086539 PCT/US2O08/O88493 ATTR Transthyretin (TTR) Wild-type TTRor He 122 Systemic senileamyloidosis AapoAI ApoAI Arg 26 Familial amyloidpolyneuropathy Agel Gelsolin Asn 187 Familial amyloidosis(Finnish) Acys Cystatin C Gin 68 Hereditary cerebralhemorrhage withamyloidosis (Icelandic) Αβ Amyloid β proteinprecursor (e.g. β- app695) Various: Gin 618, Alzheimer's disease Down's syndrome Hereditary cerebralhemorrhage amyloidosis(Dutch) Sporadic cerebral amyloidangiopathy Inclusion body myositis AB2M Beta2 microglobulin Associated with chronichemodialysis Acal (Pro)calcitonin (Pro)calcitonin Medullary carcinoma ofthyroid AANF Αβ SVEPa ab2m Atrial natriuretic factorβ-amyloid precursorprotein Beta2 microglobulin Focal Senile Amyloidoses:Isolated atrial amyloidBrain Seminal vesicles Prostate Keratin Primary localizedcutaneous amyloid(macular, papular) PrP Prion precursor protein(33-35 kDa cellularform) Scrapie protein27-30 kDa Sporadic Creutzfeldt-JacobDisease Kuru (transmissiblespongiform encephalopathies, priondiseases) 4 WO 2009/086539 PCT/US2008/088493 AIAPP Islet amyloidpolypeptide (IAPP) Islets of LangerhansDiabetes type II, Insulinoma Peptide hormones, fragments e.g., precalcitonin Exocrine amyloidosis,associated with APUDomas aSeminal vesicle exocrine protein

Often, fibrils forming the bulk of an amyloid deposit are derived from one or moreprimary precursor proteins or peptides, and are usually associated with sulfated 5 glycosaminoglycans. In addition, amyloid deposits may include minor proteins and peptides ofvarious types, along with other components, such as proteoglycans, gangliosides and othersugars, as described in more detail in the sections that follow. AA fibrils are composed of peptide fragments that range in size but are generally about8000 daltons (AA peptide or protein) formed by proteolytic cleavage of serum amyloid A protein 10 (SSA), a circulating apolipoprotein which is present in HDL particles and which is synthesizedin hepatocytes in response to such cytokines as interleukin (IL)-l and IL-6, as well as tumornecrosis factor a. See Husby, G. et al. Amyloid 1, 119-137 (1994). The proteolytic cleavageresults in the pathologic deposition of an -76-residue N-terminal two thirds of the SAA protein.In humans, the plasma concentration of SAA normally is ~0.1 mg/ml but can increase over 15 1,000-fold in response to an inflammatory stimulus. As part of this process, the SAA molecule undergoes proteolysis and the N-terminal cleavage product is deposited systemically as AAfibrils in vital organs, including the liver, spleen, kidneys, and adrenal glands. Deposition is alsocommon in the heart and gastrointestinal tract.

Generally, AA amyloidosis is a manifestation of diseases that provoke a sustained acute 20 phase response. Such diseases include chronic inflammatory disorders, chronic local or systemicmicrobial infections, and malignant neoplasms. AA amyloid diseases include, but are not limitedto inflammatory diseases, such as rheumatoid arthritis, juvenile chronic arthritis, ankylosingspondylitis, psoriasis, psoriatic arthropathy, Reiter’s syndrome, Adult Still’s disease, Behcet’ssyndrome, and Crohn’s disease. AA deposits are also produced as a result of chronic microbial 25 infections, such as leprosy, tuberculosis, bronchiectasis, decubitus ulcers, chronic pyelonephritis,osteomyelitis, and Whipple’s disease. Certain malignant neoplasms can also result in AA fibrilamyloid deposits. These include such conditions such as Hodgkin’s lymphoma, renal carcinoma, 5

SUBSTITUTE SHEET carcinomas of gut, lung and urogenital tract, basal cell carcinoma, and hairy cell leukemia. AAamyloid disease may also result from inherited inflammatory diseases such as FamilialMediterranean Fever. Additionally, AA amyloid disease may result from lymphoproliferativedisorders such as Castleman’s Disease. 5 AA Amyloidosis is insidious and progressive. Symptoms are generally presented in later stages of the disease. Frequently the patient is undiagnosed until significant organ damagehas occurred. AA fibrils are deposited in vital organs leading to organ dysfunction andsubsequently to death. The five year survival rate is 45-50%. Median survival after diagnosis is4-8 years. End stage Renal Disease is the cause of death in 40-60% of eases. See Gillmore J.D. 10 et al. Lancet 358:24-9 (2001).

Currently, there are no approved specific, amyloid-directed treatments for any of theamyloid diseases, including AA Amyloidosis. See Gillmore J.D. et al. Lancet 358:24-9 (2001).Where there is an underlying or associated disease state, therapy directed towards decreasing theproduction of amyloidogenic protein by treating the underlying disease. For example, current 15 treatment strategy for AA Amyloidosis is to target underlying inflammation, reducing ApoSSAlevels to below 10mg/l. Currently employed therapies include chemotherapy (cholorambucil andMTX), immuno-suppressants (azathioprine), anti-inflammatory drugs (colchicine) and TNFinhibitors. The invention thus fulfills a longstanding need for therapeutic regimes for preventingor ameliorating the effects of AA Amyloidosis.

20 SUMMARY OF THE INVENTION

The present invention provides an isolated human, humanized, or chimeric antibody, orantigen-binding fragment thereof, that specifically binds to an epitope within residues 70-76 ofhuman amyloid A peptide, for example, an epitope within residues 70-76 of SEQ ID NO: 2 or anepitope comprising residues set forth as SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, or 11. Antibodies or 25 antigen-binding fragments of the invention include those that compete for binding to humanamyloid A peptide with antibody 2A4 produced by ATCC Accession Number PTA-9662 or withantibody 7D8 produced by ATCC Accession Number PTA-9468. Additional antibodies of theinvention compete for binding to human amyloid A peptide with an antibody having a light chainvariable region set forth as residues 20-131 of SEQ ID NO: 152 or residues 20-131 of 153 and a 30 heavy chain variable region set forth as residues 20-138 of SEQ ID NO: 154. 6

SUBSTITUTE SHEET

The disclosed antibodies include humanized and chimeric versions of antibody 2A4produced by ATCC Accession Number PTA-9662 or a humanized or chimeric version ofantibody 7D8 produced by ATCC Accession Number PTA-9468.

For example, representative antibodies and antigen-binding fragments comprise a light5 chain variable region comprising one or more complementarity regions of a 2A4 light chainvariable region set forth as residues 20-131 of SEQ ID NO: 152 or one or more complementarityregions of a 7D8 light chain variable region set forth as residues 20-131 of SEQ ID NO: 153. Asanother example, representative antibodies and antigen-binding fragments comprise a light chainvariable region comprising two complementarity regions of a 2A4 light chain variable region set 10 forth as residues 20-131 of SEQ ID NO: 152 or two complementarity regions of a 7D8 lightchain variable region set forth as residues 20-131 of SEQ ID NO: 153. Additional representativeantibodies and antigen-binding fragments comprise a light chain variable region comprisingthree complementarity regions of a 2A4 light chain variable region set forth as residues 20-131of SEQ ID NO: 152 or three complementarity regions of a 7D8 light chain variable region set 15 forth as residues 20-131 of SEQ ID NO: 153. Representative humanized versions of a 2A4 or7D8 antibody comprise at least one light chain framework residue selected from the groupconsisting of L87 and L90 (Kabat numbering convention), which is occupied by Y and F,respectively, and wherein the remainder of the light chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin light chain variable region. 20 Representative antibodies and antigen-binding fragments comprise at least one light chainframework residue selected from the group consisting of+7, +14, +15, +17, +18, +50, +75,+88, + 92, and +109 (linear numbering), which is occupied by T, S, L, D, Q, K, Y, L, F, and L,respectively, and wherein the remainder of the light chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin light chain variable region. For 25 example, representative antibodies and antigen-binding fragments comprise at least one lightchain framework residue selected from the group consisting of +75 and +92 (linear numbering),which is occupied by Y and F, respectively, and wherein the remainder of the light chain variableregion is occupied by a corresponding residue in a human acceptor immunoglobulin light chainvariable region. In other representative antibodies and antigen-binding fragments of the 30 invention, the light chain variable region comprises a framework residue at +105 (linearnumbering) occupied by Q. 7 WO 2009/086539 PCT/US2008/088493

For example, antibodies and antigen-binding fragments of the invention include thosecomprising a light chain variable region comprising a framework residue at +7 (linearnumbering) occupied by T, wherein the remainder of the light chain variable region is occupiedby a corresponding residue in a human acceptor immunoglobulin light chain variable region;antibodies and antigen-binding fragments a light chain variable region comprising a frameworkresidue at +14 (linear numbering) occupied by S, wherein the remainder of the light chainvariable region is occupied by a corresponding residue in a human acceptor immunoglobulinlight chain variable region; antibodies and antigen-binding fragments a light chain variableregion comprising a framework residue at +15 (linear numbering) occupied by L, wherein theremainder of the light chain variable region is occupied by a corresponding residue in a humanacceptor immunoglobulin light chain variable region; antibodies and antigen-binding fragments alight chain variable region comprising a framework residue at +17 (linear numbering) occupiedby D, wherein the remainder of the light chain variable region is occupied by a correspondingresidue in a human acceptor immunoglobulin light chain variable region; antibodies and antigen-binding fragments a light chain variable region comprising a framework residue at +18 (linearnumbering) occupied by Q, wherein the remainder of the light chain variable region is occupiedby a corresponding residue in a human acceptor immunoglobulin light chain variable region;antibodies and antigen-binding fragments a light chain variable region comprising a frameworkresidue at +50 (linear numbering) occupied by K, wherein the remainder of the light chainvariable region is occupied by a corresponding residue in a human acceptor immunoglobulinlight chain variable region; antibodies and antigen-binding fragments a light chain variableregion comprising a framework residue at +75 (linear numbering) occupied by Y, wherein theremainder of the light chain variable region is occupied by a corresponding residue in a humanacceptor immunoglobulin light chain variable region; antibodies and antigen-binding fragments a light chain variable region comprising a framework residue at +88 (linear numbering) occupiedby L, wherein the remainder of the light chain variable region is occupied by a correspondingresidue in a human acceptor immunoglobulin light chain variable region; antibodies and antigen-binding fragments a light chain variable region comprising a framework residue at +92 (linearnumbering) occupied by F, wherein the remainder of the light chain variable region is occupiedby a corresponding residue in a human acceptor immunoglobulin light chain variable region;antibodies and antigen-binding fragments a light chain variable region comprising a frameworkresidue at +109 (linear numbering) occupied by L, wherein the remainder of the light chain 8 WO 2009/086539 PCT/US2008/088493 variable region is occupied by a corresponding residue in a human acceptor immunoglobulinlight chain variable region; and antibodies and antigen-binding fragments a light chain variableregion comprising a framework residue at +105 (linear numbering) occupied by Q, wherein theremainder of the light chain variable region is occupied by a corresponding residue in a humanacceptor immunoglobulin light chain variable region.

Human acceptor immunoglobulin light chain variable regions used in the inventioninclude human kappa subgroup 2 light chain variable region (Kabat convention), for example,human subgroup 2 light chain variable region from human germline VKIIA19/A3, such ashuman Vk light chain variable region comprising a sequence set forth as SEQ ID NO: 166 or167. In particular aspects of the invention, antibodies and antigen-binding fragments comprise alight chain variable region comprising an amino acid sequence set forth as residues 20-131 ofSEQ ID NO: 152, residues 20-131 of SEQ ID NO: 153, or set forth as SEQ ID NO: 155, 156,157, 158, 159, 160, 174, 175, or 176.

Representative antibodies and antigen-binding fragments of the invention also includethose comprising a heavy chain variable region comprising one or more complementarity regionsof a 2A4 heavy chain variable region set forth as residues 20-138 of SEQ ID NO: 154, forexample, a heavy chain variable region comprising two complementarity regions of a 2A4 heavychain variable region set forth as residues 20-138 of SEQ ID NO: 154, or a heavy chain variableregion comprising three complementarity regions of a 2A4 heavy chain variable region set forthas residues 20-138 of SEQ ID NO: 154. Representative humanized 2A4 and 7D8 antibodies andantigen-binding fragments comprise at least one heavy chain framework residue selected fromthe group consisting of H37, H49, H70, and H93 (Kabat numbering convention), which isoccupied by I, A, F, or V, respectively, and wherein the remainder of the heavy chain variableregion is occupied by a corresponding residue in a human acceptor immunoglobulin heavy chainvariable region. Representative humanized antibodies and antigen-binding fragments compriseat least one heavy chain framework residue selected from the group consisting of+10, +15, +19,+37, +49, +73, +78, +79, +80, +87, +95, +99, +119 (linear numbering), which is occupied by R,K, K, I, A, F, Q, S, Μ, N, Μ, V, or A, respectively, and wherein the remainder of the heavy chainvariable region is occupied by a corresponding residue in a human acceptor immunoglobulinheavy chain variable region. For example, representative humanized antibodies and antigen-binding fragments comprise at least one heavy chain framework residue selected from the groupconsisting of +37, +49, +73, and +99 (linear numbering), which is occupied by I, A, F, or V, 9 WO 2009/086539 PCT/US2008/088493 respectively, and wherein the remainder of the heavy chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin heavy chain variable region.

For example, antibodies and antigen-binding fragments of the invention include thosecomprising a heavy chain variable region comprising a framework residue at +10 (linearnumbering) occupied by R, wherein the remainder of the heavy chain variable region is occupiedby a corresponding residue in a human acceptor immunoglobulin heavy chain variable region;antibodies and antigen-binding fragments a heavy chain variable region comprising a frameworkresidue at +15 (linear numbering) occupied by K, wherein the remainder of the heavy chainvariable region is occupied by a corresponding residue in a human acceptor immunoglobulinheavy chain variable region; antibodies and antigen-binding fragments a heavy chain variableregion comprising a framework residue at +19 (linear numbering) occupied by K, wherein theremainder of the heavy chain variable region is occupied by a corresponding residue in a humanacceptor immunoglobulin heavy chain variable region; antibodies and antigen-binding fragmentsa heavy chain variable region comprising a framework residue at +37 (linear numbering)occupied by I, wherein the remainder of the heavy chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin heavy chain variable region;antibodies and antigen-binding fragments a heavy chain variable region comprising a frameworkresidue at +49 (linear numbering) occupied by A, wherein the remainder of the heavy chainvariable region is occupied by a corresponding residue in a human acceptor immunoglobulinheavy chain variable region; antibodies and antigen-binding fragments a heavy chain variableregion comprising a framework residue at +73 (linear numbering) occupied by F, wherein theremainder of the heavy chain variable region is occupied by a corresponding residue in a humanacceptor immunoglobulin heavy chain variable region; antibodies and antigen-binding fragmentsa heavy chain variable region comprising a framework residue at +78 (linear numbering)occupied by Q, wherein the remainder of the heavy chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin heavy chain variable region;antibodies and antigen-binding fragments a heavy chain variable region comprising a frameworkresidue at +79 (linear numbering) occupied by S, wherein the remainder of the heavy chainvariable region is occupied by a corresponding residue in a human acceptor immunoglobulinheavy chain variable region; antibodies and antigen-binding fragments a heavy chain variableregion comprising a framework residue at +80 (linear numbering) occupied by M, wherein theremainder of the heavy chain variable region is occupied by a corresponding residue in a human 10 WO 2009/086539 PCT/US2008/088493 acceptor immunoglobulin heavy chain variable region; antibodies and antigen-binding fragmentsa heavy chain variable region comprising a framework residue at +87 (linear numbering)occupied by N, wherein the remainder of the heavy chain variable region is occupied by acorresponding residue in a human acceptor immunoglobulin heavy chain variable region;antibodies and antigen-binding fragments a heavy chain variable region comprising a frameworkresidue at +95 (linear numbering) occupied by M, wherein the remainder of the heavy chainvariable region is occupied by a corresponding residue in a human acceptor immunoglobulinheavy chain variable region; antibodies and antigen-binding fragments a heavy chain variableregion comprising a framework residue at +99 (linear numbering) occupied by V, wherein theremainder of the heavy chain variable region is occupied by a corresponding residue in a humanacceptor immunoglobulin heavy chain variable region; and antibodies and antigen-bindingfragments a heavy chain variable region comprising a framework residue at +109 (linearnumbering) occupied by A, wherein the remainder of the heavy chain variable region is occupiedby a corresponding residue in a human acceptor immunoglobulin heavy chain variable region.

Human acceptor immunoglobulin heavy chain variable regions include a human gammasubgroup 3 heavy chain variable region (Kabat convention), for example, human gammasubgroup 3 heavy chain variable region comprising a sequence set forth as SEQ ID NO: 165,such as a heavy chain variable region comprising an amino acid sequence set forth as residues20-138 of SEQ ID NO: 154 or set forth as SEQ ID NO: 161, 162, or 163.

Additional representative antibodies and antigen-binding fragments comprise a lightchain variable region comprising three complementarity determining regions of a 2A4 light chainvariable region set forth as residues 20-131 of SEQ ID NO: 152 or three complementarityregions of a 7D8 light chain variable region set forth as residues 20-131 of SEQ ID NO: 153, anda heavy chain variable region comprising three complementarity regions of a 2A4 heavy chainvariable region set forth as residues 20-138 of SEQ ID NO: 154. For example, such antibodiesand antigen-binding fragments include those having a light chain variable region comprisingthree complementarity determining regions set forth as SEQ ID NOs: 168, 169, and 170, and aheavy chain variable region comprising three complementarity regions set forth as SEQ ID NOs:171, 172, and 173. As another example, such antibodies and antigen-binding fragments includethose having a light chain variable region comprising three complementarity determining regionsset forth as SEQ ID NOs: 177, 169, and 170, and a heavy chain variable region comprising threecomplementarity regions set forth as SEQ ID NOs: 171, 172, and 173. As another example, such 11 WO 2009/086539 PCT/US2008/088493 antibodies and antigen-binding fragments include those comprising a light chain variable regioncomprising an amino acid sequence set forth as residues 20-131 of SEQ ID NO: 152 or asresidues 20-131 of SEQ ID NO: 153, and a heavy chain variable region comprising an aminoacid sequence set forth as residues 20-138 of SEQ ID NO: 154. As another example, suchantibodies and antigen-binding fragments include those having a light chain variable regioncomprising an amino acid sequence set forth as SEQ ID NO: 155, 156, 157, 158, 159, 160, 174,175, or 176, and a heavy chain variable region comprising an amino acid sequence set forth asSEQ ID NO: 161, 162, or 163.

In particular aspects of the invention, an antibody or antigen-binding fragment comprisesa light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 155,and a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO:161; a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO:155, and a heavy chain variable region comprising an amino acid sequence set forth as SEQ IDNO: 162; a light chain variable region comprising an amino acid sequence set forth as SEQ IDNO: 155, and a heavy chain variable region comprising an amino acid sequence set forth as SEQID NO: 163; a light chain variable region comprising an amino acid sequence set forth as SEQID NO: 156, and a heavy chain variable region comprising an amino acid sequence set forth asSEQ ID NO: 161; a light chain variable region comprising an amino acid sequence set forth asSEQ ID NO: 156, and a heavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 162; a light chain variable region comprising an amino acid sequence set forth asSEQ ID NO: 156, and a heavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 163; a light chain variable region comprising an amino acid sequence set forth asSEQ ID NO: 157, and a heavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 161; a light chain variable region comprising an amino acid sequence set forth asSEQ ID NO: 157, and a heavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 162; a light chain variable region comprising an amino acid sequence set forth asSEQ ID NO: 157, and a heavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 163; a light chain variable region comprising an amino acid sequence set forth asSEQ ID NO: 158, and a heavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 161; a light chain variable region comprising an amino acid sequence set forth asSEQ ID NO: 158, and a heavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 162; a light chain variable region comprising an amino acid sequence set forth as 12 WO 2009/086539 PCT/US2008/088493 SEQ ID NO: 158, and a heavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 163; a light chain variable region comprising an amino acid sequence set forth asSEQ ID NO: 159, and a heavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 161; a light chain variable region comprising an amino acid sequence set forth asSEQ ID NO: 159, and a heavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 162; a light chain variable region comprising an amino acid sequence set forth asSEQ ID NO: 159, and a heavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 163; a light chain variable region comprising an amino acid sequence set forth asSEQ ID NO: 160, and a heavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 161; a light chain variable region comprising an amino acid sequence set forth asSEQ ID NO: 160, and a heavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 162; a light chain variable region comprising an amino acid sequence set forth asSEQ ID NO: 160, and a heavy chain variable region comprising an amino acid sequence set forthas SEQ ID NO: 163; SEQ ID NO: 174, and a heavy chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 161; a light chain variable region comprising an aminoacid sequence set forth as SEQ ID NO: 174, and a heavy chain variable region comprising anamino acid sequence set forth as SEQ ID NO: 162; a light chain variable region comprising anamino acid sequence set forth as SEQ ID NO: 174, and a heavy chain variable region comprisingan amino acid sequence set forth as SEQ ID NO: 163; a light chain variable region comprisingan amino acid sequence set forth as SEQ ID NO: 175, and a heavy chain variable regioncomprising an amino acid sequence set forth as SEQ ID NO: 161; a light chain variable regioncomprising an amino acid sequence set forth as SEQ ID NO: 175, and a heavy chain variableregion comprising an amino acid sequence set forth as SEQ ID NO: 162; a light chain variableregion comprising an amino acid sequence set forth as SEQ ID NO: 175, and a heavy chainvariable region comprising an amino acid sequence set forth as SEQ ID NO: 163; a light chainvariable region comprising an amino acid sequence set forth as SEQ ID NO: 176, and a heavychain variable region comprising an amino acid sequence set forth as SEQ ID NO: 161; a lightchain variable region comprising an amino acid sequence set forth as SEQ ID NO: 176, and aheavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 162; ora light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 176,and a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO:163. 13 WO 2009/086539 PCT/US2008/088493

Also provided are isolated nucleic acids encoding a human, humanized, or chimericantibody, or antigen-binding fragment thereof, that specifically binds to an epitope withinresidues 70-76 of human amyloid A peptide, including all such antibodies and antigen-bindingfragments as described herein above and as set forth in the claims. Further provided are cellsexpressing such nucleic acids.

In other aspects, the present invention provides an isolated antibody, or antigen-bindingfragment thereof, which specifically binds to an epitope comprising X1EDX2 in an aggregatedamyloid protein, wherein Xj and X2 are any amino acid. Such antibodies and antigen-bindingfragments include human, humanized, or chimeric antibodies, and antigen-binding fragmentsthereof, for example, those that specifically bind to an epitope within residues 70-76 of humanamyloid A peptide.

Additional representative antibodies and antigen-binding fragments include thosewherein Xi is Η, T, F, S, P, A, L, C, Q, R, E, K, D, G, V, Y, I, or W, and wherein X2 is T, S, E,R, I, V, F, D, A, G, M, L, N, P, C, K, Y, or Q; or X! is Η, T, F, S, P, or A and wherein X2 is T, S,E, R, I, V, F, D, or A; or Xi is Η, T, F, or A; or X2 is T, S, E, D, or A; or Xi is Η, T, F, or A andX2 is T, S, E, D, or A; or Xi is Η, T, or A and X2 is T, S, E, or A; or Xi is H or A and X2 is T, S,or A; or Xi is H and X2 is T or A; or Xi is A and X2 is S, T, E or V; or Xi is A and X2 is S, T orE; or Χχ is T and X2 is E; or Xi is F and X2 is D; or Xj is S and X2 is E, F or A; or Xj is P and X2is E, I or F. For example, such antibodies and antigen-binding fragments bind an epitopeconsisting of an amino acid sequence selected from the group consisting of GHEDT (SEQ IDNO: 3), HEDT (SEQ ID NO: 12), AEDS (SEQ ID NO: 13), AEDT (SEQ ID NO: 14), HEDA(SEQ ID NO: 15), TEDE (SEQ ID NO: 16), FEDD (SEQ ID NO: 17), SEDE (SEQ ID NO: 18),AEDE (SEQ ID NO: 19), PEDE (SEQ ID NO: 20), PEDI (SEQ ID NO: 21), PEDF (SEQ IDNO: 22), AEDV (SEQ ID NO: 23), SEDF (SEQ ID NO: 24), and SEDA (SEQ ID NO: 25); or anepitope consisting of an amino acid sequence selected from the group consisting of GHEDT(SEQ ID NO: 3), HEDT (SEQ ID NO: 12), AEDS (SEQ ID NO: 13), AEDT (SEQ ID NO: 14),HEDA (SEQ ID NO: 15), TEDE (SEQ ID NO: 16), FEDD (SEQ ID NO: 17), SEDE (SEQ IDNO: 18), AEDE (SEQ ID NO: 19), PEDE (SEQ ID NO: 20), PEDI (SEQ ID NO: 21), PEDF(SEQ ID NO: 22), SEDF (SEQ ID NO: 24), and SEDA (SEQ ID NO: 25); or an epitopeconsisting of an amino acid sequence selected from the group consisting of GHEDT (SEQ IDNO: 3), HEDT (SEQ ID NO: 12), AEDS (SEQ ID NO: 13), AEDT (SEQ ID NO: 14), HEDA(SEQ ID NO: 15), and TEDE (SEQ ID NO: 16). The disclosed epitopes may be found in an 14 WO 2009/086539 PCT/US2008/088493 aggregated amyloid protein, for example, an epitope comprising an amino acid sequence selectedfrom the group consisting of GHGAEDS (SEQ ID NO: 4), GHDAEDS (SEQ ID NO: 5),GDHAEDS (SEQ ID NO: 7), STVIEDS (SEQ ID NO: 8), and GRGHEDT (SEQ ID NO: 9); oran epitope comprising an amino acid sequence GHGAEDS (SEQ ID NO:4); or an epitopecomprising amino acids HEDT (SEQ ID NO: 12); or an epitope comprising amino acids HEDA(SEQ ID NO: 15); or an epitope comprising amino acids AEDS (SEQ ID NO: 13) or an epitopecomprising amino acids AEDT (SEQ ID NO: 14); or an epitope comprising amino acids TEDE(SEQ ID NO: 16); or an epitope comprising the amino acid sequence AEDV (SEQ ID NO: 23);or an epitope comprising the amino acid sequence SEDF (SEQ ID NO: 24) or PEDF (SEQ IDNO: 22); or an epitope of comprising an amino sequence selected from the group consisting ofPEDS (SEQ ID NO: 26), PEDL (SEQ ID NO: 27), TEDV (SEQ ID NO: 28), AEDE (SEQ IDNO: 19), SEDI (SEQ ID NO: 29) and TEDT (SEQ ID NO: 30); or an epitope comprising anamino sequence selected from the group consisting of LEDG (SEQ ID NO: 31), AEDM (SEQ IDNO: 32), HEDS (SEQ ID NO: 33), CEDD (SEQ ID NO: 34), QEDS (SEQ ID NO: 35), REDS(SEQ ID NO: 36), TEDG (SEQ ID NO: 16), QEDR (SEQ ID NO: 38), TEDL (SEQ ID NO: 39),PEDN (SEQ ID NO: 40), EEDP (SEQ ID NO: 41), LEDL (SEQ ID NO: 42), KEDA (SEQ IDNO: 43), SEDC (SEQ ID NO: 44), EEDD (SEQ ID NO: 45), SEDK (SEQ ID NO: 46), DEDD(SEQ ID NO: 47), DEDG (SEQ ID NO: 13), LEDE (SEQ ID NO: 49), GEDA (SEQ ID NO: 13),VEDF (SEQ ID NO: 51), YEDE (SEQ ID NO: 52), IEDL (SEQ ID NO: 53), WEDY (SEQ IDNO: 54), DEDW (SEQ ID NO: 55), SEDL (SEQ ID NO: 56), YEDQ (SEQ ID NO: 57), LEDW(SEQ ID NO: 58), YEDR (SEQ ID NO: 59) and PEDK (SEQ ID NO: 60).

The antibodies and antigen-binding fragments described herein include those that bind tothe amyloid protein in monomeric form with an affinity of less than about 107 M’1.Representative amyloid proteins include serum amyloid A protein (SAA), immunoglobulin lightchain protein (such as VX6 Wii and Vk), human islet amyloid precursor polypeptide (LAPP), betaamyloid peptide, transthyretin (TTR), and ApoAl.

Also provided are isolated nucleic acids encoding an antibody, or antigen-bindingfragment thereof, which specifically binds to an epitope comprising X1EDX2 in an aggregatedamyloid protein, wherein Xi and X2 are any amino acid., including all such antibodies andantigen-binding fragments as described herein above and as set forth in the claims. Furtherprovided are cells expressing such nucleic acids. 15 WO 2009/086539 PCT/US2008/088493

The present invention further provides methods of therapeutically treating orprophylactically treating a subject having AA amyloidosis using a human, humanized, orchimeric antibody, or antigen-binding fragment thereof, that specifically binds to an epitopewithin residues 70-76 of human amyloid A peptide, for example, an epitope within residues 70-76 of SEQ ID NO: 2. Subjects that may benefit from the disclosed therapeutic methods oftreating AA amyloidosis include those subjects suffering from an amyloid disease selected fromthe group consisting of rheumatoid arthritis, juvenile chronic arthritis, ankylosing spondylitis,psoriasis, psoriatic arthropathy, Reiter’s syndrome, Adult Still’s disease, Behcet’s syndrome,Crohn’s disease, leprosy, tuberculosis, bronchiectasis, decubitus ulcers, chronic pyelonephritis,osteomyelitis, Whipple’s disease, Hodgkin’s lymphoma, renal carcinoma, carcinomas of gut,lung and urogenital tract, basal cell carcinoma, hairy cell leukemia, Familial MediterraneanFever, and Castleman’s Disease. Subjects that may benefit from the disclosed prophylacticmethods include those subjects susceptible to or at risk of developing any of the foregoingdisorders.

Also provided are methods of therapeutically treating or prophylactically treating asubject having amyloidosis associated with an aggregated amyloid protein comprising the aminoacid sequence ED using an antibody or antigen-binding fragment that specifically binds to anepitope comprising XjEDX? in an aggregated amyloid protein, wherein Xi and X2 are any aminoacid. Subjects that may benefit from the disclosed therapeutic methods of treating amyloidosisassociated with an aggregated amyloid protein include those subjects suffering from AAamyloidosis, AL amyloidosis, Alzheimer’s disease, Mild Cognitive Impairment, amyloidpolyneuropathy, Mediterranean fever, Muckle-Wells syndrome, reactive systemic amyloidosisassociated with systemic inflammatory diseases, myeloma or macroglobulinemia associatedamyloidosis, amyloidosis associated with immunocyte dyscrasia, monoclonal gammopathy,occult dyscrasia, and local nodular amyloidosis associated with chronic inflammatory diseases.Subjects that may benefit from the disclosed prophylactic methods include those subjectssusceptible to or at risk of developing any of the foregoing disorders. In one aspect of theinvention, the amyloid protein comprises the sequence AEDV (SEQ ID NO: 23), and theamyloidogenic disease treated therapeutically or prophylactically using the disclosed methods isAA amyloidosis, AL amyloidosis, amyloid polyneuropathy, Mediterranean fever, Muckle-Wellssyndrome, reactive systemic amyloidosis associated with systemic inflammatory diseases,myeloma or macroglobulinemia associated amyloidosis, amyloidosis associated with 16 WO 2009/086539 PCT/US2008/088493 immunocyte dyscrasia, monoclonal gammopathy, occult dyscrasia, and local nodularamyloidosis associated with chronic inflammatory diseases.

The disclosed therapeutic and prophylactic methods are useful for treating humansubjects.

Representative indices of efficacious therapeutic treatment include slowing theprogression of amyloidosis, inhibiting deposition of amyloid fibril aggregates, and/or clearing ofamyloid fibril aggregates. Representative indices of efficacious prophylactic treatment includedelaying onset of amyloidosis and/or reducing a risk of amyloidosis.

Still further provided are methods of detecting an amyloid deposit associated with AAamyloidosis in a subject human, humanized, or chimeric antibody, or antigen-binding fragmentthereof, that specifically binds to an epitope within residues 70-76 of human amyloid A peptide,which antibody or antigen-binding fragment is bound to a detectable label, and then detecting thedetectable label in the subject. Additional methods comprise detecting an aggregated amyloidprotein comprising the amino acid sequence ED using an antibody or antigen-binding fragmentthat specifically binds to an epitope comprising X1EDX2 in an aggregated amyloid protein,wherein Xi and X2 are any amino acid. The foregoing detection methods may be used, forexample, for monitoring onset or progression of disease or therapy in any of the above-noteddiseases and disorders. As for the treatment methods disclosed herein, such monitoring may beperformed in humans as well as non-human subjects. Useful detectable labels includeradiolabels, such as 125I. In performing such detection methods, the step of detecting thedetectable label may be accomplished by non-invasive techniques, such as SPECT/CT imagingand NMR spectroscopy.

Still further provided are methods of active immunotherapy of a subject having AAamyloidosis using an agent that induces an immune response to residues 70-76 of amyloid Apeptide effective to induce an immune response comprising antibodies against residues 70-76 ofan amyloid A peptide. Representative agents for inducing the immune response include residues70-76 of amyloid A peptide or a sub fragment of at least 3 contiguous residues thereof havingfewer than 20 contiguous amino acids from an AA peptide. These methods are useful boththerapeutically and/or prophylactically for treatment of the subjects described herein above withrespect to passive immunotherapy, i.e., by administering an antibody or antigen-bindingfragment that specifically binds to residues 70-76 of amyloid A peptide. Indices of therapeuticand prophylactic efficacy are also as noted herein above with respect to passive immunotherapy. 17 WO 2009/086539 PCT/US2008/088493

The foregoing summarizes particular aspects of the invention, and additional aspects ofthe invention are described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Sequence alignment of human SAA1, human SAA2, human SAA3 andhuman SAA4.

Figure 2: Sequence alignment of human SAA1 and human AA1.

Figure 3: Sequence alignment of human SAA2 and human AA2.

Figure 4: Sequence alignment t of human SAA3 and human AA3.

Figure 5: Sequence alignment of human SAA4 and hHuman AA4.

Figure 6: Sequence alignment of human AA1, human AA2, human AA3 and human AA4.

Figure 7: Sequence alignment of the last seven residues of human AA1, hHuman AA2,human AA3 and human AA4.

Figure 8: Sequence alignment of mouse SAA1, mouse SAA2, mouse SAA3 and mouse SAA4.

Figure 9: Sequence alignment of mouse SAA1 and mouse AA1.

Figure 10: Sequence alignment of mouse SAA2 and mouse AA2.

Figure 11: Sequence alignment of mouse SAA3 and mouse AA3.

Figure 12: Sequence alignment of mouse SAA4 and mMouse AA4.

Figure 13: Sequence alignment of mouse AA1, mouse AA2, mouse AA3 mouse AA4.Figure 14: Sequence alignment of the last seven residues of mouse AA1, mouse AA2, mouse AA3 mouse AA4.

Figure 15: Sequence alignment of human SAA1 and mouse SAA1.

Figure 16: Sequence alignment of human AA1 and mouse AA1.

Figure 17: Sequence alignment of human SAA1 and mouse SAA1 Fragment.

Figure 18: Sequence alignment of human SAA1 alpha, human SAA1 beta, and human SAA1 gamma.

Figure 19: Sequence alignment of human SAA2 alpha and human SAA2 beta.

Figure 20: Sequence comparison of SAA proteins. The peptide region used to generate 2A4, 8G9 and 7D8 is shown in dashed lines. The 8 amino acid insert between positions 67 and68 in the Shar Pei sequence is indicated by the underline and arrow. Alignment performed with 18 WO 2009/086539 PCT/US2008/088493 CLUSTALW.

Figure 21: Germline sequences of Vk light chains.

Figure 22: Germline sequences of νλ light chains.

Figure 23: Amino acid sequence of VX6 Wil. 5 Figure 24: X-ray crystal of VX6 Wil showing position of Glu50-Asp51.

Figure 25: X-ray crystal of VX6 Wil showing position of Glu81-Asp82

Figure 26: Binding kinetics of Elan mAbs to synthetic VX6 Wil fibrils. BIAcore measurements of the interaction of mAbs 2A4, 7D8 and 8G9 at 6.6 nM to immobilized VX6 Wilfibrils. The calculated KD for each interaction was ~ 1 nM. 10 Figure 27: Concentration-dependent binding kinetics of mAb 7D8 to synthetic VX6 Wil fibrils. The antibody interaction at a concentration of 6.6 - 33.3 nM to immobilized νλ6 Wilfibrils was measured by BIAcore.

Figure 28: Binding kinetics of mAb 7D8 to synthetic Vx6 Wil fibrils in the presence ofthe p39 and p41 peptides. The interaction of the mAb 7D8 at 6.6 nM with immobilized V/.6 Will 15 fibrils was measured by BIAcore in the presence of peptides p39 and p41 at 1 or 20 pg/mL.

Figure 29: Reactivity of monoclonal antibodies with ΑΕλ tissue amyloid deposits. 125

Figure 30: Biodistribution of I-labeled mAb 7D8 in mice bearing a human ΑΕλamyloidoma.

Figure 31: Interaction of anti-AA of culture supernatants with murine-derived AA 20 fibrils. Results of mAb culture supernatants binding murine AA AEF. Upper and lower panelsare data on first and second culture fluid harvest, respectively.

Figure 32: SDS-PAGE analysis of protein A-purified 2A4, 8G9 and 7D8 mAbs.

Figure 33: Binding of purified mAbs to immunizing (p#39) and control peptide (p#41).Figure 34: Binding to murine AA amyloid extract (AEF). 25 Figure 35: Binding of purified mAbs to human renal AA amyloid extract.

Figures 36A-36E: Sequences of murine 2A4, 7D8, and 8G9 light chain and heavy chain variable regions (Figure 36A); sequences of humanized 2A4/8G9 and 7D8 light chainvariable regions (Figures 36B-36C); sequences of human light chain variable regions used asacceptor frameworks (Figure 36D); sequences of humanized 2A4/7D8/8G9 heavy chain variable 30 regions and human heavy chain variable region used as acceptor framework (Figure 36E).Underlining, CDRs; double underlining, leader sequences; lower case, back mutations. 19 WO 2009/086539 PCT/US2008/088493

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an isolated antibody or antigen-binding fragment thereof, whichspecifically binds to an epitope including XiEDX2 in an aggregated amyloid protein, wherein Xiand X2 are any amino acid.

Representative antibodies of the invention also include antibodies or fragments thereofthat (a) compete for binding to an epitope including XiEDX2 with a 2A4, 7D8, or 8G9 antibody;(b) bind to the same epitope including XjEDX2 as a 2A4, 7D8, or 8G9 antibody; (c) include anantigen binding domain of a 2A4, 7D8, or 8G9 antibody; or (d) include the six complementaritydetermining regions (CDRs) of a 2A4, 7D8, or 8G9 antibody.

The invention also provides an isolated antibody variable region including (a) a lightchain variable region of an antibody derived from a 2A4, 7D8, or 8G9 antibody; or (b) a heavychain variable region of an antibody derived from a 2A4, 7D8, or 8G9 antibody.

The invention also provides an isolated nucleic acid encoding an antibody light chainvariable region or heavy chain variable region including (a) a nucleotide sequence that encodes alight chain or heavy chain variable region of a 7D8, 2A4, or 8G9 antibody; (b) a nucleotidesequence that is identical to a nucleotide sequence of a 7D8, a 2A4, or an 8G9 antibody thatencodes a light chain or heavy chain variable region; (c) a nucleotide sequence that issubstantially identical to a nucleotide sequence of (a) or (b); or (d) a nucleic acid that specificallyhybridizes to a nucleic acid having a nucleotide sequence that is the complement of a nucleotidesequence of (a) or (b) under stringent hybridization conditions.

Cells expressing the antibodies and antigen-binding fragments of the present inventionare also provided. The invention further provides cells expressing nucleic acids of the invention.

The invention also includes methods of treating amyloid diseases and methods ofprophylaxis of amyloid diseases using the antibodies and antigen-binding fragments of theinvention. Currently, there are no approved specific amyloid-directed treatments for any of theamyloid diseases, including AA Amyloidosis and AL amyloidosis. See Gillmore J.D. et al.,Lancet 358:24-9 (2001). Where there is an underlying or associated disease state, therapydirected towards decreasing the production of amyloidogenic protein by treating the underlyingdisease. For example, current treatment strategy for AA Amyloidosis is to target underlyinginflammation, reducing ApoSSA levels to below 10mg/l. Currently employed therapies includechemotherapy (cholorambucil and MTX), immuno-suppressants (azathioprine), anti- 20 WO 2009/086539 PCT/US2008/088493 inflammatory drugs (colchicine) and TNF inhibitors. The invention provides pharmaceuticalcompositions and methods for treating a number of amyloid diseases, including amyloidosis,such as, for example, AA amyloidosis and AL amyloidosis. According to one aspect, theinvention includes pharmaceutical compositions that include, as an active ingredient, an agentthat is effective to induce an immune response in a patient against an amyloid component. Theagent can be a peptide comprising a fragment consisting of the amino acid sequence XiEDX2derived from an amyloid protein. The agent can be an antibody that specifically binds to anepitope comprising XiEDX2. In other embodiments, the agent can be an antigen-bindingfragment of an antibody. Such compositions will generally also include excipients and inpreferred embodiments may include adjuvants. In further preferred embodiments, the adjuvantsinclude, for example, aluminum hydroxide, aluminum phosphate, MPL™, QS-21(STIMULON™) or incomplete Freund’s adjuvant. According to a related embodiment, suchpharmaceutical compositions may include a plurality of agents effective to induce an immuneresponse against more than one amyloid component in the patient.

In a related embodiment, the agent is effective to produce an immune response directedagainst an aggregated amyloid protein, such as a fibril peptide or protein amyloid component.Preferably, such a fibril peptide or protein is derived from a fibril precursor protein known to beassociated with certain forms of amyloid diseases, as described herein. Such precursor proteinsinclude, but are not limited to, Serum Amyloid A protein (ApoSSA), immunoglobulin lightchain, immunoglobulin heavy chain, ApoAI, transthyretin, lysozyme, fibrogen a chain, gelsolin,cystatin C, Amyloid β protein precursor (β-ΑΡΡ), Beta2 microglobulin, prion precursor protein(PrP), atrial natriuretic factor, keratin, islet amyloid polypeptide, a peptide hormone, andsynuclein. Such precursors also include mutant proteins, protein fragments and proteolyticpeptides of such precursors. In a preferred embodiment, the agent is effective to induce animmune response directed against a neoepitope formed by a fibril protein or peptide, with respectto a fibril precursor protein. That is, as described in more detail herein, many fibril-formingpeptides or proteins are fragments of such precursor proteins, such as those listed above. Whensuch fragments are formed, such as by proteolytic cleavage, epitopes may be revealed that arenot present on the precursor and are therefore not immunologically available to the immunesystem when the fragment is a part of the precursor protein. Agents directed to such epitopesmay be preferred therapeutic agents, since they may be less likely to induce an autoimmune 21 WO 2009/086539 PCT/US2008/088493 response in the patient. Preferably, such agents preferentially produce an immune responsedirected against a pathological form of the amyloid protein, for example, an aggregated amyloidprotein, relative to nonpathological forms of the amyloid protein.

According to a related embodiment, pharmaceutical compositions of the inventioninclude agents directed to amyloid aggregates, such as those selected from the group including,but not limited to the following aggregated (e.g., fibril) peptides or proteins: AA, AL, ATTR,AApoAl, Alys, Agel, Acys, Αβ, AB2M, AScr, Acal, AIAPP and synuclein-NAC fragment. Thefull names and compositions of these peptides are described herein. Such peptides can be madeaccording to methods well known in the art, as described herein.

The methods comprise administering to the patient an effective dosage of an antibodythat specifically binds to an epitope comprising XiEDX2 in an amyloid protein, wherein Xi is H,T, F, S, P, A or any other amino acid residue immediately preceding ED in such amyloid protein;and wherein X2 is T, S, E, R, I, V, F, A or any other amino acid residue immediately followingED in such amyloid protein. In some methods, the patient is suffering from an amyloidosisassociated with an aggregated amyloid protein comprising the amino acid sequence ED. Someantibodies specifically bind to an epitope consisting of such XiEDX2. In some antibodies, Xj isΗ, T, F, S, P, or A and X2 is T, S, E, D, R, I, V, F or A. In some such antibodies, when Xi is H,X2 is T or A; when Xi is A, X2 is S, T, E or V; when Xi is T, X2 is E; when Xi is F, X2 is D;when Xi is S, X2 is E, F or A; and when Xi is P, X2 is E, I or F. In some antibodies, Xi is Η, T,F, S, P, or A and X2 is T, S, E, D, R, I, V, F or A, with the proviso that if Xi is A, X2 is not V. Insome antibodies, when Xi is A, X2 is S, T or E.

Some antibodies specifically bind an epitope comprising the amino acid sequenceGHEDT, (SEQ ID NO: 3), HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO 13), AEDT (SEQ IDNO: 14), HEDA (SEQ ID NO: 15), TEDE, (SEQ ID NO: 16), FEDD, (SEQ ID NO: 17), SEDE,(SEQ ID NO: 18), AEDE, (SEQ ID NO: 19), PEDE, (SEQ ID NO: 20), PEDI, (SEQ ID NO: 21),PEDF, (SEQ ID NO: 22), AEDV, (SEQ ID NO: 23), SEDF (SEQ ID NO: 24), or SEDA, (SEQID NO: 25).

Some antibodies specifically bind to a peptide comprising an amino acid sequenceselected from the group consisting of GHEDT, (SEQ ID NO: 3), HEDT, (SEQ ID NO: 12),AEDS, (SEQ ID NO: 13), AEDT, (SEQ ID NO: 14), HEDA, (SEQ ID NO: 15), TEDE, (SEQ IDNO: 16), FEDD, (SEQ ID NO: 17), SEDE, (SEQ ID NO: 18), AEDE, (SEQ ID NO: 19), PEDE, 22 WO 2009/086539 PCT/US2008/088493 (SEQ ID NO: 20), PEDI, (SEQ ID NO: 21), PEDF, (SEQ ID NO: 22), SEDF, (SEQ ID NO: 24),and SEDA, (SEQ ID NO: 25). Some antibodies specifically bind to a peptide comprising anamino acid sequence selected from the group consisting of GHEDT, (SEQ ID NO: 3, HEDT,(SEQ ID NO: 12), AEDS, (SEQ ID NO: 13), AEDT, (SEQ ID NO: 14), HEDA, (SEQ ID NO:15), and TEDE, (SEQ ID NO: 16).

Some antibodies specifically bind to an epitope within residues 70 to 76 of AA. Someantibodies specifically bind to an epitope within residues 71 to 75 of AA. Some antibodies areraised to a peptide comprising GHEDT, (SEQ ID NO: 3).

Some antibodies specifically bind to a peptide comprising the amino acid sequencePEDS, (SEQ ID NO: 26), PEDL, (SEQ ID NO: 27), TEDV, (SEQ ID NO: 28), AEDE, (SEQ IDNO: 19), SEDI, (SEQ ID NO: 29), and TEDT, (SEQ ID NO: 30). Some antibodies specificallybind to a peptide comprising the amino acid sequence LEDG, (SEQ ID NO: 31), AEDM, (SEQID NO: 32), HEDS, (SEQ ID NO: 33), CEDD, (SEQ ID NO: 34), QEDS, (SEQ ID NO: 35),REDS, (SEQ ID NO: 36), TEDG, (SEQ ID NO: 37), QEDR, (SEQ ID NO: 38), TEDL, (SEQ IDNO: 39), PEDN, (SEQ ID NO: 40), EEDP, (SEQ ID NO: 41), LEDL, (SEQ ID NO: 42), KEDA,(SEQ ID NO: 43), SEDC, (SEQ ID NO: 44), EEDD, (SEQ ID NO: 45), SEDK, (SEQ ID NO:46), DEDD, (SEQ ID NO: 47), DEDG, (SEQ ID NO: 48), LEDE, (SEQ ID NO: 49), GEDA,(SEQ ID NO: 50), VEDF, (SEQ ID NO: 51), YEDE, (SEQ ID NO: 52), IEDL, (SEQ ID NO:53), WEDY, (SEQ ID NO: 54), DEDW, (SEQ ID NO: 55), SEDL, (SEQ ID NO: 56), YEDQ,(SEQ ID NO: 57), LEDW, (SEQ ID NO: 58), YEDR, (SEQ ID NO: 59), and PEDK, (SEQ IDNO: 60).

Some antibodies specifically bind to a peptide comprising the amino acid sequenceAEDV, (SEQ ID NO: 23). Some antibodies specifically bind to a peptide comprising the aminoacid sequence SEDF, (SEQ ID NO: 24) or PEDF, (SEQ ID NO: 22). Some antibodiesspecifically bind to a peptide comprising the amino acid sequence AEDS, (SEQ ID NO: 13).Some antibodies specifically bind to a peptide comprising the amino acid sequence PEDI (SEQID NO: 21), AEDV, (SEQ ID NO 23), SEDF, (SEQ ID NO: 24), SEDA, (SEQ ID NO: 25),SEDE, (SEQ ID NO: 18), AEDE, (SEQ ID NO: 19), and PEDE, (SEQ ID NO: 20). Someantibodies bind to a peptide comprising the amino acid sequence TEDE, (SEQ ID NO: 16).

Some antibodies specifically bind to a peptide comprising the amino acid sequenceAEDV, (SEQ ID NO: 23). Some antibodies specifically bind to a peptide comprising the aminoacid sequence SEDF, (SEQ ID NO: 24) or PEDF, (SEQ ID NO: 22). Some antibodies 23 WO 2009/086539 PCT/US2008/088493 specifically bind to a peptide comprising the amino acid sequence AEDS, (SEQ ID NO: 13).Some antibodies specifically bind to a peptide comprising the amino acid sequence PEDI (SEQID NO: 21), AEDV, (SEQ ID NO 23), SEDF, (SEQ ID NO: 24), SEDA, (SEQ ID NO: 25),SEDE, (SEQ ID NO: 18), AEDE, (SEQ ID NO: 19), and PEDE, (SEQ ID NO: 20). Someantibodies bind to a peptide comprising the amino acid sequence TEDE, (SEQ ID NO: 16).

Any of the antibodies described above can be administered in the methods describedabove to treat or effect prophylaxis of a disease characterized by the deposition of an amyloidprotein, such as, for example, an amyloid protein comprising the amino acid sequence ED. Insome methods, if the amyloid protein comprises the amino acid sequence AEDV, (SEQ ID NO:23), then the antibody is not administered to treat or effect prophylaxis of Alzheimer’s disease orMild Cognitive Impairment. The amyloid protein can be any of serum amyloid A protein,immunoglobulin light chain protein, such as, for example, V76 Wil or Vk, human islet amyloidprecursor polypeptide (IAPP), beta amyloid peptide, transthyretin (TTR) or ApoAl.

Optionally, the patient is human. Optionally, the antibody specifically binds to apeptide whose residues consist of SEQ ID NOS. 4, 5, 6, 7, 8, 9, 10, or 11. Optionally, theantibody specifically binds to an epitope within residues 70-76 of (SEQ ID NO: 2). Optionally,the antibody is a human antibody, humanized antibody or chimeric antibody. Optionally, thehuman antibody is of human isotype IgGl, IgG4, IgG2 or IgG3. Optionally, the humanizedantibody is of human isotype IgGl, IgG4, IgG2 or IgG3. Optionally, the chimeric antibody is ofhuman isotype IgGl, IgG4, IgG2 or IgG3. Optionally, the antibody is a mouse antibody.Optionally, the antibody is a polyclonal antibody. Optionally, the antibody is a monoclonalantibody.

In some treatment methods, the antibody comprises two copies of the same pair of lightand heavy chains. In other methods, the antibody is a bispecific antibody comprising a first lightand heavy chain pair that specifically binds to the epitope of Αβ and a second light and heavychain pair that specifically binds to an Fc receptor on microglial cells. In other methods, a chainof the antibody is fused to a heterologous polypeptide.

Some treatment methods, the dosage of antibody is at least 1 mg/kg body weight of thepatient. In other methods, the dosage of antibody is at least 10 mg/kg body weight of the patient.

In some treatment methods, the antibody is administered with a carrier as apharmaceutical composition. In other methods, wherein the antibody is a human antibody to AA 24

SUBSTITUTE SHEET prepared from B cells from a human immunized with an AA peptide. Optionally, the humanimmunized with AA peptide is the patient. In some methods, the antibody is administeredintraperitoneally, orally, intranasally, subcutaneously, intramuscularly, topically or intravenously.

In some treatment methods, the antibody is administered by administering a5 polynucleotide encoding at least one antibody chain to the patient and the polynucleotide isexpressed to produce the antibody chain in the patient. Optionally, the polynucleotide encodesheavy and light chains of the antibody and the polynucleotide is expressed to produce the heavy and light chains in the patient.

Some of the above treatment methods further comprise administering an effective dosage10 of at least one other antibody that binds to a different epitope of AA. Some of the above treatment methods further comprise monitoring the patient for level of administered antibody in the blood ofthe patient. In other methods, the antibody is administered in multiple dosages over a period of atleast six months. In other methods, the antibody is administered as a sustained release composition. 15 The invention further provides methods of effecting prophylaxis of AA amyloidosis in a patient susceptible to AA amyloidosis. The methods comprise administering to the patient aneffective dosage of an antibody that specifically binds to an epitope within residues 70 to 76 of AA. Optionally, the patient is human. Optionally, the antibody specifically binds to a peptidewhose residues consist of SEQ ID NOS. 4, 5, 6, 7, 8, 9, 10, or 11. Optionally, the antibody 20 specifically binds to an epitope within residues 70-76 of (SEQ ID NO: 2). In some methods, thepatient suffers from an underlying amyloid disease selected from the group consisting ofrheumatoid arthritis, juvenile chronic arthritis, ankylosing spondylitis, psoriasis, psoriaticarthropathy, Reiter’s syndrome, Adult Still’s disease, Behcet’s syndrome, Crohn’s disease,leprosy, tuberculosis, bronchiectasis, decubitus ulcers, chronic pyelonephritis, osteomyelitis, 25 Whipple’s disease, Hodgkin’s lymphoma, renal carcinoma, carcinomas of gut, lung and urogenitaltract, basal cell carcinoma, hairy cell leukemia, Familial Mediterranean Fever, and Castleman’sDisease.

The invention further provides a human, humanized, or chimeric antibody thatspecifically binds to an epitope within residues 70 to 76 of AA. Optionally, the humanized 30 antibody specifically binds to an epitope within residues 70 to 76 of AA. Optionally, thehumanized antibody is a humanized version 7D8 antibody (ATCC Accession Number PTA-9468). 25

SUBSTITUTE SHEET

Optionally, the humanized antibody is a humanized version 7D29 antibody. Optionally, thehumanized antibody is a humanized version 7D19 antibody. Optionally, the humanized antibodyis a humanized version 7D47 antibody. Optionally, the humanized antibody is a humanizedversion 7D39 antibody. Optionally, the humanized antibody is a humanized version 7D66 5 antibody. Optionally, the humanized antibody is a humanized version 8G9 antibody. Optionally,the humanized antibody is a humanized version 8G3 antibody. Optionally, the humanizedantibody is a humanized version 8G4 antibody. Optionally, the humanized antibody is ahumanized version 8G51 antibody. Optionally, the humanized antibody is a humanized version8G22 antibody. Optionally, the humanized antibody is a humanized version 8G30 antibody. 10 Optionally, the humanized antibody is a humanized version 8G46 antibody. Optionally, thehumanized antibody is a humanized version 2A4 antibody (ATCC Accession Number PTA-9662).Optionally, the humanized antibody is a humanized version 2A20 antibody. Optionally, thehumanized antibody is a humanized version 2A44 antibody. Optionally, the humanized antibodyis a humanized version 2A77 antibody. Optionally, the humanized antibody is a humanized 15 version 2A13 antibody. Optionally, the humanized antibody is a humanized version 2A14antibody.

The invention further provides pharmaceutical compositions. The pharmaceuticalcompositions comprise an antibody that specifically binds to an epitope within residues 70 to 76 ofAA, and a pharmaceutically acceptable carrier. Some pharmaceutical compositions comprise a 20 human, humanized, or chimeric antibody that specifically binds to an epitope within residues 70 to76 of AA, and a pharmaceutically acceptable carrier. Other pharmaceutical compositionscomprise an antibody that specifically binds to an epitope within residues 70 to 76 of AA and apharmaceutically acceptable carrier, where the isotype of the antibody is human IgGl, and apharmaceutically acceptable carrier. In some pharmaceutical compositions the isotype of the 25 antibody is human IgG2, IgG3, or IgG4. In some pharmaceutical compositions the antibody ishuman. In some pharmaceutical compositions the antibody is humanized. In somepharmaceutical compositions the antibody is chimeric. In some pharmaceutical compositions theantibody is a polyclonal antibody. In some pharmaceutical compositions the antibody is amonoclonal antibody. 30 In some pharmaceutical compositions the antibody comprises two copies of the same

pair of light and heavy chains. In some pharmaceutical compositions the antibody is a bispecificantibody comprising a first light and heavy chain pair that specifically binds to the epitope of AA 26 WO 2009/086539 PCT/US2008/088493 and a second light and heavy chain pair that specifically binds to an Fc receptor on microglialcells. In some pharmaceutical compositions a chain of the antibody is fused to a heterologouspolypeptide. In some pharmaceutical compositions the carrier is a physiologically acceptablediluent for parenteral administration. Some pharmaceutical compositions are adapted to beadministered intraperitoneally, orally, intranasally, subcutaneously, intramuscularly, topically orintravenously. Some pharmaceutical compositions are adapted to be administered in multipledosages over a period of at least six months. Some pharmaceutical compositions are adapted tobe administered as a sustained release composition. Some pharmaceutical compositions furthercomprise at least one other antibody that binds to a different epitope of AA.

The invention provides methods of treating AA amyloidosis in a patient. The methodscomprise administering an agent that induces an immune response to AA70-76 in a regimeeffective to induce an immune response comprising antibodies against AA70-76 in a regimeeffective to induce an immune response comprising antibodies against AA70-76. In somemethods the patient is human. Optionally, the agent comprises AA70-76 or a subfragment of atleast 3 contiguous residues thereof and has fewer than 20 contiguous amino acids from an AApeptide. Optionally, the agent is a peptide having a sequence selected from the group consistingof SEQ ID NOS 4, 5, 6, 7, 8, 9, 10 and 11. and subffagments of at least 3 contiguous residuesthereof and has fewer than 20 amino acids from an AA peptide. Optionally, the agent is linked atits N and C termini to first and second heterologous polypeptides. Optionally, the agent is linkedat its N terminus to a heterologous polypeptide, and at its C-terminus to at least one additionalcopy of the N-terminal segment. In some methods the heterologous polypeptide induces a T-cellresponse against the heterologous polypeptide and thereby a B-cell response against AA. Insome methods the polypeptide further comprises at least one additional copy of AA. Optionally,the polypeptide comprises from N-terminus to C-terminus, AA, a plurality of additional copiesof AA, and the heterologous amino acid segment.

In some treatment methods the polypeptide is administered with an adjuvant thatenhances an immune response to the N-terminal segment. Optionally, the adjuvant and thepolypeptide are administered together as a composition. Optionally, the adjuvant is administeredbefore the polypeptide. Optionally, the adjuvant is administered after the polypeptide. In somemethods the adjuvant is alum. In some methods the adjuvant is MPL. In some methods theadjuvant is QS-21. In some methods the adjuvant is incomplete Freund’s adjuvant. In somemethods the immune response comprises T-cells that bind to the AA peptide as a component of 27 WO 2009/086539 PCT/US2008/088493 an MHC I or MHC II complex.

The invention provides methods of effecting prophylaxis of AA amyloidosis in apatient. The methods comprise administering an agent that induces an immune response toAA70-76 in a regime effective to induce an immune response comprising antibodies againstAA70-76 in a regime effective to induce an immune response comprising antibodies againstAA70-76. In some methods the patient is human. In some methods the patient is asymptomatic.In some methods the patient suffers from an underlying amyloid disease selected from the groupconsisting of rheumatoid arthritis, juvenile chronic arthritis, ankylosing spondylitis, psoriasis,psoriatic arthropathy, Reiter’s syndrome, Adult Still’s disease, Behcet’s syndrome, Crohn’sdisease, leprosy, tuberculosis, bronchiectasis, decubitus ulcers, chronic pyelonephritis,osteomyelitis, Whipple’s disease, Hodgkin’s lymphoma, renal carcinoma, carcinomas of gut,lung and urogenital tract, basal cell carcinoma, hairy cell leukemia, Familial MediterraneanFever, and Castleman’s Disease.

In some methods of effecting prohylaxis, the agent comprises AA70-76 or asubfragment of at least 3 contiguous residues thereof and has fewer than 20 contiguous aminoacids from an AA peptide. Optionally, the agent is a peptide having a sequence selected fromthe group consisting of SEQ ID NOS 4, 5, 6, 7, 8, 9, 10 and 11. and subfragments of at least 3contiguous residues thereof and has fewer than 20 amino acids from an AA peptide. Optionally,the agent is linked at its N and C termini to first and second heterologous polypeptides.Optionally, the agent is linked at its N terminus to a heterologous polypeptide, and at its C-terminus to at least one additional copy of the N-terminal segment. In some methods theheterologous polypeptide induces a T-cell response against the heterologous polypeptide andthereby a B-cell response against AA. In some methods the polypeptide further comprises atleast one additional copy of AA. Optionally, the polypeptide comprises from N-terminus to C-terminus, AA, a plurality of additional copies of AA, and the heterologous amino acid segment.

The invention further provides pharmaceutical compositions. The pharmaceuticalcompositions comprise an AA fragment consisting of residues beginning at residue 70 of AA andending at residue 76 of AA. Optionally, the AA fragment is linked at its C-terminus to aheterologous polypeptide. Optionally, the AA fragment is linked at its N-terminus to aheterologous polypeptide. Optionally, the AA fragment is linked at its N and C termini to firstand second heterologous polypeptides. Optionally, the AA fragment is linked at its N terminusto a heterologous polypeptide, and at its C-terminus to at least one additional copy of the N- 28 WO 2009/086539 PCT/US2008/088493 terminal segment. Optionally, the polypeptide further comprises at least one additional copy ofthe N-terminal segment. Optionally, the polypeptide comprises from N-terminus to C-terminus,AA, a plurality of additional copies of the N-terminal segment, and the heterologous amino acidsegment. In some pharmaceutical compositions the heterologous polypeptide induces a T-cellresponse against the heterologous polypeptide and thereby a B-cell response against the N-terminal segment.

Some pharmaceutical compositions further comprise an adjuvant that enhances animmune response to AA. Optionally, the adjuvant is alum. Optionally, the adjuvant is MPL.Optionally, the adjuvant is QS-21. Optionally, the adjuvant is incomplete Freund’s adjuvant.Optionally, the adjuvant further comprises GM-CSF. Optionally, the adjuvant is M-CSF.Optionally, the composition comprises greater than 10 micrograms of the polypeptide.

The invention provides methods of treating AA amyloidosis in a patient. The methodscomprise administering an agent effective to induce an immune response against a peptidecomponent of an amyloid deposit in the patient and a different agent that treats an underlyingdisease, and thereby treating AA amyloidosis in the patient. In some methods the underlyingdisease is selected from the group consisting of rheumatoid arthritis, juvenile chronic arthritis,ankylosing spondylitis, psoriasis, psoriatic arthropathy, Reiter’s syndrome, Adult Still’s disease,Behcet’s syndrome, Crohn’s disease, leprosy, tuberculosis, bronchiectasis, decubitus ulcers,chronic pyelonephritis, osteomyelitis, Whipple’s disease, Hodgkin’s lymphoma, renalcarcinoma, carcinomas of gut, lung and urogenital tract, basal cell carcinoma, hairy cellleukemia, Familial Mediterranean Fever, and Castleman’s Disease.

The invention provides methods of effecting prophylaxis of AA amyloidosis in apatient. The methods comprise administering an agent effective to induce an immune responseagainst a peptide component of an amyloid deposit in the patient and a different agent that treatsan underlying disease, and thereby treating AA amyloidosis in the patient. In some methods theunderlying disease is selected from the group consisting of rheumatoid arthritis, juvenile chronicarthritis, ankylosing spondylitis, psoriasis, psoriatic arthropathy, Reiter’s syndrome, Adult Still’sdisease, Behcet’s syndrome, Crohn’s disease, leprosy, tuberculosis, bronchiectasis, decubitusulcers, chronic pyelonephritis, osteomyelitis, Whipple’s disease, Hodgkin’s lymphoma, renalcarcinoma, carcinomas of gut, lung and urogenital tract, basal cell carcinoma, hairy cellleukemia, Familial Mediterranean Fever, and Castleman’s Disease.

The invention provides methods of screening an antibody for activity in treating a 29 WO 2009/086539 PCT/US2008/088493 patient having AA amyloidosis. The methods comprise contacting the antibody with AA peptideand determining whether the antibody specifically binds to AA, specific binding providing anindication that the antibody has activity in treating AA amyloidosis.

The invention provides methods of screening an antibody for activity in clearing abiological entity physically associated with an antigen. The methods comprise combining theantigen-associated biological entity, the antibody and phagocytic cells bearing Fc receptors in amedium; and monitoring the amount of the antigen-associated biological entity remaining in themedium, a reduction in amount of the antigen-associated biological entity indicating the antibodyhas clearing activity against the antigen. In some methods the monitoring step monitors theamount of the antigen remaining in the medium. In some methods the combining comprisesadding antigen-associated biological entity to the medium, and contacting the medium with thephagocytic cells bearing Fc receptors. In some methods the antigen-associated biological entityis provided as a tissue sample. In some methods the antigen is the biological entity. In somemethods the tissue sample comprises an amyloid deposit. Optionally, the tissue sample is fromthe patient or a mammal having AA Amyloidosis pathology. In some methods, the antigen isAA. In some methods the phagocytic cells are microglial cells. In some methods the tissuesample is selected from the group consisting of a cancerous tissue sample, a virally infectedtissue sample, a tissue sample comprising inflammatory cells, a nonmalignant abnormal cellgrowth, and a tissue sample comprising an abnormal extracellular matrix.

The invention provides methods of detecting an amyloid deposit in a patient. Themethods comprise administering to the patient an antibody that specifically binds to an epitopewithin amino acids 70-76 of AA and detecting presence of the antibody in the patient.Optionally, the antibody is labeled. Optionally, the antibody is labeled with a paramagneticlabel. Optionally, the labeled antibody is detected by nuclear magnetic resonance. Optionally,the labeled antibody is detected with SPECT/CT imaging. In some methods, the antibody lackscapacity to induce a clearance response on binding to an amyloid deposit in the patient.

The invention provides diagnostic kits. The kits comprise an antibody that specificallybinds to an epitope with residues 70-76 of AA. Some kits further comprise labeling describinguse of the antibody for in vivo diagnosis or monitoring of a disease associated with amyloiddeposits of AA in a patient. In some embodiments, the kits include instructions for use of theantibody or antigen-binding fragment thereof in detecting AA.

The invention further provides a method of diagnosing amyloidosis in a subject 30 WO 2009/086539 PCT/US2008/088493 comprising: (a) administering to the subject an antibody or antigen-binding fragment thereof thatis bound to a detectable label, wherein the antibody or fragment thereof specifically binds to anepitope comprising X1EDX2 in an aggregated amyloid protein, wherein Xi and X2 are any aminoacid; and (b) detecting the presence or absence of the bound antibody or fragment thereof,wherein the presence of the bound antibody or fragment indicates a diagnosis of AAamyloidosis.

Further provided herein is a method of treatment or prophylaxis of amyloidosis usingan antibody or antigen-binding fragment thereof, which specifically binds to an epitopecomprising X j EDX2 in an aggregated amyloid protein, wherein Xi and X2 are any amino acid.

The present invention provides an antibody or antigen-binding fragment thereof thatbinds specifically to an epitope comprising X1EDX2, in an aggregated amyloid protein, whereinXi and X2 are any amino acid. For example, Xi includes Η, T, F, S, P, A, L, C, Q, R, E, K, D, G,V, Y, I or W, such as Η, T, F, S, P, or A, or such as Η, T, F, or A. X2 includes T, S, E, R, I, V, F, D, A, G, M, L, N, P, C, K, Y, or Q, such as T, S, E, R, I, V, F, D, or A, or such as T, S, E, D, or A. In other examples, Xi is Η, T, or A and X2 is T, S, E, or A, such as Xi is H or A and X2 is T,S, or A. In yet additional examples, Xi is H and H2 is T or A; or Xi is A and X2 is S, T, E, or V,such as Xi is A and X2 is S, T, or E, or Xi is T and X2 is E, or Xi is F and X2 is D, or Xi is S andX2 is E, F, or A; or Xi is P and X2 is E, I, or F.

In particular, the epitopes include amino acid sequences such as those set forth in SEQID NO: 3 through to SEQ ID NO: 25, such as SEQ ID NOS: 3, 12, 13, 14, 15, and 16.Additional examples include SEQ ID NOS: 4, 5, 7, 8, and 9, such as SEQ ID NO: 4. Antibodiesof the invention that bind to the epitopes, such as to SEQ ID NO: 3, include the 2A4, 7D8, and8G9 antibodies.

The aggregated amyloid proteins to which antibodies of the invention bind are non-monomeric proteins. Such aggregated amyloid proteins include serum amyloid A protein(SAA), immunoglobulin light chain protein, human islet amyloid precursor polypeptide (IAPP),beta amyloid peptide, transthyretin (TTR), and ApoAl, such as SAA.

The invention further provides antibodies or antigen-binding fragments thereof that (a)compete for binding to an epitope that includes X1EDX2 with a 2A4, 7D8, or 8G9 antibody; (b)bind to the same epitope that includes X1EDX2 as a 2A4, 7D8, or 8G9 antibody; (c) have anantigen-binding domain of a 2A4, 7D8, or 8G9 antibody; or (d) include the six complementaritydetermining regions (CDRs) of a 2A4, 7D8, or 8G9 antibody. The invention also provides 31 WO 2009/086539 PCT/US2008/088493 chimeric or humanized versions of a 2A4, 7D8, or 8G9 antibody.

Representive antibodies, which specifically bind to an epitope that includes XiEDX2,also include antibodies having at least one, two, or three of the complementarity determiningregions (CDRs) of a light chain of a 2A4, 7D8 or 8G9 antibody. Antibodies of the invention,which specifically bind to an epitope that includes XiEDX2, also include antibodies having atleast one, two, or three of the CDRs of a heavy chain of a 2A4, 7D8, or 8G9 antibody. CDRs can be identified according to methods known in the art. For example,numbering systems for identifying CDRs are in common use. The Rabat definition is based onsequence variability, and the Chothia definition is based on the location of the structural loopregions. The AbM definition is a compromise between the Rabat and Chothia approaches. TheCDRs of the light chain variable region are bounded by the residues at positions 24 and 34(CDR1-L), 50 and 56 (CDR2-L), and 89 and 97 (CDR3-L) according to the Rabat, Chothia, orAbM algorithm. According to the Rabat definition, the CDRs of the heavy chain variable regionare bounded by the residues at positions 31 and 35B (CDR1-H), 50 and 65 (CDR2-H), and 95and 102 (CDR3-H) (numbering according to Rabat). According to the Chothia definition, theCDRs of the heavy chain variable region are bounded by the residues at positions 26 and 32(CDRl-Et), 52 and 56 (CDR2-H), and 95 and 102 (CDR3-H) (numbering according to Chothia).According to the AbM definition, the CDRs of the heavy chain variable region are bounded bythe residues at positions 26 and 35B (CDR1-H), 50 and 58 (CDR2-H), and 95 and 102 (CDR3-H) (numbering according to Rabat). See Martin et al. (1989) Proc. Natl. Acad. Sci. USA 86:9268-9272; Martin et al. (1991) Methods Enzymol. 203: 121-153; Pedersen et al. (1992)Immunomethods 1: 126; and Rees et al. (1996) In Sternberg M.J.E. (ed.), Protein StructurePrediction, Oxford University Press, Oxford, pp. 141-172.

The antibodies of the invention further include an antibody that binds specifically to anepitope comprising X]EDX2, in an aggregated amyloid protein, wherein Xi and X2 are any aminoacid, having variable regions derived from variable regions of a 2A4, 7D8, or 8G9 antibody.Antibodies having variable regions of 2A4, 7D8, or 8G9 antibodies are also included.

The antibodies of the invention further include chimeric antibodies, human antibodies,humanized antibodies, single chain antibodies, tetrameric antibodies, tetravalent antibodies,multispecific antibodies domain-specific antibodies, domain-deleted antibodies or fusionproteins.

Fragments of the antibodies of the invention are also provided. The fragments of the 32 WO 2009/086539 PCT/US2008/088493 invention may be Fab fragments, Fab’ fragment, F(ab’)2 fragments, Fv fragments or ScFvfragments. Such antibodies or fragments thereof can be coupled with a cytotoxic agent, aradiotherapeutic agent, or a detectable label.

The invention also provides an isolated antibody variable region comprising (a) a lightchain variable region derived from a 7D8, 2A4, or 8G9 antibody light chain variable region, or(b) a heavy chain variable region derived from a 7D8, 2A4, or 8G9 antibody light chain variableregion. Isolated variable regions are also provided having a light chain or heavy chain variableregion of a 7D8, 2A4, or 8G9 antibody. The isolated antibody variable regions are useful inantibody production.

The invention also provides isolated nucleic acids encoding an antibody light chainvariable region or a heavy chain variable region having (a) a nucleotide sequence that encodes alight chain or heavy chain variable region of a 7D8, 2A4, or 8G9 antibody; (b) a nucleotidesequence that is identical to a nucleotide sequence of a 7D8, a 2A4, or an 8G9 antibody thatencodes a light or heavy chain variable region; or (c) a nucleotide sequence that is substantiallyidentical, i.e., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99% to a nucleotide sequence of (a) or(b); or (d) a nucleic acid that specifically hybridizes to a nucleic acid having a nucleotidesequence that is the complement of a nucleotide sequence of (a) or (b) under stringenthybridization conditions, for example, final wash conditions of O.lxSSC at 65° C.

The present invention further provides cells and cell lines expressing the antibodies ornucleic acids of the invention. Representative host cells include mammalian and human cells,such as CHO cells, HEK-293 cells, HeLa cells, CV-1 cells, and COS cells. Methods forgenerating a stable cell line following transformation of a heterologous construct into a host cellare known in the art. Representative non-mammalian host cells include insect cells (Potter et al.(1993) Int. Rev. Immunol. 10(2-3):103-112). Antibodies may also be produced in transgenicanimals (Houdebine (2002) Curr. Opin. Biotechnol. 13(6):625-629) and transgenic plants(Schillberg et al. (2003) Cell Mol. Life. Sci. 60(3):433-45).

The invention also provides methods of treating or effecting prophylaxis of amyloidosisassociated using immunogenic fragments of an amyloid protein comprising XiEDX2, wherein Xiis Η, T, F, S, P, A or any other amino acid residue immediately preceding ED in such amyloidprotein; and wherein X2 is T, S, E, R, I, V, F, A or any other amino acid residue immediatelyfollowing ED in such amyloid protein. Without wishing to be bound by a particular theory, it is 33 WO 2009/086539 PCT/US2008/088493 believed that an epitope comprising XiEDX2 can become exposed when an amyloid proteinaggregates, or undergoes fibrillogenesis or otherwise enters a fibrillar structure, whether bycleavage from a larger precursor protein or by conformational change. For example,representative methods of treatment or prophylaxis of AA amyloidosis include administration ofAA 70-76 fragments or immunogenic fragments thereof. The invention also provides methodsof treating or effecting prophylaxis of amyloidosis associated with deposition of amyloid proteinusing antibodies reactive with XiEDX2 in an aggregated amyloid protein, wherein Xi is Η, T, F,S, P, A or any other amino acid residue immediately preceding ED in such aggregated amyloidprotein; and wherein X2 is T, S, E, R, I, V, F, A or any other amino acid residue immediatelyfollowing ED in such aggregated amyloid protein. Preferably, such antibodies are preferentiallyreactive with aggregated amyloid protein relative to non-pathological amyloid protein. Forexample, methods of treatment or prophylaxis of AA amyloidosis associated with AA fibrils mayinclude administration of antibodies specific for C-terminal region of AA fibrils (-residues 70-76of AA). The antibodies can inhibit formation of AA aggregates (e.g., fibrils) or result in theirdisaggregation and clearance, thus treating or effecting prophylaxis of AA amyloidosis. I. Definitions

The term "substantial identity" means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 65percent sequence identity, preferably at least 80 or 90 percent sequence identity, more preferablyat least 95 percent sequence identity or more (e.g., 99 percent sequence identity or higher).Preferably, residue positions, which are not identical differ by conservative amino acidsubstitutions.

For sequence comparison, typically one sequence acts as a reference sequence, towhich test sequences are compared. When using a sequence comparison algorithm, test andreference sequences are input into a computer, subsequence coordinates are designated, ifnecessary, and sequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., by the localhomology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for 34 WO 2009/086539 PCT/US2OO8/088493 similarity method of Pearson & Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr,Madison, WI), or by visual inspection (see generally Ausubel et al., supra). One example ofalgorithm that is suitable for determining percent sequence identity and sequence similarity is theBLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). Typically, default programparameters can be used to perform the sequence comparison, although customized parameterscan also be used. For amino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff &Henikoff, Proc. Natl. Acad. Sci. USA 89, 10915 (1989))

For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I (hydrophobic sidechains):norleucine, met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; GroupIII (acidic side chains): asp, glu; Group IV (basic side chains): asn, gin, his, lys, arg; Group V(residues influencing chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr,phe. Conservative substitutions involve substitutions between amino acids in the same class.Non-conservative substitutions constitute exchanging a member of one of these classes for amember of another.

The term "all-D" refers to peptides having > 75%, > 80%, > 85%, > 90%, > 95%, and100% D-configuration amino acids.

The term “agent” is used to describe a compound that has or may have apharmacological activity. Agents include compounds that are known drugs, compounds forwhich pharmacological activity has been identified but which are undergoing further therapeuticevaluation, and compounds that are members of collections and libraries that are to be screenedfor a pharmacological activity. “Amyloid disease” or “amyloidosis” refers to any number of disorders which have as asymptom or as part of its pathology the accumulation or formation of amyloid plaques.

An “amyloid plaque” is an extracellular deposit composed mainly of proteinaceous fibrils.Generally, the fibrils are composed of a dominant protein or peptide; however, the plaque may 35 WO 2009/086539 PCT/US2008/088493 also include additional components that are peptide or non-peptide molecules, as describedherein.

An “amyloid protein” or “amyloid peptide” is a protein or peptide capable ofundergoing cleavage, conformational change, aggregation or fibrillogenesis, resulting in theformation of pathological oligomers, amyloid fibrils, amyloid plaques and/or amyloidcomponents.

An “amyloid component” is any molecular entity that is present in an amyloid plaqueincluding antigenic portions of such molecules. Amyloid components include but are not limitedto proteins, peptides, proteoglycans, and carbohydrates.

An “anti-amyloid agent” is an agent which is capable of producing an immune responseagainst an amyloid plaque component in a vertebrate subject, when administered by active orpassive immunization techniques.

An “AA protein” or “AA peptide” refers to the form of amyloid protein A protein orpeptide formed by proteolytic cleavage of serum amyloid A protein (SAA), whether monomericor aggregated, soluble or insoluble.

An “aggregated amyloid protein” or “aggregated amyloid peptide” or “amyloidaggregate” refers to a pathological, non-monomeric, aggregated form of an amyloid protein oramyloid peptide. Aggregated amyloid proteins and amyloid peptides can be soluble or insoluble.Some aggregated amyloid proteins and aggregated amyloid peptides can form oligomers, fibrilsand/or amyloid plaques. Examples of such aggregated amyloid proteins and amyloid peptides,including fibril peptides and proteins are provided herein.

An “AA aggregate” refers to an aggregated form of AA.

Therapeutic agents of the invention are typically substantially pure from undesiredcontaminant. This means that an agent is typically at least about 50% w/w (weight/weight)purity, as well as being substantially free from interfering proteins and contaminants. Sometimesthe agents are at least about 80% w/w and, more preferably at least 90 or about 95% w/w purity.However, using conventional protein purification techniques, homogeneous peptides of at least99% w/w can be obtained. Therapeutic agents of the invention may prevent, effect prophylaxisof, or treat a disease associated with amyloid deposits.

Specific binding between two entities means the entities have a mutual affinity for eachother that is at least 10-, 100- or 100-fold greater than the affinity of either entity for a control,such as unrelated antigen or antibody to a different antigen. The mutual affinity of the two 36 WO 2009/086539 PCT/US2008/088493 entities for each other is usually at least 107 Μ'1, 108 Μ'1, 109 M'1, or IO10 M"1. Affinities greaterthan 108 M’1 are preferred.

The term “immunoglobulin” or “antibody” (used interchangeably herein) refers to anantigen-binding protein having a basic four-polypeptide chain structure consisting of two heavyand two light chains, said chains being stabilized, for example, by interchain disulfide bonds,which has the ability to specifically bind antigen. Both heavy and light chains are folded intodomains. The term “domain” refers to a globular region of a heavy or light chain polypeptidecomprising peptide loops {e.g., comprising 3 to 4 peptide loops) stabilized, for example, by β-pleated sheet and/or intrachain disulfide bond. Domains are further referred to herein as“constant” or “variable”, based on the relative lack of sequence variation within the domains ofvarious class members in the case of a “constant” domain, or the significant variation within thedomains of various class members in the case of a “variable” domain. “Constant” domains onthe light chain are referred to interchangeably as “light chain constant regions”, “light chainconstant domains”, “CL” regions or “CL” domains. “Constant” domains on the heavy chain arereferred to interchangeably as “heavy chain constant regions”, “heavy chain constant domains”,“CH” regions or “CH” domains. “Variable” domains on the light chain are referred tointerchangeably as “light chain variable regions”, “light chain variable domains”, “VL” regionsor “VL” domains. “Variable” domains on the heavy chain are referred to interchangeably as“heavy chain constant regions”, “heavy chain constant domains”, “CH” regions or “CH”domains.

The term “region” refers to a part or portion of an antibody chain and includes constantor variable domains as defined herein, as well as more discrete parts or portions of said domains.For example, light chain variable domains or regions include “complementarity determiningregions” or “CDRs” interspersed among “framework regions” or “FRs”, as defined herein.

Immunoglobulins or antibodies can exist in monomeric or polymeric form. The term“antigen-binding fragment” refers to a polypeptide fragment of an immunoglobulin or antibodybinds antigen or competes with intact antibody {i.e., with the intact antibody from which theywere derived) for antigen binding {i.e., specific binding). The term “conformation” refers to thetertiary structure of a protein or polypeptide {e.g., an antibody, antibody chain, domain or regionthereof). For example, the phrase “light (or heavy) chain conformation” refers to the tertiarystructure of a light (or heavy) chain variable region, and the phrase “antibody conformation” or 37 WO 2009/086539 PCT/US2008/088493 “antibody fragment conformation” refers to the tertiary structure of an antibody or fragmentthereof. “Specific binding” of an antibody mean that the antibody exhibits appreciable affinityfor antigen or a preferred epitope and, preferably, does not exhibit significant crossreactivity.“Appreciable” or preferred binding include binding with an affinity of at least 106, 107, 10s, 109M'1, or 1010 M4. Affinities greater than 107 M4, preferably greater than 108 M4 are morepreferred. Values intermediate of those set forth herein are also intended to be within the scopeof the present invention and a preferred binding affinity can be indicated as a range of affinities,for example, 106 to IO10 M4, preferably 107 to IO10 M4, more preferably 108 to IO10 M4. Anantibody that “does not exhibit significant crossreactivity” is one that will not appreciably bind toan undesirable entity (e.g., an undesirable proteinaceous entity). For example, an antibody thatspecifically binds to AA will appreciably bind AA but will not significantly react with non-AAproteins or peptides (e.g., non-AA proteins or peptides included in plaques). An antibodyspecific for a preferred epitope will, for example, not significantly crossreact with remoteepitopes on the same protein or peptide. Specific binding can be determined according to anyart-recognized means for determining such binding. Preferably, specific binding is determinedaccording to Scatchard analysis and/or competitive binding assays.

Antigen-binding antibody fragments are produced by recombinant DNA techniques, orby enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments include Fab,Fab’, F(ab’)2, Fabc, Fv, single chains, and single-chain antibodies. Additional antibodyfragments and effector function variants are disussed herein in the section entitled “Antibodies”.Other than “bispecific” or “bifunctional” immunoglobulins or antibodies, an immunoglobulin orantibody is understood to have each of its binding sites identical. A “bispecific” or “bifunctionalantibody” is an artificial hybrid antibody having two different heavy/light chain pairs and twodifferent binding sites. Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab’ fragments. See, e.g., Songsivilai & Lachmann, Clin.Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

The term “humanized immunoglobulin” or “humanized antibody” refers to animmunoglobulin or antibody that includes at least one humanized immunoglobulin or antibodychain (i.e., at least one humanized light or heavy chain). The term “humanized immunoglobulinchain” or “humanized antibody chain” (i.e., a “humanized immunoglobulin light chain” or“humanized immunoglobulin heavy chain”) refers to an immunoglobulin or antibody chain (i.e., 38 WO 2009/086539 PCT/US2008/088493 a light or heavy chain, respectively) having a variable region that includes a variable frameworkregion substantially from a human immunoglobulin or antibody and complementaritydetermining regions (CDRs) (e.g., at least one CDR, preferably two CDRs, more preferably threeCDRs) substantially from a non-human immunoglobulin or antibody, and further includesconstant regions (e.g., at least one constant region or portion thereof, in the case of a light chain,and preferably three constant regions in the case of a heavy chain). The term “humanizedvariable region” (e.g., “humanized light chain variable region” or “humanized heavy chainvariable region”) refers to a variable region that includes a variable framework regionsubstantially from a human immunoglobulin or antibody and complementarity determiningregions (CDRs) substantially from a non-human immunoglobulin or antibody.

The phrase “substantially from a human immunoglobulin or antibody” or “substantiallyhuman” means that, when aligned to a human immunoglobulin or antibody amino sequence forcomparison purposes, the region shares at least 80-90%, preferably 90-95%, more preferably 95-99% identity (i.e., local sequence identity) with the human framework or constant regionsequence, allowing, for example, for conservative substitutions, consensus sequencesubstitutions, germline substitutions, backmutations, and the like. The introduction ofconservative substitutions, consensus sequence substitutions, germline substitutions,backmutations, and the like, is often referred to as “optimization” of a humanized antibody orchain. The phrase “substantially from a non-human immunoglobulin or antibody” or“substantially non-human” means having an immunoglobulin or antibody sequence at least 80-95%, preferably 90-95%, more preferably, 96%, 97%, 98%, or 99% identical to that of a non-human organism, e.g., a non-human mammal.

Accordingly, all regions or residues of a humanized immunoglobulin or antibody, or ofa humanized immunoglobulin or antibody chain, except possibly the CDRs, are substantiallyidentical to the corresponding regions or residues of one or more native human immunoglobulinsequences. The term “corresponding region” or “corresponding residue” refers to a region orresidue on a second amino acid or nucleotide sequence which occupies the same (i.e., equivalent)position as a region or residue on a first amino acid or nucleotide sequence, when the first andsecond sequences are optimally aligned for comparison purposes.

The terms “humanized immunoglobulin” or “humanized antibody” are not intended toencompass chimeric immunoglobulins or antibodies, as defined infra. Although humanizedimmunoglobulins or antibodies are chimeric in their construction (i.e., comprise regions from 39 WO 2009/086539 PCT/US2008/088493 more than one species of protein), they include additional features (i.e., variable regionscomprising donor CDR residues and acceptor framework residues) not found in chimericimmunoglobulins or antibodies, as defined herein.

The term “chimeric immunoglobulin” or antibody refers to an immunoglobulin orantibody whose variable regions derive from a first species and whose constant regions derivefrom a second species. Chimeric immunoglobulins or antibodies can be constructed, forexample by genetic engineering, from immunoglobulin gene segments belonging to differentspecies.

An “antigen” is an entity (e.g., a protenaceous entity or peptide) to which an antibodyspecifically binds.

The term “epitope” or “antigenic determinant” refers to a site on an antigen to which animmunoglobulin or antibody (or antigen binding fragment thereof) specifically binds. Epitopescan be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retainedon exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods of determiningspatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensionalnuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in MolecularBiology, Vol. 66, G. E. Morris, Ed. (1996).

Representative antibodies of the invention include an antibody or fragment thereof thatspecifically binds to an epitope that includes XiEDX2 in an aggregated amyloid protein, whichbinds to the epitope including XiEDX2 that is also bound by e.g. a 2A4, 7D8, or 8G9 antibody.Antibodies that recognize the same epitope can be identified in a simple immunoassay showingthe ability of one antibody to block the binding of another antibody to a target antigen, i.e., acompetitive binding assay. Competitive binding is determined in an assay in which theimmunoglobulin under test inhibits specific binding of a reference antibody to a commonantigen, such as Αβ. Numerous types of competitive binding assays are known, for example:solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 40 WO 2009/086539 PCT/US2008/088493 (1986)); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlowand Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phasedirect label RIA using 1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phasedirect biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, such an assay involves theuse of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled testimmunoglobulin and a labeled reference immunoglobulin. Competitive inhibition is measuredby determining the amount of label bound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess. Usually, when acompeting antibody is present in excess, it will inhibit specific binding of a reference antibody toa common antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% or more.

An epitope is also recognized by immunologic cells, for example, B cells and/or Tcells. Cellular recognition of an epitope can be determined by in vitro assays that measureantigen-dependent proliferation, as determined by 3H-thymidine incorporation, by cytokinesecretion, by antibody secretion, or by antigen-dependent killing (cytotoxic T lymphocyte assay).

The term "neoepitope" refers to a new and/or unique site on an antigen to which Band/or T cells respond.

The term "neoepitope antibodies" refer to antibodies that specifically recognize a newN- or C-terminal amino acid sequence exposed by proteolytic cleavage of a molecule, but doesnot bind to such an epitope on the native (uncleaved) molecule. The term "neoepitopeantibodies" may refer to antibodies that specifically recognize a new N- or C-terminal aminoacid sequence exposed by proteolytic cleavage of SAA, but do not bind to such an epitope on thenative (uncleaved) SAA molecule. Some neoepitope antibodies bind to either soluble orinsoluble AA and result in dissociation of AA aggregates, including AA fibrils. A “neoepitopeantibody” may also be an antibody that specifically recognizes a new epitope that is onlyavailable to bind to an antibody after a protein undergoes a conformation change, for example, asin the case of AL amyloidosis and light chain, when only the light chain is expressed and formsamyloid.

The term "immunological" or "immune" response is the development of a beneficialhumoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or theirsecretion products) response directed against an amyloid peptide in a recipient patient. Such aresponse can be an active response induced by administration of immunogen or a passive 41 WO 2009/086539 PCT/US2008/088493 response induced by administration of antibody or primed T-cells. A cellular immune responseis elicited by the presentation of polypeptide epitopes in association with Class I or Class IIMHC molecules to activate antigen-specific CD4+ T helper cells and/or CD8+ cytotoxic T cells.The response may also involve activation of monocytes, macrophages, NK cells, basophils,dendritic cells, astrocytes, microglia cells, eosinophils or other components of innate immunity.The presence of a cell-mediated immunological response can be determined by proliferationassays (CD4+ T cells) or CTL (cytotoxic T lymphocyte) assays (see Burke, supra·, Tigges,supra). The relative contributions of humoral and cellular responses to the protective ortherapeutic effect of an immunogen can be distinguished by separately isolating antibodies andT-cells from an immunized syngeneic animal and measuring protective or therapeutic effect in asecond subject.

An "immunogenic agent" or "immunogen" is capable of inducing an immunologicalresponse against itself on administration to a mammal, optionally in conjunction with anadjuvant.

The term "naked polynucleotide" refers to a polynucleotide not complexed withcolloidal materials. Naked polynucleotides are sometimes cloned in a plasmid vector.

The term "adjuvant" refers to a compound that when administered in conjunction withan antigen augments the immune response to the antigen, but when administered alone does notgenerate an immune response to the antigen. Adjuvants can augment an immune response byseveral mechanisms including lymphocyte recruitment, stimulation of B and/or T cells, andstimulation of macrophages.

The term “effective dose” or “effective dosage” is defined as an amount sufficient toachieve or at least partially achieve the desired effect. The term “therapeutically effective dose”is defined as an amount sufficient to cure or at least partially arrest the disease and itscomplications in a patient already suffering from the disease. Amounts effective for this use willdepend upon the severity of the infection and the general state of the patient’s own immunesystem.

The term "patient" includes human and other mammalian subjects that receive eitherprophylactic or therapeutic treatment.

The invention provides antibodies or antigen-binding fragments thereof that specificallybind to an epitope that includes XiEDX2 in an aggregated amyloid protein, and which competesfor binding to the epitope comprising XiEDX2 with e.g., a 2A4, 7D8, or 8G9 antibody. 42 WO 2009/086539 PCT/US2008/088493

Competition between antibodies is determined by an assay in which the immunoglobulin undertest inhibits specific binding of a reference antibody to a common antigen, such as AA.Numerous types of competitive binding assays are known, for example: solid phase direct orindirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA),sandwich competition assay (see Stahli et al., Methods in Enzymology, 9:242-253 (1983)); solidphase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619 (1986)); solidphase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane,"Antibodies, A Laboratory Manual," Cold Spring Harbor Press (1988)); solid phase direct labelRIA using 1-125 label (see Morel et al., Molec. Immunol. 25(1):7-15 (1988)); solid phase directbiotin-avidin EIA (Cheung et al., Virology, 176:546-552 (1990)); and direct labeled RIA(Moldenhauer et al., Scand. J. Immunol., 32:77-82 (1990)). Typically, such an assay involvesthe use of purified antigen bound to a solid surface or cells expressing the antigen, an unlabeledtest immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition ismeasured by determining the amount of label bound to the solid surface or cells in the presenceof the test immunoglobulin. Usually the test immunoglobulin is present in excess. Antibodiesidentified by competition assay (competing antibodies) include antibodies binding to the sameepitope as the reference antibody and antibodies binding to an adjacent epitope sufficientlyproximal to the epitope bound by the reference antibody for steric hindrance to occur. Usually,when a competing antibody is present in excess, it will inhibit specific binding of a referenceantibody to a common antigen by at least 50% to 75%.

An antibody that specifically binds to an amyloid protein means an antibody that bindsto the amyloid protein with an affinity of at least 107 M*1. Some antibodies bind to the amyloidprotein with affinities between 108 M'1 and 1011 M'1.

An antibody that specifically binds to aggregated amyloid protein such as aggregatedAA without specifically binding to monomeric amyloid protein means an antibody that binds toaggregated amyloid protein, such as, for example fibrils (e.g., AA in aggregated β-pleated sheetform such as from a cadaver of a former AA Amyloidosis patient or a transgenic animal model)as described above and has at least a ten fold and usually at least 100-fold lower specific bindingaffinity for monomeric forms of the amyloid protein. For example, such an antibody might bindto soluble AA with an affinity of 109 M'1 and to plaques with an affinity less than 107 M'1. Theaffinity of such antibodies for plaques is usually less than 107 or 106 M'1. Such antibodies are 43 WO 2009/086539 PCT/US2008/088493 additionally or alternatively defined by fluorescence intensity relative to an irrelevant controlantibody (e.g., an antibody or mixture of polyclonal antibodies to a reversemer AA peptide)when the antibodies are contacted with fibrils and binding assessed by fluorescently labeling.The fluorescence intensity of antibodies that bind to soluble AA peptide without binding to 5 plaques is within a factor of five, sometimes within a factor of two and sometimesindistinguishable within experimental error from that of the control antibody.

Compositions or methods "comprising" one or more recited elements may include otherelements not specifically recited. For example, a composition that comprises AA peptideencompasses both an isolated AA peptide and AA peptide as a component of a larger 10 polypeptide sequence. II. Amyloid Diseases 1. Overview and Pathogenesis

Amyloid diseases or amyloidoses include a number of disease states having a widevariety of outward symptoms. These disorders have in common the presence of abnormal 15 extracellular deposits of protein fibrils, known as “amyloid deposits” or “amyloid plaques” thatare usually about 10-100 pm in diameter and are localized to specific organs or tissue regions.Such plaques are composed primarily of a naturally occurring soluble protein or peptide. Theseinsoluble deposits are composed of generally lateral aggregates of fibrils that are approximately10-15 nm in diameter. Amyloid fibrils produce a characteristic apple green birefringence in 20 polarized light, when stained with Congo Red dye. The disorders are classified on the basis ofthe major fibril components forming the plaque deposits, as discussed below.

The peptides or proteins forming the plaque deposits are often produced from a largerprecursor protein. More specifically, the pathogenesis of amyloid fibril deposits generallyinvolves proteolytic cleavage of an “abnormal” precursor protein into fragments. These 25 fragments generally aggregate into anti-parallel β pleated sheets; however, certain undegradedforms of precursor protein have been reported to aggregate and form fibrils in familial amyloidpolyneuropathy (variant transthyretin fibrils) and dialysis-related amyloidosis (β2 microglobulinfibrils) (Tan, et al., 1994, supra). 2. Clinical Syndromes 30 This section provides descriptions of major types of amyloidoses, including their characteristic plaque fibril compositions. It is a general discovery of the present invention that 44 WO 2009/086539 PCT/US2008/088493 amyloid diseases can be treated by administering agents that serve to stimulate an immuneresponse against a component or components of the various disease-specific amyloid deposits.As discussed in more detail in Section C below, such components are preferably constituents ofthe fibrils that form the plaques. The sections below serve to exemplify major forms of 5 amyloidosis and are not intended to limit the invention. a. AL Amyloidoses AL amyloid deposition is generally associated with almost any dyserasia of the Blymphocyte lineage, ranging from malignancy of plasma cells (multiple myeloma) to benignmonoclonal gammopathy. At times, the presence of amyloid deposits may be a primary 10 indicator of the underlying dyserasia.

Fibrils of AL amyloid deposits are composed of monoclonal immunoglobulin lightchains or fragments thereof. More specifically, the fragments are derived from the N-terminalregion of the light chain (kappa or lambda) and contain all or part of the variable (Vl) domainthereof. Deposits generally occur in the mesenchymal tissues, causing peripheral and autonomic 15 neuropathy, carpal tunnel syndrome, macroglossia, restrictive cardiomyopathy, arthropathy oflarge joints, immune dyscrasias, myelomas, as well as occult dyscrasias. However, it should benoted that almost any tissue, particularly visceral organs such as the heart, may be involved. b. Hereditary Systemic Amyloidoses

There are many forms of hereditary systemic amyloidoses. Although they are relatively 20 rare conditions, adult onset of symptoms and their inheritance patterns (usually autosomaldominant) lead to persistence of such disorders in the general population. Generally, thesyndromes are attributable to point mutations in the precursor protein leading to production ofvariant amyloidogenic peptides or proteins. Table 2 summarizes the fibril composition ofexemplary forms of these disorders. 25 Table 2

Hereditary Amyloidosesa

Fibril Peptide/Protein Genetic variant Clinical Syndrome Transthyretin and fragments(ATTR) Met30, many others Familial amyloidpolyneuropathy (FAP), (mainly peripheral nerves) Transthyretin and fragments(ATTR) Thr45, Ala60, Ser84,Metlll, Ilel22 Cardiac involvementpredominant withoutneuropathy 45 WO 2009/086539 PCT/US2008/088493 N-terminal fragment ofApolipoprotein Al (apoAI) Arg 26 Familial amyloidpolyneuropathy (FAP), (mainly peripheral nerves) N-terminal fragment ofApolipoprotein Al (AapoAI) Arg26, Arg50, Arg 60, others Ostertag-type, non-neuropathic(predominantly visceralinvolvement) Lysozyme (Alys) Thr56, His67 Ostertag-type, non-neuropathic(predominantly visceralinvolvement) Fibrogen a chain fragment Leu554, Val 526 Ostertag-type, non-neuropathic(predominantly visceralinvolvement) Gelsolin fragment (Agei) Asnl87, Tyrl87 Cranial neuropathy with latticecomeal dystrophy Cystatin C fragment Glu68 Hereditary cerebralhemorrhage (cerebral amyloidangiopathy) - Icelandic type β-amyloid protein (Αβ)derived from AmyloidPrecursor Protein (APP) Gln693 Hereditary cerebralhemorrhage (cerebral amyloidangiopathy) - Dutch type β-amyloid protein (Αβ)derived from AmyloidPrecursor Protein (APP) Ile717, Phe717, Gly717 Familial Alzheimer’s Disease β-amyloid protein (Αβ)derived from AmyloidPrecursor Protein (APP) Asn670, Leu671 Familial Dementia - probableAlzheimer’s Disease Prion Protein (PrP) derivedfrom PrP precursor protein51-91 insert Leul02, Vail67,Asnl78, Lys200 Familal Creutzfeldt-Jakobdisease; Gerstmann-Straussler-Scheinker syndrome(hereditary spongiformencephalopathies, priondiseases) AA derived from Serumamyloid A protein (ApoSSA) Familal Mediterranean fever,predominant renal involvement(autosomal recessive) AA derived from Serumamyloid A protein (ApoSSA) Muckle-Well’s syndrome,nephropathy, deafness,urticaria, limb pain Unknown Cardiomyopathy withpersistent atrial standstill Unknown Cutaneous deposits (bullous,papular, pustulodermal) aData derived from Tan & Pepys, 1994, supra.

The data provided in Table 2 are exemplary and are not intended to limit the scope of 46 WO 2009/086539 PCT/US2008/088493 the invention. For example, more than 40 separate point mutations in the transthyretin gene havebeen described, all of which give rise to clinically similar forms of familial amyloidpolyneuropathy.

Transthyretin (TTR) is a 14 kilodalton protein that is also sometimes referred to asprealbumin. It is produced by the liver and choroid plexus, and it functions in transportingthyroid hormones and vitamin A. At least 50 variant forms of the protein, each characterized bya single amino acid change, are responsible for various forms of familial amyloidpolyneuropathy. For example, substitution of proline for leucine at position 55 results in aparticularly progressive form of neuropathy; substitution of methionine for leucine at position111 resulted in a severe cardiopathy in Danish patients. Amyloid deposits isolated from hearttissue of patients with systemic amyloidosis have revealed that the deposits are composed of aheterogeneous mixture of TTR and fragments thereof, collectively referred to as ATTR, the fulllength sequences of which have been characterized. ATTR fibril components can be extractedfrom such plaques and their structure and sequence determined according to the methods knownin the art (e.g., Gustavsson, A., et al., Laboratory Invest. 73: 703-708, 1995; Kametani, F., et al.,Biochem. Biophys. Res. Commun. 125: 622-628, 1984; Pras, M., et al., PNAS 80: 539-42,1983).

Persons having point mutations in the molecule apolipoprotein Al (e.g., Gly->Arg26;Trp -> Arg50; Leu exhibit a form of amyloidosis (“Ostertag type”) characterized by deposits of the protein apolipoprotein Al or fragments thereof (AApoAI). These patients havelow levels of high density lipoprotein (HDL) and present with a peripheral neuropathy or renalfailure. A mutation in the alpha chain of the enzyme lysozyme (e.g., Ile->Thr56 orAsp->His57) is the basis of another form of Ostertag-type non-neuropathic hereditary amyloidreported in English families. Here, fibrils of the mutant lysozyme protein (Alys) are deposited,and patients generally exhibit impaired renal function. This protein, unlike most of the fibril-forming proteins described herein, is usually present in whole (unfragmented) form (Benson,M.D., etal. CIBAFdn. Symp. 199: 104-131, 1996). β-amyloid peptide (Αβ) is a 39-43 amino acid peptide derived by proteolysis from alarge protein known as beta amyloid precursor protein (βΑΡΡ). Mutations in βΑΡΡ result infamilial forms of Alzheimer’s disease, Down’s syndrome and/or senile dementia, characterized 47 WO 2009/086539 PCT/US2008/088493 by cerebral deposition of plaques composed of Αβ fibrils and other components, which aredescribed in further detail below. Known mutations in APP associated with Alzheimer's diseaseoccur proximate to the cleavage sites of β or γ secretase, or within Αβ. For example, position717 is proximate to the site of γ-secretase cleavage of APP in its processing to Αβ, and positions670/671 are proximate to the site of β-secretase cleavage. Mutations at any of these residuesmay result in Alzheimer’s disease, presumably by causing an increase the amount of the 42/43amino acid form of Αβ generated from APP. The structure and sequence of Αβ peptides ofvarious lengths are well known in the art. Such peptides can be made according to methodsknown in the art {e.g., Glenner and Wong, Biochem Biophys. Res. Comm. 129: 885-890, 1984;Glenner and Wong, Biochem Biophys. Res. Comm. 122: 1131-1135, 1984). In addition,various forms of the peptides are commercially available.

Synuclein is a synapse-associated protein that resembles an alipoprotein and isabundant in neuronal cytosol and presynaptic terminals. A peptide fragment derived from a-synuclein, termed NAC, is also a component of amyloid plaques of Alzheimer’s disease.(Clayton, et at, 1998). This component also serves as a target for immuno logically-basedtreatments of the present invention, as detailed below.

Gelsolin is a calcium binding protein that binds to and fragments actin filaments.Mutations at position 187 (e.g., AspfcAsn; AspfcTyr) of the protein result in a form ofhereditary systemic amyloidosis, usually found in patients from Finland, as well as persons ofDutch or Japanese origin. In afflicted individuals, fibrils formed from gelsolin fragments (Agel),usually consist of amino acids 173-243 (68 kDa carboxyterminal fragment) and are deposited inblood vessels and basement membranes, resulting in corneal dystrophy and cranial neuropathywhich progresses to peripheral neuropathy, dystrophic skin changes and deposition in otherorgans. (Kangas, H., etal. Human Mol. Genet. 5(9): 1237-1243, 1996).

Other mutated proteins, such as mutant alpha chain of fibrinogen (AfibA) and mutantcystatin C (Acys) also form fibrils and produce characteristic hereditary disorders. AfibA fibrilsform deposits characteristic of a nonneuropathic hereditary amyloid with renal disease; Acysdeposits are characteristic of a hereditary cerebral amyloid angiopathy reported in Iceland.(Isselbacher, et al., Harrison’s Principles of Internal Medicine, McGraw-Hill, SanFrancisco, 1995; Benson, et al., supra.). In at least some cases, patients with cerebral amyloidangiopathy (CAA) have been shown to have amyloid fibrils containing a non-mutant form of 48 WO 2009/086539 PCT/US2008/088493 cystatin C in conjunction with beta protein. (Nagai, A., et al. Molec. Chem. Neuropathol. 33:63-78, 1998).

Certain forms of prion disease are now considered to be heritable, accounting for up to15% of cases, which were previously thought to be predominantly infectious in nature. (Baldwin, 5 et al., in Research Advances in Alzheimer’s Disease and Related Disorders, John Wiley andSons, New York, 1995). In such prion disorders, patients develop plaques composed ofabnormal isoforms of the normal prion protein (PrPc). A predominant mutant isoform, PrPSc,also referred to as AScr, differs from the normal cellular protein in its resistance to proteasedegradation, insolubility after detergent extraction, deposition in secondary lysosomes, post- 10 translational synthesis, and high β-pleated sheet content. Genetic linkage has been establishedfor at least five mutations resulting in Creutzfeldt-Jacob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). (Baldwin) Methods forextracting fibril peptides from scrapie fibrils, determining sequences and making such peptidesare known in the art. (e.g., Beekes, M., etal. J. Gen. Virol. 76: 2567-76, 1995). 15 For example, one form of GSS has been linked to a PrP mutation at codon 102, while telencephalic GSS segregates with a mutation at codon 117. Mutations at codons 198 and 217result in a form of GSS in which neuritic plaques characteristic of Alzheimer’s disease containPrP instead of Αβ peptide. Certain forms of familial CJD have been associated with mutations atcodons 200 and 210; mutations at codons 129 and 178 have been found in both familial CJD and 20 FFI. (Baldwin, supra). c. Senile Systemic Amyloidosis

Amyloid deposition, either systemic or focal, increases with age. For example, fibrilsof wild type transthyretin (TTR) are commonly found in the heart tissue of elderly individuals.These may be asymptomatic, clinically silent, or may result in heart failure. Asymptomatic 25 fibrillar focal deposits may also occur in the brain (Αβ), corpora amylacea of the prostate (Αβ2microglobulin), joints and seminal vesicles. d. Cerebral Amyloidosis

Local deposition of amyloid is common in the brain, particularly in elderly individuals.The most frequent type of amyloid in the brain is composed primarily of Αβ peptide fibrils, 30 resulting in dementia or sporadic (non-hereditary) Alzheimer’s disease. In fact, the incidence ofsporadic Alzheimer’s disease greatly exceeds forms shown to be hereditary. Fibril peptides 49 WO 2009/086539 PCT/US2008/088493 forming these plaques are very similar to those described above, with reference to hereditaryforms of Alzheimer’s disease (AD). e. Dialysis-related Amyloidosis

Plaques composed of β2 microglobulin (Αβ2Μ) fibrils commonly develop in patientsreceiving long term hemodialysis or peritoneal dialysis. β2 microglobulin is a 11.8 kilodaltonpolypeptide and is the light chain of Class I MHC antigens, which are present on all nucleatedcells. Under normal circumstances, it is continuously shed from cell membranes and is normallyfiltered by the kidney. Failure of clearance, such as in the case of impaired renal function, leadsto deposition in the kidney and other sites (primarily in collagen-rich tissues of the joints).Unlike other fibril proteins, Αβ2Μ molecules are generally present in unfragmented form in thefibrils. (Benson, supra). f. Hormone-derived Amyloidoses

Endocrine organs may harbor amyloid deposits, particularly in aged individuals.Hormone-secreting tumors may also contain hormone-derived amyloid plaques, the fibrils ofwhich are made up of polypeptide hormones such as calcitonin (medullary carcinoma of thethyroid), islet amyloid polypeptide (amylin; occurring in most patients with Type II diabetes),and atrial natriuretic peptide (isolated atrial amyloidosis), sequences and structures of theseproteins are well known in the art. g. Miscellaneous Amyloidoses

There are a variety of other forms of amyloid disease that are normally manifest aslocalized deposits of amyloid. In general, these diseases are probably the result of the localizedproduction and/or lack of catabolism of specific fibril precursors or a predisposition of aparticular tissue (such as the joint) for fibril deposition. Examples of such idiopathic depositioninclude nodular AL amyloid, cutaneous amyloid, endocrine amyloid, and tumor-related amyloid. III. AA Amyloid Diseases AA amyloidosis, formerly called secondary or reactive amyloidosis because it developssecondary to a preexisting or coexisting disease. Such diseases include, but are not limited toinflammatory diseases, such as rheumatoid arthritis, juvenile chronic arthritis, ankylosingspondylitis, psoriasis, psoriatic arthropathy, Reiter’s syndrome, Adult Still’s disease, Behcet’ssyndrome, and Crohn’s disease. AA deposits are also produced as a result of chronic microbialinfections, such as leprosy, tuberculosis, bronchiectasis, decubitus ulcers, chronic pyelonephritis, 50 WO 2009/086539 PCT/US2008/088493 osteomyelitis, and Whipple’s disease. Certain malignant neoplasms can also result in AA fibrilamyloid deposits. These include such conditions as Hodgkin’s lymphoma, renal carcinoma,carcinomas of gut, lung and urogenital tract, basal cell carcinoma, and hairy cell leukemia. AAamyloid disease may also result from inherited inflammatory diseases such as FamilialMediterranean Fever. Additionally, AA amyloid disease may result from lymphoproliferativedisorders such as Castleman’s Disease. 1. Inflammatory Diseases Associated with AA Amyloidosis

Rheumatoid arthritis is a chronic systemic disease primarily of the joints. Thesymptoms of rheumatoid arthritis are marked by inflammatory changes in the synovialmembranes and articular structures (joints) and by atrophy and rarefaction (bone densitydecreases) of the bones. In late stages of rheumatoid arthritis, deformity and ankylosis(immobility of the joint) develop. A model of rheumatoid arthritis can be induced in mice or ratsby administering type II collagen in complete Freund's adjuvant.

Juvenile chronic arthritis comes in many forms; the most common being juvenilerheumatoid arthritis. It can occur in children at any age, but first appears more commonlybetween the ages of 2 and 6 years. There are 3 main types of juvenile rheumatoid arthritis,namely, pauci-articular arthritis, polyarticular arthritis, and systemic arthritis (also known asStill's disease). Pauci-articular arthritis typically affects 4 or fewer joints, usually the larger onessuch as the knees. It can be accompanied by stiffness, causing the child to limp. Polyarticulararthritis is characterized by 5 or more joints being affected, most commonly the smaller joints inthe hands and feet. Children with polyarticular arthritis often have a more severe form of thedisease. Systemic arthritis is characterized by joint swelling in combination with fever and apink rash. The joints may not start to swell until some months or years after the fevers begin. Itmay also affect internal organs such as the liver, heart, spleen and lymph nodes, and anemia iscommon. While systemic arthritis tends to abate of its own accord, a small percentage of thesechildren can have severe arthritis that continues into adulthood.

Ankylosing spondylitis is a rheumatic disease that causes arthritis of the spine andsacroiliac joints and can cause inflammation of the eyes, lungs, and heart valves. It varies fromintermittent episodes of back pain that occur throughout life to a severe chronic disease thatattacks the spine, peripheral joints and other body organs, resulting in severe joint and backstiffness, loss of motion and deformity as life progresses.

Psoriasis is a common chronic, squamous dermatosis, marked by exacerbation and 51 WO 2009/086539 PCT/US2008/088493 remissions and having a polygenic inheritance pattern. The symptoms of psoriasis are markedby the presence of rounded, dry scaling patches of various sizes, covered by a grayish white orsilvery white scales that have a predilection for the extensor surfaces, nails, scalp, genitalia andthe lumbosacral region.

Psoriatic arthropathy is a disorder in which psoriasis is linked to the development ofarthritis. The disorder can be exhibited in a variety of ways. The arthritis is generally mild andinvolves only a few joints. In a few patients, the disease is severe and usually affects the fingersand the spine. When the spine is affected, the symptoms are very much like those of ankylosingspondylitis.

Reiter's syndrome is a group of symptoms consisting of arthritis, urethritis(inflammation of the urogenital tract), conjunctivitis (inflammation of the lining of the eye), andlesions of the skin and mucous membranes. Reiter's syndrome is also referred to as reactivearthritis, which means that the arthritis occurs as a "reaction" to an infection that startedelsewhere in the body. Chlamydia trachomatis is the bacteria most often associated with Reiter'ssyndrome acquired through sexual contact. Several different bacteria are associated with Reiter'ssyndrome acquired through the digestive tract, including Salmonella, Shigella, Yersinia, andCampylobacter.

Adult Still's disease, also called Adult Onset Still's Disease is a rare inflammatorycondition that attacks internal organs, joints and other parts of the body. It can appear anddisappear suddenly. In very severe cases, adult Still’s disease becomes chronic and extremelydebilitating, causing terrible pain and stiffness. After many years, the disease cripples vitalorgans such as the heart and lungs.

Behcet’s syndrome is a multisystem disorder presenting with recurrent oral and/orgenital ulcerations, chronic relapsing uveitis that may cause blindness and neurologicimpairments. It is characterized by 4 major symptoms: oral aphthous ulcers, skin lesions, ocularsymptoms, and genital ulcerations, and occasionally by inflammation in tissues and organsthroughout the body, including the gastrointestinal tract, central nervous system, vascularsystem, lungs, and kidneys. The arthritis of Behcet’s syndrome is usually intermittent, self-limited, not deforming and localized to the knees and ankles.

Crohn's disease is a chronic granulomatous (small grain-like body or growth)inflammatory disease involving any part of the gastrointestinal tract from the mouth to anus; butcommonly involving the ileum (lower three-fifths of the small intestines) with scarring and 52 WO 2009/086539 PCT/US2008/088493 thickening of the bowel wall. The symptoms of Crohn's disease include the presence of chronicdiarrhea, increased bowel sounds, cramping, possibly evidenced by weight loss and aversion toeating. 2. Chronic Microbial Infection Diseases Associated with AA Amyloidosis

Leprosy is an infectious disease characterized by disfiguring skin sores, peripheralnerve damage, and progressive debilitation. Leprosy is caused by the organism Mycobacteriumleprae, which is not very contagious and has a long incubation period. Leprosy has two commonforms, tuberculoid and lepromatous. Both forms produce sores on the skin, but the lepromatousform is most severe, producing large, disfiguring nodules (lumps and bumps). Leptosyeventually causes peripheral neurological damage. Patients with long-term leprosy may lose theuse of their hands or feet due to repeated injury resulting from lack of sensation.

Tuberculosis is a contagious bacterial infection caused by Mycobacterium tuberculosis.The disease is characterized by the development of granulomas (granular tumors) in the infectedtissues. The lungs are primarily involved, but the infection can spread to other organs.

Bronchiectasis is an abnormal destruction and dilation of the large airways.Bronchiectasis is often caused by recurrent inflammation or infection of the airways. A classicbacterium that is seen in patients with bronchiectasis is Pseudomonas aeruginosa, which isnotoriously hard to eradicate. Repeated infections of the airways by this bacterium can lead tocolonization of the bronchi by this organism which predisposes such people to Pseudomonalpneumonias, which requires special antibiotics to treat.

Decubitus ulcer also known as pressure ulcer or bedsore is an ulceration of the skin andunderlying tissues caused by prolonged pressure over the affected area. They start as reddenedskin but gets progressively worse, forming a blister, then an open sore, and finally a crater.These ulcerations usually occur over bony prominences such as heels, coccyx area of the buttockand the back of the head.

Chronic pyelonephritis is an infection of the kidney and the ureters (ducts that carryurine away from the kidney). Pyelonephritis most often occurs as a result of urinary tractinfection, particularly in the presence of occasional or persistent backflow of urine from thebladder into the ureters or kidney pelvis.

Osteomyelitis is an acute or chronic bone infection, usually caused by bacteria. Oftenthe infection initiates in another part of the body and spreads to the bone via the blood. Whenthe bone is infected, pus is produced within the bone, which may result in an abscess. The 53 WO 2009/086539 PCT/US2008/088493 abscess then deprives the bone of its blood supply. Chronic osteomyelitis results when bonetissue dies as a result of the lost blood supply. Chronic infection can persist intermittently foryears.

Whipple’s disease is a rare condition that causes inadequate absorption of nutrientsfrom the intestinal tract due to infection of the intestine. It is caused by the bacteria, Tropherymawhippelii. Symptoms include diarrhea, intestinal bleeding, abdominal pain, loss of appetite,weight loss, fatigue, and weakness. Arthritis and fever often occur several years before intestinalsymptoms develop. Patients may experience neurological symptoms as well. Diagnosis is basedon symptoms and the results of a biopsy of tissue from the small intestine or other organs that areaffected. When recognized and treated, Whipple's disease can usually be cured. Withouttreatment, the condition is usually fatal. 3. Malignant Neoplasms Associated with AA Amyloidosis

Hodgkin’s lymphoma is a cancer of lymphatic tissue found in the lymph nodes, spleen,liver, and bone marrow. The first sign of this cancer is often an enlarged lymph node. Thedisease can spread to nearby lymph nodes and later may spread to the lungs, liver, or bonemarrow.

Renal carcinoma is cancer of the kidney. The cancerous cells are found in the lining oftubules in the kidney. The first symptom is usually blood in the urine. Sometimes both kidneysare involved. The cancer spreads easily, most often to the lungs and other organs. Renal cellcarcinoma is the most common type of kidney cancer followed by papillary renal cell carcinoma,chromophobe renal carcinoma and collecting duct renal carcinoma. About 5% of renalcarcinoma are unclassified because their appearance doesn’t fit into any of the other categories.

Carcinomas of the gut include gastrointestinal cancers such as colorectal, pancreatic,stomach and esophageal. Colorectal cancer is cancer that starts in the large intestine or therectum. Almost all colorectal cancers begin as benign polyps which, over a period of manyyears, develop into cancers. Most cases of colorectal cancer have no symptoms. Pancreaticcancer is a malignancy of the pancreas. Symptoms include abdominal pain, loss of appetite,significant weight loss and painless jaundice. Stomach cancer, also called gastric cancer, candevelop in any part of the stomach and may spread throughout the stomach and to other organs;particularly the esophagus and the small intestine. It may also spread, through the stomach wall,to nearby lymph nodes and organs such as the liver, pancreas, and the lungs, or to distant organssuch as the lymph nodes above the collar bone, the colon, and the ovaries. Stomach cancer is 54 WO 2009/086539 PCT/US2008/088493 often asymptomatic. Esophageal cancer is malignancy of the esophagus. Symptoms includedysphagia (difficulty swallowing), pain and substantial weight loss.

Carcinomas of the lung are a cancer of the lungs characterized by the presence ofmalignant tumours. There are two main types of lung cancer: non-small cell lung cancer andsmall cell lung cancer. Symptoms depend on the specific type of cancer, but may includechronic cough, coughing up blood, shortness of breath, wheezing, chest pain, loss of appetite,weight loss and fatigue.

Carcinomas of the urogenital tract include but are not limited to prostate cancer,bladder cancer, endometrial cancer, cervical cancer and ovarian cancer. Prostate cancer involvesa malignant tumor growth within the prostate gland. Symptoms may include frequent urination,difficulty starting and maintaining a steady stream of urine, blood in the urine, painful urination,difficulty achieving erection or painful ejaculation. Bladder cancer refers to any of several typesof malignant growths of the urinary bladder. Symptoms include blood in the urine, frequenturination, painful urination, and urinary urgency. Endometrial cancer involves cancerous growthof the endometrium (lining of the uterus). It mainly occurs after menopause, and presents withvaginal bleeding. Cervical cancer is a malignancy of the cervix. The early stages of cervicalcancer may be completely asymptomatic. Vaginal bleeding may indicate the presence ofmalignancy. In advanced stages, metastases may be present in the abdomen, lungs or elsewhere.Ovarian cancer is a malignant neoplansm of the ovaries. Ovarian cancer symptoms are oftenvague and non-specific, which include vague lower abdominal discomfort, sense of pelvicheaviness, abnormal menstrual cycle, vaginal bleeding, weight gain or loss, nonspecificgastrointestinal symptoms. Ovarian cancers shed cancer cells that often implant on the uterus,bladder, bowel, and lining of the bowel wall. These cancer cells can begin forming new tumorgrowths before cancer is even suspected.

Basal cell carcinoma is a slow-growing skin tumor involving cancerous changes inbasal skin cells. Symptoms include skin lesions located on the face, ear, neck, chest, back, orscalp; visible blood vessels in the lesion or adjacent skin; and persistent, non-healing sores. Thiscancer usually remains local and almost never spreads to distant parts of the body, but it maycontinue to grow and invade nearby tissues and structures, including the nerves, bones, andbrain.

Hairy ceil leukemia is a cancer of lymphocytes (B cells) that leads to low blood counts.The disease is caused by the abnormally shaped B cells with hair-like projections. Symptoms are 55 WO 2009/086539 PCT/US2008/088493 often vague. The low blood counts caused by hairy cell leukemia can lead to infections, fatigue,and excessive bleeding.

4. Inherited Inflammatory Disease Associated with AA

Familial Mediterranean Fever is an inherited disorder characterized by recurrent fever5 and inflammation, often involving the abdomen or the lung. Symptoms include iinflammation inthe lining of the abdominal cavity, chest cavity, skin, or joints occurs, along with high fevers thatusually peak in 12 to 24 hours. Attacks may vary in severity of symptoms, and people areusually symptom free between attacks. This disease is very rare. Risk factors include a family history of familial Mediterranean Fever or having Mediterranean ancestry. 10 5. Lymphoproliferative Disorders Associated with AA Amyloidosis

Castleman’s Disease is a form of lympoproliferative disorder characterizedpathologicaly by the presence of giant lymp node hyperplasia with plasma cell infiltration.Patients with Castleman’s Disease commonly have fever, anemia, hypergammaglobulinaemia,and an increase in the serum concentrations of acute phase reactant proteins, all of which are 15 ascribed to the large amount of IL-6 produced in the lymph nodes.

IV. Serum Amyloid A

1. Human Serum Amyloid A

Serum amyloid A (SAA) is the circulating precursor of amyloid A protein, the fibrillarcomponent of amyloid deposits. The structural studies showed that the human SAA is 20 heterogeneous and represents a family of polymorphic SAA genes and protein products. TheSAA gene superfamily comprises a cluster of closely linked genes localized to lip 15.1. SeeSellar, GC et al. Genomics 19: 221-227 (1994). Four SAA genes have been described inhumans. Representative amino acid sequences of proteins encoded by the four SAA genes areillustrated by Figure 1. Two genes (SAA I and SAA2) encode acute-phase serum amyloid A (A- 25 SAA) and are coordinately induced in response to inflammation. SAA1 and SAA2 share 95%sequence identity in both coding and noncoding regions. There are alpha, beta and gammaisoforms of human SAA1 and alpha and beta isoforms of human SAA2 as illustrated by Figures18 and 19. SAA3 is a pseudogene. SAA4 encodes constitutive SAA and is minimally inducible.See Cunnane G. Bailliere’s Clin. Rheumatol. 13(4): 615-628. All human SAA/AA molecules 30 contains a theoretical calcium-binding tetrapeptide sequence, Gly-Pro-Gly-Gly, of possibleimportance for self aggregation and with extrafibrillar moieties of amyloid in fibrillogenesis. See 56 WO 2009/086539 PCT/US2008/088493

Fykse, E.M. et al. Biochem. J. 256:973-980 (1988) and Turned et al. Mol. Biol. Med. 3:387-407(1986). The N terminal portion of SAA7AA is strongly hydrophobic, probably of importance forself aggregation and other components in amyloid deposits. See Husby et al. Clin. Immunol.Immunopathol. 70(1):2-9 (1994). The sequence of each isoform of AA and its relationship to its 5 corresponding SAA isoform is illustrated by Figures 2-5. For example, human SAA1 alphaisoform has the sequence: H2N-Met-Lys-Leu-Leu-Thr-Gly-Leu-Val-Phe-Cys-Ser-Leu-Val-Leu-Gly-Val-Ser-Ser-Arg-Ser-

Phe-Phe-Ser-Phe-Leu-Gly-Glu-Ala-Phe-Asp-Gly-Ala-Arg-Asp-Met-Try-Arg-Ala-Tyr-Ser-Asp-

Met-Arg-Glu-Ala-Asn-Tyr-Ile-Gly-Ser-Asp-Lys-Tyr-Phe-His-Ala-Arg-Gly-Asn-Tyr-Asp-Ala- 10 Ala-Lys-Arg-Gly-Pro-Gly-Gly-Ala-Try-Ala-Ala-Glu-Val-Ile-Ser-Asp-Ala-Arg-Glu-Asn-Ile-Gln-Arg-Phe-Phe-Gly-His-Gly-Ala-Glu-Asp-Ser-Leu-Ala-Asp-Gln-Ala-Ala-Asn-Glu-Try-Gly-Arg-Ser-Gly-Lys-Asp-Pro-Asn-His-Phe-Arg-Pro-Ala-Gly-Leu-Pro-Glu-Lys-Tyr-OH (SEQ IDNO:1). AA, which is a proteolytic fragment of SAA, is also heterogeneous. The predominant 15 human AA peptide consists of 76 amino acids. An example of AA has the sequence: H2N-Arg-Ser-Phe-Phe-Ser-Plie-Leu-Gly-Glu-Ala-Plie-Asp-Gly-Ala-Aug-Asp-Met-Try-Arg-Ala-Tyr-Ser-Asp-Met-Arg-Glu-Ala-Asn-Tyr-Ile-Gly-Ser-Asp-Lys-Tyr-Phe-His-Ala-Arg-Gly-Asn-Tyr-Asp-Ala-Ala-Lys-Arg-Gly-Pro-Gly-Gly-Ala-Try-Ala-Ala-Glu-Val-Ile-Ser-Asp-Ala-Arg-Glu-Asn-Ile-Gln-Arg-Phe-Phe-Gly-His-Gly-Ala-Glu-Asp-Ser-OH (SEQ ID NO :2). 20 AA70-76 refers to an AA fragment beginning at residue 70 and ending at residue 76 of (SEQ ID NO:2) consisting of the sequence GHGAEDS, (SEQ ID NO: 4), or correspondingsegment from another naturally occurring AA protein from a human or other species when thesequence of that protein is maximally aligned with SEQ ID NO:2.

2. Murine Serum Amyloid A 25 In the mouse, four SAA genes have been described. Representative amino acid sequences of proteins encoded by the four murine SAA genes are illustrated by Figure 8. MouseSAA gene family comprises four members that are closely linked in the chromosome 7. Two ofthese genes encoding major mouse SAA isotypes (SAA1 and SAA2) share high sequenceidentity not only in exons but also in introns and flanking regions and are induced in 30 approximately equal quantities in response to amyloid induction models. These two isotypesdiffer in only 9 of 103 amino acid residues; however, only SAA2 is selectively deposited intoamyloid fibrils. See de Beer M.C. Biochem J. 1991 280(Pt 1): 45-49 (1991); Hoffman J.S. et al. 57 WO 2009/086539 PCT/US2008/088493 J Exp Med. 159:641-646 (1984); Shiroo M et al. ScandJ. Immunol. 26:709-716 (1987). SAA3is a minor HDL apolipoprotein and peripherally produced acute phase. SAA4 is a constitutivesubfamily that is a minor normal HDL apolipoprotein comprising more than 90% of the SAAduring homeostasis. See Stearman R.S. et al. Nucleic Acids Research, 14(2)797-809 (1986) and 5 de Beer M.C. Genomics, 34(1):139-42 (1996).

Murine AA which is a proteolytic fragment of SAA is also heterogeneous. The sequence of each murine isoform of AA and its relationship to its corresponding SAA isoform isillustrated by Figures 9-12. A sequence alignment of murine AA1, AA2, AA3 and AA4 isillustrated by Figure 13. 10 Murine AA1 is the murine equivalent of human AA1. See Figure 16. In particular, residues 69-75 of murine AA1 (GRGHEDT, SEQ ID NO: 9) are maximally aligned withresidues 70-76 of human AA1 (GHGAEDS, SEQ ID NO: 4). See also Figure 17.

3. Shar Pei Serum Amyloid A

The Shar Pei sequence is indicated in Figure 20. Interestingly, the homologous region 15 in the human SAA protein -AEDS, (SEQ ID NO: 13) contains a conserved Thr to Sersubstitution at position 76, as well as significantly different side chain of the residue at position73 (His to Ala; Fig. 1). The -AEDS, (SEQ ID NO: 13), sequence is also observed in the SharPei species of dog, a breed that is particularly susceptible to AA-amyloidosis and could provide anaturally occurring model of systemic AA in which to evaluate novel diagnostic and therapeutic 20 applications of AA amyloid-specific antibodies and other compounds. 4. The N-Terminal Segment of AA Protein Determines Its Fibrillogenic PropertyThe amyloid fibril protein AA consists of a varying long N-terminal part of the precursor protein serum AA. Evidence shows that the amyloidogenic part of the molecule is theN-terminal 10-15 amino acid long segment. Amino acid substitutions in this part of the molecule 25 may explain why only one of the two mouse SAA isoforms is amyloidogenic. See WestermarkG.T. Biochem Biophys Res Commun. 182(1):27-33 (1992). V. Other Human Amyloidogenic Proteins

The GenbanJk Accession Numbers and XiEDX2 sequences are provided below in Table3 for several human amyloidogenic proteins, including some of those listed above in Table 2. 30 58 WO 2009/086539 PCT/US2008/088493

Table 3

Human Amyloidogenic Proteins

Human amyloidogenic protein Consensus sequence GenBank Accession Number SAAI AEDS, (SEQ ID NO: 13) SAA2 AEDS, (SEQ ID NO: 13) SAA3 AEDS, (SEQ ID NO: 13) SAA4 AEDS, (SEQ ID NO: 13) anti-Sm immunoglobulin kappa light chain Vregion; monoclonal antibody 4B4 kappachain AEDV, (SEQ ID NO :23) AAB26897 immunoglobulin variable region used by theITC52 kappa light chain (subgroup V kappa II PEDS, (SEQ ID NO: 26) AAC61608 immunoglobulin variable region used by theITC48 kappa light chain (subgroup V kappaIV) AEDV, (SEQ ID NO: 23) AAC61606 anti-RhD monoclonal T125 kappa light chainprecursor SEDF, (SEQ ID NO: 24) AAW82027 immunoglobulin kappa light chain precursor AEDV, (SEQ ID NO: 23) CAA45496 immunoglobulin kappa light chain variableregion PEDF, (SEQ ID NO: 22) AAT44350 immunoglobulin kappa light chain variableregion PEDF, (SEQ ID NO: 22) AAT44349 immunoglobulin kappa light chain variableregion PEDF, (SEQ ID NO: 22) AAT44348 immunoglobulin kappa light chain PEDF, (SEQ ID NO: 22) CAA09185 immunoglobulin kappa light chain SEDF, (SEQ ID NO: 24) CAA09181 immunoglobulin kappa light chain variableregion SEDF, (SEQ ID NO: 24) AAU14891 anti-rabies SOJA immunoglobulin kappalight chain PEDF, (SEQ ID NO: 22) AAO17825 anti-streptococcal/anti-myosinimmunoglobulin kappa light chain variableregion SEDF, (SEQ ID NO: 24) AAB68786 anti-streptococeal/anti-myosinimmunoglobulin kappa light chain variableregion PEDF, (SEQ ID NO: 22) AAB68785 anti-HL A-A2/ anti-HL A-A2 8immunoglobulin kappa light chain variableregion PEDF, (SEQ ID NO: 22) AAC99644 59 WO 2009/086539 PCT/US2008/088493

Human amyloidogenic protein Consensus sequence GenBank Accession Number immunoglobulin kappa light chain V region;anti-DNA antibody 18/2 PEDF, (SEQ ID NO: 22) AAB62946 immunoglobulin kappa light chain PEDF, (SEQ ID NO: 22) BAF75949 anti-HIV-1 gpl20 immunoglobulin 48dkappa light chain PEDF, (SEQ ID NO: 22) AAR88370 immunoglobulin kappa light chain PEDL, (SEQ ID NO: 27) BAA97671 anti-Entamoeba histolytica immunoglobulinkappa light chain PEDF, (SEQ ID NO: 22) BAA82105 anti-Entamoeba histolytica immunoglobulinkappa light chain TEDV, (SEQ ID NO: 28) BAA82102 immunoglobulin kappa light chain PEDF, (SEQ ID NO: 22) AAC41705 anti-GM2 glanglioside IgM monoclonalkappa light chain variable region AEDV, (SEQ ID NO:23) AAC26480 anti-SARS-CoV immunoglobulin kappa lightchain variable region PEDV, (SEQ ID NO: 151) AAT51719 anti-SARS-CoV immunoglobulin kappa lightchain variable region PEDF, (SEQ ID NO: 22) AAT51718 immunoglobulin kappa light chain VLJregion PEDF, (SEQ ID NO: 22) BAD27502 immunoglobulin kappa light chain VLJregion SEDF, (SEQ ID NO: 24) BAD27497 anti-HIV-1 gpl20 immunoglobulin 47ekappa light chain PEDF, (SEQ ID NO: 22) AAR88378 anti-HIV-1 gpl20 immunoglobulin 16ckappa light chain PEDF, (SEQ ID NO: 22) AAR88374 anti-HIV-1 gpl20 immunoglobulin 41 lgkappa light chain SEDF, (SEQ ID NO: 24) AAR88372 immunoglobulin kappa light chain variableregion PEDF, (SEQ ID NO: 22) AAF14212 immunoglobulin kappa light chain variableregion PEDF, (SEQ ID NO: 22) AAF14211 immunoglobulin kappa light chain variableregion PEDF, (SEQ ID NO: 22) AAF14210 immunoglobulin kappa light chain variableregion PEDF, (SEQ ID NO: 22) AAF14209 immunoglobulin V-region kappa light chain PEDI, (SEQ ID NO: 21) AAR02415 immunoglobulin kappa light chain PEDF, (SEQ ID NO: 22) AAM46647 immunoglobulin kappa light chain AEDV, (SEQ ID NO: 23) AAM46643 anti-Entamoeba histolytica immunoglobulinkappa light chain PEDF, (SEQ ID NO: 22) BAA82103 60 WO 2009/086539 PCT/US2008/088493

Human amyloidogenic protein Consensus sequence GenBank Accession Number immunoglobulin light chain kappa variableregion AEDV, (SEQ ID NO:23) AAL65723 immunoglobulin light chain kappa variableregion PEDF, (SEQ ID NO: 22) AAL65718 immunoglobulin light chain kappa variableregion SEDF, (SEQ ID NO: 24) AAL65717 immunoglobulin light chain kappa variableregion SEDF, (SEQ ID NO: 24) AAL65716 immunoglobulin light chain kappa variableregion PEDF, (SEQ ID NO: 22) AAL65714 immunoglobulin light chain kappa variableregion PEDF, (SEQ ID NO: 22) AAL65713 immunoglobulin light chain kappa variableregion PEDF, (SEQ ID NO: 22) AAL65712 immunoglobulin light chain kappa variableregion PEDF, (SEQ ID NO: 22) AAL65711 immunoglobulin light chain kappa variableregion PEDF, (SEQ ID NO: 22) AAL65710 immunoglobulin light chain kappa variableregion LEDG, (SEQ ID NO: 31)PEDF, (SEQ ID NO: 22) AAL65709 immunoglobulin light chain kappa variableregion LEDG, (SEQ ID NO: 31)PEDF, (SEQ ID NO: 22) AAL65708 immunoglobulin light chain kappa variableregion PEDF, (SEQ ID NO: 22) AAL65707 immunoglobulin light chain kappa variableregion PEDF, (SEQ ID NO: 22) AAL65706 immunoglobulin light chain kappa variableregion PEDF, (SEQ ID NO: 22) AAL65705 immunoglobulin light chain kappa variableregion PEDF, (SEQ ID NO: 22) AAL65704 immunoglobulin light chain kappa variableregion PEDF, (SEQ ID NO: 22) AAL65703 immunoglobulin kappa light chain variableregion SEDF, (SEQ ID NO: 24) AAC64146 immunoglobulin kappa light chain variableregion SEDF, (SEQ ID NO: 24) AAC64144 immunoglobulin kappa light chain variableregion PEDF, (SEQ ID NO: 22) ABI64139 anti-pneumococcal capsular polysaccharideimmunoglobulin kappa light chain AEDV, (SEQ ID NO: 23) AAL04535 immunoglobulin light chain kappa variableregion AEDV, (SEQ ID NO: 23) AAL65722 61 WO 2009/086539 PCT/US2008/088493

Human amyloidogenic protein Consensus sequence GenBank Accession Number immunoglobulin light chain kappa variableregion AEDV, (SEQ ID NO: 23) AAL65720 immunoglobulin light chain V-J region PEDF, (SEQ ID NO: 22) BAA19563 immunoglobulin light chain V-J region AEDE, (SEQ ID NO: 19) BAA19562 immunoglobulin light chain V-J region AEDE, (SEQ ID NO: 19) BAA19561 immunoglobulin light chain V-J region PEDF, (SEQ ID NO: 22) BAA19560 immunoglobulin light chain V-J region PEDF, (SEQ ID NO: 22) BAA19559 immunoglobulin light chain V-J region AEDV, (SEQ ID NO :23) BAA19558 immunoglobulin light chain V-J region PEDI, (SEQ ID NO: 21) BAA 19556 immunoglobulin kappa light chain variableregion PEDF, (SEQ ID NO: 22) AAA71907 immunoglobulin kappa light chain variableregion AEDV, (SEQ ID NO: 23) AAA71905 immunoglobulin G1 Fab light chain variableregion AEDV, (SEQ ID NO: 23) BAF49281 immunoglobulin G1 Fab light chain variableregion PEDF, (SEQ ID NO: 22) BAF48998 immunoglobulin G1 Fab light chain variableregion PEDF, (SEQ ID NO: 22) BAF48996 kappa light chain V-region AEDM, (SEQ ID NO: 32) CAA37675 immunoglobulin G1 Fab light chain variableregion SEDF, (SEQ ID NO: 24) BAF48994 immunoglobulin G1 Fab light chain variableregion PEDF, (SEQ ID NO: 22) BAF48992 Ig kappa chain precursor V-J-C region AEDV, (SEQ ID NO: 23) A53261 Ig kappa chain precursor V region AEDV, (SEQ ID NO: 23) A49137 Ig kappa chain precursor V-I region SEDI, (SEQ ID NO: 29) PN0445 Ig kappa chain precursor V-III region (EVI-15) PEDF, (SEQ ID NO: 22) A32274 Ig kappa chain V-IV region (Dep) AEDV, (SEQ ID NO: 23) A34153 Ig kappa chain V-IV region (Fue) AEDV, (SEQ ID NO: 23) B34153 Ig kappa chain V-II region (Pec) AEDV, (SEQ ID NO: 23) C34153 Chain L, Igg Fab Fragment (Cd25-Binding). AEDA, (SEQ ID NO: 62) 1MIM_L Chain H, Igg Fab Fragment (Cd25-Binding). HEDS, (SEQ ID NO: 33) 1MIM_H Ig mu chain C region, secreted splice form CEDD, (SEQ ID NO: 34) MHHU immunoglobulin kappa-chain VJ region AEDV, (SEQ ID NO: 23) AAA58923 62 WO 2009/086539 PCT/US2008/088493

Human amyloidogenic protein Consensus sequence GenBank Accession Number recombinant monoclonal antibody IgM 12kappa light chain variable region PEDF, (SEQ ID NO: 22) ABA41551 immunoglobulin light chain AEDE, (SEQ ID NO: 19) CAA65054 immunoglobulin light chain lambda variableregion AEDE, (SEQ ID NO: 19) AAL65769 immunoglobulin light chain lambda variableregion AEDE, (SEQ ID NO: 19) AAL65767 immunoglobulin light chain lambda variableregion AEDE, (SEQ ID NO: 19) AAL65765 immunoglobulin light chain lambda variableregion TEDE, (SEQ ID NO: 16) AAL65764 immunoglobulin light chain lambda variableregion AEDE, (SEQ ID NO: 19) AAL65763 immunoglobulin light chain lambda variableregion SEDE, (SEQ ID NO: 18) AAL65762 immunoglobulin light chain lambda variableregion SEDE, (SEQ ID NO: 18) AAL65761 immunoglobulin light chain lambda variableregion SEDE, (SEQ ID NO: 18) AAL65760 immunoglobulin light chain lambda variableregion AEDE, (SEQ ID NO: 19) AAL65759 immunoglobulin light chain lambda variableregion AEDE, (SEQ ID NO: 19) AAL65758 immunoglobulin light chain V-J region PEDF, (SEQ ID NO: 22) BAA19563 immunoglobulin light chain V-J region AEDE, (SEQ ID NO: 19) BAA19562 immunoglobulin light chain V-J region AEDE, (SEQ ID NO: 19) BAA19561 immunoglobulin light chain V-J region PEDF, (SEQ ID NO: 22) BAA19560 immunoglobulin light chain V-J region PEDF, (SEQ ID NO: 22) BAA19559 immunoglobulin light chain V-J region AEDV, (SEQ ID NO: 23) BAA19558 immunoglobulin light chain V-J region PEDI, (SEQ ID NO: 21) BAA19556 30-lambda immunoglobulin light chainvariable region AEDE, (SEQ ID NO: 19) AAK95335 PREDICTED: similar to Low affinityimmunoglobulin gamma Fc region receptor ΙΙ-a precursor (Fc-gamma Rll-a) (FcRII-a) (IgG Fc receptor ΙΙ-a) (Fc-gamma-RIIa) (CD32 antigen) (CDw32) QEDS, (SEQ ID NO: 35) XP_001129584 63 WO 2009/086539 PCT/US2008/088493

Human amyloidogenic protein Consensus sequence GenBank Accession Number Fc fragment of IgG, high affinity la, receptor(CD64) REDS, (SEQ IDNO:36)TEDG, (SEQ ID NO: 37)QEDR, (SEQ ID NO: 38) NP_000557 Fc fragment of IgG, low affinity lib, receptorfor (CD32) isoform 2 QEDS, (SEQ ID NO: 35) NP 001002273 XP 943944 Fc fragment of IgG, low affinity lib, receptorfor (CD32) iso form 1 QEDS, (SEQ ID NO: 35) NP 003992 Fc fragment of IgG, low affinity lib, receptorfor (CD32) isoform 4 QEDS, (SEQ ID NO: 35) NP_001002275 Fc fragment of IgG, low affinity lib, receptorfor (CD32) isoform 3 QEDS, (SEQ ID NO: 35) ΝΡ001002274 ΧΡ 001129592 Fc fragment of IgG, high affinity lb, receptor(CD64) isoform a QEDR, (SEQ ID NO: 38) NP_001017986 Fc fragment of IgG, high affinity lb, receptor(CD64) isoform b QEDR, (SEQ ID NO: 38) NP_001004340 XP_496386 Fc fragment of IgG, low affinity Ila, receptor(CD32) QEDS, (SEQ ID NO: 35) NP_067674 XP_943942 low affinity immunoglobulin gamma Fcregion receptor III-B precursor TEDL, (SEQ ID NO: 39)PEDN, (SEQ ID NO: 40)EEDP, (SEQ ID NO: 41) NP_000561 Fc fragment of IgG, low affinity Ilia,receptor for (CD 16) TEDL, (SEQ ID NO: 39)PEDN, (SEQ ID NO: 40)EEDP, (SEQ ID NO: 41) NP_000560 XP_001133750 Low affinity immunoglobulin gamma Fcregion receptor ΙΙ-a precursor (Fc-gammaRH-a) (FcRII-a) (IgG Fc receptor ΙΙ-a) (Fc-gamma-RIIa) (CD32 antigen) (CDw32) QEDS, (SEQ ID NO: 35) P12318 Low affinity immunoglobulin gamma Fcregion receptor III-B precursor (IgG Fcreceptor III-1) (Fc-gamma RHI-beta) (Fc-gamma RHIb) (FcRIIIb) (Fc-gamma RIII)(FcRIII) (FcR-10) (CD16b antigen) TEDL, (SEQ ID NO: 39)PEDN, (SEQ ID NO: 40)EEDP, (SEQ ID NO: 41) 075015 Low affinity immunoglobulin gamma Fcregion receptor III-A precursor (IgG Fcreceptor ΙΠ-2) (Fc-gamma RHI-alpha) (Fc-gamma Rllla) (FcRIIIa) (Fc-gamma RIII)(FcRIII) (FcR-10) (CD16a antigen) TEDL, (SEQ ID NO: 39)PEDN, (SEQ ID NO: 40)EEDP, (SEQ ID NO: 41) P08637 High affinity immunoglobulin gamma Fcreceptor I precursor (Fc-gamma RI) (FcRI)(IgG Fc receptor I) (CD64 antigen). REDS, (SEQ ID NO: 36)TEDG, (SEQ ID NO: 37)QEDR, (SEQ ID NO: 38) P12314 64 WO 2009/086539 PCT/US2008/088493

Human amyloidogenic protein Consensus sequence GenBank Accession Number IGHG1 immunoglobulin heavy constantgamma 1 (Glm marker) AEDT, (SEQ ID NO: 14) Q6PJA4 apoAI [Homo sapiens] LEDL, (SEQ ID NO: 42) CAA01253 apolipoprotein C-III precursor [Homosapiens] AEDA, (SEQ ID NO: 62) NP 000031 apolipoprotein A-IV precursor [Homosapiens]. AEDV, (SEQ ID NO: 23) NP_000473 gelsolin (amyloidosis, Finnish type) [Homosapiens] TEDT, (SEQ ID NO: 30)KEDA, (SEQ ID NO: 43)SEDC, (SEQ ID NO: 44)QEDL, (SEQ ID NO: 63) CAM20459 gelsolin (amyloidosis, Finnish type) [Homosapiens] TEDT, (SEQ ID NO: 30)KEDA, (SEQ ID NO: 43)SEDC, (SEQ ID NO: 44)QEDL, (SEQ ID NO: 63) CAI14413 gelsolin (amyloidosis, Finnish type), isoformCRAc [Homo sapiens]. TEDT, (SEQ ID NO: 30)KEDA, (SEQ ID NO: 43)SEDC, (SEQ ID NO: 44)QEDL, (SEQ ID NO: 63) EAW87491 gelsolin (amyloidosis, Finnish type), isoformCRAb [Homo sapiens] TEDT, (SEQ ID NO: 30)KEDA, (SEQ ID NO: 43)SEDC, (SEQ ID NO: 44)QEDL, (SEQ ID NO: 63) EAW87490 gelsolin (amyloidosis, Finnish type), isoformCRAa [Homo sapiens] TEDT, (SEQ ID NO: 30)KEDA, (SEQ ID NO: 43)SEDC, (SEQ ID NO: 44)QEDL, (SEQ ID NO: 63) EAW87489 amyloid precursor protein; APP [Homosapiens]. AEDV, (SEQ ID NO: 23) AAB23646 amyloid precursor protein; APP [Homosapiens]. AEDV, (SEQ ID NO:23) AAB19991 amyloid peptide AEDV, (SEQ ID NO: 23) AAA51768 65 WO 2009/086539 PCT/US2008/088493

Human amyloidogenic protein Consensus sequence GenBank Accession Number Amyloid beta A4 protein precursor (APP)(ABPP) (Alzheimer disease amyloid protein)(Cerebral vascular amyloid peptide) (CVAP)(Protease nexin-II) (PN-II) (APPI) (PreA4)[Contains: Soluble APP-alpha (S-APP-alpha); Soluble APP-beta (S-APP-beta); C99;Beta-amyloid protein 42 (Beta-APP42);Beta-amyloid protein 40 (Beta-APP40); C83;P3(42); P3(40); Gamma-CTF(59) (Gamma-secretase C-terminal fragment 59) (Amyloidintracellular domain 59) (AID(59)) (AICD-59); Gamma-CTF(57) (Gamma-secretase C-terminal fragment 57) (Amyloid intracellulardomain 57) (AID(57)) (AICD-57); Gamma-CTF(50) (Gamma-secretase C-terminalfragment 50) (Amyloid intracellular domain50) (AID(50)) (AICD-50); C31]. EEDD, (SEQ ID NO:45)SEDK, (SEQ ID NO: 46)DEDD, (SEQ ID NO: 47)DEDG, (SEQ ID NO: 48)AEDV, (SEQ ID NO: 23) P05067 APP protein [Homo sapiens]. EEDD, (SEQ ID NO: 45)SEDK, (SEQ ID NO: 46)DEDD, (SEQ ID NO: 47)DEDG, (SEQ ID NO: 48) AAH65523 APP protein [Homo sapiens]. EEDD, (SEQ ID NO: 45)SEDK, (SEQ ID NO: 46)DEDD, (SEQ ID NO: 47)DEDG, (SEQ ID NO: 48) AAH04369 amyloid beta (A4) precursor protein(protease nexin-II, Alzheimer disease) [Homo sapiens]. EEDD, (SEQ ID NO: 45)SEDK, (SEQ ID NO: 46)DEDD, (SEQ ID NO: 47)DEDG, (SEQ ID NO: 48)AEDV, (SEQ ID NO: 23) AAW82435 Calcitonin SEDE, (SEQ ID NO: 18) AAA58403 calcitonin precursor SEDE, (SEQ ID NO: 18) AAA35501 preprocalcitonin [Homo sapiens] SEDE, (SEQ ID NO: 18) CAA25103 Preprocalcitonin SEDE, (SEQ ID NO: 18) AAA51913 Calcitonin precursor [Contains: Calcitonin;Katacalcin (Calcitonin carboxyl-terminalpeptide) (CCP) (PDN-21)] SEDE, (SEQ ID NO: 18) P01258 calcitonin isoform CALC A preproprotein[Homo sapiens]. SEDE, (SEQ ID NO: 18) NP_001029124 calcitonin isoform CALCA preproprotein[Homo sapiens]. SEDE, (SEQ ID NO: 18) NP_001732 66 WO 2009/086539 PCT/US2008/088493

Human amyloidogenic protein Consensus sequence GenBank Accession Number calcitonin isoform CGRP preproprotein[Homo sapiens]. SEDE, (SEQ ID NO: 18) NP_001029125 Calcitonin gene-related peptide 1 precursor(Calcitonin gene-related peptide I) (CGRP-I)(Alpha-type CGRP). SEDE, (SEQ ID NO: 18) P06881 atrial natriuretic factor LEDE, (SEQ ID NO: 49) AAA35528 atrial natriuretic factor propeptide [Homosapiens]. LEDE, (SEQ ID NO: 49) CAA25700 atrial natriuretic factor LEDE, (SEQ ID NO: 49) 1101403A Atrial natriuretic factor precursor (ANF)(Atrial natriuretic peptide) (ANP)(Prepronatriodilatin) (CDD-ANF) [Contains:Cardiodilatin-related peptide (CDP)]. LEDE, (SEQ ID NO: 49) P01160 atrial natriuretic peptide LEDE, (SEQ ID NO: 49) AAA35529 keratin [Homo sapiens] GEDA, (SEQ ID NO: 50) AAB30058 keratin [Homo sapiens]. VEDF, (SEQ ID NO: 51)YEDE, (SEQ ID NO: 52) CAA31695 Keratin IEDL, (SEQ ID NO: 53)GEDA, (SEQ ID NO: 50) AAB59562 Keratin, type II cytoskeletal 6C (Cytokeratin-6C) (CK 6C) (K6c keratin) (Cytokeratin-6E)(CK 6E) (Keratin K6h). VEDL, (SEQ ID NO: 64)YEDE, (SEQ ID NO: 52)LEDA, (SEQ ID NO: 65) P48668 fibrinogen [Homo sapiens] WEDY, (SEQ ID NO: 54) CAA50740 fibrinogen alpha subunit precursor [Homosapiens]. DEDW, (SEQ ID NO: 55)SEDL, (SEQ ID NO: 56)YEDQ, (SEQ ID NO: 57)SEDG, (SEQ ID NO: 66)LEDW, (SEQ ID NO: 58) AAC97142 Fibrinogen alpha chain [Homo sapiens] DEDW, (SEQ ID NO: 55)SEDL, (SEQ ID NO: 56)YEDQ, (SEQ ID NO: 57)SEDG, (SEQ ID NO: 66) AAI01936 Fibrinogen alpha chain [Homo sapiens] DEDW, (SEQ ID NO: 55)SEDL, (SEQ ID NO: 56)YEDQ, (SEQ ID NO: 57)SEDG, (SEQ ID NO: 66) AAH98280 67 WO 2009/086539 PCT/US2008/088493

Human amyloidogenie protein Consensus sequence GenBank Accession Number fibrinogen alpha chain, isoform CRA_b[Homo sapiens]. DEDW, (SEQ ID NO: 55)SEDL, (SEQ ID NO: 56)YEDQ, (SEQ ID NO: 57)SEDG, (SEQ ID NO: 66)LEDW, (SEQ ID NO: 58) EAX04926 fibrinogen alpha chain, isoform CRAc[Homo sapiens]. DEDW, (SEQ ID NO: 55)SEDL, (SEQ ID NO: 56)YEDQ, (SEQ ID NO: 57)SEDG, (SEQ ID NO: 66) EAX04928 fibrinogen alpha chain, isoform CRA_a[Homo sapiens] DEDW, (SEQ ID NO: 55)SEDL, (SEQ ID NO: 56) EAX04924 prion protein precursor; PRNP [Homosapiens] YEDR, (SEQ ID NO: 59) AAC62750 Major prion protein precursor (PrP) (PrP27-30) (PrP33-35C) (ASCR) (CD230 antigen) YEDR, (SEQ ID NO: 59) P04156 prion protein preproprotein [Homo sapiens]. YEDR, (SEQ ID NO: 59) NP_000302 prolactin [Homo sapiens] PEDK, (SEQ IDNO: 60) CAA38264 Prolactin [Homo sapiens]. PEDK, (SEQ ID NO: 60) AAH88370 VI. Amyloid Peptides for Active Immunization

Therapeutic agents for use in the methods of the invention are immunogenic peptides,such as AA peptides and AL peptides, that on administration to a patient generate antibodies thatspecifically bind to one or more epitopes comprising X1EDX2, such as, for example, epitopes 5 between residues 70-76 of AA (“AA agents”). Additional examples of agents includeimmunogenic peptides that comprise a fragment consisting of XiEDX2 derived from otheramyloid proteins (“XiEDX2 fragments”), such as AL Vk fragments consisting of the amino acidsequence PEDI, (SEQ ID NO: 21), PEDF, (SEQ ID NO: 22), AEDV, (SEQ ID NO: 23), SEDF,(SEQ ID NO: 24), or SEDA, (SEQ ID NO: 25), and AL VX fragments consisting of the amino 10 acid sequence SEDE, (SEQ ID NO: 18), AEDE, (SEQ ID NO: 19), TEDE, (SEQ ID NO: 16) orPEDE, (SEQ ID NO: 20). An AL VX fragment consisting of the amino acid sequence FEDD,(SEQ ID NO: 17) may also be used. Some suitable amyloid proteins include Serum amyloid Aprotein, immunoglobulin light chain protein, human islet amyloid precursor polypeptide (IAPP),beta amyloid peptide, transthyretin (TTR), ApoAl and other amyloid proteins listed in Table 1 15 and which comprise the sequence XiEDX2. In some agents Xi is Η, T, F, S, P, A or any other 68 WO 2009/086539 PCT/US2008/088493 amino acid residue immediately preceding ED in an amyloid protein; and X2 is T, S, E, R, I, V,F, D, A or any other amino acid residue immediately following ED in such amyloid protein. Insome agents, Xi is Η, T, F, S, P, or A and X2 is T, S, E, D, R, I, V, F or A. In some such agents,when Xi is Η, X2 is T or A; when Xj is A, X2 is S, T, E or V; when Xi is T, X2 is E; when Xi isF, X2 is D; when Xi is S, X2 is E, F or A; and when Xi is P, X2 is E, I or F. In some agents, Xi isΗ, T, F, S, P, or A and X2 is T, S, E, D, R, I, V, F or A, with the proviso that if Xi is A, X2 is notV. In some agents, when Xi is A, X2 is S, T or E.

Some agents comprise the amino acid sequence GHEDT, (SEQ ID NO: 3), HEDT,(SEQ ID NO: 12), AEDS, (SEQ ID NO: 13), AEDT, (SEQ ID NO: 14), HEDA, (SEQ ID NO:15), TEDE, (SEQ ID NO: 16), FEDD, (SEQ ID NO: 17), SEDE, (SEQ ID NO: 18), AEDE,(SEQ ID NO: 19), PEDE, (SEQ ID NO: 20), PEDI, (SEQ ID NO: 21), PEDF, (SEQ ID NO: 22),AEDV, (SEQ ID NO: 23), SEDF, (SEQ ID NO: 24) or SEDA, (SEQ ID NO: 25). Some agentsconsist of an amino acid sequence selected from the group consisting of GHEDT, (SEQ ID NO:3, HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO: 13), AEDT, (SEQ ID NO: 14), HEDA, (SEQID NO: 15), TEDE, (SEQ ID NO: 16), FEDD, (SEQ ID NO: 17), SEDE, (SEQ ID NO: 18),AEDE, (SEQ ID NO: 19), PEDE, (SEQ ID NO: 20), PEDI, (SEQ ID NO: 21), PEDF, (SEQ IDNO: 22), AEDV, (SEQ ID NO: 23), SEDF, (SEQ ID NO: 24), or SEDA, (SEQ ID NO: 25),linked to a carrier to form a conjugate. Some agents comprise the amino acid sequence GHEDT,(SEQ ID NO: 3, HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO: 13), AEDT, (SEQ ID NO: 14),HEDA, (SEQ ID NO: 15), TEDE, (SEQ ID NO: 16), FEDD, (SEQ ID NO: 17), SEDE, (SEQ IDNO: 18), AEDE, (SEQ ID NO: 19), PEDE, (SEQ ID NO: 20), PEDI, (SEQ ID NO: 21), PEDF,(SEQ ID NO: 22), AEDV, (SEQ ID NO: 23), SEDF, (SEQ ID NO: 24), or SEDA, (SEQ ID NO:25). Some agents consist of an amino acid sequence selected from the group consisting ofGHEDT, (SEQ ID NO: 3, HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO: 13), AEDT, (SEQ IDNO: 14), HEDA, (SEQ ID NO: 15), TEDE, (SEQ ID NO: 16), FEDD, (SEQ ID NO: 17), SEDE,(SEQ ID NO: 18), AEDE, (SEQ ID NO: 19), PEDE, (SEQ ID NO: 20), PEDI, (SEQ ID NO: 21),PEDF, (SEQ ID NO: 22), SEDF, (SEQ ID NO: 24) and SEDA, (SEQ ID NO: 25), linked to acarrier to form a conjugate. Some agents comprise an amino acid sequence selected from thegroup consisting of GHEDT, (SEQ ID NO: 3, HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO:13), AEDT, (SEQ ID NO: 14), HEDA, (SEQ ID NO: 15) and TEDE, (SEQ ID NO: 16).

Preferred AA fragments are human AA1 (HAA1) alpha isoform residues 70-76(GHGAEDS, SEQ ID NO:4), HAA1 beta isoform residues 70-76 (GHDAEDS, SEQ ID NO:5), 69 WO 2009/086539 PCT/US2008/088493 HAA1 gamma isoform residues 70-76 (GHDAEDS, SEQ ID NO: 5), HAA2 alpha and betaisoforms residues 70-76 (GHGAEDS, SEQ ID NO: 4), HAA3 residues 70-76 (GDHAEDS, SEQID NO:7), HAA4 residues 78-84 (STVIEDS, SEQ ID NO:8), mouse AA1 (MAA1) residues 69-75 (GRGHEDT, SEQ ID NO:9), MAA2 residues 69-75 (GRGHEDT, SEQ ID NO: 9), MAA3residues 62-68 (GHGAEDS, SEQ ID NO:10), and MAA4 residues 76-82 (NHGLETL, SEQ IDNO: 11) or subfragments of at least three contiguous amino acids of any of these. Some AAfragments contain no residues of an AA amyloidosis peptide other than the segment designatedabove. Other AA fragments contain additional flanking residues from an AA amyloidosispeptide but contain no more than 20 or preferably no more than 10 contiguous residues in totalfrom an AA amyloidosis peptide. Additional preferred X]EDX2 and AL fragments includeGHEDT, (SEQ ID NO: 3), HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO: 13), AEDT, (SEQID NO: 14), HEDA, (SEQ ID NO: 15), and TEDE, (SEQ ID NO: 16).

Therapeutic agents for use in the methods of the invention also include immunogenicAA peptides that on administration to a patient generate antibodies that specifically bind to N-terminal epitopes of AA. Preferred agents induce an immunogenic response directed to anepitope within residues 1-15 of human AA.

Preferably, the fragment of AA or AL or other agents such as X]EDX2 fragmentsadministered lack an epitope that would generate a T-cell response to the fragment. Generally,T-cell epitopes are greater than 10 contiguous amino acids. Therefore, preferred fragments ofamyloid proteins such as AA or XiEDX2 fragments are of size 4-10 or preferably 7-10contiguous amino acids; i.e., sufficient length to generate an antibody response withoutgenerating a T-cell response. Absence of T-cell epitopes is preferred because these epitopes arenot needed for immunogenic activity of fragments, and may cause an undesired inflammatoryresponse in a subset of patients (Anderson et al., (2002) J. Immunol. 168, 3697-3701; Senior(2002) Lancet Neurol. 1, 3).

Preferred AA fragments are human AA1 (HAA1) alpha isoform residues 70-76(GHGAEDS) (SEQ ID NO: 4), HAA1 beta isoform residues 70-76 (GHDAEDS) (SEQ IDNO:5), HAA1 gamma isoform residues 70-76 (GHDAEDS, SEQ ID NO: 5), HAA2 alpha andbeta isoforms residues 70-76 (GHGAEDS, SEQ ID NO: 4), HAA3 residues 70-76 (GDHAEDS)(SEQ ID NO:7), HAA4 residues 78-84 (STVIEDS) (SEQ ID NO:8), mouse AA1 (MAA1)residues 69-75 (GRGHEDT) (SEQ ID NO:9), MAA2 residues 69-75 (GRGHEDT, SEQ ID NO:9), MAA3 residues 62-68 (GHGAEDS) (SEQ ID NO: 10), and MAA4 residues 76-82 70 WO 2009/086539 PCT/US2008/088493 (NHGLETL) (SEQ ID NO: 11) or sub fragments of at least three contiguous amino acids of anyof these. Some AA fragments contain no residues of an AA amyloidosis peptide other than thesegment designated above. Other AA fragments contain additional flanking residues from anAA amyloidosis peptide but contain no more than 20 or preferably no more than 10 contiguousresidues in total from an AA amyloidosis peptide. Additional preferred XiEDX2 and ALfragments include GHEDT, (SEQ ID NO: 3), HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO:13), AEDT, (SEQ ID NO: 14), HEDA, (SEQ ID NO: 15), and TEDE, (SEQ ID NO: 16).

Analogs of the natural AA amyloidosis, AL amyloidosis, and other amyloidosispeptides can also be used to induce an immune response in the methods and compositions of theinvention. Analogs including allelic, species and induced variants. Analogs of AA induceantibodies that specifically bind with a natural AA 70-76 peptide. Some such analogs fail toinduce antibodies that specifically binds to epitopes outside AA70-76. Analogs of AA typicallydiffer from naturally occurring peptides at up to 30% of amino acid positions by up to 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 position changes. Each deletion or substitution of a natural amino acid residueis considered a position change as is the insertion of a residue without substitution. Amino acidssubstitutions are often conservative substitutions.

Some analogs of AA or AA fragments or AL or AL fragments or other amyloid proteinfragments such as X] EDX2 fragments also include unnatural amino acids or modifications of Nor C terminal amino acids at one, two, five, ten or even all positions. For example, the naturalaspartic acid residue can be replaced with iso-aspartic acid. Examples of unnatural amino acidsare D, alpha, alpha-disubstituted amino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline,gamma-carboxyglutamate, epsilon-N,N,N-trimethyllysine, epsilon-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,omega-N-methylarginine, β-alanine, ornithine, norleucine, norvaline, hydroxproline, thyroxine,gamma-amino butyric acid, homoserine, citrulline, and isoaspartic acid. Some therapeutic agentsof the invention are all-D peptides, e.g., all-D AA or all-D AA fragments, and all-D peptideanalogs. Some therapeutic agents of the invention are 90% all-D peptides, e.g., 90% all-D AA or90% all-D AA fragments, and 90% all-D peptide analogs. Some therapeutic agents of theinvention are 80% all-D peptides, e.g., 80% all-D AA or 80% all-D AA fragments, and 80% all-D peptide analogs. Fragments and analogs can be screened for prophylactic or therapeuticefficacy in transgenic animal models in comparison with untreated or placebo controls asdescribed below. 71 WO 2009/086539 PCT/US2008/088493 AA, AL, their fragments, and analogs and XiEDX2 fragments and their analogs can besynthesized by solid phase peptide synthesis or recombinant expression, or can be obtained fromnatural sources. Automatic peptide synthesizers are commercially available from numeroussuppliers, such as Applied Biosystems, Foster City, California. Recombinant expression can bein bacteria, such as E. coli, yeast, insect cells or mammalian cells. Procedures for recombinantexpression are described by Sambrook et al., Molecular Cloning: A Laboratory: Manual(C.S.H.P. Press, NY 2d ed., 1989.)

Therapeutic agents also include longer polypeptides that include, for example, animmunogenic fragment of AA peptide, AL peptide or an XiEDX2 fragment, together with one ormore other amino acids flanking the AA peptide, AL peptide or X]EDX2 fragment on one or oneor both sides. For example, preferred agents include fusion proteins comprising a segment ofAA, AL or XiEDX2 fragment fused to a heterologous amino acid sequence that induces a helperT-cell response against the heterologous amino acid sequence and thereby a B-cell responseagainst the AA segment, AL segment or XiEDX2 fragment. One or more flanking heterologousamino acids can also be used to cap an AA or AL peptide or XiEDX2 fragment to protect it fromdegradation in manufacture, storage or use. Such polypeptides can be screened for prophylacticor therapeutic efficacy in animal models in comparison with untreated or placebo controls asdescribed below. Therapeutic agents of the invention include an immunogenic fragment of AAor AL or XiEDX2 fragment flanked by polylysine sequences. The polylysine sequences can befused to the N-terminus, the C terminus, or both the N- and C-terminus of AA or AL or animmunogenic fragment of AA or AL or X]EDX2 fragment. The AA or AL peptide, XiEDX2fragment, analog, active fragment of AA or other polypeptide can be administered in associatedor multimeric form or in dissociated form. Therapeutic agents also include multimers ofmonomeric immunogenic agents.

In a further variation, an immunogenic fragment of AA or AL or XiEDX2 fragment canbe presented by a virus or a bacterium as part of an immunogenic composition. A nucleic acidencoding the immunogenic peptide is incorporated into a genome or episome of the virus orbacteria. Optionally, the nucleic acid is incorporated in such a manner that the immunogenicpeptide is expressed as a secreted protein or as a fusion protein with an outer surface protein of avirus or a transmembrane protein of a bacterium so that the peptide is displayed. Viruses orbacteria used in such methods should be nonpathogenic or attenuated. Suitable viruses includeadenovirus, HSV, Venezuelan equine encephalitis virus and other alpha viruses, vesicular 72 WO 2009/086539 PCT/US2008/088493 stomatitis virus, and other rhabdo viruses, vaccinia and fowl pox. Suitable bacteria includeSalmonella and Shigella. Fusion of an immunogenic peptide to HBsAg of HBV is particularlysuitable.

Therapeutic agents also include peptides and other compounds that do not necessarilyhave a significant amino acid sequence similarity with AA or AL or X]EDX2 fragment butnevertheless serve as mimetics of AA or AL or XiEDX2 fragment and induce a similar immuneresponse. For example, any peptides and proteins forming β-pleated sheets can be screened forsuitability. Anti-idiotypic antibodies against monoclonal antibodies to AA or AL or otheramyloidogenic peptides such as or X1EDX2 fragments can also be used. Such anti-id antibodiesmimic the antigen and generate an immune response to it (see Essential Immunology (Roit ed.,Blackwell Scientific Publications, Palo Alto, 6th ed.), p. 181). Agents other than AA peptidesshould induce an immunogenic response against one or more of the preferred segments of AAlisted above (e.g., AA70-76 or GHEDT, (SEQ ID NO: 3) or an AL or XiEDX2 fragment listedabove, such as, for example, HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO: 13), AEDT, (SEQID NO: 14), HEDA, (SEQ ID NO: 15) and TEDE, (SEQ ID NO: 16).

Preferably, such agents induce an immunogenic response that is specifically directed toone of these segments without being directed to other segments of AA or AL or amyloid proteinfrom which the Xi EDX2 fragment was derived.

Random libraries of peptides or other compounds can also be screened for suitability.Combinatorial libraries can be produced for many types of compounds that can be synthesized ina step-by-step fashion. Such compounds include polypeptides, beta-turn mimetics,polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds,heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines andoligocarbamates. Large combinatorial libraries of the compounds can be constructed by theencoded synthetic libraries (ESL) method described in Affymax, WO 95/12608, Affymax, WO93/06121, Columbia University, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO95/30642 (each of which is incorporated by reference for all purposes). Peptide libraries can alsobe generated by phage display methods. See, e.g., Devlin, W0 91/18980.

Combinatorial libraries and other compounds are initially screened for suitability bydetermining their capacity to specifically bind to antibodies or lymphocytes (B or T) known to bespecific for AA or other amyloidogenic peptides. For example, initial screens can be performed 73 WO 2009/086539 PCT/US2008/O88493 with any polyclonal sera or monoclonal antibody to AA or AL or a fragment thereof or to anXiEDX2 fragment. Compounds can then be screened for specifically binding to a specificepitope within AA (e.g., AA70-76 or GHEDT, (SEQ ID NO: 3) or AL or to an X1EDX2 fragmentlisted above, such as, for example, HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO: 13), AEDT,(SEQ ID NO: 14), HEDA, (SEQ ID NO: 15) and TEDE, (SEQ ID NO: 16).

Compounds can be tested by the same procedures described for mapping antibodyepitope specificities. Compounds identified by such screens are then further analyzed forcapacity to induce antibodies or reactive lymphocytes to AA or AL or fragments thereof or to anX1EDX2 fragment. For example, multiple dilutions of sera can be tested on microtiter plates thathave been precoated with AA or AL or a fragment thereof or an XiEDX2 fragment and astandard ELISA can be performed to test for reactive antibodies to AA or AL or the fragment orto the X1EDX2 fragment. Compounds can then be tested for prophylactic and therapeuticefficacy in transgenic animals predisposed to amyloidosis, such as, for example, AAAmyloidosis or AL amyloidosis. The same screening approach can be used on other potentialagents, analogs of AA, analogs of AL and longer peptides, including fragments of AA, AL andX1EDX2 fragments, described above. VII. Conjugates

Some agents for inducing an immune response contain the appropriate epitope forinducing an immune response against AA but are too small to be immunogenic. In this situation,a peptide immunogen can be linked to a suitable carrier molecule to form a conjugate whichhelps elicit an immune response. A single agent can be linked to a single carrier, multiple copiesof an agent can be linked to multiple copies of a carrier, which are in turn linked to each other,multiple copies of an agent can be linked to a single copy of a carrier, or a single copy of anagent can be linked to multiple copies of a carrier, or different carriers. Suitable carriers includeserum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin,ovalbumin, tetanus toxoid, or a toxoid from other pathogenic bacteria, such as diphtheria, E. coli,cholera, or H. pylori, or an attenuated toxin derivative. T cell epitopes are also suitable carriermolecules. Some conjugates can be formed by linking agents of the invention to animmunostimulatory polymer molecule (e.g., tripalmitoyl-S-glycerine cysteine (PamsCys),mannan (a manose polymer), or glucan (a beta 1—>2 polymer)), cytokines (e.g., IL-1, IL-1 alphaand beta peptides, IL-2, gamma-INF, IL-10, GM-CSF), and chemokines (e.g., MIP1 alpha and 74 WO 2009/086539 PCT/US2008/088493 beta, and RANTES). Immunogenic agents can also be linked to peptides that enhance transportacross tissues, as described in O'Mahony, WO 97/17613 and WO 97/17614. Immunogens maybe linked to the carries with or with out spacers amino acids (e.g., gly-gly).

Some conjugates can be formed by linking agents of the invention to at least one T cellepitope. Some T cell epitopes are promiscuous while other T cell epitopes are universal.Promiscuous T cell epitopes are capable of enhancing the induction of T cell immunity in a widevariety of subjects displaying various HLA types. In contrast to promiscuous T cell epitopes,universal T cell epitopes are capable of enhancing the induction of T cell immunity in a largepercentage, e.g., at least 75%, of subjects displaying various HLA molecules encoded bydifferent HLA-DR alleles. A large number of naturally occurring T-cell epitopes exist, such as, tetanus toxoid(e.g., the P2 and P30 epitopes), Hepatitis B surface antigen, pertussis, toxoid, measles virus Fprotein, Chlamydia trachomitis major outer membrane protein, diphtheria toxoid (e.g.,CRM 197), Plasmodium falciparum circumsporozite T, Plasmodium falciparum CS antigen,Schistosoma mansoni triose phosphate isomersae, Escherichia coli TraT, and Influenza virushemagluttinin (HA). The immunogenic peptides of the invention can also be conjugated to theT-cell epitopes described in Sinigaglia F. et al., Nature, 336:778-780 (1988); Chicz R.M. et al., J.Exp. Med., 178:27-47 (1993); Hammer J. et al., Cell 74:197-203 (1993); Falk K. et al.,Immunogenetics, 39:230-242 (1994); WO 98/23635; Southwood S. et al. J. Immunology;160:3363-3373 (1998); and, Giannini,G. et al. Nucleic Acids Res. 12: 4063-4069 (1984), (eachof which is incorporated herein by reference for all purposes). Further examples include:

Influenza Hemagluttinin: HA307-319

Malaria CS: T3 epitope EKKIAKMEKASSVFNV, (SEQ ID NO: 67).

Hepatitis B surface antigen: HBsAgi9_28 FFLLTRILTI, (SEQ ID NO: 68).

Heat Shock Protein 65: hsp65i53-i7i DQSIGDLIAFAMDKVGNFG, (SEQ ID NO: 69). bacille Calmette-Guerin QVHFQPLPPAWKL, (SEQ ID NO: 70).

Tetanus toxoid: TT830-844 QYIKANSKFIGITEL, (SEQ ID NO: 71).

Tetanus toxoid: TT947-967 FNNFTVSFWLRVPKVSASHLE, (SEQ ID NO: 72). HIV gpl20 T1: KQIINMWQFVGKAMYA, (SEQ ID NO: 73).

Tetanus toxoid: TT947-967 FNNFTVSFWLRVPKVSASHLE 75 WO 2009/086539 PCT/US2008/088493 HIV gpl20 IT. KQIINMWQEVGKAMYA.

Alternatively, the conjugates can be formed by linking agents of the invention to atleast one artificial T-cell epitope capable of binding a large proportion of MHC Class IImolecules., such as the pan DR epitope ("PADRE"). PADRE is described in US 5,736141, WO95/07707, and Alexander J et al., Immunity, 1:751-761 (1994) (each of which is incorporatedherein by reference for all purposes). A preferred PADRE peptide is AKXVAAWTLKAAA,(SEQ ID NO: 74), (common residues bolded) wherein X is preferably cyclohexylalanine tyrosineor phenylalanine, with cyclohexylalanine being most preferred.

Immunogenic agents can be linked to carriers by chemical crosslinking. Techniques forlinking an immunogen to a carrier include the formation of disulfide linkages using N-succinimidyl-3-(2-pyridyl-thio) propionate (SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC) (if the peptide lacks a sulfhydryl group,this can be provided by addition of a cysteine residue). These reagents create a disulfide linkagebetween themselves and peptide cysteine resides on one protein and an amide linkage throughthe epsilon-amino on a lysine, or other free amino group in other amino acids. A variety of suchdisulfide/amide-forming agents are described by Immun. Rev. 62, 185 (1982). Other bifunctionalcoupling agents form a thioether rather than a disulfide linkage. Many of these thio-ether-forming agents are commercially available and include reactive esters of 6-maleimidocaproicacid, 2-bromoacetic acid, and 2-iodoacetic acid, 4-(N-maleimido-methyl)cyclohexane-l-earboxylic acid. The carboxyl groups can be activated by combining them with succinimide or1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.

Immunogenicity can be improved through the addition of spacer residues (e.g., Gly-Gly) between the Th epitope and the peptide immunogen of the invention. In addition tophysically separating the Th epitope from the B cell epitope (i.e., the peptide immunogen), theglycine residues can disrupt any artificial secondary structures created by the joining of the Thepitope with the peptide immunogen, and thereby eliminate interference between the T and/or Bcell responses. The conformational separation between the helper epitope and the antibodyeliciting domain thus permits more efficient interactions between the presented immunogen andthe appropriate Th and B cells.

To enhance the induction of T cell immunity in a large percentage of subjectsdisplaying various HLA types to an agent of the present invention, a mixture of conjugates with 76 WO 2009/086539 PCT/US2008/088493 different Th cell epitopes can be prepared. The mixture may contain a mixture of at least twoconjugates with different Th cell epitopes, a mixture of at least three conjugates with different Thcell epitopes, or a mixture of at least four conjugates with different Th cell epitopes. The mixturemay be administered with an adjuvant. 5 Immunogenic peptides can also be expressed as fusion proteins with carriers (i.e., heterologous peptides). The immunogenic peptide can be linked at its amino terminus, itscarboxyl terminus, or both to a carrier. Optionally, multiple repeats of the immunogenic peptidecan be present in the fusion protein. Optionally, an immunogenic peptide can be linked tomultiple copies of a heterologous peptide, for example, at both the N and C termini of the 10 peptide. Optionally, multiple copies of an immunogenic peptide can be linked to multiple copiesof a heterologous peptide, which are linked to each other. Some carrier peptides serve to inducea helper T-cell response against the carrier peptide. The induced helper T-cells in turn induce aB-cell response against the immunogenic peptide linked to the carrier.

Some examples of fusion proteins suitable for use in the invention are shown below. 15 Some of these fusion proteins comprise segments of AA linked to tetanus toxoid epitopes such as described in US 5,196,512, EP 378,881 and EP 427,347. Some fusion proteins comprisesegments of AA linked to at least one PADRE peptide described in US 5,736,142. Someheterologous peptides are promiscuous T-cell epitopes, while other heterologous peptides areuniversal T-cell epitopes. In some methods, the agent for administration is simply a single fusion 20 protein with an AA segment linked to a heterologous segment in linear configuration. Thetherapeutic agents of the invention can be represented using a formula. For example, in somemethods, the agent is multimer of fusion proteins represented by the formula 2X, in which x is aninteger from 1-5. Preferably x is 1, 2 or 3, with 2 being most preferred. When x is two, such amultimer has four fusion proteins linked in a preferred configuration referred to as MAP4 (see 25 US 5,229,490).

The MAP4 configuration is shown below, where branched structures are produced byinitiating peptide synthesis at both the N terminal and side chain amines of lysine. Dependingupon the number of times lysine is incorporated into the sequence and allowed to branch, theresulting structure will present multiple N termini. In this example, four identical N termini have 30 been produced on the branched lysine-containing core. Such multiplicity greatly enhances theresponsiveness of cognate B cells. In the examples below, Z refers to an immunogenic fragmentof AA, AL or an XjEDX2 fragment, and Zl-4 refer to immunogenic fragment(s) of AA, AL or an ΊΊ WO 2009/086539 PCT/US2008/088493 X1EDX2 fragment. The fragments can be the same as each other or different.

<img img-format="tif" img-content="drawing" file="IL206641AD00021.tif" id="idf0001" />

KA

Other examples of fusion proteins include: Z-Tetanus toxoid 830-844 in a MAP4 configuration:Z-QYIKANSKFIGITEL, (SEQ ID NO: 71) Z-Tetanus toxoid 947-967 in a MAP4 configuration: Z-FNNFTVSFWLRVPKVSASHLE, (SEQ ID NO: 72) Z-Tetanus toxoid 830-844 in a MAP4 configuration:Z-QYIKANSKFIGITEL, (SEQ ID NO: 71) Z-Tetanus toxoid 830-844 + 947-967 in a linear configuration: Z-QYIKANSKFIGITELFNNFTVSFWLRVPKVSASHLE, (SEQ IDNO: 75). PADRE peptide (all in linear configurations), wherein X is preferably cyclohexylalanine, tyrosine or phenylalanine, with cyclohexylalaninebeing most preferred-Z: AKXVAAWTLKAAA-Z, (SEQ ID NO: 74). Z x 3-PADRE peptide: Z-Z-Z-AKXVAAWTLKAAA, (SEQ ID NO: 74). Z - ovalbumin 323-339 in a linear configuration: Z-ISQAVHAAHAEINEAGR, (SEQ ID NO: 76).

Further examples of fusion proteins include: AKXVAAWTLKAAA-Z-Z-Z-Z, (SEQ ID NO: 74).Z-AKXVAAWTLKAAA, (Z-(SEQ ID NO: 74).PKYVKQNTLKLAT-Z-Z-Z, (SEQ ID NO: 77). Z-PKYVKQNTLKLAT-Z, (SEQ ID NO: 77). Z-Z-Z-PKYVKQNTLKLAT, (SEQ ID NO: 77). 78 WO 2009/086539 PCT/US2008/088493 Z-Z-PKYVKQNTLKLAT, (Z-Z-(SEQ ID NO: 77) Z-PKYVKQNTLKLAT-EKKIAKMEKASSVFNV-QYIKANSKFIGITEL-FNNFTVSFWLRVPKVSASHLE-(SEQ ID NO: 78) Z-Z-Z-QYIKANSKFIGITFL-FNNFTVSFWLRVPKVSASHLE, (SEQ IDNO: 79). Z-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE-Z, (SEQ ID NO:79). QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE-Z, (SEQ ID NO:79) Z-QYIKANSKFIGITEL, (SEQ ID NO: 71) on a 2 branched resin: fragments can be the same as each other or different.

Lys-Gly-Cys

The same or similar carrier proteins and methods of linkage can be used for generatingimmunogens to be used in generation of antibodies against AA or an immunogenic fragment ofAA, AL or an XjEDX2 fragment. For example, AA or an immunogenic fragment of AA, AL oran XiEDX2 fragment linked to a carrier can be administered to a laboratory animal in theproduction of monoclonal antibodies to AA or an immunogenic fragment of AA, AL or anXjEDX2 fragment. VIII. Nucleic Acid Encoding Therapeutic Agents

Therapeutic agents of the invention also include nucleic acids. Immune responses against amyloid deposits can also be induced by administration of nucleic acids encoding segments of AA peptide, and fragments thereof, other peptide immunogens such as XiEDX2fragments, or antibodies and their component chains, such as antibodies 2A4, 8G9 and 7D8,used for passive immunization. Such agents for use in the methods of the invention includenucleic acids encoding AA peptides that on administration to a patient generate antibodies thatspecifically bind to one or more epitopes between residues 70-76 of AA, AL or nucleic acidsencoding peptides comprising XiEDX2 fragments. Such agents for use in the methods of the WO 2009/086539 PCT/US2008/088493 invention also include nucleic acids encoding antibodies that specially bind to a C-terminalneoepitope of AA or to XiEDX2. In particular, such nucleic acids encode antibodies thatspecifically bind to HAA1 alpha isoform within residues 70-76 (GHGAEDS, (SEQ ID NO: 4),HAA1 beta isoform within residues 70-76 (GHDAEDS, (SEQ ID NO: 5), HAA1 gamma 5 isoform within residues 70-76 (GHDAEDS, (SEQ ID NO: 5), HAA2 alpha and beta isoformswithin residues 70-76 (GHGAEDS, (SEQ ID NO: 4), HAA3 within residues 70-76 (GDHAEDS,(SEQ ID NO: 7), HAA4 within residues 78-84 (STVIEDS, (SEQ ID NO: 8), mouse AA1(MAA1) within residues 69-75 (GRGHEDT, (SEQ ID NO: 9), MAA2 within residues 69-75(GRGHEDT, (SEQ ID NO: 9), MAA3 within residues 62-68 (GHGAEDS, (SEQ ID NO: 4), and 10 MAA4 within residues 76-82 (NHGLETL, (SEQ ID NO: 11). Such nucleic acids can be DNAor RNA. Additional preferred nucleic acids encode antibodies that specifically bind to HEDT,(SEQ ID NO: 12), AEDS, (SEQ ID NO: 13), AEDT, (SEQ ID NO: 14), HEDA, (SEQ ID NO:15) or TEDE, (SEQ ID NO: 16) or other X1EDX2 peptides listed above. A nucleic acid segmentencoding an immunogen is typically linked to regulatory elements, such as a promoter and 15 enhancer, that allow expression of the DNA segment in the intended target cells of a patient. Forexpression in blood cells, as is desirable for induction of an immune response, promoter andenhancer elements from light or heavy chain immunoglobulin genes or the CMV majorintermediate early promoter and enhancer are suitable to direct expression. The linkedregulatory elements and coding sequences are often cloned into a vector. For administration of 20 double-chain antibodies, the two chains can be cloned in the same or separate vectors. Thenucleic acids encoding therapeutic agents of the invention can also encode at least one T cellepitope. The disclosures herein which relate to the use of adjuvants and the use of carriers applymutatis mutandis to their use with the nucleic acids encoding the therapeutic agents of thepresent invention. 25 A number of viral vector systems are available including retroviral systems (see, e.g.,

Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993)); adenoviral vectors (see, e.g.,Bett et al., J. Virol. 67, 5911 (1993)); adeno-associated virus vectors (see, e.g., Zhou et al., J.Exp. Med. 179, 1867 (1994)), viral vectors from the pox family including vaccinia virus and theavian pox viruses, viral vectors from the alpha virus genus such as those derived from Sindbis 30 and Semliki Forest Viruses (see, e.g., Dubensky et al., J. Virol. 70, 508-519 (1996)), Venezuelanequine encephalitis vims (see US 5,643,576) and rhabdoviruses, such as vesicular stomatitisvims (see WO 96/34625) and papillomavimses (Ohe et al., Human Gene Therapy 6, 325-333 80 WO 2009/086539 PCT/US2008/088493 (1995); Woo et al., WO 94/12629 and Xiao &amp;amp; Brandsma, Nucleic Acids. Res. 24, 2630-2622(1996)). DNA encoding an immunogen, or a vector containing the same, can be packaged intoliposomes. Suitable lipids and related analogs are described by US 5,208,036, 5,264,618,5,279,833 and 5,283,185. Vectors and DNA encoding an immunogen can also be adsorbed to orassociated with particulate carriers, examples of which include polymethyl methacrylatepolymers and polylactides and poly(lactide-co-glycolides), see, e.g., McGee et al., J. MicroEncap. (1996).

Gene therapy vectors or naked DNA can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, nasal,gastric, intradermal, intramuscular, subdermal, or intracranial infusion) or topical application(see e.g., US 5,399,346). Such vectors can further include facilitating agents such as bupivacine(US 5,593,970). DNA can also be administered using a gene gun. (See Xiao &amp;amp; Brandsma,supral) The DNA encoding an immunogen is precipitated onto the surface of microscopic metalbeads. The microprojectiles are accelerated with a shock wave or expanding helium gas, andpenetrate tissues to a depth of several cell layers. For example, The Accel™ Gene DeliveryDevice manufactured by Agacetus, Inc. Middleton WI is suitable. Alternatively, naked DNAcan pass through skin into the blood stream simply by spotting the DNA onto skin with chemicalor mechanical irritation (see WO 95/05853).

In a further variation, vectors encoding immunogens can be delivered to cells ex vivo,such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates,tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of thecells into a patient, usually after selection for cells which have incorporated the vector. IX. Adjuvants

Immunogenic agents of the invention, such as peptides, are sometimes administered incombination with an adjuvant. The adjuvant increases the titer of induced antibodies and/or thebinding affinity of induced antibodies relative to the situation if the peptide were used alone. Avariety of adjuvants can be used in combination with an immunogenic fragment of AA, to elicitan immune response. Preferred adjuvants augment the intrinsic response to an immunogenwithout causing conformational changes in the immunogen that affect the qualitative form of theresponse. Preferred adjuvants include aluminum hydroxide and aluminum phosphate, 3 De-O- 81 WO 2009/086539 PCT/US2008/088493 acylated monophosphoryl lipid A (MPL™) (see GB 2220211 (RIBI ImmunoChem ResearchInc., Hamilton, Montana, now part of Corixa), RC-529 (Corixa, Hamilton, Montana).STIMULON™ QS-21 is a triterpene glycoside or saponin isolated from the bark of the QuillajaSaponaria Molina tree found in South America (see Kensil et al., in Vaccine Design: TheSubunit and Adjuvant Approach (eds. Powell &amp;amp; Newman, Plenum Press, NY, 1995); US PatentNo. 5,057,540), (Aquila BioPharmaceuticals, Framingham, MA). Other adjuvants are oil inwater emulsions (such as squalene or peanut oil), optionally in combination with immunestimulants, such as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91(1997)), pluronic polymers, and killed mycobacteria. Another adjuvant is CpG (WO 98/40100).Adjuvants can be administered as a component of a therapeutic composition with an active agentor can be administered separately, before, concurrently with, or after administration of thetherapeutic agent.

A preferred class of adjuvants is aluminum salts (alum), such as alum hydroxide, alumphosphate, alum sulfate. Such adjuvants can be used with or without other specificimmunostimulating agents such as MPL or 3-DMP, QS-21, polymeric or monomeric aminoacids such as polyglutamic acid or polylysine. Another class of adjuvants is oil-in-wateremulsion formulations. Such adjuvants can be used with or without other specificimmunostimulating agents such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(r-2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy propylamide (DTP-DPP) THERAMIDE™), or other bacterial cellwall components. Oil-in-water emulsions include (a) MF59 (WO 90/14837), containing 5%Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE) formulated into submicron particles using a microfluidizer such as Model 110Ymicrofluidizer (Microfluidics, Newton MA), (b) SAF, containing 10% Squalene, 0.4% Tween80, 5% pluronic-blocked polymer L121, and thr-MDP, either microfluidized into a submicronemulsion or vortexed to generate a larger particle size emulsion, and (c) RIBI™ adjuvant system(RAS), (Ribi ImmunoChem, Hamilton, MT) containing 2% squalene, 0.2% Tween 80, and oneor more bacterial cell wall components from the group consisting of monophosphoryllipid A(MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS 82 WO 2009/086539 PCT/US2008/088493 (DETOX™).

Another class of preferred adjuvants is saponin adjuvants, such as STIMULON™ (QS-21, Aquila, Framingham, MA) or particles generated therefrom such as ISCOMs(immunostimulating complexes) and ISCOMATRIX. Other adjuvants include RC-529, GM-CSF and Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA). Otheradjuvants include cytokines, such as interleukins (e.g., IL-1 a and β peptides,, IL-2, IL-4, IL-6,IL-12, IL13, and IL-15), macrophage colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), ehemokines,such as MIPla and β and RANTES. Another class of adjuvants is glycolipid analoguesincluding N-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each of which issubstituted in the sugar residue by an amino acid, as immuno-modulators or adjuvants (see USPat. No. 4,855,283). Heat shock proteins, e.g., HSP70 and HSP90, may also be used asadjuvants.

An adjuvant can be administered with an immunogen as a single composition, or can beadministered before, concurrent with, or after administration of the immunogen. Immunogenand adjuvant can be packaged and supplied in the same vial or can be packaged in separate vialsand mixed before use. Immunogen and adjuvant are typically packaged with a label indicatingthe intended therapeutic application. If immunogen and adjuvant are packaged separately, thepackaging typically includes instructions for mixing before use. The choice of an adjuvantand/or carrier depends on the stability of the immunogenic formulation containing the adjuvant,the route of administration, the dosing schedule, the efficacy of the adjuvant for the species beingvaccinated, and, in humans, a pharmaceutically acceptable adjuvant is one that has beenapproved or is approvable for human administration by pertinent regulatory bodies. Forexample, Complete Freund's adjuvant is not suitable for human administration. Alum, MPL andQS-21 are preferred. Optionally, two or more different adjuvants can be used simultaneously.Preferred combinations include alum with MPL, alum with QS-21, MPL with QS-21, MPL orRC-529 with GM-CSF, and alum, QS-21 and MPL together. Also, Incomplete Freund's adjuvantcan be used (Chang et al., Advanced Drug Delivery Reviews 32, 173-186 (1998)), optionally incombination with any of alum, QS-21, and MPL and all combinations thereof. X. Passive Administration Of Antibodies

Therapeutic agents of the present invention include antibodies that specifically bind to 83 WO 2009/086539 PCT/US2008/088493 to an epitope comprising XiEDX2 in an aggregated amyloid protein, wherein Xi is Η, T, F, S, P,A or any other amino acid residue immediately preceding ED in such aggregated amyloidprotein; and wherein X2 is T, S, E, R, I, V, F, A or any other amino acid residue immediatelyfollowing ED in such aggregated amyloid protein, including epitopes within amyloid peptidessuch as AA. The antibodies used for passive administration can be antibodies that bind to C-terminal or N-terminal epitopes of AA. Other amyloid proteins in addition to Serum amyloid Aprotein include serum amyloid A protein, immunoglobulin light chain protein, such as, forexample, VX6 Wil or Vk, human islet amyloid precursor polypeptide (IAPP), beta amyloidpeptide, transthyretin (TTR) and ApoAl, as well as others listed in Table 1 above. AA is formed by proteolytic cleavage of SAA. Preferred antibodies specifically bind toneoepitopes of AA which form upon proteolytic cleavage of SAA. Preferred antibodies speciallybind to a C-terminal neoepitope of AA, especially, such antibodies specifically bind to HAA1alpha isoform within residues 70-76 (GHGAEDS, SEQ ID NO:4), HAA1 beta isoform withinresidues 70-76 (GHDAEDS, SEQ ID NO:5), HAA1 gamma isoform within residues 70-76(GHDAEDS, SEQ ID NO: 5), HAA2 alpha and beta isoforms within residues 70-76(GHGAEDS, SEQ ID NO: 10), HAA3 within residues 70-76 (GDHAEDS, SEQ ID NO:7),HAA4 within residues 78-84 (STVIEDS, SEQ ID NO:8), mouse AA1 (MAA1) within residues 69- 75 (GRGHEDT, SEQ ID NO:9), MAA2 within residues 69-75 (GRGHEDT, SEQ ID NO: 9),MAA3 within residues 62-68 (GHGAEDS, SEQ ID NO: 10), and MAA4 within residues 76-82(NHGLETL, SEQ ID NO: 11). Some antibodies only bind to an epitope within one of thesepeptides. Other antibodies bind to epitopes within more than one of these peptides. Forexample, some antibodies specifically bind to a GHGAEDS, (SEQ ID NO: 4) peptide and aGHDAEDS, SEQ ID NO: 5) peptide. Some antibodies bind to a GHGAEDS, SEQ ID NO: 4)peptide without specifically binding to a GHDAEDS, SEQ ID NO: 5) peptide. Binding to atleast one of the human AA peptides is preferable. Binding to at least one of the human AApeptides and a corresponding mouse peptide is useful in that the same antibody can be tested in amouse model and subsequently used in humans. Some preferred antibodies specifically bind toepitopes within HAA1 alpha isoform residues 71-76, 72-76, 73-76, 74-76, 70-75, 70-74, 70-73, 70- 72, 71-75, 72-75, 73-75, 71-74, 71-73, 72-74, or MAA1 residues 70-75, 71-75, 72-75, 73-75,69-74, 69-73, 69-72, 69-71, 70-74, 71-74, 72-74, 70-73, 70-72. Such antibodies typicallyspecifically bind to amyloid deposits but may or may not bind to soluble AA. When an antibody 84 WO 2009/086539 PCT/US2008/088493 is said to specifically bind to an epitope within specified residues, such as HAA1 alpha isoformresidues 70-76 of for example, what is meant is that the antibody specifically binds to apolypeptide containing the specified residues (i.e., residues 70-76 of HAA1 alpha isoform in thisan example). Such an antibody does not necessarily contact every residue within residues 70-76of HAA1 alpha isoform. Nor does every single amino acid substitution or deletion with inresidues 70-76 of HAA1 alpha isoform necessarily significantly affect binding affinity. Suchneoepitope antibodies bind to AA but not to SAA. Epitope specificity of an antibody can bedetermined, for example, as described by WO 00/72880.

The antibodies used for passive administration can be antibodies to N-terminal epitopesof AA. Preferred antibodies specifically bind to a N-terminal neoepitope of AA, especially, suchantibodies specifically bind to HAA1 residues 1-15 (RSFFSFLGEAFDGAR, SEQ ID NO. 80),HAA2 residues 1-15 (RSFFSFLGEAFDGAR, SEQ ID NO. 80), HAA3 residues 1-15(QGWLTFLKAAGQGAK, SEQ ID NO: 81), HAA4 residues 1-15 (ESWRSFFKEA, (SEQ IDNO: 82), MAA1 residues 1-15 (GFFSFVHEAFQGAGD, SEQ ID NO: 83), MAA2 residues 1-15(GFFSFVHEAFQGAGD, SEQ ID NO: 83), MAA3 residues 1-9 (EAGQGSRD, (SEQ ID NO:84), and residues 1-14 MAA4 (WYSFFREAVQGTWD, SEQ ID NO: 85). Some antibodies onlybind to an epitope within one of these peptides. Other antibodies bind to epitopes within morethan one of these peptides. For example, some antibodies specifically bind to aRSFFSFLGEAFDGAR, SEQ ID NO: 80) peptide and a QGWLTFLKAAGQGAK, SEQ ID NO:81) peptide. Some antibodies bind to a RSFFSFLGEAFDGAR, SEQ ID NO: 80) peptidewithout specifically binding to a QGWLTFLKAAGQGAK, SEQ ID NO: 81) peptide. Bindingto at least one of the human AA peptides is preferable. Binding to at least one of the human AApeptides and a corresponding mouse peptide is useful in that the same antibody can be tested in amouse model and subsequently used in humans.

Some antibodies specifically bind to an epitope consisting of such XiEDX2 Preferablysuch antibodies specifically bind to such epitope in an aggregated amyloid protein. Some ofsuch antibodies preferentially specifically bind to an aggregated amyloid protein relative to themonomeric form of such amyloid protein. In some antibodies, Xi is Η, T, F, S, P, or A and X2 isT, S, E, D, R, I, V, F or A. In some such antibodies, when Xi is H, X2 is T or A; when Xi is A,X2 is S, T, E or V; when Xi is T, X2 is E; when Xi is F, X2 is D; when Xi is S, X2 is E, F or A;and when Xi is P, X2 is E, I or F. In some antibodies, Xi is Η, T, F, S, P, or A and X2 is T, S, E,D, R, I, V, F or A, with the proviso that if Xi is A, X2 is not V. In some antibodies, when Xi is 85

SUBSTITUTE SHEET A, X2 is S, T or E.

Some antibodies specifically bind an epitope comprising the amino acid sequenceGHEDT, (SEQ ID NO 3), HEDT, (SEQ ID NO: 12), AEDS, (SEQ ID NO: 13), AEDT, (SEQ IDNO: 14), HEDA, (SEQ ID NO: 15), TEDE, (SEQ ID NO: 16), FEDD, (SEQ ID NO: 17), SEDE, 5 (SEQ ID NO: 18), AEDE, (SEQ ID NO: 19), PEDE, (SEQ ID NO: 20), PEDI, (SEQ ID NO: 21),PEDF, (SEQ ID NO: 22), AEDV, (SEQ ID NO: 23), SEDF, (SEQ ID NO: 24) or SEDA, (SEQID NO: 25).

Some antibodies specifically bind to a peptide comprising an amino acid sequenceselected from the group consisting of GHEDT, (SEQ ID NO: 3), HEDT, (SEQ ID NO: 12), 10 AEDS, (SEQ ID NO: 13), AEDT, (SEQ ID NO: 14), HEDA, (SEQ ID NO: 15), TEDE, (SEQ IDNO: 16), FEDD, (SEQ ID NO: 17), SEDE, (SEQ ID NO: 18), AEDE, (SEQ ID NO: 19), PEDE,(SEQ ID NO: 20), PEDI, (SEQ ID NO: 21), PEDF, (SEQ ID NO: 22), SEDF, (SEQ ID NO: 24)and SEDA, (SEQ ID NO: 25). Some antibodies specifically bind to a peptide comprising anamino acid sequence selected from the group consisting of GHEDT, (SEQ ID NO: 3), HEDT, 15 (SEQ ID NO: 12), AEDS, (SEQ ID NO: 13), AEDT, (SEQ ID NO: 14), HEDA, (SEQ ID NO:15) and TEDE, (SEQ ID NO: 16).

Some antibodies are raised to a peptide comprising GHEDT, (SEQ ID NO: 3), such as,for example, 2A4, 7D8 and 8G9, or are humanized or chimeric versions thereof.

Antibodies can be polyclonal or monoclonal. Polyclonal sera typically contain mixed 20 populations of antibodies specifically binding to several epitopes along the length of AA.However, polyclonal sera can be specific to a particular segment of AA, such as residues 70-76of HAA1 alpha isoform. Preferred antibodies are chimeric, or humanized (see Queen et al.,Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989) and WO 90/07861, US 5,693,762, US5,693,761, US 5,585,089, US 5,530,101 and Winter, US 5,225,539), or human (Lonberg et al., 25 WO93/12227 (1993); US 5,877,397, US 5,874,299, US 5,814,318, US 5,789,650, US 5,770,429, US 5,661,016, US 5,633,425, US 5,625,126, US 5,569,825, US 5,545,806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741 (1991)). Analternative approach for humanizing an antibody, also known as veneering, is described in US6,797,492. Several mouse antibodies of different binding specificities are available as starting 30 materials for making humanized antibodies.

Representative humanized antibodies are humanized version 7D8 antibody (ATCC

Accession Number PTA-9468), humanized version 7D29 antibody, humanized version 7D19 86

SUBSTITUTE SHEET antibody, humanized version 7D47 antibody, humanized version 7D39 antibody, humanizedversion 7D66 antibody, humanized version 8G9 antibody, humanized version 8G3 antibody,humanized version 8G4 antibody, humanized version 8G51 antibody, humanized version 8G22antibody, humanized version 8G30 antibody, humanized version 8G46 antibody, humanizedversion 2A4 antibody (ATCC Accession Number PTA-9662), humanized version 2A20antibody, humanized version 2A44 antibody, humanized version 2A77 antibody, humanizedversion 2A13 antibody, and humanized version 2A14 antibody. Hybridomas that produce the7D8 antibody (JH80 7D8.29.19.47) and the 2A4 antibody (JH80 2A4.20.44077) were depositiedon September 4, 2008, and on December 17, 2008, respectively, with the American Type CultureCollection (ATCC), currently located at 10801 University Boulevard, Manassas, VA20110-2209, under the provisions of the Budapest Treaty for the International Recognition of theDeposit of Microorganisms for the Purpose of Patent Procedure (“Budapest Treaty”). TheATCC has assigned the hybridoma producing 7D8 ATCC Accession No.PTA-9468, and thehybridoma producing 2A4 ATCC Accession No. PTA-9662.

Human isotype IgGl is preferred for antibodies to the C terminal region of AA becauseof it having highest affinity of human isotypes for the FcRI receptor on phagocytic cells. Someantibodies specifically bind to AA with a binding affinity greater than or equal to about ΙΟ7, 108,109, or 101θΜ_1.

Active immunization with fragments of AA can be combined with passiveadministration of antibodies. Examples of specific combinations include AA fragmentscomprising HAA1 alpha isoform residues 70-76 with antibodies that specifically bind to epitopewithin HAA1 alpha isoform residues 70-76; AA fragments comprising HAA1 alpha isoformresidues 70-76 with antibodies that specifically bind to epitope within HAA1 alpha isoformresidues 71-76; AA fragments comprising HAA1 alpha isoform residues 70-76 with antibodiesthat specifically bind to epitope within HAA1 alpha isoform residues 72-76; AA fragmentscomprising HAA1 alpha isoform residues 70-76 with antibodies that specifically bind to epitopewithin HAA1 alpha isoform residues 73-76; AA fragments comprising HAA1 alpha isoformresidues 70-76 with antibodies that specifically bind to epitope within HAA1 alpha isoformresidues 74-76; AA fragments comprising HAA1 alpha isoform residues 70-76 with antibodiesthat specifically bind to epitope within HAA1 alpha isoform residues 70-75; AA fragmentscomprising HAA1 alpha isoform residues 70-76 with antibodies that specifically bind to epitope 87 WO 2009/086539 PCT/US2008/088493 within HAA1 alpha isoform residues 70-74; AA fragments comprising HAA1 alpha isoformresidues 70-76 with antibodies that specifically bind to epitope within HAA1 alpha isoformresidues 70-73; AA fragments comprising HAA1 alpha isoform residues 70-76 with antibodiesthat specifically bind to epitope within HAA1 alpha isoform residues 70-72; AA fragmentscomprising HAA1 alpha iso form residues 70-76 with antibodies that specifically bind to epitopewithin HAA1 alpha isoform residues 71-75; AA fragments comprising HAA1 alpha isoformresidues 70-76 with antibodies that specifically bind to epitope within HAA1 alpha isoformresidues 72-75; AA fragments comprising HAA1 alpha iso form residues 70-76 with antibodiesthat specifically bind to epitope within HAA1 alpha isoform residues 73-75; AA fragmentscomprising HAA1 alpha isoform residues 70-76 with antibodies that specifically bind to epitopewithin HAA1 alpha isoform residues 73-75; AA fragments comprising HAA1 alpha isoformresidues 70-76 with antibodies that specifically bind to epitope within HAA1 alpha isoformresidues 71-74; AA fragments comprising HAA1 alpha iso form residues 70-76 with antibodiesthat specifically bind to epitope within HAA1 alpha isoform residues 71-73; AA fragmentscomprising HAA1 alpha iso form residues 70-76 with antibodies that specifically bind to epitopewithin HAA1 alpha isoform residues 72-74. Additionally, AA fragments comprising HAA1alpha isoform residues 71-76, 72-76, 73-76, 74-76, 70-75, 70-74, 70-73, 70-72, 71-75, 72-75, 73-75, 71-74, 71-73, 72-74 may be combined with antibodies that specifically bind to an epitopewithin HAA1 alpha isoform residues 71-76, 72-76, 73-76, 74-76, 70-75, 70-74, 70-73, 70-72, 71-75, 72-75, 73-75, 71-74, 71-73, 72-74. AA fragments comprising HAA1 alpha isoformresidues 70-76, HAA1 beta isoform residues 70-76, HAA1 gamma isoform residue 70-76,HAA2 alpha and beta iso forms residues 70-76, MAA1 residues 69-75, MAA2 residues 69-75, orMAA3 residues 62-68 may be combined with antibodies that specifically bind to an epitopewithin HAA1 alpha isoform residues 70-76, HAA1 beta isoform residues 70-76, HAA1 gammaisoform residue 70-76, HAA2 alpha and beta isoforms residues 70-76, MAA1 residues 69-75,MAA2 residues 69-75, or MAA3 residues 62-68.

Some of the antibodies described above do not specifically bind the monomeric orprecursor form of the amyloid protein. Some of such antibodies specifically bind to a neoepitopegenerated upon cleavage of a precursor protein resulting in an amyloid protein. For example,some antibodies specifically bind to the C-terminal residues of mouse AA fibrils -HEDT, (SEQID NO: 12), but do not specifically bind to a peptide that extends into the non-amyloid portion ofSAA (GHEDTMADQE, SEQ ID NO: 61). Some antibodies specifically bind to a 88

SUBSTITUTE SHEET conformational epitope. Some of such conformational epitopes are linear. Some of suchconformational epitopes are exposed when an amyloid protein enters an aggregated (e.g.,fibrillar) structure or becomes partially denatured. Examples of such antibodies include murinemonoclonal antibodies 2A4 (ATCC Accession Number PTA-9662), 8G9 (ATCC Accession 5 Number_) and 7D8 (ATCC Accession Number PTA-9468), human, humanized and chimeric forms thereof, other antibodies that specifically bind to the same epitope as 2A4, 8G9or 7D8, and antigen-binding fragments of any such antibodies. Some antibodies specificallybind to an amyloid protein comprising the amino acid sequence ED. Some antibodies specificallybind to an amyloid protein selected from the group consisting of immunoglobulin light chain 10 protein, human islet amyloid precursor polypeptide (IAPP), beta amyloid peptide, transthyretin(TTR) and ApoAl.

The basic antibody structural unit is known to comprise a tetramer of subunits. Eachtetramer is composed of two identical pairs of polypeptide chains, each pair having one “light”(about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each 15 chain includes a variable region of about 100 to 110 or more amino acids primarily responsiblefor antigen recognition. The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. 1. Antibodies

The invention includes intact antibodies and antigen-binding antibody fragments, as 20 well as pegylated antibodies and antibody fragments, as well as antibodies with altered (e.g.,reduced or eliminated) effector function, for example, antibodies comprising mutations orsubstituted residues in the Fc region. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab’)2 tri-Fab’, Fab’, Fv, scFv, di-Fab’ fragmentswhich can be generated by treating the antibody with an enzyme such as pepsin or produced by 25 art-recognized recombinant engineering techniques. Additional antigen-binding fragments ofantibodies of the invention include therapeutic antibody fragments, including pegylated antibodyfragments, such as PEGylated Fab’ and PEGylated di-Fab’. Examples of effector functionmutants are described in U.S. Patent No. 5,624,821, which is incorporated by reference herein inits entirety. Some antibodies have reduced binding affinity for Fc gamma RI receptor. Effector 30 function mutant antibodies include antibodies comprising mutations in the hinge region. Somemutant IgG antibodies comprise a mutation in the heavy chain constant region at one or more ofpositions 234, 235, 236, 237, 297, 318, 320 and 322. In some antibodies one or more of residues 89 WO 2009/086539 PCT/US2008/088493 234, 236 and 237 are substituted with alanine. In some antibodies, residue 235 is substituted withglutamine. In some antibodies, residue 297 is substituted with alanine. In some antibodies,residues 318, 320 and 322 are substituted with alanine. In some antibodies, residue 318 issubstituted with valine. In some antibodies, residue 322 is substituted with glutamine.Antibodies with enhanced effector function include antibodies single S239D and I332E and thedouble and triple mutants S239D/I332E and S239D/I332E/A330L (Kabat numbering). 2. Polyclonal Antibodies

Polyclonal antibodies can be prepared as described above by immunizing a suitablesubject with an immunogen. The antibody titer in the immunized subject can be monitored overtime by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA)using immobilized target antigen. If desired, the antibody molecules directed against the targetantigen can be isolated from the mammal (e.g., from the blood) and further purified by wellknowm techniques, such as protein A Sepharose chromatography to obtain the antibody, e.g.,IgG, fraction. At an appropriate time after immunization, e.g., when the anti-antigen antibodytiters are highest, antibody-producing cells can be obtained from the subject and used to preparemonoclonal antibodies by standard techniques, such as the hybridoma technique originallydescribed by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J.Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem .255:4980-83; Yeh et al. (1976) Proc.Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75}. For thepreparation of chimeric polyclonal antibodies, see Buechler et al. U.S. Patent No. 6,420,113. 3. Monoclonal Antibodies

Any of the many well known protocols used for fusing lymphocytes and immortalizedcell lines can be applied for the purpose of generating a monoclonal antibody (see, e.g., G. Galfreet al. (1977) Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J.Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra}. Moreover, the ordinarilyskilled worker will appreciate that there are many variations of such methods which also wouldbe useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the samemammalian species as the lymphocytes. For example, murine hybridomas can be made byfusing lymphocytes from a mouse immunized with an immunogenic preparation of the presentinvention with an immortalized mouse cell line. Preferred immortal cell lines are mousemyeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterinand thymidine ("HAT medium"). Any of a number of myeloma cell lines can be used as a fusion 90 WO 2009/086539 PCT/US2008/088493 partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/0-Agl4 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitivemouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").Hybridoma cells resulting from the fusion are then selected using HAT medium, which killsunfused and unproductively fused myeloma cells (unfused splenocytes die after several daysbecause they are not transformed). Hybridoma cells producing a monoclonal antibody of theinvention are detected by screening the hybridoma culture supernatants for antibodies that bind atarget antigen, e.g., Αβ, using a standard ELISA assay. 4. Recombinant Antibodies

Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonalantibody can be identified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) with a target antigen to therebyisolate immunoglobulin library members that bind the target antigen. Kits for generating andscreening phage display libraries are commercially available (e.g., the Pharmacia RecombinantPhage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage DisplayKit, Catalog No. 240612). Additionally, examples of methods and reagents particularlyamenable for use in generating and screening antibody display library can be found in, forexample, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. PCT International Publication No.WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT International Publication No. WO92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCTInternational Publication No. WO 92/01047; Garrard et al. PCT International Publication No.WO 92/09690; Ladner et al. PCT International Publication No. WO 90/02809; Fuchs et al.(1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85;Huse etal. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkinset al. (1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al.(1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl.Acad. Sci. USA 88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554. 5. Chimeric and Humanized Antibodies

Additionally, recombinant antibodies, such as chimeric and humanized monoclonalantibodies, comprising both human and non-human portions, which can be made using standard 91 WO 2009/086539 PCT/US2008/088493 recombinant DNA techniques, are within the scope of the invention.

The term “humanized immunoglobulin” or “humanized antibody” refers to an immunoglobulin or antibody that includes at least one humanized immunoglobulin or antibodychain (i.e., at least one humanized light or heavy chain). The term “humanized immunoglobulinchain” or “humanized antibody chain” (i.e., a “humanized immunoglobulin light chain” or“humanized immunoglobulin heavy chain”) refers to an immunoglobulin or antibody chain (i.e.,a light or heavy chain, respectively) having a variable region that includes a variable frameworkregion substantially from a human immunoglobulin or antibody and complementaritydetermining regions (CDRs) (e.g., at least one CDR, preferably two CDRs, more preferably threeCDRs) substantially from a non-human immunoglobulin or antibody, and further includesconstant regions (e.g., at least one constant region or portion thereof, in the case of a light chain,and three constant regions in the case of a heavy chain). The term “humanized variable region”(e.g., “humanized light chain variable region” or “humanized heavy chain variable region”)refers to a variable region that includes a variable framework region substantially from a humanimmunoglobulin or antibody and complementarity determining regions (CDRs) substantiallyfrom a non-human immunoglobulin or antibody.

The phrase “substantially from a human immunoglobulin or antibody” or “substantiallyhuman” means that, when aligned to a human immunoglobulin or antibody amino sequence forcomparison purposes, the region shares at least 80-90%, 90-95%, or 95-99% identity (/.e, localsequence identity) with the human framework or constant region sequence, allowing, forexample, for conservative substitutions, consensus sequence substitutions, germlinesubstitutions, backmutations, and the like. The introduction of conservative substitutions,consensus sequence substitutions, germline substitutions, backmutations, and the like, is oftenreferred to as “optimization” of a humanized antibody or chain. The phrase “substantially froma non-human immunoglobulin or antibody” or “substantially non-human” means having animmunoglobulin or antibody sequence at least 80-95%, preferably at least 90-95%, morepreferably, 96%, 97%, 98%, or 99% identical to that of a non-human organism, e.g., a non-human mammal.

Accordingly, all regions or residues of a humanized immunoglobulin or antibody, or ofa humanized immunoglobulin or antibody chain, except the CDRs, are substantially identical tothe corresponding regions or residues of one or more native human immunoglobulin sequences.The term “corresponding region” or “corresponding residue” refers to a region or residue on a 92 WO 2009/086539 PCT/US2008/088493 second amino acid or nucleotide sequence which occupies the same (i.e., equivalent) position asa region or residue on a first amino acid or nucleotide sequence, when the first and secondsequences are optimally aligned for comparison purposes.

The term “significant identity” means that two polypeptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 50-60% sequence identity, preferably at least 60-70% sequence identity, more preferably at least 70-80% sequence identity, more preferably at least 80-90% sequence identity, even more preferablyat least 90-95% sequence identity, and even more preferably at least 95% sequence identity ormore (e.g., 99% sequence identity or more). The term “substantial identity” means that twopolypeptide sequences, when optimally aligned, such as by the programs GAP or BESTFITusing default gap weights, share at least 80-90% sequence identity, preferably at least 90-95%sequence identity, and more preferably at least 95% sequence identity or more (e.g., 99%sequence identity or more). For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequence comparison algorithm,test and reference sequences are input into a computer, subsequence coordinates are designated,if necessary, and sequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., by the localhomology algorithm of Smith &amp;amp; Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman &amp;amp; Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson &amp;amp; Lipman, Proc. Nat!. Acad. Sci. USA 85:2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, WI), or by visual inspection (see generally Ausubel et al., Current Protocols inMolecular Biology). One example of algorithm that is suitable for determining percent sequenceidentity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J.Mol. Biol. 215:403 (1990). Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information (publicly accessible through theNational Institutes of Health NCBI internet server). Typically, default program parameters canbe used to perform the sequence comparison, although customized parameters can also be used.For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an 93 WO 2009/086539 PCT/US2008/088493 expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff &amp;amp; Henikoff, Proc. Natl.Acad. Sci. USA 89:10915 (1989)).

Preferably, residue positions which are not identical differ by conservative amino acidsubstitutions. For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I (hydrophobic sidechains): leu,met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidicside chains): asp, glu; Group IV (basic side chains): asn, gin, his, lys, arg; Group V (residuesinfluencing chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe.Conservative substitutions involve substitutions between amino acids in the same class. Non-conservative substitutions constitute exchanging a member of one of these classes for a memberof another.

Preferably, humanized immunoglobulins or antibodies bind antigen with an affinity thatis within a factor of three, four, or five of that of the corresponding non-humanized antibody.For example, if the nonhumanized antibody has a binding affinity of KT9 M, humanizedantibodies will have a binding affinity of at least 3 x KT8 M, 4 x 10~8 M, 5 x KT8 M, or 10’9 M.When describing the binding properties of an immunoglobulin or antibody chain, the chain canbe described based on its ability to “direct antigen (e.g., Αβ) binding”. A chain is said to “directantigen binding” when it confers upon an intact immunoglobulin or antibody (or antigen bindingfragment thereof) a specific binding property or binding affinity. A mutation (e.g., abackmutation) is said to substantially affect the ability of a heavy or light chain to direct antigenbinding if it affects (e.g., decreases) the binding affinity of an intact immunoglobulin or antibody(or antigen binding fragment thereof) comprising said chain by at least an order of magnitudecompared to that of the antibody (or antigen binding fragment thereof) comprising an equivalentchain lacking said mutation. A mutation “does not substantially affect (e.g., decrease) the abilityof a chain to direct antigen binding” if it affects (e.g., decreases) the binding affinity of an intactimmunoglobulin or antibody (or antigen binding fragment thereof) comprising said chain by onlya factor of two, three, or four of that of the antibody (or antigen binding fragment thereof)comprising an equivalent chain lacking said mutation.

The term “chimeric immunoglobulin” or antibody refers to an immunoglobulin orantibody whose variable regions derive from a first species and whose constant regions derivefrom a second species. Chimeric immunoglobulins or antibodies can be constructed, for 94 WO 2009/086539 PCT/US2008/088493 example by genetic engineering, from immunoglobulin gene segments belonging to differentspecies. The terms “humanized immunoglobulin” or “humanized antibody” are not intended toencompass chimeric immunoglobulins or antibodies, as defined infra. Although humanizedimmunoglobulins or antibodies are chimeric in their construction (i.e., comprise regions frommore than one species of protein), they include additional features (i.e., variable regionscomprising donor CDR residues and acceptor framework residues) not found in chimericimmunoglobulins or antibodies, as defined herein.

Such chimeric and humanized monoclonal antibodies can be produced by recombinantDNA techniques known in the art, for example using methods described in Robinson et al.International Application No. PCT/US86/02269; Akira, et al. European Patent Application184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European PatentApplication 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabillyet al. U.S. Patent No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al.(1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liuet al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Cane. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449;and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science229:1202-1207; Oi et al. (1986) BioTechniques 4:214; Winter U.S. Patent 5,225,539; Jones etal.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988)J. Immunol. 141:4053-4060. Therapeutic agents also include antibody mimetics such ascomplementarity determining region (CDR) mimetics. 6. Human Antibodies from Transgenic Animals and Phage Display

Alternatively, it is now possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (Jr) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutant mice results in theproduction of human antibodies upon antigen challenge. See, e.g., U.S. Patent Nos. 6,150,584;6,114,598; and 5,770,429.

Fully human antibodies can also be derived from phage-display libraries (Hoogenboomet al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991)). Chimeric 95 WO 2009/086539 PCT/US2008/088493 polyclonal antibodies can also be obtained from phage display libraries (Buechler et al. U.S.Patent No. 6,420,113). 7. Bispecific Antibodies, Antibody Fusion Polypeptides, and Single-ChainAntibodies

Bispecific antibodies (BsAbs) are antibodies that have binding specificities for at leasttwo different epitopes. Such antibodies can be derived from full length antibodies or antibodyfragments (e.g. F(ab)’2 bispecific antibodies). Methods for making bispecific antibodies areknown in the art. Traditional production of full length bispecific antibodies is based on thecoexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains havedifferent specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce apotential mixture of different antibody molecules (see, WO 93/08829 and in Traunecker et al.,EMBO J., 10:3655-3659 (1991)).

Bispecific antibodies also include cross-linked or “heteroconjugate” antibodies. Forexample, one of the antibodies in the heteroconjugate can be coupled to avidin, the other tobiotin or other payload. Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed inU.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

In yet another aspect, the antibody can be fused, chemically or genetically, to a payloadsuch as a reactive, detectable, or functional moiety, for example, an immunotoxin to produce anantibody fusion polypeptide. Such payloads include, for example, immunotoxins,chemotherapeutics, and radioisotopes, all of which are well-known in the art.

Single chain antibodies are also suitable for stabilization according to the invention.The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) with a linker, which allows each variable region to interface with each other andrecreate the antigen binding pocket of the parent antibody from which the VL and VH regionsare derived. See Gruber et al., J. Immunol., 152:5368 (1994).

It is understood that any of the foregoing polypeptide molecules, alone or incombination, are suitable for preparation as stabilized formulations according to the invention. XI. Subjects Amenable To Treatment

Subjects or patients amenable to treatment include individuals at risk of disease but not 96 WO 2009/086539 PCT/US2008/088493 showing symptoms, as well as patients presently showing symptoms. Therefore, the presentmethods can be administered prophylactically to the general population without the need for anyassessment of the risk of the subject patient. The present methods are especially useful forindividuals who do have a known genetic risk autoimmune disorders. Such individuals includethose having relatives who have experienced this disease and those whose risk is determined byanalysis of genetic or biochemical markers.

Patients suffering from AA amyloidosis can be asymptomatic for a prolonged period oftime. Therefore, clinical diagnosis of AA amyloidosis is often delayed or missed until theamyloid deposits are extensive. For those patients who are symptomatic, it is estimated that only53% of the cases are diagnosed. See L.E.K. Consulting, Independent Market Research (2003).

The invention provides methods useful to treat or effect prophylaxis of a diseasecharacterized by the deposition of an amyloid protein, such as, for example, the diseasesdescribed above, including those listed in Table 1. Some methods are useful to treat or effectprophylaxis of a disease characterized by the deposition of an amyloid protein comprising theamino acid sequence ED. In some methods, if the amyloid protein comprises the amino acidsequence AEDV, then the antibody is not administered to treat or effect prophylaxis ofAlzheimer’s disease or Mild Cognitive Impairment. The amyloid protein can be any of theamyloid proteins described above, including those listed in Table 1, such as, for example, serumamyloid A protein, immunoglobulin light chain protein, such as, for example, VX6 Wil or Vk,human islet amyloid precursor polypeptide (IAPP), beta amyloid peptide, transthyretin (TTR) orApoAl.

The present methods are especially useful for individuals who do have a known risk of,are suspected to have, or have been diagnosed with AA amyloidosis or AL amyloidosis. Suchindividuals include but are not limited to those having chronic inflammatory diseases, inheritedinflammatory diseases, and chronic microbial infections, such as rheumatoid arthritis, juvenilechronic arthritis, ankylosing spondylitis, psoriasis, psoriatic arthropathy, Reiter’s syndrome,Adult Still’s disease, Behcet’s syndrome, Crohn’s disease, Familial Mediterranean Fever,leprosy, tuberculosis, bronchiectasis, decubitus ulcers, chronic pyelonephritis, osteomyelitis,Whipple’s disease, myeloma, macroglobulinemia, immunocyte dyscrasia, monoclonalgammopathy, occult dyscrasia. Chronic inflammatory and infectious conditions are prerequisiteto the development of AA amyloidosis and AL amyloidosis manifested by local nodular 97 WO 2009/086539 PCT/US2008/088493 amyloidosis can be associated with chronic inflammatory diseases. Individuals who do haveknown risk of AA amyloidosis also include but are not limited to those having malignantneoplasms as Hodgkin’s lymphoma, renal carcinoma, carcinomas of gut, lung and urogenitaltract, basal cell carcinoma, andhairy cell leukemia. Additionally, individuals who do haveknown risk of AA amyloidosis also include but are not limited to those havinglymphoproliferative disorders such as Castleman’s Disease.

In both asymptomatic and symptomatic patients, treatment can begin at any time beforeor after the diagnosis of the underlying AA or AL amyloid diseases. Treatment typically entailsmultiple dosages over a period of time. Treatment can be monitored by assaying antibody,activated T-cell (a side effect) or B-cell responses to the therapeutic agent (e.g., AA peptide), oremploying radiolabeled SAP Scintigraphy over time. If the response falls, a booster dosage isindicated. XII. Treatment Regimes

In general, treatment regimes involve administering an agent effective to induce animmunogenic response to an amyloid protein, and preferably to an aggregated form of suchamyloid protein, such as, for example, AA or AL. Preferably an immunogenic fragment of AAor AL or an XiEDX2 fragment is administered to a patient. In prophylactic applications,pharmaceutical compositions or medicaments are administered to a patient susceptible to, orotherwise at risk of, amyloidosis such as AA Amyloidosis or AL amyloidosis, in an amountsufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease,including physiological, biochemical, histologic and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting during development of thedisease. In therapeutic applications, an agent is administered to a patient suspected of, or alreadysuffering from such a disease in a regime comprising an amount and frequency of administrationof the agent sufficient to cure, or at least partially arrest, or inhibit deterioration of the symptomsof the disease (physiological, biochemical, histologic and/or behavioral), including itscomplications and intermediate pathological phenotypes in development of the disease. In somemethods, administration of agent reduces or eliminates early symptomology in patients that havenot yet developed characteristic AA or AL Amyloidosis pathology. An amount adequate toaccomplish therapeutic or prophylactic treatment is defined as a therapeutically- orprophylactically-effective dose. A combination of amount and dosage frequency adequate to 98 WO 2009/086539 PCT/US2008/088493 accomplish the therapeutic or prophylactic treatment is defined as a therapeutically- orprophylactically-effective regime. In both prophylactic and therapeutic regimes, agents areusually administered in several dosages until a sufficient immune response has been achieved. Adosage and frequency of administrations adequate to accomplish therapeutic or prophylactictreatment is defined as a therapeutically- or prophylactically-effective regime. Typically, thepatient's immune response is monitored and repeated dosages are given if the immune responsestarts to wane. The immune response can be monitored by detecting antibodies, for example, toAA or AL in the blood in the patient or detecting levels of, for example, AA or AL.

Effective doses of the agents and compositions of the present invention, for thetreatment of the above described conditions vary depending upon many different factors,including means of administration, target site, physiological state of the patient, whether thepatient is human or an animal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human but nonhuman mammals includingtransgenic mammals can also be treated. Treatment dosages need to be titrated to optimizesafety and efficacy. The amount of immunogen depends on whether adjuvant is alsoadministered, with higher dosages being required in the absence of adjuvant. The amount of animmunogen for administration sometimes varies from 1-500 pg per patient and more usuallyfrom 5-500 pg per injection for human administration. Occasionally, a higher dose of 1-2 mgper injection is used. Typically at least 10, 20, 50 or 100 pg is used for each human injection.The mass of immunogen also depends on the mass ratio of immunogenic epitope within theimmunogen to the mass of immunogen as a whole. Typically, IO’3 to 10~5 micromoles ofimmunogenic epitope are used for microgram of immunogen. The timing of injections can varysignificantly from once a day, to once a year, to once a decade. On any given day that a dosageof immunogen is given, the dosage is greater than 1 pg/patient and usually greater than 10 pg/patient if adjuvant is also administered, and greater than 10 pg/patient and usually greater than100 pg/patient in the absence of adjuvant. A typical regimen consists of an immunizationfollowed by booster injections at time intervals, such as 6 week intervals. Another regimenconsists of an immunization followed by booster injections 1, 2 and 12 months later. Anotherregimen entails an injection every two months for life. Alternatively, booster injections can beon an irregular basis as indicated by monitoring of immune response.

Doses for nucleic acids encoding immunogens range from about 10 ng to 1 g, 100 ng to 99 WO 2009/086539 PCT/US2008/088493 100 mg, 1 gg to 10 mg, or 30-300 gg DNA per patient. Doses for infectious viral vectors varyfrom 10-100, or more, virions per dose.

For passive immunization with an antibody (in combination therapies), the dosageranges from about 0.0001 to 100 mg/kg, 0.5 to less than 5 mg/kg, and more usually 0.01 to 5 5 mg/kg, 0.5 to 3 mg/kg, of the host body weight. For example dosages can be 1 mg/kg bodyweight or 10 mg/kg body weight or within the range of 1-10 mg/kg or in other words, 70 mg or700 mg or within the range of 70-700 mg, respectively, for a 70 kg patient. As an additionalexample, dosages can be less than 5 mg/kg body weight or 1.5 mg/kg body weight or within therange of 0.5 to 1.5 mg/kg, preferably at least 1.5 mg/kg. An exemplary treatment regime entails 10 administration once per every two weeks or once a month or once every 3 to 6 months. In somemethods, two or more monoclonal antibodies with different binding specificities areadministered simultaneously, in which case the dosage of each antibody administered fallswithin the ranges indicated. Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, monthly or yearly. Intervals can also be irregular as 15 indicated by measuring blood levels of antibody to AA in the patient. In some methods, dosageis adjusted to achieve a plasma antibody concentration of 1-1000 gg/ml and in some methods25-300 pg/ml. Alternatively, antibody can be administered as a sustained release formulation, inwhich case less frequent administration is required. Dosage and frequency vary depending onthe half-life of the antibody in the patient. In general, human antibodies show the longest half 20 life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. Thedosage and frequency of administration can vary depending on whether the treatment isprophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administeredat relatively infrequent intervals over a long period of time. Some patients continue to receivetreatment for the rest of their lives. In therapeutic applications, a relatively high dosage at 25 relatively short intervals is sometimes required until progression of the disease is reduced orterminated, and preferably until the patient shows partial or complete amelioration of symptomsof disease. Thereafter, the patent can be administered a prophylactic regime.

Agents for inducing an immune response can be administered by parenteral, topical,intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or 30 intramuscular means for prophylactic and/or therapeutic treatment. The most typical route ofadministration of an immunogenic agent is subcutaneous although other routes can be equally 100 WO 2009/086539 PCT/US2008/088493 effective. The next most common route is intramuscular injection. This type of injection is mosttypically performed in the arm or leg muscles. In some methods, agents are injected directly intoa particular tissue where deposits have accumulated, e.g., intracranial injection. Intramuscularinjection or intravenous infusion is preferred for administration of antibody (in combinationtherapies). In some methods, particular therapeutic antibodies are injected directly into thecranium. In some methods, antibodies are administered as a sustained release composition ordevice, such as a MEDIPAD™ device.

Agents of the invention are often administered as pharmaceutical compositionscomprising an active therapeutic agent, i.e., and a variety of other pharmaceutically acceptablecomponents. See Remington's Pharmaceutical Science (15th ed., Mack Publishing Company,Easton, Pennsylvania, 1980). The preferred form depends on the intended mode ofadministration and therapeutic application. The compositions can also include, depending on theformulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceutical compositions for animal orhuman administration. The diluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants, or nontoxic,nontherapeutic, nonimmunogenic stabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan, polylactic acids,polyglycolic acids and copolymers (such as latex functionalized SEPHAROSE™, agarose,cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates(such as oil droplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

For parenteral administration, agents of the invention can be administered as injectabledosages of a solution or suspension of the substance in a physiologically acceptable diluent witha pharmaceutical carrier that can be a sterile liquid such as water oils, saline, glycerol, or ethanol.Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pHbuffering substances and the like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, forexample, peanut oil, soybean oil, and mineral oil. In general, glycols such as propylene glycol or 101 WO 2009/086539 PCT/US2008/088493 polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Antibodiescan be administered in the form of a depot injection or implant preparation which can beformulated in such a manner as to permit a sustained release of the active ingredient. Anexemplary composition comprises monoclonal antibody at 5 mg/mL, formulated in aqueous 5 buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted to pH 6.0 with HC1.Compositions for parenteral administration are typically substantially sterile, isotonic andmanufactured under GMP conditions of the FDA or similar body.

Typically, compositions are prepared as injectables, either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to 10 injection can also be prepared. The preparation also can be emulsified or encapsulated inliposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhancedadjuvant effect, as discussed above (see Langer, Science 249, 1527 (1990) and Hanes, Advanced-Drug Delivery Reviews 28, 97-119 (1997). The agents of this invention can be administered inthe form of a depot injection or implant preparation which can be formulated in such a manner as 15 to permit a sustained or pulsatile release of the active ingredient.

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

For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in 20 the range of 0.5% to 10%, preferably l%-2%. Oral formulations include excipients, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, and magnesium carbonate. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25%-70%. 25 Topical application can result in transdermal or intradermal delivery. Topical administration can be facilitated by co-administration of the agent with cholera toxin ordetoxified derivatives or subunits thereof or other similar bacterial toxins (See Glenn et al.,Nature 391, 851 (1998)). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking or expression as a fusion 30 protein.

Alternatively, transdermal delivery can be achieved using a skin path or usingtransferosomes (Paul et al., Eur. J. Immunol. 25, 3521-24 (1995); Cevc et al., Biochem. Biophys. 102 WO 2009/086539 PCT/US2008/088493

Acta 1368, 201-15 (1998)). XIII. Combinational Drug Therapy Treatment Regimes

Combination therapy according to the invention may be performed alone or inconjunction with another therapy to treat or effect prophylaxis of AA amyloidosis. Combinationtherapy according to the invention may also be performed in conjunction with another therapywhich treats or effects prophylaxis of an underlying amyloid disease such as inflammatorydiseases, chronic microbial infections, malignant neoplasms, inherited inflammatory diseases,and lymphoproliferative disorders. There are large numbers of treatments available incommercial use, in clinical evaluation and in pre-clinical development, which could be selectedfor use with the presently disclosed invention for effecting prophylaxis and treatment of AAamyloidosis by combination drug therapy. Such treatments can be one or more compoundsselected from, but not limited to several major categories, namely, (i) non-steroidal anti-inflammatory drugs (NSAIDs; e.g., detoprofen, diclofenac, diflunisal, etodolac, fenoprofen,flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenameate, mefenamic acid,meloxicam, nabumeone, naproxen sodium, oxaprozin, piroxicam, sulindac, tolmetin, celecoxib,rofecoxib, aspirin, choline salicylate, salsalte, and sodium and magnesium salicylate); (ii)steroids (e.g., cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone,prednisone, triamcinolone); (iii) DMARDs, i.e., disease modifying antirheumatic drugs (e.g.,cyclosporine, azathioprine, methotrexate, leflunomide, cyclophosphamide, hydroxychloroquine,sulfasalazine, D-penicillamine, minocycline, and gold); or (iv) recombinant proteins (e.g.,ENBREL® (etanercept, a soluble TNF receptor) and REMICADE® (infliximab) a chimericmonoclonal anti-TNF antibody).

The duration of the combination therapy depends on the type of underlying diseasebeing treated, the age and condition of the patient, the stage and type of the patient's disease, andhow the patient responds to the treatment. The doctor can observe the therapy's effects closelyand make any adjustments that are needed. Additionally, a person having a greater risk ofdeveloping AA Amyloidosis (e.g., a person who is genetically predisposed or previously had aninflammatory disorder or other underlying diseases) or AL amyloidosis may receive prophylactictreatment to inhibit or delay the development of AA AL aggregates such as fibrils.

The dosage, frequency and mode of administration of each component of thecombination can be controlled independently. For example, one compound may be administered 103 WO 2009/086539 PCT/US2008/088493 orally three times per day, while the second compound may be administered intramuscularlyonce per day. Combination therapy may be given in on-and-off cycles that include rest periods.The compounds may also be formulated together such that one administration delivers bothcompounds. The combination of the invention can also be provided as components of a 5 pharmaceutical pack. The drugs can be formulated together or separately and in individualdosage amounts. Each compound is admixed with a suitable carrier substance, and is generallypresent in an amount of 1-95% by weight of the total weight of the composition.

The composition may be provided in a dosage form that is suitable for oral, parenteral(e.g., intravenous, intramuscular, subcutaneous), rectal, transdermal, nasal, vaginal, inhalant, or 10 ocular administration. Thus, the composition may be in form of, e.g., tablets, capsules, pills,powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes,ointments, creams, plasters, drenches, delivery devices, suppositories, enemas, injectables,implants, sprays, or aerosols. The pharmaceutical compositions may be formulated according toconventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of 15 Pharmacy, (19th ed.) ed. A. R. Gennaro, 1995, Mack Publishing Company, Easton, Pa. andEncyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999,Marcel Dekker, N.Y. XIV. Methods of Monitoring or Diagnosing AA or AL Amyloidosis

Methods of monitoring or diagnosing AA or AL amyloidosis include measuring the 20 plasma concentrations of SAA and C-reactive protein, performing tissue biopsy (renal, rectal,gastric, gingival, fat, salivary, labial glands) and histology with congo red staining and/orimmunostaining with specific antibodies directed against AA or AL aggregates such as fibrils.The invention provides methods of detecting an antibody response against AA peptide in apatient suffering from or susceptible to AA Amyloidosis. The methods are particularly useful 25 for monitoring a course of treatment being administered to a patient. The methods can be used tomonitor both therapeutic treatment on symptomatic patients and prophylactic treatment onasymptomatic patients. Some methods entail determining a baseline value of an antibodyresponse in a patient before administering a dosage of an immunogenic agent, and comparingthis with a value for the immune response after treatment. A significant increase (i.e., greater 30 than the typical margin of experimental error in repeat measurements of the same sample,expressed as one standard deviation from the mean of such measurements) in value of the 104 WO 2009/086539 PCT/US2008/088493 antibody response signals a positive treatment outcome (i.e., that administration of the agent hasachieved or augmented an immune response). If the value for the antibody response does notchange significantly, or decreases, a negative treatment outcome is indicated. In general,patients undergoing an initial course of treatment with an immunogenic agent are expected toshow an increase in antibody response with successive dosages, which eventually reaches aplateau. Administration of agent is generally continued while the antibody response isincreasing. Attainment of the plateau is an indicator that the administered of treatment can bediscontinued or reduced in dosage or frequency.

In other methods, a control value (i.e., a mean and standard deviation) of an antibodyresponse is determined for a control population. Typically the individuals in the controlpopulation have not received prior treatment. Measured values of the antibody response in apatient after administering a therapeutic agent are then compared with the control value. Asignificant increase relative to the control value (e.g., greater than one standard deviation fromthe mean) signals a positive treatment outcome. A lack of significant increase or a decreasesignals a negative treatment outcome. Administration of agent is generally continued while theantibody response is increasing relative to the control value. As before, attainment of a plateaurelative to control values in an indicator that the administration of treatment can be discontinuedor reduced in dosage or frequency.

In other methods, a control value of antibody response (e.g., a mean and standarddeviation) is determined from a control population of individuals who have undergone treatmentwith a therapeutic agent and whose antibody responses have reached a plateau in response totreatment. Measured values of antibody response in a patient are compared with the controlvalue. If the measured level in a patient is not significantly different (e.g., more than onestandard deviation) from the control value, treatment can be discontinued. If the level in apatient is significantly below the control value, continued administration of agent is warranted.If the level in the patient persists below the control value, then a change in treatment regime, forexample, use of a different adjuvant, fragment or switch to passive administration may beindicated.

In other methods, a patient who is not presently receiving treatment but has undergonea previous course of treatment is monitored for antibody response to determine whether aresumption of treatment is required. The measured value of antibody response in the patient canbe compared with a value of antibody response previously achieved in the patient after a 105 WO 2009/086539 PCT/US2008/088493 previous course of treatment. A significant decrease relative to the previous measurement (i.e.,greater than a typical margin of error in repeat measurements of the same sample) is anindication that treatment can be resumed. Alternatively, the value measured in a patient can becompared with a control value (mean plus standard deviation) determined in a population ofpatients after undergoing a course of treatment. Alternatively, the measured value in a patientcan be compared with a control value in populations of prophylactically treated patients whoremain free of symptoms of disease, or populations of therapeutically treated patients who showamelioration of disease characteristics. In all of these cases, a significant decrease relative to thecontrol level (i.e., more than a standard deviation) is an indicator that treatment should beresumed in a patient.

Some methods employ iodine-123-labeled or iodine-125-labeled serum amyloid Pcomponent (123I-SAP or 125I-SAP) Scintigraphy. 123I-SAP or 125I-SAP is intravaneously injectedinto patients and viewed with gamma camera. Radiolabeled SAP Scintigraphy is a usefulmethod to monitor the progression of amyloidosis in patients and evaluate treatments. It isspecific for amyloid and can be used to quantitatively monitor the location and amount ofamyloid deposits in patients. 123I-SAP and 125I-SAP do not accumulate in healthy subjects or innon-amyloid patients. Radiolabeled SAP scintigraphy can be used to monitor dynamic turnoverof amyloid, and can assess the efficacy of treatments aimed at regressing amyloid deposits.Further, radiolabeled SAP Scintigraphy is non-invasive and provides whole body scan. Methodsof the invention entail determining a baseline value of an antibody response in a patient beforeadministering a dosage of an agent, and comparing this with a value for the immune responseafter treatment in a patient. A significant increase (i.e., greater than the typical margin ofexperimental error in repeat measurements of the same sample, expressed as one standarddeviation from the mean of such measurements) in value of the antibody response signals apositive treatment outcome (i.e., that administration of the agent has achieved or augmented animmune response). If the value for the antibody response does not change significantly, ordecreases, a negative treatment outcome is indicated. In general, patients undergoing an initialcourse of treatment with an immunogenic agent are expected to show an increase in antibodyresponse with successive dosages, which eventually reaches a plateau. Administration of agentis generally continued while the antibody response is increasing. Attainment of the plateau is anindicator that the administered of treatment can be discontinued or reduced in dosage orfrequency. 106 WO 2009/086539 PCT/US2008/088493

The tissue sample for analysis is typically blood, plasma, serum, mucous orcerebrospinal fluid from the patient. The sample is analyzed for indication of an immuneresponse to any form of AA or AL peptide. The immune response can be determined from thepresence of antibodies that specifically bind to AA or AL peptide. Antibodies can be detected ina binding assay to a ligand that specifically binds to the antibodies. Typically the ligand isimmobilized. Binding can be detected using a labeled anti-idiotypic antibody.

In combination regimes employing both active and passive administration, analogousapproaches can be used to monitor levels of antibody resulting from passive administration.

Methods of diagnosing amyloidosis can also be employed by, e.g., administering to asubject an antibody or antigen-binding fragment thereof, that is bound to a detectable label,wherein the antibody or fragment thereof specifically binds to an epitope including XiEDX2 inan aggregated amyloid protein, wherein Xi and X2 are any amino acids, and detecting thepresence or absence of the bound antibody or fragment thereof. Detection of the bound antibodyor fragment supports a diagnosis of amyloidosis. Antibodies and fragments useful in thediagnosis of amyloidosis include the disclosed antibodies of the invention.

The diagnostic antibodies or fragments of the invention can be administered, by e.g.,intravenous injection into the body of a patient, or directly into the brain by intracranial injection.The antibody dosage is readily determined by one skilled in the art. Typically, the antibody islabeled, although in some methods, the antibody is unlabeled and a secondary labeling agent isused to bind to the antibody. The choice of label depends on the means of detection. Forexample, a fluorescent label is suitable for optical detection. Use of paramagnetic labels issuitable for tomographic detection without surgical intervention. Radiolabels may be usedincluding 211At, 212Bi, 67Cu, 125I, 131I, mIn, 32P, 2I2Pb, 186Re, 18sRe, 153Sm, "”TC&gt; or 90Y. Suchlabels may be detected using PET or SPECT or other suitable technique.

Diagnosis may also be performed by comparing the number, size, and/or intensity oflabeled loci, to corresponding baseline values. The base line values can represent the meanlevels in a population of undiseased individuals. Baseline values can also represent previouslevels determined in the same patient. For example, baseline values can be determined in apatient, and measured values thereafter compared with the baseline values. An increase in valuesrelative to baseline signals supports a diagnosis of AA amyloidosis.

The diagnostic methods of the invention may be used to diagnose amyloidosis diseasesincluding AA amyloidosis, AL amyloidosis, Alzheimer’s disease, Mild Cognitive Impairment, 107 WO 2009/086539 PCT/US2008/088493 amyloid polyneuropathy, Mediterranean fever, Muckle-Wells syndrome, reactive systemicamyloidosis associated with systemic inflammatory diseases, myeloma or macroglobulinemiaassociated amyloidosis, amyloidosis associated with immunocyte dyscrasia, monoclonalgammopathy, occult dyscrasia, or local nodular amyloidosis associated with chronicinflammatory diseases. XV. Animal Models of AA Amyloidosis AA amyloidosis can be induced experimentally in mice in which SAA concentrationsare markedly increased by injection of silver nitrate, casein, or lipopolysaccharide. These agentsstimulate the production of cytokines. See Skinner et al. Lab Invest. 36:420-427 (1997) andKisilevsky et al. Bailliere’s Clin. Immunol. Immunopathol. 8(3) 613-626 (1994). Within 2 or 3weeks after the inflammatory stimulus, animals develop systemic AA deposits, as found inpatients with AA Amyloidosis. This lag phase is dramatically shortened when mice are given,concomitantly, an intravenous injection of protein extracted from AA amyloid-laden mousespleen or liver. See Axelrad et al. Lab Invest. 47(2):139-46 (1982). The amyloidogenicaccelerating activity of such preparations was termed “amyloid enhancing factor” (AEF).Lundmark et al. reports that the active principle of AEF is unequivocally the AA fiber itself.Further, they demonstrated that this material is extremely potent, being active in doses less than1 ng, and that it retained its biologic activity over a considerable length of time. Notably, theAEF was also effective when administered orally. They concluded that AA and perhaps otherforms of amyloidosis are transmissible diseases, akin to the prion-associated disorders. SeeLundmark et al. Proc. Nat. Acad. Sci. 99: 6979-6984 (2002). AA amyloid can also be induced in transgenic strains of mice carrying the humaninterleukin 6 gene under the control of the metallothionein-I promoter resulting in markedlyincreased concentrations of SAA and developing amyloid in the spleen, liver and kidneys by 3months of age. At the time of death at about 8-9 months, organs from these transgenic mice haveextensive amyloid deposits. See Solomon et al., Am. J. Pathol. 154(4):1267-1272 (1999).

The Transgenic Rapidly Induced Amyloid Disease (TRIAD) transgenic mouse model isan improvement to the above described transgenic mouse model. TRIAD mice carry the humaninterleukin 6 gene under the control of the H-2L° histocompatibility promoter. Adminstration ofAEF to 8-week old TRIAD mice results in prominent spenic and hepatic AA amyloid depositswithin 3 to 4 weeks. Subsequently, this process progresses to other organs, leading to death 4-6 108 WO 2009/086539 PCT/US2008/088493 weeks later. The development of the systemic amyloidosis is accelerated compared to the above-described transgenic mouse model. See University of Tennessee Research Corporation, WO01/77167, Pharmacopeia, WO 95/35503 and Scripps, WO 95/30642 Wall et al. Amyloid 12(3):149-156 (2005) (each of which is incorporated by reference for all purposes). 5 The common marmoset (Callithrix jacchus) is a small New World primate native to

Brazil that has been used extensively in biomedical research. Ludlage et al. reports that commonmarmoset wrere found to have amyloid deposits in one or more organs, including the liver,adrenal glands, kidneys, and intestine. The authors posit that hereditary factors might beresponsible for the development of AA amyloidosis in this primate. In this regard, the common 10 marmoset could serve as a unique experimental model for study of the pathogenesis and therapyof AA and other systemic amyloid disorders. See Ludlage et al. Vet Pathol 42:117-124 (2005).

The Shar Pei species of dog, a breed having an AA sequence with the -AEDS motifand that is particularly susceptible to AA-amyloidosis, provides a naturally occurring model ofsystemic AA in which to evaluate novel diagnostic and therapeutic applications of AA amyloid- 15 specific antibodies and other compounds.

EXAMPLES

Example I. AA Fragments.

Peptides corresponding to amino acids 71-75 - GHEDT, as described by Yamamotoand Migita Proc. Natl. Acad. Sci. USA 82:2915-2919 were synthesized by AnaSpec, San Jose, 20 CA, USA. Polyclonal antibodies (Pab) AA were raised and the immunoglobulin fractionisolated, as previously described by Bard, F. et al., (2000) Nat. Med. 6, 916-919.

Example II. Immunogen for Preparation of Murine Antibodies.

The epitope used was GHEDT, (SEQ ID NO: 3) with a CG linker at its N terminus. Thepeptide EPRB-39 which contains the epitope is coupled to sheep anti mouse antibody. EPRB-39 25 is obtained from Anasec, San Jose, CA. The antibodies produced appear to be neoepitopespecific because they don't specifically bind to a peptide that spans the region GHEDTIADQE,(SEQ ID NO: 89).

Example III. Immunization Procedures.

Six-week-old A/J mice were intraperitoneal injected with 50 ug EPRB-39/sheep anti- 30 mouse IgG with Complete Freund’s Adjuvant (CFA) followed by Incomplete Freund’s adjuvant(IFA) once every other week for a total of three injections. Three days before fusion, the tail 109 WO 2009/086539 PCT/US2008/088493 vein was injected with 50 ug EPRB-39 SAM IgG in 90 ul PBS. The titer was estimated at1/10000 from ELISA with high background JH80 is the fusion number for EPRB-39. The following is a list of the clones andlimiting dilution clones that are active: 7D8.29.19.47*, 39, 66 IgG2bk 8G9.3.4.51.22*, 30, 46 IgG2b k 2A4.20.44.77*, 13, 14 IgG2b k 5 7D47, 8G9 and ΊΚΠ indicate preferred subclones. The antibodies produced appear tobe neoepitope specific because they don't react with a peptide that spans the C-terminus cleavagesite of SAA.

Example IV. Antibody Binding to Aggregated and Soluble AA. 10 Serum titers (determined by serial dilution) and monoclonal antibody binding to aggregated AA were performed by ELISA as previously described by Schenk D. et al., (1999)Nature 400, 173-177. Soluble AA refers to the AA fibrils sonicated in dimethyl sulfoxide.Serial dilutions of antibody were incubated with 50,000 cpm of I25I-AA overnight at roomtemperature. 50 μΐ of a slurry containing 75 mg/ml protein A sepharose (Amersham 15 Bioseiences, Uppsala, Sweden)/200 pg rabbit anti-mouse IgG (H+L) (Jackson ImmunoResearch,West Grove, PA, USA) was incubated with the diluted antibodies for 1 hr at room temperature,washed twice, and counted on a Wallac gamma counter (PerkinElmer Life Science, Grove, IL,USA). All steps were performed in radioimmunoassay buffer consisting of 10 mM Tris, 0.5 MNaCl, 1 mg/ml gelatin, and 0.5% Nonidet P-40, pH 8.0. 20 Example V. Analysis of VA6 Wil Structure.

The sequences of the expressed human Vk and νλ immunoglobulin light chain germline genes are as illustrated in Figures 21 and 22. With exception of the Kla, X3a, andA3c subgroups there is a Glu-Asp residue pairing at positions 81 and 82 in all, Vk and νλgermline gene sequences (Figures 21 and 22). In addition, a second germline encoded Glu-Asp 25 pairing at positions 50 and 51 is unique to VX6 germline gene. Thus, VX6 Wil contains both the50-51 and 81-82 Glu-Asp pairs. The side chains of residues 50 and 51 are both accessible on thesurface of VX6 Wil, as shown by x-ray crystallography (Figure 24). In contrast, only the Glu81side chain is surface exposed and the Asp82 side chain is partially buried and appears to interact 110 WO 2009/086539 PCT/US2008/088493 (either by electrostatic interactions or H-bonding) with the side chains of Lys79 and Arg61(Figure 25).

Based on these analyses of the x-ray crystal structure and the relative availability of theGlu-Asp side chains Applicants conclude that the buried Glu81 becomes accessible as thedomain enters an aggregated (e.g., fibrillar) structure (or becomes partially denatured), thusexposing what is otherwise a hidden, cryptic epitope.

Example VI. Analysis of Anti-AA Monoclonal Antibody Binding To VX6. A. Surface Plasmon Resonance

Surface plasmon resonance was used to establish the binding kinetics of severalmonoclonal antibodies with VX6 Wil fibrils and monomer. At a concentration of 6.6 nM all 3antibodies bound to the immobilized synthetic VX6 Wil fibrils with a KD of ~ 1 nM - a valuecomparable to that found for their reactivity with murine AA fibrils (Figure 26). The deflection(expressed in RU) during the binding phase was similar for mAbs 7D8 and 2A4 but was 50%lower for 8G9. This suggests that the density of this antibody on the fibrils was lower than theother 2 reagents, as the calculated affinities were similar for all 3 antibodies. An IgGl mAbserved as a control and exhibited no binding to VX6 Wil fibrils.

Titration of the mAb 7D8 over the range of 6.6 nM to 33.3 nM produced the expecteddecrease in the maximal deflection associated with kon (Figure 27). In general, the bindingkinetics were similar at each concentration, although in these pilot experiments the KD value for7D8 at 26.6 nM did differ from that obtained at the other concentrations.

To assess the specificity of the reaction and ensure that the binding of the mAbs withthe fibrils occurred via the classic F(ab)-antigen interaction (as opposed to Fc-mediated bindingor non-specific adsorption), binding data were acquired in the presence of the immunogenpeptide (p39) at 20 and 1 pg/mL (Fig. 8). Peptide p41 which does not bind the mAb 7D8 at lowconcentrations, served as a control. In the presence of 20 pg/mL p41 peptide, the bindingkinetics for mAb 7D8 with VX6 Wil fibrils were identical to 7D8 alone. In contrast, theimmunogen peptide p39 at 1 pg/mL caused a &gt; 2-fold decrease in the extent of binding as judgedby the deflection of the measured signal (Figure 28). Inhibition of fibril binding by 7D8 wasalmost completely inhibited when 20 pg/mL of p39 peptide was used. These data indicated thatmAb 7D8 bound fibrils via the F(ab) region of the molecule inasmuch as this interaction couldbe completely inhibited by the immunogen peptide.

Ill WO 2009/086539 PCT/US2O08/O88493

The reactivity of the mAh 7D8 with VX6 monomer immobilized on a chip wasexamined using the BIAcore. The antibody did not react with the monomeric protein. Thesedata indicate that the binding site recognized by the mAb 7D8 is present on fibrils, but not on thesoluble precursor protein, implying that the antigen is conformational or cryptic in nature. 5 B. Immunohistochemistry

Immunohistochemistry was performed as follows: 6pm-thick sections, cut fromformalin-fixed, paraffin embedded blocks, were subjected to and antigen retrieval by incubationwith CitraPlus (BioGenex, San Ramon, CA) for 30 min at 90°C. Tissues were immunostainedwith a 3 pg/mL solution of mAbs 2A4, 7D8, or 8G9. The IgG2a mAb TY11 served as a control. 10 A HRPO-conjugated horse anti-mouse Ig antibody (ImmPRESS Universal Reagent, VectorLabs, Burlingame, CA) was used as the secondary reagent. Slides were developed using 3,3’-diaminobezidene (Vector Labs) and examined using a Leica DM500 microscope. The interactionof the monoclonal antibodies with ALk and ΑΤλ amyloid tissues deposits was also studied usingimmunohistochemistry. As illustrated in Figure 29, amyloid deposits in a patient’s thyroid gland 15 which were composed of λ2 fragments were immunostained by 7D8, 2A4 and 8G9. The areas ofreactivity correlated with the amyloid deposits, indicated by the green-gold birefringence seen inthe Congo red-stained tissue section. The most impressive reactivity was achieved with mAbs7D8 and 2A4 mAbs while 8G9, although positive, was considerably weaker. These qualitativedata correspond well with the BIAcore analyses in which 8G9 bound less to the Vk6 Wil fibrils 20 than the other 2 reagents (Figure 26). The isotype matched mAb TY11 that served as a controlexhibited no amyloid immunoreactivity.

The amino acid sequence of this λ2 protein (SEQ ID NO: 86) (shown below) containsthe germline encoded Glu and Asp residues at position 81 and 82, respectively. 1 II 22 2?d 35

SSWIQPF5 V5GSFRQTVS ISCS-SSSSlu wIQQLRSSSP 45 55 £5 72 S3 tCVLm'DD-LL F&amp;amp;G7SSSF5S SSCS-GTSAS LSIRGLQSSD· SSDIYCSSs® S3

S3LSSL 25 Examination of an ALk amyloid tissue deposit revealed 2A4, and to a lesser degree the 7D8 and 8G9, to have positive reactivity. Again there was concordance between theimmunostaining and birefringent, congophilic amyloid regions. The TY11 mAb was unreactive. 112 WO 2009/086539 PCT/US2008/088493 C. Radioimaging of AL Amyloidoma Using 125I-Labeled 7D8

The experimental in vivo model of AL amyloidoma was used to study if radiolabeled mAb 7D8 would image human AL amyloid. The radiolabelling efficiency of 7D8, as determined by SDS-PAGE, revealed that both the IgH and IgL chains incorporated the 1-125 label, and no 5 evidence of bands associated with fragmentation or aggregation were observed. SPECT/CT125 imaging of a mouse bearing an induced AL amyloidoma revealed that the I-labeled antibody localized to the induced, dorsally-located amyloid mass, as evidenced by accumulation of the radiolabeled antibodies in the amyloid, relative to amyloid-free tissues (e.g., liver, heart, spleen, and kidneys). Radioloabeled irrelevant IgG mAb did not accumulate in the mass; however free 10 radioiodide was observed accumulating in the thyroid, indicative of the catabolism and125 dehalogention of the IgG antibody. The distribution of the I-7D8 mAb in the amyloidoma-bearing mice was quantified by measuring the activity associated with the amyloid mass ascompared to that of the liver, spleen, kidney, stomach, heart, and lung. These data confirmed theSPETC/CT imaging study. At 72-h post injection (at which time the images were acquired and 15 the tissues harvested), the amyloidoma contained ~8% ID which is ~ 4-fold higher than that seenin the liver - the site of mAb catabolism- and the heart where residual blood-pool activity wouldexpected to be high. The activity shown in the lung was due to the mode of euthanasia (data notshown).

To confirm the biodistribution data, the amyloidoma as well as the liver, spleen, heart, 20 and kidneys were harvested and tissue sections prepared for autoradiographic analysis.Radiolabelling was performed as follows: The 7D8 antibody was labeled with 2 mCi of 125 reductant-free I (Perkin Elmer) using limiting amounts of Chloramine T and suspended in PBScontaining 5 mg/ml of bovine serum albumin (BSA/PBS). Unbound isotope and proteinaggregates were removed by size-exclusion liquid chromatography through an Ultrogel AcA34 25 column (Amersham Pharmacia). Fractions containing IgG monomer were pooled for imagingexperiments. The radiochemical yield was - 50%, providing a specific activity of ~ 25 pCi/pg. 125 I-labeled mAb was subjected to SDS/PAGE (10% gels) in the presence or absence of areducing agent and analyzed with a Cyclone phosphor-imager. In accordance with the SPECTimaging and biodistribution measurements, the autoradiographs confirmed significant 125 30 accumulation of I-7D8 in the amyloidoma, relative to the liver. There was no evidence of 125 uptake of radiolabeled antibody I-7D8 in any other organs (other than the expected hepatic 113 WO 2009/086539 PCT/US2008/088493 activity associate with catabolism of the antibody). Although mAb 7D8 was relatively uniformlydistributed throughout the bulk of the amyloid mass, a moderately higher density was observedin the peripheral areas at the abdomen-amyloid boundary. There was no uptake of theradiolabeled control IgG in any organs. 5 D. Summary and Conclusions

Surface plasmon resonance, immunohistochemistry and in vivo radioimaging establish that AA-reactive antibodies 2A4, 7D8, and 8G9 bind AL amyloid and fibrils (Kd ~ I nM)derived from immunoglobulin light chains. This interaction likely occurs at the highly-conserved Glu and Asp amino acids at position 81 and 82, respectively, which form a cryptic 10 linear epitope that becomes exposed only when the amyloidogenic light chain is incorporatedinto fibrils.

Example VII. ELISA Analysis Demonstrates Antibody Binding to XiEDX2 Peptides. BIAcore analysis was performed to evaluate binding of antibodies 2A4, 7D8 and 8G4 on peptides of various sequences. As shown below in Table 4, the antibodies were found to react15 with peptides having the sequence XiEDX2. Interestingly, the antibodies did not react withpeptides having additional C-terminal residues. This suggests that the antibodies specificallybind to a neoepitope generated cleavage of SAA to generate a free C-terminal end However, asdemonstrated in Example V, the free end is not essential for binding of these antibodies to VX6

Wil, but rather the XiEDX2 domain adopts a conformation favorable to binding to the antibodies20 as it enters an aggregated (e.g., fibrillar) structure (or becomes partially denatured), exposing an otherwise hidden, cryptic epitope.

Table 4

Antibody Peptide pos/neg 2A4(39) CGGHEDT, (SEQ ID NO 87) POS 40 CGGAEDS, (SEQ ID NO: 88) pos 41 GHEDTIADQE, (SEQ ID NO: 89) NEG 64 CGGAEDT, (SEQ ID NO: 90) POS 65 CGGHADT, (SEQ ID NO: 91) WEAK 66 CGGHEAT, (SEQ ID NO: 92) NEG 67 CGGHEDA, (SEQ ID NO: 93) POS 68 CGGHEDTM, (SEQ ID NO: 94) NEG 114 WO 2009/086539 PCT/US2008/088493

69 CGGHEDTM A, (SEQ ID NO: 95) NEG

70 CGGHEDTMAD, (SEQ ID NO: 96) NEG 71 CGGHED, (SEQ ID NO: 97) FALSE POS?

7d8 (39) CGGHEDT, (SEQ ID NO: 87) POS

40 CGGAEDS, (SEQ ID NO: 88) POS

41 GHEDTIADQE, (SEQ ID NO: 89) NEG

64 CGGAEDT, (SEQ ID NO: 90) POS

65 CGGHADT, (SEQ ID NO: 91) NEG

66 CGGHEAT, (SEQ ID NO: 92) NEG

67 CGGHEDA, (SEQ ID NO: 93) POS

68 CGGHEDTM, (SEQ ID NO: 94) NEG

69 CGGHEDTM A, (SEQ ID NO: 95) NEG

70 CGGHEDTMAD, (SEQ ID NO: 96)NEG

71 CGGHED, (SEQ ID NO: 97) NEG

8g4 (39) CGGHEDT, (SEQ ID NO: 87) POS

40 CGGAEDS, (SEQ ID NO: 88) POS

41 GHEDTIADQE, (SEQ ID NO: 89) NEG

64 CGGAEDT, (SEQ ID NO: 90) POS

65 CGGHADT, (SEQ ID NO: 91) NEG

66 CGGHEAT, (SEQ ID NO: 92) NEG

67 CGGHEDA, (SEQ ID NO: 93) WEAK 68 CGGHEDTM, (SEQ ID NO: 94) FALSE +? 69 CGGHEDTMA, (SEQ ID NO: 95) FALSE+?

70 CGGHEDTMAD, (SEQ ID NO: 96) NEG

71 CGGHED, (SEQ ID NO: 97) NEG 115 WO 2009/086539 PCT/US2008/088493

Example VIII. Immunohistochemical Analysis of Mouse AA.

The reactivity of supernatants from hybridomas expressing antibodies 2A4, 8G9 and 7D8 to murine AA splenic and hepatic amyloid deposits (the principal sites of amyloiddeposition) was documented immunohistochemically. For these studies, sections of tissue 5 harvested from a TRIAD mouse with extensive AA amyloid in the liver and spleen (as evidencedby green birefringent Congophilic deposits) were stained with the mAb-containing supernatants.All 3 bound to the hepatic and splenic amyloid. In contrast, there was no reactivity with culturesupernatants derived from irrelevant hybridomas. The capability of the amyloid using 2A4, 8G9and 7D8 to immunostain amyloid in fresh (unfixed), OCT-embedded murine liver and spleen 10 was tested. There was evidence that the mAbs retained their ability to bind AA amyloid in thehepatic sinusoid. In addition, the antibody reactivity with splenic tissue was easier to interpret,and the perifollicular amyloid was intensely immunostained. To demonstrate that the mAbs wasspecifically bound AA amyloid, the mAb supernatants at a 1:25 dilution were preincubated with50 pg/mL of either peptide #39 (p#39) or #41 (p#41) for lh at room temperature. With 15 formalin-fixed tissue as a substrate, the p#39 peptide (50 pg/mL) significantly inhibited theamyloid reactivity of both 2A4 and 7D8 mAbs (the results with 8G9 are pending). In contrast,the p#41 peptide was ineffective. Comparable results were obtained with fresh tissues.

Example IX. Immunohistochemical Analysis Of Human AA.

Comparison of the amino acid sequence of mouse and human SAA from position 73-76 20 reveals 2 identical residues, a conserved Ser to Thr substitution, and a non-conserved Ala to Hisexchange. To test if the 2A4, 8G9 and 7D8 mAbs would cross-react with human AA amyloiddeposits, we tested their reactivity to human AA-containing kidney, adrenal, ovary and liver. Inall eases, the mAb supernatants immunostained the amyloid deposits. In ovarian tissue, the p#39peptide effectively blocked the binding of the mAbs to the perivascular AA amyloid, whereas the 25 p#41 peptide did not inhibit this reaction.

Example X. Interaction Of Anti-AA Of Culture Supernatants With Murine-Derived AA Fibrils.

The interaction of 2A4, 8G9 and 7D8 mAbs with AA amyloid was initially tested by ELISA and the data, given in Figure 31, analyzed using SigmaPlot (SPSS Ine.). Each pointrepresents the mean ± SE (n = 3). A culture supernatant from an irrelevant hybridoma was used 30 as a control (Ctrl Culture Sup). There was an extremely low signal-noise ratio and the resultsshowed that the first harvest contained more mAb relative to the second, as evidenced by thegreater absorbance signal relative to the control supernatant. (In addition, the 116 WO 2009/086539 PCT/US2008/088493 immunohistochemical reactivity of the day 1 material was greater than the day 2 samples).Although the SE values were large, it appeared from these data that the binding affinity of 2A4,8G9 and 7D8 was approximately equivalent with reactivity absent after ~ 1:64 dilution. Thebinding data also suggest that the capacity, i.e., the amount of mAb bound, varied with 5 7D8&gt;8G9&gt;2A4; however, these data were not corrected for mAb concentration and in subsequent studies this trend was not observed. Because of the low signal and high variabilityfound with the culture supernatants and to determine more accurately the relative binding affinityof the mAbs for murine and human AA amyloid fibrils (as well as to provide material for in vivobiodistribution studies) it was necessary to isolate the mAbs by protein A affinity 10 chromatography. The purity of the isolated mAbs was established SDS-PAGE using 10%acrylamide gels under reducing and non-reducing conditions (Figure 32). Samples in lanes 1-4treated with mercaptoethanol, lanes 5-9 without. Gel was stained with Coomassie blue: mAb8G9, lanes 1 and 6; mAb 2A4, lanes 2 and 7; mAb 7D8, lanes 3 and 8; SP2/0 controlsupernatant, lanes 4 and 9; blank, lane 5. Protein Mr markers (Std) are, form top to bottom: 176, 15 119, 75, 49, 39, 25 and 19 kDa. The interaction of the purified mAbs with immunizing peptide p#39, control peptide (p#41), murine and human AA extracts were determined by ELISA asdescribed above. These data were analyzed by fitting a sigmoidal curve using the SigmaPlotsoftware and the mAb concentration at 50% saturation (EC50), determined (Table 5).

Table 5 'EC5G values for purified mAb binding

Substrate

mAb Human AA Mouse AA (AEF) Peptide 39 Peptide 41 3G9 31.7 nM 5.64 nM 4.0 nM »100 nM 2A4 26.4 nM 4.09 nM 3.4 nM » 100 nM 7D8 13.3 nM 1.84 nM 2.3 nM » 100 nM 20 117 WO 2009/086539 PCT/US2008/088493

The interaction of the 3 mAbs with peptide p#39 exhibited saturable binding with EC50values in the low nanomolar range (see above Table 5). In contrast, even at the highestconcentration of mAb used (100 nM) there was little detectable binding to the p#41 peptide(Figure 33 - Each point represents the mean ± SE, (n = 3 at each concentration)). These dataconfirmed the immunohistochemical results described above, i.e., that peptide p#39 was capableof completely blocking the binding of the mAbs to AA amyloid laden tissues. The calculatedEC50s for the binding of each mAb with p#39 peptide were essentially identical as was the casewhen a murine AA amyloid extract was used as the substrate (Figure 34 - Each point representsthe mean ± SE (n = 3 at each concentration)). The calculated EC50 values for the mAbs bindingto mouse AA extract were essentially identical to those obtained when the p#39 peptide was usedas the substrate (Figure 34; Table 5). In contrast, when human AA amyloid extract was driedonto the wells of the microplate, the EC50 values were between 5 and 7x lower than thatobserved for mouse AA and peptide p#39 (Figure 35 - Each point represents the mean ± SE (n =3 at each concentration); Table 5). Because the EC50 value for 7D8 mAb binding was the lowestof the 3 antibodies tested, Applicants selected this reagent for in vivo co-localization andimaging studies. The 2 amino acid substitutions in the human SAA sequence with respect to themurine protein affected the EC50 values. While not wishing to be bound by a particular theory,Applicants attribute the higher EC50 for the human AA to a poorer “fit” of the amino acid sidechains in the antigen binding site, however, this effect corresponds to only a 5-fold decrease inthe relative affinity when the amyloid extracts are surface adsorbed, as in the ELISA.Furthermore, these data support the observation that all 3 mAbs bound to both murine andhuman tissue AA amyloid deposits.

Example XI. Competitive Binding Of Mabs To Mouse And Human AA Amyloid.

To determine the effect, if any, of potential denaturation when adsorbed to the surfaceof the microtiter well, the reactivity of the 2A4, 8G9 and 7D4 was evaluated using a competitionELISA in which murine or human AA amyloid extract was used as a soluble competitor for theinteraction of the mAbs with surface-bound AA extract.

In all cases, soluble (non-adsorbed) AA amyloid fibrils of both human and mouseorigin were capable of competing for the 3 mAbs, indicating that the epitope recognized by thereagents is not dependent upon the partial denaturation that results from surface adsorption. Ingeneral, the murine AA (AEF) extract was a better competitor than the human AA (Table 6). 118 WO 2009/086539 PCT/US2008/088493

Table 6 ΊΟ&amp;amp;ΰ values {ug/mL) for mAb binding to AA amyloid mAb Human AA* Mouse AA (AEF)* 8G9 &gt;119.5 17.3 2A4 &gt;211.7 14.7 7D8 &gt;881.1 26.8 fHuman AA amyloid in solution competing for adsorbed mouse AA (AEF); ^Mouse AA (AEF) in solution competing for adsorbed human AA amyloid extracton plate.

The IC50 values (concentration of AA (by weight) that reduced the mAb binding by50%) for murine AEF in solution were - 20 pg/mL, whereas for human AA the values were 6- to 5 44-fold greater (in contrast, the FC50s for human AA were only 7-fold lower than those formouse AA). This may reflect the fact that, when in solution, the epitope on the amyloid fibrils isless accessible in human AA preparations as compared to murine AA.

As expected, the 7D8 mAb that exhibited the highest relative affinity for the human andmurine AA fibrils when they were surface-adsorbed required the highest concentration of AA 10 amyloid to achieve competition.

Example XII. Radiolabeled MAb 7D8.

The radiolabeling efficiency of 7D8 was determined by SDS-PAGF. Reduced andnative mAb were analyzed and the proteins visualized using a phosphor imager. Both the IgHand IgL chains incorporated the 1-125 label, and no evidence of bands associated with 15 fragmentation or aggregation were observed.

Example XIII. Imaging of AA amyloid using 125I-labeled 7D8.

To study the in vivo localization of radiolabeled mAb 7D8 three groups of mice wereused: transgenic IL-6; AgNO3/AEF induced, and amyloid-lacking controls (WT). TheSPFCT/CT imaging revealed that the 125I-7D8 mAb localized to murine AA amyloid deposits in 20 the spleen and liver, as evidenced by the accumulation of the radiolabeled mAb in these tissuesrelative to the control mouse, which showed only low blood pool activity in the liver and freeiodide the thyroid gland.

In contrast to these mice, the AgNO3-injected mouse showed thyroid uptake of free 119 WO 2009/086539 PCT/US2008/088493 iodide, some hepatic activity, but the major site of 125I-7D8 binding was seen at the site of s.c.AgNO3 injection (the lower right dorsal area). The activity in this area is clearly circumscribedby the x-ray-attenuating silver solution as seen by CT. The 7D8 mAb has been shown to bind toAA amyloid deposits in both the liver and spleen in the presence of circulating sAA in theTRIAD mouse, as evidenced in the SPECT images. A. Biodistribution Of 125I-7D8 In Mice. 48 h post-injection of 125I-7D8 there was radioactivity in the blood pool, whichaccounted for the relatively high uptake In the lung (which fill with blood when the mice aresacrificed). Of note, the hepatosplenic accumulation of mAb in the IL-6 mouse is indicative ofthe presence of amyloid. The SPECT/CT images confirmed the distribution of the mAb in theseorgans. 72 h post-injection the blood pool values have changed little as evidenced by theunchanged activity in the heart and lung relative to the mice sacrificed at 48 h, due to therelatively long Ti/2bio for this mAb (~60 h). There was significant accumulation of theradiolabeled mAb in the IL-6 mouse, which correlated with the SPECT images that wereacquired showing impressive splenic and, to a lesser degree, hepatic uptake. Of the other organs,most important was the liver (which is the site of catabolism of IgG and the source of sAAduring the acute phase response). In the WT mice, with no inflammatory challenge or amyloid,the liver contained &lt; 6% ID/g, which is comparable to the kidney and heart where the blood poolcontributes almost exclusively to the signal. B. Autoradiographic And Histochemical Analyses.

In order to determine if the increased hepatic accumulation of 125I-7D8 in the IL-6 andAgNO3 mice resulted from amyloid uptake, catabolic clearance or binding to newly synthesizedsAA, liver as well as other tissues were subjected to autoradiographic analysis.

Based on the SPECT imaging and biodistribution measurements, it was presumed thatthe greatest amount of amyloid in the transgenic IL-6 mice was in the liver and spleen. Thissupposition was confirmed in the Congo red-stained sections in which significant amyloid wasobserved throughout the red pulp as well as in the perivascular regions and sinusoids of the liver.Additional, more discreet birefringent deposits were present in the kidneys and heart. Thedistribution of the 125I-7D8 within these tissues correlated well with the Congo red and AA-reactive material. There was no accumulation in hepatocytes that were devoid of amyloid.

Based on the biodistribution data, the AgNO3-treated mouse had more uptake of 125I-7D8 in the liver than the spleen, which was unexpected since this is not the normal pattern of 120 WO 2009/086539 PCT/US2008/088493 accumulation of AA in such animals. Congo red-staining revealed small amounts of amyloid ina single perifollicular region in the spleen (upper right comer) and extensive hepatic perivasculardeposits both of which were evident in the autoradiographs. Additionally, the s.c. site of theAgNCh injection was seen in the SPECT images to have a significant concentration of 12ti-7D8 5 (we also have observed this when radioiodinated SAP was used as the imaging agent). This sitedoes not contain amyloid (i.e., Congo red-birefringent material); however, it was immunostainedby anti-AA mAb. Without wishing to be bound to a particular theory, it is possible that the mAb7D8 localizes to sites of inflammation or “pre-amyloid” (as well as mature amyloid deposits). Incontrast to the impressive accumulation of 7D8 in the organs of the IL-6 mouse, the tissues of the 10 control mice were found to have little or no tracer in any organ other than the blood pool. Noamyloid was found in Congo red-stained sections of any organ of these controls. C. Pharmacokinetics of 125I-7D8.

After injection of the radiolabeled 7D8 antibody, the rate of disappearance of themolecule was determined and the half-life determinations summarized in Table 7. These results 15 indicated that the Ty2bi0 of 7D8 was ~ 60 h, consistent with that of an IgG2b murine mAb (note,7D8 is of the IgG2b subclass). The slightly more rapid clearance of the 125I-7D8 in the IL-6(TRIAD) mice was not considered significant. Based on these data, retention of the mAb bytissue amyloid, as evidenced in the SPECT data, over 72 h does not influence the excretion rate.

Table 7 half-fife analyses for -25I-7D8 in mice

Mouse A (S.E.) K (S.E. x IO’4} R5^ ^12 bio b/2 e/r IL-6,48h 191.7 (2.96) 0.0117(7.0) 0.98 59.2 h 56.2 IL-6, 72h 175.2(3.99) 0.012 (8.9) 0.97 57.7 h AgNO3, 48h 181.0(1.99) 0.0106(4.9) 0.99 65.3 h 61.1 AgNO3, 72h 174.1 (2.97} 0.0112(5.8) 0.98 62.2 h Ctrl, 48h 185.1 (3.19} 0.01Q8 (7.6) 0.98 64.3 h 61.3 Ctrl, 72h 185.1 (3.09) 0.0109 (5.6) 0.98 63.7 h Method of Identifying Agents that Prevent or Treat Amyloidosis

Transgenic or TRIAD Mouse. Procedures for preparation of agents are described in Schenk et 121 WO 2009/086539 PCT/US2008/088493 al. Nature 400:173-177. Agents are emulsified 1:1 (v/v) with complete Freund’s adjuvant for thefirst immunization of transgenic mice, followed by a boost in complete Freund’s adjuvant at 2weeks and monthly thereafter. PBS injections followed the same schedule and mice wereinjected with 1:1 mix of PBS/adjuvant for control. The life span of the transgenie mice iscompared to determine whether the agents are effective in preventing AA Amyloidosis byincreasing the life of the animal. 2. Histopathology. For light and polarizing microscopy, 4- to 6-pm-thick tissuesections were cut and stained with hematoxylin and eosin (HE) and a freshly prepared alkalineCongo red solution, respectively. For electron microscopy, sections were embedded in Epon(Ted Pella, Redding, CA), sectioned, and examined with a JEOL 100S transmission electronmicroscope. See Ludlage et al. Vet Pathol 42:117-124 (2005). 3. Immunohistochemistry. Paraffin-embedded tissue sections (6-pm-thick) were cuton a microtome, mounted on poly-L-lysine-coated slides, dried overnight at room temperature,and deparaffinized. Immunostaining was performed using the avidinbiotin complex (ABC-elite)technique as described previously. The primary antibodies were mouse anti-human amyloid A(Accurate Chemical and Scientific Corporation, Westbury, NY) and anti-mouse SAA polyclonalantisera. Affinity-purified horse anti-mouse immunoglobulin-G (IgG) horseradish peroxidaseconjugate (Vector Laboratories, Burlingame, CA) or goat anti-rabbit, -mouse, or -rat IgG horse-radish peroxidase conjugates (BioRad Laboratories, Richmond, CA) were used as the secondaryantibodies. 4. SAA Quantitation by ELISA. SAA concentrations were measured by an enzyme-linked immunosorbent assay (ELISA) using the Multispecies SAA ELISA kit according todirections supplied by the manufacturer (Biosource, Camarillo, CA). Standard curves wereprepared using known amounts of human SAA protein and absorbance was measured at 405 nmwith a model 4450 BioRad plate reader (Fullerton, CA). 5. Radiolabeled SAP Scintigraphy Turnover Studies in Mice. SAP was oxidativelyiodinated with 125I (2-5 MBq/mg) by using N-bromosuccinimide. 6-12 weeks old mice received2-10 pg of 125I -SAP in 200 pL intravenously. Precisely measured tail bleeds (0.01-0.04 g) weretaken at specific time intervals and trichloroacetic acid-precipitable radioactivity was counted inthe same run at the end of each experiment together with standard aliquots of the injected tracer.Pepys et al. Proc Natl. Acad. Sci. USA 91:5602-5606 (1994). 6. Radiolabeled SAP Scintigraphy Turnover and Imaging Studies in Man. SAP for 122 WO 2009/086539 PCT/US2008/088493 use in man was isolated from the plasma of a single normal accredited donor and was oxidativelyiodinated with 125I (2-5 MBq/mg) or 123I (110 MBq/50 pg of protein) by using N-bromosuccinimide. After injection of 123I SAP, data were acquired and processed on an IGEStarcam gamma camera (IGE Medical Systems, Slough, U.K. Clearance of 125I-labeled SAPwas studied in healthy individuals and patients suffering from AA amyloidosis. Pepys et al. ProcNatl. Acad. Sci. USA 91:5602-5606 (1994) 7. Amyloid Extraction and Purification. The methods used to extract amyloid fromtissue were as described by Pras et al. See Pras et al. J. Clin. Invest. 47:924-933 (1968) In brief,a portion of liver or tissues from other organs obtained at necropsy and maintained at -80 C washomogenized with cold saline in an ice bath using an Omni-Mixer (Omni International,Waterbury, CT). The extract was centrifuged at 10,000 rpm for 30 minutes at 4 C and the pelletreextracted twice more with cold saline, once with 0.1 M sodium citrate Tris-buffered saline, pH8.0, and then again with saline until the A280 of the supernatant was &lt;0.10. The resultant pelletwas homogenized with cold distilled water, and the mixture centrifuged at 35,000 rpm for 3hours at 4 C. The pellet obtained from the water extract was then lyophilized. 8. Surface Plasmon Resonance. Binding kinetics were measured on a BIAcore Xinstrument. Fibrils prepared from the Vx6 Wil were sonicated briefly with a probe sonicator andthen coupled to a CM-5 chip using amine chemistry, as per the BIAcore protocol. This processutilizes EDC and NHS to activate the carboxyl groups on the chip for coupling with free aminogroups on the fibrils. Coupling was conducted in a NaOAc buffer, pH 4.0 at a concentration of100 pg/mL, The control channel was “mockcoupled” and both channels were reacted withethanolamine to saturate unreacted sites. Approximately 16,000 RU of Vx6 Wil fibrils werecoupled.

Sensograms were run in HBS-EP buffer from BIAcore at 20 pL/min in the Fcl (Vx6Wil fibrils) minus Fc-2 (control) mode. Samples containing mAb or mAb plus peptide inhibitorswere injected (70 pL) and the sensograms collected using the delayed-wash function for 200 sec.Data were analyzed in the BIAevalutation software, using the 1:1 Langmuir model with mass-action correction. 9. MicroSPECT/CT. Two cohorts of 3 mice each were injected s.c. with 50 mg of human AL amyloid extract between the scapulae. After 7 days, one group of mice received an iv 125 tail vein injection of - 300 pCi of I-labeled mAh 7D8. The second group were administered 123 WO 2009/086539 PCT/US2008/088493 and equal quantity of murine mAb MOPC 31C as a control. After 72 hr, the mice were sacrificedby isoflurane overdose and SPECT/CT images acquired. To provide vascular contrast-enhancement in the CT images, mice were given a 200-pL iv dose of Fenestra VC™ (AdvancedResearch Technologies, Montreal, Canada) 5 min prior to scanning. 5 SPECT data were collected with a microCAT II + SPECT dual modality imaging platform (Siemens Preclinical Solutions, Knoxville, TN), capable of submillimeter spatialresolution when equipped with a 0.5 mm-pore diameter pinhole collimator. When imaging, the 2 detectors (composed of a 50 mm-diameter Hamamatsu R2486-02 multi-anode photo-multiplier 2 tube coupled to a 1 χ 1 x 8 mm Csl (Tl) crystal array arranged on a 1.2 mm grid) were10 positioned -45 mm from the center of rotation. Each SPECT dataset comprised 45 projectionscollected over 360° during the course of -50 min. Images were reconstructed using an implementation of the expectation maximization-maximum likelihood (EM-ML) algorithm.

After collection of SPECT data, high-resolution CT images were obtained. The microCAT II scanner has a circular orbit cone beam geometry, equipped with a 20-80 kVp15 microfocus x-ray source, and captures a 90 mm x 60 mm field of view using a 2048 x 3072 CCDarray detector, optically coupled to a minR phosphor screen via a fiber-optic bundle. Each CTdataset, composed of 360 projections at 1° azimuths, was acquired in 8 min. Images werereconstructed in real-time on isotropic 77-pm voxels using an implementation of the Feldkamp backprojection algorithm. 20 To facilitate co-registration of the reconstructed SPECT and CT images, Co-57 sealed sources were placed on the imaging bed. The microSPECT and CT datasets were visualized andco-registered manually with a 3-D image analysis software package (Amira, Version 3.1:Mercury Computer Systems). 10. Biodistribution. Samples of liver, spleen, kidney, heart, lung, and implanted 25 amyloid tumors (i.e., amyloidoma) were harvested from the mice and placed into tared vials, weighed, and the radioactivity measured. The primary index values were expressed as %injected dose/g tissue (% ID/g). 11. Autoradiography. 6 pm-thick sections cut from formalin-fixed, paraffin- 125 embedded blocks of tissue obtained from mice sacrificed 72 h post-injection of I-7D8 were 30 placed on Probond microscope slides (Fisher Scientific), dipped in NTB-2 emulsion (EastmanKodak), stored in the dark, and developed after a 24-h exposure. The sections were counter- 124 WO 2009/086539 PCT/US2008/088493 stained with hematoxylin and eosin (H&amp;amp;E), cover-slipped using Permount (Fisher Scientific),and examined by light microscopy. In addition, consecutive slides were stained with alkalineCongo red and viewed under cross-polarized illumination. Finally, a third slide wasimmunostained using as primary reagent our AA-reactive mAh. Digital camera microscopicimages were taken and evaluated using an image analysis software package (Image Pro Plus,Media, Cybernetics).

Example XIV. Preparation Of Humanized 2A4 And 7D8 Antibodies.

Humanized 2A4, 7D8, and 8G9 antibodies were prepared by grafting of murine 2A4,7D8, and 8G9 CDRs onto human acceptor frameworks according to techniques known in the art.Back mutations were made to reduce antigenicity while preserving binding affinity. The lightchain and heavy chain variable regions of murine 2A4 are set forth as residues 20-131 of SEQ IDNO: 152 and as residues 20-138 of SEQ ID NO: 154, respectively. The light chain and heavychain variable regions of 7D8 are set forth as residues 20-131 of SEQ ID NO: 153 and asresidues 20-138 of SEQ ID NO: 154, respectively. The light chain variable regions of murine2A4 and 8G9 are identical to each other and differ from the light chain variable region of 7D8 ina single residue in CDR1. The heavy chain variable regions of each of 2A4, 7D8, and 8G9 areidentical.

The variable kappa (Vk) of 2A4 and 7D8 belong to mouse subgroup 2, whichcorresponds to human subgroup 2 and the variable heavy (Vh) to mouse subgroup 3c whichcorresponds to human subgroup 3 (Rabat et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition. NIH Publication No. 91-3242). CDR-L1 includes 16 residues andbelongs to canonical class 4 in Vk. CDR-L2 includes 7 residues and belongs to class 1 in Vk.CDR-F3 includes 9 residues and belongs to class 1 in Vk. See Martin AC, Thornton JM. (1996)J Mol Biol. 263, 800-15. The leucine at position 27 in the 7D8 is rather unusual, and theglutamine in 2A4 is more usual. A model shows the sidechain is on the surface of the bindingsite, and therefore should be important for antigen binding. CDR-H1 includes 5 residues andbelongs to class 1, and CDR-H2 includes 19 residues and belongs to class 4 (Martin &amp;amp; Thornton,1996). CDR-H3 has no canonical classes, but the 8 residue loop probably has a kinked baseaccording to the rules of Shirai et al. (1999) FEBS Lett. 455, 188-97. This is conserved in amodel although the conformation of the apex of CDR-H3 may be different. The residues at theinterface between the Vk and Vh domains are the ones commonly found for 2A4 Vk, 7D8 Vkand 2A4 Vh. 125 WO 2009/086539 PCT/US2008/088493 A search was made of the PDB database (Deshpande et al. (2005) Nucleic Acids Res.33: D233-7) to find structures which would guide the choice of back mutations. A search of thenon-redundant protein sequence database from NCBI allowed selection of suitable humanframeworks into which to graft the murine CDRs. For Vk, a human kappa light chain withNCBI accession code BAC01562 (gi:21669075) (SEQ ID NO: 166) was chosen. This has thesame length CDR-L3 and belongs to human germline VKIIA19/A3 and human kappa subgroup2. A similar framework which only differed in the J-region was also found with NCBI accessioncode BAC01733 (gi:21669417) (SEQ ID NO: 167). BAC01562 was used as a framework for2A4 Vk, and BAC01733 was used as a framework for 7D8 Vk. For Vh, human Ig heavy chainAAC51024 (gi:1791061) (SEQ ID NO: 165) was used. See Gias et al. (1997) Clin. Exp.Immunol. 107: 372-380. This belongs to human germline VH3-72 and human heavy subgroup 3.

Representative humanized 2A4 light chain variable regions are set forth as SEQ IDNOs: 155, 156, and 157. Representative humanized 7D8 light chain variable regions are setforth as SEQ ID NOs: 158, 159, 160, 174, 175, and 176. Representative humanized 2A4/7D8heavy chain variable regions are set forth as SEQ ID NOs: 161, 162, and 163. See Figures 36A-36E.

Representative humanized antibodies of the invention include antibodies having a lightchain variable region selected from one of residues 20-131 of SEQ ID NO: 152, residues 20-131of SEQ ID NO: 153, and SEQ ID NOs: 155, 156, 157, 157, 159, 160, 174, 175, and 176; and aheavy chain variable region selected from one of residues 20-138 of SEQ ID NO: 154 and SEQID NOs: 161, 162, and 163.

Example XV. Therapeutic Effects Of MAb 2A4 In Mice With Severe Systemic AAAmyloidosis.

The therapeutic efficacy of mAb 2A4 was evaluated in H2/huIL-6 mice with severesystemic amyloidosis. The transgenic W/huYL-6 mice, which constitutively express a humanIL-6 trans gene, are prove to rapid and irreversible systemic AA amyloidosis. In a first andsecond study, mice treated with isotype-matched mAb TY-11, which has no reported activity inmice, was used as a control. Before administering the amyloid enhancing factor to induce AA,H2/huIL-6 mice were sampled and bled via the retro-orbital sinus, serum prepared, and the sAAconcentration determined using a commercially available ELISA kit. Representative values wereas follows: 2196.7 gg/mL, 823.91 gg/rnL, 1415.00 gg/mL, 1673.01 gg/mL, 814.53 gg/mL, 126 WO 2009/086539 PCT/US2008/088493 1088.18 gg/mL, 736.34 gg/mL, 1546.35 gg/mL, 953.70 gg/mL, 886.46 gg/mL, mean = 1213.4 ±478 gg/mL.

At the start of the second study (week 0), H2/huIL-6 mice were injected iv with 100 ggof amyloid enhancing factor (AEF). After induction of AA pathology by injecting AEF, the 5 mice were administered 5 injections of 100 gg subcutaneously in alternate limbs of mAb 2A4(13 animals) or TY11 (11 animals). The therapy was initiated at approximately 1 week postAEF injection. The survival of animals in each treatment group was plotted and analyzed. Theresults are shown in Table 7. Only 45% of the mAb TY11-treated mice survived to the end ofthe study. In contrast, none of the 2A4-treated mice were lost over the course of the study. 10 Analysis of the survival data using standard methods showed a significant difference in thesurvival curves (P0.0025) in both groups. The median survival of the TY11-treated mice wascalculated to be 41 days, comparable to that observed in a prior study (38.5 days).

Table 7

Percentage of animals surviving Days post injection TY 11-treated 2A4-treated 0 100.00 100.00 22 81.82 100.00 33 72.73 100.00 37 63.64 100.00 41 45.45 100.00 42 45.45 100.00 15 At week 6, post-AEF, mice were bled and sacrificed, and their organs harvested for further analysis. For quantification of amyloid in liver and spleen, Congo red birefringence wasvisualized microscopically under cross-polarized illumination and digitally recorded. The areaof birefringent material was determined by selecting (using a spectral segmentation method) andquantifying the amyloid-associated pixels. The amyloid burden index (ABI), a measure of 20 amyloid content, was expressed as the percentage area occupied by amyloid in each organ.Quantification of amyloid in the livers and spleens of 2A4 and TY11-treated mice revealed nosignificant difference between the two treatments. However, the TY 11-treated mice thatsurvived to day 42 for comparison with 2A4-treated mice were those that did not develop a 127 WO 2009/086539 PCT/US2008/088493 morbid degree or distribution of AA amyloid to thereby result in morbidity. The hepatosplenicamyloid burden is also monitored during the course of the survival study to assess an increase inamyloid burden that correlates with morbidity.

In a third study, mAb 2A4 was compared to the isotype-matched mAb JH70, which has5 no reported reactivity in mice. In addition blood chemistry and other parameters were monitoredthroughout the treatment period. Male and female H2/huIL-6 mice bom between 8/1/08 and9/7/08 were used in this study. Twenty three female mice and 16 male mice were bled via theretro-orbital sinus. Whole blood was used for chemical characterization of blood urea nitrogen(BUN) and alanine aminotransferase (ALT) to measure renal and hepatic function by using the 10 VetScan VS2 (Abaxis, Union City, CA). The serum concentration of 12 other proteins andanalytes were simultaneously measured. A complete blood count (CBC) was performed usingthe VetScan HM5 platform. In addition, each mouse was administered a low dose (-50-60 tuCi)of radioiodinated human serum amyloid P component (125I-SAP) in 5 mg/mL bovine serumalbumin to assess the amyloid burden of the mice prior to initiation of the disease process. The 15 percent of 125I-SAP retained at 24 h post-injection (pi) was measured by placing each mouse intoa dose calibrator. Retention of 125I-SAP greater than that observed in non-transgenic (control)mice was indicative of amyloid disease. Finally, serum was used to measure the concentration ofserum amyloid protein A (sAA) using a commercial ELISA assay. A summary of thesepretreatment data, selected blood chemistry values, and the treatments given to each mouse are 20 shown below in Tables 8 and 9.

Table 8

Summary Of Pre-Treatment Data And MAb Therapy For Each Animal

Mouse # sAA cone.(gg/niL) Sex 125i-sapretention (%) Therapy(Group No.) 3488 360 F 8/1/08 9 2A4 (1) 3489 996 F 8/1/08 29 2A4 (1) 3490 472 F 8/1/08 10 2A4 (1) 3492 2068 M 8/1/08 13 2A4 (1) 3493 1740 M 8/1/08 11 JH70 (1) 3494 1272 M 8/1/08 10 JH70 (1) 3495 1436 M 8/1/08 13 JH70(l) 3496 2080 M 8/1/08 9 2A4 (1) 3498 268 M 8/1/08 9 2A4 (1) 3500 700 F 8/11/08 11 JH70(l) 3501 ND F 8/11/08 9 JH70 (1) 3503 1040 F 8/11/08 11 JH70 (1) 128 WO 2009/086539 PCT/US2008/088493 T504™ _ F 8/11/08 10 35131 4400 M 8/13/08 60 2A4(1) 35141 4400 M 8/13/08 40 2A4 (1) 3515 2800 M 8/13/08 13 2A4 (1) 3521 1480 M 8/18/08 11 2A4 (1) 3524 1680 M 8/18/08 9 2A4 (1) 3549 720 F 9/6/08 9 2A4 (2) 3550 760 F 9/6/08 9 2A4 (2) 35522 0 F 9/6/08 11 2A4 (2) 3553 1160 F 9/6/08 12 2A4 (2) 3558 1660 M 9/6/08 9 JH70 (2) 3559 3520 M 9/6/08 12 JH70 (2) 3562 1312 F 9/6/08 11 JH70 (2) 3563 1120 M 9/6/08 9 JH70 (2) 3564 2512 M 9/6/08 11 2A4 (2) 3565 1960 M 9/6/08 10 2A4 (2) 3567 1880 F 9/6/08 12 2A4 (2) 3570 792 F 9/7/08 13 2A4 (2) 3573 700 F 9/7/08 8 2A4 (2) 35772 0 F 9/7/08 10 2A4 (2) 35782 0 F 9/7/08 9 2A4 (2) 3579 1120 F 9/7/08 10 2A4 (2) 35802 0 F 9/7/08 8 JH70 (2) 3581 700 F 9/7/08 9 JH70 (2) 3582 1680 F 9/7/08 9 JH70 (2) 3583 804 F 9/7/08 9 JH70 (2) 3584 1040 F 9/7/08 14 JH70 (2) 1, homozygous IL-6 animals with high sAA levels and amyloid disease early in life. 2, wild type mice without circulating sAA and no amyloid disease. 125I-SAPretention in these animals is considered normal and reflecting no amyloid burden. 5 Table 9

Normal Values For Blood Chemistry Parameters In H2/huIL-6 Mice BUN (mg/dL) GLU _______ ALT (U/L) ALB Jg/dL) TP GLOB Jg/dLl F M F M F M F M F M F M Mean 21.1 23.8 144.7 151.2 37.6 42.3 2.5 1.9 5.6 6.2 3.1 4.4 SD 4.0 2.7 14.0 17.6 16.3 24.3 0.3 0.4 0.2 0.6 0.4 0.6 n 18 13 18 13 18 13 18 13 18 13 18 13 High 28.0 30.0 184.0 179.0 79.0 105.0 3.0 2.6 6.0 7.4 3.7 5.8 Low 15.0 20.0 126.0 119.0 21.0 23.0 2.0 1.2 5.1 5.5 2.6 3.4 Median 20.0 24.0 143.0 154.0 32.5 32.0 2.4 1.9 5.6 6.0 3.2 4.3 BUN, blood urea nitrogen; GLU, glucose; ALT, alanine aminotransferase; ALB, albumin;TP, total serum protein; GLOB, immunoglobulin; F, female; M, male; SD, standarddeviation; n is the number of mice used to determine the values. 129 WO 2009/086539 PCT/US2008/088493

At the start of the third study (week 0), all of the all the H2/huIL-6 mice received 100pg iv of amyloid enhancing factor (1 mg/mL). One week thereafter, therapy began and eachmouse was administered 100 pg of either mAh 2A4 or JH70 sc as outlined in Table 8. The mAhinjections continued weekly for 7 weeks. 5 At 2 wk post-AEF, CBC, blood chemistry, and serum sAA measurements were made using blood collected via the retro-orbital sinus. At this time also, the mice in group 1 wereadministered ~ 60 pCi of 125I-SAP in BSA as before, to assess the accumulation of amyloid asevidenced by the retention of the radiolabeled SAP. Several of the animals showed an adverseeffect of extreme distress, and therefore, evaluation of amyloid burden using 125I-SAP was 10 discontinued. Results of selected blood chemistry parameters, acquired 2 wk post-AEF areshown in Table 10

Table 10 ............................(mg/dL)__________________(mg/dL).......................................................(g/dL).............................................(g/dL)............. F M F M F M F M F M F M Mean 31.4 52.1 145.1 129.8 33.9 63.3 2.3 1.8 6.5 8.1 4.2 6.2 SD 24.3 39.1 16.6 25.6 6.9 30.6 0.3 0.5 1.0 1.7 1.1 1.5 n 15 13 15 13 15 13 15 13 15 13 15 12 High 100.0 159.0 177.0 178.0 46.0 134.0 2.7 3.0 8.6 11.7 7.0 9.6 Low 16.0 20.0 104.0 82.0 22.0 32.0 1.7 1.0 5.2 6.0 3.1 4.5 Median 22.0 31.0 150.0 120.0 32.0 54.0 2.3 1.7 6.5 7.5 4.0 6.0 BUN, blood urea nitrogen; GLU, glucose; ALT, alanine aminotransferase; ALB, albumin; TP, 15 total serum protein; GLOB, immunoglobulin; F, female; M, male; SD, standard deviation; n isthe number of mice used to determine the values.

At 8 weeks post-AEF, the mice were bled a final time and immediately thereafter wereadministered ~ 200 pCi of 125I-SAP using 5% normal mouse serum as carrier. In response to this 20 treatment, a few animals showed some unusual behavior that abated within 30 min. Twenty fourhours later, the mice were injected with x-ray CT contrast agent (~ 200 pL iv in the tail vein) andwere then sacrificed by isoflurane overdose. Single photon emission (SPECT) and x-ray (CT)tomographic images of each animal were acquired. The organs were harvested and the amountof radioactivity in each sample was calculated and expressed as % injected dose per gram of 25 tissue. Additionally, a portion of each tissue was fixed overnight in buffered formalin inpreparation for sectioning and microscopic analysis.

During the 7 wk therapy study, 2 mice were found dead and 3 mice were sacrificed 130 WO 2009/086539 PCT/US2008/088493 because they were deemed unlikely to survive overnight and had a poor body condition score (&lt;2; associated with &gt; 15% weight loss). Mice that experienced an adverse reaction to 125I-SAPinjection and 1 mouse that was sacrificed due to complications that arose from a retro-orbitalbleed were not evaluated as part of the survival analysis. The survival of the mice in each mAb 5 treatment group is shown in Table 11.

Table 11

Percentage of animals surviving Days post injection TY11-treated 2A4-treated 0 100.00 100.00 41 100.00 42 100.00 53 85.71 100.00 55 71.43 100.00 56 64.29 100.00 57 64.29 100.00

Approximately 65% of the mAb JH70-treated mice that were assessable survived to theend of the study. In contrast, none of the 2A4 mice that were assessable died during the 57 days. 10 Analysis of the survival data using the standard methods demonstrated a significant difference inthe survival curves (P=0.015 using Mantel-Cox test and P=0.016 using Grehan-Breslow-Wilcoxon test).

The final blood chemistry data were analyzed according to the therapy that each mousereceived. Because of differences in the mean parameter values associated with male and female 15 H2/huIL-6 mice (at the time of sacrifice, BUN levels in female mice were higher for both 2A4-treated and JH70-treated mice), only the female mice that survived are included in Table 12below. 20 131 WO 2009/086539 PCT/US2008/088493

Table 12 BUN GLU ALT (U/L) ALB TP (g/dL) GLOB __..... ..........(fflg/dL) JM.L}__ JS^L)_ 2A4 JH70 2A4 JH70 '™2A4~ ΤίΉ7(Γ 2A4 JH70 "'"2A4"”jH70" 2A4 JH70 Mean 60.7 73.3 107.8 100.1 45.5 119.7 2.3 2.2 9.2 9.1 7.0 7.1 SD 27.2 25.7 27.0 13.3 6.2 123.1 0.5 0.6 1.5 1.5 2.0 2.1 n 6.0 7.0 6.0 7.0 6.0 7.0 6.0 7.0 6.0 7.0 6.0 7.0 High 95.0 120.0 160.0 123.0 52.0 381.0 2.9 3.0 11.7 11.9 10.1 10.6 Low 17.0 36.0 83.0 83.0 35.0 33.0 1.5 1.2 7.2 7.5 4.3 5.3 Median 66.5 70.0 99.5 98.0 46.5 65.0 2.2 2.1 9.1 8.9 7.1 6.2 BUN, blood urea nitrogen; GLU, glucose; ALT, alanine aminotransferase; ALB, albumin; TP,total serum protein; GLOB, immunoglobulin; F, female; M, male; SD, standard deviation; n isthe number of mice used to determine the values.

Mice treated with 2A4 showed decreased serum blood urea nitrogen (BUN) and alanineaminotransferase (ALT) levels when compared to mice treated with JH70. BUN and ALT aremarkers of renal and hepatic function, respectively, and their reduced levels indicate that organfunction may have been better preserved by 2A4 treatment. 10 132