HK1128928A - Anti-amyloid antibodies, compositions, methods and uses - Google Patents

Description

Anti-amyloid antibodies, compositions, methods and uses

The present application is a divisional application of "anti-amyloid antibodies, compositions, methods and uses" of chinese patent application 200480014789.0 filed 3, 26/2004.

Technical Field

The present invention relates to antibodies, including specified portions or variants, specific for at least one beta-amyloid (amyloid) protein or fragment thereof, as well as anti-idiotypic antibodies, and nucleic acids, complementary nucleic acids, vectors, host cells, and methods of making and using, including therapeutic formulations, administration, and devices, encoding such anti-amyloid antibodies.

Background

Alzheimer's Disease (AD) is a degenerative brain disease that is clinically characterized by progressive loss of memory, cognition, reasoning, judgment, and emotional stability, which gradually leads to profound mental deterioration and ultimately death. AD is a very common cause of progressive mental disorders (dementia) in the elderly, and is believed to represent the fourth most common medical cause of death in the united states. AD has been observed in races and ethnicities worldwide, and has raised major current and future public health concerns. It is estimated that the disease currently affects about 2 to 3 million individuals in the united states alone. AD is currently incurable. No treatment is currently known to be effective in preventing AD or reversing its symptoms and course.

The brain of an individual with AD shows characteristic lesions called senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits in blood vessels) and neurofibrillary tangles. A large number of these lesions, especially amyloid plaques and neurofibrillary tangles, are commonly found in some regions of the human brain of AD patients that are important for memory and cognitive function. Fewer of these lesions in a more localized anatomical distribution are also found in the brains of most elderly people without clinical AD. The brains of individuals with trisomy 21 (down's syndrome), diffuse Lewy body disease, and hereditary cerebral hemorrhage with amyloidosis of the dutch-type (HCHWA-D) are also characterized by amyloid plaques and amyloid angiopathy.

The major components of amyloid plaques are the various amyloid beta (a β) peptides, which result from the cleavage of the β -Amyloid Precursor Protein (APP). Although there has been intense scientific debate in the past as to whether plaques and tangles are the cause or only the result of alzheimer's disease, recent findings suggest that amyloid plaques are causative precursors or factors. In particular, it has been found that the production of a β peptides can result from mutations in the gene encoding amyloid precursor protein which, when subjected to normal processing, will not produce a β peptides. The identification of mutations in the amyloid precursor protein gene that cause familial early-onset alzheimer's disease most strongly suggests that amyloid metabolism is a central event in the pathogenic process of the disease. It is currently believed that normal (non-pathogenic) processing of the APP protein occurs by cleavage by the "α -secretase" (secretase), which cleaves between amino acids 16 and 17 of the a β peptide region of the protein. It is also believed that the pathogenic processing occurs in part by the "beta-secretase" which cleaves at the amino terminus of the a β peptide region of the precursor protein. It is also believed that beta amyloid may be associated with other neurological and certain cardiovascular diseases.

Accordingly, there is a need to provide anti-amyloid antibodies or fragments, and improvements to known antibodies or fragments thereof, that overcome one or more of these problems.

Disclosure of Invention

The present invention provides isolated human, primate, rodent, mammalian, chimeric, humanized and/or CDR-grafted anti-amyloid antibodies, immunoglobulins, fragments, cleavage products and other specified portions and variants thereof, as well as anti-amyloid antibody compositions, encoding or complementary nucleic acids, vectors, host cells, compositions, formulations, devices, transgenic animals, transgenic plants, and methods of making and using the same, as described herein and capable of generating combinations with those known in the art.

The invention also provides at least one isolated anti-amyloid antibody as described herein. Antibodies according to the invention include any protein or peptide comprising a molecule comprising at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one Ligand Binding Portion (LBP), such as, but not limited to, a Complementarity Determining Region (CDR) of a heavy or light chain (e.g., comprising at least one of SEQ ID NOS: 42-47, 53-58, 63-68, 73-78) or a ligand binding portion thereof, a heavy or light chain variable region (e.g., comprising at least one of 10-125 contiguous amino acids of at least one of SEQ ID NOS: 1-30, or at least one of FR1, FR2, FR3, FR4 or fragments thereof as described in table 1, and optionally further comprising at least one substitution, insertion or deletion as provided in figures 1-41), a heavy or light chain constant region (e.g., comprises the nucleotide sequence shown in SEQ ID NOS: 31-41, or at least one of CH1, hinge 1, hinge 2, hinge 3, hinge 4, CH2, CH3 or a fragment thereof as described in table 1, and optionally further comprising at least one substitution, insertion, or deletion provided in figures 1-41), a framework region, or any portion thereof that can be incorporated into an antibody of the invention. The antibodies of the invention may include or be derived from any mammal, such as, but not limited to, a human, a mouse, a rabbit, a rat, a rodent, a primate, or any combination thereof, and the like.

In one aspect the invention provides an isolated nucleic acid molecule comprising, complementary to, or hybridising to a polynucleotide encoding a specific anti-amyloid antibody, said nucleic acid molecule comprising at least one specific sequence, domain, portion or variant thereof. The invention also provides recombinant vectors containing the anti-amyloid antibody nucleic acid molecules, host cells containing the nucleic acids and/or recombinant vectors, and methods of making and/or using these antibody nucleic acids, vectors, and/or host cells.

The invention also provides at least one anti-amyloid antibody or specified portion or variant comprising at least one amyloid binding sequence and the amino acid sequence of SEQ ID NOS: at least 10-384 contiguous amino acids of at least one of 1-41, or at least one of FR1, FR2, FR3, FR4, CH1, hinge 1, hinge 2, hinge 3, hinge 4, CH2, CH3, or fragments thereof, as described in table 1, optionally further comprising at least one substitution, insertion, or deletion as provided in fig. 1-41 or as known in the art.

At least one antibody of the invention binds to at least one specific epitope specific for at least one amyloid protein, subunit, fragment, portion or any combination thereof. The at least one epitope may comprise at least one antibody binding region comprising at least a portion of the protein, preferably consisting of at least 1-5 amino acids of at least a portion of the protein, such as, but not limited to, at least one functional, extracellular, soluble, hydrophilic, external or cytoplasmic domain of the protein, or any portion thereof.

The at least one antibody may optionally contain at least one specific portion of at least one Complementarity Determining Region (CDR) (e.g., CDR1, CDR2, or CDR3 of the heavy or light chain variable region), and optionally further contain at least one constant or variable framework region or any portion thereof. The at least one antibody amino acid sequence may optionally further contain at least one specific substitution, insertion or deletion as described herein or as known in the art.

The present invention also provides at least one isolated anti-amyloid antibody as described herein, wherein said antibody has at least one activity, such as, but not limited to, a well known amyloid assay. The anti-amyloid antibody may thus be screened for the corresponding activity according to well-known methods, such as, but not limited to, at least one biological activity against amyloid.

The invention also provides at least one amyloid anti-idiotype antibody directed against at least one amyloid antibody of the invention. Anti-idiotype antibodies include any protein or peptide comprising a molecule comprising at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one Ligand Binding Portion (LBP), such as, but not limited to, a Complementarity Determining Region (CDR) of a heavy or light chain, or a ligand binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework region, or any portion thereof that can be incorporated into an antibody of the invention. The antibodies of the invention may include or be derived from any mammal, such as, but not limited to, a human, a mouse, a rabbit, a rat, a rodent, a primate, and the like.

In one aspect the present invention provides an isolated nucleic acid molecule comprising, complementary to, or hybridising to a polynucleotide encoding at least one amyloid anti-idiotype antibody, said nucleic acid molecule comprising at least one specific sequence, domain, portion or variant thereof. The invention also provides recombinant vectors comprising nucleic acid molecules encoding said amyloid anti-idiotype antibodies, host cells comprising said nucleic acids and/or recombinant vectors, and methods of making and/or using these anti-idiotype antibody nucleic acids, vectors and/or host cells.

The present invention also provides a method of expressing at least one anti-amyloid antibody or amyloid anti-idiotype antibody in a host cell comprising culturing a host cell as described herein under conditions wherein the at least one anti-amyloid antibody is expressed in detectable and/or recoverable amounts.

The present invention also provides at least one composition comprising (a) an isolated anti-amyloid antibody encoding nucleic acid and/or antibody as described herein; and (b) a suitable carrier or diluent. The carrier or diluent may optionally be pharmaceutically acceptable according to well known carriers or diluents. The composition may optionally further comprise at least one additional compound, protein or composition.

The present invention also provides at least one anti-amyloid antibody method or composition for administration of a therapeutically effective amount to modulate or treat at least one amyloid-related condition in a cell, tissue, organ, animal or patient, and/or prior to, subsequent to or during said related condition, as known in the art and/or as described herein.

The invention also provides at least one composition, device and/or method of delivery of a therapeutically or prophylactically effective amount of at least one anti-amyloid antibody according to the invention.

The present invention also provides at least one anti-amyloid antibody method or composition for diagnosing at least one amyloid-related condition in a cell, tissue, organ, animal or patient, and/or prior to, subsequent to or during said related condition, as known in the art and/or as described herein.

The invention also provides at least one composition, device and/or method of delivery for diagnosing at least one anti-amyloid antibody according to the invention.

In one aspect, the invention provides at least one isolated mammalian anti-amyloid antibody comprising a heavy chain variable region comprising SEQ ID NO: 48 or 49.

In another aspect, the invention provides at least one isolated mammalian anti-amyloid antibody comprising (i) the amino acid sequence of SEQ ID NOS: 42-44, all heavy chain Complementarity Determining Region (CDR) amino acid sequences; or (ii) SEQ ID NOS: 45-47, all light chain CDR amino acid sequences.

In another aspect, the invention provides at least one isolated mammalian anti-amyloid antibody comprising a polypeptide having the sequence of SEQ ID NOS: 42-47, or a light chain CDR of the amino acid sequence of at least one of the following.

In one aspect, the invention provides at least one isolated mammalian anti-amyloid antibody comprising SEQ ID NO: 59 or 60.

In another aspect, the invention provides at least one isolated mammalian anti-amyloid antibody comprising (i) the amino acid sequence of SEQ ID NOS: 53-55, all heavy chain Complementarity Determining Region (CDR) amino acid sequences; or (ii) SEQ ID NOS: 56-58 in the sequence listing.

In another aspect, the invention provides at least one isolated mammalian anti-amyloid antibody comprising a polypeptide having the sequence of SEQ ID NOS: 53-58, and at least one heavy or light chain CDR of an amino acid sequence of at least one of.

In one aspect, the invention provides at least one isolated mammalian anti-amyloid antibody comprising SEQ ID NO: 69 or 70.

In another aspect, the invention provides at least one isolated mammalian anti-amyloid antibody comprising (i) the amino acid sequence of SEQ ID NOS: 63-65, all heavy chain Complementarity Determining Region (CDR) amino acid sequences; or (ii) SEQ ID NOS: 66-68, and a light chain CDR amino acid sequence.

In another aspect, the invention provides at least one isolated mammalian anti-amyloid antibody comprising a polypeptide having the sequence of SEQ ID NOS: 63-68, or a light chain CDR.

In one aspect, the invention provides at least one isolated mammalian anti-amyloid antibody comprising SEQ ID NO: 79 or 80.

In another aspect, the invention provides at least one isolated mammalian anti-amyloid antibody comprising (i) the amino acid sequence of SEQ ID NOS: 73-75 of all heavy chain Complementarity Determining Region (CDR) amino acid sequences; or (ii) SEQ ID NOS: 76-78, and all light chain CDR amino acid sequences.

In another aspect, the invention provides at least one isolated mammalian anti-amyloid antibody comprising a polypeptide having the sequence of SEQ ID NOS: 73-78, or a light chain CDR of the amino acid sequence of at least one of the amino acids.

In one aspect, the invention provides at least one isolated mammalian anti-amyloid antibody comprising at least one human CDR, wherein the antibody specifically binds to a polypeptide selected from the group consisting of SEQ id nos: 50 of amino acids 2-7, 3-8, 33-42 or 34-40.

In another aspect, the invention provides at least one isolated mammalian anti-amyloid antibody comprising at least one human CDR, wherein the antibody specifically binds to a polypeptide comprising SEQ id no: 50 to at least one epitope of the complete amino acid sequence.

The at least one antibody may optionally further comprise at least one characteristic selected from the group consisting of: (i) to be selected from at least 10^-9M、10^-10M、10^-11M or 10^-12The affinity of at least one of M binds amyloid; and/or (ii) substantially neutralize at least one activity of at least one amyloid protein. Also provides a braidAn isolated nucleic acid encoding at least one isolated mammalian anti-amyloid antibody; an isolated nucleic acid vector comprising the isolated nucleic acid; and/or a prokaryotic or eukaryotic host cell containing said isolated nucleic acid. The host cell may optionally be at least one selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma or lymphoma cells, or any derived, immortalized or transformed cell thereof. Also provided are methods of producing at least one anti-amyloid antibody comprising translating a nucleic acid encoding the antibody in vitro, in vivo or in situ, whereby the anti-amyloid antibody is expressed in detectable or recoverable amounts.

Also provided are compositions comprising at least one isolated mammalian anti-amyloid antibody and at least one pharmaceutically acceptable carrier or diluent. The composition may optionally further comprise an effective amount of at least one compound or protein selected from at least one detectable marker or reporter, an anti-infective drug, a Cardiovascular (CV) system drug, a Central Nervous System (CNS) drug, an Autonomic Nervous System (ANS) drug, a respiratory drug, a Gastrointestinal (GI) tract drug, a hormonal drug, a drug for fluid or electrolyte balance, a hematologic drug, an anticancer drug, an immunomodulating drug, an ocular, otic or nasal drug, a topical drug, a nutraceutical drug, etc., a TNF antagonist, an antirheumatic drug, a muscle relaxant, an anesthetic, a non-steroidal anti-inflammatory drug (NTHE), an analgesic, an anesthetic, a sedative, a local agent, a neuromuscular blocker, an antimicrobial, an antipsoriatic agent, a corticosteroid, a anabolic steroid, an erythropoietin, an immunological (immunization) anesthetic, an anesthetic, a hormone, a pharmaceutical composition, a method of treating a, Immunoglobulins, immunosuppressants, growth hormones, hormone replacement drugs, radioprotective drugs, antidepressants, antipsychotics, stimulants, asthma drugs, beta agonists, inhaled steroids, epinephrine or analogs, cytokines, or cytokine antagonists.

The invention also provides an anti-idiotype antibody or fragment that specifically binds to at least one isolated mammalian anti-amyloid antibody of the invention.

Also provided is a method of diagnosing or treating an amyloid-related condition in a cell, tissue, organ or animal comprising (a) contacting or administering a composition comprising an effective amount of at least one isolated mammalian anti-amyloid antibody of the present invention to the cell, tissue, organ or animal. The method may optionally further comprise administering an effective amount of 0.001-50 mg/kg of cells, tissue, organ or animal per 1-24 hours, 1-7 days, 1-52 weeks, 1-24 months, 1-30 years (or any range or value therein). The method may optionally further comprise contacting or administering by at least one means selected from parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar (intraceleeblar), intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal. The method may optionally further comprise administering, prior to, simultaneously with, or after the contacting or administering, at least one composition comprising an effective amount of at least one compound or protein selected from at least one anti-infective drug, Cardiovascular (CV) system drug, Central Nervous System (CNS) drug, Autonomic Nervous System (ANS) drug, respiratory tract drug, Gastrointestinal (GI) tract drug, hormonal drug, drug for fluid or electrolyte balance, hematologic drug, anti-cancer drug, immunomodulatory drug, ocular, otic or nasal drug, topical drug, nutraceutical, and the like. The method may optionally further comprise applying at least one composition prior to, simultaneously with, or after (a) contacting or applying, comprising an effective amount of at least one compound or protein selected from at least one detectable label or reporter, a TNF antagonist, an antirheumatic, a muscle relaxant, an anesthetic, a non-steroidal anti-inflammatory drug (NTHE), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteroid, a anabolic steroid, an erythropoietin, an immunological agent, an immunoglobulin, an immunosuppressant, a growth hormone, a hormone replacement drug, a radioprotective drug, an antidepressant, an antipsychotic, an stimulant, an asthma drug, a beta agonist, an inhaled steroid, an epinephrine or analog, a cytokine, or a cytokine antagonist.

Also provided is a medical device containing at least one isolated mammalian anti-amyloid antibody of the invention, wherein the device is adapted to contact or administer the at least one anti-amyloid antibody by at least one means selected from parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavity, intracavitary, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.

Also provided is an article of manufacture for human pharmaceutical or diagnostic use comprising packaging material and a container comprising a solution or lyophilized form of at least one isolated mammalian anti-amyloid antibody of the invention. The article of manufacture may optionally comprise a container that is part of a parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery device or system.

Also provided is a method of producing at least one isolated mammalian anti-amyloid antibody of the invention, the method comprising providing a host cell or a transgenic animal or a transgenic plant or a plant cell capable of expressing a recoverable amount of the antibody. The present invention also provides at least one anti-amyloid antibody produced by the above method.

The invention also provides any of the inventions described herein.

Drawings

FIGS. 1-41 show the heavy/light chain variable/constant region proto sequence, framework/subdomain and substitutions. For each region, multiple sequence alignments of known sequences were performed and prototype sequences were generated. The framework, CDR and hinge regions are labeled with boxes. Prototype sequence residues are numbered for each amino acid position. A list of amino acid substitutions or gaps (indicated by "-") was observed at each prototype position in the aligned sequences and the number of times they occurred is shown below each prototype sequence residue.

FIG. 1 depicts the Vh1 heavy chain variable region proto-sequence, framework and substitutions.

FIG. 2 depicts the Vh2 heavy chain variable region proto sequence, framework and substitutions.

Fig. 3 depicts Vh3a heavy chain variable region prototype sequence, framework and substitutions.

Fig. 4 depicts Vh3b heavy chain variable region prototype sequence, framework and substitutions.

Fig. 5 depicts Vh3c heavy chain variable region prototype sequence, framework and substitutions.

Fig. 6 depicts the Vh4 heavy chain variable region prototype sequence, framework and substitutions.

Fig. 7 depicts the Vh5 heavy chain variable region prototype sequence, framework and substitutions.

Fig. 8 depicts the Vh6 heavy chain variable region prototype sequence, framework and substitutions.

Fig. 9 depicts the Vh7 heavy chain variable region prototype sequence, framework and substitutions.

FIG. 10 depicts the kappa 1-4 light chain variable region proto-sequence, framework and substitutions.

FIG. 11 depicts the kappa 2 light chain variable region prototypic sequence, framework and substitutions.

FIG. 12 depicts the kappa 3 light chain variable region prototypic sequence, framework and substitutions.

FIG. 13 depicts the kappa 5 light chain variable region proto sequence, framework and substitutions.

FIG. 14 depicts the kappa New1 light chain variable region prototype sequence, framework and substitutions.

Fig. 15 depicts the kappa New2 light chain variable region prototype sequence, framework and substitutions.

FIG. 16 depicts the lambda 1a light chain variable region proto sequence, framework and substitutions.

FIG. 17 depicts the lambda 1b light chain variable region proto sequence, framework and substitutions.

Fig. 18 depicts the λ 2 light chain variable region proto sequence, framework and substitutions.

FIG. 19 depicts the lambda 3a light chain variable region proto sequence, framework and substitutions.

Fig. 20 depicts the λ 3b light chain variable region proto sequence, framework and substitutions.

Fig. 21 depicts the λ 3c light chain variable region proto sequence, framework and substitutions.

Fig. 22 depicts the λ 3e light chain variable region proto sequence, framework and substitutions.

Fig. 23 depicts the λ 4a light chain variable region proto sequence, framework and substitutions.

Fig. 24 depicts the λ 4b light chain variable region proto sequence, framework and substitutions.

Fig. 25 depicts the λ 5 light chain variable region proto sequence, framework and substitutions.

Fig. 26 depicts the λ 6 light chain variable region proto sequence, framework and substitutions.

Fig. 27 depicts the λ 7 light chain variable region proto sequence, framework and substitutions.

Fig. 28 depicts the λ 8 light chain variable region proto sequence, framework and substitutions.

Fig. 29 depicts the λ 9 light chain variable region proto sequence, framework and substitutions.

Fig. 30 depicts the lambda 10 light chain variable region proto sequence, framework and substitutions.

FIG. 31 depicts the IgA1 heavy chain constant region prototype sequence, subdomain, and substitution.

FIG. 32 depicts the IgA2 heavy chain constant region prototype sequence, subdomain, and substitution.

FIG. 33 depicts IgD heavy chain constant region proto-sequence, subdomain and substitutions.

Figure 34 depicts IgE heavy chain constant region prototype sequence, subdomain and substitutions.

FIG. 35 depicts IgG1 heavy chain constant region proto sequence, subdomain, and substitution.

FIG. 36 depicts IgG2 heavy chain constant region proto sequence, subdomain, and substitution.

FIG. 37 depicts IgG3 heavy chain constant region proto sequence, subdomain, and substitution.

FIG. 38 depicts IgG4 heavy chain constant region proto sequence, subdomain, and substitution.

FIG. 39 depicts IgM heavy chain constant region proto sequence, subdomain and substitutions.

FIG. 40 depicts Ig kappa c light chain constant region prototype sequences and substitutions.

Figure 41 depicts Ig λ c light chain constant region prototype sequences and substitutions.

Fig. 42 shows the spotted membrane probed with C701 mAb. The human a β sequence was scanned with peptides offset by one amino acid. Sequence of the region:

1.AEFRHDSGYEVH(SEQ ID NO：83)；

2.EFRHDSGYEVHH(SEQ ID NO：84)；

3.IIGLMVGGVVIA(SEQ ID NO：85)；

4.IGLMVGGVVIA(SEQ ID NO：86)；

5.IGLMVGGVVI(SEQ ID NO：87)；

6.IGLMVGGVV(SEQ ID NO：88)；

7.IGLMVGGV(SEQ ID NO：89)；

8.IGLMVGG(SEQ ID NO：90)；

9.LMVGGV(SEQ ID NO：91).

figure 43 shows the membrane of spots detected by C705 mAb. The human a β sequence was scanned with peptides offset by one amino acid. Sequence of the region:

1.DAEFRHDSGYEVHHQ(SEQ ID NO：92)；

2.AEFRHDSGYEVHHQ(SEQ ID NO：93)；

3.DAEFRHDSGYEVH(SEQ ID NO：94)；

4.EFRHDSGYEVHH(SEQ ID NO：95)；

5.EFRHDSGYEVH(SEQ ID NO：96)；

6.DAEFRHDSGY(SEQ ID NO：97)；

7.DAEFRHDSG(SEQ ID NO：98)；

8.AEFRHDSG(SEQ ID NO：99)；

9.EFRHDSG(SEQ ID NO：100)；

10.EFRHDS(SEQ ID NO：101).

fig. 44 shows the spotted membrane probed by C706 mAb. The human a β sequence was scanned with peptides offset by one amino acid. Sequence of the region:

1.DAEFRHDSGYEVHHQ(SEQ ID NO：102)；

2.AEFRHDSGYEVHHQ(SEQ ID NO：103)；

3.AEFRHDSGYEVHH(SEQ ID NO：104)；

4.AEFRHDSGYEVH(SEQ ID NO：105)；

5.DAEFRHDSGYE(SEQ ID NO：106)；

6.AEFRHDSGYE(SEQ ID NO：107)；

7.DAEFRHDSG(SEQ ID NO：108)；

8.AEFRHDSG(SEQ ID NO：109)；

9.DAEFRHDSG(SEQ ID NO：110)；

10.AEFRHD(SEQ ID NO：111).

figure 45 shows the spotted membrane probed with C707 mAb. The human a β sequence was scanned with peptides offset by one amino acid. Sequence of the region:

1.EFRHDSGYEVHHQKL(SEQ ID NO：112)；

2.FRHDSGYEVHHQKL(SEQ ID NO：113)；

3.EVHHQKLVFFAEDV(SEQ ID NO：114)；

4.YEVHHQKLVFFA(SEQ ID NO：115)；

5.VFFAEDVGSNKGA(SEQ ID NO：116)；

6.KLVFFAEDVGSN(SEQ ID NO：117)；

7.EDVGSNKGAIIG(SEQ ID NO：118)；

8.FFAEDVGSNKG(SEQ ID NO：119)；

9.SNKGAIIGLMV(SEQ ID NO：120)；

10.FAEDVGSNKG(SEQ ID NO：121)；

11.KGAIIGLMVG(SEQ ID NO：122)；

12.LVFFAEDVG(SEQ ID NO：123)；

13.EDVGSNKGA(SEQ ID NO：124)；

14.KLVFFAED(SEQ ID NO：125)；

15.DVGSNKGA(SEQ ID NO：126)；

16.EVHHQKL(SEQ ID NO：127)；

17.QKLVFFA(SEQ ID NO：128)；

18.RHDSGY(SEQ ID NO：129)；

19.SGYEVH(SEQ ID NO：130)；

20.GVVIAT(SEQ ID NO：131).

FIG. 46 shows A β binding to anti-A β mAb C705₃₈。

FIG. 47A shows the use of A β₃₈The binding quantification map of (a) against the ordering of the-a β mAbs.

FIG. 47B shows the use of A β ₄₂The binding quantification map of (a) against the ordering of the-a β mAbs.

FIG. 48 shows anti-A.beta.mAbs against A.beta.in PC12 cells₄₂Effects of induced toxicity.

Detailed Description

The present invention provides isolated, recombinant and/or synthetic anti-amyloid human, primate, rodent, mammalian, chimeric, humanized or CDR-grafted antibodies and amyloid anti-idiotype antibodies, as well as compositions and encoding nucleic acid molecules comprising at least one polynucleotide encoding at least one anti-amyloid antibody or anti-idiotype antibody. The invention also includes, but is not limited to, methods and devices for making and using such nucleic acids and antibodies and anti-idiotype antibodies, including diagnostic and therapeutic compositions.

As used herein, "anti-beta-amyloid antibody", "anti-amyloid antibody portion", or "anti-amyloid antibody fragment", and/or "anti-amyloid antibody variant", and the like, include any protein or peptide comprising a molecule comprising at least a portion of an immunoglobulin molecule, such as, but not limited to, a Complementarity Determining Region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework region, or any portion thereof, or at least a portion of an amyloid receptor or binding protein, which may be incorporated into an antibody of the invention. Such antibodies optionally also affect specific ligands, such as but not limited to wherein such antibodies modulate, decrease, increase, antagonize, agonize, moderate, mitigate, block, inhibit, abrogate, and/or interfere with at least one amyloid activity or binding, or amyloid receptor activity or binding, in vitro, in situ, and/or in vivo. By way of non-limiting example, a suitable anti-amyloid antibody, specified portion or variant of the present invention may bind to at least one amyloid protein, or a specified portion, variant or domain thereof. Suitable anti-amyloid antibodies, specified portions or variants may also optionally affect at least one amyloid activity or function, such as, but not limited to, RNA, DNA or protein synthesis, amyloid release, amyloid receptor signaling, membrane amyloid cleavage, amyloid activity, amyloid production and/or synthesis.

An antibody may comprise one or more of at least one CDR, at least one variable region, at least one constant region, at least one heavy chain (e.g., γ 1, γ 2, γ 3, γ 4, μ, α 1, α 2, δ, ε), at least one light chain (e.g., κ and λ), or any portion or fragment thereof, and may further contain interchain and intrachain disulfide bonds, a hinge region, glycosylation sites that may be separated by a hinge region, and a heavy chain and a light chain. Light chains typically have a molecular weight of about 25Kd, and heavy chains typically range from 50K to 77 Kd. Light chains can be in two different forms or isotypes: kappa (. kappa.) and lambda (. lamda.) exist, which can be combined with either heavy chain type. All light chains have at least one variable region and at least one constant region. IgG antibodies are considered to be a common antibody structure and have two intrachain disulfide bonds in the light chain (one in the variable region and one in the constant region) and 4 intrachain disulfide bonds in the heavy chain, and these bonds surround a peptide loop of about 60-70 amino acids, which constitutes a "domain" of about 110 amino acids in the chain. IgG antibodies can be divided into four classes: IgG1, IgG2, IgG3, and IgG 4. Each immunoglobulin class has a different function. The following table summarizes reported examples of physicochemical properties for each immunoglobulin class and subclass.

Properties of	IgG1	IgG2	IgG3	IgG4	IgM	IgA1	IgA2	SIgA	IgD	IgE
											Heavy chain	γ1	γ1	γ1	γ1	μ	α1	α2	α1/α2	δ	E
Mean serum concentration (mg/ml)	9	3	1	0.5	1.5	3.0	0.5	0.05	0.03	0.00005
											Sedimentation constant	7s	7s	7s	7s	19s	7s	7s	11s	7s	8s
Molecular weight (X10)3)	146	146	170	146	970	160	160	385	184	188
											Half-life period (Tian)	5-30	5-30	2-10	5-30	5-15	2-10	2-10	1-10	1-10	1-10
% intravascular distribution	45	45	45	45	80	42	42	Trace amount of	75	50
											Sugar (%)	2-3	2-3	2-3	2-3	12	7-11	7-11	7-11	9-14	12

The following table summarizes non-limiting examples of antibody effector functions for human antibody classes and subclasses.

Effector function	IgG1	IgG2	IgG3	IgG4	IgM	IgA	IgD	IgE
									Complement fixation	++	+	+++	-	+++	-	-	-
Placental transfer	+	+	+	+	-	-	-	-
									Binding to Staph A	+++	+++		+++	-	-	-	-
Binding to Strep G	+++	+++	+++	+++	-	-	-	-

Thus, the type of antibody or fragment thereof used in the present invention may be selected based on the characteristics and functions desired for a particular therapeutic or diagnostic use, such as, but not limited to, serum half-life, intravascular distribution, complement fixation, and the like.

Antibody diversity is generated by at least 5 mechanisms, including (1) the use of multiple genes encoding antibody portions; (2) somatic mutations, e.g., mutations in the original V gene during B-cell ontogeny to produce different V genes in different B-cell clones; (3) somatic recombination, e.g., recombination of gene segments J1-Jn during B-cell ontogeny to join the major portions of V-region genes; (4) gene conversion, in which fragments of DNA from many pseudo-V regions can be copied into a V region to alter the DNA sequence; and (5) nucleotide additions, e.g., where the V and J regions are cleaved prior to ligation, additional nucleotides may be inserted to encode additional amino acids. Non-limiting examples include, but are not limited to, (i) selection/recombination of germline-derived vk, J and ck regions into B cell clones to produce kappa chains; (ii) selection/recombination of V λ, J and C λ regions from the germ line into B cell clones to generate λ chains; (iii) v _HD1-D30 and J_H1-J_H6 to form a functional VDJ gene encoding the heavy chain variable region. The above mechanisms act in a synergistic manner to generate antibody diversity and specificity.

The term "antibody" is also intended to include antibodies, digested fragments, specified portions or variants thereof, including antibody mimetics or antibody portions that comprise structures and/or functions that mimic antibodies or specified fragments or portions thereof, including single chain antibodies and fragments thereof. Functional fragments include antigens that bind mammalian amyloidBinding the fragments. For example, the invention includes antibody fragments capable of binding amyloid protein or portions thereof, including, but not limited to, Fab (e.g., by papain digestion), Fab '(e.g., by pepsin digestion and partial reduction), and F (ab')₂(e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction, and reaggregation), Fv, or scFv (e.g., by molecular biology techniques) fragments (see, e.g., Colligan, Immunology, supra).

Such fragments may be generated by enzymatic cleavage, synthesis or recombinant techniques well known in the art and/or described herein. Various truncated forms of antibodies can also be produced using antibody genes in which one or more stop codons are introduced upstream of the natural termination site. For example, the code F (ab') ₂The combined genes for the heavy chain portion were designed to include the CH encoding the heavy chain₁DNA sequence of a domain and/or hinge region. The various portions of the antibody may be chemically linked by conventional techniques or may be prepared as contiguous proteins using genetic engineering techniques.

As used herein, the term "human antibody" refers to an antibody in which substantially every portion of the protein (e.g., CDR, framework, C)_L、C_HDomains (e.g., C)_H1、C_H2、C_H3) Hinge, hinge (V)_L、V_H) Substantially non-immunogenic in humans, with only minor sequence changes or alterations. Similarly, antibodies designated primates (monkeys, baboons, chimpanzees, etc.), rodents (mice, rats, rabbits, guinea pigs, hamsters, etc.), and other mammals are such species, subgenera, genus, subfamily, family-specific antibodies. Furthermore, the chimeric antibodies of the invention may include any combination of the above. These alterations or variations optionally and preferably maintain or reduce immunogenicity in humans or other species relative to unmodified antibodies. Thus, a human antibody is different from a chimeric or humanized antibody. Has already been pointed out byHuman antibodies can be produced by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and/or light chain) genes. Further, when the human antibody is a single chain antibody, it may include a linker peptide not found in a natural human antibody. For example, the Fv may comprise a linker peptide, such as two or about 8 glycine or other amino acid residues, that links the heavy chain variable region and the light chain variable region. Such linker peptides are believed to be of human origin.

Bispecific, xenospecific, heteroconjugates or similar antibodies, which are monoclonal, preferably human or humanized antibodies having binding specificity for at least two different antigens, may also be used. In the present case, one binding specificity is for at least one amyloid protein and the other is for any other antigen. Methods for making bispecific antibodies are well known in the art. Typically, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature 305: 537 (1983)). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule is usually performed by an affinity chromatography step, which is rather laborious and the product yield is low. In e.g. WO 93/08829, us patent nos. 6210668, 6193967, 6132992, 6106833, 6060285, 6037453, 6010902, 5989530, 5959084, 5959083, 5932448, 5833985, 5821333, 5807706, 5643759, 5601819, 5582996, 5496549, 4676980, WO 91/00360, WO 92/00373, EP 03089, Traunecker et al, EMBO j.10: 3655(1991), Suresh et al, Methods in Enzymology 121: similar methods are disclosed in 210(1986), which are incorporated herein by reference in their entirety.

The anti-amyloid antibodies (also referred to as amyloid antibodies) used in the methods and compositions of the invention may optionally be characterized by high affinity for binding amyloid and optionally and preferably have low toxicity. In particular, the invention may employ antibodies, specific fragments or variants of the invention wherein the individual components, such as the variable regions, constant regions and frameworks, individually and/or collectively optionally and preferably have low immunogenicity. Antibodies that may be used in the present invention are optionally characterized in that they are capable of long-term treatment of patients, they may measurably alleviate symptoms and have low toxicity and/or acceptable toxicity. Low or acceptable immunogenicity and/or high affinity, as well as other suitable characteristics, may aid in achieving a therapeutic result. "Low immunogenicity" is defined herein as producing a significant HAHA, HACA or HAMA response in less than about 75%, preferably less than about 50%, of the treated patients, and/or causing low titers (less than about 300, preferably less than about 100 as measured by the dual antigen enzyme immunoassay) in the treated patients (Elliott et al, Lancet 344: 1125-Asn 1127(1994), incorporated herein by reference in its entirety).

Practicality of use

The isolated nucleic acids of the present invention may be used to generate at least one anti-amyloid antibody or specific variant thereof, which may be used to measure or effect in cells, tissues, organs or animals (including mammals and humans) to diagnose, monitor, modulate, treat, ameliorate, help prevent the occurrence of, or alleviate symptoms of, at least one amyloid condition selected from, but not limited to, at least one of an immunological disorder or disease, a cardiovascular disorder or disease, an infectious, malignant, and/or neurological disorder or disease, or other known or specific amyloid-related conditions.

Such methods may comprise administering to a cell, tissue, organ, animal or patient in need of such modulation, treatment, alleviation, prevention or reduction in a symptom, effect or mechanism of such modulation, treatment, alleviation, prevention or reduction an effective amount of a composition or pharmaceutical composition comprising at least one anti-amyloid antibody. Such effective amounts may include about 0.001 to 500mg/kg per single (e.g., rapid infusion), multiple or continuous administration, or to achieve a serum concentration of 0.01 to 5000 μ g/ml per single, multiple or continuous administration, or any effective range or value therein, which may be performed and determined by methods well known in the art described herein or otherwise.

Reference to

All publications or patents cited herein are incorporated herein by reference in their entirety as they show the state of the art to which the invention pertains and/or to provide a description and enablement of the present invention. Publication refers to any scientific or patent publication or any other information available in any medium, including all recorded, electronic or printed forms. The following references are incorporated herein by reference in their entirety: ausubel, et al, eds., Current protocols Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); sambrook, et al, Molecular Cloning: a Laboratory Manual, 2nd edition, Cold Spring Harbor, NY (1989); harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); colligan, et al, eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); colligan et al, Current Protocols in protein science, John Wiley & Sons, NY, NY, (1997-2001).

Antibodies of the invention

As is well known in the art, at least one anti-amyloid antibody of the invention may optionally be produced by a cell line, a mixed cell line, an immortalized cell or a clonal population of immortalized cells. See, for example, Ausubel, et al, Current protocols Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); sambrook, et al, Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor, NY (1989); harlow and Lane, antibodies, aLaboratory Manual, Cold Spring Harbor, NY (1989); colligan, et al, Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); colligan et al, Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), each of which is incorporated herein in its entirety by reference.

Human antibodies specific for human amyloid protein or fragments thereof may be generated against suitable immunogenic antigens, such as isolated and/or amyloid protein or portions thereof (including synthetic molecules such as synthetic peptides), for example, but not limited to, the amino acid sequence of SEQ ID NO: 50, 1-7, 1-40, 31-42, and 36-40. Other specific or general mammalian antibodies can be similarly generated. The preparation of immunogenic antigens and the production of monoclonal antibodies can be carried out using any suitable technique.

In one method, a suitable immortalized cell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2 SA3, 2 MAI, Sp2 SS1, Sp2 SA5, U937, MLA144, ACTIV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH3T3, HL-60, MLA144, NAIWMAA, NEURO2A, etc., or a xenogenous myeloma (heteromyelomas), fusion products thereof, or any cell or fusion cell derived therefrom, or any other suitable cell line known in the art (see, e.g., www.atcc.org, www.lifetech.com, etc.) is fused with an antibody, or a cell such as a hybridoma, such as a cell, a hybridoma, a cell, Or other immune or B cell-containing cell, or any other cell that expresses heavy or light chain constant or variable or framework or CDR sequences as endogenous or heterologous nucleic acid, recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptile, fish, mammalian, rodent, equine, ovine, caprine, ovine, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triple stranded hybrid, and the like, or any combination thereof. See, e.g., Ausubel, supra, and Colligan, Immunology, supra, Chapter II, which are incorporated herein by reference in their entirety.

Antibody-producing cells can also be obtained from peripheral blood or, preferably, spleen or lymph node of a human or other suitable animal immunized with the antigen of interest. Any other suitable host cell may also be used to express heterologous or endogenous nucleic acids encoding the antibodies, specific fragments or variants thereof of the present invention. Fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable well-known methods and cloned by limiting dilution or cell sorting, or other well-known methods. Cells producing antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).

Other suitable methods for generating or isolating antibodies of the desired specificity may be used, including, but not limited to, methods for selecting recombinant antibodies from peptide or protein libraries (e.g., but not limited to, phage, ribosome, oligonucleotide, RNA, cDNA, etc. display libraries; e.g., from Cambridge antibody Technologies, Cambridge shire, UK; Morphosys, Martinsreid/Planegg, DE; Biofoil, Aberdeen, Scotland, UK; BioInvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite; Xoma, Berkeley, CA; Ixsys; see, for example, EP 368, 684, PCT/GB 91/01134; PCT/GB 92/01755; PCT/GB 92/002240; PCT/GB 92/00883; PCT/GB 93/00605; PCT/WO 14442/014/3646; PCT/364642/WO 14442; PCT/36 90; PCT/WO 14438/36 90; PCT/369636; PCT/369635; PCT/366338/365636; PCT/; PCT/36 90; PCT/365636; PCT/; US 94/1234; WO 92/18619; WO 96/07754; (Scripps); WO96/13583, WO97/08320 (Morphosys); WO95/16027 (BioInvent); WO 88/06630; WO90/3809 (Dyax); US4,704,692 (Enzon); PCT/US91/02989 (Affymax); WO 89/06283; EP 371998; EP 550400; (Xoma); EP 229046; PCT/US91/07149 (Ixsys); or randomly generated peptides or proteins-US 5723323, 5763192, 5814476, 5817483, 5824514, 5976862, WO 86/05803, EP 590689(Ixsys, now Applied Molecular Evolution (AME), each of which is incorporated herein by reference in its entirety) or those that rely on immunization of transgenic animals (e.g., SCID mice, Nguyen et al, Microbiol. Immunol.41: 901-907 (1997); Sandhu et al, Crit. Rev. Biotechnol.16: 95-118 (1996); en et al, Immunol.93: 154-161(1998), each and related patents and applications are incorporated herein by reference in their entirety)), which are capable of generating human antibody profiles, as is well known in the art and/or described herein. These include, but are not limited to, ribosome display (Hanes et al, Proc. Natl. Acad. Sci. USA, 94: 4937-4942(May 1997); Hanes et al, Proc. Natl. Acad. Sci. USA, 95; 14130-14135(Nov 1998)); single cell antibody production techniques (e.g., the selected lymphocyte antibody method ("SLAM") (U.S. Pat. No. 5,627,052, Wen et al, J.Immunol.17: 887-892 (1987); Babcook et al, Proc.Natl.Acad.Sci.USA 93: 7843-7848 (1996)); gel microdroplet and flow cytometry (Powell et al, Biotechnol.8: 333-337 (1990); single cell system, Cambridge, MA; Gray et al, J.Imm.182: 155-163 (1995); Kenny et al, Bio/technology.13: 787-790(1995)), B cell selection (Steenbakers et al, mol. Biol.19: 125-134 (Jojobak et al, Proware, Biotech, Brocock 5, In, publication V.1988)).

Methods of engineering or humanizing non-human or human antibodies can also be used and are well known in the art. Typically, a humanized or engineered antibody has one or more amino acid residues from a non-human source, such as, but not limited to, a mouse, rat, rabbit, non-human primate, or other mammal. These human amino acid residues are often referred to as "import" residues, which are typically derived from an "import" variable, constant or other domain of a known human sequence.

Methods of engineering or humanizing non-human or human antibodies can also be used and are well known in the art. Typically, a humanized or engineered antibody has one or more amino acid residues from a non-human source, such as, but not limited to, a mouse, rat, rabbit, non-human primate, or other mammal. These human amino acid residues are often referred to as "import" residues, which are typically derived from an "import" variable, constant or other domain of a known human sequence.

"humanized antibody" refers to an antibody consisting in part or in whole of an amino acid sequence derived from the germline of a human antibody by altering the sequence of the antibody with non-human Complementarity Determining Regions (CDRs). The simplest such change may consist simply of a constant region of a human antibody replacing a murine constant region, resulting in a human/murine chimera that may be sufficiently low immunogenic to be acceptable for pharmaceutical use.

Preferably, however, the variable regions and even the CDRs of the antibody are also humanized by techniques now well known in the art. The framework regions of the variable regions are replaced with the corresponding human framework regions, leaving the non-human CDRs substantially intact, or even replacing the CDRs with sequences derived from the human genome. Fully human antibodies are produced in genetically modified mice whose immune system has been altered to correspond to the human immune system. As mentioned above, it is sufficient to use immunologically specific fragments of the antibodies, including fragments representing single chain forms, in the methods of the invention.

Humanized antibodies again refer to antibodies that contain a human framework, from non-human antibodies of at least one CDR, and the presence of any constant region basically with human immunoglobulin constant region, i.e. at least about 85-90%, preferably at least 95% of the same. Thus, all parts of the humanized antibody are substantially identical to the corresponding parts of one or more natural human immunoglobulin sequences, except for the possible CDRs. For example, a humanized immunoglobulin will typically not include chimeric mouse variable region/human constant region antibodies.

In human therapeutic applications, humanized antibodies have at least three possible advantages over non-human and chimeric antibodies:

1) Because the effector moiety is human, it can interact better with other parts of the human immune system (e.g., more efficiently destroying the target cell through complement-dependent cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC)).

2) The human immune system should not recognize the framework or C regions of the humanized antibody as foreign, and thus the antibody response to such injected antibody should be less than the antibody response to a fully foreign non-human antibody or a partially foreign chimeric antibody.

3) Injected non-human antibodies have been reported to have a shorter half-life in the human circulation than human antibodies. The injected humanized antibody will have substantially the same half-life as the naturally occurring human antibody, thereby allowing smaller and less frequent doses to be administered.

Well known human Ig sequences are disclosed, e.g.,

www.ncbi.nlm.nih.gov/entrez/query.fcgi；www.atcc.org/phage/hdb.html；www.sciquest.com/；

www.abcam.com/；www.antibodyresource.com/onlinecomp.html；

www.public.iastate.edu/～pedro/research_tools.html；www.mgen.uni-

heidelberg.de/SD/IT/IT.html；www.whfreeman.com/immunology/CH05/kuby05.htm；

www.library.thinkquest.org/12429/Immune/Antibody.html；

www.hhmi.org/grants/lectures/1996/vlab/；www.path.cam.ac.uk/～mrc7/mikeimages.html；

www.antibodyresource.com/；

mcb.harvard.edu/BioLinks/Immunology.html.www.immunologylink.com/；

pathbox.wustl.edu/～hcenter/index.html；www.biotech.ufl.edu/～hcl/；

www.pebio.com/pa/340913/340913.html；www.nal.usda.gov/awic/pubs/antibody/；

www.m.ehime-u.ac.jp/～yasuhito/Elisa.html；www.biodesign.com/table.asp；

www.icnet.uk/axp/facs/davies/links.html；www.biotech.ufl.edu/～fccl/protocol.html；www.isac-

net.org/sites_geo.html；aximtl.imt.uni-marburg.de/～rek/AEPStart.html；

baserv.uci.kun.nl/～jraats/linksl.html；www.recab.uni-hd.de/immuno.bme.nwu.edu/；www.mrc-

cpe.cam.ac.uk/imt-doc/public/INTRO.html；www.ibt.unam.mx/vir/V_mice.html；

imgt.cnusc.fr：8104/；www.biochem.ucl.ac.uk/～martin/abs/index.html；antibody.bath.ac.uk/；

abgen.cvm.tamu.edu/lab/wwwabgen.html；

www.unizh.ch/～honegger/AHOseminar/Slide01.html；www.cryst.bbk.ac.uk/～ubcg07s/；

www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm；

www.path.cam.ac.uk/～mrc7/humanisation/TAHHP.html；

www.ibt.unam.mx/vir/structure/stat_aim.html；www.biosci.missouri.edu/smithgp/index.html；

www.cryst.bioc.cam.ac.uk/～fmolina/Web-pages/Pept/spottech.html；

www.jerini.de/fr_products.htm；www.patents.ibm.com/ibm.html.

kabat et al, Sequences of Proteins of Immunological Interest, U.S. dept.health (1983), which are incorporated herein by reference in their entirety.

These imported sequences may be used to reduce immunogenicity or to reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic known in the art. Typically, some or all of the non-human or human CDR sequences are maintained, while the non-human sequences of the variable and constant regions are replaced with human or other amino acids. The antibodies may also optionally be humanized and retain high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies are optionally prepared by methods of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art. Computer programs are available that elucidate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays may allow analysis of the likely role of the residues in the functional role of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen. Generally, CDR residues are directly and most fully involved in affecting antigen binding. Humanization or engineering of the antibodies of the invention can be performed using any known method, such as, but not limited to, the following: winter (Jones et al, Nature 321: 522 (1986); Riechmann et al, Nature 332: 323 (1988); Verhoeyen et al, Science 239: 1534(1988)), Sims et al, J.Immunol.151: 2296 (1993); chothia and Lesk, j.mol.biol.196: 901(1987), Carter et al, proc.natl.acad.sci.u.s.a.89: 4285 (1992); presta et al, j.immunol.151: 2623(1993), U.S. patent nos. 5723323, 5976862, 5824514, 5817483, 5814476, 5763192, 5723323, 5766886, 5714352, 6204023, 6180370, 5693762, 5530101, 5585089, 5225539, 4816567, PCT/: US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755, WO90/14443, WO90/14424, WO90/14430, EP229246, each of which, including the references cited therein, is hereby incorporated by reference in its entirety.

Anti-amyloid antibodies can also be generated by immunizing transgenic animals (e.g., mice, rats, hamsters, non-human primates, etc.) capable of producing a human antibody profile, as described herein and/or as is well known in the art. Cells producing human anti-amyloid antibodies can be isolated from these animals and immortalized using suitable methods, such as those described herein.

Transgenic mice producing human antibody repertoires that bind human antigens can be generated by well-known methods (e.g., but not limited to, U.S. Pat. Nos. 5,770,428, 5,569,825, 5,545,806, 5,625,126, 5,625,825, 5,633,425, 5,661,016 and 5,789,650 to Lonberg et al, WO98/50433, Jakobovits et al, WO98/24893, Lonberg et al, WO98/24884, Lonberg et al, WO97/13852, Lonberg et al, WO 94/25585, Kucherlapate et al, WO 96/34096, Kucherlapate et al, EP 0463151B 1, Kucherlapate et al, EP 0710719A 1, Surani et al, U.S. Pat. No. 5,545,807, Bruggemann et al, WO 90/36, Brugmann et al, Kuugmann et al, Kucherlabe et al, EP 040579, Nature et al, (Lonberg) 1994,368,368,368,368,368,102, taylor et al, Nucleic Acids Research 20 (23): 6287-: 65-93(1995) and Fishwald et al, Nat Biotechnol 14 (7): 845, 851(1996), each of which is incorporated herein in its entirety by reference). Typically, these mice contain at least one transgene that contains DNA from at least one human immunoglobulin locus that is functionally rearranged or can undergo functional rearrangement. Endogenous immunoglobulin loci in such mice can be disrupted or deleted to eliminate the ability of the animal to produce antibodies encoded by the endogenous genes.

Screening for antibodies that specifically bind to similar proteins or fragments can be conveniently accomplished using peptide display libraries. The method includes screening a large number of peptides for individual members having a desired function or structure. Antibody screening of peptide display libraries is well known in the art. The displayed peptide sequences may be 3 to 5000 or more amino acids in length, typically 5-100 amino acids in length, and typically about 8 to 25 amino acids in length. In addition to direct chemical synthesis methods to generate peptide libraries, several recombinant DNA methods have been described. One type involves the display of peptide sequences on the surface of a phage or cell. Each bacteriophage or cell contains a nucleotide sequence encoding a particular displayed peptide sequence. Such methods are disclosed in PCT patent publication nos. 91/17271, 91/18980, 91/19818 and 93/08278. Other systems for generating peptide libraries have aspects of both in vitro chemical synthesis and recombinant methods. See PCT patent publication nos. 92/05258, 92/14843, and 96/19256. See also U.S. patent nos. 5,658,754; and 5,643,768. Peptide display libraries, vectors and screening kits are commercially available from sources such as Invitrogen (Carlsbad, Calif.), and Cambridge antibody Technologies (Cambridge, UK). See, for example, U.S. patent nos. 4704692, 4939666, 4946778, 5260203, 5455030, 5518889, 5534621, 5656730, 5763733, 5767260, 5856456, assigned to Enzon; 5223409, 5403484, 5571698, 5837500 assigned to Dyax; 5427908, 5580717 assigned to Affymax; 5885793 assigned to Cambridge anti-codey technologies; 5750373 assigned to Genentech; 5618920, 5595898, 5576195, 5698435, 5693493, 5698417 assigned to Xoma; colligan, as above; ausubel, supra; or Sambrook, as above, each of the above patents and publications, is incorporated herein by reference in its entirety.

The antibodies of the invention may also be prepared using at least one nucleic acid encoding an anti-amyloid antibody to provide transgenic animals or mammals, such as goats, cows, horses, sheep and the like, capable of producing the antibody in their milk. Such animals may be provided by well-known methods. See, for example, but not limited to, U.S. patent nos. 5,827,690, 5,849,992, 4,873,316, 5,849,992, 5,994,616, 5,565,362, 5,304,489, etc., each of which is incorporated herein by reference in its entirety.

The antibodies of the invention may also be prepared using at least one nucleic acid encoding an anti-amyloid antibody to provide transgenic plants and cultured plant cells (such as, but not limited to, tobacco and corn) that produce the antibody, specified portions thereof, or variants thereof in plant parts thereof or cultured cells thereof. As a non-limiting example, transgenic tobacco leaves expressing recombinant proteins have been successfully used to provide large quantities of recombinant proteins, for example, using inducible promoters. See, e.g., Cramer et al, curr. top. microbol. immunol.240: 95-118(1999) and the references cited therein. Transgenic maize can also be used to express mammalian proteins at commercial production levels with biological activities equivalent to those of proteins produced in other recombinant systems or purified from natural sources. See, e.g., Hood et al, adv.exp.med.biol.464: 127-147(1999) and the references cited therein. Antibodies, including antibody fragments such as single chain antibodies (scFv), have been produced in large quantities from transgenic plant seeds, including tobacco seeds and potato tubers. See, for example, Conrad et al, Plant mol. biol. 38: 101-109(1998) and references cited therein. Thus, the antibodies of the invention can also be produced using transgenic plants according to well-known methods. See also, for example, Fischer et al, biotechnol.appl.biochem.30: 99-108(Oct., 1999), Ma et al, Trends Biotechnol.13: 522-7 (1995); ma et al, Plant Physiol.109: 341-6 (1995); whitedam et al, biochem. soc. trans.22: 940-; and references cited therein. For plant expression of antibodies, see also, but not limited to, the above references are each incorporated by reference herein in their entirety.

The antibodies of the invention can be used with a wide range of affinities (K)_D) Binds human amyloid. In a preferred embodiment, at least one human mAb of the invention may optionally bind human amyloid with high affinity. For example, a human mAb can be equal to or less than about 10^-7M, such as, but not limited to, 0.1-9.9 (or any of them)Range or value) × 10^-7、10^-8、10^-9、10^-10、10^-11、10^-12、10^-13Or any range or value of K therein_DBinds human amyloid.

The affinity or avidity of an antibody for an antigen may be determined experimentally using any suitable method. (see, e.g., Berzofsky, et al, "Antibody-Antibody interactions," In Fundamental Immunology, Paul, W.E., eds., ravenPress: New York, NY (1984); Kuby, Janis Immunology, W.H.Freeman Company: New York, NY (1992); and methods described therein). The affinity of a particular antibody-antigen interaction measured may be different if measured under different conditions (e.g., salt concentration, pH). Thus, it is preferred to perform affinity and other antigen-binding parameters (e.g., K) with standard solutions of antibody and antigen, and standard buffers, such as those described herein _D、K_a、K_d) The measurement of (2).

Nucleic acid molecules

Using the information provided herein, e.g., encoding SEQ ID NOS: 42-49, 53-60, 63-70, 73-80, or a specific fragment, variant or consensus sequence thereof, or a deposited vector containing at least one of these sequences, the nucleic acid molecules of the invention encoding at least one anti-amyloid antibody may be obtained using methods described herein or known in the art.

The nucleic acid molecules of the invention may be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA produced by cloning or synthesis, or any combination thereof. The DNA may be triplex, double stranded or single stranded, or any combination thereof. A portion of at least one strand of DNA or RNA may be the coding strand, also referred to as the sense strand, or it may be the non-coding strand, also referred to as the antisense strand.

Isolated nucleic acid molecules of the invention may include nucleic acid molecules comprising an Open Reading Frame (ORF), optionally with one or more introns, such as, but not limited to, at least one specific portion of at least one CDR, such as CDR1, CDR2, and/or CDR3, of at least one heavy chain (e.g., SEQ ID NOS: 42-44, 53-55, 63-65, 73-75) or light chain (e.g., SEQ ID NOS: 45-47, 56-58, 66-68, 76-78); nucleic acid molecules comprising anti-amyloid antibodies or coding sequences for variable regions (e.g., SEQ ID NOS: 48, 49, 59, 60, 69, 70, 79 and 80), such as but not limited to SEQ ID NOS: 51. 52, 61, 62, 71, 72, 81 and 82; and nucleic acid molecules comprising nucleotide sequences that are substantially different from the above sequences, but which, due to the degeneracy of the genetic code, nevertheless encode at least one anti-amyloid protein as described herein and/or as known in the art. Of course, the genetic code is well known in the art. Thus, it is routine for the skilled person to generate these degenerate nucleic acid variants encoding a particular anti-amyloid antibody of the present invention. See, e.g., Ausubel et al, supra, and these nucleic acid variants are included in the present invention.

As indicated herein, the nucleic acid molecules of the invention containing nucleic acid encoding anti-amyloid antibodies may include, but are not limited to, the nucleic acid sequences themselves encoding antibody fragments; a coding sequence for a complete antibody or a portion thereof; coding sequences for antibodies, fragments or portions, and additional sequences, such as coding sequences for at least one signal leader sequence or fusion peptide, with or without additional coding sequences as previously described, such as at least one intron, and additional non-coding sequences, including, but not limited to, non-coding 5 'and 3' sequences, such as transcribed, non-translated sequences that function in transcription, mRNA processing, including splicing and polyadenylation signals (e.g., ribosome binding and mRNA stabilization); additional coding sequences that encode additional amino acids, e.g., amino acids that provide additional functionality. Thus, the antibody-encoding sequence may be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of a fused antibody containing an antibody fragment or portion.

Polynucleotides that selectively hybridize to polynucleotides described herein

The present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to the polynucleotides disclosed herein. Thus, the polynucleotides of this embodiment can be used to isolate, detect and/or quantify nucleic acids comprising these polynucleotides. For example, the polynucleotides of the invention can be used to identify, isolate or amplify partial or full length clones in a deposited library. In some embodiments, the polynucleotide is an isolated genomic or cDNA sequence, or is complementary to cDNA from a human or mammalian nucleic acid library.

Preferably, the cDNA library contains at least 80% of the full-length sequence, preferably at least 85% or 90% of the full-length sequence, more preferably at least 95% of the full-length sequence. The cDNA library can be normalized to increase the expression of rare sequences. Typically, but not exclusively, low or moderately stringent hybridization conditions are used for sequences having low sequence identity relative to the complementary sequence. Medium and high stringency conditions can optionally be used for sequences with greater identity. Low stringency conditions allow selective hybridization of sequences with about 70% sequence identity and can be used to identify orthologous and paralogous sequences.

Optionally, the polynucleotides of the invention will encode at least a portion of an antibody encoded by a polynucleotide described herein. The polynucleotides of the invention include nucleic acid sequences that can be used to selectively hybridize to polynucleotides encoding the antibodies of the invention. See, e.g., Ausubel, supra; colligan, as before, each of which is incorporated herein by reference in its entirety.

Construction of nucleic acids

The isolated nucleic acids of the invention can be prepared using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, or combinations thereof, which are well known in the art.

The nucleic acid may conveniently contain sequences other than the polynucleotide of the invention. For example, a multiple cloning site containing one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in the isolation of the polynucleotide. In addition, translatable sequences may be inserted to aid in the isolation of the translated polynucleotides of the invention. For example, a hexa-histidine tag sequence may provide a convenient route for purification of the proteins of the invention. The nucleic acids of the invention (excluding coding sequences) are optionally vectors, linkers, or linkers for cloning and/or expressing the polynucleotides of the invention.

Additional sequences may be added to these cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve introduction of the polynucleotide into a cell. The use of cloning vectors, expression vectors, linkers, and linkers is well known in the art (see, e.g., Ausubel, supra; or Sambrook, supra).

Recombinant method for constructing nucleic acids

The isolated nucleic acid compositions of the present invention may be obtained from biological sources using any of several cloning methods known to those skilled in the art, such as RNA, cDNA, genomic DNA, or any combination thereof. In some embodiments, oligonucleotide probes that selectively hybridize under stringent conditions to polynucleotides of the invention are used to identify a desired sequence in a cDNA or genomic DNA library. The isolation of RNA, the construction of cDNA and genomic libraries is well known to those skilled in the art (see, e.g., Ausubel, supra; or Sambrook, supra).

Nucleic acid screening and isolation method

cDNA or genomic libraries can be screened using probes based on the polynucleotide sequences of the invention, such as those disclosed herein. Probes can be used to hybridize to genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. One skilled in the art will appreciate that various degrees of hybridization stringency can be used in the assay; and either the hybridization or wash media can be stringent. As the hybridization conditions become more stringent, a greater degree of complementarity must be present between the probe and target which will form a duplex. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH and the presence of a partially denaturing solvent such as formamide. For example, hybridization stringency can be conveniently changed by changing the polarity of the reaction solution, for example, by manipulating the concentration of formamide in the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary depending on the stringency of the hybridization medium and/or wash medium. The degree of complementarity will optimally be 100%, or 70-100%, or any range or value therein. However, it will be appreciated that minor sequence changes in the probes and primers may be compensated for by reducing the stringency of the hybridization and/or wash medium.

Based on the teachings and guidance presented herein, methods of amplifying RNA or DNA are well known in the art and can be used according to the present invention without undue experimentation.

Well known methods of DNA or RNA amplification include, but are not limited to, Polymerase Chain Reaction (PCR) and related amplification methods (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 to Mullis et al, 4,795,699 and 4,921,794 to Tabor et al, 5,142,033 to Innis, 5,122,464 to Wilson et al, 5,091,310 to Innis et al, 5,066,584 to Gylensten et al, 4,889,818 to Gelfand et al, 4,994,370 to Silver et al, 4,766,067 to Biswas, 4,656,134 to Ringold), and RNA-mediated amplification using RNA against a target sequence as a template for double-stranded DNA synthesis (NASBA, U.S. Pat. No. 5,130,238 to Malek et al, under the trade name NASBA), the entire contents of which are incorporated herein by reference (see, e.g., Aubembr., e.g., supra; e.g., Sambrook, supra, e.g., supra, by reference).

For example, the sequences of the polynucleotides of the invention and related genes can be amplified directly from genomic DNA or cDNA libraries using Polymerase Chain Reaction (PCR) techniques. Nucleic acids used as probes to detect the presence of a desired mRNA in a sample, for nucleic acid sequencing, or other purposes may also be prepared using, for example, PCR and other in vitro amplification methods to clone a nucleic acid sequence encoding a protein to be expressed. Examples of techniques sufficient to instruct a skilled artisan to perform in vitro amplification methods can be found in Berger, supra, Sambrook, supra, and Ausubel, supra, and Mullis, et al, U.S. Pat. No. 4,683,202 (1987); and Innis, et al, PCR Protocols to Methods and Applications, eds., Academic Press Inc., san Diego, CA (1990). Commercially available kits for genomic PCR amplification are well known in the art. See, for example, Advantage-GC Genomic PCRKit (Clontech). In addition, for example, the T4 gene 32 protein (Boehringer Mannheim) can be used to increase the yield of long PCR products.

Synthetic methods for constructing nucleic acids

The isolated nucleic acids of the invention can also be prepared by direct chemical synthesis by well-known methods (see, e.g., Ausubel et al, supra). Chemical synthesis typically produces single-stranded oligonucleotides that can be converted to double-stranded DNA by hybridization to a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One skilled in the art will recognize that although chemical synthesis of DNA is limited to sequences of about 100 or more bases, longer sequences can be obtained by ligation of shorter sequences.

Recombinant expression cassette

The invention also provides recombinant expression cassettes comprising a nucleic acid of the invention. The nucleic acid sequences of the invention, e.g., cDNA or genomic sequences encoding the antibodies of the invention, can be used to construct recombinant expression cassettes that can be inserted into at least one desired host cell. A recombinant expression cassette will typically contain a polynucleotide of the invention operably linked to transcription initiation regulatory sequences that will direct transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters may be used to direct expression of the nucleic acids of the invention.

In some embodiments, an isolated nucleic acid that acts as a promoter, enhancer, or other element may be introduced at a suitable location (upstream, downstream, or in an intron) for a non-heterologous form of a polynucleotide of the invention, thereby up-regulating or down-regulating expression of the polynucleotide of the invention. For example, endogenous promoters may be altered in vivo or in vitro by mutation, deletion, and/or substitution.

Vectors and host cells

The invention also relates to vectors comprising the isolated nucleic acid molecules of the invention, host cells genetically engineered with said recombinant vectors, and the production of at least one anti-amyloid antibody by recombinant techniques well known in the art. See, e.g., Sambrook, et al, supra; ausubel et al, previously, each of which is incorporated herein by reference in its entirety.

The polynucleotide may optionally be linked to a vector containing a selectable marker for propagation in a host. Typically, plasmid vectors are introduced as precipitates, such as calcium phosphate precipitates, or as complexes with charged lipids. If the vector is a virus, it may be packaged in vitro using a suitable packaging cell line and then transduced into a host cell.

The DNA insert should be operably linked to a suitable promoter. The expression construct will also contain a transcription start site, a termination site, and a ribosome binding site in the transcribed region for translation. The coding portion of the mature transcript expressed by the construct will preferably include a translation start at the beginning of the mRNA to be translated and a stop codon (e.g., UAA, UGA or UAG) at the appropriate position at the end of the mRNA, with UAA and UAG being preferred for mammalian and eukaryotic cell expression.

The expression vector will preferably, but optionally, include at least one selectable marker. Such markers include, for example, but are not limited to, resistance to Methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. No. 4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017), ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. No. 5,122,464; 5,770,359; 5,827,739) in eukaryotic cells, and tetracycline or ampicillin resistance genes in culture in E.coli (E.coli) and other bacteria or prokaryotes (the above patents are incorporated by reference in their entirety). Suitable culture media and conditions for the above-described host cells are well known in the art. Suitable vectors will be apparent to the skilled person. Introduction of the vector construct into the host cell may be accomplished by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, or other well known methods. These methods are described in the art, such as Sambrook, supra, chapters 1-4 and 16-18; ausubel, as before, chapters 1, 9, 13, 15, 16.

At least one antibody of the invention may be expressed in a modified form, such as a fusion protein, and may include not only a secretion signal, but also additional heterologous functional regions. For example, additional amino acids, particularly charged amino acid regions, may be added to the N-terminus of the antibody to improve stability and persistence in the host cell during purification or during subsequent handling and storage. Likewise, a peptide moiety may be added to the antibody of the invention to facilitate purification. These regions may be removed prior to the final preparation of the antibody or at least one fragment thereof. These methods are described in many standard laboratory manuals, such as Sambrook, supra, chapters 17.29-17.42 and 18.1-18.74; ausubel, as described previously in chapters 16, 17 and 18.

A variety of expression systems are known to those skilled in the art that can be used to express nucleic acids encoding proteins of the present invention.

Alternatively, the nucleic acid of the invention may be expressed in a host cell by switching on (by manipulation) in a host containing the endogenous DNA encoding the antibody of the invention. Such methods are well known in the art, as described in U.S. Pat. Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, which are incorporated herein by reference in their entirety.

An example of a cell culture for producing an antibody, a specific portion or variant thereof is a mammalian cell. Mammalian cell systems are typically in the form of a cell monolayer, although mammalian cell suspensions or bioreactors may also be used. Many suitable host cell lines capable of expressing the entire glycosylated protein have been developed in the art and include COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL 1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610), and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2/0-Ag14, 293 cells, HeLa cells, and the like, which are readily available from, for example, American type culture Collection, Manassas, Va (www.atcc.org). Host cells include cells of lymphoid origin, such as myeloma and lymphoma cells. The host cells were P3X63Ag8.653 cells (ATCC accession number CRL-1580) and SP2/0-Ag14 cells (ATCC accession number CRL-1851). In particularly preferred embodiments, the recombinant cell is a P3X63Ab8.653 or SP2/0-Ag14 cell.

The expression vector for these cells may include one or more of the following expression control sequences, such as, but not limited to, an origin of replication; promoters (e.g., late or early SV40 promoter, CMV promoter (U.S. Pat. No. 5,168,062; 5,385,839), HSV tk promoter, pgk (phosphoglycerate kinase) promoter, EF-1. alpha. promoter (U.S. Pat. No. 5,266,491), at least one human immunoglobulin promoter, enhancers and/or processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., SV40 large T Ag polyA addition sites), and transcription termination sequences see, e.g., Ausubel et al, supra; Sambrook, et al, supra. other cells used to produce the nucleic acids or proteins of the invention are well known and/or can be obtained from, e.g., the American type culture Collection cell line and hybridoma catalog (www.atcc.org) or other well known or commercial sources.

When eukaryotic host cells are used, polyadenylation or transcription termination sequences are typically incorporated into the vector. An example of a terminator sequence is a polyadenylation sequence from the bovine growth hormone gene. Sequences for accurate splicing of transcripts may also be used. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al, J.Virol.45: 773-E781 (1983)). In addition, gene sequences that control replication in the host cell may be incorporated into the vector, as is well known in the art.

Purification of antibodies

Anti-amyloid antibodies can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to, protein a purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. See, for example, Colligan, Current Protocols in immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997 2001), e.g., chapters 1, 4, 6, 8, 9, 10, each of which is incorporated herein by reference in its entirety.

Antibodies of the invention include naturally purified products, products of chemical synthetic methods, and products produced by recombinant techniques from eukaryotic hosts, including, for example, yeast, higher plant, insect, and mammalian cells. Depending on the host used in the recombinant production method, the antibodies of the invention may be glycosylated or non-glycosylated, preferably glycosylated. These methods are described in many standard laboratory manuals, such as Sambrook, supra, sections 17.37-17.42; ausubel, as described supra, chapters 10, 12, 13, 16, 18 and 20, Colligan, Protein Science, as described supra, chapters 12-14, all of which are incorporated herein by reference in their entirety.

Anti-amyloid antibody

The isolated antibodies of the invention comprise the antibody amino acid sequences disclosed herein encoded by any suitable polynucleotide, or any isolated or prepared antibody. Preferably, the human antibody or antigen-binding fragment binds human amyloid and, thereby, partially or substantially neutralizes at least one biological activity of said protein. An antibody or a specific part or variant thereof which partially or preferably substantially neutralizes at least one biological activity of at least one amyloid protein or fragment may bind to said protein or fragment and thereby inhibit activity mediated by the binding of amyloid protein to amyloid receptors or by other amyloid-dependent or mediated mechanisms. The term "neutralizing antibody" as used herein means an antibody that inhibits amyloid-dependent activity by about 20-120%, preferably by at least about 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or more, depending on the assay. The ability of the anti-amyloid antibody to inhibit amyloid-dependent activity is preferably assessed by at least one suitable amyloid or receptor assay described herein and/or well known in the art. The human antibodies of the invention may be of any type (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and may comprise kappa or lambda light chains. In one embodiment, the human antibody comprises an IgG heavy chain or defined fragment, e.g., at least one isotype, IgG1, IgG2, IgG3, or IgG 4. Antibodies of this type can be prepared by using transgenic mice or other transgenic non-human mammals containing at least one human light chain (e.g., IgG, IgA, and IgM (e.g., γ 1, γ 2, γ 3, γ 4) transgene as described herein and/or as known in the art in another embodiment, anti-human amyloid human antibodies include IgG1 heavy chains and IgG1 light chains.

At least one antibody of the invention binds to at least one specific epitope of at least one amyloid protein, subunit, fragment, portion or any combination thereof. The at least one epitope may comprise at least one antibody binding region comprising at least a portion of the protein, preferably consisting of at least one extracellular, soluble, hydrophilic, external or cytoplasmic portion of the protein. The at least one specific epitope may comprise SEQ ID NO: 50 to at least one amino acid sequence of the entire specified portion of contiguous amino acids. As a non-limiting example, the antibodies of the invention exhibit binding to SEQ id no: 50 amino acids 2-7, 3-8, 33-42 and/or 34-40.

Typically, a human antibody or antigen-binding fragment of the invention will comprise antigen-binding regions comprising at least one human complementarity determining region (CDR1, CDR2 and CDR3) or at least one variant of a heavy chain variable region and at least one human complementarity determining region (CDR1, CDR2 and CDR3) or at least one variant of a light chain variable region. As a non-limiting example, an antibody or antigen-binding portion or variant may contain at least one polypeptide having the sequence of SEQ ID NO: 44 and/or a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 47, and a light chain CDR3 of the amino acid sequence of seq id no. In particular embodiments, the antibody or antigen-binding fragment may have an antigen-binding region comprising at least a portion of at least one heavy chain CDR (i.e., CDR1, CDR2, and/or CDR3) having the amino acid sequence of the corresponding CDR1, 2, and/or 3 (e.g., SEQ id nos: 42, 43, and/or 44; 53, 54, and/or 55; 63, 64, and/or 65; 73, 74, and/or 75). In another specific embodiment, the antibody or antigen-binding portion or variant can have an antigen-binding region comprising at least a portion of at least one light chain CDR (i.e., CDR1, CDR2, and/or CDR3) having the amino acid sequence of the corresponding CDR1, 2, and/or 3 (e.g., SEQ ID NOS: 45, 46, and/or 47; 56, 57, and/or 58; 66, 67, and/or 68; 76, 77, and/or 78). In a preferred embodiment, the three heavy chain CDRs and the three light chain CDRs of the antibody or antigen-binding fragment have the amino acid sequences of the corresponding CDRs of at least one of mabs C701, C705, C706, and C707 as described herein. Such antibodies may be prepared by chemically linking various portions (e.g., CDRs, framework) of the antibody by conventional techniques, by conventional techniques using recombinant DNA techniques, or by preparing and expressing one (i.e., one or more) nucleic acid molecule encoding the antibody using any other suitable method.

The anti-amyloid antibody may contain at least one of a heavy chain or a light chain variable region having a defined amino acid sequence. Any suitable Ig variable sequence may be used, e.g., from any subclass or any combination or fragment thereof. These sequences are well known in the art.

As non-limiting examples, representative variable sequences include those from IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, and the like, e.g., HC and LC, FR1, FR2, and/or FR3 sequences from any combination of Ig subclasses, e.g., as set forth in SEQ ID NOS: 48-49, 59-60, 69-70 and 79-80.

As another non-limiting example, in a preferred embodiment, the anti-amyloid antibody contains a heavy chain optionally having the amino acid sequence of SEQ ID NO: 48 and/or optionally at least one heavy chain variable region having the amino acid sequence of SEQ ID NO: 49 of at least one light chain variable region. In another preferred embodiment, the anti-amyloid antibody contains a heavy chain variable region optionally having seq id NO: 59 and/or optionally at least one heavy chain variable region having the amino acid sequence of SEQ id no: 60, or a pharmaceutically acceptable salt thereof. In a further preferred embodiment, the anti-amyloid antibody comprises a heavy chain optionally having the amino acid sequence of SEQ ID NO: 69 and/or optionally at least one heavy chain variable region having the amino acid sequence of SEQ ID NO: 70, or a light chain variable region of the amino acid sequence of seq id no. In another preferred embodiment, the anti-amyloid antibody contains a heavy chain optionally having the amino acid sequence of SEQ ID NO: 79 and/or optionally at least one heavy chain variable region having the amino acid sequence of SEQ ID NO: 80, or a light chain variable region of the amino acid sequence of seq id No. 80.

Antibodies that bind human amyloid and contain a defined heavy or light chain variable region can be prepared using suitable methods as are known in the art and/or described herein, such as phage display (Katsube, Y., et al, Int J mol. Med, 1 (5): 863-868(1998)) or using transgenic animals. For example, a transgenic mouse containing a functionally rearranged human immunoglobulin heavy chain transgene and a transgene containing DNA of a human immunoglobulin light chain locus that can undergo functional rearrangement can be immunized with human amyloid protein or a fragment thereof to cause the production of antibodies. If desired, antibody-producing cells can be isolated and hybridomas or other immortalized antibody-producing cells can be prepared as described herein and/or as known in the art. Alternatively, the antibody, specified portion or variant may be expressed in a suitable host cell using an encoding nucleic acid or portion thereof.

The invention also relates to antibodies, antigen-binding fragments, immunoglobulin chains, and CDRs containing amino acids that are substantially identical in sequence to the amino acid sequences described herein. Preferably, such antibodies or antigen binding fragments and antibodies containing such chains or CDRs can be of high affinity (e.g., K) _DLess than or equal to about 10^-9M) binds to human amyloid. Amino acid sequences that are substantially identical to the sequences described herein include sequences containing conservative amino acid substitutions, as well as amino acid deletions and/or insertions. Conservative amino acid substitution refers to the replacement of a first amino acid with a second amino acid having similar chemical and/or physical properties (e.g., charge, structure, polarity, hydrophobicity/hydrophilicity) as the first amino acid. Conservative substitutions include the substitution of one amino acid with another within the following group: lysine (K), arginine (R) and histidine (H); aspartic acid (D) and glutamic acid (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D, and E; alanine (a), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C), and glycine (G); F. w, and Y; C. s and T.

Amino acid code

The amino acids constituting the anti-amyloid antibody of the present invention are generally abbreviated. As is well known in The art, amino acid names may be indicated by their one-letter code, three-letter code, name, or trinucleotide codons (see, Alberts, B., et al, Molecular Biology of The Cell, third edition, Garland Publishing, Inc., New York, 1994):

Single letter codeCode	Three letter code	Name (R)	Trinucleotide codon (S)
				A	Ala	Alanine	GCA，GCC，GCG，GCU
C	Cys	Cysteine	UGC，UGU
				D	Asp	Aspartic acid	GAC，GAU
E	Glu	Glutamic acid	GAA，GAG
				F	Phe	Phenylalanine	UUC，UUU
G	Gly	Glycine	GGA，GGC，GGG，GGU
				H	His	Histidine	CAC，CAU
I	Ile	Isoleucine	AUA，AUC，AUU
				K	Lys	Lysine	AAA，AAG
L	Leu	Leucine	UUA，UUG，CUA，CUC，CUG，CUU
				M	Met	Methionine	AUG
N	Asn	Asparagine	AAC，AAU
				P	Pro	Proline	CCA，CCC，CCG，CCU
Q	Gln	Glutamine	CAA，CAG
				R	Arg	Arginine	AGA，AGG，CGA，CGC，CGG，CGU
S	Ser	Serine	AGC，AGU，UCA，UCC，UCG，UCU
				T	Thr	Threonine	ACA，ACC，ACG，ACU
V	Val	Valine	GUA，GUC，GUG，GUU
				W	Trp	Tryptophan	UGG
Y	Tyr	Tyrosine	UAC，UAU

The anti-amyloid antibodies of the invention may comprise one or more amino acid substitutions, deletions or additions from natural mutations or artificial manipulations as specified herein.

Of course, the number of amino acid substitutions that can be made by the skilled person depends on many factors, including those described above. In general, the number of amino acid substitutions, insertions, or deletions of any given anti-amyloid antibody, fragment or variant will not exceed 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, such as 1-30, or any range or value therein, as specified herein.

Amino acids essential for function in the anti-amyloid antibodies of the invention can be identified by methods well known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra, chapters 8, 15; Cunningham and Wells, Science 244: 1081-1085 (1989)). Alanine scanning mutagenesis introduces a single alanine mutation at each residue of the molecule. The resulting mutant molecules are then tested for biological activity such as, but not limited to, at least one amyloid neutralizing activity. Sites critical for antibody binding can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al, J.mol.biol.224: 899-904(1992) and de Vos, et al, Science 255: 306-312 (1992)).

The anti-amyloid antibody of the present invention may include, but is not limited to, antibodies selected from the group consisting of SEQ id nos: at least a portion, sequence or combination of 5 to all contiguous amino acids of at least one of 42-47, 53-58, 63-68 or 73-78.

The anti-amyloid antibody may also optionally include a peptide having SEQ ID NOS: 48. 49, 59, 60, 69, 70, 79 and 80, or a pharmaceutically acceptable salt thereof.

In one embodiment, the amino acid sequence of the immunoglobulin chain or portion thereof (e.g., variable region, CDR) is identical to SEQ ID NOS: 48. the amino acid sequence of the corresponding strand of at least one of 49, 59, 60, 69, 70, 79, and 80 has about 70-100% identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or any range or value therein). For example, the amino acid sequence of the light chain variable region may be identical to SEQ ID NO: 49. 60, 70 or 80, or the amino acid sequence of heavy chain CDR3 can be compared to the amino acid sequence of SEQ ID NO: 48. 59, 69 or 79. Preferably, 70-100% amino acid identity (i.e., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or any range or value therein) is determined using a suitable computer algorithm known in the art.

In SEQ ID NOS: 48. exemplary heavy and light chain variable region sequences are provided in 49, 59, 60, 69, 70, 79 and 80. The antibody of the invention, or a specific variant thereof, may contain any number of contiguous amino acid residues from the antibody of the invention, wherein the number is selected from an integer from 10-100% of the number of contiguous residues in the anti-amyloid antibody. Optionally, the contiguous amino acid subsequence is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more amino acids in length, or any range or value therein. Furthermore, the number of subsequences may be any integer selected from 1 to 20, such as at least 2, 3, 4 or 5.

As will be understood by those skilled in the art, the present invention includes at least one biologically active antibody of the present invention. The specific activity of a biologically active antibody is at least 20%, 30% or 40%, preferably at least 50%, 60% or 70%, most preferably at least 80%, 90% or 95% -1000% of the specific activity of the natural (non-synthetic), endogenous or related and known antibodies. Methods for measuring and quantifying enzyme activity and substrate specificity are well known to those skilled in the art.

Modified antibodies

In another aspect, the invention relates to human antibodies and antigen-binding fragments disclosed herein that are modified by covalent attachment of an organic moiety. Such modifications can result in antibodies or antigen-binding fragments with improved pharmacokinetic properties (e.g., increased serum half-life in vivo). The organic moiety may be a linear or branched hydrophilic polymeric group, a fatty acid group, or a fatty acid ester group. In particular embodiments, the hydrophilic polymeric group may have a molecular weight of about 800 to about 120,000 daltons, and may be a polyalkanediol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), a sugar polymer, an amino acid polymer, or polyvinylpyrrolidone, and the fatty acid or fatty acid ester group may contain about 8 to about 40 carbon atoms.

The modified antibodies and antigen-binding fragments of the invention may contain one or more organic moieties covalently bound, directly or indirectly, to the antibody. Each organic moiety bound to an antibody or antigen-binding fragment of the invention may independently be a hydrophilic polymeric group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" includes monocarboxylic acids and dicarboxylic acids. The term "hydrophilic polymeric group" as used herein refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than in octane. Thus, the invention includes antibodies modified by covalent attachment of polylysine. Hydrophilic polymers suitable for modifying the antibodies of the invention may be linear or branched and include, for example, polyalkanediols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG, and the like), sugars (e.g., dextran, cellulose, oligosaccharides, polysaccharides, and the like), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartic acid, and the like), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide, and the like), and polyvinylpyrrolidone. Preferably, the hydrophilic polymer modifying the antibody of the invention has a molecular weight of about 800 to about 150,000 daltons as a separate molecular entity. For example, PEG may be used ₅₀₀₀And PEG_20,000Wherein the subscript is the average molecular weight of the polymer in daltons. The hydrophilic polymeric group may be substituted with 1 to about 6 alkyl, fatty acid, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared by using a suitable method. For example, polymers containing amine groups can be coupled to carboxyl groups of fatty acid or fatty acid ester groups, and activated carboxyl groups on fatty acids or fatty acid esters (e.g., activated with N, N-carbonyldiimidazole) can be coupled to hydroxyl groups on the polymer.

Fatty acids and fatty acid esters suitable for modifying the antibodies of the invention may be saturated or may contain one or more units of unsaturation. Fatty acids suitable for modifying antibodies of the invention include, for example, n-dodecanoic acid (C)₁₂Lauric acid), n-tetradecanoic acid (C)₁₄Myristic acid), n-octadecanoic acid (C)₁₈Stearic acid) N-eicosanoic acid (C)₂₀Arachidic acid), n-behenic acid (C)₂₂Behenic acid), n-triacontanoic acid (C)₃₀) N-tetracontanoic acid (C)₄₀) Cis-delta 9-octadecanoic acid (C)₁₈Oleic acid), all-cis- Δ 5, 8, 11, 14-eicosatetraenoic acid (C)₂₀Arachidonic acid), suberic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid esters include monoesters of dicarboxylic acids containing linear or branched lower alkyl groups. The lower alkyl group may contain 1 to about 12, preferably 1 to about 6 carbon atoms.

Modified human antibodies and antigen-binding fragments can be prepared using suitable methods, such as by reaction with one or more modifying reagents. As used herein, "modifying agent" refers to a suitable organic group (e.g., hydrophilic polymer, fatty acid ester) that contains an activating group. An "activating group" is a chemical moiety or functional group that can react with a second chemical group under suitable conditions to form a covalent bond between a modifying agent and the second chemical group. For example, amine-reactive activating groups include electrophilic groups, such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl ester (NHS), and the like. The activating group that reacts with thiol includes, for example, maleimide, iodoacetyl, acryloyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. The aldehyde functional group can be coupled to an amine-or hydrazide-containing molecule, and the azide group can react with the trivalent phosphorus group to form a phosphoramidate or phosphorimide linkage. Suitable methods for introducing suitable activating groups into molecules are well known in the art (see, e.g., Hermanson, G.T., Bioconjugate techniques, Academic Press: San Diego, Calif. (1996)). The activating group can be directly bonded to an organic group (e.g., hydrophilic polymer, fatty acid ester) or through a linker moiety, e.g., divalent C ₁-C₁₂Group bound organic group at said divalent C₁-C₁₂In which one or more carbon atoms may be interrupted by hetero atoms, e.g. oxygen, nitrogen, or thioAnd (4) replacing. Suitable linker moieties include, for example, tetraethylene glycol, - (CH)₂)₃-、-NH-(CH₂)₆-NH-、-(CH₂)₂-NH-and-CH₂-O-CH₂-CH₂-O-CH₂-CH₂-O-CH-NH-. The modifying reagent containing a linker moiety can be prepared, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxyl group. Boc protecting groups can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can couple another carboxyl group as described, or can be reacted with maleic anhydride and cyclized to yield an activated maleimide (maleimido) derivative of the fatty acid (see, e.g., Thompson, et al, WO92/16221, the entire teachings of which are incorporated herein by reference).

The modified antibodies of the invention can be produced by reacting a human antibody or antigen-binding fragment with a modifying agent. For example, organic moieties can be attached to antibodies in a non-site specific manner by using amine-reactive modifying reagents, such as NHS esters of PEG. The modified human antibody or antigen-binding fragment can also be prepared by reducing disulfide bonds (e.g., intrachain disulfide bonds) of the antibody or antigen-binding fragment. The reduced antibody or antigen-binding fragment can then be reacted with a thiol-reactive modifying reagent to produce a modified antibody of the invention. Using suitable methods, such as reverse proteolysis (Fisch et al, Bioconjugate chem., 3: 147-: modified human antibodies and antigen-binding fragments containing an organic portion that binds to a specific site of an antibody of the invention can be prepared by the methods described in San Diego, CA (1996).

Anti-idiotypic antibodies directed against anti-amyloid antibody components

In addition to monoclonal or chimeric anti-amyloid antibodies, the invention also relates to anti-idiotypic (anti-Id) antibodies specific for these antibodies of the invention. An anti-Id antibody is an antibody that recognizes a unique determinant that is normally associated with the antigen binding region of another antibody. An anti-Id antibody is prepared by immunizing an animal of the same species and genotype (e.g., mouse line) from which the Id antibody is derived with the antibody or CDR-containing region thereof. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody and produce an anti-Id antibody. anti-Id antibodies can also be used as "immunogens" to induce an immune response in another animal, thereby generating what is known as an anti-Id antibody.

Amyloid antibody composition

The present invention also provides at least one anti-amyloid antibody composition comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6 or more anti-amyloid antibodies as described herein and/or as known in the art, provided in a non-naturally occurring composition, mixture or form. These compositions comprise a non-naturally occurring composition comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 42-49, 53-60, 63-70, 73-80, or at least one or two full length, C-and/or N-terminal deleted variants, domains, fragments or specified variants thereof of specified fragments, domains or variants. Preferred anti-amyloid antibody compositions include antibodies that are substituted as antibodies comprising SEQ ID NOS: 42-47, 53-58, 63-68, 73-78, or at least one or two full length, fragments, domains or variants of at least one CDR or LBP of a portion of a specified fragment, domain or variant thereof. More preferred compositions comprise SEQ ID NOS: 42-47, 53-58, 63-68, 73-78, or 40-99% of at least one of the specified fragments, domains or variants thereof. These composition percentages are calculated as weight, volume, concentration, molarity or molarity of a liquid or anhydrous solution, mixture, suspension, emulsion, granule, powder or colloid, as known in the art or described herein.

The composition may optionally further contain an effective amount of at least one compound or protein selected from at least one anti-infective drug, Cardiovascular (CV) system drug, Central Nervous System (CNS) drug, Autonomic Nervous System (ANS) drug, respiratory tract drug, Gastrointestinal (GI) tract drug, hormonal drug, drug for fluid or electrolyte balance, hematologic drug, anti-cancer drug, immunomodulatory drug, ocular, otic or nasal drug, topical drug, nutraceutical, statin, and the like. Such agents are well known in the art and include the formulation, indications, administration and administration of each of the agents given herein (see, e.g., Nursing2001 Handbook of Drugs, 21 st edition, Springhouse Corp., Springhouse, PA, 2001; Health Professional's Guide 2001, eds., Shannon, Wilson, Stang, Prentice-Hall, Inc, U pp Saddle River, NJ; Pharmotherapy Handbook, Wells et al, eds., Appleton & Lange, mStanford, CT, each of which is incorporated herein in its entirety by reference).

The CNS drug may be at least one selected from the group consisting of non-narcotic analgesics or at least one selected from the group consisting of antipyretics, non-steroidal anti-inflammatory drugs, narcotics or at least one opioid analgesic, sedative-hypnotics, anticonvulsants, antidepressants, anxiolytics, antipsychotics, central nervous system stimulants, anti-parkinson drugs, various central nervous system drugs. The ANS drug may be at least one selected from the group consisting of a cholinergic drug (parasympathomimetic drug), an anticholinergic drug, an adrenergic drug (sympathomimetic drug), an adrenergic blocking drug (sympathomimetic drug), a skeletal muscle relaxant, and a neuromuscular blocking drug. The at least one non-narcotic analgesic or antipyretic may be at least one selected from acetaminophen, acetylsalicylic acid, choline magnesium trisalicylate, diflunisal, magnesium salicylate. The at least one non-steroidal anti-inflammatory drug may be selected from At least one of celecoxib, diclofenac potassium, diclofenac sodium, etodolac, phenoxyphenylpropionic acid calcium, flurbiprofen, ibuprofen, indomethacin sodium trihydrate, ketoprofen, ketorolac, nabumetone, naproxen sodium naproxen, oxaprozin, piroxicam, rofecoxib, and sulindac. The at least one narcotic or opioid analgesic may be at least one selected from the group consisting of alfentanil hydrochloride, buprenorphine hydrochloride, cyclobutyloxymorphone tartrate, codeine phosphate, codeine sulfate, fentanyl citrate, fentanyl transdermal systems, transmucosal fentanyl, hydromorphone hydrochloride, meperidine hydrochloride, methadone hydrochloride, morphine sulfate, morphine tartrate, nalbuphine hydrochloride, oxycodone pectate, oxymorphone hydrochloride, analgesia hydrochloride, and naloxone hydrochloride, analgesia lactate, propoxyphene hydrochloride, propoxyphene naphthalenesulfonate, remifentanil hydrochloride, fentanyl citrate, tramadol hydrochloride. The at least one sedative hypnotic agent may be at least one selected from chloral hydrate, estazolam, fluvalin hydrochloride, pentobarbital sodium, phenobarbital sodium, secobarbital sodium, meprobamate, triazabepilene, zaleplon, and zolpidem tartrate. The at least one anticonvulsant may be selected from acetazolamide, carbamazepine, clonazepam, dipotassium chlorozene At least one of diazepam, divalproex sodium, ethosuximide, fosphenytoin sodium, gabapentin, lamotrigine, magnesium sulfate, phenobarbital sodium, phenytoin sodium (persistent), prometasone, tiagabine hydrochloride, topiramate, sodium valproate and valproic acid. The at least one antidepressant may be selected from amitriptyline hydrochloride, oxaziridine, amoxapine, bupropion hydrochloride, citalopram hydrobromide, clomipramine hydrochloride, desipramine hydrochloride, doxepin hydrochloride, fluoxetine hydrochloride, imipramine hydrochloride, oxazipramine, mirtazapine, nefazodone hydrochloride, nortriptyline hydrochloride, paroxetine hydrochloride, phenelzine sulfate, sertraline hydrochloride, sulfuric acid, and the likeAt least one of phencyclamine, trimipramine maleate and venlafaxine hydrochloride. The at least one anxiolytic may be selected from alprazolam, bupropion hydrochloride, and chlorazolHydrochloric acid chlorine nitrogenDi-potassium chlorine nitrogenAt least one of diazepam, doxepin hydrochloride, hydroxyzine pamoate, hydroxyzine hydrochloride, hydroxyzine pamoate, chlordiazepam, mephrombamate, midazolam hydrochloride and desmethoxydiazepam. The at least one antipsychotic agent may be at least one selected from chlorpromazine hydrochloride, clozapine, fluphenazine decanoate, fluphenazine enanthate, fluphenazine hydrochloride, haloperidol decanoate, haloperidol lactate, clindamine hydrochloride, closerpine succinate, thiamphenicol besylate, molindone hydrochloride, olanzapine, perphenazine, piperacillin, chlorpromazine mesylate, quetiapine fumarate, vesnaton, thioridazine hydrochloride, thiothixene hydrochloride, trifluoperazine hydrochloride. The at least one central nervous system stimulant may be at least one selected from the group consisting of amphetamine sulfate, caffeine, dextroamphetamine sulfate, doxorfin hydrochloride, methamphetamine hydrochloride, methylphenidate hydrochloride, modafinil, pimoline, and phentermine hydrochloride. The at least one antiparkinsonian drug may be at least one selected from the group consisting of amantadine hydrochloride, benztropine mesylate, biperiden hydrochloride, biperiden lactate, bromocriptine mesylate, carbidopa-levodopa, entacapone, levodopa, tiopronin mesylate, peramivir dihydrochloride, lepirole hydrochloride, selegiline hydrochloride, tolcapone, trihexyphenidyl hydrochloride. The at least one miscellaneous CNS drug may be selected from riluzole, bupropion hydrochloride, donepezil hydrochloride, fluoropiperidine, fluvoxamine maleate, lithium carbonate, lithium citrate, naratriptan hydrochloride, nicotine polacrilex, nicotine transdermal system, propofol, rizatriptan benzoate, sibutramine hydrochloride monohydrate, and tematriptan succinate At least one of a tan, a hydrochloric acid monobactaridam, a zolmitriptan (see, for example, Nursing 2001 Drug Handbook 337-530).

The at least one cholinergic agent (e.g., parasympathomimetic) may be at least one selected from bethanechol chloride, tenascin chloride, neostigmine bromide, neostigmine methylsulfate, physostigmine salicylate, pyridostigmine bromide. The at least one anticholinergic agent can be at least one selected from atropine sulfate, dicyclomine hydrochloride, changning, hyoscyamine sulfate, propantheline bromide, scopolamine butylbromide, and scopolamine hydrobromide. The at least one adrenergic agent (sympathomimetic agent) can be at least one member selected from the group consisting of dobutamine hydrochloride, dopamine hydrochloride, metahydroxylamine tartrate, norepinephrine bitartrate, neosynephrine hydrochloride, pseudoephedrine hydrochloride, and pseudoephedrine sulfate. The at least one adrenergic blocker (sympatholytic agent) may be at least one selected from dihydroergotamine mesylate, ergotamine tartrate, dimethylargoline maleate, propranolol hydrochloride. The at least one skeletal muscle relaxant may be at least one selected from baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine hydrochloride, darunavir sodium, methocarbamol, tizanidine hydrochloride. The at least one neuromuscular blocker may be at least one selected from the group consisting of antomopine, cisatracurine dicarbonate, doxorammonium chloride, micaronium chloride, pancuronium bromide, pipecuronium bromide, rapacunium bromide, rocuronium bromide, succinylcholine chloride, tubocurarine chloride, vecuronium bromide (see, for example, pages 531-84 of Nursing 2001 Drug Handbook).

The anti-infective agent may be at least one or at least one antiprotozoal agent selected from amebiase, anthelmintic, antifungal, antimalarial, antitubercular or at least one anti-leprosy agent, aminoglycoside, penicillin, cephalosporin, tetracycline, sulfonamide, fluoroquinolone, antiviral agent, macrolide anti-infective agent, miscellaneous anti-infective agent. The CV drug may be at least one selected from inotropic drugs, antiarrhythmic drugs, antianginal drugs, antihypertensive drugs, antilipidemic drugs, and miscellaneous cardiovascular drugs. The CNS drug may be at least one selected from the group consisting of non-narcotic analgesics or at least one selected from the group consisting of antipyretics, non-steroidal anti-inflammatory drugs, narcotics or at least one opioid analgesic, sedative-hypnotics, anticonvulsants, antidepressants, anxiolytics, antipsychotics, central nervous system stimulants, anti-parkinson drugs, miscellaneous central nervous system drugs. The ANS drug may be at least one selected from the group consisting of a cholinergic drug (parasympathomimetic drug), an anticholinergic drug, an adrenergic drug (sympathomimetic drug), an adrenergic blocking agent (sympathomimetic drug), a skeletal muscle relaxant, and a neuromuscular blocking agent. The respiratory tract drug may be at least one or at least one antitussive, miscellaneous respiratory related drug selected from antihistamines, bronchodilators, expectorants. The gastrointestinal drug may be at least one or at least one adsorbent selected from antacids or at least one antiflatulent, digestive enzyme or at least one gallstone solubilizer, antidiarrheal, laxative, antiemetic, antiulcer. The hormone drug may be at least one or at least one anabolic steroid selected from corticosteroids, androgens, estrogens or at least one progestin, gonadotropins, antidiabetic drugs or at least one glucagon, thyroid hormone antagonists, pituitary hormones, parathyroid-like drugs. The fluid and electrolyte balancing medicament may be at least one or at least one replacement solution selected from a diuretic, an electrolyte, an acidifying agent or at least one basifying agent. The blood drug may be at least one selected from the group consisting of a blood-replenishing drug, an anticoagulant, a blood derivative, and a thrombolytic enzyme. The anticancer agent may be at least one selected from the group consisting of alkylating drugs, antimetabolites, antibiotic-type anticancer agents, anticancer agents that alter hormone balance, and miscellaneous anticancer agents. The immunomodulating drug may be at least one or at least one toxoid, antitoxin or at least one anti-snake toxin selected from an immunosuppressant, a vaccine, an immune serum, a biological response modifier. The ophthalmic, otic and nasal agents may be at least one selected from ophthalmic anti-infective agents, ophthalmic anti-inflammatory agents, miotic agents, ophthalmic vasoconstrictors, miscellaneous ophthalmic, otic, nasal agents. The topical drug may be at least one or at least one pediculicide selected from anti-infective agents, miticides, topical corticosteroids. The nutraceutical may be at least one selected from vitamins, minerals, or calories. See, for example, Nursing 2001 Drug Handbook, supra.

The at least one amebiase or anti-protozoan agent may be at least one selected from the group consisting of atoquarone, chloroquine hydrochloride, chloroquine phosphate, metronidazole hydrochloride, pentamidine isethionate. The at least one anthelmintic may be at least one selected from the group consisting of mebendazole, pyrantel pamoate, thiabendazole. The at least one antifungal agent may be at least one selected from amphotericin B, amphotericin B cholesteryl sulfate complex, amphotericin B lipid complex, amphotericin B liposome, fluconazole, flucytosine, micro-sized (microsize) griseofulvin, ultra-micro-sized griseofulvin, itraconazole, ketoconazole, nystatin, terbinafine hydrochloride. The at least one antimalarial may be at least one selected from chloroquine hydrochloride, chloroquine phosphate, doxycycline, hydroxychloroquine sulfate, mefloquine hydrochloride, primaquine phosphate, pyrimethamine, and sulfadoxine. The at least one antituberculotic or antileprosy drug may be at least one selected from the group consisting of chlorphenazine, cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin, rifampin, rifapentine, streptomycin sulfate. The at least one aminoglycoside may be at least one selected from amikacin sulfate, gentamicin sulfate, neomycin sulfate, streptomycin sulfate, tobramycin sulfate. The at least one penicillin may be at least one selected from the group consisting of amoxicillin/clavulanate potassium, amoxicillin trihydrate, ampicillin sodium, benzylpenicillin trihydrate, ampicillin sodium/penicillosulfone sodium, chlorophenicillin sodium, dicloxacillin sodium, mezlocillin sodium, carbethoxynaphthalene penicillin sodium, oxacillin sodium, benzathine penicillin G, penicillin G potassium, procaine penicillin G, penicillin G sodium, penicillin V potassium, piperacillin sodium/tazobactam sodium, carbothifencillin sodium, carbothiphenol penicillin sodium/clavulanate potassium. The at least one cephalosporin may be at least one selected from cefaclor, cefadroxil, cefazolin sodium, cefdinir, cefapine hydrochloride, cefixime, cefmetazole sodium, cefonicid sodium, cefoperazone sodium, cefotaxime sodium, cefotetan disodium, cefoxitin sodium, cefpodoxime proxetil, cefprozil, ceftazidime, cefbupivam, cefazolin sodium, ceftriaxone sodium, cefuroxime axetil, cefuroxime sodium, cefalexin hydrochloride, cephalexin monohydrate, cephradine, loracarbef. The at least one tetracycline may be at least one selected from demeclocycline hydrochloride, doxycycline calcium, doxycycline hydrochloride, doxycycline monohydrate, minocycline hydrochloride, tetracycline hydrochloride. The at least one sulfa drug may be at least one selected from the group consisting of sulfamethoxazole, sulfadiazine, sulfamethoxazole, sulfacetazole. The at least one fluoroquinolone may be at least one selected from the group consisting of alatrofloxacin mesylate, ciprofloxacin, enoxacin, levofloxacin, lomefloxacin hydrochloride, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin mesylate. The at least one fluoroquinolone may be at least one selected from the group consisting of alatrofloxacin mesylate, ciprofloxacin, enoxacin, levofloxacin, lomefloxacin hydrochloride, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin mesylate. The at least one antiviral agent may be at least one selected from the group consisting of abacavir sulfate, acyclovir sodium, amantadine hydrochloride, amprenavir, cidofovir, delavirdine mesylate, didanosine, efavirenz, famciclovir, fomivirsen sodium, foscarnet, ganciclovir, indonazivir sulfate, lamivudine/azidothymidine, nelfinavir mesylate, nevirapine, oseltamivir phosphate, ribavirin hydrochloride, ritonavir, saquinavir mesylate, stavudine, famciclovir hydrochloride, zalcitabine, zanamivir, azidothymidine. The at least one macrolide anti-infective agent may be at least one selected from azithromycin, clarithromycin, dirithromycin, erythromycin base, erythromycin estolate, erythromycin ethylsuccinate, erythromycin lactobionate, erythromycin stearate. The at least one miscellaneous anti-infective agent may be at least one selected from aztreonam, bacitracin, chloramphenicol sodium succinate, clindamycin hydrochloride, clindamycin palmitate hydrochloride, clindamycin phosphate, tyline and cilastatin sodium, meropenem, nitrofurantoin macrocrystals, nitrofurantoin microcrystals, quinupristin/dalfopristin, spectinomycin hydrochloride, trimethoprim, vancomycin hydrochloride (see, e.g., pages 24-214 of Nursing2001 Drug Handbook).

The at least one inotropic agent may be at least one selected from the group consisting of amiloride lactate, digoxin lactate and milrinone lactate. The at least one antiarrhythmic agent is selected from adenosine, amiodarone hydrochloride, atropine sulfate, bromophenylethylamine, diltiazem hydrochlorideAt least one of propyramide, propyramide phosphate, esmolol hydrochloride, flecainide acetate, ibutilide fumarate, lidocaine hydrochloride, mexiletine hydrochloride, moraxezine hydrochloride, phenytoin sodium, procainamide hydrochloride, propafenone hydrochloride, propranolol hydrochloride, quinidine bisulfate, quinidine gluconate, quinidine polygalacturonate, quinidine sulfate, sotalol, tocacamide hydrochloride, and verapamil hydrochloride. The at least one anti-angina agent is selected from the group consisting of amlodipine besylate, nitroso-pentyl, bepridil hydrochloride, diltiazem hydrochlorideAt least one of isosorbide dinitrate, isosorbide mononitrate, metoprolol, nitrapyrin hydrochloride, nifedipine, nitroglycerin, propranolol hydrochloride, verapamil and verapamil hydrochloride. The at least one antihypertensive agent may be selected from acebutolol hydrochloride, amlodipine besylate, atenolol, benazepril hydrochloride, betaxolol hydrochloride, bisoprolol fumarate, candesartan cilexetil, captopril, quinacrine hydrochloride, carvedilol, clonidine hydrochloride, diazoxide, diltiazem hydrochloride Methanesulfonic acid, methanesulfonic acidDoxazosin, enalapril maleate, eprosartan mesylate, felodipine, fenlodolan mesylate, fosinopril sodium, guanabenz acetate, guanabenole sulfate, guanfacine hydrochloride, hydralazine hydrochloride, irbesartan, isradipine, labetalol hydrochloride, lisinopril, losartan potassium, methyldopa hydrochloride, metoprolol succinate, metoprolol tartrate, minoxidil, at least one of moxiflopril hydrochloride, nadroproxol, nitrapyrin methyl hydrochloride, nifedipine, nisoldipine, sodium nitroprusside, cyclopentadin sulfate, perindopril erbumine, phentolamine mesylate, propranolol hydrochloride, quinapril hydrochloride, ramipril, telmisartan, terazosin hydrochloride, tiamulol maleate, trandolapril, valsartan, and verapamil hydrochloride. The at least one antiliposomal agent may be at least one selected from the group consisting of atorvastatin calcium, cerivastatin sodium, cholestyramine, colestipol hydrochloride, fenofibrate (micronized), fluvastatin sodium, gemfibrozil, lovastatin, nicotinic acid, pravastatin sodium, and simvastatin. The at least one miscellaneous CV Drug may be at least one selected from the group consisting of abciximab, alprostadil, albutamine hydrochloride, cilostazol, clopidogrel bisulfate, dipyridamole, epifetidide, methoxamine hydrochloride, pentoxifylline, ticlopidine hydrochloride, tirofiban hydrochloride (see, e.g., Nursing 2001 Drug Handbook, page 215-.

The at least one antihistamine can be at least one selected from the group consisting of brompheniramine maleate, cetirizine hydrochloride, chlorpheniramine maleate, chlorpheniramine fumarate, cyproheptadine hydrochloride, diphenhydramine hydrochloride, methylammonium hydrochloride, loratadine, promethazine hydrochloride, theaipramine, and triprolidine hydrochloride. The at least one bronchodilator may be at least one selected from the group consisting of salbutamol, salbutamol sulfate, aminophylline, atropine sulfate, ephedrine sulfate, epinephrine bitartrate, epinephrine hydrochloride, ipratropium bromide, isoproterenol hydrochloride, isoproterenol sulfate, levalbuterol hydrochloride, metaproteline sulfate, choline theophylline, pirbuterol acetate, salmeterol xinafoate, terbutaline sulfate, and theophylline. The at least one expectorant or antitussive may be at least one selected from benzonatate, codeine phosphate, codeine sulfate, dextromethorphan hydrobromide, diphenhydramine hydrochloride, guaifenesin, hydromorphone hydrochloride. The at least one miscellaneous respiratory Drug may be at least one selected from the group consisting of acetylcysteine, beclomethasone dipropionate, berlittan, budesonide, calfactant, cromolyn sodium, alfa deoxyribonuclease, cycloprostenol sodium, flunisolide, fluticasone propionate, montelukast sodium, ledocromil sodium, palivizumab, triamcinolone acetonide, zafirlukast, zileuton (see, e.g., Nursing 2001 Drug Handbook, page 585-.

The at least one antacid, adsorbent or antiflatulent agent may be at least one selected from the group consisting of aluminum carbonate, aluminum hydroxide, calcium carbonate, magnesium aluminum hydroxide, magnesium oxide, simethicone, and sodium bicarbonate. The at least one digestive enzyme or gallstone solubilizer may be at least one selected from pancreatin, pancrelipase, ursodiol. The at least one antidiarrheal agent may be at least one selected from the group consisting of attapulgite, bismuth subsalicylate, calcium polyacrylic resin, diphenoxylate hydrochloride or atropine sulfate, loperamide, octreotide, tincture of opium, and tincture of opium (camphor-containing). The at least one laxative may be at least one selected from bisocodyl, calcium polyacrylic resin, cascara sagrada, a rhamnus fragrans fluid extract, a rhamnus fluid extract, castor oil, calcium sulfosuccinate, sodium sulfosuccinate, glycerin, lactulose, magnesium citrate, magnesium hydroxide, magnesium sulfate, methyl cellulose, mineral oil, polyethylene glycol or electrolyte solution, psyllium, senna, sodium phosphate. The at least one antiemetic agent may be at least one selected from chlorpromazine hydrochloride, dimenhydrinate, dolasetron mesylate, dronabinol, grelor hydrochloride, chlorobenzazine hydrochloride, metoclopramide hydrochloride, ondansetron hydrochloride, perphenazine, mepiquat chloride edisylate, mepiquat chloride maleate, promethazine hydrochloride, scopolamine, thiotepa promazine maleate, trimethobenzamide hydrochloride. The at least one antiulcer agent may be at least one selected from cimetidine, cimetidine hydrochloride, famotidine, dacron, misoprostol, nizatidine, omeprazole, rabeprazole sodium, ranitidine bismuth citrate, ranitidine hydrochloride, sucralfate (see, e.g., pages 643-95 of Nursing 2001 drug handbook). The at least one corticosteroid may be at least one member selected from the group consisting of betamethasone, betamethasone acetate or betamethasone sodium phosphate, cortisone acetate, dexamethasone, fluorometholone acetate, dexamethasone sodium phosphate, fludrocortisone acetate, hydrocortisone acetate, hydrocortisone cypionate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, methylprednisolone sodium succinate, prednisolone acetate, prednisolone sodium phosphate, prednisolone tert-butyl ethyl ester, prednisone, triamcinolone acetonide.

The at least one androgen or anabolic steroid may be at least one member selected from the group consisting of danazol, fluoxymethyltestosterone, methyltestosterone, nandrolone decanoate, nandrolone phenylpropionate, testosterone cypionate, testosterone enanthate, testosterone propionate, and testosterone transdermal systems. The at least one estrogen or progestin may be at least one selected from the group consisting of esterified estrogen, estradiol cypionate, estradiol/norethindrone acetate transdermal systems, estradiol valerate, estrogen (conjugate), estropipate, ethinyl estradiol and ethinyl estradiol, ethinyl estradiol and norethindrone diacetate, ethinyl estradiol and norethindrone, ethinyl estradiol and levonorgestrel, ethinyl estradiol and norethindrone acetate and iron fumarate, levonorgestrel, medroxyprogesterone acetate, medroxyprogesterone and norethindrone, norethindrone acetate, norethindrone methyl, progesterone. The at least one gonadotropin may be at least one selected from the group consisting of ganirelix acetate, ganarelin acetate, cistrelin acetate, menopausal gonadotropin. The at least one antidiabetic agent or glucaon may be at least one selected from the group consisting of acarbose, chlorpropamide, glimepiride, glipizide, glucagon, glyburide, insulin, metformin hydrochloride, miglitol, pioglitazone hydrochloride, repaglinide, rosiglitazone maleate, troglitazone. The at least one thyroid hormone may be at least one selected from levothyroxine sodium, sodium iodothyronine, liotrix, thyroid. The at least one thyroid hormone antagonist may be selected from methimazole, potassium iodide (saturated solution), propylthiouracil, and radioactive iodine (sodium iodide)¹³¹I) And a concentrated iodine solution. The at least one pituitary hormone may be at least one selected from the group consisting of corticotropin, tetracoseptide corticotropin, desmopressin acetate, leuprolide acetate, long-acting corticotropin, human methionine auxin, somatropin, vasopressin. The at least one parathyroid-like Drug may be at least one selected from the group consisting of calcitriol, calcitonin (human), calcitonin (salmon), calcitriol, dihydrotachysterol, disodium etidronate (see, e.g., Nursing 2001 Drug Handbook, page 696-796).

The at least one diuretic may be at least one diuretic selected from acetazolamide, acetazolamide sodium, amiloride hydrochloride, bumetanide, chlorthalidone, sodium ethacrynate, ethacrynic acid, furosemide, hydrabamide, indapamide, mannitol, metolazone, spironolactone, torsemide, triamterene, urea. The at least one electrolyte or replacement solution may be at least one selected from the group consisting of calcium acetate, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium glucoheptonate, calcium gluconate, calcium lactate, calcium phosphate (dibasic), calcium phosphate (tribasic), dextran (high molecular weight), dextran (low molecular weight), hydroxyethyl starch, magnesium chloride, magnesium sulfate, potassium acetate, potassium bicarbonate, potassium chloride, potassium gluconate, ringer's injection (lactate), sodium chloride. The at least one acidifying or basifying agent may be at least one selected from sodium bicarbonate, sodium lactate, tris (hydroxymethyl) aminomethane. (see, e.g., Nursing 2001 drug handbook, page 797-.

The at least one blood-tonifying agent may be at least one selected from ferrous fumarate, ferrous gluconate, ferrous sulfate (anhydrous), iron dextran, iron sorbitol, polysaccharide-iron complex, and sodium ferric gluconate complex. The at least one anticoagulant may be at least one selected from the group consisting of aclidinium sodium, dalteparin sodium, danaparoid sodium, enoxaparin sodium, heparin calcium, heparin sodium, warfarin sodium. The at least one blood derivative may be at least one selected from the group consisting of albumin 5%, albumin 25%, anti-hemophilia a factor, anti-inhibitor coagulant complex, anti-thrombin III (human), factor IX complex, plasma protein fraction. The at least one thrombolytic enzyme may be at least one selected from alteplase, anistreplase, reteplase (recombinant), streptokinase, urokinase. (see, e.g., Nursing 2001 Drug Handbook, pages 834-66).

The at least one alkylating agent may be at least one selected from busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, ifosfamide, lomustine, mechlorethamine hydrochloride, melphalan hydrochloride, streptozotocin, temozolomide, thiotepa. The at least one antimetabolite may be at least one selected from the group consisting of capecitabine, cladribine, cytarabine, floxuridine, fludarabine phosphate, fluorouracil, hydroxyurea, mercaptopurine, methotrexate sodium, thioguanine. The at least one antibiotic anticancer agent may be at least one selected from bleomycin sulfate, actinomycin D, daunorubicin citrate liposome, daunorubicin hydrochloride, doxorubicin hydrochloride liposome, epirubicin hydrochloride, idarubicin hydrochloride, mitomycin, pentostatin, plicamycin, and valrubicin. The at least one anticancer agent that alters hormonal balance may be at least one selected from the group consisting of anastrozole, bicalutamide, estramustine phosphate, exemestane, flulibam, riluzole, letrozole, leuprolide acetate, megestrol, nilutamide, tamoxifen citrate, testolactone, and toremifene citrate. The at least one miscellaneous anticancer agent may be at least one selected from asparaginase, bacillus calmette-guerin (BCG) (live intravesical), dacarbazine, docetaxel, etoposide phosphate, gemcitabine hydrochloride, irinotecan hydrochloride, mitotane, mitoxantrone hydrochloride, paclitaxel, asparaginase, porfimer sodium, procarbazine hydrochloride, rituximab, teniposide, topotecan hydrochloride, trastuzumab, tretinoin, vinblastine sulfate, vincristine sulfate, vinorelbine (see, e.g., page 867 and 963 of Nursing 2001 Drug Handbook).

The at least one immunosuppressive agent may be at least one selected from azathioprine, basiliximab, cyclosporine, daclizumab, lymphocyte immunoglobulin, murine monoclonal antibody-CD 3, mycophenolate mofetil, mycophenolate hci, sirolimus, tacrolimus. The at least one vaccine or toxoid may be selected from the group consisting of BCG vaccine, cholera vaccine, diphtheria and tetanus toxoid (adsorbed), adsorbed diphtheria and tetanus toxoid and acellular pertussis vaccine, diphtheria and tetanus toxoid and whole cell pertussis vaccine, Haemophilus b conjugate vaccine, hepatitis A vaccine (inactivated), hepatitis B vaccine (recombinant), influenza vaccine 1999-&B (purified surface antigen), influenza vaccine 1999-&B (subviral or purified subviral), influenza vaccine 1999-&B (whole virions), japanese encephalitis virus vaccine (inactivated), Lyme disease vaccine (recombinant OspA), measles and mumps and rubella virus vaccine (live attenuated), measles virus vaccine (live attenuated), meningococcal polysaccharide vaccine, mumps virus vaccine (live), plague vaccine, pneumococcal vaccine (multivalent), polio virus vaccine (inactivated), polio virus vaccine (live, oral, trivalent), rabies vaccine (adsorbed), rabies vaccine (human diploid cells), rubella and mumps virus vaccine (live), rubella virus vaccine (live, attenuated), tetanus toxoid (adsorbed), tetanus toxoid (fluid), typhoid vaccine (oral), typhoid vaccine (parenteral), Typhoid Vi polysaccharide vaccine, varicella virus vaccine, and vaccine for yellow fever One of them is less. The at least one antitoxin or antisnake toxin may be at least one selected from the group consisting of black widow spider antitoxin, pallas (Crotaldae) antitoxin (multivalent), diphtheria antitoxin (horse), and golden coral snake (Micrurus fuscus) antisnake toxin. The at least one immune serum may be selected from cytomegalovirus immunoglobulin (intravenous), hepatitis B immunoglobulin (human), intramuscular immunoglobulin, intravenous immunoglobulin, rabies immunoglobulin (human), intravenous respiratory syncytial virus immunoglobulin (human), Rh₀(D) Immunoglobulin (human), intravenous Rh₀(D) Immunoglobulin (human), tetanus immunoglobulin (human), varicella-zoster immunoglobulin. The at least one biological response modifier may be at least one selected from the group consisting of alfuzin, recombinant human erythropoietin alpha, filgrastim, glatiramer acetate for injection, interferon alfacon-1, interferon alpha-2 a (recombinant), interferon alpha-2 b (recombinant), interferon beta-1 a, interferon beta-1 b (recombinant), interferon gamma-1 b, levamisole hydrochloride, oprelvekin, and sargrastim. (see, e.g., Nursing 2001 Drug Handbook, page 964-1040).

The at least one ophthalmic anti-infective agent may be at least one selected from bacitracin, chloramphenicol, ciprofloxacin hydrochloride, erythromycin, gentamycin sulfate, ofloxacin 0.3%, polymyxin B sulfate, sulfacetamide sodium 10%, sulfacetamide sodium 15%, sulfacetamide sodium 30%, tobramycin, vidarabine. The at least one ophthalmic anti-inflammatory agent may be at least one selected from dexamethasone, dexamethasone sodium phosphate, sodium diclofenac 0.1%, flurbiprofen, ketorolac, prednisolone acetate (suspension) and prednisolone sodium phosphate (solution). The at least one miotic may be at least one selected from the group consisting of chloroacetylcholine, carbachol (intraocular), carbachol (topical), iodoethephon thiocholine, pilocarpine hydrochloride, pilocarpine nitrate. The at least one pupillary releasing agent may be at least one selected from atropine sulfate, cyclopentolate hydrochloride, epinephrine hydrochloride, phenylephrine cycloboronate, homatropine hydrobromide, neosynephrine hydrochloride, scopolamine hydrobromide, and tropicamide. The at least one ophthalmic vasoconstrictor may be at least one selected from naphazoline hydrochloride, oxymetazoline hydrochloride, tetrahydrozoline hydrochloride. The at least one miscellaneous ophthalmic agent may be at least one selected from the group consisting of alaclonidine hydrochloride, betaxolol hydrochloride, brimonidine tartrate, carteolol hydrochloride, dipivefrine hydrochloride, dorzolamide hydrochloride, emetine ester, sodium fluorescein, ketotifen fumarate, latanoprost, levobunolol hydrochloride, metiprolol hydrochloride, sodium chloride (hypertonic), and tiamulol maleate. The at least one otic agent may be at least one selected from boric acid, urea hydrogen peroxide, chloramphenicol, triethanolamine polypeptide oleate-condensates. The at least one nasal Drug may be at least one selected from beclomethasone dipropionate, budesonide, ephedrine sulfate, epinephrine hydrochloride, flunisolide, fluticasone propionate, naphazoline hydrochloride, oxymetazoline hydrochloride, neofolin hydrochloride, tetrahydrozoline hydrochloride, triamcinolone acetonide, xylometazoline hydrochloride (pages 1041-97 of Nursing 2001 Drug Handbook).

The at least one topical anti-infective agent may be at least one selected from acyclovir, amphotericin B, azelaic acid cream, bacitracin, butoconazole nitrate, clindamycin phosphate, clotrimazole, econazole nitrate, erythromycin, gentamicin sulfate, ketoconazole, sulfamylon acetate, metronidazole (topical), miconazole nitrate, mupirocin, naftifine hydrochloride, neomycin sulfate, nitrofurazone, nystatin, silver sulfadiazine, terbinafine hydrochloride, troconazole, tetracycline hydrochloride, tioconazole, tolnaftate. The at least one sarcoptic or pediculicide may be at least one selected from crotamiton, lindane, permethrin, pyrethrin. The at least one topical corticosteroid can be at least one member selected from the group consisting of betamethasone dipropionate, betamethasone valerate, clobetasol propionate, prednisolone, desoximetasone, dexamethasone sodium phosphate, diflorasone diacetate, fluocinonide, fluocinolone acetonide, fluticasone propionate, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone valerate, mometasone furoate, triamcinolone acetonide (see, e.g., pages 1098 and 1136 of Nursing 2001 Drug Handbook).

The at least one vitamin or mineral may be selected from vitamin A, vitamin B complex, cyanocobalamin, folic acid, hydroxocobalamin, leucovorin calcium, niacin, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin C, vitamin D, cholecalciferol, ergocalciferol, vitamin D analogues, 1-alpha-hydroxyvitamin D₂Parocalcitol, vitamin E, vitamin K analogs, vitamin K₁Sodium fluoride, sodium fluoride (topical), trace elements, chromium, copper, iodine, manganese, selenium, zinc. The at least one calorie may be selected from the group consisting of amino acid infusions (crystallized), amino acid infusions dissolved in glucose, amino acid infusions and electrolytes dissolved in glucose, amino acid infusions for liver failure, amino acid infusions for high metabolic stress, glucose, fat emulsions, medium chain triglycerides (see, e.g., pages 1137-63 of Nursing 2001 Drug Handbook).

The anti-amyloid antibody composition of the present invention may further comprise at least one any suitable and effective amount of a composition or pharmaceutical composition comprising at least one anti-amyloid antibody for use in a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy, which composition may optionally further comprise at least one TNF antagonist (such as, but not limited to, a TNF chemical or protein antagonist, a TNF monoclonal or polyclonal antibody or fragment, a soluble TNF receptor (e.g., p55, p70 or p85) or fragment thereof, a fusion polypeptide, or a small molecule TNF antagonist, e.g., TNF binding protein I or II (TBP-I or TBP-II), nerelimama, infliximab, enteracept, CDP-571, af-870, afelec, CDP-cit, etc.), an antirheumatic (e.g., methotrexate, acethiogold, glucethiol, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalazine), muscle relaxants, anesthetics, non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blockers, antimicrobials (e.g., aminoglycosides, antifungals, antiparasitics, antivirals, carbapenems, cephalosporins, fluoroquinolones, macrolides, penicillins, sulfonamides, tetracyclines, another antimicrobial), antipsoriatics, corticosteroids, anabolic steroids, diabetes-related agents, minerals, nutrients, thyroid agents, vitamins, calcium-related hormones, antidiarrheals, antitussives, antiemetics, antiulcers, laxatives, anticoagulants, erythropoietin (e.g., recombinant human erythropoietin alpha), At least one of filgrastim (e.g., G-CSF, recombinant human granulocyte colony stimulating factor), sargrastim (GM-CSF, leukol), an immunological agent, an immunoglobulin, an immunosuppressive agent (e.g., basiliximab, cyclosporine, daclizumab), a growth hormone, a hormone replacement drug, an estrogen receptor modulator, a mydriatic agent, a cycloplegic, an alkylating agent, an antimetabolite, a mitotic inhibitor, a radioprotective agent, an antidepressant, an antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, an agonist, donepezil, monocidazolidine, an asthma drug, a beta agonist, an inhaled steroid, a leukotriene inhibitor, methylxanthine, cromolyn, epinephrine or analog, an alfa deoxyribonuclease (lmmozyme), a cytokine, or a cytokine antagonist. Non-limiting examples of such cytokines include, but are not limited to, any of IL-1 through IL-23. Suitable dosages are well known in the art. See, for example, Wells et al, eds, Pharmacotherapy Handbook, second edition, apple and Lange, Stamford, CT (2000); PDRPHARMACOPEIa, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA (2000), each of which is incorporated herein in its entirety by reference.

Such anti-cancer or anti-infective drugs may also include toxin molecules that are complexed, conjugated to, co-formulated with, or co-administered with at least one antibody of the present invention. The toxin may optionally act to selectively kill pathological cells or tissues. The pathological cell can be a cancer or other cell. These toxins may be, but are not limited to, purified or recombinant toxins or toxin fragments containing at least one functional cytotoxic domain of a toxin, e.g., selected from at least one ricin, diphtheria toxin, snake venom toxin, or bacterial toxin. The term toxin may also include any naturally occurring, mutated or recombinant bacterial or viral produced endotoxins and exotoxins that may produce any pathological condition leading to death in humans and other mammals, including toxin shock. These toxins may include, but are not limited to, enterotoxigenic heat labile endotoxin (LT), heat stable enterotoxin (ST), Shigella cytotoxin (Shigella), Aeromonas enterotoxin (Aeromonas) enterotoxin, toxic shock syndrome toxin-1 (TSST-1), Staphylococcal enterotoxin (Staphyloccal) A (SEA), B (SEB) or C (SEC), Streptococcal enterotoxin (Streptococcus) and the like. Such bacteria include, but are not limited to, enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (e.g., serotype 0157: H7 strain), Staphylococcus species (e.g., Staphylococcus aureus (Staphylococcus aureus), Staphylococcus pyelogenes, Shigella species (e.g., Shigella dysenteriae), Shigella flexneri (Shigella flexneri), Shigella pallida (Shigella boydii), and Shigella sonnei (Shigella sonnei)), Salmonella species (e.g., Salmonella typhi, Salmonella cholerae-sui, Salmonella enteritidis), Clostridium species (Clostridium) species (e.g., Clostridium perfringens, Clostridium difficile), Clostridium species (e.g., Clostridium botulinum), Clostridium difficile (Clostridium botulinum), Clostridium species (Clostridium botulinum), heliobacter pyrulori), Aeromonas species (e.g., Aeromonas sobria (Aeromonas sobria), Aeromonas hydrophila (Aeromonas hydrophila), Aeromonas caviae (Aeromonas caviae)), pleisomonas thermoaminolytics, yersinia enterocolitica (yersinia enterocolitica), vibrio (vibrio) species (e.g., vibrio cholerae (vibrio cholera), vibrio parahaemolyticus (vibrio parahaemolyticus)), Klebsiella (Klebsiella) species, Pseudomonas aeruginosa (Pseudomonas aeruginosa), and streptococcus (streptococcus) strains see, e.g., Stein, editoria, INTERNAL MEDICINE, third edition, pages 1-13, litters, Brown, co, 1990); evans et al, eds, BacterialInfections of Humans: epidemic and Control, second edition, page 239-; mandell et al, Principlex and Practice of Infections Diseases, third edition, Churchill Livingstone, New York (1990); berkow et al, eds, The Merck Manual, 16 th edition, Merck and co., Rahway, n.j., 1992; wood et al, FEMS microbiology immunology, 76: 121-134 (1991); marrack et al, Science, 248: 705-711(1990), the contents of which are incorporated herein by reference in their entirety.

The anti-amyloid antibody compound, composition or combination of the present invention may further contain at least one suitable adjuvant such as, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants and the like. Pharmaceutically acceptable adjuvants are preferred. Non-limiting examples and methods of preparing such sterile solutions are well known in the art, such as, but not limited to, Gennaro, eds, Remington's Pharmaceutical Sciences, 18 th edition, Mack Publishing Co, (Easton, Pa.) 1990. Pharmaceutically acceptable carriers may be routinely selected which are suitable for the mode of administration, solubility and/or stability of the anti-amyloid antibody, fragment or variant composition, as is well known in the art or as described herein.

Pharmaceutical excipients and additives useful in the present invention may include, but are not limited to, proteins, peptides, amino acids, lipids, and sugars (e.g., sugars, including mono-, di-, tri-, tetra-, and oligosaccharides; derivatized sugars, such as sugar alcohols, aldonic acids, esterified sugars, and the like; and polysaccharides or sugar polymers), which may be present alone or in combination, and constitute 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin, such as Human Serum Albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/antibody components that may also play a role in buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

Sugar excipients suitable for use in the present invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; and sugar alcohols such as mannitol, xylitol, maltitol (maltotol), lactitol (lactitol), xylitol sorbitol (glucitol), inositol, and the like. Preferred sugar excipients for use in the present invention are mannitol, trehalose, and raffinose.

The anti-amyloid antibody composition may further comprise a buffer or a pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; tris, Tris hydrochloride, or phosphate buffer. Preferred buffering agents for use in the present compositions are organic acid salts, such as citrate salts.

In addition, the anti-amyloid antibody compositions of the present invention may include polymeric excipients/additives such as polyvinylpyrrolidone, ficols (polymeric sugars), dextran binders (e.g., cyclodextrins, such as 2-hydroxypropyl- β -cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates, such as "TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

These and other additional well-known pharmaceutical excipients and/or additives for use in The anti-amyloid antibody, partial or variant compositions according to The present invention are well known in The art, for example, as exemplified by "Remington: The Science & Practice of Pharmacy", 19 th edition, Williams & Williams, (1995), and "Physician's Desk Reference", 52 th edition, Medical Economics, Montvale, NJ (1998), The disclosures of which are incorporated herein by Reference. Preferred carrier or excipient materials are sugars (e.g., sugars and sugar alcohols) and buffers (e.g., citrate) or polymeric agents.

Preparation

As indicated above, the present invention provides stable formulations suitable for pharmaceutical or veterinary use, preferably phosphate buffered saline or selected salts, as well as preservative solutions and formulations containing a preservative, and multi-purpose preservative formulations containing at least one anti-amyloid antibody in a pharmaceutically acceptable formulation. Preservative formulations contain at least one well-known preservative or a preservative optionally selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof in an aqueous diluent. Any suitable concentration or mixture may be used, as is known in the art, such as, for example, 0.001-5%, or any range or value therein, such as, but not limited to, 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.4, 3.6, 3.4, 3.6, 3.7, 4.4, 4.6, 4, 4.4, 4, 4.6, 4, 4.6, or any range therein. Non-limiting examples include, but are not limited to, 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkyl parabens (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.075, 0.02, 0.05, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75%, etc.).

As indicated above, the present invention provides an article of manufacture comprising packaging material and at least one bottle containing a solution of at least one anti-amyloid antibody optionally in an aqueous diluent with a defined buffer and/or preservative, wherein the packaging material comprises a label indicating that such a solution can be stored for 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or more. The invention also includes an article of manufacture comprising packaging material, a first bottle containing lyophilized at least one anti-amyloid antibody, and a second bottle containing an aqueous diluent of a defined buffer or preservative, wherein the packaging material comprises a label that instructs a patient to reconstitute the at least one anti-amyloid antibody with the aqueous diluent to form a solution that can be stored for 24 hours or more.

The at least one anti-amyloid antibody used according to the present invention may be produced by recombinant methods, including production from mammalian cells or transgenic preparations, or may be purified from other biological sources as described herein or as known in the art.

The range of at least one anti-amyloid antibody in the product of the invention includes amounts that upon reconstitution (if a wet/dry system) yields a concentration of about 1.0 μ g/ml to about 1000mg/ml, however lower and higher concentrations are feasible and depending on the intended delivery vehicle, for example, solution formulations will differ from transdermal patch, pulmonary, transmucosal or osmotic or minipump methods.

Preferably, the aqueous diluent optionally further comprises a pharmaceutically acceptable preservative. Preferred preservatives include those selected from phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof. The concentration of preservative used in the formulation is a concentration sufficient to produce an antimicrobial effect. The concentration depends on the preservative selected and can be readily determined by the skilled person.

Other excipients, for example, isotonic agents, buffers, antioxidants, preservative enhancers may optionally and preferably be added to the diluent. Isotonic agents, such as glycerol, are generally used in known concentrations. Physiologically tolerable buffers are preferably added to provide improved pH control. The formulation may cover a wide range of pH, such as from about pH 4 to about pH 10, preferably in the range of about pH 5 to about pH 9, and most preferably in the range of about 6.0 to about 8.0. Preferably, the formulations of the present invention have a pH of about 6.8 to about 7.8. Preferred buffers include phosphate buffered saline, most preferably sodium phosphate, especially Phosphate Buffered Saline (PBS).

Other additives, such as pharmaceutically acceptable solubilizers, e.g., Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymer), and PEG (polyethylene glycol) or nonionic surfactants, e.g., polysorbate 20 or 80 or poloxamers 184 or 188,polyls, other block copolymers, and chelating agents such as EDTA and EGTA may optionally be added to the formulation or composition to reduce aggregation. These additives are particularly useful if the formulation is to be administered using a pump or a plastic container. The presence of a pharmaceutically acceptable surfactant can reduce the tendency of the protein to aggregate.

The formulations of the present invention may be prepared by a method comprising mixing at least one anti-amyloid antibody with a preservative selected from phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof, in an aqueous diluent. The at least one anti-amyloid antibody is mixed with a preservative in an aqueous diluent using conventional dissolution and mixing methods. To prepare a suitable formulation, for example, at least one measured amount of anti-amyloid antibody in buffer is combined with a desired preservative in buffer in an amount sufficient to provide the desired concentration of protein and preservative. Those skilled in the art will recognize variations of this method. For example, the order of the components added, whether additional additives are used, the temperature and pH at which the formulation is prepared are all factors that can be optimized with respect to concentration and mode of administration used.

The claimed formulation may be provided to the patient as a clear solution or as two bottles containing one bottle of lyophilized at least one anti-amyloid antibody reconstituted in a second bottle containing water, preservatives and/or excipients, preferably phosphate buffer and/or saline in an aqueous diluent and selected salts. A single solution vial or two vials to be reconstituted may be reused multiple times and may satisfy one or more cycles of patient treatment, thereby providing a more convenient treatment protocol than currently available.

The claimed product may be used for administration over a period of time ranging from immediately to 24 hours or more. Thus, the presently claimed article provides significant advantages to patients. The formulations of the present invention may optionally be safely stored at temperatures of about 2 ℃ to about 40 ℃ and retain the biological activity of the protein for extended periods of time, allowing the packaging label to indicate that the solution may be stored and/or used for periods of 6, 12, 18, 24, 36, 48, 72, or 96 hours or more. Such labels may include use for up to 1-12 months, half a year, and/or two years if a preservative diluent is used.

The solution of at least one anti-amyloid antibody of the present invention may be prepared by a method comprising mixing at least one antibody in an aqueous diluent. Mixing is carried out using conventional dissolution and mixing methods. To prepare a stable diluent, for example, at least one measured amount of antibody in water or buffer is combined in an amount sufficient to provide the desired concentration of protein and optionally a preservative or buffer. Those skilled in the art will recognize variations of this method. For example, the order of the components added, whether additional additives are used, the temperature and pH at which the formulation is prepared are all factors that can be optimized with respect to concentration and mode of administration used.

The claimed product may be provided to the patient as a clear solution or as two vials containing one vial of lyophilized at least one anti-amyloid antibody reconstituted with a second vial containing an aqueous diluent. A single solution vial or two vials to be reconstituted may be reused multiple times and may satisfy one or more cycles of patient treatment, thereby providing a more convenient treatment protocol than currently available.

The claimed product may be provided to the patient indirectly, by providing the pharmacy, clinic or other such institution and facility with a clear solution or two vials containing one vial of lyophilized at least one anti-amyloid antibody reconstituted with a second vial containing an aqueous diluent. The size of the clarified solution in this case may be up to 1 liter or more, thereby providing a larger reservoir (reservoir) from which smaller portions of the at least one antibody solution may be retrieved one or more times for transfer into smaller bottles and provided by the pharmacy or clinic to their customers and/or patients.

Known devices containing these single vial systems include those pen injector devices for delivering solutions, such as BD Pens, BDPen，And, andGenotronormHumatroRoferonJ-tipNeedle-Freefor example, by Becton Dickensen (Franklin Lakes, NJ, www.bectondickenson.com), Disetronic (Burgdorf, Switzerland, www.disetronic.com; Bio Ject, Poland, Oregon (www.bioject.com); National Medical Products, Weston Medical (Peterborough, UK, www.weston-medical.com), Medi-JectThose produced or developed by Corp (Minneapolis, MN, www.mediject.com). Known devices containing dual vial systems include those pen injector systems for reconstituting lyophilized drugs in a cartridge for delivering a reconstitution solution, such as

The presently claimed product includes packaging materials. The packaging material provides the conditions under which the product can be used, in addition to the information required by the regulatory agency. The packaging material of the present invention provides patient instructions directing the patient to reconstitute the at least one anti-amyloid antibody in an aqueous diluent to form a solution and to use the solution of the dual vial, wet/dry product over a period of 2-24 hours or more. For single bottle solution products, the label indicates that such solutions can be used over a period of 2-24 hours or more. The presently claimed product may be used for human pharmaceutical use.

The formulations of the present invention may be prepared by a method comprising mixing at least one anti-amyloid antibody and a selected buffer, preferably a phosphate buffer containing saline or a selected salt. The mixing of the at least one anti-amyloid antibody and the buffer in the aqueous diluent is carried out using conventional dissolution and mixing methods. To prepare a stable formulation, for example, a measured amount of at least one of water or buffer is mixed in water with a desired buffer in an amount sufficient to provide the desired concentration of the protein and buffer. Those skilled in the art will recognize variations of this method. For example, the order of the components added, whether additional additives are used, the temperature and pH at which the formulation is prepared are all factors that can be optimized with respect to concentration and mode of administration used.

The claimed stable or preserved formulation may be provided to a patient as a clear solution or two bottles containing one bottle of lyophilized at least one anti-amyloid antibody reconstituted with a second bottle containing a preservative or buffer and excipients dissolved in an aqueous diluent. A single solution vial or two vials to be reconstituted may be reused multiple times and may satisfy one or more cycles of patient treatment, thereby providing a more convenient treatment protocol than currently available.

Other formulations or methods of stabilizing anti-amyloid antibodies may result in clear solutions other than lyophilized powders containing the antibody. In a non-clear solution, there are formulations containing suspensions of particles, which are anti-amyloid antibodies containing structures of different sizes and are known as microspheres, microparticles, nanoparticles, nanospheres or liposomes. Such relatively uniform substantially spherical particle formulations containing an active agent can be formed by contacting an aqueous phase containing the active ingredient and a polymer with a non-aqueous phase, and then evaporating the non-aqueous phase to cause the particles to agglomerate from the aqueous phase, as taught in U.S.4,589,330. Porous microparticles can be prepared as taught in U.S.4,818,542 using a first phase containing an active agent and a polymer dispersed in a continuous solvent and removing the solvent from the suspension by lyophilization or dilution-extraction-precipitation. Preferred polymers for such formulations are natural or synthetic copolymers or polymers selected from the group consisting of gleatin agar, starch, arabinogalactans, albumin, collagen, polyglycolic acid, polylactic acid, glycolide-L (-) lactide poly (epsilon-caprolactone, epsilon-caprolactone-lactic acid copolymer, epsilon-caprolactone-glycolic acid copolymer, poly (beta-hydroxybutyric acid), polyethylene oxide, polyethylene, poly (alkyl 2-cyanoacrylates), poly (hydroxyethyl methacrylate), polyamides, poly (amino acids), poly (2-hydroxyethyl DL-aspartamide), poly (ester urea), poly (L-phenylalanine/ethylene glycol/1, 6-diisocyanatohexane) and poly (methyl methacrylate), especially preferred polymers are polyesters, such as polyglycolic acid, polylactic acid, glycolide-L (-) lactide poly (epsilon-caprolactone, epsilon-caprolactone-lactic acid copolymer, and epsilon-caprolactone-glycolic acid copolymer solvents for dissolving the polymer and/or active agent include water, hexafluoroisopropanol, methylene chloride, tetrahydrofuran, hexane, benzene, or hexafluoroacetone sesquihydrate.

Dry powder formulations can be obtained by methods other than lyophilization, such as removal of the solvent by spray drying or by evaporation or by precipitation of the crystalline composition, followed by one or more steps of removal of the aqueous or non-aqueous solvent. The preparation of spray-dried antibody formulations is taught in U.S.6,019,968. Antibody-based dry powder compositions can be produced by spray drying a solution or slurry of the antibody in a solvent and optional excipients under conditions to provide a respirable dry powder. The solvent may include polar compounds such as water and ethanol which are easily dried. Antibody stability can be enhanced by spray drying under anaerobic conditions, such as under a nitrogen blanket or by using nitrogen as the drying gas. Another relatively dry formulation is a dispersant having a microstructure with many pores dispersed in a suspension medium which typically contains a hydrofluoroalkane propellant as taught in WO 9916419. The stabilized dispersion can be administered to the lungs of a patient using a metered dose inhaler. Equipment for commercial production of spray-dried drugs is produced by Buchi ltd.

At least one anti-amyloid antibody in a stable or preserved formulation or solution described herein may be administered to a patient according to the present invention by a variety of delivery methods, including Subcutaneous (SC) or Intramuscular (IM) injection; transdermal, pulmonary, transmucosal, implantation, osmotic pump, cartridge, micropump, or other methods as understood by those of skill in the art and as known in the art.

Therapeutic applications

The present invention also provides methods of using at least one amyloid antibody of the present invention to modulate or treat at least one amyloid-related disease in a cell, tissue, organ, animal or patient, as known in the art or described herein.

The present invention also provides methods of modulating or treating at least one amyloid-related disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of obesity, immune-related diseases, cardiovascular disease, infectious disease, malignant disease, or neuropathy. Such amyloid-associated diseases may include, but are not limited to, any amyloidosis, systemic amyloidosis, Alzheimer's Disease (AD), sporadic Alzheimer's disease, familial Alzheimer's disease, lewy body variant Alzheimer's disease, prion diseases, primary systemic amyloidosis, secondary systemic amyloidosis, compact systemic amyloidosis, monoclonal protein systemic amyloidosis, active systemic amyloidosis, hereditary apoA1 amyloidosis, hereditary lysozyme amyloidosis, insulin-related amyloid protein, Finnish-type familial amyloidosis, familial subepithelial amyloidosis, familial non-neuropathic amyloidosis, British dementia, hereditary cerebral amyloid angiopathy, and Alzheimer's disease, Hemodialysis-associated amyloidosis, familial amyloid polyneuropathy, familial amyloidotic polyneuropathy, adult-onset diabetes mellitus, type II diabetes mellitus, hereditary renal amyloidosis, pituitary amyloidosis, injection-localized amyloidosis, medullary carcinoma, atrial amyloidosis, isolated atrial amyloidosis, hereditary cerebral amyloid angiopathy, hereditary fibrinogen alpha-chain amyloidosis, Parkinson's disease, Huntington's chorea, spongiform encephalopathy, prion-associated transmissible spongiform encephalopathy, Amyotrophic Lateral Sclerosis (ALS), familial amyotrophic lateral sclerosis, chronic obstructive pulmonary disease, and the like.

The present invention also provides methods of modulating or treating at least one neurological or amyloid-related disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of: neurodegenerative diseases, multiple sclerosis, migraine, AIDS dementia complex, demyelinating diseases, such as multiple sclerosis and acute transverse myelitis; extrapyramidal and cerebellar disorders, such as lesions of the spinal cortical system; a disorder of the basal ganglia or a disorder of the cerebellum; hyperkinetic movement disorders such as Huntington's disease and senile chorea; drug-induced movement disorders, such as drug-induced movement disorders that block CNS dopamine receptors; hypokinetic movement disorders, such as parkinson's disease; progressive supranuclear palsy; structural lesions of the cerebellum; spinocerebellar degeneration, such as spondylotic ataxia, friedreich's ataxia, cerebellar cortical degeneration, multiple system degeneration (multiple systems degeneration) (Mencel, Dejerine-Thomas, Shi-Drager, and Machado-Joseph); systemic disorders (Refsum's disease, blood beta-lipoprotein deficiency, ataxia, telangiectasia, and mitochondrial multisystem disorders); demyelinating core disorders, such as multiple sclerosis, acute transverse myelitis; and disorders of the motor unit, such as neurogenic muscular atrophy (anterior horn cell degeneration, such as amyotrophic lateral sclerosis, spinal muscular atrophy in infancy, and spinal muscular atrophy in adolescents); alzheimer's disease; middle-aged Down syndrome; disseminated Lewy body disease; lewy-type senile dementia; Wernicke-Korsakoff syndrome; chronic alcoholism; Creutzfeldt-Jakob disease; subacute sclerosing panencephalitis, Hallerrorden-Spatz disease; and Dementia pugilistica (Dementia pugilistica), and the like. Such methods may optionally comprise administering to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy an effective amount of a composition or pharmaceutical composition comprising at least one TNF antibody or specified portion or variant. See, for example, Merck Manual, 16 th edition, Merck & Company, Rahway, NJ (1992).

The present invention also provides methods of modulating or treating at least one immune or amyloid-related disease in a cell, tissue, organ, animal or patient, including but not limited to at least one of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, gastric ulceration, seronegative arthropathy, osteoarthritis, inflammatory bowel disease, ulcerative colitis, systemic lupus erythematosus, antiphospholipid syndrome, iridocyclitis/uveitis/optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis/wegener's granulomatosis, sarcoidosis, orchitis/vasectomy reversal processes, allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergic contact dermatitis, chronic obstructive pulmonary disease, allergic conjunctivitis, allergic pneumonia, transplantation, organ transplant rejection, graft-versus-host disease, systemic inflammatory response syndrome, sepsis syndrome, gram-positive sepsis, gram-negative sepsis, culture-negative sepsis, fungal sepsis, neutropenic fever, urosepsis, meningococcemia, trauma/hemorrhage, burn injury, ionizing radiation exposure, acute pancreatitis, adult respiratory distress syndrome, rheumatoid arthritis, alcohol-induced hepatitis, chronic inflammatory diseases, sarcoidosis, Crohn's disease, sickle cell anemia, diabetes, nephropathy, atopic disease, hypersensitivity reactions, allergic rhinitis, hay fever, perennial rhinitis, conjunctivitis, endometriosis, asthma, urticaria, systemic anaplaxis, dermatitis, pernicious anemia, hemolytic disease, thrombocytopenia, Transplant rejection of any organ or tissue, kidney transplant rejection, heart transplant rejection, liver transplant rejection, pancreas transplant rejection, lung transplant rejection, Bone Marrow Transplant (BMT) rejection, skin allograft rejection, cartilage transplant rejection, bone transplant rejection, small intestine transplant rejection, fetal thymus implant rejection, parathyroid transplant rejection, xenograft rejection of any organ or tissue, allograft rejection, anti-receptor hypersensitivity, Graves 'disease, Raynoud's disease, insulin resistant diabetes type B, asthma, myasthenia gravis, antibody-mediated cytotoxicity, type III hypersensitivity, systemic lupus erythematosus, POEMS syndrome (polyneuropathy, organ megaly, endocrinopathy, monoclonal proteopathy, and cutaneous change syndrome), polyneuropathy, organ megaly, endocrinopathy, bone marrow transplant rejection, bone graft rejection, bone marrow transplant rejection, bone graft rejection, Monoclonal gammopathy, dermato logical syndrome, antiphospholipid syndrome, pemphigus, scleroderma, mixed connective tissue disease, idiopathic Addison's disease, diabetes, chronic active hepatitis, primary biliary cirrhosis, vitiligo, vasculitis, post MI cardiotomy syndrome, hypersensitivity IV, contact dermatitis, hypersensitivity pneumonitis, allograft rejection, intracellular organism-induced granuloma, drug sensitivity, metabolism/spontaneity, Wilson's disease, hemochromatosis, alpha-1-antitrypsin deficiency, diabetic retinopathy, lymphoma goiter, osteoporosis, hypothalamus-pituitary-adrenal axis assessment, primary biliary cirrhosis, thyroiditis, encephalomyelitis, cachexia, cystic fibrosis, neonatal chronic lung disease, Chronic Obstructive Pulmonary Disease (COPD), familial hepatolytic myelocytosis, dermatological conditions, psoriasis, alopecia, nephrotic syndrome, nephritis, glomerulonephritis, acute renal failure, hemodialysis, uremia, toxicity, preeclampsia, okt3 treatment, anti-cd 3 treatment, cytokine treatment, chemotherapy, radiation therapy (e.g., including, but not limited to, asthenia, anemia, cachexia, etc.), chronic salicylates, and the like. See, for example, the Merck Manual, 12-17 th edition, Merck & Company, Rahway, NJ (1972, 1977, 1982, 1987, 1992, 1999), Pharmacotherapy Handbook, Wells et al, second edition, Appletonand Lange, Stamford, Conn. (1998, 2000), each of which is incorporated herein in its entirety by reference.

The present invention also provides methods of modulating or treating at least one cardiovascular or amyloid-related disease in a cell, tissue, organ, animal or patient, including but not limited to at least one of the following: cardiac stunning syndrome, myocardial infarction, congestive heart failure, stroke, ischemic stroke, bleeding, atherosclerosis, restenosis, diabetic arteriosclerosis, hypertension, arterial hypertension, renovascular hypertension, fainting, shock, cardiovascular syphilis, heart failure, pulmonary heart disease, primary pulmonary hypertension, arrhythmia, atrial ectopic beating, atrial flutter, atrial fibrillation (sustained or paroxysmal), post-perfusion syndrome, cardiopulmonary bypass inflammatory response, turbulent or multi-acquired atrial tachycardia, regular stenotic QRS tachycardia, specific arrhythmia, ventricular fibrillation, bundle of his arrhythmia, atrioventricular block, bundle branch block, myocardial ischemic disorder, coronary artery disease, angina petoris, myocardial infarction, cardiomyopathy, dilated congestive cardiomyopathy, Restrictive cardiomyopathy, valvular heart disease, endocarditis, pericardial disease, cardiac tumors, aortic and peripheral aneurysms, aortic dissection, aortic inflammation, obstruction of the abdominal aorta and its branches, peripheral vascular disorders, obstructive arterial disorders, peripheral atherosclerotic disease, thromboangiitis obliterans, functional peripheral arterial disorders, Raynaud's phenomenon and disease, acrocyanosis, erythromelalgia, venous disease, venous thrombosis, varicose veins, arteriovenous fistulas, lymphederma, lipoedema, unstable angina, reperfusion injury, post-pump syndrome (postpump syndrome), ischemia-reperfusion injury, and the like. Such methods may optionally comprise administering to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy an effective amount of a composition or pharmaceutical composition comprising at least one anti-amyloid antibody.

The present invention also provides methods of modulating or treating at least one infectious disease or amyloid-related disease in a cell, tissue, organ, animal or patient, including but not limited to at least one of the following: acute or chronic bacterial infections, acute and chronic parasites or infectious processes including bacterial, viral and fungal infections, HIV infection/HIV neuropathy, meningitis, hepatitis (e.g., type a, type b, type c, etc.), septic arthritis, peritonitis, pneumonia, epiglottitis, escherichia coli 0157: h7, hemolytic uremic syndrome/thrombolytical thrombocytopenic purpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, mycobacterium tuberculosis, mycobacterium intracellulare, pneumocystis carinii pneumonia, pelvic inflammatory disease, orchitis/epididymitis, legionella, Lyme disease, influenza a, EB virus, viral-associated hepatocytic syndrome, encephalitis/aseptic meningitis, and the like.

The invention also provides methods of modulating or treating at least one malignant or amyloid-related disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of leukemia, Acute Lymphoblastic Leukemia (ALL), acute lymphocytic leukemia, B-cell, T-cell or FAB ALL, Acute Myeloid Leukemia (AML), acute myeloid leukemia, Chronic Myelogenous Leukemia (CML), Chronic Lymphocytic Leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, tumor-related syndrome/hypercalcemia syndrome of malignancy, malignant tumor-associated syndrome/hypercalcemia syndrome, malignant tumor-associated syndrome, and/or malignant tumor-associated disease in a cell, tissue, organ, animal or patient, Solid tumors, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary non-polyposis cancer, hodgkin's lymphoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, metastatic disease, cancer-related bone resorption, cancer-related bone pain, and the like. Any of the methods of the present invention may comprise administering to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy an effective amount of a composition or pharmaceutical composition comprising at least one anti-amyloid antibody. Such methods may optionally further comprise co-administration or combination therapy to treat such diseases or disorders, wherein administration of the at least one anti-amyloid antibody, specific portion or variant thereof further comprises administration prior to, concurrently with and/or after its administration of at least one selected from the group consisting of: at least one TNF antagonist (e.g., without limitation, a TNF chemical or protein antagonist, a TNF monoclonal or polyclonal antibody or fragment, a soluble TNF receptor (e.g., p55, p70, or p85) or fragment thereof, a fusion polypeptide, or a small molecule TNF antagonist, e.g., TNF binding protein I or II (TBP-I or TBP-II), nerelimomab, infliximab, enteracept, CDP-571, CDP-870, afelomab, lenercept, etc.), a antirheumatic (e.g., methotrexate, acethiomeglumine, glucosothiadine, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflulomide, sulfasalazine), a muscle relaxant, an anesthetic, a non-steroidal anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial (e.g., aminoglycosides), a local anesthetic, a neuro antagonist, Antifungal agents, antiparasitic agents, antiviral agents, carbapenems, cephalosporins, fluoroquinolones, macrolides, penicillins, sulfonamides, tetracyclines, another antimicrobial agent), antipsoriatic agents, corticosteroids, anabolic steroids, diabetes-related agents, minerals, nutrients, thyroid agents, vitamins, calcium-related hormones, antidiarrheals, antitussives, antiemetics, antiulcers, laxatives, anticoagulants, erythropoietin (e.g., recombinant human erythropoietin α), filgrastim (e.g., G-CSF, recombinant human granulocyte colony stimulating factor), sargrastim (GM-CSF, leukocyteine), immunological agents, immunoglobulins, immunosuppressive agents (e.g., basiliximab, cyclosporins, daizelimumab), growth hormones, hormone replacement drugs, estrogen receptor modulators, mydriatic agents, Cycloplegics, alkylating agents, antimetabolites, mitotic inhibitors, radioprotective agents, antidepressants, antimanics, antipsychotics, anxiolytics, hypnotics, sympathomimetics, stimulants, donepezil, amantadine, asthma drugs, beta agonists, inhaled steroids, leukotriene inhibitors, methylxanthines, cromolyn, epinephrine or analogs, alfa-deoxyribonuclease (Pulmozyme), cytokines, or cytokine antagonists. Suitable dosages are well known in the art. See, for example, Wells et al, eds, Pharmacotherapy Handbook, second edition, apple and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA (2000); nursing2001 Handbook of Drugs, 21 st edition, Springhouse Corp., Springhouse, PA, 2001; health Professional's Drug Guide 2001, eds, Shannon, Wilson, Stang, preptic-Hall, Inc, Upper Saddle River, NJ, each of which is incorporated herein in its entirety by reference.

TNF antagonists (also including at least one antibody, specific portions and variants thereof of the present invention) suitable for the compositions, combination therapies, co-administrations, devices and/or methods of the present invention include, but are not limited to, anti-TNF antibodies, antigen-binding fragments thereof, and receptor molecules that specifically bind TNF; compounds that prevent and/or inhibit TNF synthesis, TNF release or its effect on target cells, such as thalidomide, tenidap, phosphodiesterase inhibitors (e.g., pentoxifylline and ciclopirox), A2b adenosine receptor agonists and A2b adenosine receptor enhancers; compounds that prevent and/or inhibit TNF receptor signaling, such as mitogen-activated protein (MAP) kinase inhibitors; compounds that block and/or inhibit membrane TNF cleavage, such as metalloproteinase inhibitors; compounds that block and/or inhibit TNF activity, such as Angiotensin Converting Enzyme (ACE) inhibitors (e.g., captopril); and compounds that block and/or inhibit TNF production and/or synthesis, such as MAP kinase inhibitors.

As used herein, a "tumor necrosis factor antibody," "TNF α antibody," or fragment, and the like, reduces, blocks, inhibits, eliminates, or interferes with TNF α activity in vitro, in situ, and/or preferably in vivo. For example, suitable TNF human antibodies of the invention can bind TNF α and include anti-TNF antibodies, antigen-binding fragments thereof, and specific mutants or domains thereof that specifically bind TNF α. Suitable TNF antibodies or fragments may also reduce, block, abrogate, interfere with, prevent and/or inhibit TNF RNA, DNA or protein synthesis, TNF release, TNF receptor signaling, membrane TNF cleavage, TNF activity, TNF production and/or synthesis.

The chimeric antibody cA2 consists of a high affinity neutralizing antigen-binding variable region of the mouse anti-human TNF α IgG1 antibody (designated a2) and the constant region K immunoglobulin of human IgG 1. The human IgG1Fc region increased the allogeneic antibody effector function, increased circulating serum half-life and reduced the immunogenicity of the antibody. The avidity and epitope specificity of chimeric antibody cA2 was derived from the variable region of murine antibody a 2. In a specific embodiment, a preferred source of nucleic acid encoding the variable region of murine antibody a2 is the a2 hybridoma cell line.

Chimeric a2(cA2) neutralized the cytotoxic effects of native and recombinant human TNF α in a dose-dependent manner. The affinity constant of chimeric antibody cA2 was calculated to be 1.04X 10 based on the binding assay of chimeric antibody cA2 and recombinant human TNF α¹⁰M^-1. A preferred method for determining monoclonal antibody specificity and affinity by competitive inhibition can be found in Harlow, et al, antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988; colligan et al, eds., Current protocols Immunology, Greene Publishing Assoc. and Wiley Interscience, New York, (1992-; kozbor et al, immunol. today, 4: 72-79 (1983); ausubel et al, Current Protocols in Molecular Biology, WileyInterscience, New York (1987-; and Muller, meth. enzymol, 92: 589 found in 601(1983), which are incorporated herein by reference in their entirety.

In a specific embodiment, the murine monoclonal antibody a2 is produced by a cell line designated c 134A. The chimeric antibody cA2 was generated from a cell line designated c 168A.

Additional examples of monoclonal anti-TNF antibodies that can be used in the present invention are described in the art (see, e.g., U.S. Pat. No. 5,231,024; Mller, a. et al, Cytokine 2 (3): 162-169 (1990); U.S. Pat. No. 07/943,852 (filed on 9/11/1992); rathjen et al, International publication No. WO 91/02078 (published 2/21 1991); rubin et al, EPO patent publication No. 0218868 (published 22/4 1987); yone et al, EPO patent application No. 0288088 (26/10 1988); liang, et al, biochem. biophysis. res. comm.137: 847-854 (1986); meager, et al Hybridoma 6: 305-311 (1987); fendly et al Hybridoma 6: 359-369 (1987); bringman, et al, Hybridoma 6: 489-507 (1987); and Hirai, et al, J.Immunol.Meth.96:57-62(1987), which are incorporated herein by reference in their entirety.

TNF receptor molecules

Preferred TNF receptor molecules for use in the present invention are those that bind TNF α with high affinity (see, e.g., Feldmann et al, International publication No. WO 92/07076 (published 30.4.1992); Schall et al, Cell 61: 361-. Specifically, 55kDa (p55 TNF-R) and 75kDa (p75TNF-R) TNF cell surface receptors are useful in the present invention. Truncated forms of these receptors containing the extracellular domain (ECD) of the receptor or a functional portion thereof (see, e.g., Corcoran et al, Eur. J. biochem. 223: 831-840(1994)) may also be used in the present invention. Truncated forms of the TNF receptor containing ECD have been detected in urine and serum as 30kDa and 40kDa TNF α inhibitory binding proteins (Engelmann, H. et al, J.biol.chem.265: 1531-1536 (1990)). TNF receptor multimeric molecules and TNF immunoreceptor fusion molecules and derivatives and fragments or portions thereof are additional examples of TNF receptor molecules for use in the methods and compositions of the present invention. The TNF receptor molecules useful in the present invention are characterized in that they are capable of treating patients for a long period of time and are good to greatly alleviate symptoms and have low toxicity. Low immunogenicity and/or high affinity, as well as other undefined properties, may contribute to the achieved therapeutic result.

The TNF receptor multimeric molecules for use in the present invention contain all or a functional portion of the ECD of two or more TNF receptors linked by one or more polypeptide linkers or other non-peptide linkers, such as polyethylene glycol (PEG). The multimeric molecule may further comprise a signal peptide of a secreted protein to direct expression of the multimeric molecule. These multimeric molecules and methods for their production have been described in U.S. application No. 08/437,533 (filed 5/9/1995), the contents of which are incorporated herein by reference in their entirety.

The TNF immunoreceptor fusion molecules used in the methods and compositions of the present invention include at least a portion of one or more immunoglobulin molecules and all or a functional portion of one or more TNF receptors. These immunoreceptor fusion molecules can be assembled as monomers, or hetero-or homo-multimers. The immunoreceptor fusion molecule may also be monovalent or multivalent. An example of such a TNF immunoreceptor fusion molecule is a TNF receptor/IgG fusion protein. TNF immunoreceptor fusion molecules and methods for their production have been described in the art (Lesslauer et al, Eur.J.Immunol.21: 2883-2886 (1991); Ashkenazi et al, Proc.Natl.Acad.Sci.USA 88: 10535-minus 10539 (1991); Peppel et al, J.exp.Med.174: 1483-minus 1489 (1991); Kolls et al, Proc.Natl.Acad.Sci.USA 91: 215-minus 219 (1994); Butler et al, Cytokine6 (6): 616-623 (1994); Baker et al, Eur.J.Immunol.24: 2040-minus 2048 (1994); Beutler et al, U.S. Pat. No. 5,447,851; and U.S. App. No. 08/442,133(1995, published 5-16), each of which is incorporated herein by reference in its entirety). Methods for generating immunoreceptor fusion molecules can also be found in Capon et al, U.S. Pat. nos. 5,116,964; capon et al, U.S. patent No. 5,225,538; and Capon et al, Nature 337: 525-531(1989), the contents of which are incorporated herein by reference in their entirety.

Functional equivalents, derivatives, fragments or regions of a TNF receptor molecule refer to portions of a TNF receptor molecule, or portions of the sequence of a TNF receptor molecule encoding a TNF receptor molecule, of sufficient size and sequence to functionally resemble a TNF receptor molecule that may be used in the present invention (e.g., bind TNF α with high affinity and have low immunogenicity). Functional equivalents of TNF receptor molecules may also include modified TNF receptor molecules that are functionally similar to the TNF receptor molecules used in the present invention (e.g., bind TNF α with high affinity and have low immunogenicity). For example, a functional equivalent of a TNF receptor molecule can contain a "SILENT" codon or one or more amino acid substitutions, deletions or additions (e.g., one acidic amino acid for another acidic amino acid; or one codon encoding the same or a different hydrophobic amino acid for another codon encoding a hydrophobic amino acid). See, Ausubel et al, Current Protocols in Molecular-Biology, Greene publishing Assoc. and Wiley-Interscience, New York (1987-.

Cytokines include any well-known cytokine, see, e.g., copew cytokines. Cytokine antagonists include, but are not limited to, any antibody, fragment or mimetic, any soluble receptor, fragment or mimetic, any small molecule antagonist, or any combination thereof.

Therapeutic treatment

Any of the methods of the present invention may include a method of treating an amyloid-mediated disease comprising administering to a cell, tissue, organ, animal or patient in need of such modulation, treatment or treatment an effective amount of a composition or pharmaceutical composition comprising at least one anti-amyloid antibody.

Such a method may optionally further comprise co-administration or combination therapy to treat such diseases or disorders, wherein the administration of the at least one anti-amyloid antibody, specific portion or variant thereof further comprises prior to, simultaneously with and/or after the administration thereof, administration of at least one agent selected from the group consisting of: anti-infective drugs, Cardiovascular (CV) system drugs, Central Nervous System (CNS) drugs, Autonomic Nervous System (ANS) drugs, respiratory tract drugs, Gastrointestinal (GI) tract drugs, hormonal drugs, drugs for fluid or electrolyte balance, hematological drugs, anticancer drugs, immunomodulatory drugs, ocular, otic or nasal drugs, topical drugs, nutraceuticals, and the like, at least one TNF antagonist (such as, but not limited to, a TNF antibody or fragment, a soluble TNF receptor or fragment thereof, a fusion polypeptide, or a small molecule TNF antagonist), antirheumatic drugs (such as methotrexate, acethiogulin, glucoraphanin, etazothioprine, etanercept, aurothiomalate, hydroxychloroquine sulfate, leflunom, sulfasalazine), muscle relaxants, anesthetics, non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, anti-inflammatory drugs (NSAIDs), and the like, Neuromuscular blockers, antimicrobials (e.g., aminoglycosides, antifungals, antiparasitics, antivirals, carbapenems, cephalosporins, fluoroquinolones, macrolides, penicillins, sulfonamides, tetracyclines, another antimicrobial), antipsoriatics, corticosteroids, anabolic steroids, diabetes-related agents, minerals, nutrients, thyroid agents, vitamins, calcium-related hormones, antidiarrheals, antitussives, antiemetics, antiulcers, laxatives, anticoagulants, erythropoeitins (e.g., recombinant human erythropoietin α), filgrastims (e.g., G-CSF, recombinant human granulocyte colony stimulating factor), sargrastims (GM-CSF, leukocyteines), immunizing agents, immunoglobulins, immunosuppressive agents (e.g., basiliximab, cyclosporins, daclizumab), growth hormones, Hormone replacement drugs, estrogen receptor modulators, mydriatic agents, cycloplegics, alkylating agents, antimetabolites, mitotic inhibitors, radioprotective agents, antidepressants, antimanics, antipsychotic agents, anxiolytic agents, hypnotics, sympathomimetics, stimulants, donepezil, monocidazine, asthma drugs, beta agonists, inhaled steroids, leukotriene inhibitors, methylxanthines, cromolyn, epinephrine or analogs, alfa-deoxyribonuclease (Pulmozyme), cytokines or cytokine antagonists. Such agents are well known in the art and include the formulation, indications, administration and administration of each of the agents given herein (see, e.g., Nursing 2001Handbook of Drugs, 21 st edition, Springhouse Corp., Springhouse, PA, 2001; Health Professional's Drug Guide 2001, eds., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper saddleriver, NJ; PharmotherapapyHandbook, Wells et al, eds., Appleton & Lange, mStanford, CT, all of which are incorporated herein by reference in their entirety every other place).

Typically, treatment of the pathological condition is achieved by administering an effective amount or dose of at least one anti-amyloid antibody composition which, in total, average range from at least about 0.01 to 500mg of at least one anti-amyloid antibody per kg of patient per dose, preferably at least about 0.1 to 100 mg of antibody per kg of patient per single or multiple administrations, depending on the specific activity contained in the composition. Alternatively, effective serum concentrations may include 0.1-5000 μ g/ml serum concentration per single or multiple administrations. Suitable dosages are well known to medical practitioners and will, of course, depend on the particular disease state, the specific activity of the composition being administered, and the particular patient undergoing treatment. In some cases, in order to achieve a desired therapeutic amount, repeated administrations must be provided, i.e., repeated single administrations of a specifically monitored or metered dose are administered, wherein the single administrations are repeated until a desired daily dose or effect is achieved.

Preferred dosages may optionally include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 94, 96, 97, 98, 99, 100 mg/kg or any range thereof, or to achieve 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 9.9, 9.0, 14.0, 14.5, 15.5, 9, 20, 9.0, 9.5, 9, 15.5, 9, 5, 15.0, 9, 5, 15.5, 15, 5, 9, 15.0, 9, 5, 9, 15.5, 15.0, 9, 15.5, 15, 9, 15, 5, 15.0, 15, 5, 15, 5, 15.0, 15, 9, 15.5, 15, 9, 15, 15.0, 15, 20, 15.0, 15, 9, 15, 15.0, 15, 15.5, 15, 20, 15, 5, 15, 5, 20, 5, 15, 20, 15, 9, 5, 900. 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, and/or 5000 μ g/ml serum concentration per single or multiple administrations, or any range, value, or fraction thereof.

Alternatively, the dose administered may vary depending on well-known factors such as the pharmacokinetic profile of the particular agent, and the mode and route of administration thereof; age, health and weight of the recipient; the nature and extent of the symptoms, the type of current treatment, the frequency of treatment, and the desired effect. Typically, the dosage of active ingredient may be from about 0.1 to 100mg/kg body weight. Generally, 0.1 to 50, and preferably 0.1 to 10, mg/kg body weight/administration or sustained release forms are effective to achieve the desired results.

As a non-limiting example, treatment of a human or animal may be provided as a single or periodic dose of at least one antibody of the invention, said dose being from 0.1 to 100mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg/day on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively on day 1, 2, 3, 4, or 100 mg/kg/day, or alternatively on day, 5. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks, or alternatively or additionally, at least one year of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years, or any combination thereof, using single, infusion, or repeated doses.

Dosage forms (compositions) suitable for internal administration typically contain from about 0.001 mg to about 500 mg of active ingredient per unit or container. In such pharmaceutical compositions, the active ingredient will generally be present in an amount of about 0.5-99.999% by total weight of the composition.

For parenteral administration, the antibodies can be prepared as solutions, suspensions, emulsions, granules, powders or lyophilized powders in combination with or separately from a pharmaceutically acceptable parenteral carrier. Examples of such carriers are water, saline, ringer's solution, dextrose solution, and 1-10% human serum albumin. Liposomes and non-aqueous carriers, such as fixed oils, may also be used. The carrier or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by known or suitable techniques.

Suitable pharmaceutical carriers are described in the standard reference book Remington's pharmaceutical Sciences, a.osol, recent edition.

Alternative administration

Many well-known and developed ways may be used according to the present invention for administering a pharmaceutically effective amount of at least one anti-amyloid antibody according to the present invention. Although pulmonary administration is used in the following description, other modes of administration may be used in accordance with the present invention to achieve suitable results.

The amyloid antibodies of the present invention may be delivered in a carrier, as a solution, emulsion, colloid, or suspension, or as a dry powder using any of a variety of devices and methods suitable for administration by inhalation or other means described herein or known in the art.

Parenteral formulations and administration

Formulations for parenteral administration may contain, as common excipients, sterile water or saline, polyalkylene glycols, such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. Aqueous or oily suspensions for injection may be prepared according to known methods using suitable emulsifying or wetting agents and suspending agents. The injectable agents may be nontoxic, parenterally acceptable diluents, such as aqueous solutions in solvents, or sterile injectable solutions or suspensions. As a usable carrier or solvent, water, ringer's solution, isotonic saline, etc. may be used; as a general solvent, or suspending solvent, sterile fixed oils may be used. For this purpose, any type of non-volatile oils and fatty acids may be used, including natural or synthetic or semi-synthetic fatty oils or fatty acids; natural or synthetic or semisynthetic mono-, di-or triglycerides. Parenteral administration is well known in the art and includes, but is not limited to, conventional injection means, pneumatic needle-less injection devices as described in U.S. patent No. 5,851,198, and laser perforation devices as described in U.S. patent No. 5,839,446, which are incorporated herein by reference in their entirety.

Alternative delivery

The invention also provides for the administration of at least one anti-amyloid antibody by parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, bolus, vaginal, buccal, sublingual, intranasal, or transdermal means. The at least one anti-amyloid antibody composition may be prepared for parenteral (subcutaneous, intramuscular or intravenous) or any other administration, in particular in the form of a liquid solution or suspension; for vaginal or rectal administration, particularly in semi-solid forms, such as, but not limited to, creams and suppositories; for buccal or sublingual administration, such as, but not limited to, tablet or capsule form; or intranasal administration, such as, but not limited to, in the form of a powder, nasal drops or aerosol or certain agents; or transdermal administration, such as, but not limited to, gels, ointments, lotions, suspensions or patch delivery systems, it has chemical enhancers, such as dimethyl sulfoxide, to modify the skin structure or increase the Drug concentration in transdermal patches (juninger, et al, "Drug performance Enhancement"; hsieh, d.s., eds., 59-90 (Marcel Dekker, inc. new York 1994, which is incorporated herein by reference in its entirety), or an oxidizing agent, which enables the application of protein or peptide containing formulations to the skin (WO 98/53847), or the application of an electric field to create transient transport pathways, such as electroporation, or to increase the mobility of charged drugs through the skin, such as iontophoresis, or the application of ultrasound, such as sonophoresis (U.S. patent nos. 4,309,989 and 4,767,402) (the disclosures and patents of which are incorporated herein by reference in their entirety).

Pulmonary/nasal administration

For pulmonary administration, it is preferred that at least one anti-amyloid antibody composition is delivered in a particle size effective to reach the lower airways of the lung or sinuses. According to the present invention, the at least one anti-amyloid antibody may be delivered by any of a variety of inhalation or nasal devices known in the art for administration of a therapeutic agent by inhalation. Such devices capable of depositing aerosolized formulations in the sinus cavities or alveoli of a patient include metered dose inhalers, nebulizers, dry powder generators, nebulizers, and the like. Other devices suitable for pulmonary or nasal administration of the guide antibody are also well known in the art. All such devices may use formulations suitable for administration of the antibody dispensed as an aerosol. Such aerosols may consist of solutions (aqueous and non-aqueous) or solid particles. Metered dose inhalers likeMetered dose inhalers typically use a propellant gas and require actuation during inhalation (see, e.g., WO 94/16970, WO 98/35888). Dry powder inhalers like Turbuhaler marketed by inlae Therapeutics^TM(Astra)，(Glaxo)，(Glaxo)，Spiros^TMInhaler (Dura) device andpowder inhalers (Fisons) use breath actuation of mixed powders (US4668218 Astra, EP 237507Astra, WO 97/25086Glaxo, WO 94/08552Dura, US 5458135 Inhale, WO 94/06498 Fisons, which are incorporated herein in their entirety by reference). Atomizer image AERx ^TM Aradigm、Atomizers (Mallinckrodt), and AcornNebulizers (Marquest Medical Products) (US 5404871 Aradigm, WO 97/22376), the entire contents of which are incorporated herein by reference, produce aerosols from solutions, while metered dose inhalers, dry powder inhalers, and the like produce small particle aerosols. These specific examples of commercially available inhalation devices are intended to be representative of the specific devices suitable for the practice of the present invention and are not intended to limit the scope of the present invention. Preferably, the composition comprising at least one anti-amyloid antibody is administered by dry powder inhaler or nebulizer. There are several desirable features of an inhalation device for administering at least one antibody of the present invention. For example, delivery by an inhalation device is advantageously reliable, reproducible and accurate. The inhalation device may optionally deliver small dry particles, for example, less than about 10 μm, preferably about 1-5 μm, for good respirability.

Amyloid antibody composition applied as a spray

A spray comprising an amyloid antibody composition may be generated by pressurizing a suspension or solution of at least one anti-amyloid antibody through a nozzle. The nozzle size and configuration, applied pressure, and liquid feed rate can be selected to achieve the desired output and particle size. Electrospray can be generated, for example, by an electric field in combination with capillary or nozzle feed. Advantageously, the particle size of the particles of the at least one anti-amyloid antibody composition delivered by the nebulizer is less than about 10 μm, preferably from about 1 μm to about 5 μm, most preferably from about 2 μm to about 3 μm.

Formulations of the at least one anti-amyloid antibody composition suitable for use with a nebulizer typically include the at least one anti-amyloid antibody composition at a concentration of about 0.1mg to about 100mg per ml of solution or mg/gm, or any range or value therein, of the antibody composition, such as, but not limited to,. 1,. 2,. 3,. 4,. 5,. 6,. 7,. 8,. 9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100mg/ml or mg/gm in an aqueous solution. The formulation may include agents such as excipients, buffers, isotonicity agents, preservatives, surfactants, and preferably, zinc. The formulation may also include excipients or agents for stabilizing the antibody composition, such as buffers, reducing agents, total protein or sugars. The total protein used to prepare the antibody composition includes albumin, protamine, and the like. Typical sugars used to formulate antibody compositions include sucrose, mannitol, lactose, trehalose, glucose, and the like. The antibody composition formulation may also include a surfactant that can reduce or prevent surface-induced aggregation of the antibody composition by spraying of the solution in forming an aerosol. A variety of conventional surfactants can be used, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. The amount will generally be from 0.001 to 14% by weight of the formulation. Particularly preferred surfactants for use in the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20 and the like. Other reagents known in the art for the preparation of proteins, such as amyloid antibodies, or specific portions or variants may also be included in the formulation.

Administration of amyloid antibody compositions by nebulizer

The antibody composition is administered by a nebulizer, such as a jet nebulizer or an ultrasonic nebulizer. Typically, in a jet atomizer, a pressurized air source is used to generate a high velocity gas jet through an orifice. As the gas expands through the nozzle, a low pressure zone is created which draws the antibody composition solution through a capillary tube connected to a liquid reservoir. The liquid flow from the capillary tube shears into unstable filaments or droplets as it exits the tube, thereby generating an aerosol. A range of configurations, flow rates and baffle types can be used to produce the desired performance characteristics from a given spray atomizer. In ultrasonic atomizers, high frequency electrical energy is used to generate the mechanical energy of vibration, usually with piezoelectric transducers. The energy is transferred to the formulation of the antibody composition, either directly or through a coupling fluid, thereby generating an aerosol containing the antibody composition. Advantageously, the particles of the antibody composition delivered by the nebulizer have a particle size of less than about 10 μm, preferably from about 1 μm to about 5 μm, most preferably from about 2 μm to about 3 μm.

Formulations of at least one anti-amyloid antibody suitable for use with a jet or ultrasonic nebulizer typically comprise a concentration of about 0.1mg to about 100mg of the at least one anti-amyloid antibody per ml of solution. The formulation may include agents such as excipients, buffers, isotonicity agents, preservatives, surfactants, and preferably, zinc. The formulation may also include excipients or agents for stabilizing at least one anti-amyloid antibody composition, such as buffers, reducing agents, total protein or sugars. The total protein used for preparing at least one anti-amyloid antibody composition includes albumin, protamine, and the like. Typical sugars for preparing at least one anti-amyloid antibody include sucrose, mannitol, lactose, trehalose, glucose, and the like. The at least one anti-amyloid antibody composition formulation may further comprise a surfactant that may reduce or prevent surface-induced aggregation of the at least one anti-amyloid antibody by spraying of the solution in forming an aerosol. A variety of conventional surfactants can be used, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. The amount will generally be from 0.001 to 4% by weight of the formulation. Particularly preferred surfactants for use in the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20 and the like. Other reagents known in the art for the preparation of proteins, such as antibody proteins, may also be included in the formulation.

Administration of amyloid antibody compositions by metered dose inhalers

In a Metered Dose Inhaler (MDI), a propellant, at least one anti-amyloid antibody, and any excipients or other additives are included in a canister as a mixture comprising a liquefied pressurized gas. Actuation of the metering valve releases the mixture as an aerosol, preferably containing particles in a size range of less than about 10 μm, preferably from about 1 μm to about 5 μm, most preferably from about 2 μm to about 3 μm. The desired aerosol particle size can be obtained by formulation using antibody compositions produced by a variety of methods well known to those skilled in the art, including jet milling, spray drying, critical point condensation, and the like. Preferred metered dose inhalers include those manufactured by 3M or Glaxo and which use hydrofluorocarbon propellants.

The formulation of the at least one anti-amyloid antibody for use with a metered dose inhaler device will generally comprise a finely divided powder containing the at least one anti-amyloid antibody suspended in a propellant as a suspension in a non-aqueous medium, for example, by the aid of a surfactant suspension. The propellant may be any conventional material used for this purpose, such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, or hydrocarbons, including trifluorochloromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1, 1, 1, 2-tetrafluoroethane, HFA-134a (hydrofluorocarbon-134 a), HFA-227 (hydrofluorocarbon-227), and the like. Preferably, the propellant is a hydrofluorocarbon. The surfactant may be selected to stabilize at least one anti-amyloid antibody as a suspension in the propellant, to protect the active agent from chemical degradation, and the like. Suitable surfactants include sorbitan trioleate, soy lecithin, oleic acid, and the like. In some cases, it is preferred to use a solution aerosol of a solvent such as ethanol. Additional reagents known in the art for the preparation of proteins, such as proteins, may also be included in the formulation.

One skilled in the art will recognize that the methods of the present invention may be practiced by pulmonary administration of at least one anti-amyloid antibody composition using a device not described herein.

Oral formulations and administration

Oral formulations rely on co-administration of adjuvants (e.g., resorcinol and nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether) to artificially increase the permeability of the intestinal wall, and co-administration of enzyme inhibitors (e.g., pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DFF) and tryptase) to inhibit enzymatic degradation. Formulations for delivery of hydrophilic agents, including proteins and antibodies and combinations of at least two surfactants, intended for oral, buccal, mucosal, nasal, pulmonary, vaginal transmembrane, or rectal administration are taught in U.S.6,309,663. The active ingredient compound for oral administration in solid dosage form may be mixed with at least one additive including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, arginates, chitin, chitosan, pectin, gum tragacanth, gum acacia, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, and glycerides. These dosage forms may also contain other types of additives, for example, inactive diluents, lubricating agents such as magnesium stearate, parabens, preservatives such as sorbic acid, ascorbic acid, alpha-tocopherol, antioxidants such as cysteine, disintegrating agents, binding agents, thickening agents, buffering agents, sweetening agents, flavoring agents, and the like.

Tablets and pills can be further processed into enteric-coated formulations. Liquid preparations for oral administration include emulsion, syrup, elixir, suspension and solution preparations permitted for medical use. These formulations may contain inactive diluents commonly used in the art, such as water. Liposomes have been described as drug delivery systems for insulin and heparin (U.S. patent No. 4,239,754). Recently, microspheres of artificial polymers of mixed amino acids (proteoid) have been used to deliver drugs (U.S. Pat. No. 4,925,673). In addition, the carrier compounds described in U.S. Pat. No. 5,879,681 and U.S. Pat. No. 5,5,871,753 are well known in the art for oral delivery of bioactive agents.

Mucosal formulations and administration

Formulations for oral administration of a biologically active agent encapsulated in one or more biocompatible polymer or copolymer excipients, preferably a biodegradable polymer or copolymer, provide microcapsules which, due to the appropriate size of the resulting microcapsules, result in absorption by the follicular lymphocyte tract (folliculi lymphatic aggregati) of the animal, otherwise known as "peyer's patches" or "GALT", and which do not lose effectiveness as the active agent has passed through the gastrointestinal tract. Similar follicular lymphocyte bundles can be found in the bronchi (bronchei tubs) (BALT) and large intestine. Such tissues are commonly referred to as mucosa-associated lymphoreticular endothelial cell tissue (MALT). For absorption through mucosal surfaces, compositions and methods of administering at least one anti-amyloid protein include emulsions containing a plurality of submicron particles, mucoadhesive macromolecules, bioactive peptides, and an aqueous continuous phase that facilitates absorption through mucosal surfaces by achieving mucoadhesion of the emulsion particles (U.S. patent No. 5,514,670). Suitable mucosal surfaces to which the emulsions of the present invention are applied may include corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, gastric, intestinal and rectal routes of administration. Formulations for vaginal and rectal administration, e.g., suppositories, may contain as excipients, for example, polyalkylene glycols, petrolatum, cocoa butter, and the like. Formulations for intranasal administration may be solid and contain, as an excipient, for example, lactose, or may be aqueous or oily solutions of nasal drops. For oral administration, excipients may include sugars, calcium stearate, magnesium stearate, pregelatinized starch, and the like (U.S. patent No. 5,849,695).

Transdermal formulations and administration

For transdermal administration, at least one anti-amyloid antibody is encapsulated in a delivery device, such as a liposome or polymeric nanoparticle (nanoparticle), microparticle, microcapsule, or microsphere (collectively referred to as microparticles unless otherwise specified). Many suitable devices are known, including microparticles made from synthetic polymers such as polyhydroxy acids, polyorthoesters, polyanhydrides, and polyphosphazenes of polylactic acid, polyglycolic acid, and copolymers thereof, and natural polymers such as collagen, polyamino acids, albumin and other proteins, alginates and other polysaccharides, and combinations thereof (U.S. Pat. No. 5,814,599).

Extended administration and formulation

It is sometimes desirable to deliver a compound of the invention to a subject by a single administration over an extended period of time, for example, a week to a year. A variety of sustained release, depot (depot) or implant dosage forms can be utilized. For example, the dosage form may contain pharmaceutically acceptable non-toxic salts of compounds having a low degree of solubility in body fluids, e.g., (a) acid addition salts using polybasic acids such as phosphoric, sulfuric, citric, tartaric, tannic, pamoic, alginic, polyglutamic, naphthalene monosulfonic or naphthalenedisulfonic acids, polygalacturonic acids, and the like; (b) salts with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like, or organic cations derived from N, N' -dibenzyl-ethylenediamine or ethylenediamine; or (c) a combination of (a) and (b), e.g., a zinc tannate salt. Furthermore, the compounds of the present invention or, preferably, the relatively insoluble salts such as those just described, may be formulated in a gel suitable for injection, for example, in an aluminum monostearate gel with, for example, sesame oil. Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts, and the like. Another type of sustained release depot formulation for injection would contain a compound or salt dispersed to be encapsulated in a slowly degrading, non-toxic, non-antigenic polymer such as the polylactic acid/polyglycolic acid polymer described in U.S. patent No. 3,773,919. The compound or preferably the relatively insoluble salt as described above may also be formulated into cholesterol-based silicone rubber pellets, which are particularly useful for animals. Other Sustained Release, depot or implant formulations, e.g., gas or liquid liposomes, are known in the art (U.S. Pat. No. 5,770,222 and "suspended and Controlled Release Drug Delivery Systems", j.r. robinson ed., Marcel Dekker, inc., n.y., 1978).

Having generally described the present invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration only and are not intended to be limiting.

Example 1: cloning and expression of amyloid antibodies in mammalian cells

Typical mammalian expression vectors contain at least one promoter element that mediates the initiation of transcription of the antibody coding sequence encoding the heavy and light chain variable regions adjacent to the coding sequence of the known constant region, and signals required for transcription termination and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences, and interfering sequences, which are flanked by donor and acceptor sites for RNA splicing. High efficiency transcription can be achieved with the early and late promoters from SV40, the Long Terminal Repeat (LTRS) from retroviruses, e.g., RSV, HTLVI, HIVI, and the early promoter of Cytomegalovirus (CMV). However, cellular elements (e.g., the human actin promoter) may also be used. Suitable expression vectors for use in the practice of the present invention include, for example, vectors such as pIRESlne o, pRetro-Off, pRetro-On, PLXSN or pLNCX (Clonetech Labs, Palo Alto, CA), pcDNA3.1(+/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hygro (+/-) (Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Mammalian host cells that may be used include human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese Hamster Ovary (CHO) cells.

Alternatively, the gene may be expressed in a stable cell line containing the gene integrated into the chromosome. Co-transfection with selectable markers such as dhfr, gpt, neomycin or hygromycin allows for the identification and isolation of transfected cells.

The transfected gene can also be amplified to express the encoded antibody in large quantities. The DHFR (dihydrofolate reductase) marker can be used to develop cell lines carrying hundreds or even thousands of copies of the gene of interest. Another useful selectable marker is Glutamine Synthase (GS) (Murphy, et al, biochem. J.227: 277-. Using these markers, mammals are grown on selective media and cells with the highest resistance are selected. These cell lines contain amplified genes integrated into the chromosome. Chinese Hamster Ovary (CHO) and NSO cells are commonly used for the production of antibodies.

Expression vectors pC1 and pC4 contain fragments of the strong promoter (LTR) and CMV-enhancer of the Rous sarcoma virus (Cullen, et al, Molec. Cell. biol. 5: 438-447(1985)) (Boshart, et al, Cell 41: 521-530 (1985)). Multiple cloning sites, for example, having restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate cloning of the gene of interest. The vector also contains the 3' intron of the rat preproinsulin gene, polyadenylation, and termination signals.

Cloning and expression in CHO cells

The vector pC4 was used to express amyloid antibodies. Plasmid pC4 was derived from plasmid pSV2-dhfr (ATCC accession No. 37146). This plasmid contains the mouse DHFR gene under the control of the SV40 early promoter. Chinese hamster ovary or other cells transfected with these plasmids that lack dihydrofolate activity can be selected by growing on selection media (e.g., alpha minus MEM, Life Technologies, Gaithersburg, MD) supplemented with the chemotherapeutic agent methotrexate. Amplification of the DHFR gene in cells resistant to Methotrexate (MTX) has been described in detail (see, e.g., F.W.Alt, et al, J.biol.chem.253: 1357-1370 (1978); J.L.Hamlin and C.Ma, biochem.et Biophys.acta1097: 107-143 (1990); and M.J.Page and M.A.Sydenham, Biotechnology 9: 64-68 (1991)). Cells grown in increasing concentrations of MTX develop resistance to the drug by overproducing the target enzyme DHFR (due to the amplification of the DHFR gene). If a second gene is linked to the DHFR gene, the gene is usually co-amplified and overexpressed. It is well known in the art that this method can be used to develop cell lines carrying more than 1000 copies of the amplified gene. Subsequently, when methotrexate is withdrawn, a cell line is obtained that contains the amplifiable gene integrated into one or more chromosomes of the host cell.

Plasmid pC4 contains a strong promoter for the Long Terminal Repeat (LTR) of the Rous sarcoma virus (Cullen, et al, Molec. Cell. biol. 5: 438-447(1985)) for expression of the gene of interest and a fragment isolated from the enhancer of the immediate early gene of human Cytomegalovirus (CMV) (Boshart, et al, Cell 41: 521-530 (1985)). Downstream of this promoter are BamHI, XbaI and Asp718 restriction enzyme cleavage sites which allow gene integration. This plasmid contains the 3' intron of the rat preproinsulin gene and a polyadenylation site after these cloning sites. Other high efficiency promoters may also be used for expression, for example, the human b-actin promoter, the SV40 early and late promoters, or long terminal repeats from other retroviruses, for example, HIV and HTLV 1. The Tet-Off and Tet-On gene expression systems of Clontech and similar systems are useful for expressing amyloid in mammalian cells in a regulated manner (M.Gossen, and H.Bujard, Proc.Natl.Acad.Sci.USA 89: 5547-5551 (1992)). For polyadenylation of mRNA, other signals from, for example, human growth hormone or globin genes may also be used. Stable cell lines carrying the gene of interest integrated into the chromosome can also be selected after co-transfection with a selection marker such as gpt, G418 or hygromycin. It may be advantageous to use more than one selectable marker at the beginning, e.g., G418 plus methotrexate.

Plasmid pC4 was digested with restriction enzymes and then dephosphorylated using calf intestinal phosphatase by methods well known in the art. The vector was then separated from a 1% agarose gel.

In one set of experiments, DNA sequences encoding whole amyloid antibodies were used according to well-known method steps, e.g., as set forth in SEQ ID NOS: 51 or 52, which correspond to the sequences given in SEQ ID NOS: 48 or 49, the HC and LC variable regions of an amyloid antibody of the invention. Isolated nucleic acids encoding suitable human constant regions (i.e., HC and LC regions) are also used in this construct. In another set of experiments, the sequences corresponding to the sequences as set forth in SEQ ID NOS: 59 or 60 of the HC and LC variable regions set forth in SEQ ID NOS: 61 or 62. Also useful are nucleic acids corresponding to the nucleic acids as set forth in SEQ ID NOS: 69 or 70 in SEQ ID NOS: 71 or 72, and a DNA sequence corresponding to the sequence given in SEQ ID NOS: 79 or 80 in SEQ ID NOs: 81 or 82.

The isolated variable and constant region encoding DNA and the dephosphorylated vector are then ligated using T4DNA ligase. Coli HB101 or XL-1 Blue cells are then transformed and bacteria containing the fragment inserted in plasmid pC4 are identified using, for example, restriction enzyme analysis.

Chinese Hamster Ovary (CHO) cells lacking an active DHFR gene were used for transfection. Mu.g of expression plasmid pC4 was co-transfected with 0.5. mu.g of plasmid pSV2-neo using lipofectin. Plasmid pSV2neo contains a dominant selectable marker, the neo gene from Tn5, which encodes a gene that confers resistance to a group of antibiotics including G418. Cells were seeded in alpha minus MEM supplemented with 1. mu.g/ml G418. After 2 days, cells were trypsinized and seeded into alpha minus MEM hybridoma clone plates (Greiner, Germany) supplemented with 10, 25, or 50ng/ml methotrexate and 1. mu.g/ml G418. After about 10-14 days, single clones were trypsinized and plated in 6-well dishes or 10ml flasks using varying concentrations of methotrexate (50nM, 100nM, 200nM, 400nM, 800 nM). Clones grown at the highest concentration of methotrexate were then transferred to new 6-well plates containing even higher concentrations of methotrexate (1mM, 2mM, 5mM, 10mM, 20 mM). The same procedure was repeated until clones were obtained which grew at a concentration of 100-200 mM. For example, the expression of the desired gene product can be analyzed by SDS-PAGE and Western blotting or by reverse phase HPLC analysis.

Binding kinetics of human anti-human amyloid antibodies

ELISA analysis demonstrated that antibodies purified from these host cells bind amyloid in a concentration-dependent manner. In this case, the avidity of the antibody for its cognate antigen (epitope) is measured. BIAcore analysis of human antibodies yielded quantitative binding constants and revealed that several human monoclonal antibodies had very high affinities, K_DIs 1 × 10^-9To 9X 10^-12。

Conclusion

The human amyloid-reactive IgG monoclonal antibody of the present invention was produced. Human anti-amyloid antibodies were further characterized. Several antibodies were produced with a 1X 10⁸To 9X 10¹²The affinity constant of (c). The high affinity of these fully human monoclonal antibodies makes them suitable for therapeutic applications in amyloid-dependent diseases, pathologies or related conditions.

Example 2: expression and purification of amyloid protein or antibody in E.coli

The bacterial expression vector pQE60 was used for bacterial expression in this example (QIAGEN, inc., Chatsworth, CA). pQE60 encodes ampicillin antibiotic resistance ("Ampr") and contains a bacterial origin of replication ("ori"), an IPTG inducible promoter, a ribosome binding site ("RBS"), six codons encoding histidine residues that allow for affinity purification using the nickel-nitrilo-triacetic acid ("Ni-NTA") affinity resin sold by QIAGEN, inc. These elements are arranged such that a DNA fragment encoding a protein or antibody can be inserted such that a protein or antibody is produced having six His residues covalently linked to the carboxy terminus of the protein or antibody (i.e., a "6 × His tag"). However, protein or antibody coding sequences may optionally be inserted to prevent translation of the six His codons, thus, producing a protein or antibody without a 6 XHis tag.

Encoding a desired portion of an amyloid antibody, e.g., SEQ ID NOS: 48. HC and LC variable regions represented in 49, 59, 60, 69, 70, 79 and 80, SEQ ID NOS: HC CDRs represented in 42-44, 53-55, 63-65 and 73-75, SEQ ID NOS: the LC CDRs represented in 45-47, 56-58, 66-68 and 76-78, optionally also containing part or all of the coding sequences of known human constant regions and optionally and preferably lacking a hydrophobic leader sequence, are amplified from the deposited cDNA clones using PCR oligonucleotide primers based on the given sequence that anneal to the amino terminus of the DNA sequence encoding the desired portion of amyloid protein or antibody or to the sequence 3 ' of the cDNA coding sequence of the deposited construct, additional nucleotides containing restriction sites that facilitate cloning in the pQE60 vector are added to the 5 ' and 3 ' sequences, respectively.

For cloning of amyloid or antibodies, the 5 'and 3' primers have nucleotides corresponding to a part of the coding sequence of the amyloid or antibody or complementary thereto according to well known method steps. One skilled in the art will of course appreciate that the 5' primer initiation point in the protein or antibody coding sequence may be altered to amplify a desired portion of the intact protein or antibody, which is shorter or longer than the mature form.

The amplified amyloid nucleic acid fragment and vector pQE60 were digested with appropriate restriction enzymes, and the digested DNAs were ligated together. Insertion of amyloid DNA into the restrictive pQE60 vector places the amyloid or antibody coding region (including its associated stop codon) downstream of the IPTG-inducible promoter and in frame with the initiating AUG codon. The relevant stop codon prevents translation of the 6 histidine codon downstream of the insertion point.

Standard methods are used, such as Sambrook, et al, 1989; the ligation mixture was transformed into competent E.coli cells by the method described in Ausubel, 1987-1998. Coli strain M15/rep4 contains multiple copies of plasmid pREP4, which expresses the lac repressor and confers kanamycin resistance ("Kan"), M15/rep4 was used to implement the illustrative examples described herein. This strain is only one of many suitable strains for expressing amyloid or antibodies and is commercially available from QIAGEN, Inc. Transformants can be identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA was isolated from resistant colonies and the nature of the cloned DNA was confirmed by restriction analysis, PCR and DNA sequencing.

Clones containing the desired construct were grown overnight ("O/N") in liquid culture in LB medium supplemented with ampicillin (100. mu.g/ml) and kanamycin (25. mu.g/ml). The O/N cultures were used to inoculate large media at a dilution of about 1:25 to 1: 250. The cells were grown to an optical density ("OD 600") of 0.4 to 0.6 at 600 nm. isopropyl-b-D-thiogalactopyranoside ("IPTG") was then added to a final concentration of 1mM to induce transcription from the lac repressor sensitive promoter by inactivation of the lacI repressor. The cells were then cultured for a further 3 to 4 hours. Cells were then harvested by centrifugation.

The cells were stirred in 6M guanidine hydrochloride (pH8) at 4 ℃ for 3-4 hours. Cell debris was removed by centrifugation and the supernatant containing amyloid was dialyzed against 50mM sodium acetate buffer (pH6) supplemented with 200mM NaCl. Alternatively, the protein or antibody can be successfully refolded by dialyzing the protein or antibody against 500mM NaCl, 20% glycerol, 25mM Tris/HClpH7.4 containing a protease inhibitor.

If an insoluble protein is produced, the protein is made soluble according to well known process steps. After renaturation, the protein or antibody is purified by ion exchange, hydrophobic interaction and size exclusion chromatography. Alternatively, pure amyloid or antibody is obtained using an affinity chromatography step, such as an antibody column. The purified protein or antibody is stored at 4 ℃ or frozen at-40 ℃ to-120 ℃.

Example 3: cloning and expression of amyloid polypeptides in baculovirus expression systems

In this illustrative example, the DNA of a clone encoding the antibody (e.g., containing SEQ ID NOS: 51-52, 61-62, 71-72, or 81-82) was inserted into Baculovirus using plasmid shuttle vector pA2GP to express amyloid antibody using a Baculovirus leader sequence and standard Methods described in Summers, et al, A Manual of Methods for Baculoviral Vectors and Cell Culture products, Texas Agricultural Experimental expression Bulletin No.1555 (1987). The expression vector contains the strong polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcMNPV), followed by the secretory signal peptide (leader peptide) of baculovirus gp67 protein or antibody and convenient restriction sites such as BamHI, XbaI and Asp 718. The polyadenylation site of simian virus 40 ("SV 40") is used for efficient polyadenylation. For easy selection of recombinant viruses, this plasmid contains the β -galactosidase gene from e.coli, under the control of a weak drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted gene is flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to produce viable viruses expressing the cloned polynucleotide.

As will be readily understood by those skilled in the art, other baculovirus vectors such as pAc373, pVL941 and pAcIM1 may be substituted for the above vectors, provided that the construct provides appropriate targeting signals for transcription, translation, secretion, etc., including signal peptides and in-frame AUG (if desired). Such vectors are described, for example, in Luckow, et al, Virology 170: 31-39.

The cDNA sequence encoding the amyloid antibody of the deposited or other clone lacking the AUG start codon and the naturally associated nucleotide binding site is amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of the gene. Non-limiting examples include 5 'and 3' primers having a portion of the coding sequence corresponding to amyloid or an antibody or complementary thereto, e.g., as set forth in SEQ ID NOS: 48-49 for C705 is as given in SEQ ID NOS: 59-60 for C706 as shown in SEQ ID NOS: 69-70, for C707 is SEQ ID NOS: 79-80.

The amplified fragments were separated from 1% agarose gel using a commercially available kit (e.g., "Geneclean," BIO 101inc., La Jolla, CA). The fragment was then digested with the appropriate restriction enzymes and purified again on a 1% agarose gel. This fragment is referred to herein as "F1".

The plasmid is digested with the corresponding restriction enzymes and optionally dephosphorylated using calf intestinal phosphatase, which can be performed using conventional methods well known in the art. The DNA was then separated from the 1% agarose gel using a commercially available kit ("Geneclean" BIO 101inc., La Jolla, CA). This vector DNA is referred to herein as "V1".

Fragment F1 and dephosphorylated plasmid V1 were ligated together with T4DNA ligase. Coli HB101 or other suitable E.coli hosts such as XL-1Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells are transformed with the ligation mixture and plated onto culture plates. Bacteria containing plasmids with the human amyloid gene were identified using a PCR method in which one of the primers used to amplify the gene and a second primer were within the vector, so that only those bacterial colonies containing amyloid gene fragments showed amplification of the DNA. The sequence of the cloned fragment was confirmed by DNA sequencing. This plasmid is referred to herein as pBac amyloid.

Using Felgner, et al, proc.natl.acad.sci.usa 84: 7413-7417(1987) using 1.0. mu.g of commercially available linearized baculovirus DNA ("Baculogold ^TMbaculovirus DNA ", Pharmingen, San Diego, Calif.) was co-transfected with 5. mu.g of plasmid pBacamyloid. Mix 1 μ g Baculogold^TMViral DNA and 5 μ g of plasmid pBac amyloid were mixed in sterile wells of a microtiter plate containing 50 μ l of serum-free Grace's medium (life technologies, inc., Rockville, MD). After that time, the user can use the device,add 10. mu.L Lipofectin and 90. mu.L Grace's medium, mix and incubate for 15 minutes at room temperature. The transfection mixture was then added dropwise to Sf9 insect cells (ATCC CRL1711) seeded in 35mm tissue culture dishes containing 1ml of serum-free Grace's medium. The plate was shaken back and forth to mix the freshly added solution. The plates were then incubated at 27 ℃ for 5 hours. After 5 hours, the transfection solution was removed from the plate and 1ml of Grace's insect medium supplemented with 10% fetal bovine serum was added. The plate was returned to the incubator and incubation continued at 27 ℃ for 4 days.

Four days later, the supernatant was collected and plaque assay was performed according to a known method. Agarose gels with "Blue Gal" (Life Technologies, inc., Rockville, MD) were used to allow easy identification and isolation of clones expressing Gal, which produced Blue-stained plaques. (A detailed description of this type of "plaque assay" can also be found on pages 9-10 of the user guide for insect cell culture and baculovirus distributed by Life Technologies, Inc., Rockville, MD). After appropriate incubation, blue-stained plaques are picked with a micropipette tip (e.g., Eppendorf). Agar containing the recombinant virus was then resuspended in a microfuge tube containing 200 μ LGrace's medium, and the suspension containing the recombinant baculovirus was used to infect Sf9 cells seeded in 35mm petri dishes. The supernatants of these dishes were harvested four days later and stored at 4 ℃. The recombinant virus is called V-amyloid.

To confirm the expression of the amyloid gene, Sf9 cells were grown in Grace's medium supplemented with 10% heat-inactivated FBS. Cells were infected with the recombinant baculovirus V-amyloid at a multiplicity of infection ("MOI") of about 2. After 6 hours, the medium was removed and replaced with SF900 II medium (available from, e.g., life technologies, inc., Rockville, MD) without methionine and cysteine. If a radiolabeled protein or antibody is desired, 5mCi 35-S methionine and 5mCi 35S-cysteine (available from Amersham) are added after 42 hours. Cells were further incubated for 16 hours and then harvested by centrifugation. The proteins or antibodies in the supernatant as well as intracellular proteins or antibodies were analyzed by SDS-PAGE and then by autoradiography (if radiolabeled). Microsequencing of the amino acid sequence of the amino terminus of the purified protein or antibody can be used to determine the amino terminal sequence of the mature protein or antibody and thereby determine the cleavage point and length of the secreted signal peptide.

Example 4: characterization of linear epitopes of anti-human amyloid beta antibodies

Introduction to the design reside in

Alzheimer's Disease (AD) is a progressive dementia, the pathology of which is characterized by hyperphosphorylated tau in neurons and extracellular deposition of β -amyloid (A β) plaques in the brain. The formation of a β plaques is caused by the overproduction of highly self-aggregating 42 amino acid peptides of the Amyloid Precursor Protein (APP), or by inefficient clearance. APP or A beta ₄₂The normal function of the peptide is not known, but A.beta.is considered₄₂The category is associated with AD. Abeta (beta)₄₂Can self-aggregate rapidly to form oligomeric structures that progress to fibrils and eventually form plaques. These plaques are hallmarks of AD pathology.

Therapeutic intervention against disruption of the amyloid cascade has been elucidated in transgenic AD animal models using active vaccination with a β peptide or peripheral administration of a β -specific monoclonal antibodies. The rationale for these approaches relies on immune system-mediated plaque and/or a β clearance. The mechanism of plaque clearance has been proposed to be through direct binding of antibodies to plaques in the brain, thereby activating microglial phagocytic deposition of a β by Fc receptors. Alternatively, peripherally administered antibodies may bind to circulating parts, thereby altering the homeostasis of a β concentrations between the central nervous system and plasma and promoting a β efflux from the brain.

Method

Peptide synthesis on cell membranes

Membranes were purchased from Intavis (Bergisch Gladbach, germany). Fluorenylmethoxycarbonyl (Fmoc) amino acids and N-Hydroxybenzotriazole (HBOT) were from Novabiochem (Meudon, France). N, N' -diisopropylCarbodiimides (DIC) are from Fluka (Germany). N, N' -Dimethylformamide (DMF) and N-methylpyrrolidinone-2 (NMP) were obtained from applied biosystems. Rink resin was purchased from Advanced Chem Tech. Peptide synthesis of A β -KMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVML (SEQ ID NO: 50) -was performed according to Frank R. (2002) using Auto Spot Robot ASP 222(Abimed GmbH, Germany) as described previously (Kramer et al, 1994). The membranes used are derivatized with polyethylene glycol spacer arms of length 8 to 10 ethylene glycol units (amino-PEG) ₅₀₀-UC Sheet, load: 400nmol/cm²) (Intavis AG, Lot AC 112050900). The grid was created by spotting of C-terminal beta-alanine. All peptides were N-acetylated and each single point yielded approximately 20nmol of peptide.

Membrane detection and regeneration

After an overnight saturation step in SuperBlock blocking buffer (Pierce), protein G purified antibody (1. mu.g/ml, incubated for 2 hours at room temperature) was added to the membrane. After washing the membranes, a 1:10,000 dilution of horseradish peroxidase conjugated goat anti-mouse IgG (Jackson immunoresearch laboratory Inc.) was incubated for 1 hour at room temperature. Bound antibody was detected by ECL plus kit (Amersham) which produced a positive indication of antibody binding at the spot.

Results

The cellulose-bound overlapping peptide sets spanning the primary sequence of human a β were probed with anti-a β IgG antibodies C701, C705, C706 and C707. Linear peptide epitopes can be identified for these monoclonal antibodies using peptide scanning (15-mer to 6-mer with 1 amino acid shift).

The spotted membrane results indicated that the C701 recognition sequence LMVGGV (fig. 42). C705 specifically recognized the N-terminal sequence EFRHDS (FIG. 43). C706 specifically recognized the N-terminal sequence AEFRHD (FIG. 44). C707 recognizes the central domain and the C-terminal sequence GLMVGGVVIA (FIG. 45).

Conclusion

In summary, epitope mapping of anti-a β monoclonal antibodies using a spotted membrane showed that these antibodies recognize linear epitopes. Two mAbs C705 and C706 are specific for the A.beta.N-terminal sequence. One mAbC701 recognizes the C-terminal sequence of a β. Interestingly, mAb C707 binds to the central domain and C-terminal sequence of a β.

Example 5: ligand binding of anti-human amyloid beta antibodies

Method

BIAcore 3000, CM5 sensor surface, amine coupling kit, HEPES buffered saline (HBS, 10mM HEPES, 150mM NaCl, pH 7.4 with 3mM EDTA and 0.005% Tween-20) and 10mM sodium acetate pH 4.5 were purchased from BIAcore, Inc. anti-A.beta.monoclonal antibody (100. mu.g/mL) was dialyzed against HBS diluted 1:10 with water. The dialyzed mAb solution was then diluted 1:10 into 10mM sodium acetate pH 4.5. The CM5 sensing surface was balanced with HBS in BIAcore 3000. Each antibody was immobilized on the flow cells using the immobilization wizard provided in the operating software and the protocol provided in the amine-coupled kit. The guide was set to 2500RU to which the antibody was immobilized. Typically, 2000-3000RU is actually fixed.

Results

Data were collected as a function of time for each mAb response (figure 46). Report points were collected from sensorgram 10 seconds before peptide injection, 10 seconds before completion of the association phase, and 60 seconds into the dissociation phase. This data was then used to determine the binding stoichiometry and measure the stability (fraction of bound peptide remained on the sensor surface after 60 seconds). By assuming 1RU ═ 1pg peptide (or protein)/mm ²Making it possible to perform these calculations.

Due to the potential multimerization state of the 1-38 and 1-42 peptides, quantitative analysis of binding constants was not possible. However, qualitative analysis is possible. Figure 47 arranges mabs as a function of binding ratio and the fraction remaining on the surface after 60 seconds reflects the stability of the complex. According to this analysis, C705 and C707 appear to be able to bind to "monomeric" peptides, whereas C701 and C706 require some aggregation of the peptides before they bind.

Example 5: oligomeric neutralization of anti-human amyloid beta antibodies

Method

Production of A.beta.according to published protocol (Klein, 2002)_1-42Oligomer formulations. Briefly, 1mg of human A β was administered_1-42(California peptide, cat. No. 641-15) was monomerized in 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFIP) and 0.45mg aliquots were added to non-silanized microcentrifuge tubes. HFIP was allowed to evaporate overnight at room temperature in a fume hood. If HFIP remains, it is removed at speed-vac for 10 minutes. Then 5mM A.beta.stock solution was prepared by adding 20. mu.l of anhydrous DMSO (Hybri-Max, Sigma) to 0.45mg of the monomerized peptide membrane. Then, 980. mu.l of ham's F12 medium (BioSource, Inc) was added to produce a 100. mu.M solution of the oligomer. The resulting solution was incubated at 4 ℃ for 24 hours. After incubation, the oligomer solution was centrifuged at 14,000 Xg for 10 minutes at 4 ℃. The resulting supernatant was carefully recovered and used as a 100. mu.M solution of the oligomer for cytotoxicity studies.

Rat PC12 cells (ATCC) were plated at 20,000 cells/well in F12K medium (1% horse serum, 1% Pen/Strep) in collagen-coated 96-well plates and allowed to incubate at 37 ℃ and 5% CO₂Adherence was performed overnight. The medium was refreshed with F12K immediately before the start of the assay. All A.beta.antibodies (C700, C701, C705-707) and the commercially available mouse anti-A.beta.antibody 6E10(Signet, Cat. No. 9320-05) were diluted to 5.6. mu.g/10. mu.l in sterile water. Then 5 mu M A beta_1-42The oligomers were preincubated with each antibody at 4 ℃ for 2 hours. Then, the oligomer and antibody combination was added to the pool and incubated at 37 ℃ for 24 hours. In this experiment, 5% ethanol was used as a positive control for cytotoxicity. Cell viability was assessed by adding 10. mu.l of MTT reagent (Roche, # 1-465-007) to each well and allowed to incubate for 4 hours. Viable cells reduce MTT reagent to formazanSalt crystals. The crystals were dissolved overnight in the buffer provided (Roche) and then read on a spectrophotometer at 550nm-690 nm.

Results

Testing of A β monoclonal antibodies against A β Using the rat PC12 cell line₄₂The ability of the oligomer to be toxic. Toxicity was measured using the MTT assay. This assay measures cell proliferation and viability. The MTT assay also represents a measure of cellular mitochondrial function, since mitochondrial dehydrogenase activity is required to reduce MTT dye to formazan Salt crystals, read on a spectrophotometer. When comparing the PC12 cells treated with the vehicle to the cells treated with 5. mu. M A. beta. oligomers, A.beta.₄₂MTT reduction after oligomer exposure was typically reduced by 40-50%, as shown in FIG. 48. Anti-human Abeta antibodies were tested for protection against Abeta₄₂The ability of the oligomer to be toxic. After preincubation of Α β oligomers with anti-human Α β antibodies, they were exposed to neuronal-like PC12 cells.

Exposure of cells to only 5 μ M oligomers resulted in a 27.3% reduction in cell viability compared to vehicle controls. All anti-human a β antibodies were able to confer neuroprotection to PC12 cells after 2 hours of preincubation with the oligomers. C705 and C706 completely prevented A.beta.in PC12 cells₄₂Oligomer-induced toxicity. Commercially available mouse monoclonal Ab antibody 6E10 did not protect cells at the concentrations tested (560 μ g/ml).

It will be understood that the invention may be practiced otherwise than as specifically described in the foregoing specification and examples.

Many modifications and variations of the present invention are possible in light of the above teachings and are therefore within the scope of the appended claims.

TABLE 1

Sequence listing

<110>Mercken，Marc；Benson，Jacqueline M.

<120> anti-amyloid antibodies, compositions, methods and uses

<130>CEN5021 PCT

<140>PCT/US04/09522

<141>2004-03-26

<150>US 60/458,474

<151>2003-03-28

<150>US 60/458,469

<151>2003-03-28

<150>US 60/458,509

<151>2003-03-28

<150>US 60/458,510

<151>2003-03-28

<160>131

<170>PatentIn version 3.3

<210>1

<211>125

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(125)

<223> Vh1 heavy chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(31)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(32)..(32)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(33)..(46)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(47)..(47)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(48)..(79)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(80)..(80)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(81)..(125)

<223> framework 4

<400>1

<210>2

<211>124

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(124)

<223> Vh2 heavy chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(30)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(31)..(31)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(32)..(45)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(46)..(46)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(47)..(78)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(79)..(79)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(80)..(124)

<223> framework 4

<400>2

<210>3

<211>100

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(100)

<223> Vh3a heavy chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(31)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(32)..(32)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(33)..(46)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(47)..(47)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(48)..(79)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(80)..(80)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(81)..(100)

<223> framework 4

<400>3

<210>4

<211>102

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(102)

<223> Vh3b heavy chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(30)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(31)..(31)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(32)..(45)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(46)..(46)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(47)..(78)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(79)..(79)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(80)..(102)

<223> framework 4

<400>4

<210>5

<211>101

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(101)

<223> Vh3c heavy chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(30)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(31)..(31)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(32)..(45)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(46)..(46)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(47)..(79)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(80)..(80)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(81)..(101)

<223> framework 4

<400>5

<210>6

<211>108

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(108)

<223> Vh4 heavy chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(33)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(34)..(34)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(35)..(48)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(49)..(49)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(50)..(81)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(82)..(82)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(83)..(108)

<223> framework 4

<400>6

<210>7

<211>132

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(132)

<223> Vh5 heavy chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(31)

<223>MISC_FEATURE

<220>

<221>MISC_FEATURE

<222>(32)..(32)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(33)..(46)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(47)..(47)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(48)..(79)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(80)..(80)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(81)..(132)

<223> framework 4

<400>7

<210>8

<211>125

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(125)

<223> Vh6 heavy chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(30)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(31)..(31)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(32)..(45)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(46)..(46)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(47)..(78)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(79)..(79)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(80)..(125)

<223> framework 4

<400>8

<210>9

<211>91

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(91)

<223> Vh7 heavy chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(30)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(31)..(31)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(32)..(45)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(46)..(46)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(47)..(78)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(79)..(79)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(80)..(91)

<223> framework 4

<400>9

<210>10

<211>93

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(93)

<223> kappa 1_4 light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(24)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(25)..(25)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(26)..(40)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(41)..(41)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(42)..(73)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(74)..(74)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(75)..(93)

<223> framework 4

<400>10

<210>11

<211>92

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(92)

<223> kappa 2 light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(23)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(24)..(24)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(25)..(39)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(40)..(40)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(41)..(72)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(73)..(73)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(74)..(92)

<223> framework 4

<400>11

<210>12

<211>91

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(91)

<223> kappa 3 light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(23)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(24)..(24)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(25)..(39)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(40)..(40)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(41)..(72)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(73)..(73)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(74)..(91)

<223> framework 4

<400>12

<210>13

<211>85

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(85)

<223> kappa 5 light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(23)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(24)..(24)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(25)..(39)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(40)..(40)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(41)..(72)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(73)..(73)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(74)..(85)

<223> framework 4

<400>13

<210>14

<211>79

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(67)

<223> kappa New1 light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(17)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(18)..(18)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(19)..(33)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(34)..(34)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(35)..(66)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(67)..(67)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(68)..(79)

<223> framework 4

<400>14

<210>15

<211>77

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(65)

<223> kappa New2 light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(15)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(17)..(31)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(32)..(32)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(33)..(64)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(65)..(65)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(66)..(77)

<223> framework 4

<400>15

<210>16

<211>98

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(98)

<223> lambda 1a light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(22)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(23)..(23)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(24)..(38)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(39)..(39)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(40)..(71)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(72)..(72)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(73)..(98)

<223> framework 4

<400>16

<210>17

<211>99

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(99)

<223> lambda 1b light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(23)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(24)..(24)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(25)..(39)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(40)..(40)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(41)..(72)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(73)..(73)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(74)..(99)

<223> framework 4

<400>17

<210>18

<211>99

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(72)

<223> lambda 2 light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(22)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(23)..(23)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(24)..(38)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(39)..(39)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(40)..(71)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(72)..(72)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(73)..(99)

<223> framework 4

<400>18

<210>19

<211>107

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(107)

<223> lambda 3a light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(22)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(23)..(23)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(24)..(38)

<223> framework 2vv

<220>

<221>MISC_FEATURE

<222>(39)..(39)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(40)..(71)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(72)..(72)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(73)..(107)

<223> framework 4

<400>19

<210>20

<211>93

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(93)

<223> lambda 3b light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(22)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(23)..(23)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(24)..(39)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(40)..(40)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(41)..(72)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(73)..(73)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(74)..(93)

<223> framework 4

<400>20

<210>21

<211>98

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(98)

<223> lambda 3c light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(22)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(23)..(23)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(24)..(38)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(39)..(39)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(40)..(71)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(72)..(72)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(73)..(98)

<223> framework 4

<400>21

<210>22

<211>98

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(98)

<223> lambda 3e light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(22)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(23)..(23)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(24)..(38)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(39)..(39)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(40)..(71)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(72)..(72)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(73)..(98)

<223> framework 4

<400>22

<210>23

<211>94

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(94)

<223> lambda 4a light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(22)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(23)..(23)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(24)..(38)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(39)..(39)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(40)..(71)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(72)..(72)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(73)..(94)

<223> framework 4

<400>23

<210>24

<211>95

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(95)

<223> lambda 4b light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(22)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(23)..(23)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(24)..(38)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(39)..(39)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(40)..(71)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(72)..(72)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(73)..(95)

<223> framework 4

<400>24

<210>25

<211>88

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(75)

<223> lambda 5 light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(22)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(23)..(23)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(24)..(39)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(40)..(40)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(41)..(74)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(75)..(75)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(76)..(88)

<223> framework 4

<400>25

<210>26

<211>101

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(101)

<223> lambda 6 light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(22)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(23)..(23)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(24)..(38)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(39)..(39)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(40)..(73)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(74)..(74)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(75)..(101)

<223> framework 4

<400>26

<210>27

<211>89

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(72)

<223> lambda 7 light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(22)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(23)..(23)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(24)..(38)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(39)..(39)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(40)..(71)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(72)..(72)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(73)..(89)

<223> framework 4

<400>27

<210>28

<211>89

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(89)

<223> lambda 8 light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(22)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(23)..(23)

<223> complementarity determining region 1(CDR1), X is 5-25(14) arbitrary amino acids.

<220>

<221>MISC_FEATURE

<222>(24)..(38)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(39)..(39)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(40)..(71)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(72)..(72)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(73)..(89)

<223> framework 4

<400>28

<210>29

<211>91

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(91)

<223> lambda 9 light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(22)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(23)..(23)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(24)..(38)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(39)..(39)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(40)..(79)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(80)..(80)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(81)..(91)

<223> framework 4

<400>29

<210>30

<211>87

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(87)

<223> lambda 10 light chain variable region

<220>

<221>MISC_FEATURE

<222>(1)..(22)

<223> framework 1

<220>

<221>MISC_FEATURE

<222>(23)..(23)

<223> complementarity determining region 1(CDR1), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(24)..(38)

<223> framework 2

<220>

<221>MISC_FEATURE

<222>(39)..(39)

<223> complementarity determining region 2(CDR2), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(40)..(71)

<223> framework 3

<220>

<221>MISC_FEATURE

<222>(72)..(72)

<223> complementarity determining region 3(CDR3), X is any amino acid.

<220>

<221>MISC_FEATURE

<222>(73)..(87)

<223> framework 4

<400>30

<210>31

<211>354

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(354)

<223> IgA1 heavy chain constant region

<220>

<221>MISC_FEATURE

<222>(1)..(102)

<223>CH1

<220>

<221>MISC_FEATURE

<222>(103)..(121)

<223> hinge

<220>

<221>MISC_FEATURE

<222>(122)..(222)

<223>CH2

<220>

<221>MISC_FEATURE

<222>(223)..(354)

<223>CH3

<400>31

<210>32

<211>340

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(340)

<223> IgA2 heavy chain constant region

<220>

<221>MISC_FEATURE

<222>(1)..(102)

<223>CH1

<220>

<221>MISC_FEATURE

<222>(103)..(108)

<223> hinge

<220>

<221>MISC_FEATURE

<222>(109)..(209)

<223>CH2

<220>

<221>MISC_FEATURE

<222>(210)..(340)

<223>CH3

<400>32

<210>33

<211>384

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(384)

<223> IgD heavy chain constant region

<220>

<221>MISC_FEATURE

<222>(1)..(101)

<223>CH1

<220>

<221>MISC_FEATURE

<222>(102)..(135)

<223> hinge 1

<220>

<221>MISC_FEATURE

<222>(136)..(159)

<223> hinge 2

<220>

<221>MISC_FEATURE

<222>(160)..(267)

<223>CH2

<220>

<221>MISC_FEATURE

<222>(268)..(384)

<223>CH3

<400>33

<210>34

<211>497

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(497)

<223> IgE heavy chain constant region

<220>

<221>MISC_FEATURE

<222>(1)..(103)

<223>CH1

<220>

<221>MISC_FEATURE

<222>(104)..(210)

<223>CH2

<220>

<221>MISC_FEATURE

<222>(211)..(318)

<223>CH3

<220>

<221>MISC_FEATURE

<222>(319)..(497)

<223>CH4

<400>34

<210>35

<211>339

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(339)

<223> IgG1 heavy chain constant region

<220>

<221>MISC_FEATURE

<222>(1)..(98)

<223>CH1

<220>

<221>MISC_FEATURE

<222>(99)..(113)

<223> hinge

<220>

<221>MISC_FEATURE

<222>(114)..(223)

<223>CH2

<220>

<221>MISC_FEATURE

<222>(224)..(339)

<223>CH3

<400>35

<210>36

<211>326

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(326)

<223> IgG2 heavy chain constant region

<220>

<221>MISC_FEATURE

<222>(1)..(98)

<223>CH1

<220>

<221>MISC_FEATURE

<222>(99)..(110)

<223> hinge

<220>

<221>MISC_FEATURE

<222>(111)..(219)

<223>CH2

<220>

<221>MISC_FEATURE

<222>(220)..(326)

<223>CH3

<400>36

<210>37

<211>377

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(377)

<223> IgG3 heavy chain constant region

<220>

<221>MISC_FEATURE

<222>(1)..(98)

<223>CH1

<220>

<221>MISC_FEATURE

<222>(99)..(115)

<223> hinge 1

<220>

<221>MISC_FEATURE

<222>(116)..(130)

<223> hinge 2

<220>

<221>MISC_FEATURE

<222>(131)..(145)

<223> hinge 3

<220>

<221>MISC_FEATURE

<222>(146)..(160)

<223> hinge 4

<220>

<221>MISC_FEATURE

<222>(161)..(270)

<223>CH2

<220>

<221>MISC_FEATURE

<222>(271)..(377)

<223>CH3

<400<37

<210>38

<211>327

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(327)

<223> IgG4 heavy chain constant region

<220>

<221>MISC_FEATURE

<222>(1)..(98)

<223>CH1

<220>

<221>MISC_FEATURE

<222>(99)..(110)

<223> hinge

<220>

<221>MISC_FEATURE

<222>(111)..(220)

<223>CH2

<220>

<221>MISC_FEATURE

<222>(221)..(327)

<223>CH3

<400>38

<210>39

<211>476

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(476)

<223> IgM heavy chain constant region

<220>

<221>MISC_FEATURE

<222>(1)..(104)

<223>CH1

<220>

<221>MISC_FEATURE

<222>(105)..(217)

<223>CH2

<220>

<221>MISC_FEATURE

<222>(218)..(323)

<223>CH3

<220>

<221>MISC_FEATURE

<222>(324)..(476)

<223>CH4

<400>39

<210>40

<211>107

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(107)

<223> light chain kappa constant region (IgKc)

<400>40

<210>41

<211>107

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(107)

<223> light chain lambda constant region (Ig lambda)

<400>41

<210>42

<211>5

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(5)

<223> Heavy Chain (HC) Complementarity Determining Region (CDR)1

<400>42

<210>43

<211>17

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(17)

<223>HC CDR2

<400>43

<210>44

<211>8

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(8)

<223>HC CDR3

<400>44

<210>45

<211>16

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(16)

<223> Light Chain (LC) Complementarity Determining Region (CDR)1

<400>45

<210>46

<211>7

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(7)

<223>LC CDR2

<400>46

<210>47

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(9)

<223>LC CDR3

<400>47

<210>48

<211>136

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>SIGNAL

<222>(1)..(19)

<223> Signal peptide

<220>

<221>MISC_FEATURE

<222>(20)..(49)

<223> Framework Region (FR)1

<220>

<221>MISC_FEATURE

<222>(50)..(54)

<223>CDR 1

<220>

<221>MISC_FEATURE

<222>(55)..(68)

<223>FR2

<220>

<221>MISC_FEATURE

<222>(69)..(85)

<223>CDR 2

<220>

<221>MISC_FEATURE

<222>(86)..(117)

<223>FR 3

<220>

<221>MISC_FEATURE

<222>(118)..(125)

<223>CDR 3

<220>

<221>MISC_FEATURE

<222>(126)..(136)

<223> FR4/J region

<400>48

<210>49

<211>133

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>SIGNAL

<222>(1)..(20)

<223> Signal peptide

<220>

<221>MISC_FEATURE

<222>(21)..(43)

<223>FR1

<220>

<221>MISC_FEATURE

<222>(44)..(59)

<223>CDR1

<220>

<221>MISC_FEATURE

<222>(60)..(74)

<223>FR2

<220>

<221>MISC_FEATURE

<222>(75)..(81)

<223>CDR2

<220>

<221>MISC_FEATURE

<222>(82)..(113)

<223>FR3

<220>

<221>MISC_FEATURE

<222>(114)..(122)

<223>CDR3

<220>

<221>MISC_FEATURE

<222>(123)..(133)

<223> FR4/J region

<400>49

<210>50

<211>42

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(42)

<223> known beta amyloid sequence

<400>50

<210>51

<211>408

<212>DNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(1)..(408)

<223>C701HC

<220>

<221>sig_peptide

<222>(1)..(57)

<220>

<221>misc_feature

<222>(58)..(147)

<223>FR1

<220>

<221>misc_feature

<222>(148)..(162)

<223>CDR1

<220>

<221>mjsc_feature

<222>(163)..(204)

<223>FR2

<220>

<221>misc_feature

<222>(205)..(255)

<223>CDR2

<220>

<221>misc_feature

<222>(256)..(351)

<223>FR3

<220>

<221>misc_feature

<222>(352)..(375)

<223>CDR3

<220>

<221>misc_feature

<222>(376)..(408)

<223> FR4/J region

<400>51

<210>52

<211>399

<212>DNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(1)..(399)

<223>C701 LC

<220>

<221>sig_peptide

<222>(1)..(60)

<220>

<221>misc_feature

<222>(61)..(129)

<223>FR1

<220>

<221>misc_feature

<222>(130)..(177)

<223>CDR1

<220>

<221>misc_feature

<222>(178)..(222)

<223>FR2

<220>

<221>misc_feature

<222>(223)..(243)

<223>CDR2

<220>

<221>misc_feature

<222>(244)..(339)

<223>FR3

<220>

<221>misc_feature

<222>(340)..(366)

<223>CDR3

<220>

<221>misc_feature

<222>(367)..(399)

<223> FR4/J region

<400>52

<210>53

<211>7

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(7)

<223>HC CDR1

<400>53

<210>54

<211>16

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(16)

<223>HC CDR2

<400>54

<210>55

<211>13

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(13)

<223>HC CDR3

<400>55

<210>56

<211>16

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(16)

<223>LC CDR1

<400>56

<210>57

<211>7

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(7)

<223>LC CDR2

<400>57

<210>58

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(9)

<223>LC CDR3

<400>58

<210>59

<211>142

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(142)

<223>C705 HC

<220>

<221>SIGNAL

<222>(1)..(19)

<220>

<221>MISC_FEATURE

<222>(20)..(49)

<223>FR1

<220>

<221>MISC_FEATURE

<222>(50)..(56)

<223>CDR1

<220>

<221>MISC_FEATURE

<222>(57)..(70)

<223>FR2

<220>

<221>MISC_FEATURE

<222>(71)..(86)

<223>CDR2

<220>

<221>MISC_FEATURE

<222>(87)..(118)

<223>FR3

<220>

<221>MISC_FEATURE

<222>(119)..(131)

<223>CDR3

<220>

<221>MISC_FEATURE

<222>(132)..(142)

<223> FR4/J region

<400>59

<210>60

<211>132

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(132)

<223>C705LC

<220>

<221>SIGNAL

<222>(1)..(19)

<220>

<221>MISC_FEATURE

<222>(20)..(42)

<223>FR1

<220>

<221>MISC_FEATURE

<222>(43)..(58)

<223>CDR1

<220>

<221>MISC_FEATURE

<222>(59)..(73)

<223>FR2

<220>

<221>MISC_FEATURE

<222>(74)..(80)

<223>CDR2

<220>

<221>MISC_FEATURE

<222>(81)..(112)

<223>FR3

<220>

<221>MISC_FEATURE

<222>(113)..(121)

<223>CDR3

<220>

<221>MISC_FEATURE

<222>(122)..(132)

<223> FR4/J region

<400>60

<210>61

<211>426

<212>DNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(1)..(426)

<223>C705 HC

<220>

<221>sig_peptide

<222>(1)..(57)

<220>

<221>misc_feature

<222>(58)..(147)

<223>FR1

<220>

<221>misc_feature

<222>(148)..(168)

<223>CDR1

<220>

<221>misc_feature

<222>(169)..(210)

<223>FR2

<220>

<221>misc_feature

<222>(211)..(258)

<223>CDR2

<220>

<221>misc_feature

<222>(259)..(354)

<223>FR3

<220>

<221>misc_feature

<222>(355)..(393)

<223>CDR3

<220>

<221>misc_feature

<222>(394)..(426)

<223> FR4/J region

<400>61

<210>62

<211>396

<212>DNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(1)..(396)

<223> C705 wipe LC

<220>

<221>sig_peptide

<222>(1)..(57)

<220>

<221>misc_feature

<222>(58)..(126)

<223>FR1

<220>

<221>misc_feature

<222>(127)..(174)

<223>CDR1

<220>

<221>misc_feature

<222>(175)..(219)

<223>FR2

<220>

<221>misc_feature

<222>(220)..(240)

<223>CDR2

<220>

<221>misc_feature

<222>(241)..(336)

<223>FR3

<220>

<221>misc_feature

<222>(337)..(363)

<223>CDR3

<220>

<221>misc_feature

<222>(364)..(396)

<223> FR4/J region

<400>62

<210>63

<211>5

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(5)

<223>HC CDR1

<400>63

<210>64

<211>17

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(17)

<223>HC CDR2

<400>64

<210>65

<211>10

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(10)

<223>HC CDR3

<400>65

<210>66

<211>10

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(10)

<223>LC CDR1

<400>66

<210>67

<211>7

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(7)

<223>LC CDR2

<400>67

<210>68

<211>8

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(8)

<223>LC CDR3

<400>68

<210>69

<211>138

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(138)

<223>C706 HC

<220>

<221>SIGNAL

<222>(1)..(19)

<220>

<221>MISC_FEATURE

<222>(20)..(49)

<223>FR1

<220>

<221>MISC_FEATURE

<222>(50)..(54)

<223>CDR1

<220>

<221>MISC_FEATURE

<222>(55)..(68)

<223>FR2

<220>

<221>MISC_FEATURE

<222>(69)..(85)

<223>CDR2

<220>

<221>MISC_FEATURE

<222>(86)..(117)

<223>FR3

<220>

<221>MISC_FEATURE

<222>(118)..(127)

<223>CDR3

<220>

<221>MISC_FEATURE

<222>(128)..(138)

<223> FR4/J region

<400>69

<210>70

<211>128

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(128)

<223>C706 LC

<220>

<221>SIGNAL

<222>(1)..(22)

<220>

<221>MISC_FEATURE

<222>(23)..(45)

<223>FR1

<220>

<221>MISC_FEATURE

<222>(46)..(55)

<223>CDR1

<220>

<221>MISC_FEATURE

<222>(56)..(70)

<223>FR2

<220>

<221>MISC_FEATURE

<222>(71)..(77)

<223>CDR2

<220>

<221>MISC_FEATURE

<222>(78)..(109)

<223>FR3

<220>

<221>MISC_FEATURE

<222>(110)..(117)

<223>CDR3

<220>

<221>MISC_FEATURE

<222>(118)..(128)

<223> FR4/J region

<400>70

<210>71

<211>414

<212>DNA

<213> Intelligent (Homosapiens)

<220>

<221>misc_feature

<222>(1)..(414)

<223>C706 HC

<220>

<221>sig_peptide

<222>(1)..(57)

<220>

<221>misc_feature

<222>(58)..(147)

<223>FR1

<220>

<221>misc_feature

<222>(148)..(162)

<223>CDR1

<220>

<221>misc_feature

<222>(163)..(204)

<223>FR2

<220>

<221>misc_feature

<222>(205)..(255)

<223>CDR2

<220>

<221>misc_feature

<222>(256)..(351)

<223>FR3

<220>

<221>misc_feature

<222>(352)..(381)

<223>CDR3

<220>

<221>misc_feature

<222>(382)..(414)

<223> FR4/J region

<400>71

<210>72

<211>384

<212>DNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(1)..(384)

<223>C706 LC

<220>

<221>sig_peptide

<222>(1)..(66)

<220>

<221>misc_feature

<222>(67)..(135)

<223>FR1

<220>

<221>misc_feature

<222>(136)..(165)

<223>CDR1

<220>

<221>misc_feature

<222>(166)..(210)

<223>FR2

<220>

<221>misc_feature

<222>(211)..(231)

<223>CDR2

<220>

<221>misc_feature

<222>(232)..(327)

<223>FR3

<220>

<221>misc_feature

<222>(328)..(351)

<223>CDR3

<220>

<221>misc_feature

<222>(352)..(384)

<223> FR4/J region

<400>72

<210>73

<211>5

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(5)

<223>HC CDR1

<400>73

<210>74

<211>17

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(17)

<223>HC CDR2

<400>74

<210>75

<211>5

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(5)

<223>HCCDR3

<400>75

<210>76

<211>16

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(16)

<223>LC CDR1

<400>76

<210>77

<211>7

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(7)

<223>LC CDR2

<400>77

<210>78

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(9)

<223>LC CDR3

<400>78

<210>79

<211>133

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(133)

<223>C707HC

<220>

<221>SIGNAL

<222>(1)..(19)

<220>

<221>MISC_FEATURE

<222>(20)..(49)

<223>FR1

<220>

<221>MISC_FEATURE

<222>(50)..(54)

<223>CDR1

<220>

<221>MISC_FEATURE

<222>(55)..(68)

<223>FR2

<220>

<221>MISC_FEATURE

<222>(69)..(85)

<223>CDR2

<220>

<221>MISC_FEATURE

<222>(86)..(117)

<223>FR3

<220>

<221>MISC_FEATURE

<222>(118)..(122)

<223>CDR3

<220>

<221>MISC_FEATURE

<222>(123)..(133)

<223> FR4/J region

<400>79

<210>80

<211>133

<212>PRT

<213> Intelligent (Homo sapiens)

<220>

<221>MISC_FEATURE

<222>(1)..(133)

<223>C707 LC

<220>

<221>SIGNAL

<222>(1)..(20)

<220>

<221>MISC_FEATURE

<222>(21)..(43)

<223>FR1

<220>

<221>MISC_FEATURE

<222>(44)..(59)

<223>CDR1

<220>

<221>MISC_FEATURE

<222>(60)..(74)

<223>FR2

<220>

<221>MISC_FEATURE

<222>(75)..(81)

<223>CDR2

<220>

<221>MISC_FEATURE

<222>(82)..(113)

<223>FR3

<220>

<221>MISC_FEATURE

<222>(114)..(122)

<223>CDR3

<220>

<221>MISC_FEATURE

<222>(123)..(133)

<223> FR4/J region

<400>80

<210>81

<211>399

<212>DNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(1)..(399)

<223>C707HC

<220>

<221>sig_peptide

<222>(1)..(57)

<220>

<221>misc_feature

<222>(58)..(147)

<223>FR1

<220>

<221>misc_feature

<222>(148)..(162)

<223>CDR1

<220>

<221>misc_feature

<222>(163)..(204)

<223>FR2

<220>

<221>misc_feature

<222>(205)..(255)

<223>CDR2

<220>

<221>misc_feature

<222>(256)..(351)

<223>FR3

<220>

<221>misc_feature

<222>(352)..(366)

<223>CDR3

<220>

<221>misc_feature

<222>(367)..(399)

<223> FR4/J region

<400>81

<210>82

<211>399

<212>DNA

<213> Intelligent (Homo sapiens)

<220>

<221>misc_feature

<222>(1)..(399)

<223>C707LC

<220>

<221>sig_peptide

<222>(1)..(60)

<220>

<221>misc_feature

<222>(61)..(129)

<223>FR1

<220>

<221>misc_feature

<222>(130)..(177)

<223>CDR1

<220>

<221>misc_feature

<222>(178)..(222)

<223>FR2

<220>

<221>misc_feature

<222>(223)..(243)

<223>CDR2

<220>

<221>misc_feature

<222>(244)..(339)

<223>FR3

<220>

<221>misc_feature

<222>(340)..(366)

<223>CDR3

<220>

<221>misc_feature

<222>(367)..(399)

<223> FR4/J region

<400>82

<210>83

<211>12

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>83

<210>84

<211>12

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>84

<210>85

<211>12

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>85

<210>86

<211>11

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>86

<210>87

<211>10

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>87

<210>88

<211>9

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>88

<210>89

<211>8

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>89

<210>90

<211>7

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>90

<210>91

<211>6

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>91

<210>92

<211>15

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>92

<210>93

<211>14

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>93

<210>94

<211>13

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>94

<210>95

<211>12

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>95

<210>96

<211>11

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>96

<210>97

<211>10

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>97

<210>98

<211>9

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>98

<210>99

<211>8

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>99

<210>100

<211>7

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>100

<210>101

<211>6

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>101

<210>102

<211>15

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>102

<210>103

<211>14

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>103

<210>104

<211>13

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>104

<210>105

<211>12

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>105

<210>106

<211>11

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>106

<210>107

<211>10

<212>PRT

<213> Artificial

<220>

<223> peptide

<210>108

<211>9

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>108

<210>109

<211>8

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>109

<210>110

<211>9

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>110

<210>111

<211>6

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>111

<210>112

<211>15

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>112

<210>113

<211>14

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>113

<210>114

<211>14

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>114

<210>115

<211>12

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>115

<210>116

<211>13

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>116

<210>117

<211>12

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>117

<210>118

<211>12

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>118

<210>119

<211>11

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>119

<210>120

<211>11

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>120

<210>121

<211>10

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>121

<210>122

<211>10

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>122

<210>123

<211>9

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>123

<210>124

<211>9

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>124

<210>125

<211>8

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>125

<210>126

<211>8

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>126

<210>127

<211>7

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>127

<210>128

<211>7

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>128

<210>129

<211>6

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>129

<210>130

<211>6

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>130

<210>131

<211>6

<212>PRT

<213> Artificial

<220>

<223> peptide

<400>131

Claims (10)

1. At least one isolated mammalian amyloid antibody comprising a heavy chain variable region comprising SEQ id no: 69-70 and at least one variable region of at least one heavy chain and at least one light chain.

2. At least one isolated mammalian amyloid antibody comprising (i) the amino acid sequence of SEQ id no: 63-65, at least two of a heavy chain Complementarity Determining Region (CDR) amino acid sequence of at least one of; or (ii) SEQ ID NOS: 66-68.

3. At least one isolated mammalian amyloid antibody comprising a heavy chain variable region having the amino acid sequence of SEQ id no: 69-70 in a sequence of amino acids of at least one of the heavy or light chain CDRs.

4. At least one isolated mammalian amyloid antibody that binds to a polypeptide comprising a polypeptide having the amino acid sequence of seq id NOS: 63-68 binds to the same amyloid polypeptide region.

5. An isolated nucleic acid vector comprising an isolated nucleic acid encoding at least one isolated mammalian amyloid antibody of any one of claims 1-4.

6. A prokaryotic or eukaryotic host cell containing an isolated nucleic acid encoding at least one isolated mammalian amyloid antibody of any one of claims 1-4.

7. The host cell according to claim 6, wherein said host cell is at least one selected from the group consisting of COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, or lymphoma cells, or any derived, immortalized or transformed cell thereof.

8. A method of producing at least one amyloid antibody comprising translating a nucleic acid encoding at least one isolated mammalian amyloid antibody of any one of claims 1-4 under in vitro, in vivo or in situ conditions, whereby the amyloid antibody is expressed in detectable or recoverable amounts.

9. A composition comprising at least one isolated mammalian amyloid antibody according to any one of claims 1-4 having at least one human CDR, wherein said antibody specifically binds to a polypeptide comprising SEQ ID NO: 50 to at least one epitope of the entire amino acid sequence, said composition further comprising at least one pharmaceutically acceptable carrier or diluent.

10. Medical device containing at least one amyloid antibody according to any one of claims 1-4, wherein the device is adapted to contact or administer said at least one amyloid antibody by a mode selected from the group consisting of: parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavity, intracavitary, cerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.