AU2015249034B2 - Treatment and prophylaxis of amyloidosis - Google Patents

Abstract

Methods useful for effecting prophylaxis or treatment of amyloidosis, including AA Amyloidosis and AL amyloidosis, by administering peptides comprising neoepitopes, such as AA fragments from a C-terminal region of AA, and antibodies specific for neoepitopes of aggregated amyloid proteins, for example, antibodies specific for the C terminal region of AA fibrils. Antibodies for inhibition of formation and/or increasing clearance of amyloid deposits in a patient thus effecting prophylaxis or treating amyloid disease.

Description

TREATMENT AND PROPHYLAXIS OF AMYLOIDOSIS

The present application is a divisional application of Australian Application No. 2008345022, which is incorporated in its entirety herein by reference.

CROSS-REFERENCES TO RELATED APPLICATIONS

Priority is claimed to U.S. Provisional Application No. 61/095,932, filed, September 10,2008, and to U.S. Provisional Application No. 61/007,544, filed December 28, 2007, each of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

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

BACKGROUND OF THE INVENTION

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Amyloidosis is a general term that describes a number of diseases characterized by the existence of pathological forms of amyloid proteins, often involving extracellular deposition of protein fibrils, which form numerous “amyloid deposits” or “amyloid plaques,” which may occur in local sites or systematically. These deposits or plaques are composed primarily of a naturally occurring soluble protein or peptide, assembled into extensive insoluble deposits ΙΟΙ 00 pm in diameter in a variety of tissue sites. The deposits are composed of generally lateral aggregates of fibrils that are approximately 10-15 nm in diameter. Amyloid fibrils produce a characteristic apple green birefringence in polarized light, when stained with Congo Red dye. Generally, the fibrillar composition of these 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 larger precursor protein. More specifically, the pathogenesis of amyloid aggregates such as fibril deposits generally involves proteolytic cleavage of an “abnormal” precursor protein into fragments that aggregate into anti-parallel β pleated sheets.

The fibrillar composition of these deposits is an identifying characteristic for the various forms of amyloid disease. For example, intracerebral and cerebrovascular deposits composed primarily of fibrils of beta amyloid peptide (β-ΑΡ) are characteristic . consequence of long term hemodialysis treatment. Mmc rcoc5H.lv. pri<^8i>Hoci3K:idi'''d^e^e^. such 8¾ CreurzfcidkUtcob dise^d·* iiave'also been r^e0.gft%^d'^-§iid^q!id·. tlfeeasgsv. in general.. primary amylaidoses of the disease are chuMelen/wl b\ :he preset n.e «Ί :“amyloid light chain-type* fAL-type) protein fibfils. -ό named for die ho»YH*iog> <n die V reeminsd region of She AL fibrils to the variable fragment of isniHUisoclebuhn light cl-:00 {kappa or lambda)

The various forms of disease have been divided into classes, rnostis on the basis m whether the aniyloidosis is associated with an underlying .systematic illness, Thus, certain disorders arc considered to be primary amylnidoscs, in which there is no evidence for preexisting or cocxisimig disease. In secondary or reactive (AA type: amyloidosis char;ictcn.r.ed l\v the presesee deposinoo: of amyloid protein. A (AA) fibrils, there Is an nnderlybig or associated: chronic inflammatory or infections disease state. •Heredofamilial umvfoidoses nn.o have «Associated neuropathic, renal, or eurdioscircular deposits of the ATTR transthyretin syne. Other heredofamilial arnyloidoses include other syndromes and may have different a soy Send components ie.g.. familial Mcddemuieun Sever which is characterised by AA fibrils). Other forms of amyloidosis include local forms, characterised by focal, often tumor-like deposits that occur in isolated organs. Other any kudoses arc associated with aging, and arc commonly charactcneed by plague formation in the bean or brain. Also comtnon are amyloid deposits associated with long term hemodisiy*.fs These and other forms of amyloid disease arc summarised m Table I flan. S.Y. and Pepys, flistopathology 25:-403-114. 100-4: Harrisons Handbook of bucmal Medicine. I ΥΪ! Ed,, issclhacher, K..I.. et a!., eds. McGraw-Hill, San francs see. i AM; and are described tn ITS. Parent ’so\. 6.^75.4 41. ii.SvO.fef. 6.M 3./-45. o.vl\%4. and 6,936.2-16. winch are incorporated by refere-nre herein in their entirety

’Seminal vesicle exocrine protein ()tic·;fibrils forming the bulk of an amyloid deposit arc derived from one or more primary precursor proteins, or peptides, and are usually associated with su! fitted glyeoisarninoelycans. In addition, amyloid deposits may include minor proteins and napndes of various types, along with other components, such as proteoglycans, ganghosides and other sugars, as described in more detail in the sections that foilosv. ΛΛ fibrils are composed of peptide Payments that range In si/.e but arc generally about 8000 ballons ίΛΛ peptide or protein} formed by proteolytic cleavage of serum amyloid A protein {SSAh a ciwaLvong apolipoprotein which is present in ΠΓ.Η. particles and which is symhesiecd in hepatoeytes in response to such cytokines as interleukin HI>1 and IL~6. as well as motor necrosis (actor h. Sec Busby, G, et as. Amv>oiJ l, ilo-li" 11094} The proteolytic cleavage results in the pathology deposition of an -76-residue N-terminal two thirds of the SAA protein, hi Inunans, the plasma concentration of SAA normally is -0 I mg mi but eats increase over 1,000-told in response to an inflammatory stimulus. As parr of this process, the S.AA molecule undergoes proteolysis and the iV-tcrminai cleavage product is deposited systemic ally as Λ A fibrils in vital organs,; mi tiding, the liver, spleen, kidneys, and adrenal glands Deposition is also common in the heart and gastrointestinal tract.

Generally, ΛΛ arnyioidosH is a manifestation of diseases that provoke a sustained acute phase response. Seek diseases include chrome inflammatory disorders, ehronie local or systemic microbial infections, and malignant neoplasms, A.A amyloid diseases include, but are not limited to mil aroma tory diseases, such as rheumatoid arthritis, juvenile chronic arthritis, ankylosing spondylitis, psoriasis, psoriatic arthropathy, Rotter's syndrome, Adult Still's disease, Behcet's syndrome, and Crohn's disease. ΛΛ deposits are also produced as a result of ehronie microbial infections, such as leprosy, tuberculosis, bronchiectasis. decubitus ulcers, chronic pyelonephritis, osteomyelitis, and Whipple's disease Certain malignant neoplasms can also result in Λ.Α fibril amyloid deposits. These include such conditions such as Hodgkin's lymphoma, renal carcinoma. carcinomas of gut, lung and urogenital tract, basal cell carcinoma, and hairy cell leukemia. AA amyloid disease may also result from inherited inflammatory diseases such as Familial Mediterranean Fever. Additionally, AA amyloid disease may result from lymphoproliferative disorders such as Castleman’s Disease. AA Amyloidosis is insidious and progressive. Symptoms are generally presented in later stages of the disease. Frequently the patient is undiagnosed until significant organ damage has occurred. AA fibrils are deposited in vital organs leading to organ dysfunction and subsequently to death. The five year survival rate is 45-50%. Median survival after diagnosis is 4-8 years. End stage Renal Disease is the cause of death in 40-60% of cases. See Gillmore J.D. et ah, Lancet 358:24-9 (2001).

Currently, there are no approved specific, amyloid-directed treatments for any of the amyloid 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 the production of amyloidogenic protein by treating the underlying disease. For example, current treatment strategy for AA Amyloidosis is to target underlying inflammation, reducing ApoSSA levels to below 10mg/l. Currently employed therapies include chemotherapy (cholorambucil and MTX), immuno-suppressants (azathioprine), anti-inflammatory drugs (colchicine) and TNF inhibitors.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

SUMMARY OF THE INVENTION

According to a first aspect, the invention provides an isolated antibody or antigenbinding fragment that specifically binds to a neoepitope exposed in an aggregated light chain immunoglobulin protein, wherein the neoepitope comprises the sequence X1EDX2 and wherein Xi is Η, T, F, S, P or A and wherein X2 is T, S, E, D, R, I, V, F or A.

According to a second aspect, the invention provides a method of therapeutically or prophylactically treating a subject having AL amyloidosis comprising administering to the subject an effective dosage of the antibody or antigen-binding fragment of the invention.

According to a third aspect, the invention provides a method of detecting an amyloid deposit associated with AL amyloidosis in a subject comprising: (a) administering to the subject the antibody or antigen-binding fragment of the invention, wherein the antibody or antigen-binding fragment is bound to a detectable label; and (b) detecting the detectable label in the subject.

According to a fourth aspect, the invention provides use of the antibody or antigenbinding fragment of the invention in the manufacture of a medicament for treatment or prophylaxis of AL amyloidosis in a subject.

According to a fifth aspect, the invention provides use of the antibody or antigenbinding fragment of the invention, wherein the antibody or antigen-binding fragment is bound to a detectable label, in the manufacture of a medicament for detecting an amyloid deposit associated with AL amyloidosis in a subject.

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

The disclosed antibodies include humanized and chimeric versions of antibody 2A4 produced by the hybridoma deposited under ATCC Accession Number 9662 or a humanized or chimeric version of antibody 7D8 produced by the hybridoma deposited under ATCC Accession Number 9468.

For example, representative antibodies and antigen-binding fragments comprise a light chain variable region comprising one or more complementarity determining regions of a 2A4 light chain variable region set forth as residues 20-131 of SEQ ID NO: 152 or one or more complementarity regions of a 7D8 light chain variable region set forth as residues 20-131 of SEQ ID NO: 153. As another example, representative antibodies and antigen-binding fragments comprise a light chain variable region comprising two complementarity determining regions of a 2A4 light chain variable region set forth as residues 20-131 of SEQ ID NO: 152 or two complementarity determining regions of a 7D8 light chain variable region set forth as residues 20131 of SEQ ID NO: 153. Additional representative antibodies and antigen-binding fragments comprise a light chain variable region comprising three complementarity determining regions of a 2A4 light chain variable region set forth as residues 20-131 of SEQ ID NO: 152 or three complementarity regions of a 7D8 light chain variable region set forth as residues 20-131 of SEQ ID NO: 153. Representative humanized versions of a 2A4 or 7D8 antibody comprise at least one light chain framework residue selected from the group consisting of L87 and L90 (Rabat numbering convention), which is occupied by Y and F, respectively, and wherein the remainder of the light chain variable region is occupied by a corresponding residue in a human acceptor immunoglobulin light chain variable region. Representative antibodies and antigen-binding fragments comprise at least one light chain framework 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 a corresponding residue in a human acceptor immunoglobulin light chain variable region. For example, representative antibodies and antigen-binding fragments comprise at least one light chain 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 variable region is occupied by a corresponding residue in a human acceptor immunoglobulin light chain variable region. In other representative antibodies and antigenbinding fragments of the invention, the light chain variable region comprises a framework residue at +105 (linear numbering) occupied by Q.

Fijr example.. antibodies and antigen-binding fnigmems of the urvenuou inektdc those comprising a light chain variable region compiling a framework residue at *? tlinear wfsvbobssgl occupied by 1\ wherein -he remainder of tin; light chain variable rector; is occupied by a corresponding residue in a human acceptor immunoglobulin light chain variable region; antibodies and anf gmi-bmdmg fragments a light chant variable region comprising a framework residue at bid (linear numbering) occupied by S, wherein the remainder of the light chain variable region is occupied by a corresponding residue in a human acceptor immunoglobulin light chain variable region; anuboebes and amigeri-hiudiug fragment' a light chant variable region comprising a framework residue <n rl5 (linear numbering) occupied by L, wherein the . remainder of the light chant variable region is occupied by a corresponding residue ut ti human acceptor immunoglobulin light chain variable region; antibodies and antigen-binding fragments a tight chant variable region comprising a framework tesidue at -H"? shr scar numbering) occupied by LX wherein -he remainder of she light chain variable region is occupied by o conv-sponding residue in a human acceptor immunoglobulin light chain variable region; antibodies and amsgsn-binding fragments a light chain variable region comprising a framework residue at -mb ;linear numbering) occupied by Q. wherein the remainder of the light chaitt variable region is occupied by a corresponding residue tit a human acceptor immunoglobulin lieu! chain variable region; antibodies and antigen-binding fragments a light chain variable region comprising a framework residue at 150 {linear numbering» occupied b\ K, wherein the rentalnder of the light chain variable region is occupied by a corresponding residue in a human acceptor immunoglobulin belts chain variable region; antibodies and entlgem-bindmg fragments a light chant variable region comprising, a framework residue at ; ~:5 {linear numbering) occupied by Y, wherein the remainder of the light chain variable region Is occupied by a corresponding residue in a human acceptor immunoglobulin light chain variable region; antibodies asni amsgcri-biriding fragments a light chain variable region comprising a framework residue ut -t*k {linear numbering) occupied by 1., wherein the remainder of the light chain variable region is occupied by ;t corresponding residue in a human acceptor immunoglobulin light chain variable region; antibodies and antigenbinding fragments a light chain variable region comprising a framework residue at -¾ (linear numberings occupied by F, wiser cm the remainder of the light dans; variable region is occupied by a corresponding residue in a human accentor immunoglobulin light chain variable region; antibodies' and antigen-binding fragments a light chain variable region eo.mptismg a framework residue at Ή Ob (linear numbering) occupied by 1.. wherein the remaindei of the light chain variable region 5¾ occupied by a corresponding residue m a human acceptor immunoglobulin light chain variable region; and antibodies and *imigco-N riding fragments a light chain variable region comprising a framework residue at t Ϊ05 (linear nurnbenng) occupied by Q, therein the remainder of the light chain variable region is occupied by a corresponding residue in a human acceptor immunoglobulin light chain variable region.

Human acceptor immunoglobulin light chant variable teutons used m the invention include human kappa subgroup 2 light chain variable region - Rabat conventionh for example·, human subgroup 2 light chain variable region from human gcnnlirse VK11AI 9y\3. such as tun nun Vk lupin chain varttdde region comprising a sequence set forth as SEQ ID NO: fo-t* or 167. In particular aspects of the invention, antibodies and antigen-binding fragments comprise a tight chain vanablc region eompriHug an arnino acid sequence set forth as residues 20-131 of SEQ ID NO: 152. residues 20-131 of SEQ ID NO· 153. or set forth as SliiQ ID NO: 155. 156, 157. 158. 15m 160. Γ·4, 175, or 176.

Representative antibodies and aniigcn-biridmg fragments of the invention also include those comprising a heavy chain variai.de region comprising one or more complementarity regions of a 2Ad heavy chain variable region set forth as residues 20-13s of SEQ ID NO: 153, for -example, a heavy chain variable region comprising two completnenlanty region» of a 2A4 heavy chain variable region set forth as residues 20-138 of SEQ ID NO; )54, ora heavy chain variable :-region comprising three complementarity regions of a 2A4 heavy chant variable region set forth as residues 20-138 of SEQ ID NO; 154. Representative humanized 2A4 and ""'DF antibodies and antigen-bitiditu: fragments comprise at least one heavy chain framework residue selected from the group consisting of 1127, 114b, H?0, and live (Rabat numbering convention), which ts occupied bv I, A, F. or V, respectively, and wherein the remainder of the heavy chain variable region is occupied by a corresponding residue in a human -acceptor tmmunoglobabn heavy chain variable region. Representative humanized antibodies ami antigen-binding fragment comprise at least one heavy chain framework residue selected from the group consisting of - Iff -15. W, *37, -dfo --'Ή, -t78, *79, i pp, -i-87, -95. t99. t)!(linear numbering), which is occupied by R, K, R„ 1, Λ, F. Q. S. Μ N, Μ, V, or A. respectively, and wherein the remainder of the heavy chain variable region is occupied by u corresponding residue tn a human acceptor tmmtmoglobulin heavy chain variable region. For example, representative hntrunuaed antibodies and antigenbinding fragments comprise at leas·', one heavy chain framework residue selected from the group consisting of -*W. *49, *73, and *99 (linear numbering), which is occupied by 1, P, or V, respectsvely, and wherein the remainder of the heavy chant variable region is occupied by a corresponding residue in a human acceptor immunoglobulin heavy chain variable region, for example, antibodies and antigen-binding fragment* of the invention include those comprising a heavy chairs variable region comprising a framework residue at -HO (linear lumbering} occupied by R., wherein the remainder of the heavy chain variable region is occupied by a corresponding residue in a human acceptor immunoglobulin heavy chain variable region; .antibodies and antigen-binding fragments a heavy chain variable region comprising a framework residue at -IS {linear numbering} occupied by K, wherein the remainder of the heavy chain variable region is occupied by a corresponding residue in a human acceptor immunoglobulin heavy chain variable region; antibodies and anugen-bmding fragments a heresy chain variable region comprising a framework residue at t)9 (linear numbering) occupied by K. wherein the remainder of the heavy chain variable region is occupied by a corresponding residue in a human acceptor immunoglobulin heavy chain variable region; antibodies and antigen-binding fragments . a heavy elutin variable region comprising a framework residue &amp;t -M? (linear numbering) occupied by h wherein the remainder of the heavy chain variable region is occupied by a corresponding residue in a human acceptor immunoglobulin heavy chain variable region; antibodies and anUgcn-bmdmg fragments a heavy chain variable region comprising a framework residue at -Mb (linear numbering) occupied by A. wherein the remainder of the heavy chain variable region ss occupied by a cosvesponding residue ;n a human, acceptor immunoglobulin heavy chum variable region, antibodies arid antigen-binding fragments a heasy chain variable region comprising a framework residue at -?.> tlinear numbering) occupied by F.· wherein the .remainder of the heavy chain wiriable region is occupied by a corresponding residue m a human acceptor immunoglobulin heavy chain variable region; antibodies and antigen-binding fragments . a heavy chain variable region comprising a framework residue at *78 (linear numbering) occupied by Q, wherein the retnainder of' the heavy chain variable region is occupied by a corresponding residue in a human acceptor inununogiobulin heavy chain variable region; antibodies and antigen-binding fragment* a heavy chain variable region comprising a framework·· residue at *7y (linear numbering) occupied by S, wherein the remainder of the heavy chain variable region is occupied by a corresponding residue to a human acceptor urernmoglobuun heavy chain variable region: antibodies and antigen-binding fragments a heavy chain variable region comprising a framework residue at MO (linear numbering) occupied by M, wherein the remainder of the heavy chain variable region is occupied by a corresponding residue in a human acceptor immunoglobulin heavy chain variable region; antibodies and anugcu-bmding fragments a heavy chain variable region comprising a framework residue at a 8'· (linear numbering) occupied by N, wherem Ore remainder of Ore heavy chain variable region i.« occupied by a corresponding residue in a human acceptor immunoglobulin heavy chain variable region; antibodies and aungen«binding fragments a heavy chain variable region comprising a framework residue at -95 (linear numbering) occupied by My wherein tfe fgeijftftd&amp;r pf |h.t?· -H^Vy chain varobie region is occupied by a corresponding residue in a human acceptor immunoglobulin heavy chain variable region; antibodies and antigen-binding fragment*' a heavy chain variable region comprising a framework residue at *bv (linear mimhcring·)·occupied by Vh wherein the remainder of tin. heavy cl tain variable region is occupied by a corresponding residue in a human acceptor immimoglolHilin heavy chum variable region; and antibodies and antigen-binding fragments a heavy chain variable region comprising a framework residue si HOO (linear numbering .1 occupied by /V wherein the remainder of the heavy chain variable region is occupied by a corresponding residue in a human acceptor immunoglobulin hoasy chain variable region.

Human acceptor immunoglobulin heavy drain variable regions include a human gamma subgroup 3 heavy chain variable region (Kabaf convention), for example... human gamma subgroup 3 heavy chain variable region comprising s. sequence set forth as SEQ ID NO; 165, suclt as a heavy chain variable region comprising an amino acid sequence set forth as residues 20*138 ofSEQ ID NO; 154 or set forth as SEQ ID NO' lot. DC, or lob

Additional representative antibodies and antigen-bind mg fragments comprise a light chain variable region comprising three complementarity determining regions of a 2A4 light chain variable region set forth as residues 20-131 of SEQ ID NO; 152 or three complementarity regions of 3 ?D8 'ught chain variable region set forth as residues 20-1 31 of SEQ ID NO: i 53. and . tt heavy chain v ariable region comprising three coraplcmcmariiy regions of a 2A4 heavy chain variable region set forth us rew dues 20-138 of SEQ ID NO; 154, For example, such am I hod lea and antigen-binding fragments include those having a light chain variable region composing three complementarity- determining regions set forth as SEQ ID NOs: U-y 169, and P0, and a heavy chain variable region comprising three complementarity regions set forth as Shi} ID NOs1 171, 172, and 173. As another example, such antibodies and antigen-binding fragments include those having a light chain variable region comprising three complementarity determining regions sol forth as Sf.Q ID NO.-: 1 160, and PO, and a heavy chain variable region comprising three complementarity regions set forth as SEQ ID NCN: 171. I 72, .and : 73. Ah another example, sueh antibodies and antsycre-hindinc h;u.n;en?s include those composing a bight chair; variable region comprising an amino acid sequence so? forth as residues 21)-131 of SEQ ID NO; 132 or »$ residues 20-131 of SEQ ID NO. 153, and a heavy chain v a Sable morion comprising an ammo acid x'quenee sen forth as residues 20-13b of SEQ ID NO. 154. As another example, such .antibodies and antigen-binding fragments irscinde those having a light chair; variable morion comprising an amino acid sequence set forth as SEQ ID NO: 155. 15b. 15". 15S, )59. ί60, P4, 175. or I?{\ and a heavy chain variable region comprising an emitnt acid sequence set forth as SEQ Li) NO; 161, DO. or i63 1st particular aspects of the invention, an antibody or antigen-hind mg fragment comprises •a light chain variable region comprising an amino acid sequence set forth, as SEQ ID NO- 155.. and a heavy chain variable melon comprising an amine; aetd sequence set forth as SEQ ID NO: 161; a light chain variable region comprising an ammo acid sequence sc? forth as SEQ ID NO; 155, and a heavy chain variable region comprising an amino at id sequence set ioah as SEQ ID NO; 102; a light chain variable region comprising art amino a ad sequence set forth as SEQ ID NO: 155, and a heavy chain variable region comprising an moino acid sequence set forth as SEQ ID NO: 103: a light chain variable region comprising an amino aclel sequence set north >ts SEQ ID NO: 156. a.nd a heavy chair; variable region comprising an amino eeid sequence set forth as SEQ) If) NO; 161; a light chain variable region comprising an amine* acid sequence set forth as SEQ ID NO; 156, and a heavy chain variable region comprising art amino acid sequence set forth as SEQ ID NO; 162; a light chain variable region comprising cat ammo acid sequence set forth as SEQ) ID NO: 1.26. and a heavy chain variable region comprising an amino acid sequence sc! forth as SEQ ID NO' 163; a light chain variable region comprising an amino acul seqneuee set forth as SEQ ID NQ; )$?, and a heavy chain variable region comprising an amino acid sequence set forth a* SEQ ID NO; 161; y light chain variable region comprising an ammo acid sequence sot forth as SEQ ID NO: 127, and a heavy chain variable region comprising an amino acid sequence sc? tend as 56:31} ID NO: 162; a Huht chair: variable region corn prist os' an amino acid sequence set forth as SEQ ID NO: 15", and a heavy chain variable region comprising an amino add sequence set forth as SEQ ID NO; 163: a light chain variable region comprising ;m ammo add sequence set fort!; as SEQ ID NO; 15 x, and a heavy chain variable region composing an ammo uetd sequence set forth as SEQ' ID NO. 161: a light chain variable region comprising an arm no acid sequence set forth as SE.Q) ID NO; 158, and a heavy chain variable region comprising an nmum scut sequence set forth as SEQ ID NO' 162; a light chain variable region comprising an amino aed sequence set forth as S!:'0 ID NO: 15N and a heavy chain variable region comprising an ammo acid sequence Net forth .½ SEQ ID NO. lO: a light chain \ unable region comprising an amino arid sequence set forth u-> SEQ ID MO; 15m and a heav s chain % a π aide region comprising an amino acid sequence ^et Ieoh as SEQ ID NO' Ibl; u light chain variable region comprising an ammo acid sequesue set forth a a SE.Q ID NO; 159, and a heavy On a in variable region comprising an amino acui sequence sea forth as SE.Q ID NO; UC; a light chain variable melon comprising an ammo mad sequence set lorth as SEQ ID NO; I5m and a heaw chain variable region comprising nn amino acid sequence act torth as SI:-Q ID NO. lb?·: a light chain \ unable region emuprising an ammo acid sequence sol forth as SEQ ID NO' IbO, and a hem v chain variable region comprising an amino acid sequence set forth as ShQ ID NO; If I. a light chain v.u nmle region comprising an ammo acid sequence set ion.lt as Si:.'Q ID NO; I60, and a hem, \ chain \ amdeIe melon comprising an ammo mad sequence set forth as Sil.'Q ID NO. 16a; a main chain variable region comprising an amino acid sequence set forth us SEQ ID NO; I(>b, mid a heavy chain variable region comprising an amino acid sequemc sei forth as SEQ ID Nil.}· MQ; SFQ ID NO' I”4.. and a heavy chain vurhfole. region comprising an memo acid sequence set forth as SE.Q 1D NO- lot; a light chain < arufoie region comprising an amnio acid sequence set forth as NEQ ID NO; 174, and a heavy chain mumble region comprising an amino acid sequence set forth as SE.Q ID NO; 167; t* light chain variable region comprising an ammo acid sequence set lord- as ShQ ID NO: 1 m, and u heavy chain variable region comprising an ammo acid sequence set forth as SEQ ID NO: 16.1, a light chain variable region comprising an ammo acid sequence set forth a.-' SE.Q ID NO. Γ'5, and a hears chum variable region comprising an amino acid scqucui.e set forth as SEQ ID NO: iόI; a light cham variable region comprising an amino acid sequence set ford: as SEQ ID NO. 175, and a hcavs· chant sariahie region comprising at: arm no acid sequence set. forth as SEQ ID NO: NS.?; a light chain variable region comprising an ammo acid sequence set forth as SEQ ID NO'· 175, and a heavy chain sarOble region comprising an ammo acid sequence set forth as SEQ Hi) NO: 563; a light chum variable region comprising mi amino acid sequence set forth as SE Q ID NO: i ?:\ and a heavy chain variable region comprising an ammo acid sequence set forth as ShQ ID NO: Inl; a light chain variable region comprising an amino acid sequence set forth as SE.Q ID NO. Pm and a heavy chain v ariable region comprising an ammo acid sequence set forth ns SEQ ID NO: I Ed; or a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: l76, and a heavy chain variai.de region comprising an amino acid sequence set forth as SEQ) ID NO. 165.

Also presided arc isolated nucleic acids cnoodin>; a human, 'in.unarna.cd.. or chimeric .antibody, or antigen-binding fragment thereof, thut specifically hinds re* aft epitope within residues "0-'“6 of human amyloid A peptide, including ait such antibodies and antigen-binding fragments as described herein «hose and as set forth in the claims. Further pros sou! «re celts e\pre-wing sttt h nuolc-e acids.

In other aspects, the present invention provides «η Notated antibody, or antigen-binding fragment Hereof, which specifically binds to an epitope comprising XiEDXv sn an aggregated amyloid protein, therein Xt and X:- are any ammo aosd. Such antibodies and aotigen-bmdmg •fragments include human, humanized. or chimeric antibodies, and antigen-binding fragments thereof, for example, llto.se that specifically bind So an epitope wiihm residues 70-76 of human amyloid Λ peptide.

Additional representative ouibodios and amhum-bindiue fragments include those oherein X; is Η, Γ, E. 8, r, Λ, L, ( , 0, F·. b, R, IX O. V, v, I, o- A, and wherein \; ts I, S, 1:., R, L V, F, D, A, G, Μ, I., K, P, C, K. V. or Q, or X · ts Η, T, F, S. P, or \ and wherein X, is 1', 8, F, R. I, V. K D, or .V. or Xj ts Η, I, l·, or Λ. or 8.: ss Γ, S, it, D, or At or X: ts Η, Γ, F. or A and X: is Γ. S, 17, D. or At or Xj is Η. T. or A and X: is If 8, f, or A; or X; ts H or .A and X; ts T, 8. or A, or X; is 11 and X.- is T or A; or Xj ts A and X.: N 8. T, f ot V; oi X; Is A unci X.· is 8, T or if; or X} is T and .X.: is F; or X; is F and X.: is D; or NX is N and X·: is 1::, 1- or A. or X; is P and X.· is F, I or F For example, such antibodies and antigen-binding fragments bind an epitope consisting of an amino acid .mquenee selected from the group consisting of ΟΗΗΠ1 (SFQ ID NO- .0. HF.DT 181:.() ID NO: ΓΝ, AFDS (81:.() ID NO. U), AEDT (SEQ ID NO' 14} Hi:.DA (8170 ID NO: 15), 1XDI: (SFQ ID NO: IF), FFDD (SEQ ID NO; 17). SE.DF (SFQ ID NO: (hi.

A EOF iSEO ID NO: 0)). PE DU: (SEQ ID NO. ,70b PFD1 *$EQ ID NQ- 7 0. PFDE (8EQ ID NO: 77). Al:DV (SEQ ID NO; 77), SFDF (SFQ ID NO: 74}. and SFD A (SFQ ID NO· 75k (M an epitonc consisting of an amino acid sequence selected from the group consisting ofGHFDT (SFQ ID MX. 7), HFDT (SFQ ID NQ- 17), AFDS (SEQ ID NO: 1.1). Λ BEX Γ (SEQ ID NO. 14c it FIX \ (8FQ ID NO: If), TEDE (SFQ ID NO; Dm FEDD (SET) ID NO; 10, SFDF. (SFQ H> NO: DO AEDE (SEQ ID NO. 10>. pr.DE (SLQ ID NO 70). PFDl (SEQ ID NO' 70. PEDF (SFQ ID NO- 77), 8EDI" (SFQ ID NO; 74). and SEDA (8F.Q ID NO. 75c or an epitope consisting of,in ammo acid sequence selected from the group consisting of 0H.FDT (SFO ID NO: 3). HFDT (SFQ ID NO. 12}. AFDS (SFQ ID NO. KG. AFDΓ tSF.Q ID NO' 14). 14FDA (SEQ ID NO; leg and I F.DE (SEQ ID NO: 10), The disclosed epitopes mm be found In an aggregated amyloid protein, for example, an epitope comprising an amino acid sequence -selected from :lie group combslhtg of GHGAETlS (SEQ ID NO' 4). GHDAEPS (SEQ ID NO: 5). CIO HA EDS (SEQ If) NO' ?t. STM LOS ISIfQ ID NO: 3 ), and GOOD if DT (SEQ ID NO: 9 V. or an epitope comprising an amino acid sequence GHGAEDS tSEQ ID NO:4); or an epitope comprising amino acids HfDT (314.} ID NO: 12); or an epitope comprising amino aeids Dlf'DA •(SEQ ID NO; 15). or an epnopc comprising amino acids AtiDS (SEQ IP NO; DO or an epitope comprising amino acids AFDT ($if*2 ID NO; Id); or an epuope comprising ammo acicD TEOE i'SEQ ID NO: In); or an epitope comprising die arnino acid sequence AsiPV (SEQ ID NO. 2a-5; or an epitope comprising the amino acid sequence SLDF (SEQ ID NO; 24) or PE DP t S if Q ID NO. deg ο- an epitope of comprising an ammo sequence selected from the group consisting of FEDS i'SEQ HD NO: efffi REDE (SEQ ID NO: 27), TEDV (\| Q ID NO, 20. AlfDI: i'SEQ ID NO; I9h SEDI (SifQ ID NO' 20 ary) !EDI SEQ ID NO: AO); or art epitope comprising an amino sequence selected front the group consisting of 1.EDO (SEQ ID NO. 3 0, AEDM tSEQ ID NO: 32g HEPS (SEQ ID NO; 33), CEDD (SEQ ID NO 34). QEDS (SEQ ID NO' 35), REDS {SEQ ID NO. >6). TlfDO (SEQ ID NO. Ιό), QEDR (SEQ ID NO: 33), TEDE tSEQ ID NO; 37), PEON iSEQ ID NO' 40), El:DP (SEQ ID NO; 4 0. LED!. (SEQ ID NO: 32). K.EDA tSEQ ID NO; 43), SOX' (SEQ ID NO: 44), EEDD (SEQ ID NO: 43), SifDK (SEQ ID NO; 40), DEDD i'SEQ ID NO: 40, DI'DO (SEQ ID NO: 13). LED if (SEQ ID NO. 4'D, Gif DA (SEQ ID NO: 13), M:DF (SEQ ID NO: 50, YEDE (SEQ ID NO: 52). HIDE (SEQ ID NO: 53), WEDY (SEQ ID NO: 54), DI'DW sSEQ ID NO: 55), SE.DL (SEQ ID NO: Dm 3 FDQ (SEQ ID NO. 5 0, LEDW (SEQ ID NO; 53), Y If DR (SEQ ID NO: 59) :md PlfDK (SEQ ID NQ; 60). T he antibodies and andgen-bnajum fragments described herein include those that hind to the amyloid protein in monomeric form with an affinity of Dm than about HI M Represen s a) i sc amyloid proteins include scrum amyloid A protom tSAA), iromtmog.lobulin hgh) chain protein tsueh as Yob Wit and YV>. human Edet amyloid precursor pot;·peptide GAPE), beta amyloid neptidc, transthyretin (TER), and ,ΑροΑ l.

Also provided arc isolated nucleic adds encoding an antibody, or antigen-binding. fragment thereof, which specifically binds to an epitope comprising X:EDX-: in an aggregated amyloid protein., wherein X; and X3 are any amino acid., including all such antibodies and antiuen-hindinu fragments as described herein above and as set forth h) the claims. Further V. V- W 1 . . provided arc ceils expressing such nucleic acids.

The prefer;· invention hardier provides nieihod··· of therapeutically heating or prophylacticaHy treating a subject having AT amyloidosis using a he men., humanized, or chimeric antibody, or andgtm-bhidhig fragment thereof, hint .specifically binds to tin epitope within residues 70~76 oihuman amyloid A peptide, for csample, an epitope widen residues 7(5-7o of SF.Q ID NO: .7. Subjects that may benefit from the disclosed dterapeuiie mo:hods of tresume. ΑΛ im y kudos i-, include those subjects sufic ring from an amyloid disease selected from dm group con si <u soy of rheumatoid end rids, juvenile chronic arthritic. ankylosing spondylitis, psmiaDs, ρ.sonside arthropathy, Reiter's syndrome, Aduli Sill;’·' disen.se, Boheet's syndrome, X'mhrfs disease, leprosy, tuberculosis. bronchiectasis. decubitus ulcers, chronic pyelonephritis, osteomyelitis. Whipple's disease, Hodgkin's lymphoma, renal carcinoma, carcinomas of gish lung ansi uroy.enmd tract, basal cell carcinoma, hairy eeU leukemia, Familial Mediterranean 'Fever, arid Cuadernan's Disease. Subjects that may benefit from ihe disclosed prophylactic methods include those subjects susceptible to or at risk of' developing any of the foregoing disorders.

Also provided are methods of therapeutically treating or prophvlaaiccdly treating a subject having amyloidosis associated with an aggregated amyloid protein comprising the amino acid sequence ED using an antibody or antigen-binding fragment that specifuaily binds to an epitope comprising X{EQX> in an aggregated amyloid protein, wherein Xt end .V are any amino acid Subjects that, may be-iebt from the disclosed therapeutic methods of treating amyloidosis associated with an aggregated amvlmd protein include those subjects suffering from Ah amyloidosis. AL amyloidosis, Alzheimer's disease, Mdd Cognitive impairment, amyloid ipolyneuropathy. Mediterranean fever, Muakie-Weils syndrome, reactive systemic amyloidosis associated with systemic inflammatory diseases, .myeloma or niacroglobcd in cm la associated amyloidosis, amyloidosis associated with immuriocyte dysemsia, monoclonal gamotopathy, occult dyserusiu, and local nodular amyloidosis associated with chronic inflammatory diseases. Subjects that may benefit iron'; the disclosed prophylactic methods include those subjects susceptible to or at risk of developing any of the foregoing disorders. In one aspect of the invention, the amyloid protein comprises the sequence Ab'DV (isEC) ID NO: 11 h and the amyloidogenie disease treated therapeutically or prophylaerieally using die disclosed methods is A A amyloidosis. AL amyloidosis,, amyloid polyneuropathy. Mediterranean fever, Muckic-WoOs syndrome, reactive systemic amyloidosis associated with systemic inflammatory diseases, myeloma or macrogiobtdinemia associated amyloidosis, amyloidosis associated with: immunoeyte dyserasia, monoclonal gammopathy. occult dyMrasla,: .a&amp;di ideals rtodnl&amp;r .amyloidosis'' associated with chronic hulammatory diseases.

The disclosed therapeutic and prophylactic methods are useful tor treating human subjects.

Repre-wn-atsv, indices of efficacious ihempe-tfie treatment nu.hide slowmg the pro pres moo of amyloidosis inhibiting deposition of amyloid fibril age regal Ov artd or clearing of amyloid fibril aggregates Representative indices of efficacious prophy bam meatrw.n! include delaying onset of amyloidosis anchor redt-eiug u risk of amyloidosis.

Still further provided are methods of defecting an amyloid deposit associated \\;th ΛΛ amyloidosis in a subject human,, htsmamceci, or chimeric antibody, or anugca-bmdmg fragment thereof, that specifically binds to an epitope widest residues 70- » of human amyloid A peptide, which end body or anhge-i-btttdiag fragment is bound to a detectable label, and then detecting the detectable label In the subject. Additional methods comprise detecting an aggregated amyloid protein eon-prising the amino acid sequence liiD using an antibody or antigen-lundtng fragment that spedhealh lands to an epitope comprising X<Lf>X: iu an aggregated amyloid protein, oin.-rem Xf and .v- arc any amino acid. The foregoing detection methods -nay be used, tor •example, for monitoring onset or progression of disease or therapy in any of the above-noted diseases and disorders. -\s for the treatment, methods disclosed herein, such monitoring may be performed m humans as well as non-human .subjects. Useful detectable labels include radiolabels, such as ‘ " I. In performing, such detection methods, the step of detecting the detectable label may be accomplished by nou-hivsMve techniques, such as SPECTCT imaging and NivIR spectroscopy

Still further ptosklcd are methods of active immunotherapy of a subject having ΑΛ amyloidosis using an agent that induces art immune response to residues 70-76 of amyloid A peptide effect;*.e to induce an immune response comprising anybodies against residues 7(1-76 ot an amyloid A peptide. Representative agents for inducing the immune response ineiude residues 70-76 of amyloid A popsidc or a subfragmem of at least 3 contiguous residues thereof having fewer than .70 contiguous ami.no acids front an AA peptide These methods are userid both therapeutically and/or prophytaehcaily for treatment of the subjects described herein above with respect to passive immunotherapy, ms, by administering au antibody or antigen-binding fragment that specifically binds to residues '-0-76 of amyloid A peptide. Indices of therapeutic and prophylactic efficacy are also as noted herein abuse with respect to passive immunotherapy.

Tire ioregolng .summarizes purnetd&amp;r aspects: of the thAeMiott, Md addltsonal aspects of the invention arc described herein,

BRIEF 0Β8€ΕίΙ5ΊΊΟΝ OF THE DRAWINGS

Figure I; Sequence alignment of human SAAB human SAA2. human SAA 3 un>j human SAA4,

Figure 2: Sequence alignment of human SAA 1 and human A A i.

Figure 3; Sequence ulignmefu of human SAA2 and human A A2.

Figure i: Sequence a ligntoom t of human SAAi and human Λ AS,

Figure 5. Sequence aligmneri· ofhntusn SAA4 and hHuman ΑΛ4.

Figure 6; Sequence alignment of human ΛΑΒ human A A2, human A A 3 and human ΑΛ4.

Figure ?: Sequence alignment; of die last seven AA?, human Λ AS and human AA4.

Figure A; Seqncneo alignment of mouse SAAB mouse SAA', mouse SAA? and mouse

SAAB

Figure ?; Sequence alignment of mouse SAAi and mouse A A1 Figure 10: Sequence alignment of mouse SAA2 and mouse A A'2.

Figure 1 1; Sequence alignment of mouse SAA 3 arid incase ΛΛ3.

Figure 12; Sequence align mem of mouse SAA4 and mMousc AA4.

Figure 13; Sequence alignrnenl of mouse AA B mouse AA?, mouse ΛΑ3 mau.se AA4. Figure 14; Sequence alig.rmi.erii of the last .seven residues of mouse AAL mouse AA2S mouse A A3 mouse AA4.

Figure IS; Sequence alignment of human SAA I and mouse SAAB Figure 16; Sequence alignment of human AA 1 and mouse AA i.

Figure 17; Sequence alignment of human SAAI and mouse SAAi .Fragment Figure IS; Sequence alignment of human SAA I alpha, human SAAI beta, and human SAAi -gamma.

Figure Bf Sequence alignment of human S A Λ'2 alpha and human SA \2 Feta.

Figure 20; Sequence comparison of SAA proteins. The peptide region used to generate 2.-34, ><Gv and 7D8 is shovvu in dashed lines. The S amino acid insert be;ween post-tons 67 and 6K in the Shur Pei sequence is indicated by the underline and arrow Alignment performed with eLOTALW.

Figure 21: Germline sequence^ of Yk light chains.

Figure 22: uermline sequences of V>, light chains.

Figure 23: Amino acid sequence of VA.6 Wii.

Ffpre24: AAgy crystal of VX6 Wil showing position of GlPCFAsp U

Figure 23: X-my crystal of V&amp;b Wil show ing position of Glubl -Aspki!

Figure 2tr Binding kinetics of film rnAbs to synthetic V>.« Wii rthriB. BlAcore roeasu-emems of the interaction of mAh* 2Λ4, 7D8 and 8(39 at ο ο ηM to immobiimed Υλ.6 Wd "fibrils. 'The cnkulatod TCP for each interaction eras - l nM

Figure 2?; Concent ration-dependent binding kinetics of mAh 7D* to synthetic \'}j> Wii fibrils. The antibody inieraciion at a. concentration of 6.6 - 33,3 «Μ to immobilized Υλο Wil fibrils von measured by BlAeore,

Figure 28: Binding kinetics of mAh 703 to synthetic V;6 Wil fibrils in the presence of d?e pib) and p4l peptides Tbe interaction of the mAh ~T>8 at 6.6 nM with immobilized V#6 Will fibrils was measured by BlAeore in the presence oi peglidcs p39 arid p41 at 1 or 20 gg'ml.

Figure 29: React ivttv of monoclonal antibodies with Αί.λ tissue amyloid deposits. . . . . .. . ., . . AS' . , ...... . ...... .....

Figure 3ft: iSkPstrlbyiioa of lAPeled. mAh ?ΪΜ ia ogee bpaiiug a itunifu ΑΒχ arrty ioidoma.

Figure 31: interaction. of atuFAA of culture supernatants with murine-derived A.A iibrils. Results of mAb culture supernatants binding murine ΛΑ ACF. Upper and lower panels are data on first and second culture thud harvest, ru-spcctAely.

Figaro 32: SDS-PAGF. analysis of protein A-ptai fied 2 A 4. KGv and 70s rnAbs.

Figure 33: Binding of purified rnAbs to immunising \pp9> and control peptide tpP 1 g

Figure 34: Binding to murine A A amyloid extract (AFF>.

Figure 35. Binding of purified rnAbs to human renal A A amyloid extract.

Figures 36A--36E: Sequences of murine 2ΛΑ 7D8„ and 3(39 light chain and heavy chain variable regions tFigure 36AF sequences of htrmasuxed 2A4.8G9 and 7D8 light chain variable regions (Figures 36B-36C}; sequences of human light chain variable regions used as acceptor frames orks (Figure 36D}; sequences of humanized 2/44. 703 3(39 heavy chain variable regions and human heavy chain variable region used as acceptor framework {Figure 36£). Underlining, CDRa; double .mderlitsiog, leader sequences; lower case, back mutations.

DETAILED DESCRIPTION OP T111:. INVENTION

Vhc invention provides *tn isolated antibody or antigen-binding fragment thereof. which speeiScaHy binds to an epitope whereto X;i and Xa arc any umino add.

Representative antibodies cu the invention also include antibodies or tnignwnta rncreof that {a} con-pel·:: for binding kj an epitope including X‘;EDX: with a 2 Ad. ’’DK. or h<39 antibody; (b) bind to the same epitope including X;EDX·: as &amp; 2ΛΧ Έ>8, or KGD .antibody, let include an .antigen binding domain of a 7 A-L "DK. or x&amp;} antibody; or id) include she ox complementarity detonuimrig regions (CDRs) of a 2A4, 7DN or KG9 antibody*

The Invention α I set provides ;tn isolated antibody variable rcg'ion including - a) a light chain variable region of an antibody domed from a 2Λ4, TDK, or 8G*> antibody.; or lb) a heavy chain variable region of an antibody derived from a 2 Ad. TDK. or KCD antibody.

The invention also provides an isolated nucleic acid encoding att antibody light chain variable region or heavy chain variable region including la) a nucleotide sequence that encodes a light chain or heavy chain variable region of a TDK, 2.A4, or Kl.X antibody; ibo a nucleotide sequence that is identical to a nucleotide secfnenee of a ?Db, a 2Ad, or an kG9 antibody that encodes: &amp;. light cMip: or heavy admin- vaneble: regipn; feT a .iin&amp;feofM&amp;: sequence: that he .substantially identical to a nucleotide sequence of la) or (b); or Id) a nucleic add that specifically hybridizes to a nucleic acid having a nucleotide sequence that h; the complement of a nucleotide sequence of (as or fbs under strings”!! h\ b; d-amion -, oodusoas.

Cells expressing the .monodies and amigumbinding fragments of the present invention are also provided The insomiou ftsrrber prov ide\ cells expressing immeic adds of the invention

The invention also includes methods os treating amyloid diseases and osethods of prophylaxis of amyloid diseases using the antibodies and a msec η dm; dm a fragments of the invention, ('unct-th, there me sm approved specific umvloid-direeied trennnenis he any of the amyloid diseases, including ΑΛ Amyloidosis and XL ;.un>loldosts Λ\ν Gill mime j D. e* ah, hmssw 75h.7h-° {700S). Whete there is an under]} mg or us.soctnWd disease -tate, therapy directed towards decreasing the production of sms lomogomc promin by treating the under!} ing disease l or os ample, current ireatmettt strategy lor A T \myloidoms X to target underlying hhiammmmw reducing ApoSSA levels to below lOntgl. Currently employed dwmpics melude chemotherapy ieholorantboeii and MIX), isnmifno-sifppressama la/athioprine). antiinflammatory drugs teoidsicmes and l'N|- inhibitors. The intention provides pharmaceutical compositions and nieshods for treating a msmbvr of amyloid diseases, Including amyloidosis, such as, tor usasnpie, A Λ amyloidosis and AL amyloidosis According to one aspect, the tnvemion includes pharmaceutical composiiirms that include, us an hcsssc sngredtestt, an anent that is effect is e to Induce an Imrmme response in a paticn; against an amyloid i.ouipoucnt Πκ-agent cun he a peptide comprising a tVaemem consisting of the ammo acsd v.-mu-s'ce XjbDX.' derived from an amyloid protein The agent can be an and body dun sp^.-meaHy binds to as? epitope comprising X;M)X s in other embodiments, the ayes?? cus? be an amtgen-bmdmg fragment of an antibody. SnJn vompositions sc id gesiendiy also include c\eipients ?md in preferred embodiments mas utehidc adjuvants. !n further pseferred embodiments, site ad:·as mas-, include, for example, aluminum hydroxide, a dims π cut; phosphate. Mhfy'k OS- d I (ΒΤΙνΠ.Η.ΟΝ A\s or incomplete fYo'undX adjuvant. According in a «ehied cnibodtiueni, such phannaeeitticul compositions may indude a pkm.ddy cd'«perns effective so induce ;m Imroum espouse against more than one amyloid component is? the patios??.

In a rdsued embodiment, the agent is effective to produce an imtmmc response directed against as? aggregated amyloid protein, such as a fibril peptide or pnMes'n amyloid a>nvpone;;i. Pscierably, such a fibril peptide or proieiss is devised bran a t'lhsil pseeresor protein know?· to be associwed with certain forms of amyloid diseases, as described herein. Such precursor proteins Inc.inde, but are not bruited to. Serum Amyloid Λ protests tApoSSA). imrnonoglobnlin light chain, immimoglobuiiit heavy chain, A po A I, tmtisrhyretin. lysoey me, ttbtogen o: oka is?, gclsosm, eystatin C, Amyloid jk protein precursor t P--APP), Beta,. rucroglobuhn, priori psecumor process? (FrPb aired no trim-one factor, kendo?. islet usnyioid polypeptide, a peptide hormone and synneloin. Such preevsrsors also include mutant proteins, proses·! ifugmosits and proteolytic neplides of .sneh precursors. in a preferred erohndimesrt, the agent ss eHcesivc io mduce an immune re upon ο.- directed against a ueoepitope iosnsed by u fibril protein or peptide, wish respect to a fibril precursor promts? shut is. us described is? more detad herein, many fsbrd-fortnhsg. peptides s?r proteins sax fragments of stseh precursor prorems. sneh as those listed above, When such in; go scans are formed, sneh as by pndcoSyiic cleavage, epitopes may hi.-, revealed his? are cot ρrescue on the precursor and are therefore not immunologieulB u\ niktbiv to the ητηιηηη-system vvkc;? hie tmgnumt is t part of the preeursus protem. Ago;?is din,·used ns stn;h epitopes may be preferred therapeutic age??is, since they may be less likck so induce as? autoimmune response in the patient. Preferably, .such agents preferentially pmd tsce an immune response directed against a pathological form of the amyloid protein, for example, an aggregated amyloid protein, relative to nonnadK.dogk.ai forms of the amyloid protein.

According to a related embodiment, pharmaceutical compositions of the invent ton include agents directed to amyloid aggregates, such as those selected from the group including, but not limited to the following aggregated (c.g., fibril) peptides or proteins A.A, At. ATTR, AApoAf. Alys, A gel. Acys. Aj$, ABdvf AScr. Aeaf. At ARP and synuclcro-NAC fragment, Fite full names and compositions of these peptides are described herein. Such peptides can be made according to methods well known to the art, as described herein.

The methods comprise administering to the patient an effective dosage of an ami body that specifically binds to an epitope comprising X»0>X.< to an amyloid protein, wherein X- is IT T, F, S, P. A or any other amino acid residue immediately preceding ED in such amyloid protein; and wherein X.< is T, S, E, R, I, V, F, A or any other ammo acid residue immediately to!lowing UD in such amyloid protein. In some methods, the patient is suffering from an amyloidosis associated with an aggregated amyloid protein comprising the amino acid sequence ED. Some antibodies specifically bind to an epitope consisting of such X}ED.\X In some antibodies. X: is IT. T. f. S, E, or A :md X.· Is T, S, E, D. R, 1.. V, F or A In some such asuibodies, when X» is H, XX is T or A; when X; ts A, X? sa S, Γ, If or V; when X; ts T, X; is E; when X.j is F, X; ts D; when X· is S, X: is F. F or A; and when X, is F, X> ts E, I or F. In .-,onto antibodies. Xi is Η. T. F, S. IF or A and X- is 7. fib 1:., D, R, L V, For A. with the proviso that if X» is A, XX is not V. In so;no antibodies, when X, Λ, X,. is S, 7 or E.

Some antibodies spec; flea I Is bind an epitope composing the amino acid sequence (.ill EDI., t SFQ ID NO: 3F I1EDT, MEM ID N()· 17) Λ EDS, <;SFQ ID NO 13k -31)07 tSF.Q ID NO' ED. HED.A tSFQ ID NO: )51. TEDE, iSEQ ID NO' 16), FEDD. (SFQ ID XO, P), Sfr.DE. (SEQ ID NO: I hi. AFDE. tisFQ ID NO- IM, FFDE. (SFQ ID NO. 70), PEDl, iSFQ ID NO' 31 k PFDF, fSEO ID NO; 23», ,\FDY. {SFQ ID NO: .235, SF.Df ;SH) !D NO: IMF or SEDA. iXHQ ID NO: 251.

Some antibodtes spec Hi,; a by bind to a pepude composing an amino acid sequence selected front me group consisting of 0F1EDT, t$EQ ID NO: 3F HFDT, (SEQ ID NO; 12k AEDS, (SFQ ID NO. i X». Λ IDT. t SFQ ID NO; Ms, Hi) DA, {SFO ID NO; 15k 7 EOF. tSEQ ID NO: 16k FF.DD, tSFQ ID NO: ]?}, SFDF., (SEQ ID NO: MF AFDF tSFQ ID NO: IFF PEDF. :{Sf:.Q ID NO. 20). PEDI. (.SEQ ID NO: 21). PE 1.3b. (SEQ ID NO: 22), SEDF, (SLQ ID NO: 24), .«id SEDA, (SEQ ID NO' 25). Some and bodies spceificaii) hind ιο a peptide comprising -ass anmu > acid sequence xdecied from she group emiMrine os’ Cd i.EDT, (SEQ ID NO. 3, NEDid i'SEQ ID NO· I2i, AEDS, iSEQ ID NO: I3E AEDΓ, (SFQ ID NO: M), Ft EDA, (SEQ ID NO. 15). and TED!?., (SEQ ID NO: :b)

Sonic antibodies .specifically bind to an epitope within residues 70 to 76 of AA. Some .antibodies specifically bind to an epbope v.i?lim residues "I to 33 of AA. Some uniibodies arc raised to a peptide comprising GHE.DT, ($£Q ID NO: 3s.

Some antibodies specifically bind to a peptide com pr is mg the amino acid x:q nonce P 1:138. iSEQ ID NO: 2M. PEDI., iSEQ ID NO: 27). 11:230 (SEQ ID NO: 2U. Λ 1:.1..)1:2 (SEQ ID NO: U). SF.DE {SEQ If) NO: 29), and TEDT, (SEQ ID NO: 30) Some antibodies specifically bind so a peptide comprising the amino acid sequence LUXE iSEQ ID NO: 3 i).. AEDA1, tSEQ ID NO: 32), REDS. {SEQ ID NO. 33). ¢1:.013. (SEQ ID NO; 3-1), QFDS, (SEQ ID NO: 33). REDS. (SEQ ID NO: 36). TFDG. (SEQ ID NO: 3D, QEDR, (SEQ (D NO: 3U. TEDL. (SEQ ID NO: 39). PEON, iSEQ ID NO: 40), EE DP. (SEQ ID NO: 11). LED!., (SEQ ID NO: 12). KU3A. (SEQ ID NO; 43». SEDC. (SEQ ID NO; 44». EEDD, (SEQ ID NO; 432 SEDA, (SEQ ID NO: 46). DEDD, (SEQ ID NO. 47), 1)11)0. (SEQ ID NO; 12}. I.EDI:.. (SEQ ID NO: TO, GED,\. (SEO ID NO: 50), V HDF. sSf.Q ID NO: 5 0, YEDE. (SEQ ID NO. 52). I EDI.. (SEQ ID NO, 5.3V, WFDY, (SEQ ID Mr 54), DEDW. (SEQ ID NO' 53), SEDL, (SEQ ID NO: 5U.· YEDQ.; (SEQ ID NO: 50, I.EDVv, (SEQ ID NO: 52). YE DR. {SEQ ID NO: 59). and PEON. (SEQ ID NO: 60).

Some antibodies specifically hind :o a peptide comprising (he amino acid sequence AEDV, (SEQ ID NO: 23) Some and bodies .specifically bind to u peptide comprising the amino acid .sequence SEDF, (SEQ ID NO: 24} or PEDF. (SEQ ID NO 22) Some antibodies .specifically bind to a peptide comprising the amino acid sequence AEDS. (SI3Q ID NO: 13). Some antibodies specifically hind to a peptide comprising die amino acid sequence PEDI (SEQ ID NO; 2D, AEDV, iSBQ ID NO 23). SEDF, (SEQ ID NO: 24), SEDA. (SEQ Hi) NO: 25). SEDE. (SEQ ID NO: IS), AEDE, (SEQ ID NO 19). and PEDE, (SEQ ID NO: 20). Sonic antibodies bind to a peptide comprising (he ammo acid sequence TEDE, {SEQ ID NO: 16).

Some antibodies sped Heally bind to a peptide comprising the ammo acid sequence AEDV, (SEQ) ID NO: 23 s. Some antibodies specifically bind to a pepiidc comprising the amino acid sequence SEDF, (SEQ 113 NO: 24) or PEDF. (SEQ ID NO: 22). Some antibodies specifically bind to a peptide comp; mm a the ammo actd seque-see Λ EDS ¢81:4) ID NO: EG. Nome amibodice specifics!iy bind ?o a peptide comprising rite amnio acid sequence FED! (8EQ ID NO' 2D, AF.DY, iSEQ ID KO 2d}. NEDE, (SCQ if) NO' 2-1), SEDA, iSFQ m NO' 25). SFDIN W.Q ID NO' 18 k A(:DIE (SB) H) NO. (-). and PEDE, iSFQ (0 NO. 20} Souk· antibodies bind ro a peptide comprising the amino need sequence TEDF, (810 ID NO: 16}

Am. of the antibodies described ,υχη-e tan be administered m ihc methods described above to (real or effect prophylaxis of a disease e ha on: ter Erect by ihc deposition of an amyloid protem, such as, for example, an amyloid protem composing (ho amino acid sequence E.0 hi some methods, if the amyloid protein comprises -be amino acai sequence Af.D V, tSEQ I id NO. 2.0, then the antibody is no( administered to (reat. or el fee· prop by hods of 81 rite oner's du-em-e or Mild Cognitive bn pat ms cm. The amyloid proloin can be any of so mm amyloid Λ protean. Inmtunoglobalm light chain protein, such as, for example, VXo \\dI or Yn, human islet amyloid precursor polypeptide ί ΙΛΡΡΚ beta amyloid peptide, transthyretin ΠΎΚ) or ApoAl.

Optionaliy, the patient is human. Optionally, the «nobody memfiosHy binds to .¾ peptide whose residues consist of Sbf) ID NOS. 4, 5, 6, y 8, 9, ;0, or It. Optionally, me amibody speeiilcaliy binds to an epitope wchin residues 8()-76 of (8f () Ui> NO: cl. Optionally, the antibody is a human antibody, humanised antibody or chimeric antibody, Optionally, the human amibody is of human isotype IgGF. IgGd. DG2 or IgG 8. Optionally, the human iced antibody is of human isotype Ig.G I, ig.G4. lgG2 or igG3 Optionally, the chimeric antibody Is of human isot>pc IgGF lg.G4, IgG 2 or mile, Optionally, the antibody is a mouse antibody Optionally, the antibody Is a polyclonal antibody. Optionally, the antibody is a monoclonal antibody.

In some treatment methods, the antibody comprises two copies of the same pair of light and heavy chains (n other methods, the antibody is a bixpeeifie antibody comprising a first light ami heav y chain pair that spec!beady binds (o the epitope of All end a second light and heavy chain pair that speeiilcaliy hinds to an re receptor on rmcmcmd cells. In other methods, a chain of the antibody is fused to a heterologous polypeptide

Some irentmeni methods, ihc dosage of antibody is at least 1 mg 'kg body weight of the patient. 1ft other methods* the dosage of and hods is ai least 10 rng. kg body weight of the patient.

In srime meatmen! method's, the antibody Is administered with a carrier as a phaimabowiidal.eoroposirion. In other methods, wherein the amibody Is g human antibody to AA prepared from B cells from a human immunized with an A A peptide. Optionally, the human immunized with AA peptide is the patient. In some methods, the antibody is administered intraperitoneally, orally, intranasally, subcutaneously, intramuscularly, topically or intravenously.

In some treatment methods, the antibody is administered by administering a polynucleotide encoding at least one antibody chain to the patient and the polynucleotide is expressed to produce the antibody chain in the patient. Optionally, the polynucleotide encodes heavy 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 dosage 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 of the patient. In other methods, the antibody is administered in multiple dosages over a period of at least six months. In other methods, the antibody is administered as a sustained release composition.

The invention further provides methods of effecting prophylaxis of AA amyloidosis in a patient susceptible to A A amyloidosis. The methods comprise administering to the patient an effective 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 peptide whose residues consist of SEQ ID NOS. 4, 5, 6, 7, 8, 9, 10, or 11. Optionally, the antibody specifically binds to an epitope within residues 70-76 of (SEQ ID NO: 2). In some methods, the patient suffers from an underlying amyloid disease 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, renal carcinoma, carcinomas of gut, lung and urogenital tract, basal cell carcinoma, hairy cell leukemia, Familial Mediterranean Fever, and Castleman’s Disease.

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

Optionally, the humanized antibody is a humanized version 7D29 antibody. Optionally, the humanized antibody is a humanized version 7D19 antibody. Optionally, the humanized antibody is a humanized version 7D47 antibody. Optionally, the humanized antibody is a humanized version 7D39 antibody. Optionally, the humanized antibody is a humanized version 7D66 antibody. Optionally, the humanized antibody is a humanized version 8G9 antibody. Optionally, the humanized antibody is a humanized version 8G3 antibody. Optionally, the humanized antibody is a humanized version 8G4 antibody. Optionally, the humanized antibody is a humanized version 8G51 antibody. Optionally, the humanized antibody is a humanized version 8G22 antibody. Optionally, the humanized antibody is a humanized version 8G30 antibody. Optionally, the humanized antibody is a humanized version 8G46 antibody. Optionally, the humanized antibody is a humanized version 2A4 antibody (ATCC Accession Number 9662). Optionally, the humanized antibody is a humanized version 2A20 antibody. Optionally, the humanized antibody is a humanized version 2A44 antibody. Optionally, the humanized antibody is a humanized version 2A77 antibody. Optionally, the humanized antibody is a humanized version 2A13 antibody. Optionally, the humanized antibody is a humanized version 2A14 antibody.

The invention further provides pharmaceutical compositions. The pharmaceutical compositions comprise an antibody that specifically binds to an epitope within residues 70 to 76 of AA, and a pharmaceutically acceptable carrier. Some pharmaceutical compositions comprise a human, humanized, or chimeric antibody that specifically binds to an epitope within residues 70 to 76 of AA, and a pharmaceutically acceptable carrier. Other pharmaceutical compositions comprise an antibody that specifically binds to an epitope within residues 70 to 76 of A A and a pharmaceutically acceptable carrier, where the isotype of the antibody is human IgGl, and a pharmaceutically acceptable carrier. In some pharmaceutical compositions the isotype of the antibody is human IgG2, IgG3, or IgG4. In some pharmaceutical compositions the antibody is human. In some pharmaceutical compositions the antibody is humanized. In some pharmaceutical compositions the antibody is chimeric. In some pharmaceutical compositions the antibody is a polyclonal antibody. In some pharmaceutical compositions the antibody is a monoclonal antibody.

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 bispecific antibody comprising a first light and heavy chain pair that specifically binds to the epitope of AA ami a second hgju arid heavy chain pair that specifically binds ίο an lx receptor cat microglial cells. in pharmaceutical compositions a chain of the antibody in fused to a lteteroU>goa.> polypeptide. In sot no pi tar? naeemicai compositions the carrier is a physiologtctdly acceptable diluent for parenteral administration. Some pharmaceutical compositions are adapted to be . administered i-itmpernoneaiiy orally. iurranasaity. subcutaneously frUramuseularlv, topically or intravenously. Some pharmaceutical compositions are adapted to be administered in multiple dosages over a period of a? least six months. Some pharmaceutical compositions arc adapted to be .administered as a sustained re.leu.se .cot.npo.siti.an. Some phan.naeeuti.eal compositions further comprise at least one other antibody that binds to a different epitope of AA.

The invention provides methods of treating ΛΛ amyloidosis in a patient. The methods comprise adto to (storing an agent that induces an immune response to ΛΑ70-76 in a regime ci’fectivo to induce an immune response comprising antibodies against Λ.Α70-7Ρ in a regime effective to induce an immune response comprising antibodies against A A70-76. In some methods the patient is human Optionally, the agent comprises AA70-76 or a subf-agment of at least 3 contiguous residues thereof ami has fewer than 20 contiguous amino acids from an A A peptide. Optionally, the agent is a peptide havrng a sequence selected iVom the group consisting of SHQ ID NOS 4, 5. (\ 7, 8, 9, 10 and 11. and subfragmeols ol at least 3 contiguous residues thereof and has fewer than .:0 amino acids from an AA peptide. Optionally, the agent is linked at its N and C term in t to first and second heterologous polypeptides. Optionally, die agent is linked at its N terminus to a hctefologous polypeptide, and at its C-terminus to st least one additional copy of the N-teronnul segment. In some methods the heterologous polypeptide induces a T-cdl response against the heterologous polypeptide and thereby a B-cell response against AA. In some rneth.od.s the polypeptide farther co-nprises »t least one additional copy of AA. Optionally, the polypeptide comprises from N-terminus to C-terminus, AA, a plurality of addirtonal copies of A A, ;.mu the heterologous ammo acid segment.

In some treatment methods the polypeptide, is administered with an adjuvant that enhances an Immune response to the N-termmtd segment. Optionally, the adjuvant and the polypeptide are administered together as a eon-position. Optionally, the adjuvant is administered before the polypeptide. Optionally, the adjuvant is administered utter the polypeptide in some methods the adjuvant is alum, in some methods the adjuvant A MPL. in some methods the .adjutant ss OS-21. In some methods the adjutant is incomplete Freund's adjuvant. In some methods the immune response comprises T-ceils that bind to die AA peptide as a component of an MHC 1 or MHC Π complex.

The invention provides methods of effecting prophylaxis of ΛΛ amyloidosis in 4 patient The 'methods cos uprise admin inning an agon; that induce** an immune response u* ΑΛ70-76 in a regime effective to induce an immune response comprising antibodies against A A 70-76 in a regime effect t\ e to ntdnce an immune response comprising antibodies against AA70-76. in some methods the patient is human. In some methods the patient is asymptomatic, tn some methods the patient suffers front an underlying amyloid disease selected from die group consisting of rheumatoid arthritis, juvenile chrome arthritis, ankylosing spondy litis, psoriasis, psoriatic arthropathy, Reiter's syndrome. Adult Still's disease, Behcet's syndrome, Crohn's disease, leprosy, tuberculosis, b-onehioctasts. dees Situs ulcer·', chronic pyelonephritis, osteomyelitis. Whipple's disease, Hodgkin's lymphoma, renal carcinoma, carcinomas of gut, lung and urogenital trad, basal cell carcinoma, hairy cell leukemia, Familial Mediterranean Fever, and Casdcntun's Disease, h> some methods of effecting prohylasts, die agent eotnriri-.es Α.Υ70-Ά or a subfrugmem of at least s eotPtguous residues thereof and has fever than 20 contiguous amino acids front an AA peptide. Optionally, the agent is a peptide having a sequence selected front the group cooHshug of SL7Q ID NOS 4. 5, h, 7, S. v. 10 and U. and sub fragments of at least ? contiguous residues thereof and has fever than 2.0 amino acids from an Λ A peptide. Optionally, the agent is linked at its N and C termini to first and second heterologous polypeptides. Opuonaliy, the agent is linked at its N" terminus to a heterologous polypepode, and at Its C-tenuinus to or least one additional copy of the N-termhtai segment, in some methods the heterologous polypeptide induces a !-cell response against the heterologous polypeptide and: Thereby a B~cell response against AA. In some methods the polypeptide further comprises at: least one additional copy of A A. Optionally, the polypeptide comprises from N-ternunus to C-icrurmus, Λ A. a plurality of additional copies of A A. and the heterologous arm no acid segment.

The invention further provides pharmaceutical compositions. The pharmaceutical compositions comprise an Λ.Λ fragment consisting of residues beginning m ms fine 70 of Λ A and ending at residue 76 of AA, Optionally, the AA thtgmeai is linked at Its C-terminus to a heterologous polypeptide. Optionally, the AA fragment -is linked at its Nhtermions to a heterologous polypeptide. Optionally, the AA fragment is linked at its N and F termini 10 Urst and second heterologous poUpepodev Optionally, the AA fragment is linked at its N terminus o a heterologous polypeptide, and at its C-termlnus to at least one additional copy of the N-tennuu) segment. Optionally, the polypeptide further comprises at least one addition! copy ι.Τ die N-terminal s eg mem. Optionally, the polypeptide comprises from N-term m us to ("--terminus. ΑΑ» a phtndits of additional copies of the No coni rod segment , and the heterologous amino acid segment, In some pharmaceutical compositions the heterologous polypeptide induces a T-ccil response against the heterologous polypeptide and thereby a B-eei! sespouse e gum si the N-tcirnnuil segment.

Sonic pharmaceutical compositions In a her comprise an adjuvant that CiiSaaices at? immune response to AA. Optionally, the ndju\ant is alum. Optionally, the adjuvant is MPL. 'Optionally, the adjuvant -s QS-,'1. Optionally, the adjuvant B incomplete Freund's adjuvant. Optionally, the adjuvant further composes GM-CSfu Optionally, the adjuvant is M-CSK Opttonaliy, the composition comprises greater than 10 nhemgrams of the polypeptide.

The invention provides methods of nvadng. Λ A amyloidosis it; a pan cut. The methods comprise administering an agent effective to induce an immune response against a peptide cotn pot tent of an amyloid deposit in the patient and a Jiff,: mm agent beu treats an underlying disease, and thereby treating ΛΛ amyloidosis in the patient. In munc mediods the underlying, disease is selected Srotn the group consisting of rheumatoid arthritis, juvenile chronic arthritis, ankylovug spondylitis, psoriasis, psoriuue arthropathy, Reiter's syndrome, .Adult Sts 11 s disease, Behcet's syndrome, Crohn's disease, leprosy, tuberculous, bronchiectasis, decubitus ulcers, chrome pyelonephritis, osteomyelitis, \\T;!ppk:.A disease, Hodgkin's Umphoout, renal caretnoma, carcinomas of gut. lung and urogenital tract, basal eel I carcinoma, hairy cell leukemia. Fannied Mediterranean Fever, and (.'as be man's Diem me

The invention provides medtods of effecting ptophyUiMs of A. A amyloidosis in a patient. The methods comprise admire storing an agent vine.five tr> induce an immune response against a peptide component of an amyloid deposit in the patient and .¾ different agent dam neats an underlying disease, and thereby treating A Λ amyloidosis m the patient, in ‘tome methods the underlying disease is selected front the group consisting of rheumatoid arthritis, juvenile chronic arthritis, ankylosing spondylitis, psurnous, psoriatic arthropathy, Re!tor's ondrorne Adult Still's disease, Behcet's synchonu.-, ( mho's disease, leprosy. inboreuiosK bronchiectasis. decubitus ulcers, chronic pyekniepiiohs, osteomyelitis, \Yhtopic's disease, Hodgkin's lymphoma, renal carcinoma, carcinomas of gut. lung and urogenital tract.· basal cell carcinoma, hairy cell leukemia. Faun:mi Mediterranean Fever, and Cnstieman s Disease. the Invention provides methods of screening an antibody for activity hi treating a patient having AA amyiotdosis. The methods comprise contacting the anubody with ΛΛ popude :???d determining whether the antibody specifically binds *ο ΛΛ. specific binding providing -as? utdicaUms that he antibody has activity in treating AA aotvkuubsss.

Thy invention provides methods of screening an antibody lot activity h? clearing a biological e;?bi v physically associated with an antigen. The methods comprise combining tin.; antigen-associated biological entity, the antibody and phagocytic cells bearmg Pc receptors tn κ medium; and monitoring the amount of the ami gen-assotlatcd biological entity remaining in the mediant. a seduction is? amount of die amigen-a-ssoeiaied biological entity so Aceh no the aoubodv has clearing activity a gains? the antigen. In some methods the monitoring step monitors the amount of the antigen remaining in the rricdium. \n some methods the combining comprises adding antigen-associated biological entity to the mediant, and contacting the modems with the phagocytic cells bearing Pc receptors. In some methods the antigen-associated btotogtesd entity is provided as a tissue sample, is? some methods the antigen is the biological entity, in some 'method.- the tissue sample comprises an amyloid deposit Optionally. the tissue satrtpic is front the patient or a mammal having A A Amyloidosis pathology. In some methods, the antigen is AA 1st some methods the phagocytic cells arc microglial cells, in some methods the tissue sample is selected from the group consisting of a cancerous tissue sample, a vitally -elected tissue sample, a tissue sample comprising infhtmmmorv cells, a noos'eaherumr abnormal cell growth, and a tissue sample comprising as? abnormal extracellular matrix

The invention provides methods of delecting an amyloid deposit in a patient The methods comprise administering to the partem an antibody that speciilcaily hinds to an epitope will?in amino acids "0-76 of ΛΛ and detecting presence of the antibody it? the put tern. Optionally, the antibody is labeled. Oprkmaily, the antibody is labeled with a paramagnetic label. Optio???tlly. the labeled antibody Is detected by nuclear magnetic resonance. Optionally, the labeled antibody is detected with SPf'.i" ACT imaging. In some methods, the ami hod) lacks capacity to mduee a clearance response on bmdmg to an amyloid deposit in the patient.

The invention provides diagnostic kits. Use kits eompri.se an antibody shat specific·.?!ly binds to ass epitope with residues 70-76 of A A. Some kits further comprise labeling describing :;.\0 of the antibody lor in v:\o diagnosis ?.?r monitoring of u disease associated wtth amyloid deposiis of AA its a patient in mm?·.- '..snbodisnents, the fits include .mstruetiosm for tt.se of the antibody or anhgest-bntdntg hruytimu thereof ??; detecting ΛΛ,

The ipyefetioa itifther provides a. method of :&amp;gno»mg giayloidosis, in # subject coin prising: kG administering to the subject an antibody or antigen-binding ihignierst thereof that >s bound to a detectable label, wherein die antibody or fragment mcreut sped t Rally hi neb m m. epitope comprising XJiDXj in <m aggregated amyloid protein, wherein X.· and X; are any amino:. ac>d, and ih) 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 V\ amyloidosis,

Purijter provided herein is a method of treatment or prophylaxis of amyloidosis using .an antibody or atstigen-bmdmg fragment thereof, which specifically bn ids to an epitope comprising λ',ΕΡΧ» in an aggregated antyloid prorein, wherein Xj and X. arc any ammo acid

The present invention provides an antibody or antigen-binding fragment thereof that binds specifically to an epitope comprising X > EDX>. in an aggregated amyloid protein, w·herein X( and X,: arc any amino acid. For example. Xd includim I F F, F. S, P< A. 1.,, C, Q, a, E, K, D, G, V, V, l or \V, such as H, i, l·'. S, P, or Λ, or such as H, f, F. or A. χ. me hides Γ, S, F. R, I, V, F, D. A, G, M. L, N. IX C, K, V, or Q, such as Ύ. S. F, R, L V, F, D. or A, or melt as T, S. E, D, or A in other csarnplcs, X; is Η, T. or A and X.: is Γ, S, Γ, or A, such as X· is 1! ot A and X;: is Γ, S, or A in yet additional examples. X? is H and H? is T o; A.; or Χ, is A and X> is S, T, F., or V. such as Xj is A and X; is S. 7, or E. or X; is 1' and X; is E. or Xt is F and X,· is D, or X, is S and X.> is F. r, or A: or X> is P and Xd is E, 1, or F, in parocular, the epitopes include amino acid sequences such as those set forth m SifQ ID NO. 3 through to XFQ IP NO; 25. such as SEQ ID NON: 3. 12. 13. 14, 15, and 16. Additional examples include SEQ ID NON; 4, 5. ”, R and v. such as SEQ ID NQ: 4. Antibodies of the invention that bind to the. epitopes, such as to SEQ ID MX 3. Include the 2Λ4. ?DR and FCsb antibodies.

Fhe aggregated amyloid proteins to which antibodies of the imvntjo» bind are nonmonomeric proteins. Such aggregated amyloid proteins include scrum amyloid A protein (SAA), immunoglobulin light chain protein, human islet amyloid precursor polypeptide 11A PIN Petit amyloid pepiide, transthyretin ΓΓΓΕ F and ApoA 1. such as SAA.

The invention further provides antibodies or armgen-bindiug IragntcnN thereof that, ia) compete for binding to an epitope that includes X;f:T)X; vvirfc a 2A.4, 7(>K, or HGe antibody' ·:b? bind to die same epitope that includes A:EDX.· ns a 2Λ4. 7DF, or bOd antibody; (c) have an aniigen-muding domain of a 2.Λ4, 7DH. or n< A> antibody; or (d> include the six complementarity determining regions (CPRG of a. 3Ad, /DR or PGiS antibody. The invention also provides elimfenc or humanized versions of &amp; 2A4, 70h. or KG') antibody

Represcmivo antibodies. which specifically hind to an epitope that includo.s XjEDX.-x .also include antibodies having at ieaai one. two, or three of the contpiomonturity determining regions «"DRsI of a light chain of a 2Λ-1. 7DK or H7> an!ih«dy, Antibodies of the im ention, which speed!cady bind to an epitope that includes X;ff)X., alsu include antibodies having at least one, wo. or three of the C'DRs of a heavy chain of a 7 Vf 7D>d ot 7(.1/11 antibody CDRs can he idem idee .meordma to methods known 1-¾ the an For example, numbering system.-* for :deunfyiog ODRs are in oomnu'n use. I he Kahnt deft ms so?: 1¾ bused on sequence variability, and the (hothie definition is based on the locution of the structural mop lemons The \bM definition ns a compromise between the Kahm and Cbothia approaches. I lie C'DRs of the light chant variable region are bounded by the residues at positions 7-1 and 34 (C.'DRId ). 50 ;md 5o {ODRd-I. f and So and 4>" < CR>R3-1. t according to the Rabat. Chothia, or .MAI algorithm. According to the Kuhn definition, the ( DR a ot'the homy chant tunable region are Rounded by the residues at pomma is ID and 5 7 R sC ' DR 1-1 Π, 50 and 0? t,( DR741f and ;:':5 and 107 {CDR5-H) tnumbering according m KubaD. According to the < hothtt definition, the CDRs of the heavy chain variable region me bounded by the residues at positions 7f; and 37 f {'DR I-I It. 57 and 5b iCPRe-lit, and o5 and 107 if 'DR 4-11! «numbering according to ( ho tin.at. According m the Ab\| definition, the CDRs of the beam chain s enable tegmn are bounded by the residues at positions 7«< and 35B sCDRMD, 50 end 5-S if DR7-4 Id and R5 and 107 t'CDRh-H) tnumheimg according to Κ.ιΧ,ηι. ,νν Martm et at. (I'JSO) /Vs.. .Von·' V< a./, „vf. C,vt N(V ^767-0777: Murtm <..t a! H;d>ls ,\h t/tOiX /Re: urn o/. 703' 171 153.: Pedersen et ;-J. i RCC? 1 17v and Rees et at «Ι9'·>Μ ht Sternberg MJ.F. fed j. Pm ten- Structure

Prediction. Oxford \ htiv ersiry Press. Oxford, on. Id ΐ -1 u7. the antibodies of the invention further include an antibody that binds ywemeuby to .m epdone comprising X; R.DX; m am ageregtued umykud protein, \xherein X; and X; are any amino acid, having van.tide tegions derived from variable regions of a 7Λ4, ~I>N. or hOv antibody.

Antibodies having variable regions of.;Λ-1, '/DM or f09 ami bodies :nv also included

The antibodies of the invention further include chimeric amilmdic-, human antibodies, humanized antibodies, single chain antibodies, tetntmerte 'antibodies, tettavttieru antibodies, ntu id spec the antdstdms dnm:nrnspeeif:e antibodies, dontain-dcieicd antibodies or fusion proteins.

Fragments of the antibodies of the invention are also provided, The fragments of the invention may be.· Ktb fragment*. Fab' fragment, Ρίηΐνη fragments, |·\· fragments or SeFv fragment1». Such antibodies or fragments thereof can be coupled h »h a cytotoxic agent, ή .radiotherapeutie agent, or a detectable label

The invention also provides an isolated antibody variable region comprising fa) a light chain variable region derived bon- a 7OK -Ad, os %19 a?dibody light chair: variable iegion, or ib 1 a heavy chain variable region derived from a 7DK 2/-)4, or >4)9 antibody tight eham variable region. Isolated variable regions are also provided having a light chain or heavy chain variable region of a ?DK 2Λ4.. or 8G9 antibody. Ί Ite isolated antibody v actable regions are useful in ant (body pro duel ion

The invention also provides isolated nucleic adds encoding an antibody held chum variable regiott or a heavy chain variable region having tat a nucleotide sequence that encodes a light, chair; or heavy chain variable region of a 7DK 2.-)4, or 8(% antibody, (h) a nucleotide sequence that is identical to a nucleotide sequence of a %%, a 2-)% ot an 8CV> antibody dint encodes a light or heavy chain variable region; or (·,} a nucleotide sequence that is substantially identical, U\. at least 50%, 55"«, 60%, 65",, 70°«, 75%, S0%, 85%, 87%, *H%, 89%, 70%, 91%, 92%, 93%, 94%·, 93%, 96%, 97%, 91%, 9933, 99,3%. or 99% is a aucleoddc sequence: of fa) or to); or Ith a nuclesc ae;d that specifically hybridises lo a nucleic acid having a nucleotide sequence that is the complement of a nucleotide sequence of fa) or lb) under .stringent hybridization conditions, for example, fired wash conditions of 0 1 - 83() at b.5v‘ %

The present invention further presides coils ami cell hues expressing the antibodies or nucleic acids of the in vet: lion. Representative host coils include mammalian and human cells, such as (110 cells, HEK-293 cells, HeLa cells, CV-i cells, and COS cells. Methods for generating a .stable cell line following transformation of a heterologous construct into a host cell arc known in the art. Representative uori-marnmahau host ceils include msec) cells ί Potter et a I (1993) /nr. Rev. hmwmoL 1012--3):103-112.). Antibodies may also be produced in transgenic annuals (Hoodcbtnc (2002) Cmr Opin. Biotedmoi. 13(6).625-629) and transgenic plants (Sehiliberg et ni. t'2003) (37/ 36% Life. 6)./. ^0(3):433-45). 1 he invention also provides methods of treating or etYeeting prophylaxis of amyloidosis associated using immunogenic fragments of an amyloid protein comprising XiEDX?, wherein Alts Η, T. F, S. F. A or any other amino acid residue immediately preceding F.D in such amyloid protein; and wherein X> is T, S. E, R, I, V, F, Λ ot any other amino acid residue immediately following ED in such amyloid protein. Without wishing fo be bound by a particular theory, if is

Pel iovcd that as: epitope even prising X;1.\DX; cars become exposed when an amyloid protein aggregates, or undergoes imrulogenesis or o there isc enters a lien Her s-re cut re, w hot her by cietv. age fro so a larger precursor protein os' by eombr-nabonnl chang;;. cor exampv·. represents bore methods of treatment or prophylaxis of Λ A amyloidosis include administration of Λ A 70- “6 fragments or sera smugs,sod fragments thereof The invsmtiou also pars ides methods of wealing or cdidemig prophylaxis *4 ann kudosis assoc sewd wish deposition of amyloid protein using antibodies reaUoe u tth XsR>X m m aggregated amyloid protein, wherein Xt is Η. T. F, S, P. Λ m assy mho· ammo acid residue immediately preceding HD os such aggregated amyloid protein; and wherein X·.· is T. S. E. R. f V. P. a or- any other amino add residue immediately following ED in .such aggregated amyloid protein Preferably, such antibodies are preferentially reactive with aggregated amyloid protein relative to non-palhoiogiea! amyloid proiein, for example, methods of treatment or prophylaxis of A A amyloidosis associated wuh AA fibrils may include administration of antibodies spec! tie for C· terminal region of Λ A lihrils {« residues 70-76 of A. A) The antibodies can inhibit formation of A A aggregates teg., fibrils) or result tn their dt Mggregatton: and elearaeee, thus treating or effecting, prophyf® is of M· amyl of dosia, 1. Peis· boons

The term 'htubstanisAf identity* nteana that two peptide sequence^ when, optMMfy aligned, such as by the programs GAP or BESTFIT using default gap weights, shave at least 65 percent sequence identity, preferably at least HO or 00 percent, sequence identity., more preferably at least 95 percent sequence identity or more le.ys, 99 percent sequence identity or higher). Preferably., residue positions, which are not identical differ by conservative amino acid substitutions.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a -sequence comparison algorithm, test and reference sequences arc input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. bhe sequence comparison algorithm then calculates the percent sequence identity for the text sequencers) relative t.o the reference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison east be conducted, c.g, by the local homology algorithm of Smith &amp; Waterman, .-h/v Appt. Math. 2:4X7. Π9ΧΙ), by the homology alignment ilgorilhm. of 14ed4teb»n· it Woswch, of Mol Biol. 41:4411 (1979¾ by tlw seat'eft lor .Similarity method of Pearson <&amp;. Lipman. Proe. NaiΊ. Acad Sid. USA 850444 ¢1988), by computenired implementations of these algorithms (GAP. BbSitTG PASTA, and TFASTA in ;he Wisconsin Genetics Software Package. Genetics footputer Group. 5"$ Science Dr,. Madison. Wl), or by visual inspection ( w generally Ausubcl <·/ οί. supra). One example of algorithm I ha; is suitable for determining percent sequence identity and sequence similarity ss the BLAST algorithm, which is described in Ahschnl cf ul.; ./. ,\f<>L B'nA. 215:405 - T 1 0 -1900) Software for performing BLAST analyses is publicly available through the National Center lor Biotechnology Information (http; wwsv.-mbudm.nth.gov T. Typically, default program parameters can be used vo perform the sequence comparison, although customised parameters can also be used. For aonno acid sequences, the BLASTP program uses a- defaults a word length (W) of 8, an expectation ΐIS> of )0. and the BLOSUMbe scoring matrix (sec ifqp&amp;off &amp; Bemkoff, Pr<\. ,\ad. Acad. S<.:>. I AT sd, )()9)5 ί lOxb))

For purposes of classifying amino acids .substitutions as conseivutive or noneonservauve, amino acids are grouped as follows; Grot.;·; ί {hydrophobic sidcciiain*): norlcucine. met, ala. vul, leu. ile; Group II tneutral hydrophilic side chains*: eys, scr, the; Group III {aeidie side chains): asp. glut Group IV (basic side chains*; asn, gin. his, Jys. arg; Group V (residues influencing chain orientation), glv. pro; and Group VI (aromatic side chains). irp, tyv, pbe. Conservative substitutions involve substitutions between amino acids in the same Gass. Non-consers';Uive substitutions constitute exchanging a member of one of these classes for a member of a nothe r. T he term “all-D" reicis to peptides having > 75°·,», > 80%, >. >G‘G. A 90%, .>. 95%. and 100% D-eonitgurailon amino acids.

The term "agent" is used to describe a compound that Isas or may have a pharmacological activity. Agents include compounds that are known drugs, compounds lor which pharmacological activity has been identified but which are undergoing further therapeutic evaluation, and compounds that are members of collections and libraries that are to be screened for ά pharmacological activity. "Amyloid disease" or "amyloidosis" refers to an> number of disorders winch haw as a symptom or as pad of its pathology the accumulation or .formation of amyloid plaques An "amyloid plaque" ;s an extracellular deposit composed mam Is of proteinaceous fibrils. Generally, the fibrils are composed of a dominant protein or peptide; however, the plaque may also include additional eompbtients tfeat a® peptide or aou-pepndo molecules. as described herein.

An “d-nyloid protein" or '‘amyloid pepbde' is a protein r>r n-.-pride capable of undergoing cleavage,. conformational change, aggregation or tibnllogcne.'K resulting in the formation of pathological oWgomm. amyloid fibril·;, amyloid plaques and/or amyloid components.

An “amyloid component'5 is any molecular entity that is present in an amyloid plaque including antigenic portions of such molecules. Any, loid components hvhide but arc not limited ro proteins, peptides, proteoglycans. and carbohydrates

An "anti-amyloid agent" is an agent which ts capable of producing an immune response apiuM an bitty fold: plaque component ta i vertebrate stifoeef, when administered, teg active or passive tinmunicaboit teciiuiques

An %\A protein" or "ΛΑ peptide" refers to the form of amyloid protein A protein or peptide formed by proteolytic cleavage of semir; amyloid A protein tSAAp whether monomeric or aggregated.: soluble or insoluble.

An ^aggregated amyloid proteh% or "aggregated amyloid pepdde' or "aptytold: aggregate'' refers to a pathological, non-monomeric aggregated form of an amyloid protem or amyiou.1 peptide. Aggregated amyloid proteins and amyloid peptide's ran he soluble or msoluble. Some aggregated antslaid proteins and aggregated amyloid peptides east form oligomers, fibrils and/or amyloid plaques. Examples of such aggregated amyloid proteins and amyloid peptides, including fibril peptides and proteins are provided herein.

An "A A aggregate" refers to an uggK'gated form of f A. "therapeutic agents of the invention arc typically substantially pure from umieAred contanunaut. This means dun an agent is typically at least about wAv «weight weight) punts, as well as being substantially free from interfering proteins and contaminants. Somuttmea the agents are at least about SO':···. w;w and, more preferably at least 90 or about 95% wAv parity. However, using eons anions! protein purification techniques, homogeneous peptides of at least 99% sv.-w can be obtained, "therapeutic agents of the invention may prevent, effect prophylaxis oil. or neat a disease associated with amyloid deposits,

Specific binding between two entities means the entities have a mutual affinity for each other that is at least h>, 100- or 100-told greater than the affinity of either entity for a control, suei; as unrelated tmtlgen or antibody to a different antigen. The mutual aff niry of the two comics for each other is usually at least: !0:: M'1, Ifi M' V^t Μ·'f .Affieliiesgteaier than ΙΟ" M : are preferred.

The term ''ininmnoglobolin" or "antibody" tuned tmerchamreubly herein) refers to an .aj.m.geo~hindiisg protein having a basic iota'-polypeptide chain structure consisting of mo heavy and two light chains, said chains being stabilized* Ihr example, by interchain distil fide bonds, which has the ability ns specifically bind antigen, Both heavy and light chains arc folded mto domains 1'ke term ''domain' refers to a globular region of a heavy or light chain polypeptide comprising peptide loops (e.g., comprising 3 to 4 peptide loops) stabilized, for example, by f-pleatcd sheet and or intmehuin disulfide bond. Domains arc further referred to herein as eddnshnit" or "variable", based on the rekui\ e tack of sequence variation svhhin the domains of various class members m the case of a "constant'1 domain, or the sienifeani variation wkhm toe .· . o· ·. \.· " domains of various class members ht the case of a '"variable" dor-tain. "Constant'* horn,tins on the light chair- are referred to inteirhitpgeably as "light chairs constant regions'’, "light chain constant domains", 'Of' regions or *'(.'! " domains. "Constant" domains on lilt· heavy chain are referred to interchangeably as ' heavy chain constant regions" "heavy chain constant domains1'. 'Of regions or "Of" domains, "Variable" domains on the light chain are referred to interchangeably as ‘light chain variable regions", “light chain variable domains", “VI.." regions or “VI.." domains. “Variable'' domain* on the heavy chain are referred to tmerchangeably as "heavy chant constant regions", “heavy chain constant domains", “ΟΊ' regions or "CH" domains.

The term “region" refers to a pari or portion of an antibody chain and includes eonstant or variable domains as denned herein, as well as more discrete parts or pontons of said domains. For example, light chain variable domains or regions include "complementarity delcrmirfng regions" or "CDRs" Interspersed among “framework regions" or "FRs". as defined herein. hnmunogiobulins or antibodies east exist in monomeric or polymertc form. Tito term “amigvivbmdittg fragment" refers ro a polypeptide fragment of an immunoglobulin or antibody binds antigen or competes wit.lt intact antibody with the intact antibody from which they were derived) fits antigen binging i/..- , specif c binding), t he term "conformation" refers to the tertiary structure of a protein or polypeptide te'.g, an antibody, antibody chain, domain or region thereof). For example, the phrase "light tor heavy) chain conformation" refers to the tertiary structure of a light (or heavy) chain variable region, and the phrase "antibody eon formation" or ' "anubody feagmenf eotTfermsiotf refers to tfee terilarg tometore: fef aft .tofibody or fegment ί hereof, "'Spec; tic fending" of an antibody mean that the antibody exhibits ;mpreciabie affinity lor antigen or a preferred epitope and, preferably, does not exhibit significant crossroads try, "Appreciable" or preferred binding include binding eeith an affinity of at lc.tsi Ilf. 10 , it)', 10’ ΛΓ1, or !0!" M", Affinities greater than 10 Mb preferably gt eater than 10' M" are more proffered. Values intermediate of those set forth herein arc also intended to be tylfhin the scope of the present invention and a preferred binding affinity can be indicated as a range of affinities, for example. 10f’ to I0!,i M f, preferably 10 to 10:V M more preferably 10'' to 1 U*S! M !. An antibody that "does not exhibit significant eroxsreactb ity" is one that odl not appreciably bind to an undesirable emits fe.g., an undesirable proteinaceous entity ?. for example an antibody that specifically binds to ΑΛ will appreciably bind AA but wtll not slgnificantfe react with rton-AA proteins or peptides {toys, non-AA proteins or peptides included in plaquesC .An antibody specific for a preferred epitope wife tor example, not significantly erossreaci with remote epitopes on the same protein or peptide. Specific binding can be determined according to any art-rccognfecd menus for determining such fending Preferably, specific binding h determined according to Seatchartl analysis and or competitive binding assays.

Antigen-binding antibody fragments arc produced by recombinant ON A techniques, or by eruiYmaue or chemical cleavage of intact immunoglobulins. Binding fragments include Fab, Fab’. Fmb'p, babe. Fe, single chains, and single-chain antibodies. Additional antibody fragments and effector id notion variants are dist-ssed herein m the section entitled "Antibodies". Other than "bispecifir" or '“hifunehonai" immunoglobulins m antibodies, an immunoglobulin or antibody is understood to fene each of its fendntg situs identical A "fexpocifk" or "hifunctional antibody'' fe an tub fife :1 hybrid ami bods having too different heavy tight ehaio pairs and two different binding sites. Rtspccifie antibodies can be produced by a variety of methods mefefeng fusion of hybridomas or imkmg of Fab' fragments. See. r/., SongVvilat &amp; Lachmann, (7in. by·. hiwuiiui!. "'9:31 f -.fe 1 (I'feOp Kostelny w to,, V. //eewwfe 1-lH, led7· 1553 i 199.7).

The term bum uu feed irnmenogkfeuHfe' or "inmunbed auiihoriv" refers to an immunoglobulin or antibody tha* includes si least one humanized immunoglobulin or antibody chairs (/.e.. at least one humanized light or heat s chain). Tito term ’'humanized immunoglobulin chain1' or "humanized antibody chain" t/.;·, a 'fe uni a meed immunoglobulin light chain" or "humutifeed immunoglobulin heavy ebaftffe refers to tn imbrnnoglofetlm or antibody chain |fg.s a Sight or heavy chain, respectively > (saving a variable region Thai nidudes a variable framework region subsw-h-wly from a human mow; uogkbwim os antibody and <.onqdemcmaruy dewrmmmg regimes t'CDRs) (e.g., at least one (. DR,, me Tend da, nvo (dPRs, snore preferably Three CDRs* substantially from a non·human rmnumeulobnlin or uuttbody, and ttmhcT includes cousin:0 regions W. g,. at bast one cm; slam wgiou or ponton Demon in the ease oh a bght chain, and preferably Once constant regions in the ease of a hem, v eh,no), Tin. term ''intmao fried ywwwde region" (o,g., ''humsnoted light chain variable region" or 'liommiwed imam chain variable region") refers to a variable region that mm mb as a variable isaoieomk region substantially from a human onmunogSohtdin or antibody and compiemvnfaniy dcmnmmne icgiona {('Diva) substantially front a non-hut nan nmrmuoghitwlm or a; id body. 1'he phrase "substantially from a. human mo'mismgkfrukri or antibody" or “snbsiandally human" means tliaf. mb on aligned n> a human immunoglobulin or antibody annuo sequence tor • com pari son purposes, the region shares at least 80--bCDm preferably ^0-*>5" :>, more pscferubly ’:'fr-fr)'-·, identity ids'., los.al sequence identity) with the huso an fra mem oi'h or corwfant region sequence, a how mg, for example, for aonsem mwe substitutions, consensus sequence substh'uTioriS, germ line aulwfinmoiw, backmuTaimns. and the like. The i-urmiuciion of conservative substitutions, consensus sequence substitutions, gernuine substitutions, back mutations, and the like, is often referred to ns “opbmwiwofr' of a huma π iced antibody or .chain The phrase substantially from a -ion-human iinronnoglobolin m wufrody" or "substantially non-human" means having an smnumogiobudn or antibody sequence at least 80-‘>5°·.·, preferably -8)-05-,.. mow preferably, Sfrfr0"%, OR'fr,. or 09½ identical to That of a ton-human organism, mg,, a non-huntan mammal

Accordingly, ail regions or residues of a hrmummed imrmtnouiobulin or antiltody, or of a human wed or: mm loglobmm or antibody chain, except possibly she CDRs, arc substantially identical to the corresponding regions or residues of one or more native human immunoglobulin sequences. The term “corresponding region" or "corresponding residue" refers 10 a region or residue on a second amino acid or nucleotide sequence which occupies the same the., equivalunr) position .½ a region or residue on a. first amino acid or nucleotide sequence, when the firm and second sequences arc optimally aligned tor comparison purposes.

The terms "humanized immunoglobulin'* or ''humanized antibody'' are not intended to encompass chimeric immunoglobulins or antibodies, as defined /-5/.½. Although humanized imsniMogiobullns or antibodies arc chimeric In their construction (/.<-„ compose regions from ivsoic the;t one specie* of protein}, they indudc additional features (its., variable regions comprising donor CDR residues and acceptor framework residues} not found in chimeric iinwaneekibubas or antibodies, as deihted herein

The ternt 'Vhlmenc immurjogiobitlirr or antibody refers to an immunoglobulin or antibody whose variable regions derive mom a first aperies and whose constant regions derive from a second species, On meric immutu>giobujms or antibodies can be constructed, for oMtnpie by genetic engineering, from Immunoglobulin gene segments belonging to different species. \St ""antigen" an emus t,..y. a protenacciuts entity or peptide) to which an antibody specifically hinds.

The term "epitope" or "antigenic determinant" refers *o a site on an, antigen to which an inunutioglobulm or antibody tor antigen binding fragment thereof* specifically binds l:‘pttopes can be formed both morn contiguous amino acids or noncontiguous amino uvids juxtaposed by tertiary folding of a protect Epitopes formed from contiguous amino acids arc typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary (biding are typically hist on treatsnent with denaturing solvents. At; epitope typically includes at least 3. 4., s, 6. '·, 8, 9. 10, 1 L IT 13, 14 or IS amino a cuds in a uuniue spatial conformation. Methods of determining spatial cor:formation of epitopes include, for example, v-ray crysndlography and X-btmc-nAonul nuclear magnettc resonance See, e.g., Ερΐηψ·.' rio/yvny ProiocoI\ he in S/olecniur

Vol. 66, Cl. E. Morris, ml {16*96*.

Representative antibodies of the invention include an -antibody or hurwere thereof that specifically binds to an epitope that includes X; Γ f>X; in an aggregated amyloid protein, which binds to the epitope including XiEDNb that is also botsnd by »t ,?A-h 7Ob, or 8Q0 antibody, Antibodies that recognize the same epitope east be identified in a Ample immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen, be.. a competitive binding assay. Competitive binding is determined it* an as-my in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen, such as Afi Numerous types of competitive binding assays arc known, ibr example: solid plta.se direct or indirect radioimmunoassay (RIA p solid -phase direct or Indirect enzyme immunoassay (ElAb sandwich competition assay I see Stahls a e/b 3 i /7s >J\ in /meivno/ogy 9:342 11,;-'hse: solid phase direct biouu-avidm Ε1Λ isc;: Kirkland ¢/ u/.. J. /nmm,a.·,·/ 137:3614 {ί 9K6VK solid phase.· dirvor labeled assay, solid phase direct labeled sandwich as.xav (see Harlow and Lana .4 ro/U ><.//< v; A Lulhirutiny ManuaL Cold Spring Harbor' Press (HS8;K sohd phase : direct label RIA «sins: 1-125 label (see Morel e/ at.. λίοΐ. Immunol. 2d 1).7 ί 19**)); solid phase direct biotin-a e-ditt El A (Cheung et aL, imo/ogy 1?ό;54ο {1990)). and direct Libeled RIA. i'Moldenhaeer <:? «/.. ScauJ J. Immunol 32'77 (19901) Typically. such an ass;;} involves the use oipunikd antigen bound to a solid surface or cells bearing either oh these, as; unlabclcd test IrnaunogUihubn and a labeled reference immunoglobulin. Compotibve inhibition is measured by determining the amount oh label bound to tire solid surface or cells in Lina presence oh the fesl immunoglobulin. Usually the test immunoglobulin is present in excess. Usually, when a competing antibody is present in excess, if will inhibit specific binding of a reference antibody to 8.common antigen b\ at least 50-55%. 55-00"··., 60-05%, ο5··70% 70-'75% or more. A;r epitope is also recognized by immunologic cells, for example. B cells anchor T cells. Cellular recognition of an epitope can be determined by 9; vino assays that measure antigen-dependent proliferation. d •.berm bind b>' 'Ή-thyrriidine incorporation, by cytokine secretion, by antibody secretion, or by iiruigen-depcndcnt killing (cytotoxic T lymphoevtc assay).

The term "neoepiiopo" refers to a new <md-or unique site on an antigen to which B and/or I cells respond. ihe term "neocpiiopo antibodies" refer to antibodies dun specifically recognize a new N~ or C-nenrunul ammo acid sequence exposed by prmcolync cleavage oi a molecule, but does not bind to wait an epitope on the native dmeL-aved) molecule. The term 'ueoephupe antibodies" may refer to amibodios tlun specific,illy recognize :·. new N- or C ecu renal amino acid sequence exposed hs protcob, tie a Lao. age of SA.\, hut do not bind to stab an epitope on the native tut·».leaved} SAA mohs.de. Some neoepnope amibudics bind to either soluble or insoluble A Λ and result in dissociation of Λ Λ aggregates, mdnding ΑΛ fibrils. \ "neoepuope antibody" m«v also b-e au antibody bun specified!) reeognsz.es a new epitope diat is only available to hmd to an antibody abor a protein Ufideuy.xw a tvuformauon change. for example, as in the ease of Λl amyloidosis and light chain, when only the light chain is expressed and terms amyloid.

The term "irrmumoiogicaU or "immune" response is the development of a beneficial humoral {antibody mediated) and. or a cellular f mediated by amigcu-spccific I cells or their secretion products; response directed against an amyloid peptide m a recipient patient. .Such a response tan be an active response induced by administration of immunogen or a passive response induced by administration of antibody or pruned I'-eelis. A colluhr immune .espouse A elicited by the presentation of polypeptide epitopes in association with Class I or Class II MI1C molecules to activate am icon-specific f'D4 T helper cells and/or CD* ’ cytotoxic T ceils. The response may also involve· activation of monocytes, macrophages, NK cells, basophils, dendritic colls, astrocytes, microglia cells, eosinophils of other components of innate immunity The presence of a eel I-mediated immunological response can be determined by proliferation assays (CO-T T cells) or CTL {cytotoxic T lymphocyte) assays isee Burke, χΐψηκ Ttggcs, supra). The relative contributions of humoral and cellular responses to the protective or therapeutic ctrect of an immunogen can he distinguished by separately isolating antibodies and T-eeiia from an immunized syngeneic animal and measuring protective or therapeutic effect in a second subject.

An "immunogenic agent" or "immunogen'’ is capable of inducing an immunological • response against Itself on administration to a mammal, optionally in conjunction with an . adjuvant.

The term "naked polynucleotide1' refers to u polynucleotide not vomplexed wtth •colloidal materials. Naked polynucleotides are sometimes cloned In a plasmid vector.

The term, "adjuvant" refers to a compound that when administered its conjunction with an antigen augments the Immune, response to the antigen, but when administered alone does not generate an immune response to the antigen. Adjuvants can augment an immune response by several mechanisms including lymphocyte recruitment; stimulation of B and-br T cells, and stimu!at ioη οf «acrophages.

The term "effective dose’' or ''effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect The toon "therapeutically effective dose” is defined as an amount sufficient to cure or at least partially arrest the disease and its •complications in a patient, already suffering from the disease. Amounts effective for this use will depend upon the severity of the infection and the general state of the patient's own immune system.

The term "patient'1 includes human and other mammalian subjects that receive either prophylactic or therapeutic· treatment.

The invention provides antibodies or amigeu-hinding fragments thereof that specifically bind to an epitope that includes X;EBX; in an aggregated amyloid protein, and which competes for binding to the epitope comprising XT&amp;DX·» with e.y., a 2Λ-1, 7DS. or 8Gb antibody.

Competition between antibodies is determined by an asw.y in \%bioh she immtmoglobulm under tee; inhibits .specific binding of a reference •-mi; body to a common am mere such as AA Numerous types of competitive biodine assess arc known, tor vxasople solid phase direct or indirect mdtohTsvnunoassay (RIAr solid phase direct or indirect enzyme immunoassay (irL\k sandwich competition assay face Stahls es at,, t A-/AAs hr fjr'vtmdog*'. A 2-I.7-,:5c <i%rss. -olid phase direct hiotln-avidin Ε1Λ i see Kirkland ct al„ A /.me,ran-· >/ [.) AdoUr-An p> t UAiVig sol so phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lasse. "Antibodies, A Laboratory Manual” Cold Spring Harbor Press (1988)); xolid phase tlh-ectf label III A using 1-125 label (see Morel et ah, Xfokv Itumunol 25(11.7-15 Π98Κ}); solid phase direct biotin-avtdm El A (Cheung ct al... Virology 176.546-552 (1900)}; and direct labeled RIA (Moldeohuuer et ah, Sotnu.«/. hrununoi. 32;?? A2 (19905). Typically, such an assay involve*» the use of purified antigen bound to a solid surface or cells expressing the antigen, an unlabeled tes! ImmurtogiohuHn and a labeled reference immunoglobulin. Competitive inhibition is measured by determining, the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin Is present In excess. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur. Usually, when a competing antibody is present in excess, it will irthibii specific binding of a reference antibody to a common antigen by at least 50% to 75%.

An antibody that specifically binds to an amyloid protein means an antibody that binds lo the amyloid protein with an affinity of at least 10 NT!. Some antibodies bind to the amyloid protein with atfirmlcs between 1 O'" M'1 and 10" M '

An antibody that specifically binds to aggtegated amyloid protein such as aggregated AA without specifically binding to monomeric amyloid protein means an antibody that binds to aggregated amyloid protein, such as, fo? example iiboU te.g., A A in aggregated io-pimaed sheet form such as from a cadaver of a former A Λ Amyloidosts patient or a transgenic animal model· as described above and has al least a ten fold and usually at least 100··Ibid lower specific binding .affinity for monomeric forms of the amyloid protein, for example, such an antibody might bind to soluble Λ A with an affinity of 10" M" mid to plaques with an affinity less than 10 M The affinity of such antibodies for plaques is usually less than It) or 10" M Such antibodies are . additionally or altcmam dy defined by fluoresoencvt intensity relatis'c to an irrelevant control .antibody u'g.. an antibody or mb tore of polyclonal antibodies to a reversemer AA peptide) when the and bodies arc contacted v, i rh fibrils sod binding assessed by dsoressendy labeling. Tbs; ihiotcseeuce intensity of antibodies that bind to soluble ΛΛ peptide v oho at binding io plaques is within a faelor of five, sometimes within a factor of two and sometimes ImUxtingutiduible within experimental error from that of the control antibody.

Compositions or methods "comprising'' one or metre recited elements nan include other elements not specifically recited, Ftn example, a eomposnion that comprises AA peptide encompasses both, uu isolated AA peptide and AA peptide as a component of a larger polypeptide sequence. if, Am\ ioid Diseases |. Overview and Puthogcnesi*-

Amyloid diseases or amylotdoscs include a number of disease slates hating a tvidc variety of outward symptoms. These disorders have In common the presence of abnormal extracellular deposits of protein fibrils, known as '''amyloid deposits' or "amyloid plaques"' that are usually about 10Ί00 urn an diameter and are localized to specific' organs or tissue regions. Such plaques are composed primarily of a naturally occurring soluble protein or peptide. These insoluble deposits arc composed of generally lateral aggregate* of fibrils that arc approximately 10-15 nm so diameter. Amyloid fibrils produce a chaructcvistk· apple green birefringence in pole need light, o km stained with Congo Red dye. The disorders are classified on the basts of the major ftbnl eosoponciUs tomtit tg the p bus to: deposits, a*, dismissed be bee "( he peptides or proteins forming the plaque deposits are often produced front a larger precursor protein. More sped ideally, the pathogenesis of amyloid fibril deposits generally involves proteolytic cleavage of an "abnormal" precursor panda into fragments. These fragments generally aggregate into .inti-parallel j.> pleated sheets; however, certain undegraded fort ns of precursor protein have been reported to aggregate and form fibrils in familial amyloid polyneuropathy A a Hunt transthyretin fibrils) and dialysis-related amyloidosis Cj3- mictoglobtnin fibrils* t Tan, w a/., 190-1. \npra), .1.1. Clinical Syndromes

This section provides descriptions of major types of utnylotdoses. Including their characteristic plaque fibril compositions. H is a general discover} of the present invention that amyloid diseases can be treated bv administering agents that .sen. e to Misnniate an immune response against a component or components of the variou > disease-specific amyloid deposits As discussed in more detail in Section (" below., such components ate rnvfonsbB constituents of the librils that form the plaques. The sections below sene to exemplify major forms of amyloidosis and are not intended to kmt! the invention. a. At Amyioidoscs

At amyloid deposition is generally associated with almost any uyscrasia of the B lymphocyte lineage, ranging front malignancy of plasma ceils (multiple myelomas to benign •monoclonal gam-mo path)·. At times, the presence of amyloid deposits may he a primary indicator of the underlying dysemaia.

Hint of At amyloid deposes are composed of monoclonal immunoglobulin light chains or fragments thereof. More sped Heal ly, the Ifagnvms are derived from the X-tenninai region m th·.· light chain {kappa or lambdas and corsair all or pan of she venable s'V; 5 domain thereof. Deposits general lx occur in tits' mesenehy rnal nasties, causing peripheral and ann-nomie neuropathy, carps! men el syndrome, macroglo.vba, restrictive eatshomyopathy, arthropathy of large joints, immune dy sera as as, m\ elomas. us well as occult dyscrastas. Howcxcv, si .should be noted rhiii almost as iv tisvue, paoieulark usceral organs such a a the heart, may he mooted, b, Hereditary Systemic Ant) Imposes

There are musty terms of hereditarx sestet me amyletdoses .Although they are reiatiselv rare conditions, adult onset of symptoms and hie sc inheritance: panes ns {uaisiuh autosomal dominants lead to persistence of such disorders in the genera! population. Generally, the syndromes are attributable to point mat-Pious at the preenr-tot protein leading to production of variant .unyloidogenic peptides or proteins, fable .?. sum murines the fibril composition of exemplary forms of these disorders.

Table : .Hereditary A mytoidoses’’

‘’Data derived from Tan &amp; Pcpys, (994, supra..

The data provided is Table 2 are exemplary and arc not im coded to herd the scope of the invention. For exampic, more than 40 separate point ton ratio ns in the iransthv reim gene have 'been described u!1 of which give rise to clinically similar forms of ranviliul amyloid poly neuropathy. frsnathyretin (TTR) is· a 14 kdodalton protein that is aKo sometimes referred to us nreaiburoin, U is produced by the liser and choroid plexus, and h functions in transporting thyroid hormones and Gramm A, At least 50 variant forms of the proto ' c.wh characterised by a singly sxnino acid change, arc responsible for virions ibrrfts of iirnllql amyloid: polyneuropathy. For example, substitution of proline tor leucine at. possum; 55 result1' in a particularly progressive term of neuropathy; substitution of methionine for leucine at position 1 I I resulted in a severe cardiopathy in Danish pat tents Amyloid deposit1' isolated front heart tissue of patients with systemic amyloidosis have revealed that ihe deposits are composed of a heterogeneous mixture of TTR and fragments thereof collectively referred io as ATTR. the full length sequences of which have been charactered, AMR fibril components can be extracted from such plaques arid their structure and sequence determined according to the methods known in the an se.g., Gustavssort, A.: m al, l aborafory Invest. 73: 7Q3-70R, 1995; Kameutni, F . er oL Biochcm. Biophys. Res. Cornrnim Fed'. OAMv'.'M, 1954:. Pr.i.x, M , et al , FN.AS 5(): 539-45, 1985 9

Persons having point mutations in the molecule apohpopmucio Λ1 te,g., Giy~?Arg3h, Trp Arg50; Leo Arg(>0; exhibit a form of imylosdosis t"Ox;c0¾ type'’} eluiraetcnccd by deposits of the protest; upolipoprotein Al os' fragments thereof l-XApoAFs These patterns have low levels of high density lipoprotein {HDL't and presem with a peripheral neuropathy or renal fa i hue A. mutation to tbs alpha chain of The enpyrae lysoxyme fme., Ilsh^TfirST; or Asp'-HNsd/s is the basis of another form of OstcrtagAype nort-neuropatlue hereditary amyloid reported in Fnglixh i,mhfies, Here, llhnls of the mutant hsoityme protein t Alys- xre deposited, astd pstiem's generally exhibit· impaired renal function. T ills protein, unlike most of the flhrib forming proteins described herein, is usually prevent in whole uiniVagmenmd} form <Benson, M L>„ e?.·.;/. ( IBA Fdn. Syntp. 199' 104-131, !!>opv. jfamylold peptide (Afl) is a 59-43 ammo acid peptide derived by proteolysis from a large protein known as Iveta amyloid precursor prolem φ.ΑΡΡ) Mutations h; jH.APR result in familial forms of Alzheimer's disease, Down's syndrome and 'nr senile dementia, charaemciacd by cerebral deposition of plaques composed of Ap fibrils and other components, which me described in further detail below. Known mutations in APP associated with Alzheimer's disease occur proximate to the cleavage sites of β or y secret ase. or within Αβ. For example., position 71? is proximate to the site of p-secrctase cleavage of APP in its processing to All and positions 670 6'/! are proximate to the site of β-sccrerasc cleavage. Mutations at an> of these residues may resui? in Alzheimer's disease, presumably by causing an increase the amount of the 42 43 amino acid form of Λβ generated from API'. The structure and sequence of Αβ peptides of various lengths arc well known in the art. Such peptides can he made according to methods ;known in the art {e.g. Gi turner and Wong. Biochcm Biophys. Res. Conan. 129; FH5-A90. 1%4; Glcnner and Wong. Biochcm Biophys. Res ('oatm. 122; 1131-:135. I%4). In addition, vinous forms of the peptides are commercially available:.

Svnueletn ts a synapse-associated protein that resembles an a lipoprotein and is abundant in neuronal cytosol and presynaptie terminals. A peptide fragment derived from a-synueletn, tconed NAC. is also a component of amyloid plaques of Alzheimer's disease. (Clayton, et ah. 199s) Hus component also serves as a target lb; immunologically-based treatments of the present hivcntion.. as .detailed bciovv .

Gelsoun is a calcium binding protein that binds to und i'raemeuts act in ti laments. Mutations at posh ion ) f " (e.g., A.sp~iFAsm Asp-ATyn of the protein result tn a form of hereditary systemic amyloidosis, usually found in patients from Finland, as well as persons of Dutch or Japanese origin, in afflicted individuals, fibrils termed front yd softs fragments (AgeF>, usually consist of amino acids 3-245 {% kDa earbosytermhuii fragment) and are deposited 1st blood vessels and basement membranes, resulting in corneal dystrophy and cranial neuropathy which progresses to peripheral neuropathy, dystrophic skin changes and deposition in other organs. {Kangas. f(.. *·/«/. Human Mol. Genet 5(9¾. 123?·· 124.1 19%¾.

Other mutated protein*. such as mutant alpha chain of Fibrinogen tAfibA) and rnutasU eysauin Γ (Acys) also foon fibrils and produce chaructcrtstfc hereditary disorders. AnbA fibrils form deposits charaderisue of a turn neuropathic hereditary amyloid with renal disease; Acys deposits are characteristic of a hereditary cere but: amyloid angiopathy reported in Iceland, tlssclbaehe;. et af. Hum son's Principles of haemal Medicine. McGraw-Hill, San Francisco. 1995; Benson, et a/., <upra.). in at least some eases, patterns with cerebral amyloid angiopathy (CAA.t has e been shown to have amyloid fibrils containing a non-mutant form of ey statin €: in txMj.unet.ioh. with. beta pmteih, (Naggi, A,, p:&amp;l Moles. |1ϊ0^Κ-δίΐΐ#ρΛ^1-. 32: ov'M, ! 90K }

Cerrain farms of prion disease are now considered to he horitabk:. accounting lor up to 157 i> of cases, which were previous!}·· thought to he predominantly inkxtioux m nature. {Baldwin, pt al« m fymareh .S^wmnes in MzkgirH&amp;r a D&amp;gAse ·$?# iMs&amp;f'tiern Joltst Wiley end

Sons- Non York. I dps}, in such prion disorders, patients develop plaques composed of .abnormal isoiorms of the normal prion protein tPrPG, A predominant mutant mo form. PrP'v. also referred to as ASer. differs from the normal cellular protein in its rest star me to protease degradation, insolubility after detergent cxrraetion. deposition in secondary· lyeosomox, posttranslational synthesis, and high |>p leafed sheet content. Genetic linkage has from established for at least ίο e mutations resulting tn Creutifeldt-jticob disease ·< JDf Gerstmaon-Strausslcr-hchdnker syndrome (GSSt, and fatal unkind insomnia tFFh. (Baldwin) Methods for extracting fibril peptides from scrapie fibrils, determining sequences and making snoh -peptides are known in the art. {cm.. Bockcs. M, et oL i. Gen. Virol A>: 2567. ?;.. jOoyy

For example, one form of GSS has been linked to a PrP mutation at eodort 102. while lekmccphafic GSS segregates with a mutation m codon 117. Mutations si codons h)S and 7! 7 result in a form of GSS in which ncitrltie plaques characteristic of AlaheknerY disease contain FrP instead of Aja peptide. Certain forms of familial· CjfMtave· been "associated with mutations at codon.' 200 and 2 10; mutations at codons 120 and I 7b have been found in both familial CJD and F FI. i Ba Id win, mpm). e. Senile Systemic Amyloidosis

Amyloid deposition, either systemic or focal, increase·) with age. For example, ftbriks of wild type transthyretin (TTR) are commonly found in the heart tissue of elderly individuals. These may he. asymptomatic, clinically silent, or may result in heart fat I tire. Asymptomatic fibrillar focal deposits mas also occur In dte bruin lApy corpora amylacca of the prostate (Ai.F mlcrodobnlink joints and seminal t esteles, d. Cerebral .Amyloidosis

Focal deposition of amyloid is common in tint brain, particularly in elderly individuals. The most frequent type of amyloid in the brain is composed primarily of AB peptide fibrils, result mg in dementia or sporadic mon-hcrcditary} Alzheimer A disease. In fact, the incidence of sporadic Alzheimer's disease greatly exceeds forms shown to be hereditary. Fibril peptides forming these plaques are very .similar Hi ihbsh. #itfc· refet-^ee- $$ hs^te| forms o f ΛI zhe« m c r s d tsease {A D). e. Dialysis-related Amyloidosis

Plaques composed of IT oucmglobulin fAjyjM) fibrils commonly develop m patients receiving long term hcmsx&amp;tlysis or peritoneal dialysis. j5: microgiobuhn is u 11.8 kilodnkon polypeptide and is the light chain of (.lass I MHC antigens, which are present on all nucleated cells Under norma I circumstances, it i\ continuously '-hod front cell ntembmuw" and is normally filtered by the kidney, Failure ol clearance, such as m Ihe case of impasted runal function, leads to deposition in the kidney and other sites fprimarih in collagen-fids tissues of the joints). Unlike other fibril proteins, AfcM molecules ard generally present in ibrhti4tf iltd· fibrils. (Benson, supra}. f. Hormonc-dcnwd Ants kudoses

Endocrine organs may harbor amykno deposits, particularly in aged individuals. Hormone-secreting Honors nosy also contain hormone-derived amyloid plaque", the fibrils of which are marie up of polypeptide hormones such as eakitonm need id lory carets to mu of the thsrotdb islet arnvioid polypeptide tamylin: oecwvmg us rno-u patients wuh Type il diabetes), and atria) natriuretic peptide «isolated atrial imyloitiosts). sen nonces and A rueones of these proteins are well know n in the art. g. M ised laneous A my iotdoses

There are a variety of other forms of amyloid disease that are normally manuest us tocai Ded deposits of amyloid, in gene run these diseases are probably the result ol the local teed production and/or lack of catabolism of .specific fibril precursors or a predisposition of a particular tissue i'suelt as the joint) for fibril deposition. Examples of such idiopathic deposition include nodular Al amyloid, cutaneous amyloid, endocrine amyloid, and tumor-related amyloid. 11.1.. A.A Amyloid Diseases Λ.Λ amyloidosis, formerly called secondary or reactive amyloidosis because it develops secondary to a precasting or coexisting disease. Such diseases include, but arc not limited to inflammatory diseases, such as rheumatoid arthritis, juvenile chronic arthritis, ankylosing spondylitis, psoriasis, psoriatic arthropathy, Reiter's syndrome. Adult Still's disease, Behcet's syndrome, and Crohn's disease. ΑΛ deposits are also produced as a result of chronic microbial infections, such as leprosy,. tuberculosis, bronchiectasis, decubitus ulcers, chronic pyelonephritis* osteomyelitis, and \Vhippie's disease. ( ertmn malic-inn- neoplasms can also result m \Λ fibrd amyloid deposits. These me Uric such condition.·» a,-» Hodgkin's lymphoma. renal cat si nos run carcinomas of pus, lung and urogenital tract, based: red carcinoma, and heirs cell leukemia. A A. amyloid disease may also result from -nbemed mimvrnnatory diseases sus'h as Familial Mediterranean Fever. Additionally, Λ Λ amyloid disease sees result mom lymnfx>pr$niieraPve disorders such as ( a.sUeman's Disease. I. Indaromnfory Diseases Associated v.iih ΛΛ An!\ k>ido.os

Rhennuitotd as throe s is ;i ehsome *w stormc disease pr.-mufe of the joints The symptoms of rlv mourn id arththts are marked by tnit-enmamry ehane.es in the sy ran ad membranes and articular structures yeanst and ip, atrophy and snseiaction i:>no densuy deer-, a v.;,; of the hones In hue stapes of rheumatoid arthritis. deumnnw and <ud,-> I os A a immobility of the joint} dot chap Λ model of rheumatoid arthritis eat: he induced ;n mice or rats by adntunvtenog ty pe II eolHecn lit complete Freund's adnoant juvenile chronic .nth; tits »otnes m many tonus, the most , ommon boiae tin otitic i heuntaioid .nthnlis. |t can noun it- children at any ape. hut Fh-u appears ;store commonly between the apes off and o years There are ^ nusn types of |o rent re shea masted artitrins, namely, pauei-utticuku urihrio·.. pol>articular sirthrut», and v.stetrue utfntitfs tako known ts StHlk disease). Ρηΐη. ο,η'ΐνίο,Ο' arthritis typ^aily affects -I or fewer joints, usually the ksrperones such as the Dices It .an be aceo-upainod by softness. ·,austtn? the :hub to limp Polyauden'tor arthritis is diyraetwcd by “> or mom iomts hen to aHected tnost commonly die smaller joints nt the haitds at id feet. Children wait pokuriiadur arthritis often base -t nine severe form of the disease. Systemic sola As s. ehar.tcter;.'c-j by until swdlmg m combination with few and a nink rash. The joints may no; star: to swell until some months or y earn a lie; the revets hep in. h may also affect iviternal organ» stub as tb>- Iwer, heart, spleen and lymph nodes, and anemia is common. While '-osteon·, arthritis tends to abate id'its own accord, a natal; percentage of these children can h:w e see ere arihnb·- that:cm mutes into adulthood

Ankylosing spon-dy litts is a rheumatic disease that causes arthritis of the spine and sacroiliac joints and stmt -..«use inflammation of the eyes, lungs, and heart valves. It varies from micrron'lesn episodes of back pam that occur throughout life to a sesere chrome disease that attacks the --pme, peripheral joints and othet body organs, resulting in severe jotnl and back stiffness, !ov. of motion and detbrtotiy as hfe psogressek.

Pscsriasid ts a common clmmc:, Sqisarnous dermatosis* roarfeed by MacerhatiiM -M# emissions and basing e poiygome iniieriu.·ice pattern The symptoms of psoriasis arc marked b> the presence of rounded, dry staling patches of various sizes, covered by a grayish white or silvers while scales flwjt base a predilection ior the extensor surfaces, nails, scalp, genitalia and the lumbosacral region.

Psoriatic anh top a ih\ is a disorder in winch psoriasis is linked to foe development of arthritis The disorder can be c>. hr hi ted in a variety of ways The arthritis Is generally mihl and involves only a few joints. In a few patterns, the disease h; severe and usually affects tfte fingers and site spine. When the spine is affected, the symptoms are rosy much hke those of ankylosing spend y I his

Renefs syndrome is a croup of symptoms consisting of arthritis, urethritis; {inO&amp;inmation of the urogenital tract>» conjunctivitis tinflammation of the lining of the eye), and lesions of the skin and mucous membranes. Render's syndrome is also referred to as reactive arthritis, which recasts that the arthritis occurs as a ''reaction” to an infection that slatted dsowhsre in the body . CkhmydiiiimckoMatis is the bacteria most often assoeiated with Reiter’s syndrome acquired through sexual contact, Several different bacteria are associated with Reiter’s syndrome acquired through the digestive tract, Including '$higgMety lenihria, and, C$ni0l&amp;ikwt&amp;^ Λ dull Still’s disease, also called Adult Onset Still’s Disease is a rare inflammatory condition ilu.it attacks internal organs, joints and other parts of the body. It can appear and disappear suddenly, hi very severe cases, adult Still's disease becomes chronic and extremely debilitating, causing terrible pain and soilness. After many years, the disease cripples vital organs ,,uch as -he heart and lungs.

Behcet'« syndrome is a ;mritixyai.em disorder presenting with recurrent oral and/or genital ulcerations, chronic relapsing uveitis that may cause blindness and neurologic impairments, h is characterised by 4 major symptoms' ora: aphthous ulcers, sktn lesion-;, ocular symptoms, and genital ulcerations, and occasionally by iriHamonition in tissues and organs throughout the body, including the gusiroimestin:.u tract, central nervous system, vascular system, lungs, and kidneys 'she arthritis of Behcet's syndrome is usually mtermitient., self-limned, not delbrmmg and localised to the knees and ankles.

Crohn's disease is a chronic granulomatous (small grain dike body or growth) mfuimsoatorv disease h;\ob, nig any part of the gastrointestinal tract from the mouth to tutus, but common's involving the ileum (lower tlwee-fifths ol the small intestines} with scarring and thickening of me bowel widi. The symptoms of Crohn's disease include the presence of chrome diarrhea. increased bowel sounds, emtinins;, possibly evidenced by weighs, loss unci aversion so eating. 2. Chronic Microbial Infection Diseases Associated with A A Amyloidosis

Leprosy -s an infections disease crease sen.red by dssnguneg skat sores, peripheral nerve damage, and progressive debilitation. Leprosy is caused by rite organism tfy^ohuch-niftu fcpt\i<\ which is mn very contagions and has a long incubation period. Leprosy has two common forms* tuberculoid and lepromatous Both forms produce sores on the skin, but the lepromatous form is most severe, producing large, disfiguring nodules {lumps and humps). Leprosy eyebtb&amp;lly causes peripheral nemoiogicat damage·.·. Patients with.iotig-lgmi leprosy omy iPsecihC use of their hands or feet due to repeated injury resulting from luck of sensation

Ttdiereulosis: is a contagious bacterial: inieeiieu caused. by The disease is characterized by the development of granulomas igranular tumors* in the infeefvd tissues. The lungs are primarily involved, but the infection can spread to other organs.

Brouehieeta.ws A an abnormal destruction and dilation of the large airways Bronchiectasis Is often caused by recurrent inflammation or infection of the airways Λ classic bacterium that is ;,cert in pattern- with bronchieciasre is P^cnJonuma^ rere.grv rer which is notoriously hard to eradicate. Repeated infections of the airway·* by this bacterium can lead to colonization of the bronchi by this organism w hich predisposes such people to P^uJanamo! /ν?νηηηηη’η·.. which requires special met A retire to neat.

Decubitus ulcer also known as pressure uh.ee or bedsore is an ulceration of the skin and underlying tissues caused by prolonged pressure ovet the alTeetcd area They soul as reddened skin but gets progressively worse, forming a blister, then an open sore, and finally a crater These ulcerations usually occur over bony prominences such re heels, coceys area of the buttock into the buck of the head.

Chronic pyelonephritis is ut infection of the kidney and the ureters {duets that carry urine away from the kidney). Pyelonephritis most omen occurs a* s result of urinary tract iniection, particularly in the presence of occasional or persistent backflow of urine from the bladder nuo the ureters or kidney pelvis.

Osteomyelitis is an acute1 or chronic bone infection, usually caused by bacteria. Often the hdeetion In mates tn another mot of the bods and spreads to the bone vta the blood. When the hone is infected, pus is produced within the bone, which may result. In an abscess. The· abscess dice deprives the bone of ns blood supply. Chrome osteomyelitis results when hone tissue dies as a result of the lost blood supply. Chronic infection cun persist intermittently for years.

Whipple's disease is a mm cos id mem diet ucuses inadequate absorption of nutrients frotn the intestinal trn-..t due to nuAtioo of the mresone It ts caused by the baaerla. 'in.iphcrviiiu nSymptom,·* include dtanhea, mfostund Heeding, abdominal pain, loss of appetite, weight loss, fatigue, end weakness. Arthritis and tever often occur several years before intestinal symptoms develop, iWienis nu\ vspenenee neurological .symptoms as well. Diagnosis is bused on symptoms and the results of a biopsy ot tissue from the .small intestine or other organs that are affected. When recognized and treated. Whipples disease can usually be cured. Wttbo-.n treMmem. the condition is usually fatal. d. Malignant Neoplasms Associated with AA Amyloidosis

Hodgkin's lymphoma is a euneor of lymphatic tissue found in the lymph nodes, spleen, liver, and hone marrow. The first sign of this cancer Is often »n enlarged lymph node. The disease can spread to nearby lymph nodes and iafec may spread to the lungs, liver, or bone AtatTitw,

Renal carcinoma is cancer of the kidney. The cancerous ceils are found in the lining of tubules in the kidney. The first symptom is usually blood in the urine. Sometimes both kidneys arc involved. Hie cancer spreads easily, most often to the Uuigs and other organs. Renal cell carcinoma is the most common type of kidney cancer followed by papillary reran eel! carcinoma, chromophobe renal carcinoma and collecting duct renal carcinoma About 5H of renal carcinoma are unclassified because their appearance doesn't ill into any of the other categories. C arcinomas of rite gut include gastrointestinal cancers such, as colorectal, pancreatic, stomach and esophageal. Colorectal cancer is cancer dun starts in the large intestine or the rectum. Almost all colorectal cancers begin as benign polyps winch, over a period of many years;, develop Into cancers, Most cases of coioreeM cancer hay® tto symptoms, Faaereaifc cancer 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, can develop in any part o! the. stomach and may spread throughout the stomach- and to other organs; particularly the esophagus and the small uueshne It may also spread, through the stomach wall, to nearby lymph nodes and organs such as the lively pancreas, and the lungs, or to distant organs sucli as the lymph nodes above the collar bone, the colon, and the osanes. Stomach cancer is often asymptomatic Esophageal cancer is malignancy of the esophagus. Symptoms fflcinda dysphagia {difficulty swallowing}, pairs and substantia] weight loss.

Carcinomas of the lung are a cancer of rite lungs character!red by the presence of malignant tumours- There arc two main types of lung cancer. non-small cell lung cancer and small cell lung cancer. Symptoms depend on I he specific type of cancer, but may include . chrome cough- coughing tip blood, shortness of breath, wheextng- chest pam. loss of appetite, weigh! loss and fatigue.

Carcinomas of the urogenital tract include hut arc not limited to prostate cancer, bladder cancer- endometrial cancer, cert teal cancer and ovarian cancer. Prostate cancer invokes a malignant tumor' gmvt-h within the prostate gland. Symptoms may include frequent urirsaUon-difflculty starting and maintaining a steady stream of mine., blood in the urine, painful urination, difficulty achieving erection or paiuit.il ejaculation. Bladder cancer refers to any of several types of malignant growths of the urinary hiaddvr. Symptoms include blood tn the urine, frequent urination, painful urination, and urinary urgency Endometrial caucer involves cancerous grow th of the endometrium {lining of the uterus,!, it mainly occurs after menopause, and presents wait vaginal bleeding. Cervical cancer is a malignancy of the ccrvh. The early stages of ccrs leal cancer may be completely asymptomatic. Vaginal bleeding may indicate the presence of •malignancy. In advanced stages, mctast&amp;scs may be present in the abdomen, lungs or elsewhere. Ovarian cancer is a malignant neopumsm of the ovaries 0%an<m cancer symptoms are often vague and non-specific, which include vague lower abdominal discomfort, sc ewe of pclv;e heaviness, abnormal menstrua! cycle, vaginal bleeding, weight gain or loss, nonspecific gastrointestinal symptoms. Qvmian cancers shed cancer ceils that often implant on the uterus, bladder, bowel, and lining of the bowel wall. These cancer cells can begin terming new tumor growths before cancer is even suspected.

Basal cell carcinoma is a slow-growing skin tumor involving cancerous changes m .basal skin cells. Symptoms include skin lesions located ort the fare. car. neck, chest, hack, or scalp; visible blood vessels in the lesion or adjacent skin, and persistent, non-healing sores. This cancer usually remains local and almost never spreads to distant parts of the body, but U may continue to grow and invade nearby tissues and structures, including the nerves, bones, and 'brain.

Many cell leukemia ns a cancer of lymphocytes ill cells} dun leads to low blood counts. The'dfeea.se is caused by the abnormally shaped 8 cells with hair-like projections, Symptoms are often vague The low blood dodijis. dau&amp;dd 'fey Isakf'.obJ:} ish*|Sth.i&amp;£#h. Id»# Τ||%»§:, and oscesslve bleeding. 4. Inherited Imlumroafors Disease Associated wuh ΑΛ

Familial Mediterranean Fever is an inherited disordc; chat ac. termed by recurrent finer and inflammation, often involving the abdomen o; the bun* S',mpioma include iinftammufton in the lining of the abdominal cavity, ckc.-w cavity, skin, -m joints oecins, along with high fevers that usually peak in 12 to 21 hour1·. Aback'; mas t.iry in severity of symptoms. and people arc usually symptom tree between Ataeks 11: is disc eve ts scry race. Risk factors include a family history of familial Mediterranean rover or hat·sue Mediterranean ancestry 5. Lymphoprolifemtho Disordcts Assoc coed with Λ Λ Amyloidosis

Cast leman's Disease is a form of iympopro!i ferattve disorder characterised pathologicaly by the presence of giant lynap node hyperplasia with plasm;·; cell infiltration. Patients with CVitientan's Disease commonly have fever, anemia, hyperaanraraglobulhtaemla, and an increase in the scram concentration-* of acute phase reactant proteins, all of winch are ascribed to the huge amount of 1L-6 produced in she lymph nodes.

IV, Seram Amyloid A

1. Human Seram Amyloid A

Serum amyloid A t'S.AA} is the circulating precursor of amyloid .A protect, the fibrillar component of amyloid deposits. The structural studies showed that the human SAA is heterogeneous and represents a family of polymorphic SAA genes and protein products. The SAA gene superfamily comprises u cluster of closely iirtked genes 1 oca heed to 1 IplS.l. See Sellar, GC el al. GAno/uk y 19: 221-22”· s 19944, Four SAA genes have been described m humans. Representative ami.no acid sequences of pro;cm- encoded by the lour SAA genes are illustrated by figure 1. Two genes 1.9.4.4/ and 0.4.42? encode acute-phase serum amyloid Λ tA S.AA1 and are eoordinately induced in response to inflammation. 0,4 4/ and .Sl4.be .share 95*·» sequence identity in both coding and noncodtng regions 7here arc alpha, beta and gamma isotorms οΠ toman SAA I and alpha and beta isofornts of human SAA 2 as illustrated by figures IS and 19. ,S',f4e is a pscudogenc. SAA4 encodes constitutive SAA and is mire really inducible. SVc Cnnnanc G. Bui Were \ O/n, RhanmiutL I3(<lt: 1412-028. AH human SAA-A A molecules contains a theoretical calcium-binding tcfrapeptnle sequence, Gly-Pro-Gly-Gly, of possible ioigortaaeo fhr self aggregation and with extrafibrillar moieties of amyloid in Sbriilogenesis. See ibimc, I'..Μ. Cl al. 8i')ch<-m. J. 256.-)73^0 $ 1988 s arid Turned tt al. Mol Biol. Mai .ViX’MO? {19861, The N terminal portion ofSAAΆΑ is strongly hydrophobic., probably of importance for self aggregation ami oilier components so amyloid deposits. ,S\o Hu sby of al. Clin biiniiwol· immfruyuifkoL ‘·:0ί i ;-.N9 t1994k The sequence of cad's isoform of A.A and id relationship to its corresponding SAA isofi >rrn is illustrated by Figures NS. for exes epic, human SAAl alpha -soiorm lias the sequence H;N Aden LysG..cml.cm ihrTby -Lcu- V'al -Phe-f'ys-bs/v -Leii· Vul4.eu-GiyAGb$erNer-Argm;er·· 'Pke-Pho~Scr-Phc4Am-Giy-G!u-Ak-H}e~Asp-Giy-AGa-Arg-A*p4'1ei~Try-Afg~Aia-Ts r-Ser-Asp-iView\rg-GliKAI^Asn4'yoUeTily~$er-AHp4..ys-Tyr4'‘hedlis-Aia“ AΓg4uyΛ^n4ye"AsρΛ!iJ^ ΛU-L ys~ \fg4!i!y-Pro~Gly-GK - A la-Try - Ala-Ala-G Us-YaM ie-Scr-Asp-Λ In-A rg-Gi a-.-Vm-! ic-(bn- A;'g4die--Phcb’liv--His-Gb-'-'Ala'-Glu--Asp-Gc5'-4..cU'o\kr· Α·ιΡ'ί'ϊΙϊΐ··Λ1;.ί··Α9ι··Λ^!;''(Ι1ο·'ί!·ν··ΟΚ Ar^Scr4})y4oyvAxp4>ro-Axn4-bvPi;c~Ai>j-Pa>-A)aA}ly-i.cu--p5xvGhj4.y.s-Tyr-OH fSii:0 IP 'NO: I ?. A A, which is a proteolytic fragment of SAA, is also heterogeneous. The predominanf human Λ A peptide consists of 7o amino acids. A si example oi A.A has the sequersoe: Vi^N-Arg-Ser-Phc-Phc-Scr-PbC'LcU'Gly-OHoAia'Pnc-Asp-Oly'Aia-Arg'Asp-Mct-Try-Arg-Ak'· Tyr-Scr-Asp-Mei-Arg-'GUi-Ala-Asn-Tyr-lle-Gly-Ser-Asp-Lyii'I'yr-phc-i its-Ala-Arg-Gly-Asn-Tvr-Asp-Abi-Ala-l.ys-.\rg-Gly-l'>sx>-Giy-Cily Ahs-fry-Ala-A b-G: u V ;.G He- Ser-Asp-Λ fi-.ory-Glu~Asn -1 le-GhwVrg-Phc-Pfre-Gly-His-Gly-A la-GIis-Asp-Scr-OI 1 *;$EQ ID NO:2 b

AA'dlGb refers to an A A fragment beginning at residue '?(> and ending ;n residue ?P of (SEQ ID NOG) consisting of die sequence GRGAEDS. (Sb.Q H> NO: 4>„ or correspond mg segment from another naturally occurring A A protem from a human or other species veheu Use sequence of that proiein Is maximally aligned with SfiQ ID NOG

c Man no Serum Amyloid A

In the mouse, four SAA genes have been described. Reprosemnmc ammo acid sequences of proteins encoded by die four murine SAA genes are i has timed by (figure 8. Mouse S.A A gene family comprises four members that are closely linked in the chromosome ?. i vm of these genes encoding major mouse SAA isotypes f$AAl and SAAeq share high sequence identity not only in exons but also in imrons and flanking regions and aye induced in .approximately equal quantities in response to amyloid induction models. These Nso i.sotypes differ in only 9 of I Of ammo acid residues: honema only SAAd is sdeedrely deposited into amyloid fibrils. Nee de Beer M.C. BiorhanJ. I v9; 380( Id G: 4s..qo t igqt ^ Hoffman hS, of. d. J Exp Med. i 59:641 --646 (1984*; Shiroc Μ or. a! Si and J hmmm<d. 24.^99--716 «1987). SAA3 a minor HDL apolipoprotein and peripherally produced acme phase, bAA-1 is u constitutive subhnmK (has is a minor normal HDL anoliponroiein comprising more «’nan 90% of the SAA during homeostasis, See Steam?»» R.S, et pi, NugMfe AeMs Me&amp;fcf&amp;hj· 14C3P9 a809 6i9h6:| and de Beer M.C. G< nondex, 3-h I )· 1:6)-42 Π996Τ

Marine AA which is a proteolytic fragment of SAA is also heterogeneous. The sequence oh each murine isoiurm of Λ A and its relatioo-fmp to its corresponding SAA Rolorm is dhrurakxi bs Figures 9-12. Λ sequence alignment of murine AAl. ΛΛ2, AA3 and ΛΛ4 «s -illustrated by Figure 13

Murine AA = ο dm muon. eqmvaieot of 1 ηη·,η* \At See f igure 16. In particular, residues 69---¾ of murine Λ A1 iGRGHFOT. SFQ ID NO: 9) are maximally auvmed- with residues 70-76 of human AA 1 (GF1GAHDS, SEQ ID NO: 4). See a ho Figure 17.

3, Soar Pci Scrum Amyloid A

The Sear Pci sequence 1¾ indicated in f igure 20 Intejusrmglv, she homologous region in the human SAA protein -AF.PS. (SF.Q I'D NO- 13) contains a conserved fur to Scr substitution at position '76, ss wed as significantly different side chant of the residue at position 73 - His lo -Mat Fig. IK The -ACDS, (Si'iQ ID NO. 13), sequence is also observed In the Soar Pei species of dog, a breed that is particularly susceptible to Λ A-amyloidosis and could provide a naturally occurring mode! of systemic AA in which to evaluate nos cl diagnostic and therapeutic application.·» of A A amyloid-specific antibodies and other compounds. 4. The N-Terminal Segment of ΛΛ Proletn Determines Its Fihrtllogenie Property

The amyloid fibril protein AA consists of a varying hum N-terminal pan of the precursor protein .serum AA. Evidence shows that the amyloidogenic par? of the molecule Is the N-iorn'tinai !0-! 5 amino acid long segment. Amino acid substitutions in this part of the molecule may explain why indy one of the two ntousc SAA tsommts ss amyloidogonic. See Weslemutrfe G. Th Biochem Acs Cvmnw&amp; 182 (1);27-33 (;3 992), ¥> Other Human Antyloidoeenic Proteins

The Gcnbank Accession Numbers and XiEDX? sequences are provided belosv lit Table 3 tor several human aroyloidogenic proteins, including some of those listed above in fable 2. i .ihb 3

Human Amyloidogcnis. iVofeinn

VI. Amyloid Peptides for Active

Therapeutic agents for use in the methods of the invention are immunogenic pcnPde.s. such as Λ Λ peptides and AL peptides, that on administration to a patient pen ora re rnmbodies that specifically bind to 0·«: or more epitopes comprising AtPDX-, si.u.h a*., tor esampie, epitopes bemecn residues "0-76 of ΑΛ p\\A agents "X Additional examples of agents include umnunogemc peptides dad comprise a fragment consisting of XdhDX - derived front other amyloid proteins p'XjOXX; fragments"; such as AL VT fragments consisting of the amino acid sequence PBPb iSEQ ID NO: 2!f PL'Dr'. (S£Q ID NO; 2:.). Ab'DY, (Sff.Q ID NO; 3). SEDE, t'SEQ ID NO; 2d), or SEDA, fSIX) ID NO. 75s, and Ai. \'λ fragments consisting of the amino acid sequence XEDE. (SE'Q ID NO' if). AiiDE. fSE'.Q ID NO: 10). Π DE. (SE'Q ID NO; Do nr PEDF, t'SF.Q if) NO: 20?. An Ai. Va fragment consisting of the amino acid sequence f HDD. tSEQ ID NO. 17) may also be used. Nome suitable amyloid protons include Serum amyloid Λ protein, immunoglobulin light chain protein, human islet amyloid precursor polypeptide 11 AFP}, beta amyloid peptide, transthyretin U'TID, Λρο.Μ anti other amyloid proteins listed in Table 1 and 'Vbieh comprise tiro sequence X:E'DX-. in some agents X; 1¾ Η. T, F, S. P„ A or any other amino acid residue immediately p? eroding f.\D in an amyloid protein; and .'< ns T. S, I?... R, L V . F. D, A or any other us-vino acid residue iromediurely following ED in such amyloid protein. its sOiiic agents, Xj is H, Id. F. S. id or A and Xd is T, S, id D. R, 1.. V, F or A. in some such agents, when X; is H, X; is T or A, when X; is A, X.· is S, T, E or V. when X, is Id X.> is id when Xt is F, X> is D, when X< is S, Xj ss Ed Γ or A; and wiser» X: is id X- is Cd 1 or Id in sons.; agents, X., is H. "id F, S, id or A and X: is E S, Id IX R< E V, |: or Λ. with the proviso -hat if X s ts A, X; is not Vd in some agents, when X» is A. X;.· is 8, T or id

Some agents comprise the amino acid sequence GHEDT, (SEQ ID NO; 3j, HEDT, iSEO ID NO' I2E A EDS. ί SEQ ID NO; led Ad Did {SEQ ID NO: id), H F.P.A. (SFQ ID NO; 151. TEDid iSEQ ii.) NO. 16), FEDD, (SEQ ID NO' :?). SEDE, (SEQ ID NO: IE), AEDid (SEQ ID NO: 10), PEDF. (SEQ ID NO: 20}.; PEDE tSEQ ID NO: 21X PE Did tSEQ fD NO: 22), AEDV, {SEQ ID NO- 251. SEDF, tSEQ ID NO; 241 or SEDA. {SEQ ID NO' 25). Some agents consist oh an amino acid sequence selected from the group consisting of GHEDT, tSEQ FD NO, 5, HEDT, (SEQ ID NO- 12). Λ EDS, {SEQ ID NO: 15), AEDT, (SEQ ID NO: 14:, HEDA, tSEQ ID NO: 15), TEDid {SEQ ID NO: DO, FEDD. (SEQ ID NO: 17», SEDE, (SEQ ID NO' IS). AEDid (SEQ ID NO: 19). REDE, (SEQ ID NO: 20), PEDi, (SEQ ID NO: 2 i). PEDF. (SEQ ID NO; 22?, AEDVd (SEE) ID NO: 25), SEDE, (SEQ ID NO: 24), or SEDA. (SEQ ID NO: 25k linked to a carrier to form a conjugate. Some agents comprise the amino acid sequence GHEDT, (SEQ ID NO. 5, HEDT. tSEQ ID NO' 12:, A EDS. (SFQ fD NO. 15), AEDT, ISEO ID NO; )4), HEDA, sSEQ ID NO: 15), EE.Did ·;SEQ ID NO: 16). i:EDO, (SEQ ID NO; Γ), Si":Did tSEQ ID NO: IS).. AEDid (SEQ ii.) NO; IS). PEDE, (SFQ ID NO; 20s, PED1. (SEQ ID NO, 21:, PEDF, (SEQ ID NO- 22:, AEDV, tSEQ ID NO. 2Q. Sr Did (SEQ ID NO. 24:, or SEDA, tSEQ ID NO. 25). Some agents consist of an ammo acid sequence selected front the group consisting of GHEDT, (SEQ ID NO' X HEDT, (SEQ ID NO; 12), AEDS, (SB,) ID NO. 15), AdDl', (SEQ ID NO: 14), HEDA. (SEQ ID NO: 15). TEDtd (SEQ ID NO: 16), FEDD, (SEQ ID NO; : 7(, SEDE, (SEQ ID NO: ISK AEDid (SEQ ID NO- IS), PEDE, (SFQ ID NO; 20}, PEDE (SEQ ID NO; 21 k PEDF, tSEQ ID NO: 22), SEDF, (SEQ ID NO; 2-1} and SEDA, (SEQ ID NO: 251, linked io a carrier to form a conjugate Sonic agents comprise an a mi no acid sequence selected front the group consisting of GHEDT, tSEQ ID NO: 5, HEDT, (SEQ ID NO' 12), AEDS, tSEQ ID NO; 15), AEDT. (SFQ ID NO' :4), HEDA. (SEQ ID NO' 15) and I EOF, (SEQ ID NO: 16),

Pros erred ΛΛ fragments are human A AI (ΗΛΛΠ alpha Isoform residues 70-76 (GHGAEDS, SEQ ID NO:-i), HA As beta isot'orm residues Ά)··7ό : id HD A EDS. SEQ ID NO;S), ΗΛΛΙ gamma tsofVnm residues "0-M {GHDAHDS. SEQ !D NO' 5 k ΗΛΑ2 alpha and beta u-sOionns residues :0- ;6 (GRGA EOS, SEQ ID NO- 4k HA.A5 reskruos 70-/0 (GEKHAEDS, >?EQ if> NO:?k HA AO residues ?*-S4 (STVIEDS, SEQ ID NON}. mouse A \ i (MAA 1) reside -975 (GKGHED1. SEQ ID NO»}, \] Α.Λ2 roiducs 00-''5 tGKGHEDr, SEQ ID NO. 9j. ΜΛΛ3 residues M-6E (GHGAEDS. SEQ ID NO:lOk ami ΜΑΛ4 residues 76-0.: iNRGI.E'l l . SEQ ID NOD i) or subfragmcrnts of at least’ three contiguous ammo acids of any of these. Some Λ A fragments contain no residues of an AA amyloidosis peptide other than the segment designated above Other ΛΑ fragments contain additional flunking residues from an A A -tmyloido.sks peptide but contain no move than ,20 or preferably no more than 10 contiguous residues in total .from at: Λ A amyloidosis peptide. Additional preferred XjE.DX·; and Al. fragments include GMi:J .)1, (SEQ IP NO: .Ο. ΠΕΡΙ, (SEQ ID NO: :2 c A EDS, tSHQ ID NO; IS}. Λ ED H (SEQ ID NO: 14}, 11 EDA. (SEQ ID NO: 15).. and TEDE. (SEQ ID NO. ffi) iherapeukc agents for use in the methods of the invention also include immunogenic AA peptides that on administration to a patient generate antibodies boat specifically bit id to N-tcirninal epitopes of ΛΛ. Preferred agents induce an immunogenic response directed to an epitope: wife® residues 1.-1.5 of human. A A,

Preferably, ihe fragment of A A or Al, or other agents such as X; f DXs fragments administered lack an epitope that would generate a 1-cell response io ihe fragment. Generally, 7-cod epitopes arc greater than 10 contiguous amino acids. Therefore, preferred fragments of amyloid proteins such as .AA or XjEDX·.· fragments arc of sGc 1-10 or preferably "M0 eofiiiguous ammo acids; ;.e., sufficient length to generate an snUbodv respottse without ^generating a T-cell msporme. absence of T-ecIi epitopes is preferred because these epitopes are not needed lor immunogenic activity of fragment*. and may cause an undesired inflammatory response m a subset of patients i Anderson et a!.„ {2002),/ {miuufiol· Ms, .)607-3 A) D Senior (2002.1 l.ancei .Mm®. G 3 k

Preferred ΛΛ fragments are human AA 1 lBA.:\l) alpha iso form residues 70-76 (GHGAEDS) iSEQ ID NQ- -D, HA ΛI Iveta isot'orm residues 70..76 (GHDAHDS) (SEQ ID NO.5}. HAA1 gurnma isoform residues 70-76 (GHDAHDS. SHE) ID NQ: 51, ΗΑΛ.7 alpha and beta isoforms residues 70-76 iGHG.AEDS. SEQ ID NO. 4}. HA A3 residues 70-76 (GDHAHDS · (SEQ ID NO: k H A A 4 residues MM (Nl \ 1 E.DS 1 (61::13 ID NON}, mouse AA1 (MAA!) residues 69-75 (GRGHEDT) (SEQ ID NO:9k MAA2 residues 69-75 i GRGHEDT. SEQ ID NO: Ok M A A3 residues 62-66 (GHGAEDS) iSHQ ID NO; 10k and ΜΛΛ4 residues 76-S2 (NHGLHTL) {SEQ ID NO.11} or subfragmenis of -it lotust three contiguous ammo aotds of any of these. Some AA fragments contain no residues of no ΛΑ amyloidosis pepude other than the seyrnen: designated above. Other A Λ fragments contain additional if rut long residues front an ΆΑ amyloidosis peptide bul contain no more titan 20 o.r preferably no snore than 10 contiguous residues in total front an A A aroyloklosi-s pepude. Additional preferred XsfiDX;. and At. fragments include GHEDT, (SEQ ID NO; 3k HBDE (SEQ ID NO: 12k ABDS. tSBQ ID NO: Ιοί. AEPT.. (SEQ ID NO; Hh I if.DA. (SEQ ID NO: 150 and TEDE. tSEQ ID NO' IM.

Analogs of die natural Λ A amvloidoius. AL amyloidosis, and miser amyloidoMs peptides can also be used to Induce an Immune response in the methods and compositions ol the invention, Analogs including allelic, »pec-es and tndtse-ed varueua Analogs of A .A uuluec antibodies that specebeally bind with a natural AA 70-76 peptide. Some such analogs fan to induce antibodies that specifically binds to epitopes outside AA70-?b. Analogs of AA typically differ front natumily occurring peptides at up to 30% of amino acid positions by up to E 2. 3, 4, 5, 6, 7. A 0 or 10 position change's. Each deletion or sub-utultost of a natural a so i no acid residue D considered a position change as ss die insertion of a residue without suosdiuoon Ann no acid» substitutions are often conservative stibstimtiops,

Nome analogs of ΑΛ os' Λ A fragments or At. or At. fragments os'other asm losd protein fragments sue I: as .N.;EDNA fragments also include unnatural amino acids or modi Heat sons of N or C terminal amino acids at one, two, five, ten m even alt positions For example, the naruraj aspartic acid residue can be replaced veil iso-asru.nl:c acid. Examples of unnatural ammo audn are D. alpha, alpha-disubsotuted .amino acids. N-alkyi ; urn no acids, lactic acid, 'Miydrosyprolisi'.,. gamma-ea rbosy gl > tramate, epsi ion-- N, N, N -rnmethy I lysi ne, epsilon-M-ace! y 11 yssne, O'phosphosei'inc. N-aeesylserinc. N-fonttylrnethiontne, 3--meihylh:stidhtc, .Ahydrosylysme, omcga-N-mei hy iargin tne, % alanine, orni ih u to, rtoneuot tup nerval use, Its- duos proiu'se., thyroxine., gumma-amino butyric acid, Itomosenue, eitruilme, and isouspanie aetd, Nome merapeum; agents of the insentiou use edf-D peptides, eg, ail-D AA or ali-D ΛΑ fragments, and a 114') peptide analogs. Some therapeutic agents of the no. ention are '30% al!--D peptides, e.g,, %)% all-D AA or :·Λ>0% ali-D A Λ fragnsents, and 00*-« ali-D peptide analogs Some therapeutic agent» of the invention arc 80”« all-D peptides, e.g., sir’,, ali-D ΛΛ or 80% all-D AA fragments. and N0% all-D peptide analogs fragments and analogs can be. screested for prophylactic or therapeutic efficacy in transgenic ummai models in comparison with untreated or placebo controls as described below. A A. A L, ihoif fragments, and analogs and XjEDX.: fragments and their analogs can be synthesized by solid phase peptide synthesis or recombinant expression, or can he obtained front natural sources. Automatic peptide synthesizers ere commercially uvaitabU; fro in numerous suppliers, such us Applied Biosystems, Foster City, California, Recombinant expression can be in bacteria, such as L. cob, yeast, insect ceils or mammalian cells. Procedures for recombinant .expression are described by Sxmbrook ct al., Mokxuhir f'hmn^: A Lulmmitity Mtirntfri (CS.H.P. Press, NY Ζύ ed„ Ifofo.}

Therapeutic agents also itrehado longer polypeptides that include, for example, an immunogenic fragment oi'.AA peptide, AL peptide or an XdfDXb fragment, together with one or more other atnino acids flanking the A A peptide, AL peptide or X,EDX? fragtrient on one or one or both sides. For example, preferred agents include fusion proteins composing a «egment of AA. AI. or X(I:!DX; fragment fused to a heterologous amino acid sequence that induces a helper T-ccll resixntse against the heterologous amino acid sequence and thereby a 8-cell response against the A A segment. AL segment or XJfDX.· fragment. One or more flanking heterologous annuo acids cast also he used to cap an AA or Ah peptide or XYdYT.· fragment to protect it front degradation in manufaettire, storage or use Such polypeptide1· cars be screened for prophylactic or therapeutic efficacy in animal models in comparison ugh untreated o? placebo controls as described below, Therapeutic agents of the invention include an immunogenic fragment of A A or A 1., or X;F.DX^ fragment Hanked by polylysine sequences. The poly lysine .sequences cat; be fused to the N-terminus, the C terminus, or both the N- and C-terrmnus of AA or XL or att immunogenic fragment of \A or Ah or XTl.UXs fragment. The AA or Al peptide, Xtl.’OX· fragment, analog, active fragment of \A or other polypeptide can be udtninistered itt associated or multi meric form or in dissociated form Therapeutic agents also include muiumers of monomeric immunogenic agents.

In a further variation, an immunogenic fragment of AA or AL or Xfo.DX·· fragment east be presented by a sums or a bacterium as part of an immunogenic composition. A nucleic acid encoding the immunogenic peptide is incorporated mm a genome or epRome of she virus or bacteria. Optionally, the nut:loir, acid Is incorporated in such a manner that the immunogenic peptide is expressed as a secreted protein or as a fusion protein with- an outer surface protein of a urns or a trtnismembr&amp;ne protein of a bacterium so that the peptide is displayed. Viruses or bacteria used in such methods should be non pathogen ic or attenuated. Suitable viruses include adenovirus, HSV, Venezuelan equine encephalitis virus and other alpha viruses, vesicular stomatitis virus, and oiha· rlmfedo viruses, vaccinia atEt fowl pox:, Suitalsic teciena tHeiod&amp; ' Salmonella and Shigella, fusion of an immunogenic peptide to HBsAg of HBY is particularly suitable.

Thcrapcuue agents also include peptides and other compounds that do not n-.ees'.anis have a significant anti no acid sequence similarity with AA or Ai or Xif.DX.·; fragment %gt neterthdcss serve as rnimetics αί AA or Al. or X<EDX.t fragment amt induce a similar immune response. For example, any peptides and proteins forming fi-pleated sheets cats be -'ctvened for stu lability. Anti-idiotypic antibodies against monoclonal antibodies to A A or Al. or other amyloidogenio peptides such a.s or XtEDX; fragments can also be used. Such antt-ld antibodies mimic the antigen and generate an immune response to It (see Essential Immunology (Rod ed.. Blackwell Scientific Pub! teal ions, Palo .A Ho, Mh ed. }, p. 18 b. Agents other than Λ A peptldes shoald induce an immunogenic response against one or more of the preferred segments of AA listed above fe g... ΛΑ70-76 or GBEDT. (SLQ ID NO. H or an AL or X;b.PX- fragment listed above, such as, tor example, HHDT. (SEQ ID NO: ID, A EDS. tSEQ ID NO; 13}, AED'MSEQ ID NO: Mb HI:DA, (SfQ ID NO: IS? and TEPL (SEQ IP NO’ 16).

Preferably, such agents induce :m immunogenic response that is specifically directed to one of these segments without being, directed to other segments of A A or Al or amyloid protein horn winch the XiED.X·· (ragmen! was derived.

Random libraries of peptides or other compounds can also be screened tor suitability. Combinatorial libraries ear: be produced for many typos of compounds that can be synthesized tit a step-by-step lashton. Such compounds include polypeptides, beta-turn munches, polysaccharides, phospholipid*, hormones, prostaglandins, steroids, aromatic compounds, 'heterocyclic compounds, benzodiazepines, oligomeric N--substituted gly tines and oligoearbamates Large combinatorial libraries of the compounds cat: be const me tod by the encoded synthetic libraries t LSI.) method described in Ally max, WO 95‘12606, Aflymas, WO M3 06*21, Columbia University, WO M+'OSCRG Pharmacopeia, VvO Op3330.3 and Senpps, WO VS ‘30642 {each of which is incorporated by reference for all purposes). Peptide Itbraries can also be generated by phage display methods. See, e,g., Devlin, WO Hi ;MV80,

Combinatorial libraries and other coot pounds are Initially screened for suitability by determining their capacity to specifically bind to antibodies or Kmphoeytcs fB or Th It now o to be specific for A A or other arnylotdogenie peptides, for example, initial screens can he performed \V;H! any poivdoegl -sen or monoclonal antibody to ΛΛ or AL or a fragment thereof or to an X{EDX,= fragment, Compounds can then be screened for specifically binding m a specific .epitope within A A ίο g., A A"?0-7o or GL1EDT, i$£Q ID NO', 5j or AL or to an X : EDX^.traumeM listed above, such as, for example. HEDT, (SEQ ID NO. lei, AftXS. iSLQ ID NEE !>}, AFDT, t Sf.Q ID NO: 14s, HE DA. (SEQ ID NO: 15} and 1 DDE, «SEQ ID NO: lb).

Compounds can be tested by the same procedures described tor mappmg antibody epitope specificities. C'umpoundx Identified by such screens are then further analysed for eupHCUv to induce anti bodies or reactNe lymphocytes to A A or AL or fragments thereof or to an X iE[.)X fragment. For example, multiple dilution·-; of sera can he tested on mierotiter plates that have bees- precoated with AA or AL or a fragment thereof or an X:LD..K> fragment and a standard E.USA can ire performed to test tor reactive antibodies to ΛΛ or ,AL or tite fragment or to the X;EDX,· fragment. Compounds can then be tested for prophylactic and therapeutic efficacy in transgenic animals predisposed to umy loidosis, such as, for example, A A Amykucktsix or AL amyloidosis. The same screening approach can be used on other potential agents, analogs of AA, analogs of AC. and longer peptides, including fragments of A A, Al. and .X'iBDXT fragments, described above VI1. Conjugates borne agents for i-iducing at; immune response contain the appropriate epitope for inducing an immune response against Λ.Λ but ate too small m be immunogenic. In bus situation, a peptide immunogen can he linked to a suitable currier molecule to form a conjugate which kelps elicit an immune response. A. single agent can be linked to a single carrier, multiple copies of an agent can be linked to multiple copies of a carrier, which are in turn linked to each other, multiple copies of an agent ear; be linked in a single copy of a carrier, or a single copy of an agent can be linked to multiple copies of a carrier, or different carriers. Suitable carriers include serum albumins, keyhole limpet hemooyusiin, immunoglobulin molecules, thyroglohidin, ;.n albumin, tetanus toxo;d, or a toxoid from other pathogenic bacteria, such as diphtheria, E. coil cholera, or H. pyhr.\ or an at ten timed toxin derivative. T eeh epitopes arc also suitable carrier molecules. Some conjugates cun be formed by unking agents of the invention to an immunostlmuhitory polymer molecule (c.g.. tripe! maw f-S-glycerine cysteine 1 ptmnCysg munnan (a manosc polymer), or glue an (a beta 1 -w; polymer)}. eyAikitiw py,;, 11.-1, if.-I alpha and beta peptides, 111,-2. gamma-INF, Ό>!% GM~€'SBf, and ehhsnbihlhes Ml'PI alp ha and beta, and R ANTES). Immunogenic agents can also be linked to pcpudc.s iluit enhance transport across tissues, ns described in O’Mahony, WO 5?7·Ί76Η* and WO 9?·’! ‘Mid, immunogens may be linked to the carries with or with oat spacers annuo acids . giy-gk).

Some conjugates, can be formed by linking agents of the invention to at least one T cell epitope. Some T ceil epitopes are promiscuous while other T cell epitopes are universal Promiscuous T cell epitopes are capable oi enhancing she induction of f cJ: immunity in a wade variety of subjects displaying various RLA types, tn contrast to promiscuous T cell epitopes, universal 1' cell epitopes are eapabte of enhancing the induction of T >_eil immunity in a large percentage, e g., at least 75^ of subjects displaying various HLA molecules encoded by different idf.Λ-DR alleles. A large number of naturally occurring T-eHl epitopes ex As, such as. Tetanus toxoid feu... she Pc and P30 epitopes), Hepatitis B aarfacc antigen, pertussis, toxoid, measles virus P protein, Ohlarrwdia rraehomitls major outer membrane protein, diphtheria toxoid te.g., ORkHsRv, Plasmodium falciparum cireumsporozne 1. iHasmodiuni falciparum €S antigen. Schisiosoma mansoni tnose phosphate Bomersae. Escherichia cob TraT, and tnOuenra virus hcmuglutUnm I HA) The immunogenic peptides of the invention cuts also be conjugated to the T-ecll epitopes described tn Sinigugku F. ct ah. Suture, 3o<r?7x-'?80 i 1988?; Chiex R.M et aL ,F iv>.p. Med., !?K;2?-4": flOOg Hammer J. et <?L, Cell 74 )97..2(0 DOfO); F*ik K, et 0., bmmmog.eneties, 39:230-2-)2 {190-1y WO °s 23635', Souriwvood S. et al. .1. immunology; I60-.3.AO-3373 t1098?, and, Gianoini.G. et al. Nucleic Acids Res 12: 4063-4069 {1984), teach of which is incorporated herein by reference for ail purposes). Further examples include; buluen/.n Homaglurtinm; H .-W- .¾s,

Malaria Cl'S: !3 epitope ERKIAKivlEK ASSYFNV, (SFQ ID NCr 67).

Hepatitis B surface antigen: HBsAg,^ FFLlTRiiT!, tSEO ID NO. ok).

Heat Shock Protein W hsp65:=M"t IWIGDUAEAMI>KVGNfc'G.<SEQ ID NO. 69). baeille faimettc-Guerin QYHFQPLPRAVYKL fSFQ ID NO· 70).

Tetanus toxoid: TTv QYliCANSKJFlGlTEL· (SPQ ID NO' 7 i s.

Tetanus toxoid: 11'·,- -." FNNFTVSFvVi.RVPKVSASULF, iSEQ ID NO: 72). HIV gp120 'Π. KQHNMWQEVOKAMVA. iSEQ ID NO: 73)

Tetanus toxoid: TO,,- FNNTTV STAVERVPR VS .ASHti:.

HIV gpi.20 Tl: KQOMMWQh V (7ΚΔΜ Y7V

Alternatively. the conjugates can be formed by linking agents of she invention to a? least one artificial T-ccii epitope capable of binding a targe proportion of MH(.' Class II molecules., such tts the pan DR epitope ("PADRE” h PADRU is described in US 5,736141, WO dS/07707, and Alexander j el aL Immunity, 1:75 i-7ί'ί (1004) (each οΓ which is incorporated herein by reference for all purposes'). Λ preferred PADRE peptide is AKXVAAWTLKAAΛ, (SPQ ID NO: 74), (common residues bolded) wherein X is preferably eyelohexylaIaninc tyrosine or phenylalanine. with cyclobexy lah-mine being most preferred, immunogenic agents can be linked to carrier.* by chemical cross! inking. Techniques for Imkirtg an immunogen to a, carrier include the: formation of disulfide fiftkapta nling Xu sueenbnbdyb V< 2-pYridyMhio; propionate (SPDP) and st-eeimmidyl 44 N·· msilcimidomelhyIkydohexane-1 -carfeoxylate (SMCCi t if the peptide lacks ft stflthydryi group, this can be. provided by addition of a cysteine residue). These reagents create a disulfide linkage between themselves and peptide cysteine resides on one protein and an anode linkage through the epsilon-amino on a lysine, or other free amino group in other amino acids. -\ variety of such disu tilde'anode·· iormsng agents arc described by fniiiiun. Re v. 67. 175 i )442), Other hifunctional coupling agent- form a thtoethet rasher than a disulfide linkage. Many of these ihloodhew forming agents arc commercially available and include reactive esters of o-ntaletmiuoeaprole acid, 2-hromoacciic acid, and 2-iodoa.cetie acid.. •b.i\s.maleinvldo-rnetb.Yhcyclobc>:ane· i carboxylic acid. The carboxyl groups can be activated by combining them with succimmidc or I -hydiOxyl-a-nittFO-d-sulfonic·' acid, sodium salt.

Irorminogonicity can be improved through the addition of spacer residues leg,, Oly~ Giy? between the Ί\ epitope and the peptide immunogen of the Invention, hi addition to physically separating the Tt; epitope from the B cell epitope . the peptide immunogen}. the glycine residues can disrupt any artificial secondary structure* created by the joining of the ΊΑ epitope with the peptide immunogen, and thereby eliminate interference between the T amber B ceil responses. The conformational separation between iho helper epitope and the antibody eliciting domain thus permits more efficient interactions between the presented immunogen and the appropriate ‘1 :. and B cells

Vo enhance the induction of T cell immunity in a large percentage of subjects displaying rntrlons HLA types to an agent of the present invention, a .mixture of conjugates with different Π, coll epitopes can be prepared. The mixture may concurs a mixture ref cl I oast two conjugate'; with different Ί\ cell epitopes, a mixture ot'yf least throe conjugates with different Th cell epitopes, or a. mhluve of at least, four conjugate* with different Tj, cell epitopes. The mix tore may be administered with an adjuvant.

Immunogenic peptides can also K- expressed as fusion protests with earners ih'.e 'heterologous peptides). The immunogenic peptide can be linked at ns amino terminus. its carboxyl terminus. or both to a earner. Optionally, multiple repeats of die immunogenic penode can be present to the fusion protein Optionally, ,tn unmnnogeme pepode can be nuked to rmbtipO copies of a hetcroiogou'- peptide. for example, at both the \ and C t-.rrmm oh she peptide Optionally, multiple copies of an immunogenic peptide ean he finked to multiple copses of u heterologous pent-dc o h=v!; are linked to -mob other, Some carrier peptidvs setae to induce a helper T-cell resputise against the carder peptide. I he ind-tet J hdpos l-cells to turn induce a B-eell respoitsc .gainst the tmmunogenk pcptwU. linked to the Career.

Somc. s'- am pies of fusion proteins suitable for use to tb,· tnwmion are shown below Some of these tusion proteins compete segments of ΛΛ linked to tetanus townd epuope* such as described in s W 5,; vo,; l A I P .USAs 1 and L'P 12X34 7. Some lesion proteins comprise segments of Λ Λ linked to at least one PADRlf peptide deset shed Su OS >,Xh\ i -XT S^me heterologous peptides are promiscuous I'·tell epuopes while other heterologous peptides nr·.· tttitversal T-ccll epitopes. In some methods, the agent lor administration is simply a -etude ie-oeu protein with an Λ Λ segment linked to a heterologous segment in Imcur configuration. The ds crunch tie agents of the invention can be represented using a formula. For example.. itt some methods, the agent is multimer of fusion proteins repa\senied by the lormufi; 2\ m which x is an integer front 1--5. Preferably \ is 1, 2 or 5. with 2 being most preferred. When x is two., such a multimer has four fusion proteins linked in a preferred configuration referred to as MAN tsec US 5,229,490).

The MAP-! configuration is shown below, where branched structures are. produced by initiating peptide synthesis u! both the N terminal and side chain amines of lysine. Depending tnon the number of tin«> lysine is Incorporated into the sequence and allowed to branch, the resulting structure wifi present multiple N termini. In this example, lour «lent tea I N termini ha-, e been produced o:a the hrancited lysine-comaming eot'e. Such multiplicity greatly enhances the response eness of cognate B cells. In fisc examples below, 7. refers to art immunogenic irugnsent of ΛΛ, Ak. or an Xh EDX-: fragment, and A 1--4 refer to immunogenic fragment-s) of Λ a, A f m an iX-iEiS^irugtncm Hsc iMgoi&amp;tits emt boifee sardeis eaeh eiietor diflfereiii·

Either examples of fusion proteins include; £-Tetanus toxotd 830-844 in s ΜΛΡ4 configuration; Z~Q Y ί K A N S K F1 ΟIT P 1.., (SBO ID NO; 71) X-Totanua toxoid 94 7-907 in a ΜΛΡ4 configuration: Z-FN'NFTVSFWLRVPKVSASHU4 i$l:Q ID NO: 72) I.-Tetanus toxoid 830-404 in a MAP4 configuration: X-QY i K AN SK PI (is f TEL. ?SFQ ID NO; 70 Z-Tetanus toxoid 4)0--844 )-947-967 in a linear configuration: X~QY I N.ANSivFiGtF E LF N'NFTYSF \V L R V PR YS A Sli L E, iS.F.Q ID NO' 75).

INy'IRF peptide tad in linear configurations), wherein X 0 prt-ferabD evelohexGaDnine, syrnsine or pheuviaiamne, v- iih uydohexvudanu-e being most prokrred-Z; AN.XΥΛΛWTLR A AA-Z. (SEQ ID NO: 74). Z x 3-PADRE peptide: Z"Z"Z" A K.X FA Λ WT 1 ΚΛΑΑ, (SEQ ID NO: 7% Z - ova)bums·t 323-339 In a linear configuration;

Z-IFQA VFI AA HA II: 1'NF.AGR, (SEQ If) NO: 7oF

Further examples of fuxlon proieGx in--bade: AKXVAAWO ΚΛΑΑ-Χ-Ζ-Χ-Χ, (SEQ ID NO: 74). X-ARXVAAΧΎΙ..Κ.ΛΛΛ, iX-tSFQ IF) NO; 74) PRYYKQNU.RF.AT-X-X-Z, (SEQ ID NO: 7 7). Z-Pfv YVXQNTI..N.LAT-X, (SFQ ID NO' 77). X~X-X.-PR.YYKOYUXlA.T (SEQ ID NO; 77). X-Z-Pk VVKQNTLK. LA T, {X-Z-tS LQ ID NO: 77} Z-PKWKQhfaKLAT-EKKIAKMFXASSYFNV.QYIKAK$KFIGITEL* Γ NNFTVSF WI.RVPKYSASI It fMSHQ ID NO- 7*> X-Z-Z- QYIKANSKF IGITf: L-FNNI'7 VSFW.I..RV PN VSASHLE. (XFQ ID NO- /-5¾. X-QYj KANSK* IGil'ILCFN NPTYSf WLR VPKVSASH UAXS iWNO: QYI KAN SKJKIGfT E U Ί N NFTVSF W L RVPKVSAnH L E-Z. <SE.Q ID NO: ?9t X,~Q YIΚΛNS KEIG ITLf, tXLQ ID NO. 7>) on a 1 branched rosin: fragments can be ffee a® as cacli other or dlfeont

The same or similiU earner proteins astd method* oHinkage can be used for generating immunogens to bo used in generation of antibodies against ΑΛ or an immunogenic fragment of ΛΛ. ΛΙ. or an Χ,ΕΡΧ: fragment. E'or example, AA or an immunogenic fragment of ΛΛ, ΛI. or ail X;FOX’: fragment linked to a carrier can be a d ου ni stared to a laboratory animal in the production of monoclonal ami bodies to ΛΛ or an anmenogeme fragment of AA, Al. or an XjldDX; fragment, VIII. Nucleic Acid Encoding Thertpeefle Agedts

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 XfFD.XX fragments, o? antibodies and their component chains, such ns antibodies 2Λ4. 8G9 and ?DH.: used tor passive imnumizanon. Such agents for use ur the methods- of the invention include nucleic acids encoding AA peptides that on administration to a patient generate antibodies that 'Specifically bind to sine or more epitopes between residues "0-76 of AA, AL or nucleic acids encoding peptides comprising XXEDX-· fragments Such agents (or use in the methods of the invention also moludc nucleic a os (In encoding antibodies Thai specially bind to a C-terminal neoepliopc of ΑΛ or so X:EDX>. hi particular, such nucleic acid* encode antibodies that specifically bind u.;; \ I A Ο 1 alpha isoform within residues 70-7 6 (GHGAleDS, (SEQ if) 'NO-. d}. Τ1ΛΑ1 bom isolorm muhin residues 70-70 (GliDAEDS, (SEQ ID NO. 5V. HAA! aamma be norm within residues “0-76 fGHDAEDS. (SEQ ID NO: $s, He A 7 alpha and beta No forms wilhm residues 70-76 (GHGAEDS, {SEQ ID NO: 4h HAA3 within residues 70-76 (GDHAEDS. (SEQ ID NO: ?k ΜΑ.Λ4 within residues 78-8-4 (STV1EDS, ISEQ ID NO: 3), motive AAI (M.\A ί} welkin residues 60-75 (GRGE1EDT. (SEQ ID NO' νϊ. VIAA2 within residues 60-75 (GRGHEDT, (SEQ ID NO: 9). At.A A3 w khan s'Csidues 65 -60 (GHGAEPS, (SEQ ID NO; -IT and ΜΛΛ4 within residues 76-H2 {NMOLE TL. (SEQ U> NO: Hr Sack nucleic acids can be ON A or RNA. Additional profened nucleic acids encode amlbodiea thus specs See! K hind (o HEDld (SEQ ID NO. Icy A EDS, iSEO H> NO: 13 i, Λ EDI'. (SEQ ID NO; ME HEDA, (SEQ ID NO: 15) or TED.E, (SEQ ID NO, 16) or oihe* XdEDX; peptides Hated above. A nucleic acid segment .encoding an immunogen is typically linked to regulatory elements,, such as a promoter and enhancer, that allow expression, of the DMA segment tn the intended target cells of a patient, for expression in blood ceils, as is desirable for induction of an immune response, promoter and enhancer elements from light or heavy chain inirnunoglobuhn genes or (he (MV major intermediate early promoter and enhancer are suitable to direct expression. The linked regulatory elements and coding sequence* are afters cloned us to « vector For ,κί ministration of double-chain urvbhmbes, the two chains can be cloned t.n the same or separate vectors The nucleic seals encoding therapeutic agents of the invention can also encode at least one T coll epitope. The disclosure* herein which relate to the use of adjuvants and the use of carriers apply mmuis mutandis to rheir use with the nucleic acids encoding the lh.erapct.iiic agents of the present invention Λ number of viral vector systems arc. available including retroviral systems Air. v.y., I. tovrie sod Turner Cur. Ορια. (kua\ Dewlap. 3. 102-109 < 1993)): adenoviral vectors Gee. s..g.. Sen et al.../. 17m/. 67, 5911 (10)3)).. adeno-associated virus vetrors (see, e.„g., Zhou ct uL, J. E.ks>: ,UV<7. 179. IK67 (1994)}.. viral vectors from rhe pox family including vaccinia vims and the avian pox viruses, viral vectors from the alpha virus genus such as those derived from Sindbis and Semliki Forest Viruses (see., e,q.. Dubcnxky ct al.,,./, DA,·/ 70, 503-519 Π9%)}, Veneatdan equine encephalitis virus Gee OS 5.6-17576) and rhabdo\iru;'Cs, such us vesicular stomatitis virus (see WO 96 34625) ;5nd papillomaviruses (One ct a].. Huuinu Geee Therapy 6, 325-333 (1995); Woo e( aL,: WO 94/121)29 And Xiao &amp;. Bmiidso% Mmfeid ,4eMv, IfeU 24, 2420-2622 {i 996)}. DNA encoding nn mummogen, or a vector containing she same. cat: he packaged into liposomes, Suitable lipids and related analog.' arc described by US 5.20X.024, 5,264,0lb, 5-2/4,//2 and 5.2>U .185, Yoenjrs and DNA encoding an immunogen can a!.so be adsorbed to or . associated -with particulate earners, examples of which include poly methyl methacrylate polymers and poiyiaettdcs and polytlactiuc-co-glseolidcs). see, r,.g.. McGee es el, A .sfu ;·<> Εηαψ 09%),

Gene therapy vectors or naked. ΡΝΛ can be delivered in vivo h> administration to an individual patient, typically by systemic administration ύ·.ο . intravenous, isurnpcritoneal. nasal gastric, imrudermal, intramuscular, subdermal or intracranial infusion) or topical application (sVe cap, US 5,299,240) Such vectors can further include facilitating agents such ;-w buptvucme I US 5,5i!X9'/0}. ON A can also be administered using a gene gun. tScc Xiao &amp; 8ran-jsrna, vu/ii'i?,;· l he DNA encoding an irnnnmogert i.s precipitated onto the surface of rrsicroscopic metal heeds. The mtcropmiceiilcs are accelerated whit a shock wave or expanding helium gas, mui pcnetrale /issues to a depth of several ceil layers, Por example, The Aecel^: Gene Delivery: Device manufactured by Agacctns. Inc. Middleton Vvl is suitable. Alternatively, naked DNA can pass through skin into the blood stream .«imply by spotting the DNA onto skit; with chemical or mechanical irritation (see WO 95/05853).

In a further variation, vectors encoding immunogens tr.au be delivered to ceils es vivo, such as cells cxplanted from an individual pat tent (¢..--,9-, lymphocytes, bone marrow asptm?cs, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a pahent, usually alter selection for c-...-.1 is ν,-ineh have incorporated the vector. DC, Adjuvants immunogenic agents of the invention: such as peptides, arc sometimes administered in combination with tut adjuvant. The adjuvant increases the (her of induced antibodies and/or dtc binding affinity of induced antibodies relative to the situation if the peptide wem used alone. Λ variety of udjnvu-tis can be used in combination with an immunogenic fragment of A A, to elicit an immune response. Preferred adjuvants augment the intrinsic response to an immunogen without causing conformational changes in the immunogen that affect the qualitative form of the response. Preferred adjuvants include aluminum hydroxide and aluminum phosphate. 2 Dc-O-race kited monophosphoryl kpso A {MPL{we GB 22202U (RJBt imninnoChom Research Inc., Hank Hon, Montana, now pan* of (.'oris; a), R( -524 (Conxa. Hermkosm Montana } STlMl.?i.ONiS! QS--2 ; la a triumpeue glycoside or saponin isolated iron; He; bark of the Quilinia Sa|-H>;uuna Medina tree found is South America Gee Kcns.il er oL, is Vavritn· DoJgn: Da· Subunit ra;;/ -UipiVtu-i! H/>/cawed {eds, Powell &amp; Newman, Plenum Press. NY, i!>->5t, US Pa cent No 5,05'l540y fAqusla BioPhansaceuhcels, Framingham, MA), Other adjuvants arc oil is water emulsions {such as squaleno or peanut oil), optkmstlty in combination with 'immune stimulants, such as monophosphotyi lipid Λ gwv Stoute c; η/., K. Ertyl. J. Λ33b, 8PMI Π 997)),-plurcmic polymers, and killed mycobacteria. Another adjuvant is CpO tWO IW). Adjuvants can be administered ax a component of a the-apes?ic composition with art active agent or cast be administered separately, before, coneurremiy with, or a her administration of the : her open tic agent.

A preferred class of Ml to, a ms is a heron urn salts (alum's, .such a·, alum hvdroxkie, alum phosphate, Yura sulfate. Such adjuvants can be used with or without other specific irmsmnosiirmilating agents sue It as MPf. or 3- DMP, OS-21. polymeric or monomeric craw, so adds such us polyglufamk acid or polyiysino. Another class of adjuvants Is oilon-water emuMop: formuktionp. Spelt adjuvhois mxi bo psod with or without other xpeeifk immunosrimuiuting agents such as mommy! peptides · c.g., Nracciyhouran;G--L--thr-on;yu-D-isoglutarninc i uh.r-MDPt, N~acctyt-noi'muraniyt-L*ala'nyi'-D-isogiuiarninc fuor-MDP), N-aectylmuriimyi-L-nlanyt-D-isogiutaminy!-L.-atau!nc-2-( t'-i'dipuiounsyl-sn-giyccm-.j - hydroxyphosphoryloxyKHhylantiiiC ΐ'ΜΤΡ--ΡΪ:Λ No-iCCoHghoiSi-HontyhNo-KWCyhritiruxnyhL-'A.hrO isoghi-l .-Aiu-dipuimuosY propylanudc (DTP-DPP; THPR AMI HI' Ί, or other bacterial cell wall components. Oiiomwuter emulsions Include (a) MF59 tWO b0"M83'H, containing 5'*« Sip lake ic, 0.:11, Tween 80, and 0.5"*·. Span 15 t optional t v containing various amounts of ΜΓΡ-Pk) formulated into submicron panicles using a raicrofkudiccr such as Model MOV rmercHhedracT iMicroOuidies. Newton \IAj, (hi SAP, conic;rung i0?« Sqttatene. 0.4'hi Tween 80, 5h> piuronic-biockcil polymer U21, and thr-MDP, either no craubn diced into a submicron emulstott or vortexed to generate a larger particle sure emulsion, and fej R.tBFv' adjuvant ssstern fRA.Si, (Ribt tmtmtnoChem, Hamilton, ΥΓΠ containing 2% sguYcne. 0,2 A Twees; Kb, and one or more bacterial cell wall components from the group eonseemg οί η; o nop hasp h ray lipid ,Λ tMPL t. trehalose dimycoiatc (TDM), and ceil wall skeleton (CWS's, preferably MPL CWS (detox:»').

Another class of preferred adjuvants is saponin adjuvants, such as XTlMU1.0Nis: <QS-2!, Aqmh, Framingham, MAI ns particles generated there frmn such as ISCOMs (iranuimtshmulaltng eomplc.xe.s) and ISCOMATRIX Other adiisvank include RC-thXT GM-CSF and ( ompicte l-'ieund's Adjuxant ΑΤ,Μ and Incomplete ftvund’s Adjuvant UFA?. Other . adjuvants Include c>tokines. such as Interleukins p.-gr,. It··] >x and |j peptides,. IL-4, iL 6. 'll -12, IDLE and II.-IdK toacropbuge colony -nimulatinq factor tM-<‘SB. granulocyte·· macrophage colon) stimulating fact or (GM-i. SFf tinner o>,-co>'s4 factor Π'ΝΓ'κ cbemokinex. such as MIPki and fi and RANTES. Another class of adjuvants is glyeolipid analogues including N--glyco^yiamides, N-glyco$y!umss and N-g)yeos> Icarbamates. each oF which is •wbAeuted in the sugar residue by an amino acids a\ immus-o-moduiaiors or adjuvants (sa- IAS Pat. No. 4,855,2b.)?. Heat .shock proteins, v.,g., HSP70 and HSP^O, may also be used as adjuvants.

An adjuvant can be ad mint.stored with an immunogen as a .single composition, or can be administered before, concurrent with, or after administration of the immunogen. Immunogen and adjuvant can be packaged and supplied in the same vlad or can be packaged in .separate vials and mixed before use. immunogen and adjuvant are typically packaged with a label indicating the intended therapeutic application If immunogen and adjuvant are packaged separately, the packaging typically includes instructions for mixing before use. The choice of an adjuvant and/or carrier depends on the stability of the immunogenic formulation containing the adjuvant, the route of admin tsiratlon, the dosing schedule, the efficacy of the adjuvant for the species feme, vaccinated, and, in humans, a pharmaceutically acceptable adjuvant is one that lias been approved or is improvable for human administration by pertinent regulatory bodies. For example. Complete Round» adjuvant Is not suitable for human administration. Alum, MPL and QS-21 are preferred. Optionally, two or score dliferent adjuvants van be used simultaneously. Preferred com Hina items include alum with MFL. alum with QS-2K MPL with QSOI« MPL or RC-529 with (ΊΜ-CSF. and alum. Qv 21 and MPL together. Also,. Incomplete.Frctmd'sadjuvant car; be u.'Cd {(.’hung at Advance*! Drug Delivery AwL-as 32, l?3-|x<> i 1998)1, optionally in combination with any of alum. QS-.?. !, and MPL and ail combinations thereof. .X. Pass s', e Adtnmislration Of Antibodies

Therapeutic agents of Ihe present mvesuion include antibodies that specifically hind to to an epitope comprising. .XsEDX; in an aggregated amyloid piotein. whorem Xt ns Η. T, F. S, P, A or any other asnino acid residue immediately preceding ED in such aggregated amyloid protein; and wherein K> X T.. X. E, IX I, Y, F. A or any other amino acid n.-stdue immediately following ED in such aggregated amyloid protent, including, epitopes within amyloid peptides such as A A. 1 he antibodies used for passive administration can be urn* bodies that hind to G. terminal or N-rcrminai epitopes of ΛΛ. Other amyloid protems m addition to Serum amyloid A protein include serum amyloid A protein, immunoglobulin light chain protein, such as. tor example. Va6 Wil or Vk. human islet amyloid precursor polypeptide OAPP). beta amyloid peptide, iransthvretitt HTR) and ApoAl. as well as others listed m Table 1 shove. ΑΛ Is formed by proteolytic e kin age of S.AA. Preferred antibodies specifically bind to neoepi topes of A A which term upon proteolytic cleavage ofXAA. Preferred antibodies spec only bind to a C-terminal neoepitope of ΑΛ, especially, such ami bodies specifically bind ίο HAAI alpha iso form within residues 70-76 (GHGAEDS, SEQ ID NO:4T HAAI beta isoform wnhin residues 70-70 (GHDAbDX, SEQ ID NO:5), HAAI gumma isofosm wait in residues "0--'O (GEICfAOXS, SEQ ID NO; 5), HA Ac alpha and beta iso fori ns widest residues 70-70 i GHGAEDS, SEQ ID NO; 10). HA \3 withm ressdmm 70- "6 s G DH A ET>S, SEQ ID NO;?). Η.ΑΛ 1 within residues ?*X-I {ST VI EDS, SEE) ID NO XT mouse .ΑΛΙ i XIA A Π within residues 69- 75 (GIIGHEDT. SEQ ID NO:9), ΜΑΛ2 within residues 69-75 tGRGHEDT. SEQ ID NO. M.-V\3 within residues 67-AS (GHGAEDS, SEQ ID NO-10). end ΜΛΛ4 wuhin residues "6-X2 (NEIGLETL SEQ ID NO:lit. Sente aniibodie- only bind to an epitope within one of these peptides. Other antibodies bind to epitopes within snore than one ot dun-c peptides. E'er example, some antibodies specifically bind to a GHGAEDS.. I SEQ ID NO- -I) peptide ami a GHDAEDS. SEQ ID NO: 5) peptide. Some antibodies hind to a GHGAEDS. SEQ) ID NO: 4) peptide without ewe;beady binding, to a GHDAEDS, SEQ ID NO: 5s peptide. Binding to as least one of the human ΛΛ peptides is pro ter able. Binding to as leas! one of the human A Λ peptides iissd a corresponding mou&amp;e peptide is use in i in thtss the same antibody can be tented in a mouse model and aubsequeufiy used in humans. Seine preferred am;bodies specifically bind to epitopes within ΗΛA1 alpha isefonn residues 71-76, 72-70, 73-7A 74-76, 70-75, 7 0-7-1, 70-7.3, 70- λ;, ?|.?5, 7.2-75, 71-74, 71-7.1. 7.7-74, or MAAI residues 70-75, 71-75, 7.2-75, 73Q5. 69-74, 69-75, 64-72, 69-1' I, "Ό-'-d. 71-4, 'G--''4, 7-)-73, 70--72. Such antibodies typically specifically bind to amyloid deposits but rnay or may not bind so soluble AA. When an antibody is .said to .specificdliy bind to an epitope within specified residues, such as ΕΙΑΛ I alpha isofemi residues 70-76 of tor dxampiQ what is meant. is that, the antibody sitooilcalb Mb# to a polypeptide containing the specified residues tie.,, residue's "0-T ofHAAl alpha i.sofomt in this an example). Such an antibody dees not necessarily contact cuts iesiduc within residues 70 Qo of HA AI fdpha isotbnr:. Nor does esery single amino acid substitution or debt ton o ith in residues 70-76 of HA AI alpha isofonn necessarily significantly a fleet binding affinity. Stteh ncnepltopc antibodies hind to A.A hut not to SAA. Epitope specificity of an antibody can he determined, .fbt example, as described by WO 00 ,'72SXt.i,

The antibodies used for passive administration can be antibodies to N-terminal epitopes of A A. Preferred antibodies .specifically bind to a N-fennina! neoepitopc of AA, especially, such antibodies specifically bind to ΗAAI residues 1-15 tRSFFSFLGE AFDGAR, SEQ ID NO. SO), HAA2 residues 1-15 (RSFfSfLGEAfDGAR., SEQ ID NO. SO). HA A.) residues 1-15 (QGWiFIPLKAAGQGAK, SEQ ID NO; SI), ΗΛ.Α4 residues :--15 (ESWR.SFEK.EA, iSiX) ID NO: Nat. MAAi residues IΊ5 iGFFSFVEUfAFQG AGO, SFQ ID NO: S3), MAA2 residues M5 (GEFSFVHEAFQGAGD. SFQ ID NO: ED. ΝΊΑΑ3 residues Kb (EAGQGSRD. (SFQ ID NO: S-4). and residues DM MAA4 (WYSFFRFAVQGTWD. SEQ ID NO: *5) Some antibodies only bind to an epu'ope w ohm one of these peptnkw. Other antibodies Dud to cpuojvs oidsm move than one of iheA pen?ides. For esample, some antibodies speeTcuik bind to a RSEFSFLGPAPDG AR, SFQ ID NO SO» p. pfsde .urn a QGWI ITU\A \O0‘) \k, SFQ ID NO: s I} peptide. Sonii. antibodies Find to a RSFI'SElGl \ΠΧ? Ms, SFQ U> N'O NO} pjpude without specsEcaiE binding m a QQ’A I I'll K Μ\(,ΧΧ'ΚΤν, SFQ ID V.» 61} peptide Binding to at iea-t ouc of she human \ A pep-setes t\ pi clemHe Rnsstnsg to tu lea'W ossc of the Imnnm A.A peptides and u eorroyn>n-.hnc, mouse peptide -s smese; tn tltat titc same antibody c.us In. tcsii.d its a mouse raod’l and snKequcnth used m humans.

Some amd'-ndies -pecdie,;U\ bmd v- sis': epuope conseomy of-uett .TED.V· Preferably such antibodies vpcs'ilk'ulK bus si -o -neb epitope m at; aggregated amyloid ptoieit; borne of such antibodies ptesetentiall·, spcedTNK bind to jut uggiegafed annsoid protent ?el,ns\c to the monomeric form of sneb .msylc-M protein In sot no antibodies, X ;s Π. Γ I, X, P or A and S'; is T. S. F. D, R. L V, I or \ In -a ins. such antibodies, when is If .A- m for \. o he si X, ;> A. X; is S. T, F nr \ when \j us T \ - is F; w ben X, ;s F. \ is D, when X: ;s X. X. s- Γ. f ο?' A; and when X; is F, V ts I I or F. In wan·..· wwl-ndsc,-., S; >\ ί I, l F, X, R, or \ and X; is I X. F, D. R, f V, E or A with the p?o<.;so than if X: is A X- os not \ In snnte antibodies, \shen Xf is A, X2 is S, T or E.

Some antibodies specifically bind an epitope comprising 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 antibodies specifically bind to a peptide comprising 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 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 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) 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 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 70-76 of 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, US 5,693,761, US 5,585,089, US 5,530,101 and Winter, US 5,225,539), or human (Lonberg et al., W093/12227 (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)). An alternative approach for humanizing an antibody, also known as veneering, is described in US 6,797,492. Several mouse antibodies of different binding specificities are available as starting materials for making humanized antibodies.

Representative humanized antibodies are humanized version 7D8 antibody (ATCC Accession Number 9468), humanized version 7D29 antibody, humanized version 7D19 antibody, humanized version 7D47 antibody, humanized version 7D39 antibody, humanized version 7D66 antibody, humanized version 8G9 antibody, humanized version 8G3 antibody, humanized version 8G4 antibody, humanized version 8G51 antibody, humanized version 8G22 antibody, humanized version 8G30 antibody, humanized version 8G46 antibody, humanized version 2A4 antibody (ATCC Accession Number 9662), humanized version 2A20 antibody, humanized version 2A44 antibody, humanized version 2A77 antibody, humanized version 2A13 antibody, and humanized version 2A14 antibody. Hybridomas that produce the 7D8 antibody (JH80 7D8.29.19.47) and the 2A4 antibody (JH80 2A4.20.44077) were depositied on September 4, 2008, and on December 17, 2008, respectively, with the American Type Culture Collection (ATCC), currently located at 10801 University Boulevard, Manassas, VA20110-2209, under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure (“Budapest Treaty”). The ATCC has assigned the hybridoma producing 7D8 ATCC Accession No. 9468, and the hybridoma producing 2A4 ATCC Accession No. 9662.

Human isotype IgGl is preferred for antibodies to the C terminal region of AA because of it having highest affinity of human isotypes for the FcRI receptor on phagocytic cells. Some antibodies specifically bind to AA with a binding affinity greater than or equal to about 107, 10s, 109, or 1010ΜΛ

Active immunization with fragments of AA can be combined with passive administration of antibodies. Examples of specific combinations include AA fragments comprising HAA1 alpha isoform residues 70-76 with antibodies that specifically bind to epitope within HAA1 alpha isoform residues 70-76; AA fragments comprising HAA1 alpha isoform residues 70-76 with

antibodies that specifically bind to epitope within HAA1 alpha isoform residues 71-76; AA fragments comprising HAA1 alpha isoform residues 70-76 with antibodies that specifically bind to epitope within HAA1 alpha isoform residues 72-76; AA fragments comprising HAA1 alpha isoform residues 70-76 with antibodies that specifically bind to epitope within HAA1 alpha isoform residues 73-76; AA fragments comprising HAA1 alpha isoform residues 70-76 with

antibodies that specifically bind to epitope within HAA1 alpha isoform residues 74-76; AA fragments comprising HAA1 alpha isoform residues 70-76 with antibodies that specifically bind to epitope within HAA1 alpha isoform residues 70-75; AA fragments comprising HAA1 alpha isoform residues 70-76 with antibodies that specifically bind to epitope width; ΗΛΑΙ alpha iaoform residues "0—4, \A irugmenta comprcnug H.AAI alpha laotofn's residues "O-AS wdh antibodies th.» -p-e-ohc;hi> bind m epitope vclihtn ΗΑΛ1 alpha isciorm residues 70-“V ΛΛ (Vagmema comm'Ding HaA; alpha isofcum residues 70-?*' a, nh antibodies that specifically bind to epitope within ΗΛΑΙ alpha 1 so form residues At-Ah AA fragments t.\>t uprising HA. AI alpha isofurm residues 70-76 w hh anti boon, a non apachh ally Land to epiume malan ΗΛΑΙ alpha iaoform residues Ή-'·.0.; A A fragmems corripriMng H'VAI alpha iaoibrm residues "0- Aj ohh antibodies mm speed icali> brad m spitope within HAA I alphas isciorm residues 72~“5t Λ.\ (Vayrnents comprising ΗΛΛΙ alpha isoform marines 70-76 sbth antd'odsea hat .specifically bind to epitope within HA AI alphas Ho form residues >%?>. \.A fragments cmuprising Η,ΑΛΙ alpha isoform re.-unues 70-76 w stb antibodies that spaa:heady hied to epitope within ΗΑΛ1 alpha iaoform residues 7 d a A fray mere a comprtaing ΗΑΛ1 alpha iso loop residues ?0-7o wbh antibodies that specifically bind to epitope within HA.M alpha Hoibrm residues 7|.'/4; Λ A fmgmems comprising ΗΛΛ1 alpha isotorm residues A) mg with antibodlea that spew ;fu. ally bind to epitope width·; ΗΛΑ1 alpha inform residues 7{-7.b Λ A fragments composing ΠΑΛΙ alpha isoiorm residues 70-76 w;th an ft bod tea that spemftcaily hind to epitope within 11 Ads I aloha iso form residues 7.7.-7-1. Addi-iorndiy, Λ Λ fragments comprising H.AAI alpha isoform residues 71-70, --2-76, 73-Μ. M-M, 70-75, 70-74, 70-73, 70-72, A-eh, m'-M, 7373, "H-"··-:, 71-A-i, '73-7-1 rna> be conjoined with antibodies that apeciibalty bind to an epitope wititin ΗΛΑ I alpha taotbrm residues 71-76, Abb-,. 73~?o, M-M 70-75. ^O-m. 70-7.3, 70-72,

71-75, 72-75, 73-"5. 71-74, 7f-73. 72-7-4. A A fragments composing HAAS alpha isoform residues ?0-~6. ΗΛΑ; beta is-, ho rut residues 70-76, H.AAI yarn mu iso form residue "'0-76, H.AA2 alpha and beta isoforms residues 70-76, Al A A ; icaiduea 6-).'A, ΜΑΛ2 residues 69-75. or M.AA.a residues 62-68 mas be corn hi su'd with antibodies that speeineaib, bind to an epitope within ΗΑΛΙ alpha iso form residues 7()-76, Η AA 1 beta isoform residues ?0-7o. HAAS gamma iso-orm residue 70-7(>, HaA .2 alpha and b-eta -soiorma residues 70-76, ΜΛ.-Μ residues 60-75. ΜΛ A2 residues 654-73. or MAA3 residues p2-6X

Some of the antibodies described above do not specifically bind the monomeric or precursor form of the amy toid protein. Some of such antibodies specifically bind to a neoepitopc generated -upon cleavage of a precursor protein rest-ding tn an amyloid protein. For example, some antibodies specifically bind to the C uermiuai residues of mouse A A fibrils -II EDI, (SL'Q ID NO. ! 2). but do not specifically bmd to a peptide that us tends into the non-amyloid portion of SAA {GHEDTMADQE, Sb'Q ID AO: M}. borne amt bodies specifically bind to a conformational epitope. Some of such conformational epitopes are linear. Some of such conformational epitopes are exposed when an amyloid protein enters an aggregated (e.g., fibrillar) structure or becomes partially denatured. Examples of such antibodies include murine monoclonal antibodies 2A4 (ATCC Accession Number 9662), 8G9 and 7D8 (ATCC Accession Number 9468), human, humanized and chimeric forms thereof, other antibodies that specifically bind to the same epitope as 2A4, 8G9 or 7D8, and antigen-binding fragments of any such antibodies. Some antibodies specifically bind to an amyloid protein comprising the amino acid sequence ED. Some antibodies specifically bind to an amyloid protein selected from the group consisting of immunoglobulin light chain 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. Each tetramer 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 chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. 1. Antibodies

The invention includes intact antibodies and antigen-binding antibody fragments, as 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 or substituted residues in the Fc region. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab’)2 tri-Fab’, Fab’, Fv, scFv, di-Fab’ fragments which can be generated by treating the antibody with an enzyme such as pepsin or produced by art-recognized recombinant engineering techniques. Additional antigen-binding fragments of antibodies of the invention include therapeutic antibody fragments, including pegylated antibody fragments, such as PEGylated Fab’ and PEGylated di-Fab’. Examples of effector function mutants are described in U.S. Patent No. 5,624,821, which is incorporated by reference herein in its entirety. Some antibodies have reduced binding affinity for Fc gamma RI receptor. Effector function mutant antibodies include antibodies comprising mutations in the hinge region. Some mutant IgG antibodies comprise a mutation in the heavy chain constant region at one or more of positions 234, 235, 236, 237, 297, 318, 320 and 322. In some antibodies one or more of residues .734, 274 and 2 7“ u-e xubsthuted with aktnme. In some A: bodies, residue 275 is substituted w;?b gbusiTune. In sonic uribbodww residue 297 is snbstutncd with alanine In some antibodies, residues 718, 7-70 end 777 ere substituted wid; akmate. In some antibodies, residue 718 is substituted wid; saline, in some antibodies, residue 322 is substituted with glutamine. Antibodies with enhanced effector Seme non include antibodies sonde S239D and 13321:. end the double and triple mutants S239D 1332E and S239D/I332ir. 'a.)3QL (Rabatmrmbendgj, 2, ihxlyekuuu .Antibodies

Potyciooa; antibodies can be prepared as described above by unrnunwino a suitable subject with nn immunogen. The antibody titer in the immunized subject' can be monitored over time by standard teehntqt.es, such as with an enzyme linked immunosorbent assay «ELISA) using Itnmobuiecd target antigen, if desired, the antibody molecules directed against the target antigen cart be isolated hont the mammal (e.g., from the bloodi and further purified by well known techniques, such ns protein Λ Sepbaro.sc chromatography to obtain the antibody, e.g\, IgG, fraction. At an appropriate bmc after immure cation. *·.<>.. when the rn'ihuunsgcn antibody filers arc highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies In standard techniques, such as the hyhridoma technique originally described by Kohler and Mtisiein { B>75> Sutmc 256:491.49?) (see also. Brown w uL (198! 1 J\ himnmoi. i 2 ?;539.46; Brown v; «'/. (19801.0 Βίο/. Chew .255:-8-^0--83: Vche/u/, (19'h>)/hue. 77;λ;/ Ar<j<J Si'i CS.-ί 77,-3927-31; and Veit rf »/. (1982; Inf. ,/. Gnuc-r 29:269-75). For the preparation ol’ebimenc polyclonal antibodies, see Bucehlesv?;?/. U.S Patent No 6,420,1 I V 3. Monoclonal .Antibodies

Any of the muse, well known ptmuenN used for fnstng lymphocytes and humortalLed ceil lines can be applied for the purpose of gs-ucrating a monoclonal antibody (see, eg , Cl. O-dtrc of >:,;/ 11977? .-Vjfii/Y cow5.3)52; Getter <.> al. 3Gw\-,0.-- C< // (huv;., cited vu/uv, I erner, huh J Biot. .1 /;';/.. cited .o«;vu: Kenneth, tbu.m , t *??//·; a/Gw coed su/mR. Mm cover, die ordmunh skilled worker wdl appreciate that the·s' are m no. summons of such methods w Itich also would be useful, typically, the immortal cell line to g . a me.doma cell ImM is derived from the .o.unr mammalian species as the lymphocytes, for example, murine hyhridoma··- can he made by fusing lymphocytes from a mouse imrntnuned wurh an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell hues that are sensitive to culture medium con burn ng hypoxauthine, aminopterin and thymidine (’’RAT medium''). Any of a number of myeloma cell linos am be used as a fusion partner according to stand-.ltd techniques, eg:;... the P3-NSI. I-Ag4-L P3-v63-Ag8.o53 or Sp2 O-4gl4 myeloma lines. These myeloma hues are available: trorn ATCC. Typically, HAT-scnsitivc mouse myeloma eeU>i are fuserl to mouse splenocytcs using polyethylene. glycol ffPBCPW Bybndoma cells resulting front the fusion arc (best selected using MAT medium, which, kills tot fused and nnproduciivdy fused myeloma cells (unty-ed splenoeytes die after several days because they arc not transformed). Hybndoma cells producing a monoclonal antibody of the invention are detected by screening the hybndoma culture supernatants lor antibodies that bind a target antigen, e..g., Αβ. usings standard BLISA assay. 4, Recomb i re; η t Λ n i bod ies

Altemarive to preparing monoclonal anttlxxiy-sccretmg hybridoma**, a monoclonal antibody can he identified and isolated by screening a recomb 5 mu a combinatorial tmtuunoglobuim library (c.g., an amibods phage display library) with a target and gen to thereby isolate immunoglobulin librarx members -hat bind the mrew antigen. Kits for generating and screening phage display libraries are commercially available tog., the Pharmacia Hn-omhi/iattf PhiiAutibady System. Catalog No. 27-9400-01; and the Mmmgeoe .WOelr5M /-Vmgv Ουρίαν Kip Catalog No. 740M2). Additionally., examples of methods and reagents particularly amenable for use in generating and screening antibody display library cun be found in, for example, Ladnerg«/. U.8. Patent No. 0,.:23,409: Rang mo/. PC? International Publication No. WO 92 I kb 19; Dower om. PCX International Publication No, WO 91 17271: Winter w a/. PC'f International Publication WO 92'20791; Mark I and 0/ <<!. PCX international Publieation No. WO 9215679: Breitling a ui. PC'T Intentoiorsal PubHmukm Vv'O 93 01288.: McCatYeriy ¢ / n/. PCX International Publication No. Vv'O 92 01047. Garrard *;/ af. PCX International Publication No. WO 92/0969(): iudner ο ο.ί. PCT InvermuionO Publication No. WO 90 02809; Fuchs et u/. (1991) B>o.-Ta:Km>bi^\· 9:1370-1372: Hay 0 0/. 0992) than. Antihixi. //tiri'A /?><;./ 3X1-85; Huso or a!. (190-9 NM.wv 216:12 75-128i; Griflitt* <·/ ul. ( 1993) £9//49,/ 12; "25-754; Hu* kins i'! at (1992)9. .1/1.;/. Βίοι. 226.889-896; Clarkson at. (1991) Katun' 352‘624-628. Goan c/ ¢//. 0992) Proa. W. AnU Sri. USA 99:3576-3580; Ciarrad d xi 0990 tib'Tpchmh&amp;v 9:i3?3· 1377: Hoogcnboom O 0/. (1991) A ere. Sax’ /6·: s. 19:41.13-4 I 37; Barbas O al. t 199() proc. Sad. Acad. Sci USA 88:7978.7982, and McCaflcrty m 0/ Katun 11990) 348-552154. 5. Chimeric and Humanmed Antibodies

Additionally. recombinant antibodies, such as chimeric and humaowed monoclonal antibodies, comprising both hitman and non-human portions, which can he made using standard reeorttbinant DMA techniques. arc within the scope of the invention

The term “humanized immunoglobulin" or “htmvam/ed refers; te »s immunoglobulin or antibody that include-. m least one humanized immunoglobulin or antibody chain t/.e.. at least one humanized light or heavy chain.!. The torn·; "humanized immunoglobulin chain'' or “humanized antibody chain'' a ' hnnu.au/od immunoglobulin light, chain" or “humanized immunoglobulin heavy chain") refers lo an immunoglobulin or antibody chain Ife.. n light or heavy chain, respectively) having a variable region that includes a variable framework region substantially horn a human inununogiobuhn or antibody and complementarity determining regions (f'DRs} {<.\.g,, at least one i'DR, preferably tun C'DRs. more preferably three ODRs't substaniiidly from a nor:-hunnm immunoglobulin or antibody, retd further includes constant regions tree., at least one constant region or portion thereof, in the case of a light chain, and three con at a rtf regions ist the case of a heavy chain). The term "humanized variable region" tV.g., “humanized light John variable region" or 'Itsmafthied heavy chain variable region'') refers >'o a variable region that includes a variable framework region substantially from a human Immunoglobulin or antibody and complementarity determining regions (CDRs) substantially farm a non-hitman immunoglobulin or antibody

The phrase “substantially from a human immunoglobulin or antibody" or “substantially human ' menus that, when aligned to a human mmiunogkd'ibm or antibody amino sequence for comparison purposes, the region shares at least SO-bO'w, d0-95i!e. or o5-db% identity {;.*·.< local sequence identify) with hm human framework or uOUMant regain sequence, allowing, for example, tor conservative substiruflons. consensus sequence substitution*. gertriline substitutions, baekrnutation.s. and the like. The introduction of conservative substitutions, consensus sequence substitutions, germline substitutions, haibrntnutions. and the like, is often referred to as “optimization" of a humanized antibody or chain. The phrase ".substantially from a non-human immunoglobulin or antibody' or “substanficily non-human" means having an immunoglobulin or antibody sequence at least hO-°S%, preferably at least RO-Aew. more preferably. do";, 97'1··.. df'M, or On·1..., identical to that of a non-human organism. c,g., a nonhuman mammal

Accordingly, «It regions or residues of a humanized immunoglobulin or antibody, or of a humanized immunoglobulin or antibody chain, except the ODRs, are substantially identical to the corresponding, regions or residues of one or mote nath-e human immunoglobulin sequences. The term “corresponding region" or "corresponding residue"' refers to a region or residue on a second amino acid or nuclooude sequence which occupies ihc same ii ;/., equivalent, position as a region or residue on a first amino acid or nucleotide sequence, when the ftrst and second sequences are optimally aligned for comparison purposes.

The term ‘'significant identity" means that two polypeptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at toast 5()-ό0% sequence identity, preferably at least 60-70% sequence identity, more preferably at least 7060% sequence identity. more preferably at least 80-90% sequence identity, even more preferably at. least 9f}-9$% sequence identity, and even more preferably at least 95% sequence identity or nx>;v {i.y.. 9;t% sequence identity or more? The term ''substantia; identity" means that two polypeptide sequences, when optimally aligned, such as by the programs GAP or BP.STHT using default gap weights, share at least 89-90% sequence identity,, preferably at least Τ>%5:!;. sequence identity, and more preferably at least 95% sequence identity or tno-c (e.g. °9V:·.., sequence identity or more). For sequence comparison, typically one sequence acts as a reference sequence, to winch test sequences arc compared. When using a sequence comparison algorithm.: rest and reference sequences are input Into a computer, subsequence coordinates are designated, tf necessary., and sequence algorithm progrant parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequences) relative to the reference sequence, based on the designated program parameters.

Optimal alignment of sequences ibr comparison can be conducted, eg., by the local homology algorithm of Smith &amp; Waterman. A A. bo/?/, /6//6. e:4H2 Π98Π. by the homology abgument algorithm of Needieman &amp; Wynsch,,/. Λ6,7 .66,7. -15:7-0 {1979), by the search for similarity method of Pearson ά Shipman, pr,,,.·. :\\u'/. Acad. Sn. ISA 85:2444 (1988 f, by computerised implementations of these algorithms {GAP, BEST PIT. PASTA, and TfASTA in the Wisconsin Genetics Software Package, Genetics Computer Group. 575 Science Dr.< Madison, Wl), or by visual inspection iwv gcw-rjiiy Aunubei ft a!.. Current Proaoeok tn Molecular· Biology). One example of algorithm that is suitable Ibr determining percent wqueuee identity and .sequence similarity is the BLAST .algorithm- which is described in Abschw w,//,./, .)/;>/. Bh>.·. .715:-10.¾ (1990). Software tor petforming BLAST analyses is publicly :x\tillable through the National (.'enter for Biotechnology Information {.publicly accessible through the National bust meres of Health NCBI internet server). Typically, default program parameters man be used to perform the sequence comparison, although customised parameters cun also he used. For amino acid sequences, the BLAST!3 program uses as defaults u word length (W) of 3, an expectation t ii.> of 10, and the BLOSPM 62.Hbhifcoff Pm&amp;:NatL A cud. Sec CSa 89-10015 (HJ^iji. ihmerably, residue position*· w hied are not identical diner by censers arise annuo sc id substitution*. For purposes of classifying ammo acids substitutions as conservative or uofK'tmserxtUive, amino acids are gwnmeo as follows· Group 1 (by dt'uphobie sidechaiuxf leu. met, ;da, miL leu, do; Group 11 (neutral hydrophilic side chainsr: cys. ser. thn Group ill (acidic side chaired' asp. gtu: Group IV (basic side chains}; mm, pin, h;s, iys. arg; Group V (residues influencing chain orientation); gly, pro. and Group VI (duomatic side chains}, up, tsc, pice. GonsereauY·,.· substitutions involve sacs via in does between arm no acids in the .same cla..->s. Nonconservative substitutions constitute exchanging a member of" one of these Gasses tor a member Of Mother.

Preierably, humanized imrmsnogloimlinx or antibodies bind antigen v-, uh an affinity that is within a factor of three, four, or foe of that of the corresponding non·humanized antibody. For example, if the noohmuamaed antibody has a binding affinity of 10" M, human mod antibodies will base a binding affinity of at least 3 < 10” M, ;1 x 10 ' M, 5 x I0' M, or 10' M. When describing the binding properties of an immunoglobulin or antibody chain, the chain can be described based on its ability to "direel antigen (t.y . Ab) binding" A chain is said to "direct antigen binding'' whet? it confers upon an intact mumumgloiwim or antibody tor antigen binding fragment thereof) a specific hauling property or binding affinity. A mammon (dog,, a backmutation) is said to substantially aficivt the ability of a heavy or Hghi chain is.? direct antigen binding tf ii affects (o.g., decreases? the binding affinity of an intact mmmnogmbuhn or antibody (or a mi mm bmdmg fragment thereof} comprising said chair; by ai least an order of magnitude compared to that oS’the antibody tor antigen binding fragment thereof} comprising an ccjuixulcnt chain lucking said mutation. A mutation "does not substantially affect (c.g., decrease 1 the ability of a chain to direct antigen binding" if u affects dee reuses'} the binding affinity of an imam immunoglobulin or antibody (or antigen bmdmg fragment thereof') composing said chain b\ only a factor oi two. three, or tour of that of the antibody tor antigen binding fragment thereof} ecmqmsnm an eon 1--. a lent chain jacking said roufaiiosi.

The term "chimeric immunoglobulin" or antibody refers to an Immunoglobulin or antibody whose variable regions derive from a first spemes and whose constant reasons dew.c iVom a second .species. Chimeric irnnutnogiobniins or antibodies cm; be constructed, tor example by genetic engineering, tVorn immunoglobulin gene segments bdemgmg to different .specif. The icrms^ Mut muni zed immunoglobulin" or "humanized antibody" am nor intended to encompass cbini-eric intmmK-giobuhns or ainibodios, ax defined tfdra. Although hnnenneed immunoglobulins or antibodies are chimeric rn their construction (be., com price regions from more than one spcci-e* of prorein}, they include additional foam re--- Hi.·., variable mgions comprising donor COR residues and acceptor framework residues) not found in chimeric immtioogluhulma or antibodies., as defined herein.

Such chimeric and humanized monoclonal antibodies can he produced by recombinant DMA techniques known in Uiv art, tor example using methods described in Robinson et at. international Application No. PCT/US86/02269; Akira,, et at. Eiuropejts Patent Application 18-4,187; TanjgucSn. M., European Patent Application 171,49-:1: Morrison et al burotvan Parent Application 17.>,494; Neuberger a' oi PCT internationai Publication "No. WO 80. Oi Am; Cahills et at. fJ,$. P&amp;ient No. 4,8 16,867: Cabiily et ai. Europerm Patent .Application 17.8,073: Better et ah Π988) Se/efux 240:1041-104 3: Liu es at. (1987) Proc. Nad. Acod. Set l-Adi K4:8434-344,}; Liu i f («'. (1,/, haain>'.>>!. (3^8521-3526. Sun <. ? at. { 1987) Proc. Sail. Acad. Si t. UNA 84:2:4-2IS; Nislumma ef at. 119x7» Can< Re\. 47.9^9-1005: Wood end. 11988} Nature 314:44(--440: and Slam-, m -d. 11088),/. Nad. ('em er Iasi. SO. 1588-15.50): Morrison, S, 1.,. (iO-85) Scicace 329. 1702-120·; Oi et ai i I08M /6o/m /md/em 4:2 14, Winter U.S. Patent 5,7.25,589; Jones et ai 119ko) ’wow J21:552-575: Vc-hoey m a" at. < 1988) Sr mere 230:1534; and Bcidler c< at. (1088) a tateeacd. 14).4058-40(4). Therapendc a gems also include antibody mimetics such as oomph.rnenun ivy uemnmning region li'DRs mimem>, 6, Human .Antibodies bom Transgenic Animals raid Phage Display

AhernatbeK. it is now possibh.. to produce transgenic animals leg., mice} dun arc capable, upon uurnuni/arion, of producitig a full repertoire of human antibodies in the. absence of endogenous smmimoglohubi; production. Lor example, it Dus been described that the hornoaygous dcieiiou of the antibod}' hem, y-ciimri iommg region t.h·) gene m ebimerie and gcrni-Hne mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human ueim-line immunoglobulin gene array in such yerm-hme mutant mice results in (he production of human antibodies upon antigen challenge. See, u.ig, ITS. Patau Nos. 6,150.584: 6,114.,598: ami 5.770.47.9.

Fully human antibodies can also be derived from phage-display libraries (iloogenboom v? o/., .1. Mol. Biol, 227:88] (1941 g Marks ,.(a! , J. Mol. 844.. 222'58l-W>7 {199}4. Chimeric polyclonal antibodies oars also ho obtained from phage d I-.play binaries ; Bucehier rt or l S Patent No. 6,431X1131, 7. Bsapcoiflo Antibodies. Antibody fusion Polypeptides, and Single-Chain Anti todies RBpcoirr a rm bodies ilia A ha s are antibodies fa as bar a binding specificities for at least two different eprtopes. Such antibodies can 6c ale π a of from It; Π length anybodies or antibody fragments {c,z ihahVd hiapoesfic nmibodiem, Methods for soaking hispcelfie antibodies arc known in die art bridhionu: production of full length hr-poohao antibodies is based ms she coex press ire of two immunoglobulin heavy chain-light chans pairs, where she wo chains have different specificities tMilktetn or <,>/., Nature, 305 537-536 U°S3)s Because of iho random assortment of immunoglobulin heavy and light chains, these hybndomu.s tunadromus) produce a. potential nsiNp-re of different antibody molecules (toe, WO 63 ObWb.·1 and in frtsunecker or u/., fc'MBO .1., ;0;3s05-3fAO (i op in. 1:6specific antibodies also moissrle eroas-iinked or 'hervroconiugale'' antibodies. For example, ora: of the antibodies m the heteruconingutc can be coupled so avidsn. the oboes' so biotin os' other payload Hcseroansjugurc antibodies may be mode using any convenient croxv linking methods. Suitable eross-l inking agents arc well ks>own m the ass. and arc disclosed in la.is. Pat, Ns.;. --Lb ox/WO,, along w ith a number of cross-linking techniques.

In yet smother aspect, the antibody can be fused, cheoueaby or ges-ci scully. to a payload such as a reactive, detectable. or functional moiety. -or exainnL·, att immunoioxin to produce ait antibody fission polypeptide. Such poyloads suciiide. for example. immutsoioxuss, ehemodierapcishos, and radioisotopes, all of which arc well-known in site ass

Single chain antibodies are also suitable for stabi I i ration according to the invention. The fragments comprise a heavy-chain variable domain fv'f h umrseeied to a light-chain variable domain (VI} with a linker, which allows each variable region so interface wish each other and recreate she antigen bisxiing pocket of she parent antibody from which the VI. and VH regions are derived. See Gruber ¢:/a/., 3 Immunol., 132:3368 (l =364¾.

Is is understood that any of the foregoing polypeptide molecules, sxlooc or in combination: are suitable for preparation as stabilised formulations accordhxg so she sn>ention. XI, Subjects Amenable To Treatment

Subjects or patients amenable to treatment includeindividuals at risk of disease but not ..showing symptoms, as well as patients presently showing symptoms. Therefore, the present methods can be administered prophylactiealiy to the general population without the need for any assessment of the risk of the subject patient. The present methods are especially useful for individuals who do have a known generic risk autoimmune disorders. Such individuals include· those having relatives who have experienced this disease and those whose «sf: is determined by analysis of genetic os· biochemical markers.

Patients suffering from A A s;}ny!oido#s-d^.|0^syt#td^id.fdr^:pi^Jo«ifCt.f$d^di:df •time. Therefore, disncal diagnosis of ΛΑ amyloidosis is often delayed or missed unit) the tons hold deposits are extensive. For those patients who are symptomatic, it is estimated that only S.fA of the cases are diagnosed, .ve I...!::. K. Consulting, independent Market Research (2003».

The invention provides methods useful to treat or effect prophylaxis of a disease charactermed by the deposition of an amyloid protein, such &amp;s, for example, the diseases described above, including those listed in Table I, Some methods arc useful to treat or effect prophylaxis of a disease characterized by the deposition of an amyloid protein comprising the amino acid sequence ED. In some methods, if the amyloid protein comprises the ammo acid sequence AEDV, then the antibody is not administered to treat or effect prophylaxis of •Alzheimer's disease or Mild Cognitive Impairment, The amyloid protein can be any of the amyloid proteins described above, including those listed in Table 1. such as, foi example, serum amyloid Λ protein, immunoglobulin light chant protein, such as. for example, v/,6 Wil or Vk, human islet amyloid precursor polypeptide tIAPP), beta amyloid peptide, transthyretin (TTR) or Λρο I.

The present methods arc especially useful for Individuals who do have a known risk of are suspected to have, or have been diagnosed with A A amyloidosis or A i. amyloidosis. Such individuals Include but are not limited to those hut lug chronic foilsmneeorv diseases, inherited Inflammatory diseases, artel chronic microbial infections, such as rheumatoid arthritis, juvenile chronic arthritis, anks losing spondylitis, psoriasis, psoriatic arthropathy, Reiter's syndroms'. Adult .Still:s disease. Behcet's syndrome, Crohn's dfoease. Familial Mediterranean Fever, leprosy, tuberculosis, bronchiectasis, decubitus ulcers, chronic pyelonephritis, osteomyelitis. Whipple's disease, myeloma, macroglobu 1 men iia. l-rununocyte tiy.wrasiu, monoclonal gammopathv, occult dyscMsia. Chronic· ndlurnnutnry and foicetious conditions are prerequisite to the development of A A amyloidosis end At. amyloidosis manifested by local nodular umyloidosi* can be associated with chrome inflammatory diseases. Individuals who do have known it'd·, of AA amyloidosis also include hut arc not limited to those having malignant .neoplasms as Ilodgkln's iymnhoma, reus! carcinoma, carcinomas of gut. long and urogenital tract, basal cell carcinoma, andhuiry cell leukemia. Additionally, individuals who do have 'knonti risk of ΛΑ amyloidosis ;jIso Include but a to not limited to those having 'iymphoproi iicr.duc disorders such as CusUernan's Disease. in both asymptomatic tmd symptomatic patients, treatment can begin at any time before or after the diagnosis of the underlying A Λ or At amyloid diseases. Treatment typically entails, multiple dosages over a period of time. Treatment cast ho monitored by assaying umibody. activated l~eclt {a side effect* o; B-ecll responses to the therapeutic agent (v.g., AA peptides, or employing radiolabeled SAP Scintigraphy over time It'the response fails, a booster dosage A Indicated XII, Treatment Regimes 1st general, treatment regimes invoke administering an agent effective to induce an immunogenic response to an amyloid protein, and preferably to an ..segregated form of such amyloid p rot cm. such as, for example. ΛΛ or AT. Preferably an immunogenic fragment os' Λ a or AI or an fragment is administered to a patient. In prophylactic applications, pharmaecutical compivsitlons or medicaments are administered to a patient susceptible to. or otherwise at risk of. amyloidosis such as A A Amyloidosis or T L amyloidosis, it! an amount sufficient 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, its complications and intermediate pathological phenotypes presenting during development of the disease. In therapeutic applications, an agent is administsred to a patient suspected of, or already suffering from such h. disease in a regime comprising an amount and frequency of administration of the agent sufficient to cure, or at least, partially arrest, or inhibit deterioration of the symptoms of the disease «physiological, biochemical, histologic and/or behavioral), including list complications and intermediate pathological phenotypes in development of the disease. In some methods, administration of agent reduces or eliminates early symptrtmoiogy in patients that have not yet developed characteristic AA or .AT Amyloidosis pathology. An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically'- or nrophylacticatly-effeclive dose. A combination of amount and dosage frequency adequate to . accomplish the therapeutic or prophylactic treatment ss defined ns a ihcmpeuticallx -· $r • pre^hy lacticaliy-dTccli vc regime. In both prophylactic ;-,nd therapeutic regimes, agents arc usually administered in sex era! dosages ntuil a sunleiem ire nos no response has been seines ed, Λ damage end frequency of administrations udeqtnno to accomplish therapeutic or prophylactic mourn enf is defined us a therapeutical h - or prophx laetmuK -effective regime Fype.afy, iI\<: patient's immune response is monitored and repeater.; dosages arc gsx on it the immune response·: stuns to wane, The immune response can be monitored bx detecting am thud ie». for example id A Λ or At. in the Mood in the patient or detecting lewis of. for example. ΛΛ or AL.

Tineciive doses of the agents and compositions of the present invention, for the treatment of the above described conditions xary depending upon many ddlefeut factors,, including means of administration, target site, physiological state of the padenr. whether the patient is human or an animal, other medications administered, and whether mmrmcnt is prophs lactic or therapeutic. Usually, the patient is a human but mm bum an mammals including transgenic mammals can also be treated. Treatment dosages need to- be titrated to optimize saiety and efficacy. The amount of immunogen depends on sv homer adjusant is also administered, with higher dosages being required In the absence of adjuvant. The amount of an immunogen for administration sometimes caries front l-500 ug per pattern and more usually from 5-500 ug per injection for human administration. Occasionally, a higher dose of 1-2 mg per injection is used. Typically at least 10, 20, 50 or 100 Up Is used for each human injection The mass of immunogen also depends on the mass ratio of immunogenic epitope within the immunogen to the mass of immunogen as a whole Typically, 10 ' to 10 " micromoles of immunogenic epitope are used for rmcrogram of-YarnImogen. The tuning of injections can sary sigmficamjy from once a day, to once a year, to once a decade. On any given day that a dosage of immunogen is given, the dosage is greater than t gg/patieni and usually g;osier than 10 jag.·' patient if adjuvant is also administered, and greater than 10 ug-patient and usually greater hum 100 ug/patiesu in the absence of adjuvant. A typical regimen consists of an immunization followed by booster injections at time Intervals, such ns 0 wed·: intervals. Another regimen consists of an mmnmiration followed by booster injections 1, 2 and 12 months knee Another regimen entails an injection every two months for life. Alternatively. booster injections can he on 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 nu to 100 mg, I μ-g to 18 .fng> or 30-3$#' ifg.et$t§ y£t§r:. from i 0* 100, or more, \ irions per Jose.

For passive immunicaHon with an antibody fin combination therapies κ the dosage, .ranges Worn about 0.000! i<> 100 mg4g, 0.5 to less than 5 mg kg. and triorc usualiy 0.GI to 5 mg, kg, 0.5 to 3 mg.kg, of the host body weight. For example dosages can be I mg'kg body-weight or 10 make body weight or within the range of 1 -; 0 mg'kg or in r>ther words, 70 mg or 700 mg or within the range of"?0—00 mg, respectively, for a 70 ug pattern. Ah an additional example, dosage» van be h;»s than 5 mglcg body weight or 1.5 mg/kg body weight or within the range of 0.5 to 1.5 mg. kg. preferably >it least 1.5 mg kg An exemplary treatment regime emails administration once per every two weeks or once a month or once every 5 to b months. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which ease die dosage of each antibody administered fells within the ranges indicated. Antibody is usually administered on multiple occasions Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular a* indicated by measuring blood levels of antibody ίο Λ A in the patient, in some methods, dosage is adjusted to achieve a plasma antibody concentration of ; - JOCK) ug.;ml atkl in some methods 35 -300 ug-ml. Alternatively, antibody can he administered as :.·, sustained release formulation, in which ease less frequent admmi.stratton is required. Dosage and frequency sure depending on the half-life of fee antibody in the patient, in general, human antibodies show the longest half life, followed by human used antibodies, chimeric antibodies, and nonhusuan antibodies The dosage and frequency of administration can very depending on whether the treatment is prophylactic or therapeutic, in prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time, Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short mens ah -s .sometimes required until progression of foe disease is reduced or terminaled, and preierabiy goal the patient shows partial or complete amelioration: of sympipnis of disease. Thereafter, the patent can be administered a prophylactic regime.

Agents for inducing an immune response can be administered by parenteral, topical, intravenous, oral, subet-tat terms. intraarterial, intracranial, intrapcrifoneal. inf ran a sal or intramuscular means for prophylactic and-or therapeutic treatment. The most typical route os administration of an immunogenic agent is subcutaneous although other ionics can be equally effective. The next most common route is intramuscular injection, Oris type of injection is most typically performed in the arm or leg muscles. in some methods, agents are injected directly into a eanicuiar tissue where deposits base accumulated, e.g., intracranial injection. Intramuscular injection of intravenous infusion is preferred for administration of anubody tin combination therapies), in some methods., nunicnlar therapeutic antibodies·» arc- injected direct!} into the .-cranium. In some methods, antibodies are administered as a sustained release composition or devteCx such as a MED!PAD1"’ dec Ice

Agents of the invention are often administered as pharmaceutical compositions comprising an active therapeutic agent, fo. and a variety of other pharmaceutically acceptable components. See /bam r; gnu? T Phci> f)nii.cntU\ii Srwf-ur {! 5th ed., Mack Publishing Company, Easton. Pennsylvania. 1980). The preferred form depends on the intended mode of administration arid therapeutic application. The compositions can also include, depending on the formulation desired, pharmaccuticaily-aeceptahie, non-toxic carriers or diluents, which arc defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological pho,sphaic-butiered saline, Ringers .solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other earners, adjuvants, or nooroxlc, nonthcrapeufic, noninmtnnogenic stabilizers and the like.

Pharmaceutical compositions can also include huge, slowly metabolixed ntaeromolecules such, as proteins, polysaccharides such as ohitasan, polylacdc acids, polyglycolic acids and copolymers (such as latex functionalized SEPS-i.AROSI:l!'\ ugarosc, cellulose, and the like), polymeric amino adds, amino acid copolymers, and lipid aggregates (such as oil droplets or hposomest. Additionally, these carriers can function as immunostimulating agents </.e., adjuvants). for parenteral administration, a gen vs of the invention can be administered as injectable dosages of a solution or suspension of the. substance in a physiologically acceptable diluent with a pharmaceutical carrier that cars be a sterile liquid such as water oils, saline, glycerol, or ethanol Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants. pH buttering substa.rn.es and the like can ho present in compositions. Other components of pharmaceutical compositions arc those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol arc preferred liquid eanictw, particularly for irhoetable solutions Antibodies can he administered in die form of a depot injection or implant preparation which can be formulated in such a manner us to penult a wnXiuned release of the active m grad sent At? exemplary composition comprises monoclonal antibody at 5 mg ml., formulated in aqueous bn Her consisting of Ή1 ntM f-histidine, 150 rnM NaCl, adp.tsted to pH 0 0 v, it It IlCVl. : Compositions for parenteral administration are typically substantially sterile, isotonic and: many met u red under GMP conditions of the PDA or similar body.

Typically, compositions arc prepared as injecrablcs, either as liquid solutions or suspensions; solid forms suitable for solution in. or suspension in. liquid vehicles prior to injection can also be prepared The preparation also cart be emulsified or encapsulated in liposomes or micro panicles such as poiylacside, polyglyeoltde, or copolymer for enhanced adjuvant effect, as discussed above tsee banger, Sck'n·.. c 249. 1.52? {1990} and Hanes, AdV'jm e<7 Drug DJh*n Revives 2b. 07-} 19 (,0707). The agents of this invention can be administered in the form of a depot injection or implant preparation which cast be formulated is- such a manner as to permit a sustained or pulsatile release of the active ingredient.

Additional formulations visitable for other modes of administration include oral, inira nasal, and pulmonary formulations, suppositories, and transdermai appl Gallons. for suppositories, binders and carriers include, for example, polyalbviene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0 5“·» to 102.», preferably PvTHa Oral formulation* include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions take tire form rd solutions, suspensions, tablets., pills, capsules, sustained release formulations or powders and contain I0%-95··'·, of active ingredient, preferably 25<}a-~0%.

Topical application can result in imnsdcrmai or inirademsal delivery. fopical administration can be facilitated by co-admttiisiraiion of the agent with cholera toxin or detoxified derivatives or subunits -hereof or other similar bnetennl toxins (Sec Glenn e/ o/.. Sawn- 391., 851 (199^)1. Co-admintsbmion can be achieved by using the components as a mixture or as linked molecules obtained by chemical cross! inking or expression a* a fusion protein.

Alternatively, iransdertnal delivery cart be achieved using a skin path or using rimsferosomes (Paul ct a I... £W·. J. (.wesum;.·/. 25. 55c I -24 1190sg <(\·νχ el. af, bVoc/ieni. /i/op/vvv. Xί11. CornbtstiUionai Drug IXcrapy 'inrun'scat Regime1, {"ombinaiion therapy according to the mvendon men· be perk stoned alone or in conjunct ion with .mother iherans to me·:» or effect prophylaxis of ·\Λ amyloidosis rombinatlon i h crap) according as die me cun'on may also ho performed to conjunction wiln another iherap* which neats or effects prop Its is sis of an underlying amyloid J mease such a a isdlummaiosy discuses,. chronic friserobtai la feet sons,. andsg:tutu neoplasms, mi ter hen ndlamotutory discuses, and hmphoptx>!iferanVe disorders, Thor·: arc large numbers of treatments available m commercial use, in clinical o\ a I nation and in prowl »»cj; development. winch emdd he aeka tod for uac with die presently de-ek>.wd in a canon her efface age prap-hyf.raia and treatment of ΛΛ ungvloidosw by , omhinadon drag, therapy. Such, treatments can he one or more cootpoitttds selected front, Inn not iimhed no several minor Onego nos. namely, tit non-ateroidai anti·· uhlinmrKaors drugs {NSAIDs; c.g., dmoprofan, dichdenao difhsnisal. etodolae, fenoprofbn, fkabiprofea. ihapmlcn. mdomethadm ketoproten. men ioieaamemc, mefenanne acid, mcloxicum. nahememte. naproxen sodium, oxaptcoin, piroxicarn, snimdac, tohnetim cclecoxth, tofeeuxih, uspirm. c holme saue\Luc, salsabe. and sodium and magnesium salicylates. (di sic raids (eg,, conlsone, dexam-cihasooe. hydrocortisone, metavlprcdinsoione, prednisolone, prednisoia., triamcinolone}; <du} DMARDs, disease modifying amirheumatte dupes teg., cyclosporine. a/nlhioprine. methotrexate. lefh.momide, cyclophosphamide, hvdro.xychloruqamc. sulthsaiaeine. D-p-eniedlannne. minocycline, and gold}; nr tic) recombinant proteins (c.u„ EMBREl.s?' (ctanercopR a -soluble TNF receptor) and RHMICADE*.^ (infliximab) a cliirncric monoclonal atui-fNF antibody;·.

The duration of the combination therapy depends on the type of underlying disease being treated, the age and condition of the patient. the stage and type of the patients disease, and Itow the patient responds to the treatment. The doctor cat; observe the therapy's effects closely and make any adjustments that are needed. Additionally, a person haying a greater risk of developing A A Amyloidosis (e.g.. a person who is gcsteiieally predisposed or previously had ait innanimatory disorder or other underlying diseases} or A.L amyloidosis may meed a prophylactic treatment to inhibit or delay the development of A A Al, aggregates such as fibnis.

The dosage, frequency and mode of admit',tstradon of each component of the cornMnatioi) can fee controlled mdependentiy.· For example, one ©orrspoutid may he admihisfered: 'orally three rimes per day, while the .second compound may be administered intramuscularly once per day, Combination therapy may be given m on-and-off cycles that include rest periods. The compounds ntav also be formulated together such .that one administration delivers boils compounds. The combination of the interdict) cm also be pros idea as components of a pharmaceutical pack. The drugs can be formulated together or separately and in individual dosage amounts. Each compound is admixed with a suitable earner substance, and is generally present in an amount of 1 -95% hy weight of the tend weight of the composition.

The composition may be provided in a dosage form that us suitable for oral, parenteral tep., intravenous, intramuscular, subcutaneous), rectal, transdermal. nasal, vaginal, inhalant, or ocular administration. Thus, the composition mas he in form of. egg, tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions- gels including hydrogels, pastes, ointments, creams, plasters, drenches, delivery devices, suppositories, enemas, injcctables, •implants, sprays, or aorosois. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice tsec. e.g.< Remington' The Science and Trucucc of Pharmacy, i I bib ed.) ed. A. IT Gennaro. 1995, Mack Publishing Company, flnston, Pa. and 'Encyclopedia of Pharmaceutical Technology, oda j. Ssvarbrick and 3. C. Boylan. 198M999. Marcel Dckker. N.Y. XIV, Methods of Monitoring or Diagnosing AA or AL Amyloidosis

Methods of monitoring or diagnosing AA or AL amyloidosis include measuring the plasma conccmraitons of SAA and Oreactive protein, performing tissue biopsy (ratal, rectal, gastric, gingival, fat, salivary, labial glands) and histology with Congo red staining and or immunotfatnmg with specific antibodies directed against AA or AL aggregates such as fibrils. 7 he in venison provides methods of detecting ass antibody response against A A peptide m a patient suffering from or susceptible to AA Amyloidosis. The methods are particularly useful for monitoring a course of meatman being administered to a patient. The methods can be used to monitor both therapeutic treatment on symptomatic patients and prophylactic treatment on asymptomatic patients. Some methods email determining &amp; baseline value of an antibody rcspott.se in a patient before administering a dosage of an immunogenic agent, end compering this with u value for the immune response a tier treatment. A significant increase (%., greater than the typical margin of experimental error in repent measurements of the same sample, expressed ax one standard deviation from: the mean of such rneasumteeitsHn value of the antibody respotssc signals a positive treatment outcome (ία , that administration of the agent has achieved or augmented an immune response), if the value for the antibody response does· not: change significantly, or decreases, a negative treatment outcome is indicated... In general, •patients undergoing an initial course of treatment with an immunogenic agent are expected to show an increase in amibody response with successive dosages, which eventually readws a plateau. Administration of agent is ecu orally continued white the anti hotly response d increasing. Attainment of the plateau is an indicator that the administered of treatment can be discontinued w reduced in dosage or frequency in other methods, a eommi value u t\, a mean and standard deviation) of an antibody response is determined (or a control population. Typically the individuals in the control population have not received prior treatment. Measured values of the amibody response in a patient after administering a therapeutic agent are then compared with the control value. A significant increase relative to the control value (c.g., greater than one standard deviation from the mean) signals a positive meatmen t outcome. A lack of significant' -mcrcasc or a decrease signals a negative treatment outcome. Administration of ageui is ye00rally couUnued while the amibody response Is increasing relative to the control value. As before, attainment of a plateau relative m control values in an indicator due the administration of treatment can he discontinued or reduced in dosage or frequency.

In other .methods, a control value of a nobody response bv.g.„ a mean and At mim'd deviation) is determined from a control population of indiv iduals win:· have undergone treatment -with a therapeutic agent and whose amibody responses have reached rt plateau in response to treatment Measured values of antibody response in a patient are compared with the control value If the measured level in a patient is not significantly different toy., more than one standard deviation) front the control value, treatment can he discontinued. If the level 1st a patient is significantly below- the control value, continued administration of agent is warranted, if she level in the pa bent persists below the control value, then a change in treatment regime, tor example, use of a different adjuvant, fragment or switch to passive administration may be indicated.

In other methods, u patient who is not presently receiving treat incut but has undergone a previous course of treatment is monitored for antibody response to determine whether a .resumption of treatment is required, t he measured value of antibody response in the patient can be compared with a value of antibody response previously achieved in the patient after a previous course of treatment. A significant decrease relative to the previous measurement iia, greater than a typical margin of error in repeat p^Mditracois '.$£'$$ .t*ai?t:&amp;· 1¾ an indication that treatment can he resumed. Alternatively. the value measured in a patient can be compared with a control value I mean plus standard deviation) determined m a population of patients after undergoing a course of treatment Alternatively, the measured value in a patient can be compared with a control value in populations of prophyinc Really treated panv-ms who remain free of symptoms of disease, or populations of therapeutically treated patients who show aired toot dor; of disease eharnctenstscs. In all of these eases, a significant decrease relative to the control level 17. <0, more than a standard deviation) is an indicator that treatment should he resumed in a patient

Some methods employ iodine·· U.V labeled or iodine· 125-htbcled serum amyloid P component f'M-SAP or 1 'l-SAP; Scintigraphy, l-SAP or ‘"1-SAP is infra vancously injected into patients and viewed with gamma, camera. Radiolabeled SAP Scintigraphy is a useful method io monitor the progression of amyloidosis in patients and evaluate treatment*, h is specific for amyloid and can be used to in.umftuuvdy monitor the location and amount of amyloid deposits in patients. :'"RSAP and *'1-SAP do not ace emulate in healthy subjects or in non-amyloid patients. Radiolabeled SAP scintigraphy cun be used to men nor dynamic turnover of amyloid, and can assess the efficacy of treatments aimed at .regressing amyloid deposits. Further, radiolabeled SAP Scintigraphy is rum-invasive and provides whole body scan. Methods of the invention entail determining a baseline value of an antibody response in a patient before administering a dosage of an agent, and comparing tins with a value lor the immune response after treatment m a patient. A significant increase the., greater than the typical margin of experimental error hi repeat measurement* of the same sample., expressed as one standard deviation iron; the mean of such measurements} in value of the antibody response signals a positive treatment outcome (/.«·., that administration of the agent has achieved or augmented an immune response). If the value for the antibody response does not change significantly, or decreases, a negative treatment outcome is indicated. In general, patients undergoing an Initial course of treatment with an immunogenic agent are expected to show an increase in antibody response with successive dosages, which eventually reaches a plateau. .Administration of agent is generally continued w hile the antibody response is increasing. Attainment of the plateau is an indicator that flic administered of treatment can be discontinued or reduced in dosage or frees!·, ;v..\

The tissue sample tor analysts is typically blood, plasma, .serum, mucous nr eorcbruspimti fluid from the pet lent, T he sample is amdyecd tor indication of an immune response fo any harm of Α.Λ or At. peptide The immune response -ran be determined fro;π ihe presence of antibodies that specifically bind ιο ΛΛ or AT peptide. Antibodies can be detected in a bind inn assay to a bga nd thsU spevha.'sby binds to the antibodies. Ty pteally ill-.: bean el R immobilized. Binding can be detected using u labeled end-idiom fee asm bode in com hi oat ion regimes employing both active and passive administration, analogous .approaches cun be used to naan:to; levels of antibody sesnltsrsg horn passive udnmustub ion

Methods of diagnosing amyloidosis can also he employed by, w g , administering fo a subject ass autihocly or anugen-b:nd:ng iv.igmcnt iheteof. that ss hound to a detectable label, wherein the a no body or fragment thereof specifics He binds to an epitope including XthDA·; m ass aggregated amyloid protein, therein X; and X> are any amino acid-, and detecting the presence or absence of the bound antibody or fragment thereof. Detection of the bound antibody or fragment supports a diagnosis of amyloidosis antibodies aw! fragments tr-eial in the diagnosis of amyloidosis include the disclosed antibodies of rise invention.

The diagnostic antibodies or fragments of the invention win he administered, by e, y.. inunxenous injection into dies body of a patient, or direedy uuo the lain a by intracranial iniacpeui. The antibody dosage is readily determined by tine skilled in the art. 1 ypiealty, the antibody is labeled, although in cm no methods, the as moody is unlabelcd atnl a secondary labeling agent is used to hind to the antibody. The choice of label depends on the means of detection. Rn example, a nuoreseent label is statable for optical detectiosa to.se of paramagnetic labels Is amiable for tomographic detection o ithoui surgical intervention. Radiolabels may be nsed including '".At. ;‘:Bi, °'Cu. p"l, "SL i!iln. r> ''‘Vb, ’'"Re, P;Re, "''Sot. or *‘\ Such labels may be detected using PhT or SFECT or other suitable teehnitjne.

Diagnosis may also be performed h\ comparing the number, size. and.or Imewere of labeled loci, to corresponding baseline values. The ba.se line -, Dues van neureseui the mean !cseis in a population of nndiseasvd individuals. Baseline \abacs can also represent previous levels determined In the same patient. For example base lute --. abacs can be determined in a patient, and measured values thereat ter coronated oids the baseline values \n increase tn values relative to baseline signals supports a diagnosis of .A A amyloidosis,

Ihe·: domsiosbe methods of the ;memion may be used to diagnose amyloidosis diseases including Λ Λ amyloidosis, -U amyloidosis, AwheaoerX disease, Mild Dognidve Impairment, amyloid polyneuropathy, Mediterranean fever. Muck ie* Wells syndrome. renctrve systemic .amyloidosis associated with systemic inflammatory diseases, myeloma or tnaertiglohulhvmia associated amyloidosis, amyloidosis associated with irnrotmocyte dyscrasia. monoclonal gammopathy, oeeulf dyscrasia. or local nodular amyloiddsi? associated with, ehmnib i n tl a m n tat o ry d i se a son, XV. Avtirnal Models of AA Amyloidosis ΛΛ amyloidosis can he induced experimentally m ntice in which SAA concentrations are markedly increased by Injection of silver nitrate, casern, or Iipopolysaccharidc. These agents Stimulate the production of cytokines. .Ska·· Skinner ct ah Lob inwsi 36:-120.-427 (1907} and Kisiicvsky et ;4. Puil/iv^e 3 Clu>. Ιααοια'οί. l>nifUfo<ipaifii>L Si 3} 613-626 i 1994). vVhhio 2 or 3 weeks after the infUmmatoty xtinuthts, mi ms. Is develop systemic A A deposits. as found in patients with Ad' Amylotdosis. I'his lag phnse is dramatically shortened when mice art given, concomitantly, am intravenous injection of protein extracted from AA amyloid-laden mouse spleen or liver. See Axe-lrad ct ah Lab lavesL 47(2):.139-46 (19X25. The aniyloidogenie accelerating activity of such preparations was termed ''amyloid onkumdng la dor'" t aE F) Lundniark et a), reports that the active principle of AFF is unequivocally the AA fiber Itself Further, they demonstrated mat this material is extremely potent, being active in doses less than I ng, and that it retained its biologic activity over a considerable length of time. Notably, the AFF was also effective when administered orally. They concluded that ΛΛ and perhaps other forms of amyloidosis are transmissible diseases, akin to the pnon-u.\socl,ucd disorders. Sec Lutidmarketal rrtu .37;,' .-fun/ „V/. 99. 6979-09X4 (2002) ΑΛ amyloid can also be induced in transgenic struttta of mice carrying the human interleukin 6 gate under the control of the rnetailoihtonein-l promoter resulting ht markedly increased conconnemons of 3?A-7 and developing amyloid in the spleen, liver and kidneys by 3 months of age .At the time of death at about s~o months, organs front these transgenic mice have extensive amyloid deposits. Ave Solomon ct si.. Am.,/. PathuL 1 34(4};1247. Γ272 11999}.

The Transgenic Rapidly induced Amyloid Disease iTRIADS transgenie mouse model is an improvement to the above described tranxgomc mouse model TRIAD msec carry the human interleukin 6 gone tinder the control of the H-2t..i: histocompatibility piomotcr. Ado-lustration of Aii.F to f-week old TRIAD mice results in prominent specie and hepatic A A amyloid ilepo-no, wi thin 3 to 4 weete. Sitbsequoh tly, this process progresses to other organs, leading To death 4»9 weeks later. The develop??! erst of the systemic amyloidosis is accelerated compared to the abovedescribed transgenic mouse model. Stv University os' Tcwnc-ssec Research Corporation. WO 01 'w167. Pharmacopeia. WO 95. 35503 and Seripp».. WO 95/306-12 Wait er at Amyloid ;2(3): MW 156 (2005 ) (each of which is incorporated by reference for all purposes).

The common marmoset (Cattitkrix jctcclnt*) is a small New World primate native to Brazil that has been used extensively in bmmcuics] research. Ludlage ct al. reports that common marmoset were 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 he responsible for die development of AA amyloidosis in this primate. In this regard, the common marmoset -could serve as a unique experimental model tor study of the pathogenesis and therapy of A A .and oilier systemic amyloid disorders. Sfe Ludlage et al. 1 ct Pat hid -12:11 '/'·· i 24(2005).

The Shar Pci species of dog. a breed 1 laving an Λ.Λ sequence, with the -AEDS motif and that is panieidarly susceptible to Α.Λ-amyioidosis. pro? Ides a naturally occurring model of systemic AA in which to evaluate novel diagnostic and therapeutic applications of AA amyloid-specific antibodies and other compound», I N AMP l 1::.5

Example 1, ΛΛ Fragments.

Peptides eorrespondlng to amino acids 71-75 ~ OH EOT. as described by Yamamoto and Migita Proa Nut!. Acad. $a\ USA 82:2915-2919 were synthesized by AnuSpcc. San Jose. CA. USA. Polyclonal antibodies (Pah) AA were raised and die immunoglobulin fraction isolated, as previously described by Bard, E et a;., (2000) Mai. Sicd. 6, 9(6.919.

Example 11. immunogen for Preparation of Murine Antibodies,

The epitope used was GHEDT, (SEQ ID NO: S) with a CG linker at its N termmus. The peptide EPRB-39 which contains the epitope is coupled to .sheep anti misuse antibody, i: PRB-39 is obtained from Auasec, San Jose. CA The ami bodies produced appear to be neoepstopc· specific because they don't specifically bind to a peptide that spans the region GHFP7IAOQE, sSEQ ID NO; K9).

Example III. Immunization Procedures.

Six-week-old A j mice were miraperifoneui injected with 50 ug EPRB-39 sheep ami-mouse i.gG with Complete Freund's Adjuvant fCFA} followed by ineomplcte Freund's adjuvant UFA) once every other week tor a. total of three injections. fhiec clays before fusion, the tail vein was injected with 50 ug I? PR B-39 SAM IgG m AMI id PITS. TfespfHff· 'Λ 'S$ttinafe<i at 1/IOOUO irocn FLIS.A with high background JH 80 is the fusion number tor LPEB-.F) The following is a list of the clones and: limiting dilution clones that are active;

"Τ.>4?„ sGO snd 2A7'V indicate pt^fenOd sukioncs. the antibodies produced appear to lx- neoepkopo specific because they dors'- react odd'! a peptide that spates the C-terminus cleavage site ot'SAA. L sample IV. Antibody Bitiding to Aggregated and Soluble ΛΛ.

Scrum titers (determined by, serial dilutions and monoclonal antibody histdistg to aggregated \ \ were performed by ELISA as previously described by Schenk D. ut al, (1 odd? Witmv -)00, ΓΜ-Γ'/ν, Soluble A.-\ refers to the A A fibrils sonicated in dimethyl s-tlfosidc. Serial dilutions of antibody wese incubated oath :5CUX>0 q>m of !ΐ Ί-ΛΛ overnight at roont temperature. A0 μ! of a slurry containing o mg'ml protein A, sephsrosc (Arocrsham Bioseiences, Irpjvmkg Sweden) 200 ug rabbit anti-mouse IgG ibGlA {Jackson imfnunoKeseareh, West Grove, FA, USA? was incubated with the diluted antibodies for 1 hr at room temperature, washed twice, and counted on a Wallae gamma, counter {Perkinhlmer Life Science. Grove, It, LSA}. AH steps were perlormed in radioimmunoassay buffer consisting of 10 n;M A rts, 0.5 M Nad, 1 mg'ml gelatin, and 0,5'% Nonidet F-40. pH H.O.

Example V. Analy-us of Ya6 Wil Structure,

The sequences of the expressed human Vk astd YX mtmunortlobt-iia light chain gorrniine genes are as illustrated in Figures 2! artd 22 With exception of die via, Xla, >3a. and X3c subgroups there is a Gin-Asp residue pairing at positions hi and 8.7 in ail, Ye and VX germ bine grate segue; tecs {Figures 21 and 221. In addition, a second germ hue encoded G hi-Asp pairing at positions 50 and 51 is unique to ΥλΑ gems line gene. ‘ihus. YXh Wtl contains both the .50-51 and S1-H2 Glu-Asp pads. The, side chums of residues 50 and 51 are both accessible on the surface of Υλο Wit, as shown by x-ray egsiakograpOy (Home 7.4V In conir,%, only the GiuSt Aide c&amp;ltt is surface exposed and the AxpSe side chain is partially busied and appears to interact (either by eleefrosMte. intgraciiods ©r Hdiondlng} %fih ih© sy© .dmlns. of fey©7§ and ;A*gt§!' (Figure 25).

Rased ou. these analyses o.flb». \-my crystal stt.tt crurc an© the roOmo ,n at lability *>1 the Ght-Λχρ side chains Appl wants conclude dun tin bunco ARM becomes accessible us ih, domain entem an aggregated ion,, tlhidian strut Uev (or become·' fuoiuth don. unr>. df thus exposing what is otherwise a hidden, cryptic epitope. F\a.mpie VI. A na lysis of A nt i- A A Monoclonal An si body Rinding To VAn A. SiuT-urc Ria&amp;nioo. Resonance.

Su{ Face nlasmon resonance was used to establish the binding hoctics of several monoclonal antibodies with Υλ6 W11 fibrils und monomer. At a concentration of 6.6 nM all 3 antibodies bound ©; die immobilized synthetic VXo Wii fibrils with u K.D of·' 1 nM a value comparable to that found for their reactivity with murine A A fibrils (Figure 26b The deflection (expressed in RU) during the binding phase was similar for ruAbs 7D8 and 2Λ4 bul \ob '50°;> lower for bed). This suggests that the density of this antibody on the fibrils was lower than the other 2 reagents, as the calculated affinities were similar for all e antibodies. An IgGI mAb served as a control and exhibited no binding to ν'λό Wii fibrils.

Titration of the mAb 708 over the range of 6.6 nM to 333 nM produced the expected decrease m the maximal deflection associated with kot> (figure 27}. In general, the binding, kinetics were similar at each concern©©;©;!, although in these pilot experiments the KD value for at eo.b nM dul diflet from Ur.a obtamctl at the other concentrations.

To assess (he specificity of the reaction and ensure that the binding of the mAbs with the fibrils occurred via the classic Fiubbuntigen interaction (a.s opposed to F;>tnediatcd binding or non-specific adsorption;, binding data were acquired tn the presence of the immunogen peptide (p.M); at 20 and I ug rn 1.. (Fig 8;. Pc pi ide p4i winch does not hind the mAb ?D8 at low concentrations, served as a control. It's the presence of 20 μ god.. p4j pepitdc, the binding kinetics for mAb 708 with VlsO Wii fibrils were identical to 708 alone. In contrast the irnrntmog.cn peptide pity at 1 ug ml., caused a > e-fold decrease in the era cm of binding ns judged by the deflection of the measured -signal (f igure 28;. Inhibition of fibril binding by 70s was almost completely inhibited when 20 μg'mL of p7v peptide was used. The*-c data indicated that mAh 708 bound fibrils via the Rah) region of the molecule inasmuch a.s loo. interaction could be completely inhibited by the immunogen peptide.

The reactivity of the mAh 7D8 with vA.b monomer immobilized on ;· chip was examined axing (he BIAeore, The antibody did not react with the monomeric protein. These data indicate that the binding site recognised by file mAb TDK is present o.u .fibrils, but no* on the soluble, precursor protein, implying that the antigen is oonforrnatlonal or; cryptic in. nature. B. ini m o no h; si oehem tst sy

IrmnnnohistochentiMfy was performed as iollowsc bjim-ihiek sections cut from . formal in.-fixod« paraffin embedded blocks, were subjected to and antigen retrieval by ineubasmn with CitraPiu.s· {BtoGcnox. Sun Ramon, ( A) for 30 min at 9CPC. Ttss-ics were inmtunostained with a 3 ,ug/ml solution ofrnAbs 7? Ad,7 DR or $G9. The lgG2a mAh TV I i served as a control A HRPO-conjugsicd horse antt-mouse Ig antibody HrwnPRESS Universal Reagent, Vector Labs. Burlingame. CA) was used as the secondary reagent Slides were developed using 3..V-diaminobezidene (Vector Labs? and examined using a Leica DM500 mitroxeopc. The hues set ton of the monoclonal antibodies wuh ALe and A l λ amyloid tissues deposits was also studied using irrmumohistoehernistry. As II lustra ted in Figure 20, amyloid deposits in a patient's thyroid gland which were composed of λ2 fragments were immnnosraincd by 7DR 2Ad and Γην areas ol reactivuy correlated with the amyloid deposits., indicated by die green-gold birefringence seen in the Congo red-stained us sue section. The most impressive reactivity was achieved with rnAbs 7f.)s and 2Λ4 rnAbs while F(iF, although positive, was considerably weaker. Hi esc qualitative data correspond well with rlie BIAeore analyses in which bCP> bound less to the VX6 Wit fibrils than the other 2 reagents f figure 26), The isotype matched mAh TV I! that served ns a control exhibited no amyloid immunorcacuvity.

The amino acid sequence, of this λ2 protein (SBQ li> NO. f(o (shown below) contains the genu line encoded Giu and Asp s-esiducs at position Kt and b.7, respectively

Examination of an A Lie amyloid tissue deposit revealed 2A4„ and to a lesser degree the and FG9, to nave positive reactivity. Again there ’was concordance between the irnmuno-staining and birefringem. congophillt: amyloid regions. The TY11 mAh was unreaedve.

C, Radioimaging of AL Arnykndoma I .sing l' Ί-Labekd 7Da The experimental m vivo model of AL am>loidomst was used to study it mdintabclct) ntAb 71¾ would image human AL amyloid. The mdioiabeUmg eflk senes of 7DS, as determined by SDS-PAOE, revealed that both the IgH and IgL chaim- mcoipmxncd rtf·/ I-12d label. aw! no evidence of bands associated with fragmentation or aggregation ttcsv ohsmed. SPEC I CT imaging of u mouse bearing an induced AL. amyloidoma revealed that the I labeled antibody ioealtiled to the induced, dorsally-loeatcd amyloid mass, as evidenced by accumulation of the radiolabeled antibodies in the amyloid, relative to amyloid-free tissues (c.g.., liver, heart, spleen, . and kidneys). Radfoloabded irrelevant IgG mAh did not accumulate in the mass; however free - radioiodidc was observed accumulating in the thyroid, mdteatis c of the catabolism and

i.} S dehahtgentioti of rtse IgG anobody. The distribution of the I DL mAh in the amyloidoma-bearing otiec was qu;mL fled by measuring rhe activity -wsoc sated with the amyloid mass as compared to that of the liver, spleen, kidney, stomach, heart, and lung. These data confirmed the SPETOCT imaging study. At ?2-h post injection tat which lime rhe images were acquired and die tissues hurtcateur the aniytoidensa contained - tU·,, ID widen is · 4~foki higher than that seen in the liver the sue of mAb catabolism-· and the heart where residual blood-pooI activity would expected ro be high The uerts tty shown in the lung was due to the mode of euthanasia {data not show ft)

To confirm the biodintribution dais, the amyloidoma as well as the it very spleen, heart, and kidney-; were harvested and tissue sections prepared for autoradiographic analysis. Radiotabelhng was performed as follows; The ~f>K ieuToey ua·- labeled with 2 mC i of reduetum-frec I (Perkin Elmers using limiting amounts of Chlorarntne T and suspended in PBS containing 5 mg ml of bovine serum albumin {BSA PBSs Unbound isotope a;id protein aggregates were removed by ske-e.xcUtsion liquid chromatography through an Uihogel Ac And column t Amcrsham Pharmacia). Fraetiona- containing. JgG monotner were pooled for imaging experiments The radiochemical yield was - SOT,. providing a .specific ucbvby of - 23 bCifug l-labclcd nt \b was subjected to SDS/PAGE (10% gels) in the presence or absence of a reducing a gem and analysed w ith a Cyclone phospbor-imagor. In accordance with the SPELT imaging and biodistribution measurements, the autoradiographs confirmed significant , . C;'S ... . . . . . accumulation of !-"OS in the amyloidoma., relative to the Ever. 1'here was no evidence of Uiftftko bf mltokbekd Mflbody·' 1-708 m boy other organs Irtiidr than the mpnetad hefatk activity associate with cafabohxrn of the antibody). Although rnAb 7DH was relatively uniformly distributed throughout the bulk of the amyloid mass. a moderately higher density was observed in the peripheral areas at the abdomen-amyloid boundary There was no uptake of the rad to labeled control IgG 1st any organs, D. Summary .arid Conclusions

Surface plasmon resonance, imruunohisiochcmistry and in m<> rsdioimuging establish that KA· reactive antibodies 2A4, 7LXA and K{» bind Ah amyloid and fibrils (Kd - I nM) derived frttm intntunoglobultn light chains. This interaction likely occurs at the highly-conserved Gin and Asp amino aeids at position K! and 82. respectively, which form a cryptic linear epitope lhat beeoru.es exposed only when the arrsyloidogeuie light chain is incorporated into fibrils.

Example Nil I' I .ISA Analysis Demonstrates Antibody Binding to XsBDX» Peptides,

BlAcotv analysis was performed to evaluate binding of antibodies 2Λ-!, 7DH and 8G-I on peptides of various sequences. As shown below tit Table 4. the antibodies were found to react with peptides having tire sequence XiBDX... interestingly, the antibodies did nor react with peptides having additional €-terminal residues. "Phis suggests that the antibodies specifically bind to a neoepifope generated cleavage of S7A to generate a free C--emninai wed I iowever, as demonstrated in Example V. the tree end is not exsenmd for binding of these antibodies to Vko Wil. but rather the XiBDX> domain adopts a eoniormatioe favorable to binding to the antibodies as it enters an aggregated te.g, Ohnliar) structure (or becomes partially denaturedh exposing an otherwise hidden, cryptic epitope. I . d, 4

Example YUI Imnmnoinstoeheniieul A.nnh x;s of Mou-w Λ Λ.

The reactivity of supernatants from hybAdomas expressing antibodies 2 Vi. 8(W and ~F>n ίο murine A A .splenic and hepatic amyloki dennsus (the principal slips pi amyloid deposition) was documented Irniwmohisiochenucaliy. for these studies, sections of tissue harvested from a TRIAD mouse with extensive ΑΛ amyloid or ilk (her and spleen ias es tdeneed o> green Ivea fn no era {'oleophilic deposits) were stumed ο uh the ntAb-conttveung supernmants All d hoc ad to the heps: he and splenic amyloid, In com rase there v as no reactivity with culture supc;nui,mls domed it on; irrelevant by brides mw. ilk·, capability of the act yield ns; op 2 AT. 8G;> and "Df to imotunostam amyloid ;n t'resh {unfixed}. OCT "embedded murine liver and spleen was iestetf The,re was evidence that the mAbs retained their ability to bind A.A amyloid m the hepatic sum sold. In adds;som the antibody reactivity with .splenic tissue was easier to mtevpreg assd l.l;c penfollietdar amyloid was intensely irnmunostained. To demonstraic that the mAbs was specsfieully bound .A.A amyloid, the mAh supernatants at a I Ad dims ion were pm men bated with SO μ garni.. of cither peptide -dSO spTbo or dal {pAsi) for Γη at room temper attire. Wsth ibnnalim· fixed tissue as a .substrate, the pAV’i peptide (50 μgmiA significantly mhibited the amyloid reactivity of both 2Ad and 2Db mAbs (the results with hCA) are pending). In contrast, d;e pod i peptide was ineffective. Comparable results were obtained with fresh tissues. {'sample IX. inmninolusiucheouod Analysis Of Human ΛΛ,

Comparison of tlte arnitto acid xerptence of mouse and human S.A.A from portion 7¾-76 reveals 2 identical residues, a conserved Sea to Thr substitution, and a non -conserved A Us to 'ills exchange. To test if tlte 2.AT bCF> and 7D* mAbs would cross-reaet with human ΑΛ amyloid deposits, we tested fheir reactrvsty to human A.V-comuunne; Kidney, adrenal. ovary ami liver. In ail cases, the nt.Ab supernatants innunnostained the amyloid deposits In os..naan ns-uxe the peA) peptide effectively blocked the binding of tlte m.Ahs to the pori vascular A A amslotu. svhereas the pA-H peptide did no? inhibit tins reaction. (example X. i;v; cruet ion Of-AntiΆΛ OfCTdntrc Supernatants With Niasme-Dertved A.A Fibrils.

The Interaction of 2AT 80S and 7Dd mAbs with A.A amyloid was initially tested by EUBA and the data, given in Figure 3L analysed using SigmuPbt {SPSS Ira..). Each point represents the mean i SE (n 3). A culture supernatant iron; an uxeievunt hvbrldoma was nsed as a control {(Ari Culture Suns, There was an extremely low signal-noise ruin; and the rest;Its showed that the fust hurtcs; contained more mAh relative to the second, as evidenced by the greater absorbance .signal relative to the control supernatant.. {in addition, the innmmohi.stochemieul react isaty of the day i material was greater -ban the da> 2 .samples} Although the Si:; values wore large, ir appeared from those daw that the binding atm-ity of 2AT K(.b) and T)H wa.s approximately egema lorn with reactivity absent after -- I M dilution. The binding data also suggest that the capacity, i.c.. the amount of mAh bound, carsed with /Dtt'-xGs'-'aAd; however, those data were not corrected for mAb concentration and hr std\scqnem studies this trend was not observed. Because of the low signal and high variability Sound with the culture supernatants and to determine more accurately the relative binding affinity of the rnΛhs for marine and human Λ.Α amyloid fiords (as well as to provide material for in vo.o biodismbuiion studies) it was necessary m isolate the mAbs by protein A affinity chromatography. The purity of the isolated mAbs was e-Uablisbed SOS-PAGB using !b% acrylamide gels under redui.mg and non-reducing conditions {figure 3hr Samples in lanes l-f moated wed; mercuptoethunol, lanes 5-0 without. Gel was stained 'with Coomassie blue; mAb HGM lanes I and ή; mAh 7 Λ 4, lanes 2 and mAh ?Ub, lanes 3 and b; Src 0 comrwl snpernamnt, lanes -{ and A blank, lane 5. Protein Mr markers tStd's are, form top tsa bottom; I o I 19. 75, 4A >-g 75 ,uid Iv kDa The mccruc?ion of the purified jnAos with tmnumleing pepude P'dav, control pop hoc (pA-i I i, murine and human ΛΛ ex mu. is were determined by bIJSA as described ubu\e. These data were anatyaed by luting a sigmoid-d curse using the SwmaPloi software and tltc rnAb concentration at 50% saturation iliCSO-, determined I fable 5},

Table 3

The imeraetion of the Λ rnAbs with peptide pee·) exhibited saturable binding with eh’’'50 values in die have nunotnolar range (see above Table 5). in contrast, even at the highest concentration of mAb used (100 nVIi there was Utile detectable binding to the pv?41 peptide '(Figure 33 - Bach norm represents the mean .·).·. SB, (n ::: 3 at each concentration!). These data confirmed the iotmunohixiochcnuca! results described above, i.e., dun peptide pd30 was capable of completely blocking the binding of the rnAbs to A A amyloid laden tissues. The calculated EC50s for the binding of each mAb with p#39 peptide w ere essentially Identical as was the ease when a murine ΛΛ amyloid extract was used as the substrate t Figure 34 - Each point represents the mean *. SB in ~ 3 at each concentration)}. The calculated EC50 values for the rnAbs binding to mouse A A extract w ere esscntiitlly identical to those obtained when the peg9 peptide was used ns the substrate (Figure 34c Table 5). In contrast, when human Λ A amyloid extract was dried onto the wells of the rnicroplaic. die IHC50 values were between 5 and ?x Sower than that observed for mouse AA and peptide p#39 (Figure 35 Each point represents the mean SF tn :: 3 at each concentration); Tabic 5). Because die hC50 value for ?Db mAb btndmg was the lowest of the 3 antibodies tested. Applicants selected mis reagent for in vivo co-localization and imaging studies. The 3 amino acid substitutions in the human SAA sequence with respect to the murine protein affected the F.C50 values. While not wishing to be bound by a particular theory. Applicants attribute the higher EC50 tor the human Λ A to a poorer An'" of the amino acid side chains in the antigen binding site, however, this effect corresponds to only a .5-fold decrease in tte relative aftimty when die. amyloid extracts are surface adsorbed, as in the El.ISA Furthermore, these data support the observation tint! ail 3 rnAbs bound to both murine and human tissue A A amUmd deposits.

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

To determine the effect, if any, of potential demituration when adsorbed to the surface of the mieromer well, the mucin ay of dre 3Λ-1. HOT and 7 Da was evaluated using u competition □..ISA in which murine or human AA amyloid extract was used as a soluble competitor for the intemerinn of the mAhs with surface··bound ΑΛ extract

In all eases, soluble mom-adsorbed} AA amyloid fibrils of both human and mouse origin were capable of competing for the 3 m.-Abs, indicating that the epitope recognized by the reagents Is not dependent upon the pa real denaturatiou that results from surface adsorption In general, the murine Λ A (AET) extract was a better competitor than the human AA i Table fit. iAbie#

/Css values {ug;mL} foem&amp;is Bmdlng i&amp;M amghM

'Human AA amyloid in solution eotr-pehny for adsorbed mouse AA (AEF}. '“Mouse AA (AEF; <n solution competing fot odeo-eect hotpgp. M. on plate

The K 50 \alues (concentration of ΛΑ (by weight! duo reduced the ntAb binding by 50X4 for murine ABF in so in don were 20 ug-rnE, whereas for human AA the values were A- ro 44-fold greater fin contrast, lltc HOIK for human ΛΛ were only "Mold lower than those for mouse ΑΛΙ Hus tony rod ecu the fact fhoi, when in solution. the epitope on the amyloid fibrils is toss access-hie m human .A. A p-epamuons as compared 10 murine A A.

As expected, the. 7D8 mAh dun exhibited the highest re A due effirnty For the human and murine ΛΛ id mis n hen they were surface-adsorbed required the highest concentration -,:4( A Λ amyloid to achieve competition.

Example XU. Radiolabeled MAb TDK

The radiolabcihtg efficiency of TDK was determined by SDS-KACJE. Reduced ».md native mAh were analyzed and the proteins visualized using a phosphor imager. Both the lull and ML chains incorporated the i-l25 label. and no evidence of bands associated with fragmentation or aggregation were observed.

Example XIII. Imaging ot A A amyloid using :“'i--labcded 70b,

To study the w vivo localization of radiolabeled mAh 7D8 three gronns of mlcc were used; transgenic tL-b; AgNO.VAEF induced, and amyloid-lacking controls (Wfi, The SPECT'CT imaging rescaled that the ""'I-7D8 mAh localized to murine AA amyloid deposits in the spleen and liver, as evidenced by the accumulation of the radiolabeled mAh in these tissues relative to ihe eonm.n mouse, which showed only low blood pool activity in the liver and free iodide the thyroid gland.

In contrast to these iftiesy. the Thytolt# offer iodide, some hopple activity, hut she major sire of *"“1-708 binding was seen at fee site of s.c AgN'03 injection {the lower right dorsal uiva), The aerivity in this area is cie»rl> ctrountscrihee .by she x-ray-atiennating stiver solution as seen In f'T The ?DH mAh has been shown to bind to ΛΛ amyloid deposits in both the User and spleen, in the presence of eircnlsnng sAA in the TRIAD mouse, as evidenced in the SRKCT images A. Biodistribunon Of *';I-7D8 In Msec. 48 h post-tniection of *" 1-708 there was radioactivity in the Hood pool, which accounted for the relanwb. high uptake 1st the lung {which nil with blood when the mice are sacrificed ϊ AH' note, the hepntoaplersic accumulation of mAh in the it.-6 mouse is indicative of the presence of asnylosd 1 he SPkCT fi’T images confirmed the distribution of the mAh m these organs, A: h post-injection the blood pool values have changed little as evidenced by the unchanged activity its the heart and lung relative to she none sacrificed a? 4b h, dec to the relatively long T; for this mAh {-60 hh There was significant accumulation of the radiolabeled mAb in the 11..-6 mouse, which correlated with she SPH1T images that were acquired show mg impwoso c splenic and, to a leaser degree, hepatic uptake. Of she other organs, most important was the bver (which is the site of catabolism of IgG and the source of sAA during the acute phase response), in the \VT mice, with no ud'iammmoi'v cha! range or amyloid, the liver contained -- 6A. it> g. which is comparable so the kidney and hean where she blood poo! contributes almost exclusively so the signal. R. Automdiugmpiuc And H Atociterrucal Analyses

In order to determine if the increased hepatic accumulation of ! "4-708 in the 11..-6 and

AgNOv mice resulted from amyloid uptake, catabolic clearance or binding to newly synthesised v\A. bver as well as other tissues were subleased to autoradiographic analysis.

Based on the SPiiiCT imaging and biodistribution measurements, is was presumed shat ihe greatest amount oi any ioid in she transgenic 11.-6 mice was m the liver end spleen. This supposition was connnncd in die Outgo red-stained sections in which significant amyloid was observed throughout the red pulp as well as in the perivascular regions and sinusoids of the User. Additional, more discreet btrefrlngent deposits were present in the kidneys and heart. The distribution of rite withm these tissues correlated well with the Congo red and ΑΛ- reuciive material. There was no accumulation in hepatocytes that were devoid of amyloid.

Based on the biodistribufion data, the AgNO-treated mouse had snore uptake of '1-7ESS in the liver than tire spleen, which: was unexpected since this is not the normal pattern of aeon rmuat ion of ΛΛ ;?t Midi animals. < ongo red-siaisnng rose;Jed small amounts of amyloid iu •a single pen follicular region in -he spleen tupper right corner! and extensoo hepatic perivascular deposit both (>f winch wore eviden; in the autoradiographs. AddltionalK. she s.e. site of me AgKCR suieet.iou was scon in the SPEC! images to have a MgmfU.oni cot kient ratio? i of ‘ i-"DS two also have observed this when radiofodmatod SAP was used as the imaging agent} I he> site aloes not contain amyloid (t.e.. Congo rad-two Tin gen; mxicnaik hoe aver, it was iinmunostumed b> ami-ΛΑ mAb Without aishtog to be bound to a particular theory, it is possible dug the mAh 7|>x localises to >o;c-< of nnhnrsmaoon o; '"pre-a;ny;οίί.Γ <as well as tnatute amshnd deposits) in contrast to the impressive ore gnu Union of 7DP in the organs of the IL-P mouse, the tissues of the control msec wore found to have htfle os- no totes'? tn any organ other than the blood pool No amyloid was fburui in Congo red-stained sections ofaby orgatt of ilWseoouiOb. C. Phariitucokiueties of:' 'l-7Db. .After injection of the radiolabeled oAK antibody, the rats' of disappearance of the molecule was determined arid the half-life determinations st.immaiic.ed in Table ?. Those results indicated that the T *>... of 7D8 vs us - 60 h, consistent with that of an IgGab murine snAb (note, 7D8 is of the IgGeb subclass}. The slightly more rapid clearance of the f l-7Dh in the 11..-6 «TRIAD} .mice \ut.s no- cousidesed significant. Based on those data, retention of the mAh by tissue an>lolcl as evidenced in the SPEC! data, over 72 h dots not influence the excretion rate. fable 7 ana/ysee fee m1~WBmmim

.1. Method of Irteoilymg Agents that Present or Treat Amyloidoais IMng: Transgenic or TRIAD Mouse. Procedures for preparation of agents are described in Schenk ci ίύ Vtuwe 400:1~5-17~. Agents are emulsified 1:I iv \ ) wuh complete ircund's adjuvant lor the 'tlrsi immunization of transgenic mice. tollow-ud by a boo·;; in complete FcctmdT adjutant a? 2 week··· and monthly theread.cr. PBS Injections toiluwed the same schedule and mice were injected with l: i mi λ of PBS.'adhn ant for control. The life span of the Oowwome once A compared to determine whether the agents are eileetAe in preventing Λ A Amyloidosis b\ incrcasinc the life of the amma.1, 2. Hislopathoiitgy for light and polarizing mictoseopw 1- to mpm-thick tissue sections were cut and stained with hematoxylin and ooxln ;HFi mid a beshls prepared alkaline Congo red solution, respectively. For ciearon microscopy. sections were embedded in Ppon {Ted Pella, Redding, CAy sectioned, and examined with a JPOL SOUS transmission dec trot; microscope. .SVv l.udlage cn ul. U/ Parfat! 42:11 7 -) 24 <2005;. 3. bwmmohiswchemi-mw. ParafTln-ernbcdded tissue sections ttd-urm ducks wore cut on a microtome, mounted on poly · t -lyMne-c oaied slides, dried overnight at room tempera mm, and depara ffinte.ed. hm me lostaining wax peribnned using titc avidinbiotin complex (ABC-e)iiei technique as described previously. The. primary antibodies were mouse anti-human amyloid A (Accurate Chemical and Scientific Corporation, Westhury, NY) and anti-mouse SAA polyclonal antisera. Affinity-perilled horse ami-mouse immunoglobu I in -G (IgG? horseradish peroxidase conjugate (Vector Laboratories. Burlingame. CAt or goat anti-rabbit, -mouse, or -rat lg<3 horseradish peroxidase conjugates (BioRud Laboratories, Richmond, C A t were used as· the secondary antibodies. 4. R.AA Quantitation by bU$A. SAA concentrations were measured by un eneyme-Unkcd immunosorbent assay iPi ISA) using the Multispecies SAA FI .ISA kit according to directions supplied by the ntanufacturer {Bioxooree, Camarillo. (' As. Standard curves were prepared using known amount·* of human SAA protein and absorbance was measured at 405 run with a model 4450 BioRud plate render ibulierfon, CA >, 5. Radiolabeled SAP Scintigraphy Tmmmer Studies in Mice SAP wax oxidatively iodinated with ΚΊ t2-5 MBcrrng) by using N-bromoxuceinirmae. 6-12 weeks old mice received 2-10 ttg of *' Ί -SAP in 200 μί., intravenous!) Precisely measured tail bleeds {0 01-0 04 gt were taken at specific time intervals and trichloroacetic· aeid-precipiotble radioactivity was counted in the .«ume run at the end of each experiment together with standard aliquots of the injected tracer. Fepys of at. .Pone N&amp;tLAmU Scl USA 01: j€02-Sb(M 11 6. Radbhsbcied SAP Scimigraphy Vuntover and Imaging Man. SAP ibt use in man isolated from the plasma of a, single normal accredkcd donor and was oxidatively animated with I (2-5 MBqmnO or !'Ί 1110 MBcphO μμ os protein* h\ u^nu V .broinosHCv-inindd·:. Xtier nneef-on r-l 1 SAP, data vere acijmred .sn<1 pn.Vvsvd on an lid 'Starcam gamma camera dub Medical Sweats, Slough, * · K Clearance of ” bi-sMed SAP was studied in. healthy individuals and patietrw suirhnng Iron- Λ \ cuw hnd>t\:\ Pepss ct at /Aa Wat AokI Set. I S i ‘-H Siii)2 όόΟΡ t i‘AD t " AnyWd b\tryeoim and Punfk'.moti, The methods coed to e.vma amslmd bom tissue a ,.-re as Peso?iPod b\ Pras el ah S>,.c Puts ei ai J i Un. /; n O 47 '>24-W'· ; j ο(Ά t m la a portion officer ot m-sues from olhor .•roans ohamed at tRvropss and nisisnAru.d at SO ( w as homogenized sxtth sold salute tn .in tee bath none an Otom-Msvet A Ami hdentatiotajL AX atcsbitry, ί I) The os tract ο m „ enut idged a? 10,000 rpm tor gf m· nines at 4 C ,md the pellet uven acted on foe naan w-d· «.old sahue. ttnec with 0 1 M sodium etna ίο ΓηΝ-ΡηΠοκΌ sabne, pH P.d, and then again with saline umd the AdM't ·,·! the survrrcrmm was -- 0 10 1 Its' resttirant pellet was homogenised cal; cold distill d watew and Hr otivare ceaO'hugeJ at 75.000 rpm lot ή hours at 4 ί Hie pellet uhouned font the v aw estwet v as then ΙνορίηΙι/,ν,Ι. 0. Senl ace PI a si non Resonance, Binding kinetics were measured on a BIX core X instrument. Fibrils prepared Font the V 6 XX :1 'acre «ontented hoefh wdh a ptohe -On-catoi and hen coupled to a CXIo chip using an one shcon v.r> as per the BhXcon; protocol I his pjoee.ss mii:/,s ΠΧ end MRS to acdraic the cm host. I groups on the chip for coupling n ith free as nine gtoups mt the fibttls Coupling r,as eon-Jusied :n ; XaO.A.e bullet, pH -1,0 at a concentrattoti ot 100 gg ntl. 1 ho control chanm. 1 ks "nutcsceupled'' and boih channels wore reacted with eihunotamine n; ,an stale ttoceaeted sites, Xppsosunatelv i 0,010! Rl of Vo WO: fibrils wre coupled.

Set'ssogrants were run in HBS-EP buffer from BlAcore ai 20 tsL.'min in the Fci |V.-0 XVii ilhrikst minus Pc-2 (control} mode. Samples containing mAh or mAb plus peptide Inhibitor,·» were injected ί70 pi..} unci the sonsognuns collected ttstttg tlte debyed-oash function for 200 sec. Data were m.-dyzed itt the Bl.Aev-duiation software, using the 1;: Langmuir model with muss-actlmt correction, 0. lVhcroSPbi.'T:(.'T> Two cohorts of i rnicc cadt were injected s.e. with 5ft mg of human At. amyloid ex tract boisvocn t.hc scapulae. After 7 days, erne group of nttcc received an is lad vein intjeetton of · 300 uC; of biabeled twAb WH. Tfefc ami equal quantity of murine mAh MOPE 3 IC as a control After 72 hr. the mice were sacrificed h> isoflu rune overdose and SPEC.'TXT I so ayes acquired To provide vascular contrast-enhancement in the Cl Images, -nice were gwen a 200-ua is dose of fenestra VCs- {Advanced Research Technologies. ivlorureai, Canada)3 min prior ;o scanning, SPEC?! data were collected with a microffAl it t SPECT dual modality in taking plMforrn {Siemens Preclmical Solution.-', Knoxville. TN), capable of submillimetcr spatial resolution when equipped with a 0.5 rare-pore diameter pinhole conirmuor. When imaging, the 2 detectors tcomposed of a 50 mm-diameter Hamamatsu R24S6-02 multi-anode photo-multiplier tube coupled to α I 1 v- f mm Cat {Tl> crystal array arranged on u 1.2 mm grid) were positioned Ά5 mm from the center of rotation. Each SPECT daiU"Ct comprised 45 projecnosts collected over 300' during fne course of -50 min. Images were reconstructed using m implementation of the expectation m a >;i m leaf mi- ma x i ra s ra likelihood {EM-Mt. ) algorithm.

After collection of SPECT data, high-resolution El images were obtained. Ike micros?AT II scanner has a circular orbit cone beam geometry, equipped with a 20-^0 kVp .mlcrofocus x-ray source, and captures a 30 mm x 00 mm held of s sew using a 204 K x 5073 ( CT> array detector,. optically coupled to a minR phosphor screen vsa a fber-opnc bundle. Bach E'T ' dataset, composed of 360 projections at C azimuths, was acquired in 5 min. Images 'were reconstructed in real-time cut isotropic ‘17-pm voxels using an implementation of the Eckikamp Itm.trprojectiot\ algoritkm.

To facilitate co-registration ol the reconstructed SPEC!' and if f images, E'o-37 sealed sources were placed or; the imaging bed The rmetoSPECΐ and CT datasets were wsuaf.eed and co-registered manually with a 3-D unage analyst·.' software package {Amira, Version 3.1. Mereury Contpoter Sy.steo.us).. 10. Biodistributiom Samples of hver, spleen, kidney, heart, lung, and implanted amyloid motors (i.e . amyioidomat were harvested from the mice and placed into fared vials, weighed, and dm radioactivity measured. The primary index values were expressed us % injected doxe/g tissue P'« IP g>. 1!.. Autoradiography, b gm-thiek sections cut from forroalm-iixed. paraffin-embedded blocks of tissue obtained from mice sacrificed 72 h post-injection of i-?OH were placed on Probond microscope slides (Fisher Scientific), dipped m Ν'ΓΒ-2 emulsion (Eastman Kodak), stored in the dark, and developed after a 24-h exposure. The sections were counter-.stained with hematoxylin and eosin (HA;l:.f cover-slipped using Ponnount (Fisher Scientific), and examined by fight micro&amp;cup>. in addition. consecutive slides were stained with alkaline Congo red and viewed under cross-pohn'ired ilhimmation Finally., a third slide was tmmunostatned using as primary icagcvst our ΛΑ-feuctKe mAb. Digital camera microscopic. Images were taken and evaluated using an Image analysis software package 1 In-age Fro Plus, Meduc Cybernetics).

Example XIV, Preparation Of (Fenianiced 2Ad And ?Dk Antibodies.

Fiinnanieed 2 AC 70 X, and d{>} antibodies were prepared by main eg of marine 2A4, 7Dh. and Kfj9 CDRs onto human acceptor frameworks according to techniques know.a in the art. Bod·; mute cons were made to reduce' antigenicity while preserving binding affinity The light chain and heavy chant variable regions of murine 2 Ad arc set forth as res tunes 20-1 31 of SFQ if) NO; 15 2 nod as residues 20-1 3 b of OEQ ID NO; 154. respectively. The light chat a and heavy chain tunable regions of 0.48 are set forth as residues 20-131 of SEO ID MO; 03 and as residues 20-1 3 k of SFC) ID NO; 154. respectively. The light chain variable regions of marine 2.Λ4 and k{ 19 are identical to each other and differ from the light chain variable region of ?Pk in a single residue in ODR.I. The heavy chain variable regions of each of 2Λ4, 7 DO. mid KGh are identical.

Ike variable kappa (Vk) οί' 2A-t and '?D<M belong to moose subgroup 2. which corresponds to hum·in subgroup 2 and the variable heavy ΐ Vh > to mouse subgroup 3c which correspond* to human subgroup 3 (Kukri et k, t BAN) Sequences of Proteins of hTntnsnologileal Interest. Fifth Edition. N1H Publication Mo, oi-3242) CORT..1 includes Ιό residues and belongs to canonical class -I ;n Vk CPR-1.2 inelndes 7 residues and belongs to class I in Vk. CDR-1..3 inelndes 9 rent dues mid Ik longs to class 1 m Vk. Set- Martin AC. Thornton JM. i 1996? j Mol Biol. 263. kOO-i.A The leucine at position 27 in the 7D8 is rather unusual, and the glutymmc in 2A4 is swore usual. A model shows the sid&amp;cham is on the surface of the binding site, and there fore should lx; important for antigen binding. CDR-HI includes 5 residues and belongs to class I. ami CDR-H2 includes 19 residues and belongs to class 4 (Martin &amp; Thornton, 1996). CDR-H3 ha* no canonical classes, but the H residue loop probably has a kinked base according to the rules of Shi rat et ah (1940) niBS Lett. 455, 188-97. This is conserved in a model although the conformation of the apex of CDR-H3 may be different. The residues at the interface between the Vk and Vh domain* arc the ones commonly found for 2 Ad Vk, 7D8 'Vk and 2A4 Vh. A .search was made of the ROB database tDeshpande ot nl, (2005) Nuelcie Acids Res 33; 0233--7} to find structures which would guide the choice of Pack mutations A search of the non-redunduot protein sequence database freer: ACB1 allowed .selection of suiiabte human frameworks into which to graft the m-tfine C'ORs. hot Vh, a human kappa light chain with NCBf accession code B.A601S67. (gr2166i!075'i t S HQ ID NO: ton's was chow re Hus has die same length CPR--L3 ami belongs to human germlinc VKflARi Λ3 and Unman kappa subgroup 2 A simitar framework which only ulffemd In dm i-region was also Ibund with NC8I accession code BAC01733 tgi:2l66v417> (SEQ ID NO; 167). BAC0I562 was u>«i as a iVamcwork for 3A3 Yk, and BAAOI "33 mas used as a framework Am 7Do V'k. For Vh. loam.an lg heavy chain Λ AC'31024 {go A'3! 061) {Sf.Q ID NO; ;65) was nsod ΛΥη (..has el at {199 7) (7 P? £.\ p. inunuivti. 107; 372-3*0. 'This belongs to human germ line VH3-A7 and human heavy subgroup 3,

Representative humanized 2Ad light chain variable regions are set forth; as SEQ ID AOs; 135. 15b. and 15Ά Repecsencmsvc Daman i red 7D6 light chain variable regions arc act forth as SEQ ID NOs. 15 A 159, 10(). P4, 175, and 176. IbeprcsenTauve humanized 2.A4 7D8 heavy cham variable region.-' are set forth; as SEQ ID AOs: 161, 162, and 163. See Figures 36Λ-36 b.

Representative hum an mod antibodies of the invention include antibodies having a light chain variable region «elected from one of residues 20-131 of SEQ ID NO: 152. residues 20-131 of SFQ ID NO' 153, and SEQ ID NOs; 155. !5f\ I5~. 157, 159, 160, 174, 175, and 176; and a heavy chain variable region selected from one of residues 20-13* of SFQ ID AO 153 and SFQ ID NOs: 161. 162., and 163.

Example XV. Therapeutic Effects Of MAi> ,M4 lift: Mice With Severe .Systemic A.A. Amyloidosis.

The therapeutic efficacy of mAh 2.Λ4 was evaluated in 142 halL-e mice with severe systemic amyloidosis. The transgenic H2 hidi.--6 mice, winch constitutive!}· express a human If.-6 tvansgene, are prove to rapid and irreversible systemic .A.A amyloidosis In a Ami and second study, mice treated with Aoiypo-mavched mAh TV-1 j, which; has no reported activity in nice, was used as a control Before administering the amyloid enhancing factor to induce Λ A, H2'hulL.~6 mice were sampled arid bled via the retro-orbital sinus, serum prepared, and site sAA concentration determined using a commercially available EUR A kit. Representative values were as lollo'As 2.196,7 ug mb, 623.91 up mb, 1315.()(: ug ml., 1673,01 Ug mb, *14 53 tig ml.. 1088.18 ug/mL, 73634 :$53:JHp^ftrL,~M§$6 ng rdL m.d#Y:::: 111:3.4* 4"8 iig/ml...

At the start of the .second study {week 0'?, H2 hnlL-η mice were I moored ;v with 100 ug of amyloid enhancing factor t ABF} Λ tier induction of ΛΑ pathology by ispechng A fifth the mice vs ere administered 5 injections of 100 gg subcutaneously in alternate limbs of mAh 2A4 (13 animals) or TV 1 i iH ammalst. The themny was initiated at approximately 1 week ix»st AEF injection. The survival of animals m each treatment group w;m plotted and uu»1y<iod The results are shown in "fable ”, Only 45υ« of site mAb TY1 1 -treated mice survived to the end of the study, In contrast, nunc of the 2A4-trentcd truce were lost over the course of the studs Analysis of the survival data using. mumixrd methods showed a significant difference in the survival curves (P': 0.0()25) in both groups. The medlar; survival of the TV! i-treated msec w as calculated to he4 1 days, comparable to that observed in a prior study (3s.5 days;.

Table 7

At week t\ post-A Oh mice vs ere bled and sacrificed, and then organs harvested for further analysis. For quantification of amyloid in liver and spleen. Congo red birefringence was visualized microscopically under cross-polarized i lb. mil nation and digitally recorded. The area of hired'lngem materia! was determined by selecting (using a spectral segmentation method) and quam; lying the amyloid-associated pixels. The amyloid burden sndex (ABit, a measure of amyloid content, was expressed us the percentage area occupied by amyloid in each organ. Quantification of amyloid in the livers and spleens of 2 A 4 and TV; I-treated mice revealed no significant diOerenee between the two treatments. However, the TV SI nrouted mice that survived to day 42 for comparison with .? A 4-treated mice wore those that did not develop a morbid degree or distribution of AA amyloid to thereby result in morbidity: The hepatosplemc amyloid burden is also monitored during the course of the survival study to assess an increase in amyloid burden bun correlates with morbidity.

In a third study, mAb 2Ad was compared to the isotype-rnatchcd mAb JH70. which has u.) reported reactivity in mice, ha addition blood cbermstrv and other parameters were monitored throughout the treatment period Male and female H2h»IL-6 once bore between Η I Ox and 0 7.OS were used in this stud) Twenty three female mice and io male once were bled \u the retro-orbital shins, Whole blood was used for chemical character] canon of blood an ',..a tmfogon ίBUNv and alanine nmiriemansterase tALTi to measure renal and hepat-e function In usiny the Vohsewu VS2 tAbaxis, Union (Ity, CAT ITe serutn concern ration of 12 other pone:re- and analytes were simultaneously measured. A complete blood count ((’BCi was performed using the Vetlscun HMS platform. In addition, each mouse was administered a low dose (- :50-60 g(.'i) of radiolodimuvd human serum amyloid P component ("M-SAPt in 5 mgwrtl. bovine scrum albumin to assess the amyloid burden of the mice prior to munition of the disease process. T he percent of ' M-SAP retained at 24 h pont-mjecrion (pi) was measured by placing each mouse into a dose calibrator. Retention of **'1*SAP greater than that observed so non Transgenic (control) mice was indicative of amyloid disease, 'finally, serutn was used to measure the concentration of serutn amyloid protein A tsAA) using a commercial h. US A. assay, Λ summary of these pretreatrrtersf dam, selected' blood chemistry values, and the treatments given to each mot use are shown below in Tables h and 9.

Table h

Summary Of Pro-Treatment Data And MAb Therapy feu bach Animal

1, homosvxous it -?·; animals with hi ah sAA levels and amyloid disease cam· in Hie, .. , · * *·' , :,. . .,. "" ., . . ,. . . , ' ^ -ζ . . 2, v\iid type mice v> ithouJ circulating »AA and ;>,:· ams-iont disease. ’’ (-SAP retention in dte.v untnuds is considered normal and reflecting no amyloid borden.

Ί able P

Normal Values Per Blood Chemistry Parameters In R2’hulL-6 Mies'

BUN., blood urea nitrogen; 01.Lb glucose; AIT. alanine aminotransferase; ALB, alburn m; TP, total scrum pro;cm. GLOB, imrmmoglobalin; F, female; M. male; SD. sturniurd deviation: n is the number of mice used *o determine the values. Λί the siart. of the third study (week Ok alt of die ait the H2hulL-o mice received 100 ug iv of amyloid enhancing factor (I mu mt'.j. One week thereat ter, therapy began and each mouse wax administered 100 μμ of minor mAh .?.A4 or JH70 sc an end mod in fable tv The mAh Injections continued weekly Lbr 7 weeks,

V; 2 w k posi-AHF, GB(. Good chemistry, and serum sAA sm.asufmnemx weso made ising blood collected via the nctvt*-orbi?uf sinus. At this tmse also, the mice m group I were administered - 60 μ(Ι ot' L>i'~SAP in B$A as before, to assess the aeauTnd-.uton of amyloid »$ evidenced by the retentior of the -adictiuheled SAP Several of the an muds showed an adverse effect of extreme distress, and therefore. «valuation of amyloid burden using :''sbSAF was discontinued. Results ot' selected blood chemistry parameters, acquired 2 wk post-AEF are shown to .Table HT

Table 10

BUN, blood urea nitrogen:. GUf, glucose; AL T, alanine aminotransferase; At B, albumin: TP., total scrum protein: GLOB, iroirumogiobahn; F, icirK.de: kt., male, SIX standard deviation: π is the nun the? of mice used to dotemhue the values

At IS weeks post-AEF, the mice were bled a final time and immediately thereafter were administered ·· 200 uCi of:'' !-SAP using 5% normal mouse sen.nrs as currier. In response to dhx treatment, a few animals .shows.·d sotue unusual behavior that abated within .M3 mm. Twenty four hours later, the rntce were injected with x-ray CT contrast agent {' 200 ul. tv in the l id velm and were then sacrificed by isodnrane overdose. Single photon emission (SPEC?) arid x-ray fCT) tomographic image* of each animal were acquired. The oeaans were harvested and the amount of radioactivity in each sample was calculated and expressed as fo injected dose per gram ot tsssue. Additionally, a portion of each tissue was fixed overnight in buffered Pm mb in in preparation tor sectioning and microscopic analysis.

During, the 7 wk therapy study. 2 mice were found dead and .1 mtee were -oscniiced because they were deemed unlikely m survive overnight and had a poor body coedition score (2: associated with > 15% weight loss), kike that experienced an adverse reaction to '"'l-SAP injection and 1 mouse fh.it was sacrificed due1 to complications dun arose from a retro-orbital bleed were not evaluated as pan of the survival analysis. The survival of the mice in each mAb treatment group is shown in Table 11,

Table 1!

Approximately 65'.% of the mAh JH?(Mrcatcd mice that were assessable survived to die cud of the study In contrast, none of the 2Ad mice dan were assessable died during the 57 days Analysis of the survival data using die standard methods demonstrated a stgnbkiml difference in the snrv isal curves fP:::0.0;S using Mantel'Cox test and P:::0.dtf> using Orehan-Brexlow-

Wikoxon tests,

The final blood chemistry data were analysed according to the therapv that each mouse received Because of differences in the mean parameter values associated with nude and female HI; hid l mice (at the time of sacrifice. SUN levels tn female mice were higher for both 2Λ4-treated arid JH?0-trealed micch ottiv the female mice that survived are included in Table 12 below.

Tabic V2

BUN. binod urea nitrogen; GLU, gluco,ve·. ALT, elnninc aonnotransferase; ALB, albumin; TP, total screen protein; GLOB, immunoglobulin; F, female, M, male; SD. .standard dc\ration; n A t he number of mice used to determine ti ts' values.

Mice treated with ~A-4 ahovsed decreased serum blood urea nitrogen (BUN) and uiamne >.uru not ran.si erase (ALT) levels when compared to mice treated with JH?0. BLN &amp;nd ALT are markers of renal and hepatic function, respond vch, and their reduced levels i mil cate that organ function may have been better preserved-by dA4 treatment.

Claims (15)

1. An isolated antibody or antigen-binding fragment that specifically binds to a neoepitope exposed in an aggregated light chain immunoglobulin protein, wherein the neoepitope comprises the sequence X1EDX2 and wherein Xi is Η, T, F, S, P or A and wherein X2 is T, S, E, D, R, I, V, F or A.

2. The antibody or antigen-binding fragment of claim 1, wherein the sequence X1EDX2 is selected from the group consisting of FEDD, SEDE, AEDE, TEDE, PEDE, PEDI, PEDF, AEDV, SEDF and SEDA.

3. The antibody or antigen-binding fragment of claim 1 or claim 2, wherein the antibody or fragment is a chimeric antibody, a human antibody, a humanized antibody, a single chain antibody, a tetrameric antibody, a tetravalent antibody, a multispecific antibody, a domain-specific antibody, a domain-deleted antibody, a fusion protein, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, or a ScFv fragment.

4. A method of therapeutically or prophylactically treating a subject having AL amyloidosis comprising administering to the subject an effective dosage of the antibody or antigen-binding fragment of any one of claims 1-3.

5. A method of detecting an amyloid deposit associated with AL amyloidosis in a subject comprising: (a) administering to the subject the antibody or antigen-binding fragment of any one of claims 1-3, wherein the antibody or antigen-binding fragment is bound to a detectable label; and (b) detecting the detectable label in the subject.

6. The method of claim 4 or claim 5, wherein the AL amyloidosis is associated with peripheral or autonomic neuropathy, carpal tunnel syndrome, macroglossia, restrictive cardiomyopathy, arthropathy of large joints, immune dyscrasia, myeloma, monoclonal gammopathy, occult dyscrasia, or a chronic inflammatory disease.

7. Use of the antibody or antigen-binding fragment of any one of claims 1-3 in the manufacture of a medicament for treatment or prophylaxis of AL amyloidosis in a subject.

8. Use of the antibody or antigen-binding fragment of any one of claims 1-3, wherein the antibody or antigen-binding fragment is bound to a detectable label, in the manufacture of a medicament for detecting an amyloid deposit associated with AL amyloidosis in a subject.

9. The use of claim 7 or claim 8, wherein the AL amyloidosis is associated with peripheral or autonomic neuropathy, carpal tunnel syndrome, macroglossia, restrictive cardiomyopathy, arthropathy of large joints, immune dyscrasia, myeloma, monoclonal gammopathy, occult dyscrasia, or a chronic inflammatory disease.

10. The method of claim 5 or claim 6, wherein the detectable label is a radiolabel.

11. The method of claim 10, wherein the radiolabel is l25I.

12. The method of any one of claims 5, 6, 10 or 11, wherein the detecting is performed by SPECT/CT imaging or NMR spectroscopy.

13. The use of claim 8 or claim 9, wherein the detectable label is a radiolabel.

14. The use of claim 13, wherein the radiolabel is 125I.

15. The use of any one of claims 8, 9, 13 or 14, wherein the detecting is performed by SPECT/CT imaging or NMR spectroscopy.