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(11) Patent Number: KE 466   
           
(45) Date of grant: 08/09/2011   

(51) Int.CI.S:G OlN 33/53

(21) Application Number: ICE/P/2010/001081

(22) Filing Date: 14/11/2008

(30) Priority data:60/988,481 16/1112007 US and 61/019,747 08/01/2008 us

(86) PCT data PCTIUSOS/083659 14/1112008 wo 2009/065054  A2 22/05/2009
 
(73) Owner:THE ROCKEFELLER UNIVERSITY of  1230 York Avenue, NewYork, NY 10021-6399, U.S.A.

(72) Inventor:RAVETCH, Jeffrey 500 Park Avenue, New York, NY 10022, U.S.A and FUKUYAMA, Hidehiro Strasboug,France

(7 4) Agent/address for correspondence:Kaplan & Stratton Advocates, P.O. Box 40111-00100, Nairobi
 
(54) Title: ANTIBODIES SPECIFIC FOR THE PROTO FIBRIL FORM OF BETA-AMYLOID PROTEIN.

(57) Abstract: Isolated antibodies have been characterized which show specific affinity to a repeating confonnational epitope of a proto fibril fonn of the human B-amyloid peptide as compared to low molecular weight fonns of B-amyloid peptide. These isolated antibodies and related pharmaceutically effective compositions may be useful in the therapeutic and/or prophylactic treatment of Alzheimer's disease by effectively blocking the ability of the protofibril fonn of B-amyloid peptide to fonn fibril fonns linked with complications associated with Alzheimer's disease. The isolated antibodies of the present invention are also useful in various diagnostic assays an associated kits.
 
ANTIBODIES  SPECIFIC  FOR  THE  PROTOFIBRIL  FORM  OF  BETA-AMYLOID

PROTEIN CROSS-REFERENCE TO RELATED APPLICATIONS

This   application   claims  priority  to  provisional  patent 5    applications:    60/988,481    filed    November    16,    2007;    and 61/019,747  filed  January  8,   2008,   the  contents  of  which  are

incorporated  by  reference.

FIELD  OF  THE  INVENTION

The    present   invention   relates   to   isolated   antibodies

10    that interact specifically with conformational epitopes of a protofibril form of human beta-amyloid peptide. These antibodies show minimal or no detectable affinity towards lower molecular weight forms of beta-amyloid peptide. The

antibodies  disclosed  herein  will  be  useful  in  the  diagnosis, 15    treatment     and/or     prevention     of     beta-amyloid     plaque deposition   associated   with   the   onset   and   progression   of

Alzheimer's  disease.

BACKGROUND  OF  THE  INVENTION

Beta-Amyloid    (A~)  peptides    are    thought    to    be    a

20    causative agent for Alzheimer's disease ("AD") through the formation of insoluble A~ peptide fibrils and deposition of these fibrils to form amyloid plaques. The formation of such plaques within the area of the brain critical for

memory and other cognitive functions is thought to lead to 25 dementia associated with this disease (see Selkoe, 1994, J.

Neuropathol. Exp. Neural. 53:438-44 7). Beta-Amyloid peptides comprise a group of peptides 39-43 amino acids long that are proteolytically processed from amyloid precursor protein (APP), by both J3-secretase and y-secretase at the

amino- and carboxyl-terminus, respectively. There are at least five distinct isoforms of APP: 563, 695, 714, 751, and 770 amino acids in length, respectively (see Wirak et al.,

1991   Science  253:323).     These   isoforms   of  APP   are  generated 5    by   alternative   splicing   of   primary   transcripts   of   the   APP gene.     Numerous   missense   mutations   have   been   identified   in APP     in     families     with     autosomal     dominant     early-onset Alzheimer's    disease.        Some    mutations    cluster    near    the secretase  cleavage  sites  and  affect  APP  metabolism  either  by

10    increasing the production or the proportion of Ar> forms (e.g., Ar> 42 ), which tends to be more fibrillogenic and to

aggregate faster than other forms. Neuronal toxicity may reside in the large molecular weight fibrils which are formed via aggregation of soluble A~ peptides into insoluble

15    fibrils and, subsequently, fibril incorporation into amyloid plaques. An intermediate fibril form is the protofibril

(PF) form, a large molecular weight oligomeric form of A~ peptides which is soluble in vitro and may be isolated as an approximately -670 kDa entity. Thus, the in vitro formation

20    of insoluble A~ peptide fibrils is the end result of the initial oligomerization of A~ peptide to form a structurally

distinct,   soluble   higher   molecular  weight   protofibril   form. These  transient  protofibrils  structures   are  precursor  to  the amyloid  fibers   responsible  for   cell  dysfunction  and  neuronal 25     loss     in    Alzheimer's     disease     (AD)     and    other     protein

aggregation  diseases.

Various treatments have been forwarded in attempts to prevent the formation of Ar> peptide, for example, inhibitors to prevent the proteolytic processing of APP. Also,

30    immunotherapy strategies such as administration of anti-Ar> antibodies (to induce clearance of amyloid deposits) or immunization with Al3 peptide antigens (to promote a humoral

response) have been enlisted in an attempt to reduce plaque size and density.

u.s.  Patent  No.   7,179,463,   issued  to   Lannfelt   et   al. , discloses    a    method    of    treating   Alzheimer's    disease    by 5    administering    an    antibody    raised    against    a    protofibril consisting   of   the   Arctic   mutation   within   the   A~  peptide coding  region.    No  exemplification  of  raised  antibodies   are presented   in   the   specification   and   no   comparison   as   to affinity   for   low  molecular   weight   forms   of   A~  peptide   are


10    presented.

U.S. Patent Nos 6,761,888 and 6,750,324, issued to Schenk et al. , disclose a series of antibodies which recognize various epitopes along the amino acid sequence of

A~ 42 • Antibodies specific for the N-terminus and mid-regions 15 of A~ 42 showed efficacy in reducing plaque both ex vivo and
il1    vivo.

Despite current knowledge in the field of treating and preventing Alzheimer's disease, there remains a need for an improved compositions and methods of treating and/or

20    preventing this disease. The compositions and methods of the present invention address and meet these needs by

disclosing    antibodies   specific   for   protofibrillar   forms   of

A~ peptide while showing minimal detectable affinity against low molecular weight forms of the A~ peptide.

25    Pharmaceutically effective compositions comprising such an antibody or antibodies will be useful in treating and/or preventing beta-amyloid plaque deposition known to be associated with the onset and progression of Alzheimer's disease.

SUMMARY  OF  THE  INVENTION

The present invention relates to an isolated antibody that shows specific binding to a conformational epitope of a protofibril form of P-amyloid peptide. The monomer of wild

5    type beta-amyloid (AP) peptide is known in the art and is shown herein as SEQ ID NO: 1. The isolated antibodies of the present invention have affinity for such a repeated conformational epitope for the larger molecular weight protofibril form of the AP peptide while showing minimal or

10    no affinity for other forms of AP peptide, such as monomer or dimer forms of AP peptide.

The present invention also relates to an isolated antibody that specifically interacts with and shows a measurable affinity to a conformational epitope of a

15    protofibril form of AP peptide, whereby the protofibril epitope is represented by an exposed region of a AP-protofibril form comprising the amino terminal portion of an exposed portion of the AP peptide.

The    present   invention   further   relates   to   an   isolated

20    antibody that specifically interacts and shows a measurable affinity to a conformational epitope of a protofibril form of AP peptide, whereby the protofibril epitope is

represented by an exposed region of a AP-protofibril form comprising amino acids 1-20 (SEQ ID N0:2) of an exposed

25    portion  of  the  AP  peptide.

The present invention also relates to an isolated antibody that specifically interacts and shows a measurable affinity to a conformational epitope of a protofibril form of AP peptide, whereby the protofibril epitope is

30    represented  by   an   exposed   region   of   a   AP-protofibril   form

which comprises amino acids 4-12 and 9-20 (SEQ ID NOS: 3 and 4, respectively) of an exposed portion of the A~ peptide.
The   present   invention   relates   in   part   to   monoclonal antibodies  13C3,   lDl  and  19A6,   and  any  affinity  matured  form 5    of   13C3,    lDl   and   19A6.      The   present   invention   further relates     to    an    antibody    which    mimics     the    functional specificity  as  described  herein  for   13C3,   lDl  and  19A6.    To this  end,   the  present  invention  also  relates  to  biologically active  fragments   and/or  mutants  of  the   13C3,   lDl,   19A6  or  a


10    13C3-, lDl-, or 19A6-like antibody, including but not necessarily limited to amino acid substitutions (e.g., as a
directed form of affinity maturation of the V" or v, regions), deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations

15    provide a basis for an antibody or antibody binding portion that results in a similar or improved version of a 13C3, lDl, 19A6 or 13C3-like antibody binding protein. In one

embodiment of this portion of the invention, the v" and VL region of 13C3 comprises the amino acid sequence as set

20 forth in SEQ ID NO: 7 (VH) and/or SEQ ID NO:S (Vc), respectively.

The present invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence which encodes the VH and/or V0 regions of a 13C3, lDl or 19A6

25    antibody; and especially an isolated nucleic acid molecule (polynucleotide) encoding a biologically relevant portion of

13C3, or affinity matured version or otherwise mutated version of 13C3, lDl or 19A6 antibody. To this end, one embodiment of the present invention relates to a nucleic

30    acid molecule which comprises a nucleotide sequence encoding the VH and V0 region of 13C, as set forth in SEQ ID NO:S


(13C3:    V"    region)    and    SEQ    ID   N0:6    (l3C3:    region),

respectively.

The present invention also relates to isolated antibodies 13C3, lDl or 19A6 as disclosed herein, antibodies

5    that specifically interact and show a measurable affinity to a conformational epitope of a protofibril form of A~ peptide.

The present invention also relates to a hybridoma capable of producing a monoclonal antibody of the present

10    invention. Particular hybridomas of the present invention include hybridomas which produce exemplified monoclonal antibodies 13C3, 19A6 and lDl, respectively.

The present invention relates to pharmaceutically

effective    compositions   which   comprise   an   isolated   antibody

15 as disclosed and further defined herein: an isolated antibody that specifically interacts with and shows a measured affinity to and ability to specifically bind to a repeating conformational epitope of a protofibril form of A~ while showing minimal or no measurable affinity to low

20 molecular weight forms of A~. These compositions may optionally comprise one or more carriers, one or more excipients, and/or one or more chemical derivatives.

The    present    invention    also    relates    to    methods    of treating   an   individual   afflicted   with   Alzheimer's   disease 25    comprising       administering       to       the       individual       a pharmaceutically   effective   composition   which   comprises   an isolated  antibody  disclosed  herein,   namely  an  antibody  that specifically   interacts   and   shows   a   measured   affinity   to   a repeating  conformational  epitope  of  a  protofibril  form  of  A~


30    peptide while showing minimal or no detectable affinity toward lower molecular weight forms of A~ peptide. These methods will provide for a therapeutic intervention so as to

reduce the amount of amyloid deposits in the brain of an individual afflicted with Alzheimer's disease. Particular embodiments of this portion of the present invention relate to methods of treating an individual afflicted with

5 Alzheimer's disease comprising administering a pharmaceutically effective composition formulated with an antibody showing specific affinity (as at least compared to low molecular weight forms of A~ peptide) to a conformational epitope of a protofibril form of A~ peptide,

10    especially whereby the protofibril epitope is represented by an exposed region of a A~-protofibril form which comprises amino acids 1-20 (SEQ ID N0:2) of an exposed portion of the

A~ peptide. Specific embodiments relating to these therapeutic and prophylactic methods disclosed herein may

15    utilize exemplified mouse monoclonal antibodies 13C3, 19A6, lDl, as well as affinity matured versions of any such

antibody, chimeric antibody, humanized antibody, human monoclonal antibody, and/or any other such antibody form known in the art, including but not limited to the antibody

20    or specific binding members reviewed herein. Any such antibody or specific binding member may be referred to within this specification as a 8 l3C3-like antibody." Thus,

a 8 l3C3-like antibody" is meant to also encompass the 13C3 monoclonal antibody disclosed herein.

25 The present invention also relates to methods of screening for and selecting compounds which may act as an inhibitor of fibril and/or senile plaque formation associated with Alzheimer's disease. Such a methodology comprises utilizing an antibody with 13C3-like


30    characteristics (e.g., specific affinity to the PF vs. LMW forms of A~ peptide) in various antibody/peptide/test oompound interaction assays in order to select a compound

which modulates the process of fibril and/or plaque formation.

The present invention further relates to diagnostic assay methods to specifically determine protofibril levels

5    within a subject or patient. Such assays may be carried out by any techniques known and available to the artisan, including but not limited to Western blots, ELISAs,

radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known

10    in the art. Thus, one embodiment of this portion of the invention relates to taking a tissue sample from a subject or patient and determining the level of PF A~ in the sample using a diagnostic kit and associated assay; whereby the kit comprises a 13C3-like antibody, thus allowing for the

15    specific determination of PF A~ levels in the tissue sample. The tissue sample for analysis is typically blood, plasma, serum, mucus or cerebral spinal fluid from the subject or

patient

To    this   end,   the   antibodies   of   the   present   invention

20    may be utilized for at least the following uses: ( 1) as a prophylactic or therapeutic agent to prevent or reduce plaque deposits associated with Alzheimer's disease, either alone or in conjunction with any available combination

therapy;    (2)   in   designing   peptide   immunogens   that   may   be 25    used     to     elicit     an     effective     antibody     response     in prophylactic  or   therapeutic   vaccination   strategies   relating to   treatment   of   Alzheimer's   disease;    (3)    to   generate   a prophylactic   or   therapeutic   anti-idiotypic   antibody    (Ab2) mimicking  the  cryptic  epitope(s)   that  bind  the  antibodies  of

30 the present invention; and, ( 4) in designing peptides derived from the complementarity determining regions (CDRs) of the neutralizing antibodies of the present invention for

use in either screening inhibitors of protofibril formation for use in prophylactic and/or therapeutic regimes and ( 4) as a diagnostic reagent to determine the level of protofibrillar A~ in serum or CSF of a patient at risk for

5    developing  AD.

It is an object of the present invention to provide for antibodies that specifically interact and show affinity to an exposed, conformational epitope of a protofibril form of

A~ peptide  which  comprises  amino  acids  1-20   (SEQ  ID  N0:2)   of

10    an  exposed  portion  of  the  A~ peptide.

It is a further object of the present invention to provide for antibodies that interact and show affinity to an exposed conformational epitope of a protofibril form of A~ peptide which comprises amino acids 4-12 and 9-20 (SEQ ID

15    N0:3,   4)   of  an  exposed  portion  of  the  the  A~ peptide.

Another object of the present invention is to provide 13C3-like antibodies which prevent or reduce A~ peptide protofibril formation linked to the deposition of plaques associated with Alzheimer's disease.

20 Another object of the present invention is to provide assays utilizing l3C3-like antibodies in antibody/peptide/test compound interaction assays to select compounds which will be useful in treating plaque deposition associated with Alzheimer's disease.


25 As used herein, "Ka" is intended to refer to the association constant of a particular antibody antigen interaction, "Kd" is intended to refer to the dissociation constant of a particular antibody-antigen interaction.

As    used    herein,    the    term    "epitope"    or    "antigenic

30    determinant" refers to a site on an antigen to which B and/or T cells respond or a site on a molecule against which an antibody will be produced and/or to which an antibody

will bind. For example, an epitope can be recognized by an antibody defining the epitope. An epitope can be either a

"linear epi tope 11 (where a primary amino acid primary sequence comprises the epitope; typically at least 3

5    contiguous amino acid residues, and more usually, at least 5, and up to about 8 to about 10 amino acids in a unique sequence) or a "conformational epitope" (an epitope wherein the primary, contiguous amino acid sequence is not the sole def~ining component of the epitope). A conformational

10    epitope may comprise an increased number of amino acids relative to a linear epitope, as this conformational epitope recognizes a three-dimensional structure of the peptide or

protein.    For  example,   when  a  protein  molecule  folds   to  form

a    three   dimensional   structure,    certain   amino   acids   and/or

15    the polypeptide backbone forming the conformational epitope become juxtaposed enabling the antibody to recognize the epitope. Methods of determining conformation of epitopes

include    but    are    not    limited    to,    for    example,    x-ray

crystallography,    two-dimensional   nuclear   magnetic   resonance

20    spectroscopy and site-directed spin labeling and electron paramagnetic resonance spectroscopy. See, for example,

Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996), the disclosure of which is incorporated in its entirety herein by reference.

25 As used herein, "specific binding" between two entities means an affinity of at least 10 6 M-1 , 10 7 M- 1 , 10 8 M-:, 10 9 M- 1 , or 10 10 M- 1 •

As used herein, "protofibrils" are protofibrillar aggregates which include spherical structures comprising A~

30    peptides that appear to represent strings of the spherical structures forming curvilinear structures.


As used herein, the term Hisolated" is used herein as it is used within the art. Namely, the state in which antibodies/specific binding members, nucleic acid molecules and the such are found. Antibodies/specific binding members

5    and nucleic acid molecules will be free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such

10    preparation is by recombinant DNA technology (practiced ill vitro) or in vivo. #Isolated" covers any form containing the identified and characterized component(s) of the present invention following removal from that initial environment. Examples, but certainly not limitations, include


15 pharmaceutical formulations, formulation with diluents, antibodies/speciflc binding members, nucleic acid molecules and portions thereof which have been modified (e.g., antibody glycosylation) either in vitro or in vivo and removed from that environment.


20 As used herein, the term "recombinant human antibody" represents a viable subset of "antibodies" generated by various means of recombinant DNA technology and non-human transgenics that are well known in the art. Such methodology is utilized to generate an antibody from one or


25    the following origins: (i) a scFv or alternative antibody isolated from a combinatorial human antibody library; ( ii) a partial or complete antibody generated from a respective

expression vector stably or transiently transfected into a host cell, preferably a mammalian host cell (e.g.,

30    subcloning :mcleotide sequences encoding VH and VL chains into an expression vector in conjunction with respective CH and CL nucleotide sequences, so as to promote expression of


a predetermined form of antibody showing specificity to the PF form of A~); and/or (iii) an antibody isolated from a non-human transgenic animal which contains human immunoglobulin genes, or by any other known methodology

5    which relies of the recombinant 'mixing and matching' of human immunoglobulin gene sequences to other DNA sequences in order to generate the human recombinant antibody of interest.

The   terms   usubject"   or   upatient"   is   meant   to   include

10 any member of the Phylum Chordata, including, without limitation, humans and other primates, including nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory

15    animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.

The  term  "treating"   or   "treatment"   of   a  disease  refers

20    to executing a protocol, which may include administering one or more drugs to a subject (human or otherwise), in an effort to alleviate signs or symptoms of the disease. Alleviation can occur prior to signs or symptoms of the disease appearing, as well as after their appearance. Thus,

25    "treating"    or    "treatment"    includes    "preventing"    or

''prevention"    of    disease.    In    the    case    of    Alzheimer's

disease,    "preventing"   or   11 preventing"   may   also   occur   in   a

situation    where   a   course   of   treatment   is   advanced  in   order

to    prevent   or   stall   onset   of   the   symptoms   associated  with

30    Alzheimer's disease. In addition, "treating" or "treatment" does not require complete alleviation of signs or symptoms,


does not require a cure, and specifically includes protocols which have only a marginal positive effect on the subject.

As  used  herein,   the  term  •active  ingredient"   refers   to a   13C3-like   antibody   which   shows   affinity   and   specificity 5     (e.g.,   specific   binding)   to   the   amino   terminal   portion   of

the  protofibril  structure  of  beta-amyloid.

As    used    herein,    the    terms    "effective    amount"    or

"pharmaceutically  effective  amount"  of  antibody,   as  provided herein,   refers   to   a   nontoxic   but   sufficient   amount   of   the 10    active  ingredient  in  order  to  provide  the  desired  biological result.    An  appropriate  "effective"  amount  in  any  individual case  may  be  determined  by  one  of  ordinary  skill  in  the  art

using  routine  experimentation.

As    used  herein,   the  terms   "pharmaceutically  acceptable"

15    or "pharmacologically acceptable" mean a material may be administered to an individual in a drug delivery device along with the formulated biological agent without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the

20 composition in which it is contained (e.g., a "pharmaceutically acceptable composition").

As used herein, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier, diluent, and

25    excipient that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is

included  under  these  phrases.

As  used  herein,   the  term  "excipient"  refers  to  an  inert

30    substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.

The term "minimal affinity" as used in comparing affinity of the antibodies for the protofibrillar form of the A~ peptide with affinity of the antibodies for other forms of A~ peptide, such as fibrils, sheet structures, and

5    low molecular weight oligomers and monomers, indicates that ratio of the affinity for the protofibrillar A~ form to the

affinity    for    other   A~  forms    is    greater    than    about    2.

Preferably, the ratio is greater than about 3, or about 4, or about 5.


10    BRIEF  DESCRIPTION  OF  THE  FIGURES

Figure l shows the process of A~ fibrillogenesis, including formation of protofibril oligomers.

Figure 2A-B show the process of A~ fibrillogenesis and purification of A~ forms at time 0 (A) and 4 hours (B) as
15    indicated by absorbance at rnAU 215 (absorbance @ 215 nm) for elution volumes. At 4 hours (B), the low molecular weight (LMW) form elutes as a -15 kDa dimer while the protofibril form size elutes at -670 kDa.

Figure 3A-B show the specificity of monoclonal

20    antibodies 13C3 (A) and 4G8 (B) for the protofibril (PF:-+-) and low molecular weight (LMW: _._) forms of A~, as indicated by optical density (OD) read at 450/650 nm for increasing concentrations of both the PF and LMW forms of

A~.

25 Figure 4A-C show data from a Biacore'" binding assay showing affinity of monoclonal antibodies 4G8 (A), 13C3 (B) and control IgG 1 (C) to varying concentrations of low molecular weight (LMW) form of A~ from 0.25 ~g/ml LMW A~ to

4.0  ~g/ml LMW  A~.

Figure 5A-B show data identifying the epitopes recognized by the anti- A~ antibodies described above. Figure 5A illustrates a Western Dot Blot analysis with monoclonal antibodies 13C3 (top panel), IDl (middle panel)

5    and 4G8 (bottom panel) against _a series of overlapping 13 amino acid peptides as described in Example 5. Figure 5B illustrates amino acid sequence of A~1-v (SEQ ID NO: 1) , as well as the predicted epitopes of monoclonal antibodies

13C3,    and  lDl.

10 Figure shows the reactivity of monoclonal antibodies l3C3 (_._) and 4GB (-+-) with SEC fractions from /PA2 supernatant secreting A~ oligomers. Protofibril (PF) and low molecular weight (LMW) fractions are indicated on the x-axis as measured by optical density (OD) read at 450/650.

15 Figures 7A-C show micrographs from thin section immuno-electron microscopy showing the affinity of monoclonal antibody 13C3 to repeated structures on A~ fibrils (B, C). Control immuno-EM is IgG 1 (A) .

Figure  8A-B  show  data  from  electron  micrographs  showing

20    reduction in plaque numbers in a representative TgCRNDS transgenic mouse after administration of control IgG,

(A)    antibody in comparison to administration of 13C3

monoclonal  antibodies   (B)  .

Figure    9A-B   show   that   treatment   of   TgCRND8   transgenic

25    mice with 13C3 monoclonal antibodies on a once a week (A) or twice weekly (B) regime result in a reduction of senile plaque formation.

Figure 10 shows the nucleotide and amino acid sequence of the cloned light and heavy chains variable regions for

30    mAb  l3C3.

Figure 11 shows that the acute peripheral administration of 13c3 in APP transgenic mice does not lead


to an increase in plasma AB unlike reference antibody 3D6 administration.

Figure 12 shows that 13C3 recognlzes human amyloid neuritic plaques (aggregated) in AD brains but not the

5    diffuse An deposits unlike the reference 3D6 anti-AB antibody.


DETAILED  DESCRIPTION  OF  THE  INVENTION

The amyloid precursor protein (APP) plays an important role in the pathogenesis of Alzheimer's disease (AD).

10    Proteolytic processing of APP by ~- and y-secretases generate A~ peptides (A~) which normally range in the length from 39 to 43 amino acids in length. The onset of Alzheimer's disease is characterized by the accumulation of oligmeric or

aggregated    forms   of   A~  in   the   brain.    The   immunological

15    compositions of the present invention are useful in treating or preventing Alzheimer's disease, for use as reagents in diagnostic assays, as well as for designing small molecule inhibitors of amyloid deposition. The 13C3-like antibodies of the present invention may be administered

20    prophylactically to the general population of a mammal, especially a human, in a contemplated pharmaceutically

acceptable formulation in an amount and/or dosage regime sufficient to eliminate, reduce or delay onset of the disease. Methods of prophylactic treatment are especially

25    warranted with individuals known to be at a genetic or familiar risk of Alzheimer's disease. Numerous genetic

markers of risk for Alzheimer's disease have been identified, including but not limited to APP mutations (e.g., the Indian mutation (Val717Phe), the Swedish

30 mutations (Lys670Asn, Met671Leu), the Hendricks mutation (Ala692Gly), the Dutch mutation (Glu693Gln), the Iranian



mutation (Thr714Ala), the German mutation (Val 715Ala), and the Florida mutation (Ile716Val), to list a few. Additional mutations which may indicate an increased risk of Alzheimer's disease include mutations in the presenilin

5    genes (PSl and PS2) and ApoE4. The present invention also relates to therapeutic intervention via pharmaceutically acceptable compositions comprising a 13C3-like antibody for individuals presently suffering from Alzheimer's disease can

be    recognized   from   characteristic   dementia,   especially   in

10    the presence of risk factors described above or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the Alzheimer's disease

symptoms and complications. Either prophylactic- or therapeutic-based treatment methods contemplated herein may

15    be used to address early or late onset Alzheimer's disease. In view of the importance of oligomeric forms of A~ in the onset of Alzheimer's disease, the present invention relates to an isolated antibody that specifically interacts and shows a measured affinity to a repeating conformational

20    epitope of a protofibril form of A~ peptide. The monomer of wild type A~ peptide (A~ 42 ; the 42 amino acid form) is known in the art and is shown herein as SEQ ID NO:l:

Asp  Ala  Glu  Phe  Arg  His  Asp  Ser  Gly  Tyr  Glu  Val  His  His  Gln

Lys    Leu  Val  Phe  Phe  Ala  Glu  Asp  Val  Gly  Ser  Asn  Lys  Gly  Ala

25    Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala (SEQ ID NO:l).

The isolated antibodies of the present invention will show affinity for a repeated conformational epitope of the larger molecular weight, oligomeric protofibrillar form of the A~

30    peptide while showing minimal affinity for other forms of A~ peptide, such as low molecular weight monomers and dimers.


The present invention also relates to an isolated antibody that interacts and shows a measurable affinity to a conformational epitope of a protofibril form of A~ peptide, whereby the protofibril epitope is represented by an exposed

5    region of a A~-protofibril form which comprises the amino terminal portion of an exposed portion of the A~ peptide.
The present invention further relates to an isolated antibody that specifically interacts and shows a measurable affinity to a conformational epitope of a protofibril form

10 of A~ peptide, whereby the protofibril epitope is represented by an exposed region of a A~-protofibril form which comprises amino acids 1-20 (SEQ ID N0:2) of an exposed portion of the A~ peptide, as follows: Asp Ala Glu Phe Arg His Asp Ser Gly Tyr G1u Val His His Gln Lys Leu Val Phe Phe

15    (SEQ  ID  N0:2).

As exemplified herein, mouse monoclonal antibodies have been identified which specifically show specific affinity for the protofibril (PF) form of the A~ peptide, while showing minimal affinity for low molecular weight species of

20    the A~ peptide. The dimer form of A~ (~15 kDa) over time polymerizes to form a soluble PF form of A~, with a molecular weight of approximately 670 kDa. Mice were immunized with this higher molecular weight PF A~. Monoclonal antibodies were screened for specificity to the

25    high molecular weight PF form of the A~ peptide while showing minimal or no ability to bind lower molecular weight forms of A~. This portion of the present invention is exemplified by the screening, isolation and characterization

of    the   13C3  series   of  monoclonal   antibodies   raised  against

30    the -6 70 kDa high molecular weight protofibril form of the A~ peptide (i.e., 13C3, lDl and 19A6). This series of


monoclonal antibodies shows the intended specificity in vitro while also reducing Alzheimer's disease-associated plaque formation in a transgenic mouse Alzheimer's disease model. Thus, in a particular embodiment of the invention,

5    the isolated antibody specifically interacts and shows a measurable affinity to a conformational epitope of a

protofibril form of A~ peptide, whereby the protofibril epitope is represented by an exposed region of a A~­ protofibril form which comprises amino acids 4-12 (SEQ ID

10    N0:3) and 9-20 (SEQ ID N0:4) of an exposed portion of the A~ peptide: Phe Arg His Asp Ser Gly Tyr Glu Val (SEQ ID N0:3); Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe (SEX ID N0:4).

One    embodiment   of   the   present   invention   relates   to   an

15    antibody which comprises a V" (SEQ ID NO: 7) and/or VL (SEQ ID NO: 5) region as disclosed for 13C3, so as to impart 13C3-like specificity to the PF versus LMW form of the A~ peptide. An additional embodiment is a 13C3-like antibody or biologically relevant fragment thereof which show

20    specificity to the PF form over the LMW form of the A~ peptide. Thus, the present invention also relates to biologically active fragments and/or mutants of the 13C3,

lDl,     19A6    or    a    13C3-like    antibody,     including    but    not necessarily  limited  to  amino  acid  substitutions   (e.g.,   as   a 25     directed    form    of    affinity    maturation    of    the    V6      or    Vc regions),    deletions,    additions,    amino   terminal   truncations and   carboxy-terminal   truncations   such   that   these   mutations provide  a   basis   for   an  antibody  or  antibody  binding  portion that   results   in   a   similar   or   improved   version   of   a   13C3,


30    lDl, 19A6 or 13C3-like antibody binding protein. As noted herein, one embodiment of this portion of the invention

related to the VH and/or V0 region of such an antibody comprising the amino acid sequence as set forth in SEQ ID NO: 7 and/or SEQ ID N0:5, respectively. The present invention notes the existence of codon redundancy which may

5    result in differing DNA molecules expressing an identical antibody or portion thereof (e.g., alternative nucleic acid

molecules encoding an identical scFv or a VH and/or VL portion of an IgG). For purposes of this specification, a sequence bearing one or more replaced codons will be defined

10 as a degenerate variation. Another source of sequence variation may occur through RNA editing. Such RNA editing may result in another form of codon redundancy, wherein a change in the open reading frame does not result in an altered amino acid residue in the expressed protein. Also

15    included within the scope of this invention are mutations either in the DNA sequence or the translated antibody which improve the ultimate physical properties of the expressed

antibody. To this end, the present invention relates to (i) affinity matured versions of a 13C3, lDl, 19A6 or any other

20 such 13C3-like antibody, and/or (ii) mutated forms of 13C3, lDl, 19A6 or any other such 13C3-like antibody, including but not limited to one or more mutations in the CDRl, CDR2 an/or CDR3 regions as generated through known affinity maturation methodology and recombinant DNA techniques known

25 for introducing site specific mutations. Thus, the isolated antibodies of the present invention are antibodies that specifically interact with a conformational epi tope of a protofibril form of AP peptide. The isolated antibodies of the present invention will show affinity for such a

30 conformational epitope for the larger molecular weight protofibrillar form of the AP peptide while showing minimal affinity for other forms of AP peptide, such as fibrils,

sheet structures, and low molecular weight oligomers and monomers.

The present invention also relates to the isolated monoclonal antibody, l3C3. This portion of the invention

5    also relates to a hybridoma which produces the monoclonal antibody, 13C3. A hybridoma which produces the monoclonal

antibody 13C3 is available under ATCC Accession No. PTA-8830.

The    present   invention   also   relates   to   the   isolated

10    monoclonal antibody, lDl. This portion of the invention also relates to a hybridoma which produces the monoclonal antibody, lDl.

The present invention also relates to the isolated monoclonal antibody, 19A6. This portion of the invention

15    also relates to a hybridoma which produces the monoclonal antibody, 19A6.

The present invention also relates to methods of screening for and selecting compounds which may act as an inhibitor of fibril and/or senile plaque formation

20 associated with Alzheimer's disease. Such methodology comprises utilizing an antibody with 13C3-like affinity to the PF form of A~ peptide in various antibody/peptide/test compound interaction assays in order to select a compound which modulates the process of fibril and/or plaque

25    formation. The compound may be a non-proteinaceous organic or inorganic molecule, a peptide (e.g., as a potential prophylactic or therapeutic peptide vaccine), a protein, DNA (single or double stranded) or RNA (such as siRNA or shRNA).

It    will   become   evident   upon   review   of   the   disclosure   and

30    teachings of this specification that any such peptide or small molecule which effectively competes with a 13C3-like


antibody for binding to the PF form of A~ peptide represents a possible lead compound relating to prophylactic or therapeutic treatment of Alzheimer's disease. To this end, interaction assays may be utilized for the purpose of high

5    throughput screening to identify compounds that occupy or interact with the 13C3 epitopes of the PF form of A~ peptide and displace the antibody.

Various antibody/antigen-based assays known in the art may be used which incorporate and rely on a 13C3-like

10    antibody of the present invention as an essential reagent in screening for compounds useful in the prophylactic or therapeutic treatment of Alzheimer's disease (e. g., a small

inorganic molecule or candidate peptide vaccine), including but not limited to an ELISA assay, a radioirnrnune assay, a

15    Western blot analysis, any homogenous assay relying on a detectable biological interaction not requiring separation or wash steps (e.g., see AlphaScreen from PerkinElmer)

and/or SPR-based technology (e.g., see BIACore)). Compounds and/or peptide vaccine candidates identified through use of

20    a 13C3-like antibody may be detected by a variety of assays. The assay may be a simple "yes/no" assay to determine whether there is a change in the ability to form the known antibody/antigen complex, or may be made quantitative in nature by utilizing an assay such as an ELISA based assay, a

25    homogenous assay, or an SPR-based assay. To this end, the present invention relates to any such assay, regardless of the known methodology employed, which measures the ability of a test compound to compete with 13C3-like antibody, to an appropriate peptide or protein mimetic of the amino terminal

30    portion  of  a  13C3  epitope  of  the  PF  form  of  A~ peptide.

The antibodies described herein may be used as the basic reagents in a number of different irnrnunoassays to

determine the presence of a A~ protofibril form in a tissue sample. Generally speaking, the antibodies can be employed in any type of immunoassay, whether qualitative or quantitative. This includes both the two-site sandwich

5    assay and the single site immunoassay of the non-competitive type, as well as in traditional competitive binding assays. One embodiment of interest, for ease of detection, and its

quantitative nature, is the sandwich or double antibody assay, of which a number of variations exist, all of which

10    are intended to be encompassed by this portion of the present invention. For example, in a typical forward sandwich assay, unlabeled antibody is immobilized on a solid substrate, e.g., microtiter plate wells, and the sample to be tested is brought into contact with the bound molecule.

15    After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen binary complex, a second antibody, labeled with a reporter molecule

capable of inducing a detectable signal, is then added and incubation is continued allowing sufficient time for binding

20    with the antigen at a different site and the formation of a ternary complex of antibody-antigen-labeled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal, which may

be    quantitated    by    comparison    with    a    control    sample

25    containing known amounts of antigen. Variations on the forward sandwich assay include the simultaneous assay, in which both sample and antibody are added simultaneously to the bound antibody, or a reverse sandwich assay in which the labeled antibody and sample to be tested are first combined,

30    incubated and added to the unlabelled surface bound antibody. These techniques are well known to those skilled in the art, and the possibility of minor variations will be


readily apparent. As used herein, "sandwich assay" is intended to encompass all variations on the basic two-site technique.

For    the   sandwich   assays   of   the   present   invention,   the

5    only limiting factor is that both antibodies have different binding specificities for the AP protofibril form. Thus, a number of possible combinations are possible. As a more specific example, in a typical forward sandwich assay, a primary antibody is either covalently or passively bound to

10    a solid support. The solid surface is usually glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinylchloride or

polypropylene.      The   solid   supports   may   be   in   the   form   of tubes,   beads,   discs   or   microplates,   or   any   other   surfaces 15     sui table    for    conducting    an    immunoassay.        The    binding processes  are  well  known  in  the  art .    Following  binding,   the solid   phase-antibody   complex   is   washed   in   preparation   for the   test   sample.     An  aliquot  of   the  body  fluid  containing  a AP  protofibril   form  to  be  tested  is  then  added  to   the   solid


20    phase complex and incubated at 25°C for a period of time sufficient to allow binding of any AP protofibril form

protein present to the antibody specific for the AP protofibril form. The second antibody is then added to the solid phase complex and incubated at 25°C for an additional

25    period of time sufficient to allow the second antibody to bind to the primary antibody-antigen solid phase complex. The second antibody is linked to a reporter molecule, the visible signal of which is used to indicate the binding of the second antibody to any antigen in the sample. By


30    "reporter molecule", as used in the present specification is meant a molecule which by its chemical nature, provides an analytically detectable signal which allows the detection of


antigen-bound    antibody.        Detection    must    be    at    least relatively    quantifiable,    to    allow    determination    of    the amount   of   antigen   in   the   sample,   this   may  be   calculated  in absolute  terms,   or  may  be  done  in  comparison  with  a  standard 5     (or  series  of   standards)   containing  a   known  normal  level   of

antigen.

The most commonly used reporter molecules in this type of assay are either enzymes or fluorophores. In the case of an enzyme immunoassay an enzyme is conjugated to the second

10    antibody, often by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are well known to the skilled artisan. Commonly used enzymes include

horseradish    peroxidase,   glucose   oxidase,   beta-galactosidase

15    and alkaline phosphatase, among others. The substrates to be used with the specific enzymes are generally chosen tor the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. For example, p-nitrophenyl phosphate is suitable for use with alkaline phosphatase

20    conjugates; for peroxidase conjugates, 1, 2-phenylenediamine or toluidine are commonly used. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labeled antibody is added to the

25    first antibody- A~ protofibril protein complex and allowed to bind to the complex, and then the excess reagent is

washed away. A solution containing the appropriate substrate is then added to the tertiary complex of antibody-antigen-labeled antibody. The substrate reacts with the

30    enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually


spectrophotometrically, to give an evaluation of the amount of antigen that is present in the serum sample.

Additionally, fluorescent compounds, such as fluorescein or rhodamine, may be chemically coupled to

5    antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome~labeled antibody absorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a


10    characteristic longer wavelength. The emission appears as a characteristic color visually detectable with a light

microscope. As in the enzyme immunoassay (EIA), the fluorescent~labelled antibody is allowed to bind to the first antibody~ A~ protofibril form protein complex. After

15    washing the unbound reagent, the remaining ternary complex is then exposed to light of the appropriate wavelength, and the fluorescence observed indicates the presence of the antigen. Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred

20    for the present method. However, other reporter molecules, such as radioisotopes, chemiluminescent or bioluminescent molecules may also be employed. It will be readily apparent to the skilled artisan how to vary the procedure to suit the

required  use.

25 In another embodiment, the sample to be tested (e.g., human blood or spinal fluid containing a A~ protofibril form) may be used in a single site immunoassay wherein it is adhered to a solid substrate either covalently or noncovalently. An unlabeled anti~ A~ protofibril protein


30    antibody is brought into contact with the sample bound on the solid substrate. After a suitable period of incubation, for a period of time sufficient to allow formation of an


antibody-antigen binary complex a second antibody, labelled with a reporter molecule capable of inducing a detectable signal, is then added and incubation is continued allowing sufficient time for the formation of a ternary complex of

5    antigen-antibody-labeled antibody. For the single site immunassay, the second antibody may be a general antibody (i.e., zenogeneic antibody to immunoglobulin, particularly anti- (IgM and IgG) linked to a reporter molecule) that is

capable of binding an antibody that is specific for the AP 10 protofibril protein form of interest.

A 13C3-like antibody may take one of numerous forms known in the art. Antibodies may take the form of any type of relevant antibody fragment, antibody binding portion, specific binding member, a non-protein synthetic mimic, or

15    any other relevant terminology known in the art which refers to an entity which at least substantially retains the

binding specificity/neutralization activity. Thus, the term nantibody" as used in any context within this specification is meant to include, but not be limited to, any specific

20 binding member, immunoglobulin class and/ or isotype (e.g., IgG 1 , IgG 2 , IgG,, IgG 4 , IgM, IgA, IgD, IgE and IgM); and biologically relevant fragment or specific binding member thereof, including but not limited to Fab, F(ab')2, Fv, and scFv (single chain or related entity). Therefore, it is

25    well known in the art, and is included as review only, that an nantibody" refers to a glycoprotein comprising at least

two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. A heavy chain is comprised of a heavy chain

30    variable region (VH) and a heavy chain constant region (CHl, CH2 and CH3 J • A light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL).


The variable regions of both the heavy and light chains comprise framework regions (FWR) and complementarity determining regions (CDR). The four FWR regions are relatively conversed while CDR regions (CDRl, CDR2 and CDR3)

5    represent hypervariable regions and are arranged from NH 2 terminus to the COOH terminus as follows: FWRl, CDRl, FWR2,

CDR2,   FWR3,   CDR3,   FWR4.     The   variable   regions   of   the   heavy and   light   chains   contain   a   binding   domain   that   interacts with    an    antigen    while,    depending    of    the    isotype,    the 10    constant     region ( s)     may     mediate     the     binding     of     the immunoglobulin  to  host   tissues  or   factors.     That   said,   also included    in    the    working    definition    of    "antibody"    are chimeric    antibodies,    humanized    antibodies,    a    recombinant antibody,    as   human   antibodies   generated   from   a   transgenic


15    non-human animal, as well as antibodies selected from libraries using enrichment technologies available to the artisan. Antibody fragments are obtained using techniques readily known and available to those of ordinary skill in the art, as reviewed below. Therefore, an "antibody" is any


20    such entity or specific binding member, which specifically binds the conformational epitope of the protofibril form of AP as described herein. Therefore, the term "antibody"

describes   an   immunoglobulin,   whether   natural   or   partly   or wholly   synthetically   produced;    any   polypeptide   or   protein 25     having    a    binding    domain    which    is,    or    is    substantially homologous   to,    an   antibody   binding   domain.      These   can   be derived   from   natural    sources,    or    they   may   be   partly   or wholly   synthetically   produced.     Examples   of   antibodies   are the   immunoglobulin   isotypes   and   their   isotypic   subclasses;


30    fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd and diabodies, as discussed without limitation, infra. It is known in the art that it is



possible to manipulate monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody Such techniques may

5    evolve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. A hybridoma or other cell producing an antibody may be subject

10 to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced. Antibodies can be modified in a number of ways, and the term "antibody" should be construed as covering any specific binding member or substance having a binding domain with the

15 required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of "antibody" including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Such an entity may be a

20    binding fragment encompassed within the term "antigen-binding portion" or "specific binding member" of an antibody including but not limited to (i) a Fab fragment, a

monovalent fragment consisting of the VL, VH, CL and CH domains; (ii) a F (ab') 2 fragment, a bivalent fragment

25    comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH domains; (iv) a Fv fragment consisting of the VL and

VH domains of a single arm of an antibody (v) a dAb fragment, which comprises a VH domain; (vi) an isolated

30    complementarity determining region (CDR); (vii) a 'scAb', an antibody fragment containing VH and VL as well as either CL or CH; and (viii) artificial antibodies based upon protein

scaffolds, including but not limited to fibronectin type III polypeptide antibodies (e.g., see U.S. Patent No. 6,703,199, issued to Koide on March 9, 2004 and PCT International Application Publication No. WO 02/32925). Furthermore,

5    although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them

to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single

10    chain  Fv  ( scFv)) .

In one embodiment, the variable light (VL) region for the isolated 13C3 or 13C3-like antibodies of the present invention may comprise a 113 amino acid peptide sequence (SEQ ID NO: 5) which is encoded by a 339 base pair

15    nucleotide  sequence   (SEQ  ID  NO:  6) :

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Gly Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro

Gly  Gln  Ser  Pro  Lys  Leu  Leu  Ile  Tyr  Thr  Val  Ser  Asn  Arg  Phe

20    Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Asn Thr Phe Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg (SEQ ID NO: 5)

GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCC

25    ATCTCTTGCAGATCTGGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTATACAGTTTCCAACCGATTT TCTGGGGTCCCGGACAGGTTCAGTGGCAGTGGATCAGGGTCAGATTTCACACTCAAGATC AGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAATACATTTGTTCCT TGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG (SEQ ID NO: 6)

30 In a further embodiment, the variable heavy (VH) region for the isolated 13C3 or 13C3-like antibodies of the present invention may comprise a 115 amino acid peptide sequence


(SEQ ID NO: 7) encoded by a 345 base pair nucleotide sequence (SEQ ID NO: 8):

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Val Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr

5    Asp Tyr Ala Met His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile Gly Val Ile Ser Thr Lys Tyr Gly Lys Thr Asn Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys Ala Arg Gly Asp Asp Gly Tyr Ser Trp

10    Gly Gln Gly Thr Ser Val Thr Val Ser Ser (SEQ ID NO: 7); CAGGTCCAGCTGCAGCAGTCTGGGCCTGAGCTGGTGAGGCCTGGGGTCTCAGTGAAGATT
TCCTGCAAGGGTTCCGGCTACACATTCACTGATTATGCTATGCACTGGGTGAAGCAGAGT

CATGCAAAGAGTCTAGAGTGGATTGGAGTTATTAGTACTAAGTATGGTAAGACAAACTAC

AACCAGAAGTTTAAGGGCAAGGCCACAATGACTGTTGACAAATCCTCCAGCACAGCCTAT

15    ATGGAGCTTGCCAGATTGACATCTGAGGATTCTGCCATCTATTACTGTGCAAGAGGGGAC GATGGTTATTCCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO:
8) .

In a further embodiment, the framework regions, FWRl, FWR2, FWR3, and FWR4, of the VL chain may be comprised of

20    amino acid set forth in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID N0:11, and SEQ ID N0:12, respectively, as follows:

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Gly (SEQ ID NO: 9);

Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr (SEQ ID NO: 10);

Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys (SEQ ID NO: 11);

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg (SEQ ID NO: 12). In a further embodiment, the complementarity

determining regions, CDR1, CDR2, and CDR3, of the VL chain may be comprised of the amino acids set forth in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, respectively, as follows:
 

Gln  Ser  Leu  Val  His  Ser  Asn  Gly  Asn  Thr  Tyr   (SEQ  ID  NO:   13);


Thr  Val  Ser   (SEQ  ID  NO:  14);

Ser  Gln  Asn  Thr  Phe  Val  Pro  Trp  Thr    (SEQ  ID  NO:   15).

In a further embodiment, the framework regions, FWRl, FWR2, FWR3, and FWR4, of the VH chain may be comprised of

5    the amino acids set forth in SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, and SEQ ID N0:19, respectively, as follows:

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Val Ser Val Lys Ile Ser Cys Lys (SEQ ID NO: 16);

Met    His  Trp  Val  Lys  Gln  Ser  His  Ala  Lys  Ser  Leu  Glu  Trp  Ile

10    Gly  Val   (SEQ  ID  NO:  1 7);

Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys Ala Arg (SEQ ID NO: 18);

Trp  Gly  Gln  Gly  Thr  Ser  Val  Thr  Val  Ser  Ser   (SEQ  ID  NO:   19).

15 In a further embodiment, the complementarity determining regions, CDRl, CDR2, and CDR3, of the VH chain may be comprised of the amino acids set forth in SEQ ID N0:20, SEQ ID N0:21, SEQ ID N0:22, respectively, as follows:

Gly  Ser  Gly  Tyr  Thr  Phe  Thr  Asp  Tyr  Ala   (SEQ  ID  NO:   20);

20    Ile Ser Thr Lys Tyr Gly Lys Thr Asn Tyr Asn Gln Lys Phe Lys Gly Lys (SEQ ID NO: 21);

Gly  Asp  Asp  Gly  Tyr  Ser   (SEQ  ID  NO:   22).

Polyclonal   or   monoclonal   antibodies    for   use   in   the

disclosed    treatment    methods    may    be    raised    by    known

25    techniques. Monospecific murine (mouse) antibodies showing specificity to a conformational epitope of a target of choice may be purified from mammalian antisera containing antibodies reactive against this region, or may be prepared

as    monoclonal   antibodies   using   the   technique   of   Kohler   and

30    Milstein (1975, Nature 256: 49~-497). Monospecific antibody as used herein is defined as a single antibody species or


multiple    antibody    species    with    homogenous    binding

characteristics, such as the mouse monoclonal antibodies exemplified herein with the 13C3 series of monoclonal

antibodies.    Hybr idoma   cells   are   produced   by   mixing   the

5    splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions which will allow

the formation of stable hybridomas. The splenic antibody producing cells and myeloma cells are fused, selected, and screened for antibody production. Hybridoma cells from

10    antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson (1973, Soft Agar

Techniques,    in    Tissue    Culture   Methods    and   Applications,

Kruse and Paterson, Eds, Academic Press). Monoclonal antibodies are produced in vivo by injecting respective

15 hydridoma cells into pristine primed mice, collecting ascite fluid after an interval of time, and prepared by techniques well known in the art.

Beyond species specific monoclonal antibodies described above, the antibodies of the present invention may also be

20 in the form of a "chimeric antibody", a monoclonal antibody constructed from the variable regions derived from say, the murine source, and constant regions derived from the intended host source (e.g., human; for a review, see Morrison and Oi, 1989, Advances in Immunology, 44: 65-92).

25    For example, the variable light and heavy DNA sequences (e.g. SEQ ID NO: 6 and 8, respectively) from the rodent

(e.g., mouse) antibody may be cloned into a mammalian expression vector. These light and heavy "chimeric" expression vectors are cotransfected into a recipient cell

30    line and selected and expanded by known techniques. This cell line may then be subjected to known cell culture techniques, resulting in production of both the light chain


and heavy chain of a chimeric antibody. Such chimeric antibodies have historically been shown to have the antigen-binding capacity of the original rodent monoclonal while significantly reducing immunogenicity problems upon host

5    administration.

A logical improvement to the chimeric antibody is the •humanized antibody," which arguably reduces the chance of the patient mounting an immune response against a therapeutic antibody when compared to use of a chimeric or

10 full murine monoclonal antibody The strategy of "humanizing" a murine Mab is based on replacing amino acid residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grafting of entire complementarity determining regions


15 (Jones et al., 1986, Nature 321: 522-526). This technology is again now well known in the art and is represented by numerous strategies to improve on this technology; namely by implementing strategies including, but not limited to,

"reshaping"    (see  Verhoeyen,   et  al.,   1988,   Science  239:   1534-

20    1536), "hyperchimerization" (see Queen, et al. , 1991, Proc. Natl. Acad. Sci. 88:2869-2873) or "veneering" (Mark, et al., 1994, Derivation of Therapeutically Active Humanized and Veneered anti-CD18 Antibodies Metcalf end Dalton, eds.

Cellular    Adlwsion:    Molecular    Definition    to    T,~erapeutic

25 Potential. New York: Plenum Press, 291-312). These strategies all involve to some degree sequence comparison between rodent and human sequences to determine whether specific amino acid substitutions from a rodent to human consensus is appropriate. Whatever the variations, the


30    central theme involved in generating a humanized antibody relies on CDR grafting, where these three antigen binding sites from both the light and heavy chain are effectively

removed from the rodent expressing antibody clone and subcloned (or "grafted") into an expression vector coding for the framework region of the human antibody. For example, utilizing the above techniques a humanized antibody

5    may be expressed wherein the CDRl, CDR2, and CDR3 regions of the variable light chain are set forth in SEQ ID NOS: 13, 14 and 15, respectively, and the CDRl, CDR2, and CDR3 regions of the variable heavy chain are set forth in SEQ ID NOS 20, 21 and 22, respectively. Therefore, a "humanized antibody"

10    is effectively an antibody constructed with only murine CDRs (minus any additional improvements generated by

incorporating one or more of the above mentioned strategies), with the remainder of the variable region and all of the constant region being derived from a human

15    source.

The   present   invention  also   relates   to   isolated  nucleic acid   molecules   and   associated   amino   acid   sequences   which relate  to  the  VH  and/or  VL  regions   of  the  13C3  antibody,   and more     specifically,     an    isolated    nucleic    acid    molecule 20     (polynucleotide)   encoding  a  biologically  relevant  portion  of 13C3,    or    affinity    matured   version    or    otherwise    mutated version   of   13C3,    lDl,    19A6   or   other   13C3-like   antibody. These    nucleic    acids    are    substantially    free    from    other nucleic    acids.       For    most    cloning    purposes,    DNA    is    a



25 preferred nucleic acid. These DNA molecules may be subcloned into an expression vector and subsequently transfected into a host cell of choice wherein the recombinant host cell provides a source for substantial levels of a relevant portion of the 13C3, lDl, 19A6 or 13C3-


30 lDl-, or 19A6-like antibody, or affinity matured version thereof. Such procedures may be used for a variety of utilities, such as generating scFvs or for co-expressing



these VE and VL chains in a mammalian expression vector system which encodes human CH and C1 regions, of say, an IgG antibody. The degeneracy of the genetic code is such that, for all but two amino acids, more than a single codon

5 encodes a particular amino acid. This allows for the construction of synthetic DNA that encodes an antibody of the present invention where the nucleotide sequence of the synthetic DNA differs significantly from the nucleotide sequences disclosed herein, but still encodes such an

10    antibody. Such synthetic DNAs are intended to be within the scope of the present invention. If it is desired to express

such synthetic DNAs in a particular host cell or organism, the codon usage of such synthetic DNAs can be adjusted to reflect the codon usage of that particular host, thus

15    leading  to  higher  levels  of  expression  of  the  an  antibody  of

the present invention. In other words, this redundancy in the various codons which code for specific amino acids is within the scope of the present invention. Therefore, this invention is also directed to those DNA sequences which

20    encode RNA comprising alternative codons which code for the eventual translation of the identical amino acid, as shown

below: A=Ala=Alanine: codons GCA, GCC, GCG, GCU; C=Cys=Cysteine: codons UGC, UGU; D=Asp=Aspartic acid: codons GAC, GAU E=Glu=Glutamic acid: codons GAA, GAG;

25 F=Phe=Phenylalanine: codons UUC, UUU; G=Gly=Glycine: codons GGA, GGC, GGG, GGU; H=His =Histidine: codons CAC, CAU; I=Ile =Isoleucine: codons AUA, AUC; AUU; K=Lys-Lysine: codons AAA, AAG; L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU;

M=Met=Methionine:   codon   AUG;   N=Asp=Asparagine:   codons   GAU, 30    GAC;      P=Pro=Proline:      codons      CCA,      CCC,      CCG,      CCU; Q=Gln=Glutamine:    codons   CAA,    CAG;    R=Arg=Arginine:    codons AGA,  AGG,   CGA,   CGC,   CGG,   CGU;  S=Ser=Serjne:   codons  AGC,   AGU,



UCA,    UCC,   UCG,   UCU;   T~Thr~Threonine:  codons  ACA,   ACC,   ACG,

ACU;    V~Val~Valine:    codons      GUA,       GUC,       GUG,       GUU;

w~Trp~Tryptophan:    codon   UGG;    Y~Tyr~Tyrosine:  codons   UAC,

UAU.    Such  recombinant  expression  vectors  may  then  be  stably

5    or transiently transfected into an appropriate cell line for the generation of alternative antibody form.

The present invention notes the existence of codon redundancy which may result in differing DNA molecules

expressing    an   identical   antibody   or   portion   thereof   (e.g.,

10    alternative nucleic acid molecules encoding an identical scFv or a VH and/or VL portion of an IgG). For purposes of this specification, a sequence bearing one or more replaced

codons will be defined as a degenerate variation. Another source of sequence variation may occur through RNA editing.

15    Such RNA editing may result in another form of codon redundancy, wherein a change in the open reading frame does not result in an altered amino acid residue in the expressed protein. Also included within the scope of this invention are mutations either in the DNA sequence or the translated

20    antibody which improve the ultimate physical properties of the expressed antibody. To this end, the present invention relates to (i) affinity matured versions of a 13C3-like antibody, including but not limited to 13C3, 19A6 and lDl, and/or (ii) mutated forms of a 13C3-like antibody, including

25    but not limited to 13C3, 19A6 and/or lDl, including but not limited to one or more mutations in the CDRl, CDR2 an/or CDR3 regions as generated through known affinity maturation methodology and recombinant DNA techniques known for

introducing    site    specific    mutation.    Such    isolated    or

30    purified nucleic acid molecules will represent the VH and/or VL portions of a 13C3-like antibody. These nucleic acids are substantially free from other nucleic acids. For most



cloning    purposes,   DNA   is   a   preferred   nucleic   acid.    These

DNA molecules may be subcloned into an expression vector and subsequently transfected into a host cell of choice wherein the recombinant host cell provides a source for substantial

5    levels of a relevant portion of a 13C3-like antibody, or affinity matured version thereof. Such procedures may be used for a variety of utilities, such as generating scFvs or for co-expressing these VH and VL chains in a mammalian expression vector system which encodes human CH and CL

10    regions,  of  say,   an  IgG  antibody.

The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain nucleic acid molecules encoding the respective heavy and/or light regions of a 13C3-like

15    antibody. These nucleic acid molecules, in whole or in part, can be linked with other DNA molecules (i.e., DNA molecules which encompass immunoglobulin genes used for generation of a recombinant human antibody) that are not naturally linked, to form "recombinant DNA molecules" which

20 encode a respective human recombinant antibody. These vectors may be comprised of DNA or RNA. For most cloning purposes DNA vectors are preferred. Typical vectors include plasmids, modified viruses, bacteriophage, cosmids, yeast artificial chromosomes, and other forms of episomal or

25    integrated DNA. It is within the purview of the skilled artisan to determine an appropriate vector for a particular gene transfer, generation of a recombinant human antibody or

other use. Methods of subcloning nucleic acid molecules of interest into expression vectors, transforming or

30    transfecting host cells containing the vectors, and methods of making substantially pure protein comprising the steps of introducing the respective expression vector into a host


cell, and cultivating the host cell under appropriate conditions are well known. The antibody (such as an IgG recombinant human antibody) so produced may be harvested from the host cells in conventional ways. Any known

5    expression vector may be utilized to practice this portion of the invention, including any vector containing a suitable promoter and other appropriate transcription regulatory

elements. The resulting expression construct is transferred into a prokaryotic or eukaryotic host cell to produce

10    recombinant protein. Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned DNA and the translation of their rnRNAs in an appropriate host. Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, blue

15    green algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of DNA

between   hosts    such   as    bacteria-yeast   or   bacteria-animal cells.       An    appropriately    constructed    expression    vector should   contain:   an   origin   of   replication   for   autonomous :?.0    replication   in   host   cells,    selectable   markers,    a   limited number   of  useful   restriction   enzyme   sites,   a   potential   for high   copy   number,    and   active   promoters.      A   promoter   is defined   as   a   DNA   sequence   that   directs   RNA   polymerase   to bind  to  DNA  and  initiate  RNA  synthesis.     A  strong  promoter


:?.5 is one which causes rnRNAs to be initiated at high frequency. Techniques for such manipulations can be found described in Sambrook, et al. (1989, Molecular Cloning. A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) are well known and available to the

30    artisan of ordinary skill in the art. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or

viruses. Commercially available mammalian expression vectors which may be suitable, include but are not limited to, pcDNA3. neo (Invitrogen), pcDNA3 .1 (Invitrogen), pCI-neo (Promega), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39

5 (New England Bioloabs), pcDNAI, pcDNAianp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO pSV2-neo (ATCC 37593) pBPV-1 (8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 31199), pRSVneo (ATCC 31198), pSV2-dhfr (ATCC

10    31146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565). Also, a variety of bacterial expression vectors are available, including but not limited to pCR2 .1 (Invitrogen), pET1 la (Novagen), lambda gtl 1 (Invitrogen), and pKK223-3

(Pharmacia).    In    addition,    a    variety    of    fungal    cell

15    expression vectors may be used, including but not limited to pYES2 (Invitrogen) and Pichie expression vector (Invitrogen). Also, a variety of insect cell expression vectors may be used, including but are not limited to

pBlueBaciii    and    pBlueBacHis2    (Invitrogen),    and    pAcG2T

20    (Pharmingen) .

Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as

E.    coli,     fungal    cells    such    as    yeast,     mammalian    cells including,     but    not    limited    to,     cell    lines    of    bovine, 25     porcine,     monkey    and    rodent    origin;     and    insect     cells. Mammalian   species   which  may   be   suitable,-26   include   but   are not   limited  to,   L   cells   L-M(TK-)    (ATCCCCL1.3),   L   cells   L-M (ATCC  CCL  1.2),   Saos-2   (ATCCHTB-85),   293   (ATCCCRL1573),   Raji (ATCC   CCL   86),    CV-1    (ATCC   CCL   70),    COS-1    (ATCC   CRL1650),


30    COS-7(ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), Cl27I (ATCC


CRL 1616), BS-C-l(ATCC CCL 26), MRC-:0 (ATCCCCL17l) and CPAE (ATCC CCL 209) .

Yet another improvement over re-engineered antibodies as reviewed above is the generation of fully human
5 monoclonal antibodies. The first involves the use of genetically engineered mouse strains which possess an immune system whereby the mouse antibody genes have been inactivated and in turn replaced with a repertoire of functional human antibody genes, while leaving other


10    components of the mouse immune system unchanged. Such genetically engineered mice allow for the natural in vivo

immune response and affinity maturation process which results in high affinity, fully human monoclonal antibodies This technology is again now well known in the art and is

15    fully detailed in various publications, including but not limited to u.s. Patent Nos. 5,939, 598; 6,075,181; 6,114,598; 6,150,584 and related family members (assigned to Abgenix, disclosing their XenoMouse technology); as well as U.S. Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;

20    5,789,650; 5,877, 397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429 (assigned to GenPharm International and available through Medarex, under the umbrella of the "UltraMab Human

Antibody Development System"). See also a review from Kellerman a:1d Green (2002, Curr. Opinion in Biotechnology

25    l3:   593-597).

Finally, techniques are available to the artisan for the selection of antibody fragments from libraries using enrichment technologies, including but not limited to phage display, ribosome display (Hanes and Pluckthun, 1997, Proc.

30    Nat. Acad. Sci. 94: 4937-4942), bacterial display (Georgiou, et al., 1997, Nature Biotechnology 1:0: 29-34) and/or yeast display (Kieke, et al., 1997, Protein Engineering 10: 1303-


1310) may be utilized as alternatives to previously discussed technologies to select single chain antibodies which specifically bind to target cytokine. Single-chain antibodies are selected from a library of single chain

5    antibodies produced directly utilizing filamentous phage technology. Phage display technology is known in the art (e.g., see technology from Cambridge Antibody Technology (CAT)) as disclosed in U.S. Patent Nos. 5, 565,332; ::,,133,'143; !J,8'11,90'1; 5,872,21:-o; !J,885,793; 5,962,255;

10    6, 140, 471; 6, 22!J, 44'1; 6, 291650; 6, 492, 160; 6, 521, 404; 6,544,731; 6,555,313; 6,582,915; 6,593, 081, as well as other U.S. family members, or applications which rely on priority filing GB 9206318, filed 24 May 1992; see also Vaughn, et al. 1996, Nature Biotechnology 14: 309-314).

15    Single chain antibodies may also be designed and constructed using available recombinant DNA technology, such as a DNA amplification method (e.g., PCR), or possibly by using a respective hybridoma eDNA as a template. Single-chain

antibodies    can    be    mono-or    bispecific;    bivalent    or

20    tetravalent. A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated

nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below.

25 The present invention further relates to an antibody-based pharmaceutical composition comprising an effective amount a 13C3-like antibody, or an affinity matured version, which provides a prophylactic or therapeutic treatment choice to inhibit fibril and/or senile plaque formation

30    associated with Alzheimer's disease. The antibody-based pharmaceutical composition of the present invention may be

formulated    by   any   number   of   strategies   known   in   the   art

(e.g., see McGoff and Scher, 2000, Solution Formulation of Proteins/Peptides: In McNally, E.J., ed. Protein Formulation and Delivery. New York, NY: Marcel Dekker; pp. 139-158; Akers and Defilippis, 2000, Peptides and Proteins as

5 Parenteral Solutions. In: Pharmaceutical Formulation Development of Peptides and Proteins. Philadelphia, PA: Talyor and Francis; pp. 145-177; Akers, et al., 2002, Pharm. Biotechnol. 14:4"1-127). A pharmaceutically acceptable composition suitable for patient administration will contain

10    an effective amount of the antibody in a formulation which both retains biological activity while also promoting maximal stability during storage within an acceptable

temperature range. The pharmaceutical compositions can also include, depending on the formulation desired,

15 pharmaceutically acceptable diluents, pharmaceutically acceptable carriers and/or pharmaceutically acceptable excipients, or any such vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect

20    the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and

Hank's solution. The amount of an excipient that is useful in the pharmaceutical composition or formulation of this

25    invention is an amount that serves to uniformly distribute the antibody throughout the composition so that it can be uniformly dispersed when it is to be delivered to a subject

in  need  thereof.     It  may  serve  to  dilute  the  antibody  to  a concentration     which     provides     the     desired     beneficial 30    palliative   or   curative    results    while    at    the    same    time minimizing   any   adverse   side   effects   that   might   occur   from too  high  a   concentration.     It  may  also  have  a  preservative


effect. Thus, for the antibody having a high physiological activity, more of the excipient will be employed. On the other hand, for any active ingredient(s) that exhibit a lower physiological activity, a lesser quantity of the

5    excipient will be employed. In general, the amount of excipient in the composition will be between about 50%

weight (w) and 99.9% w. of the total composition. If the antibody exhibits a particularly low physiological activity, the amount of excipient could be as little as 1% w. On the

10    other hand, for an antibody that has a particularly high physiological activity, the amount of excipient may be between about 98.0% and about 99.9% w. In addition, the antibody or antibodies may by administered in the form of a "chemical derivative" (a molecule that contains additional

15    chemical moieties which are not normally a part of the base molecule). Such moieties may improve the solubility, half-

life, absorption, etc. of the biological agent. Alternatively, these moieties may attenuate undesirable side effects of the antibody. Pharmaceutical compositions can

20    also include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized sepharose, agarose, cellulose, and the like), polymeric

amino   acids,    amino   acid   copolymers,    and   lipid   aggregates 25     (such   as   oil   droplets   or   liposomes) .     Additionally,    these carriers   can   function   as   immunostimulating   agents    (i.   e., adjuvants).      For   parenteral   administration,    agents   of   the invention   can   be   administered   as   injectable   dosages   of   a solution  or  suspension  of  the  substance  in  a  physiologically


30    acceptable diluent with a pharmaceutical carrier which can be a sterile liquid such as water oils, saline, glycerol, or ethanol. Additionally, auxiliary subFtances, such as


wetting   or   emulsifying   agents,    surfactants,    pH   buffering substances   and   the   like   can   be   present   in   compositions. Other  components  of  pharmaceutical  compositions  are  those  of petroleum,    animal,    vegetable,    or    synthetic    origin,    for 5    example,     peanut    oil,     soybean    oil,     and    mineral    oil. In  general,   glycols  such  as  propylene  glycol  or  polyethylene glycol    are    preferred    liquid    carriers,    particularly    for


injectable  solutions.

The  antibody  formulation  may  be  in  liquid  form  or  solid 10    form.      A   solid   formulation   is   generally   lyophilized   and brought   into   solution   prior   to   administration   for   either single  or  multiple  dosing.     The   formulations   should  not   be exposed  to  extreme  temperature  or  pH  so  as   to  avoid  thermal denaturation.       Thus,    it    is    essential    to    formulate    an 15    antibody   composition   of   the    present    invention   within    a biologically   relevant   pH   range.      A   solution   buffered   to maintain   a   proper   pH   range   during   storage   is   indicated, especially  for  liquid  formulations  stored  for  longer  periods of   time   between   formulation   and   administration.     To   date,



20    both liquid and solid formulations require storage at lower temperatures (usually 2-8°C) in order to retain stability for

longer    periods.    Formulated    antibody    compositions,

especially liquid formulations, may contain a bacteriostat to prevent or minimize proteolysis during storage, including

25    but not linuted to effective concentrations (usually <1% w/v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and/or propylparaben. A bacteriostat may be contraindicated for some patients. Therefore, a lyophilized formulation may be reconstituted in a solution either

30    containing or not containing such a component. Additional components may be added to either a buffered liquid or solid antibody formulation, including but not limited to sugars as

a cryoprotectant (including but not necessarily limited to polyhydroxy hydrocarbons such as sorbitol, mannitol, glycerol and dulcitol and/or disaccharides such as sucrose, lactose, maltose or trehalose) and, in some instances, a

5    relevant sa~t (including but not limited to NaCl, KCl or 1iCl). Such antibody formulations, especially liquid formulations slated for long term storage, will rely on a useful range of total osmolarity to both promote long term stability at temperature of 2-8°C, or higher, while also

10    making the formulation useful for parenteral injection. An effective range of total osmolarity (the total number of molecules in solution) is from about 200 mOs/1 to about 800

mOs/1. It will be apparent that the amount of a cyroprotectant, such as sucrose or sorbitol, will depend

15    upon the amount of salt in the formulation in order for the total osmolarity of the solution to remain within an appropriate range. Therefore a salt free formulation may contain from about 5% to about 25% sucrose, with a preferred range of sucrose from about 7% to about 15%, with an

20    especially preferred sucrose concentration in a salt free formulation being from 10% to 12%. Alternatively, a salt free sorbitol-based formulation may contain sorbitol within a range from about 3% to about 12%, with a preferred range from about 4% to 7%, and an especially preferred range is

25    from about 5% to about 6% sorbitol in a salt-free formulation. Salt-free formulations will of course warrant increased ranges of the respective cryoprotectant in order

to maintain effective osmolarity levels. These formulation may also contain a divalent cation (including but not

30    necessarily limited to MgC1 2 , CaC1 2 and MnC1 2 ); and a non-32 ionic surfactant (including but not necessarily limited to Polysorbate-SO (Tween 80.,), Polysorbate-60 (Tween 60®),


Polysorbate-40 (Tween 40®) and Polysorbate-20 (Tween 20®), polyoxyethylene alkyl ethers, including but not limited to Brij 58"', Brij 35®, as well as others such as Triton X-100"', Triton X 114"', NP40"', Span 8S and the Pluronic series of non-

5    ionic surfactants (e.g., Pluronic 121)). Any combination of such components, including probable inclusion of a bacteriostat, may be useful to fill the antibody-containing formulations of the present invention. The antibody

composition  of  the  present  invention  may  also  be  a  "chemical

10 derivative", which describes an antibody that contains additional chemical moieties which are not normally a part of the immunogloblulin molecule (e.g., pegylation). Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may

15    attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule.

Numerous    examples    of    various    carriers,    diluents,

excipients    and   the    such   are   known   in   the   art   and   are

disclosed  in  references  cited  herein,   as  well  as  Remington's

20    Pharmaceutical Sciences (18th ed.; Mack Publishing Company, Easton, Pa., 1990), the contents of which are incorporated herein by reference. Briefly, it will be appreciated that

suitable    carriers,    excipients,    and   other    agents   may   be incorporated  to  formulate  the  pharmaceutical  compositions  to 25    provide    improved   transfer,    delivery,    tolerance,    and    the like.      The   methods   of   incorporating   the   biological   agent and/or  additional  active   ingredient(s)   into  the  carrier  are known  to  a  person  of  ordinary  skill  in  the  art  and  depend  on the   nature   of   the   biological   agent   and   the   nature   of   the


30 carrier selected by a person practicing the current invention. Ionic binding, gel encapsulation or physical trapping inside the carrier, iontophoresis and soaking the


carrier   in  a   solution  of   the   biological   agent   are   suitable examples     contemplated    in    formulating    a    pharmaceutical composition    to    be    used    to    practice    of    the    disclosed treatment  methods.    Alternatively,   the  carrier  may  be  little 5    more    than   a    diluent    for    the    biological    agent.       These formulations    may    include    for    example,    powders,    pastes, ointments,   jelly,   waxes,   oils,   lipids,   anhydrous   absorption


bases,    oil-in-water    or    water-in-oil    emulsions,    emulsions

carbowax    (polyethylene   glycols   of   a   variety   of   molecular

10    weights), semi-solid gels, and semi-solid mixtures containing carbowax. The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age,

weight,    sex   and   medical    condition   of   the    patient;    the

15    severity of the condition to be treated; the route of administration; the renal, hepatic and cardiovascular function of the patient; and the particular biological agent thereof employed. A physician or veterinarian of ordinary

skill    can   readily   determine   and   prescribe   the   effective

20    amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentrations of drug within the range that

yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites.

25 This involves a consideration of the distribution, equilibrium, and elimination of a drug. Any of the foregoing formulations may be appropriate in treatments and therapies in accordance with the present invention, provided that the active ingredient in the formulation is not

30 inactivated by the formulation and the formulation is physiologically compatible.


The pharmaceutical compositions of the present invention may be administered to the host in any manner, strategy and/or combination available in the art in amounts sufficient to offer a therapeutic treatment against

5    Alzheimer's disease. These compositions may be provided to the individual by a variety of routes known in the art, especially parenteral routes, including but in no way limited to parenteral routes such as intravenous (IV), intramuscular (IM); or subcutaneous (SC) administration,

10    with IV administration being the norm within the art of therapeutic antibody administration. These compositions may be administered as separate or multiple doses (i.e.,

administration    of    the    antibody    at    staggered    times    by

maintaining  the  sterile  condition  of  the  formulation  through

15    the treatment regime) . The dosage regimen utilizing the compounds of the present invention is selected in accordance

with   a   variety   of   factors   including   type,    species,    age, weight,   sex  and  medical  condition  of  the  patient   (such  as  a human  patient);  the  severity  of  the  condition  to  be  treated; 20    the    route    of    administration;     the    renal,     hepatic    and cardiovascular   function   of   the   patient;   and   the   particular antibody   thereof   employed.     A  physician   or  veterinarian   of ordinary    skill    can   readily   determine    and   prescribe    the effective    therapeutic   amount    of    the    antibody.       Optimal


25 precision in achieving concentrations of antibody within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.

30    Antibodies described herein may be used alone at appropriate dosages. Alternatively, co-administration or sequential administration of other agents may be desirable. It will be



possible    to   present   a    therapeutic   dosing   regime   for    the antibodies    of    the   present    invention    in    conjunction    with administration   of    alternative   prophylactic   or    therapeutic regimes.      An    effective   dosage   regime   will   vary   depending 5    upon      many      different      factors,       including      means      of administration,    target    site,    physiological    state    of    the patient,   whether   the   patient   is   human   or   an   animal,   other medications      administered,      and     whether      treatment      is prophylactic  or   therapeutic.     For   administration  of   a   13C3-



10    like antibody, the dosage ranges from about 0. 0001 to 100 mg/kg, and more usually 0. 01 to 5 mg/kg of the host body weight. In the case of Alzheimer's disease, amyloid deposits occur in the brain, agents of the invention can also be administered in conjunction with other agents that


15    increase passage of the agents of the invention across the blood-brain barrier.

Another aspect regarding delivery and dosage regimes for a 13C3-like antibody composition of the present invention relates to drug delivery via parenteral routes,

20    which may include non-injectable and injectable devices. Typically, injectable compositions are prepared as either 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 can be

25    emulsified or encapsulated in liposomes or microparticles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above (see Langer, 1990, Science 249: 1527-1523; and Hanes, 1997, Advanced Drug

Delivery  Reviews  28:   97-119).    The   agents   of  this   invention

30    can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner

as to permit a sustained or pulsatile release of the active ingredient.

Specific embodiments include PLGA microspheres, as discussed herein and as further known in the art, as well as

5polymer-basednon-degradablevehicles    comprisingpoly
( ethylene-co-viny 1    acetate;    PEVAc)  .    Additionally,

controlled-release and localized delivery of antibody-based therapeutic products is reviewed in Grainger, et al . , 2004,

Expert    Opin.    Biol.    Ther.    4(7):    1029-1044),    hereby
10incorporatedby    reference    inits    entirety.    Suitable

microcapsules capable of encapsulating the antibody may also include hydroxymethylcellulose or gelatin-microcapsules and polymethyl methacrylate microcapsules prepared by coacervation techniques or by interfacial polymerization.

15 See PCT publication WO 99/24061 entitled "Method for Producing IGF-1 Sustained-Release Formulations," wherein a protein is encapsulated in PLGA microspheres, this reference which is hereby incorporated herein by reference in its entirety. In addition, microemulsions or colloidal drug

20    delivery systems such as liposomes and albumin microspheres, may also be used. Other preferred sustained-release

compositions   employ  a   bioadhesive   to   retain   the   antibody   at the   site   of   administration.     As   noted  above,   the   sustained-release   formulation    may   comprise    a    biodegradable   polymer 25     into   which   the   antibody   is   disposed,   which   may   provide   for non-immediate    release.        Non-injectable    devices    may    be described    herein    as    an    "implant",    "pharmaceutical    depot

implant",    "depot   implant",    "non-injectable   depot"   or   some

such   similar   term.     Common   depot   implants   may   include,   but 30    are     not     limited     to,      solid     biodegradable     and     non-biodegradable   polymer   devices   (such   as   an   extended   polymer or   coaxial   rod   shaped   device),    as   well   as   numerous   pump


systems also known in the art. Injectable devices are split into bolus injections (release and dissipation of the drug subsequent to injection), and repository or depot injections, which provide a storage reservoir at the site of

5    injection, allowing for sustained-release of the biological agent over time. A depot implant may be surgically tethered to the point of delivery so as to provide an adequate reservoir for the prolonged release of the antibody over

time.    Such  a   device   will   be   capable   of   carrying  the   drug

10 formulation in such quantities as therapeutically or prophylactically required for treatment over the pre-selected period. The depot implant may also provide protection to the formulation from degradation by body processes (such as proteases) for the duration of treatment.

15    As known in the art, the term •sustained-release• refers to the gradual (continuous or discontinuous) release of such an agent from the block polymer matrix over an extended period of time. Regardless of the specific device, the sustained-release of the 13C3-like antibody composition will result in

20 a local, biologically effective concentrations of the antibody. A sustained release of the biological agent ( s) will be for a period of a single day, several days, a week or more; but most likely for a month or more, or up to about six months, depending on the formulation. Natural or

25    synthetic polymers known in the art will be useful as a depot implant due to characteristics such as versatile degradation kinetics, safety, and biocompatibility. These copolymers can be manipulated to modify the pharmacokinetics

of    the   active   ingredient,   shield   the   agent   from   enzymatic

30    attack, as well as degrading over time at the site of attachment or injection. The artisan will understand that there are ample teachings in the art to manipulate the



properties of these copolymers, including the respective production process, catalysts used, and final molecular weight of the sustained-release depot implant or depot injection. Natural polymers include but are not limited to

5 proteins (e.g., collagen, albumin or gelatin); polysaccharides (cellulose, starch, alginates, chitin, chitosan, cyclodextrin, dextran, hyaluronic acid) and lipids. Biodegradable synthetic polymers may include but are not limited to various polyesters, copolymers of L-


ID    glutamic   acid   and   gamma   ethy 1-L-glutamate   (Sidman   et   al.,

1983, Biopolymers 22:547-556), polylactides ([PLA]; U.S. Pat. No. 3,773,919 and EP 058,481), polylactate polyglycolate (PLGA) such as polylactide-co-glycolide (see, for example, U.S. Pat. Nos. 4,767,628 and 5,654,008),

15    polyglycolide (PG), polyethylene glycol (PEG) conjugates of poly(a-hydroxy acids), polyorthoesters, polyaspirins, polyphosphagenes, vinylpyrrolidone, polyvinyl alcohol (PVA),

PVA-g-PLGA, PEGT-PBT copolymer (polyactive), methacrylates, poly(N-isopropylacrylamide), PEO-PPO-PEO (pluronics), PE0-

20    PPO-PAA copolymers, PLGA-PEO-PLGA, polyorthoesters (POE), or any combinations thereof, as described above (see, for example, U.S. Pat. No. 6,991,654 and U.S. Pat. Appl. No.

20050187631, each of which is incorporated herein by reference in its entirety, hydrogels (see, for example,

25    Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277; Langer, 1982, Chern. Tech. 12:98-105, non-degradable ethylene-vinyl acetate (e.g. ethylene vinyl acetate disks and poly (ethylene-co-vinyl acetate)), degradable lactic acid-glycolic acid copolyers such as the Lupron Depot~,

30    poly-D-(-)-3-hydroxybutyric acid (EP 133,988), hyaluronic acid gels (see, for example, U.S. Pat. No. 4,636,524), alginic acid suspensions, polyorthoesters (POE), and the



like. Polylactide (PLA) and its copolymers with glycolide (PLGA) have been well known in the art since the commercialization of the Lupron Depot~, approved in 1989 as the first parenteral sustained-release formulation utilizing

5    PLA polymers. Additional examples of products which utilize PLA and PLGA as excipients to achieve sustained-release of the active ingredient include Atridox (PLA; periodontal disease), Nutropin Depot (PLGA; with hGH), and the Trelstar Depot (PLGA; prostate cancer). Other synthetic polymers

10    included  but  are  not  limited  to  poly(c-caprolactone),   poly3-

hydroxybutyrate, poly(~-malic acid) and poly(dioxanone)]; polyanhydrides, polyurethane (see wo 2005/013936),

polyamides, cyclodestrans, polyorthoesters, n-vinyl alcohol, polyethylene oxide/polyethylene terephthalate,

15 polyphosphazene, polyphosphate, polyphosphonate, polyorthoester, polycyanoacrylate, polyethylenegylcol, polydihydropyran, and polyacytal. Non-biodegradable devices include but are not limited to various cellulose derivatives (carboxymettyl cellulose, cellulose acetate, cellulose

20    acetate propionate, ethyl cellulose, hydroxypropyl methyl cellulose) silicon-based implants (polydimethylsiloxane),

acrylic    polymers,   (polymethacrylate,   polymethylmethacrylate,

polyhydroxy (ethylmethylacrylate),   as   well   as   polyethylene-

co-(vinyl    acetate),    poloxamer,    polyvinylpyrrolidone,

25    poloxamine, polypropylene, polyamide, polyacetal, polyester, poly ethylene-chlorotrifluoroethylene,
polytetrafluoroethylene    (PTFE    or    "Teflon™"),    styrene
butadiene  rubber,   polyethylene,    polypropylene,   polyphenylene
oxide-polystyrene,            poly-a-chloro-p-xylene,

30    polymethylpentene, polysulfone and other related biostable polymers. Carriers suitable for sustained-release depot formulations include, but are not limited to, micospheres,


films, capsules, particles, gels, coatings, matrices, wafers, pills or other pharmaceutical delivery compositions. Examples of such sustained-release formulations are described above. See also U.S. Patent Nos. 6,953,593;

5    6,946,146; 6,656,508; 6,:>41,033; and 6,451,346, the contents of each which are incorporated herein by reference. The dosage form must be capable of carrying the drug formulation in such quantities and concentration as therapeutically required for treatment over the pre-selected period, and

10    must provide sufficient protection to the formulation from degradation by body processes for the duration of treatment.

For example, the dosage form can be surrounded by an exterior made of a material that has properties to protect against degradation from metabolic processes and the risk

15    of, e.g., leakage, cracking, breakage, or distortion. This can prevent expelling of the dosage form contents in an uncontrolled manner under stresses it would be subjected to during use, e.g., due to physical forces exerted upon the drug release device as a result of normal joint articulation

20 and other movements by the subject or for example, in convective drug delivery devices, physical forces associated with pressure generated within the reservoir. The drug reservoir or other means for holding or containing the drug must also be of such material as to avoid unintended

25 reactions with the active agent formulation, and is preferably biocompatible (e.g., where the dosage form is implanted, it is substantially non-reactive with respect to a subject's body or body fluids). Generally, the respective biological agent(s) is administered to an individual for at

30    least 12 hours to at least a week, and most likely via an implant designed to deliver a drug for at least 10, 20, 30, 100 days or at least 4 months, or at least 6 months or more,


as required. The 13C3-like antibody can be delivered at such relatively low volume rates, e.g., from about 0.001 ml/day to 1 ml/day so as to minimize tissue disturbance or trauma near the site where the formulation is released. The

5    formulation may be released at a rate of, depending on the specific biological agent(s), at a low dose, e.g., from

about 0.01 pg/hr or 0.1 pg/hr, 0.25 pg/hr, 1 pg/hr, generally up to about 200 pg/hr, or the formulation is delivered at a low volume rate e.g., a volume rate of from

10    about 0.001 ml/day to about 1 ml/day, for example, 0.01 micrograms per day up to about 20 milligrams per day. Dosage depends on a number of factors such as potency, bioavailability, and toxicity of the active ingredient (e.g., IgG antibody) used and the requirements of the

15    subject.                   
    These  and  other  objects,   advantages  and  features  of  the
    present   invention   will   become   apparent   to   those   persons
    skilled    intheartuponreadingthedetailsofthe
    methodology  and  compositions  as  more  fully  set  forth  below.
20        EXAMPLES           
    EXAMPLE  1:  PREPARING  THE  PROTOFIBRILLAR  FORM  OF  AMYLOID  BETA
    (A~42)                   
    A~42  synthetic    peptides    (American    Peptide    Company,
    Inc.,   CA)   were  prepared  according  to  the  method  described  by
25    Fezoui   et   al.    (Fezoui,et    al.    Amyloid    7(3):    166-178.
    (2000))Briefly,   lyophilized  A~42 was  dissolved  in  2mM  NaOH
    at   a   lmg/ml   concentration   (pH-10.5)   followed   by   sonication
    and  lyophillzation.NaOH-treated  A~ was  dissolved  in  water
    at   a   concentration   of   lmg/ml   and   filtered   with   a   0.22pm
30    ULTRAFREE-MC   filter(Millipore,    MA).    A    0. 5mg/ml    peptide

solution was buffered at the final concentration of 50mM phosphate; lOOmM sodium chloride and incubated for 4 hr. at


room temperature. To separate the protofibrillar form from the low-molecular weight proteins, the supernatant was fractionated using size-exclusion chromatography. Purified SEC fractions were then stored at 4C 0 •

5 Various forms of the AP42 protein are represented as showing its ability as a monomer or dimer to associate together to form a high-molecular weight oligomer (protofibril) (Figure 1) . Further aggregation of the soluble protofibrils creates an insoluble form of the


10    protein, whereas the protofibrils can disassociate back to a lower-molecular weight form.

To purify the protofibrillar form of A~ from the low-molecular weight proteins, samples were fractionated with an AKTA chromatography system using a Superdex 75 size-

15    exclusion column. Figure 2A shows that without incubating the AD 42 synthetic peptides at room temperature, there is no aggregation of oligomers to form the protofibrils. Figure 2B illustrates that after a 4 hr. incubation of the

AP42    synthetic  peptides,   subsequent  SEC  purification  shows  a

20    definitive  protofibril  fraction.

EXAMPLE 2: GENERATING MONOCLONAL ANTIBODIES WITH SPECIFICITY FOR PROTOFIBRILLAR AP

The 13C3, 19A6, and lDl antibodies were created by immunizing Balb/c mice with the fibrilliar A~ protein using

25    a protocol known in the art. (Harlow, et al. Cold Spring Harbor Laboratory. (1988)) Spleens were removed and fused

with SP2 myeloma cells in several 96 well plates. Fusion cultures were monitored for growth and supernatants were screened for their ability to bind the protofibrilliar

30    fraction  by  antibody-capture  immunoassays.


EXAMPLE 3: CHARACTERIZATION OF MONOCLONAL ANTIBODIES WITH SPECIFICITY FOR PROTOFIBRILLAR AP

Antibody capture assays were used to further characterize the monoclonal antibodies produced from the

5    hybridomas (13C3, 19A6, and lDl). To microtiter plates, 50ul of a 2ug/ml protofibrillar AP42 protein solution was added to each well and the plates were incubated at 4°C

overnight. After incubation, the residual antigen solution was removed and washed with PBS solution. Serial dilutions

10    of the hybr idoma supernatants were added to the plates containing the bound antigen and incubated for l hour at

room temperature. This primary antibody solution was removed and the wells were again washed with PBS solution. An enzyme-labeled secondary antibody was next added and

15    incubated for 1 hour at room temperature. After removal of the secondary antibody solution, a chromogenic substrate specific for the conjugated enzyme, was added to the react ion and the detection of the captured antibody yielded quantitative results.


20 Additionally, changing the secondary reagent to isotype-specific anti-immunoglobulin antibodies, the particular immunoglobulin isotype of each monoclonal was identified. In these experiments, commercially available anti-A~42 antibodies were used to compare the binding

25 specificity of 13C3, 19A6, and lDl monoclonal antibodies. Figures 3A and B illustrate the protofibrillar (PF) and

the low-molecular weight (LMW) forms of the A~42 peptide used to test the specificity of the 13C3 antibody in antibody capture immunoassays. Specifically, Figure 3A

30    illustrates the plot generated from the ELISAs showing that the 13C3 antibody is specific for the protofibrillar form ( PF) of Ai>42 and does not recognize the low-molecular

weight(LMW) forms of the protein. Figure 3B illustrates the ELISA data with the commercially available 4GB antibody, showing that it recognizes both the low-molecular weight and the protofibrillar forms of the A~42 protein.

5 EXAMPLE 4: SPECIFICITY OF MONOCLONAL ANTIBODIES TO THE PROTOFIBRILLAR FORM OF A~42 USING SURFACE PLASMON RESONANCE (biACORE).

The purified monoclonal antibodies listed in Table 1 (below), were immobilized to a BIAcore sensor chip in

10 accordance with publish protocols. (Nice, et al. BioEssays 21: 339-352 (1999)). The high sensitivity of the BIAcore optical response quantifies a change in reflectivity and a baseline response for the ligand alone is generated. The interaction analysis is performed as the analytes, the LMW


15 form or the PF form of A~42, are injected in solution over the sensor chip and the change in surface plasmon resonance generates a response identifying the specificity of each antibody's ability to bind LMW and PF A~42. Both the 13C3 and the 19A6 antibodies all bound to the PF form of A~ 42

20 with higher specificity than the LMW form. Of all the antibodies used in this experiment, the commercially-available antibodies showed higher specificity for the LMW

A~42 over tte PF form of A~ 42, as indicated the ratio of PF binding/ LMW binding.

25



•5

    AP 17•22    lgG2b    Senetek Inc.    228.6    340.3    1.5
6E10    AP 3•8    lgG2b    Senetek Inc.    400.1    541.1    1.4
B2E1    AP 1-17    lgG1    IBL    69.9    68.4    1.0
                       

The Surface Plasmon Resonance Analysis shown by sensorgram that 13C3 (Figure 4B) does not bind the LMW forms

of Aj3 42 protein. However, the 4G8 (Figure 4A) shows a standard association/disassociation curve for the LMW Aj342 protein. The antibody isotype control IgGl (Figure C) does not bind the LMW Aj3 as well. Automated BIAcore systems,

5    which    use    the    detection    principle    of    Surface    Plasmon

Resonance,    were   used   in   these   experiments.    The   binding

specificity data for the 19A6 antibody showed that 19A6 had a binding ratio of 5.8, which is similar to that of 13C3 at a ratio of 5.3.

10    EXAMPLE  5:  EPITOPE  MAPPING  OF  THE  13C3  ANTIBODY

Mapping the epitopes of 13C3, lDl and 19A6 was conducted using the RepliTope Microarrays system (JPT Peptide Technologies GmbH) according to published protocol.

(Korth,    et    al.    390:    74    (1997)).    Each    spot    on    the

15    microarray contains a 13 amino acid peptide of Aj342 where each shift in position on the microarray represents an amlno acid shift (fom N-term to C-term), i . e . SEQ ID NO: 23, SEQ ID NO: 24 ... SEQ ID NO: 51; and SEQ ID NO: 52. Listed below are the peptides and their exact amino acid sequence,

20    corresponding to their position on the slide array. Once the peptides are fixed to the RepliTope Microarray, the samples are incubated with the 13C3 antibody and then subsequently labeled with a secondary that is conjugated to a chemiluminescence tag of choice. The spots that yield a

25    signal represent the epitope binding sites on the protein by the antibody.

Asp  Ala  Glu  Phe  Arg  His  Asp  Ser  Gly  Tyr  Glu  Val  His   (SEQ  ID  NO:

23)
Ala  Glu  Phe  Arg  His  Asp  Ser  Gly  Tyr  Glu  Val  His  His   (SEQ  ID  NO:
30    24)

Glu  Phe  Arg  His  Asp  Ser  Gly  Tyr  Glu  Val  His  His  Gln   (SEQ  ID  NO:

25)
Phe  Arg  His  Asp  Ser  Gly  Tyr  Glu  Val  His   His   Gln  Lys   (SEQ  ID  NO:

26)
35    Arg  His  Asp  Ser  Gly  Tyr  Glu  Val  His   His  Gln  Lys  Leu   (SEQ  ID  NO:

27)


His    Asp  Ser  Gly  Tyr  Glu  Val   His   His   Gln   Lys   Leu  Val   (SEQ   ID  NO:

28)

Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe (EEQ ID NO: 29)

5    Ser  Gly  Tyr   Glu  Val  His  His  Gln   Lys   Leu  Val  Phe  Phe   (SEQ  ID  NO:

30)

Gly  Tyr   Glu  Val  His  His  Gln  Lys   Leu  Val   Phe   Phe  Ala   (SEQ   ID  NO:

31)
Tyr    Glu  Val  His  His  Gln   Lys   Leu  Val  Phe  Phe  Ala  Glu   (SEQ   ID  NO:

10    32)
Glu  Val   His   His   Gln  Lys   Leu  Val   Phe  Phe  Ala   Glu  Asp   (SEQ   ID  NO:

33)

Val  His   His   Gln  Lys  Leu  Val   Phe   Phe  Ala   Glu  Asp  Val   (SEQ   ID  NO:

34)

15    His  His   Gln  Lys   Leu  Val  Phe  Phe  Ala  Glu  Asp  Val   Gly   (SEQ   ID  NO:

35)
His   Gln   Lys  Leu  Val   Phe   Phe  Ala   Glu  Asp  Val   Gly  Ser   (SEQ   ID  NO:

36)

Gln    Lys   Leu  Val   Phe  Phe  Ala  Glu  Asp  Val   Gly  Ser  Asn   (SEQ   ID  NO:
20    37)

Lys   Leu  Val  Phe  Phe  Ala  Glu  Asp  Val   Gly  Ser  Asn  Lys   (SEQ   ID  NO:

38)

Leu  Val   Phe  Phe  Ala  Glu  Asp  Val   Gly  Ser  Asn   Lys   Gly   (SEQ   ID  NO:

39)
25    Val  Phe  Phe  Ala  Glu  Asp  Val  Gly  Ser  Asn   Lys   Gly  Ala   (SEQ   ID  NO:

40)

Phe  Phe  Ala  Glu  Asp  Val   Gly  Ser  Asn   Lys   Gly  Ala   Ile   (SEQ   ID  NO:

41)

Phe  Ala   Glu  Asp  Val  Gly  Ser  Asn   Lys   Gly  Ala   Ile   Ile   (SEQ   ID  NO:

30    42)

Ala  Glu  Asp  Val   Gly  Ser  Asn  Lys   Gly  Ala   I le   Ile  Gly   (SEQ   ID  NO:

43)
Glu  Asp  Val   Gly  Ser  Asn  Lys   Gly  Ala   Ile   Ile   Gly  Leu   (SEQ   ID  NO:

44)
35    Asp  Val   Gly  Ser  Asn   Lys   Gly  Ala   Ile   Ile   Gly  Leu  Met   (SEQ   ID  NO:

45)
Val   Gly  Ser  Asn  Lys   Gly  Ala   Ile   Ile  Gly  Leu  Met  Val   (SEQ   ID  NO:

46)
Gly    Ser  Asn  Lys  Gly  Ala   Ile   Ile   Gly  Leu  Met  Val   Gly   (SEQ  ID  NO:

(c)    47)

Ser  Asn   Lys  Gly  Ala  Ile   Ile   Gly   Leu  Met  Val   Gly  Gly   (SEQ   ID  NO:

48)
Asn  Lys   Gly  Ala   Ile   Ile   Gly  Leu  Met  Val   Gly  Gly  Val   (SEQ  ID  NO:

49)
45    Lys   Gly  Ala   Ile   Ile   Gly  Leu  Met  Val   Gly  Gly  Val  Val   (SEQ   ID  NO:

50)

Gly  Ala   Ile  Ile  Gly  Leu  Met  Val   Gly  Gly  Val  Val   Ile   (SEQ   ID  NO:

51)
Ala   Ile   Ile  Gly  Leu  Met  Val   Gly  Gly  Val  Val   Ile  Ala   (SEQ  ID  NO:
50    52)


Figure ~A illustrates a dot blot from a RepliTope Microarray experiment identifying the epitopes of the antibodies, 13C3, lDl and 4GB on the A~ 1-42 peptide. The

bound    antibody  is  represented  by  a   chemiluminescent  signal.

5    Figures   !JB   illustrates   the   A~  1-42   amino   acid   sequence

showing the polypeptide seqments of the 13C3 epi topes as they occur in the sequence. The 1Dl antibody shows the same epitopes as the 13C3 whereas the commercial 4GB antibody identifies a different epitope.

10    EXAMPLE  6:  CHARACTERIZATION  OF  13C3  SPECIFICITY

Figure    6    illustrates    fractions    from    size-exclusion

chromatography of the supernatants from the 7PA2 cell line, a secreting AI) oligomer cell line. Antibody capture assays were used to further characterize the binding of the 13C3

15    antibody   with   the   protofibrillar   and   low-molecular   weight

fractions    from    the    SEC-purified    7PA2.    To   microtiter

plates, 100ul of a 1:200 dilution of each fraction was added to each well and the plates were incubated at 4°C overnight. After incubation, the residual antigen solution was removed

20    and washed with PBS solution. Serial dilutions of the 13C3 supernatants were added to the plates containing the bound antigen and incubated for 1 hour at room temperature. This

primary antibody solution was removed and the wells were again washed with PBS solution. An enzyme-labeled secondary

25    antibody was next added and incubated for 1 hour at room temperature. After removal of the secondary antibody solution, a chromogenic substrate specific for the conjugated enzyme, was added to the reaction and the detection of the captured antibody yielded quantitative

30    results. This assay identified that the 13C3 antibody specifically recognizes only the protofibrillar fraction whereas the 4GB antibody recognizes all fractions. The 7PA2


cell line was provided by Dennis J. Selkoe, M.D. at Harvard Medical School.

EXAMPLE  7  CHARACTERIZATION  OF  13C3  REACTIVITY  BY  EM.

The    method  of   staining  was   performed  using  a   standard

5    protocol. (Brenner, et al. Biochim. Biophys. Ada 34, 103-110 (1959)). A small volume (10 microliters) of a 0.2mg/ml protofibrillar solution was applied to carbon-coated formvar grids ( 400 mesh) for 2 min. Then the grids were blocked in l%BSA and incubated with the 13C3 antibody followed by a

10    subsequent incubation with a secondary antibody conjugated to colloidal gold. The samples were negatively stained by placing on 2 successive drops of 2% phosphotungstic acid for 30 sec each. Excess stain was drawn off with filter paper,

the   grids   were   air   dried,   and   observed   on   a   JEOL   lOOCX 15    transmission    electron    microscope    at    80kV.      Images    were recorded  on  large  format   Kodak   4489   negatives  and  digitized

on  a  flat  bed  scanner.

IEM (Immuno-Electron Microscopy) images showing the binding specificity of the anti-Ai3 antibody clone 13C3 to

20    Ai342 fibers (Figures 7B and 7C), whereas the isotype control antibody, IgGl shows no binding (Figure 7A). The secondary antibody is conjugated to a colloidal gold particle.

EXAMPLE  8:  13C3  TREATMENT  OF  A  MOUSE  MODEL  OF  HUMAN  AD

The    13C3   monoclonal   antibody   was   used   to   treat   Ai3

25    plaques in an Alzheimer's Disease mouse model, TgCRND8. The mouse contains the human APP695 eDNA transgene, which

accelerates the deposition of Ai3 amyloid plaques in the mouse brain, appearing within 1 month of age. A sample group of 5 TgCRND8 mice five weeks in age were give

30 immunizations of the 13C3 monoclonal antibody at a concentration of lOmg/ kg of mouse once a week for the duration of seven weeks. A second group of 5 TgCRNDS mice,



were given the treatment course, however an isotype control IgGl antibody was administered. Experiments were repeated with treatments at twice a week instead of once a week.
Both    control   and  experimental   animals   were   sacrificed

5    at 12 weeks of age. Histological preparations of the brains revealed reductions in A~ plaques in 13C3 treated mice.

Serial sections of cryopreserved brains from TgCRNDS mice were treated with 13C3 or IgGl monoclonal antibodies. Figures SA and SB illustrate differences in the number of A~

10    amyloid  plaques  between  each  respective  antibody.

Statistical T-tests show that the 13C3 antibody treatment at once a week reduces A~ amyloid plaques in the Alzheimer Disease model (Figure 9A) . However, twice a week treatments (Figure 9B) show the same level of plaque

15    reduction.

All of the above TgCRNDS mice were obtained from Dr. David Westaway of the University of Toronto.

EXAMPLE 9: MOLECULAR CHARACTERIZATION OF THE VARIABLE REGIONS OF MAE l3C3

20 The IgG heavy chain variable region and the IgG Kappa light chain region were cloned from the 13C3 hybridoma. Both heavy and light chain sequences (Figure 10) were analyzed using VBASE2 (http://www.vbase2.org), a database of germ-line variable genes from the immunoglobulin loci of

25    human and mouse extracted from the EMBL-Bank and Ensembl data libraries. (Retter et al. Nucleic Acids Res. 33:D671-4 (2005)). Results for the analysis identified that both the heavy and light chain variable regions were from a newly

identified    immunoglobulin   but   had   73%   and   81%   identity,

30    respectively, to other immunoglobulin variable regions in the database. Also identified in these sequences against these databases were the Frame Work Regions (FWR) and the Complementarity Determining Regions (CDR) . Results were


only slightly varied when sequences were analyzed against VBASE, KABAT, and IMGT/LIGM database.

EXAMPLE 10: ACUTE PERIPHERAL ADMINISTRATION OF l3C3 IN APP TRANSGENIC ~liCE DOES NOT LEAD TO AN INCREASE IN PLASMA M

5    UNLIKE  REFERENCE  ANTIBODY  3D6  ADMINISTRATION

APP transgenic mice (Thy APPSL, age 10-14 weeks) were injected intraperitoneally at the dose of 10 mg/kg (i . e . , 300 pg/mouse) with antibodies 13C3, a control IgGl (DM4, not recognizing At) and a reference anti-At antibody 3D6

10 recognizing all conformers of At. Plasma M was quantified at time zero pre-injection, 6h, 24 h and 7days post injection in the same mice. Quantification of plasma At was performed with an immunoassay using anti-All antibody pairs not interfering with 13C3 or 3D6 binding to At.


15 Administration of 3D6, an antibody against all conformers of All, leads to a large increase in plasma M, likely by protecting All molecules from degradation. This effect was used to suggest the potential uperipheral sink" hypothesis as mechanism of action of anti-M immunotherapy


20 (Demattos et al . , 2001, PNAS 17:8850). Unlike 3D6, l3C3 administration does not lead to any increase in plasma At levels. This is consistent with the properties of 13C3, an antibody that is specific for the protofibrillar forms of All and is not recognizing the soluble mono- or oligomeric forms


25    of Af.J peptide. These forms are the likely ones present in plasma.

EXAMPLE 11: 13C3 RECOGNIZES HUMAN AMYLOID NEURITIC PLAQUES (AGGREGATED) IN AD BRAINS BUT NOT THE DIFFUSE At DEPOSITS UNLIKE THE REFERENCE 3D6 ANTI-At ANTIBODY

30 Immunohistochemistry studies were performed with 13C3 and 3D6 antibodies on human Alzheimer-diagnosed brain sections using standard techniques. Antibody immunostaining was detected with a DAB chromogen (Fig. 12) . 13C3 labels amyloid deposits with a typical morphology of mature amyloid


neuritic plaques (also called dense plaques) with a very dense core surrounded by a lighter halo or for the larger plaques a very strong staining. In adjacent brain sections, 3D6 stains many more objects than 13C3 as seen at lower

5 magnification (Fig. 12, left panels) . Further characterization at higher magnification (Fig. 12, right panels) indicated that 3D6 labels the same mature amyloid neuritic plaques as 13C3 and, in addition, numerous diffuse amyloid deposits that have been classically described using


10    anti-A£ immunolabelling. The diffuse plaques are not of fibrillar nature as described in the literature as they

cannot be detected by thioflavin S and other histological markers of fibrils (Mann, 1989, Ann. Med. 21:133). To rule out differences in sensitivity of the two antibodies,

15 similar experiments were conducted with a higher concentration (20 pg/ml) of 13C3 and again diffuse deposits could not be detected. This data is consistent with the properties of 13C3, an antibody that is specific for the protofibrillar forms of A£ and is not recognizing the

20    soluble mono- or oligomeric forms of A£ peptide unlike 3D6. INDUSTRIAL APPLICABILITY

The   invention   has   applications   in   the   treatment   and

diagnosis    of  Alzheimer's  disease.

All    publications    cited    in    the    specification,    both 25    patent     publications     and     non-patent     publications,     are indicative  of  the  level  of  skill  of  those  skilled  in  the  art to   which   this   invention   pertains.     All   these   publications are   herein   fully   incorporated   by   reference   to   the    same extent   as   if   each   individual   publication   were   specifically

30 and individually indicated as being incorporated by reference.

Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is

5    therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the following claims.




WHAT IS CLAIMED IS:

1.    An isolated antibody  that  specifically interacts lft.ij !shows 2t~~

affinity to a conformational epitope of a protofibril form of A~~p~tide, whereby the protofibril epitope is represented by an exposed region of a A~rotofibril .~

5    comprising the amino acid sequence as set forth in SEQ ID N0:2, wherein said antibody shows minimal or no affinity for monomer or dimer forms of A~ peptide.

2.    The antibody of claim 1 which is a monoclonal antibody.

3.    The monoclonal antibody of claim 2 which is a humanized monoclonal antibody or a human monoclonal antibody.

10    4.    An isolated antibody that  specifically    interacts    and  shows    a measurable
    affinity to  a conformational epitope of a protofibril    form  of    A~ peptide,    whereby the

protofibril epitope is represented by an exposed region of a A~-protofibril form comprising an amino acid sequence selected from the group consisting of SEQ ID N0:3 and SEQ ID N0:4, wherein said antibody shows minimal or no affinity for monomer or

I 5    dimer forms of A~ peptide.

5.    The antibody of claim 4 which is a monoclonal antibody.

6.    The monoclonal antibody of claim 5 which is designated 13C3.

7.    The monoclonal antibody of claim 5 which is a humanized monoclonal antibody or a human monoclonal antibody.

20    8.    The antibody of claim 4 further comprising a variable light chain comprised

of the amino acid sequence as set forth in SEQ ID N0:5, or a variable heavy chain comprised of the amino acid sequence as set forth in SEQ ID N0:7.

9. The antibody of claim 4 further comprising a variable light chain comprising a CDR! region as set forth in SEQ ID N0:13, a CDR2 region as set forth in

25    SEQ ID NO:I4, and a CDR3 as set forth in SEQ ID NO:l5, or a variable heavy chain comprised of a CDR! region as set forth in SEQ ID N0:20, a CDR2 region as set forth in SEQ ID N0:21, and a CDR3 as set forth in SEQ ID N0:22.
 

10.    A method of producing a monoclonal antibody ~~ SP.ecfficaTiy5inas'1n

vitro to a repeating conformational epitope of a protofibril fo~~~2'~~id•p~tid<:\ while showing greater affinity to a protofibril form of ~-amyloid pe~

molecular weight form of ~-amyloid peptide, comprising:

5    (a) immunizing a mammal with the protofibril form of ~-amyloid peptide;

(b)    harvesting B-cells of said mammal;

(c)    creating hybridomas from the harvested B-cells, wherein said hybridomas produce antibodies; and,

(d)    selecting hybridomas  which produce  antibodies  specifically  binding  to  the

10    protofibril form of ~-amyloid peptide while showing minimal affinity to monomer or dimer forms of ~-amyloid peptide.

11.    A method for quantifying the amount of a protofibril form of ~-amyloid

peptide in a tissue or fluid sample, comprising:

(a) obtaining the tissue or fluid sample from a subject;

15 (b) contacting the tissue or fluid sample with an antibody or fragment thereof that specifically binds to the protofibril form of ~-amyloid peptide while showing minimal affinity to low molecular weight forms of ~-amyloid peptide; and,

(d)    quantifying the amount of protofibril form of ~-amyloid peptide in the sample.

12.    The  method  of claim  11  wherein  the  antibody  is  monoclonal  antibody

20    selected from the group consisting of 13C3, lDl and 19A6.

13.    A kit for detecting protofibril form of ~-amyloid peptide while showing greater affinity to a protofibril form of ~-amyloid peptide than to a low molecular weight form of ~-amyloid peptide, comprising:

(a) an antibody or a fragment thereof, capable of specifically binding in vitro to a

25    repeating conformational epitope of a protofibril form of ~-amyloid peptide while showing minimal affinity to low molecular weight forms of ~-amyloid peptide; and,
 
(b) a reagent that binds, directly({~(indirec\11, ro' said ~r the A-i:g/n~ •

thereof.

14.    The kit of claim 13 wherein the~ monoclonal antibna,-~

from the group consisting of 13C3, lDl and 19A6.

5 15. A pharmaceutical composition comprising a variable region fragment which specifically interacts with the protofibrillar form of b-amyloid, wherein said specific interaction is characterized by a ratio of the affinity of said variable region fragment for the protofibrillar Ab form to the affinity for other Ab forms greater than about 2.

10    16. The method of claim 15 wherein the antibody is a monoclonal antibody.

17.    The method of claim 16 wherein the monoclonal antibody is an antibody selected from the group consisting of 13C3, 19A6 and lDl.

18.    The method of claim 16 wherein the monoclonal antibody is a humanized monoclonal antibody or a human monoclonal antibody.

15    19.    A hybridoma which secretes an antibody selected from the group consisting
    of 13C3, 19A6 and IDI.
    20.    An  isolated  nucleic  acid  molecule  encoding  a  variable  heavy  chain
    fragment  of monoclonal  antibody  13C3  wherein  the  variable  heavy  chain  fragment
    comprises the amino acid sequence as set forth in SEQ ID N0:7.

20 21. An isolated nucleic acid molecule encoding a variable heavy chain fragment of monoclonal antibody 13C3 wherein the nucleic acid molecule comprises the nucleotide sequence as set for in SEQ lD N0:8.

22. An expression vector for the expression of a variable heavy chain fragment of monoclonal antibody 13C3 in a recombinant host cell wherein said expression vector

25    contains the nucleic acid molecule of claim 21.

23.    A host cell which expresses a variable heavy chain fragment of monoclonal antibody 13C3 wherein said host cell contains the expression vector of claim 22.

24.    An isolated nucleic acid molecule encoding a variab'l8.liiht chain fragment of monoclonal antibody 13C3 wherein the variable light chain fragment comp-rf~es the amino acid sequence as set forth in SEQ ID N0:5.

25.    An isolated nucleic acid molecule encoding a variable light chain fragment

5    of monoclonal antibody 13C3 wherein the nucleic acid molecule comprises the nucleotide sequence as set for in SEQ ID N0:6.

26.    An expression vector for the expression of a variable light chain fragment of monoclonal antibody 13C3 in a recombinant host cell wherein said expression vector

contains the nucleic acid molecule of claim 25.

10 27. A host cell which expresses a variable light chain fragment of monoclonal antibody 13C3 wherein said host cell contains the expression vector of claim 26.

28.    An isolated variable heavy chain fragment of monoclonal antibody 13C3 which comprises the amino acid sequence set forth in SEQ ID N0:7.

29.    An isolated variable light chain fragment  of monoclonal antibody  13C3

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