Antibody Formulations and Methods

Abstract

Antibody formulations and methods useful for prophylaxis or treatment of amyloidosis, including AA amyloidosis and AL amyloidosis.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation of U.S. patent application Ser. No. 16/730,265 filed Dec. 30, 2019, which is a continuation of U.S. patent application Ser. No. 15/868,567 filed Jan. 11, 2018, now U.S. Pat. No. 10,517,830 issued 31 Dec. 2019, which is a continuation of U.S. patent application Ser. No. 15/139,628 filed 27 Apr. 2016, now U.S. Pat. No. 9,884,020 issued 6 Feb. 2018, which is a divisional of U.S. patent application Ser. No. 14/720,505, filed 22 May 2015, now abandoned, which is a divisional of U.S. patent application Ser. No. 13/660,957, filed 25 Oct. 2012, now U.S. Pat. No. 9,089,529 issued 28 Jul. 28, 2015, which claims priority to U.S. Provisional Application No. 61/551,406, filed 25 Oct. 2011, each of which is incorporated by reference herein in its entirety.

TECHNICAL FIELD

[0002] The invention resides in the technical fields of immunology and medicine.

BACKGROUND OF THE INVENTION

[0003] Amyloidosis is a general term that describes a number of diseases characterized by the existence of pathological forms of amyloid proteins, often involving extracellular deposition of protein fibrils, which form numerous "amyloid deposits" or "amyloid plaques," which may occur in local sites or systematically. These deposits or plaques are composed primarily of a naturally occurring soluble protein or peptide, assembled into extensive insoluble deposits 10-100 m in diameter in a variety of tissue sites. The deposits are composed of generally lateral aggregates of fibrils that are approximately 10-15 nm in diameter. Amyloid fibrils produce a characteristic apple green birefringence in polarized light, when stained with Congo Red dye. Generally, the fibrillar composition of these deposits is an identifying characteristic for the various forms of amyloid disease.

[0004] The peptides or proteins forming the plaque deposits are often produced from a larger precursor protein. More specifically, the pathogenesis of amyloid aggregates such as fibril deposits generally involves proteolytic cleavage of an "abnormal" precursor protein into fragments that aggregate into anti-parallel .beta. pleated sheets.

[0005] Systemic amyloidoses are a complex group of diseases caused by tissue deposition of misfolded proteins that result in progressive organ damage. The most common type, AL amyloidosis or primary amyloidosis, involves a hematological disorder caused by clonal plasma cells that produce misfolded immunoglobulin light chains. Overproduction of misfolded light chain by plasma cells results in deposits of abnormal AL protein (amyloid), in the tissues and organs of individuals with AL amyloidosis. Clinical features of AL amyloidosis include a constellation of symptoms and organ dysfunction that can include cardiac, renal, and hepatic dysfunction, gastrointestinal involvement, neuropathies and macroglossia. The mechanisms by which amyloidogenic immunoglobulin light chains result in organ dysfunction are not well characterized, however, it is hypothesized that both amyloid deposits and prefibrillar aggregates may contribute to cytotoxic effects on organs observed in patients with AL amyloidosis. AL amyloidosis is a disease entity of its own, although AL amyloidosis can occur concurrently in a small subset (up to 15%) of patients with multiple myeloma.

[0006] AL amyloidosis is a rare disorder with an estimated incidence of 8 in 1,000,000 people. Only 1200 to 3200 new cases of AL amyloidosis are reported each year in the United States. Two thirds of patients with AL amyloidosis are male and less than 5% of patients are under 40 years of age. Both the causes and origins of AL amyloidosis remain poorly understood.

[0007] Current treatment of patients with AL amyloidosis is aimed at reducing or eliminating the bone marrow disorder, i.e. the plasma cells that are responsible for producing the light chains, thereby limiting or halting the production of amyloid. The most aggressive treatment options include stem cell transplant and high-dose chemotherapy for those patients who can tolerate it. Other treatment regimens include combinations of drugs often used to treat hematological malignancies, such as melphalan, prednisone, dexamethasone and proteosome inhibitors such as bortezomib, in an attempt to reduce light chain production. There are no currently approved treatments for AL amyloidosis that directly target potentially toxic forms of the amyloidogenic proteins.

[0008] A different form of systemic amyloidosis, AA amyloidosis or secondary amyloidosis, occurs "secondarily" as a result of other illness, such as chronic inflammatory diseases (for example, rheumatoid arthritis and ankylosing spondylitis) or chronic infections (for example, tuberculosis or osteomyelitis). In secondary amyloidosis, the depositing amyloid protein is amyloid A protein, derived from an acute-phase protein serum amyloid A. The treatment of secondary amyloidosis is directed at treating the underlying illness.

[0009] Thus, there is a need for therapies to treat AA amyloidosis and AL amyloidosis, which directly target the pathological amyloid fibrils. The present invention provides pharmaceutical formulations of 2A4 and 7D8 antibodies, and chimeric and humanized versions thereof, which show high affinity binding to both AL and AA amyloids due to a shared immunogenic epitope of the pathological forms of these proteins.

SUMMARY OF THE INVENTION

[0010] The present invention provides antibody formulations useful for prophylaxis and treatment of amyloid disease. In one aspect of the invention, a pharmaceutical formulation comprises (a) a chimeric or humanized version of antibody 2A4 (ATCC Accession Number PTA-9662) or of antibody 7D8 (ATCC Accession Number PTA-9468), or fragment thereof, which specifically competes for binding to antigen with 2A4 or 7D8, and/or which is directed to an epitope comprising AEDS (SEQ ID NO: 18), wherein the antibody is present at a concentration within the range from about 1 mg/mL to about 100 mg/mL; (b) histidine buffer present at a concentration within the range from about 20 mM to about 30 mM; (c) trehalose present at a concentration within the range from about 210 mM to about 250 mM; and (d) polysorbate 20 present at a concentration within the range from about 0.005% to about 0.05% by weight; wherein the formulation is characterized by a pH within the range from about 6 to about 7. For example, representative formulations of the invention comprise an antibody having a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 4 and/or a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 5. More particularly, such a formulation can comprise an antibody having a light chain comprising an amino acid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising an amino acid sequence set forth as any one of SEQ ID NO: 14-16, for example, an antibody having a light chain comprising an amino acid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising an amino acid sequence set forth as SEQ ID NO: 15.

[0011] Additional representative formulations of the invention comprise (a) an antibody having a light chain variable region comprising three complementarity determining regions set forth as SEQ ID NOs: 6, 7, and 8, and a heavy chain variable region comprising three complementarity regions set forth as SEQ ID NOs: 9, 10, and 11; and (b) an antibody having a light chain variable region comprising three complementarity determining regions set forth as SEQ ID NOs: 12, 7, and 8, and a heavy chain variable region comprising three complementarity regions set forth as SEQ ID NOs: 9, 10, and 11.

[0012] In representative formulations of the invention, the antibody is present at a concentration within the range from about 5 mg/mL to about 15 mg/mL (e.g., about 10 mg/mL), or present at a concentration within the range from about 25-75 mg/mL (e.g., 50 mg/mL).

[0013] In other representative formulations of the invention, histidine buffer is present at a concentration of about 25 mM. The histidine buffer can comprise L-histidine and L-histidine HCl monohydrate. For example, L-histidine can be used at a concentration within the range from about 16 mM to about 22 mM and L-histidine HCl monohydrate can be used at a concentration within the range from about 4 mM to about 8 mM.

[0014] In other representative formulations of the invention, trehalose is present at a concentration of about 230 mM.

[0015] Prepared as described herein, representative formulations of the invention (a) are characterized by an osmolality of about 300 mOsm/kg; (b) comprise less than about 10% of the antibody present as an aggregate in the formulation; (c) further comprise a bulking agent; (d) are sterile; and/or (e) are stable upon freezing and thawing.

[0016] In one aspect of the invention, a formulation comprises (a) an antibody comprising a light chain comprising an amino acid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising an amino acid sequence set forth as any one of SEQ ID NOs: 14-16, and which is present at a concentration of about 10 mg/mL; (b) a histidine buffer present at a concentration of about 25 mM; (c) trehalose present at a concentration of about 230 mM; (d) polysorbate 20 present at a concentration of about 0.2 g/L; and (e) a pH of about 6.5.

[0017] In another aspect of the invention, a pharmaceutical formulation comprises (a) an antibody, which is antibody 2A4 (ATCC Accession Number PTA-9662), antibody 7D8 (ATCC Accession Number PTA-9468), or a chimeric or humanized version of antibody 2A4 or of antibody 7D8, or fragment thereof, which specifically competes for binding to antigen with 2A4 or 7D8, and/or which is directed to an epitope comprising AEDS (SEQ ID NO: 18), wherein the antibody is present at a concentration within the range from about 50 mg/mL to about 100 mg/mL; (b) a buffer; (c) a non-reducing sugar; and (d) a non-ionic surfactant. In particular examples, the antibody of the disclosed formulations comprises a light chain comprising an amino acid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising an amino acid sequence set forth as SEQ ID NOs: 15.

[0018] In another aspect of the invention, the antibody formulations are lyophilized. For example, a representative lyophilized formulation can comprise: (a) a humanized version of antibody 2A4 (ATCC Accession Number PTA-9662) or antibody 7D8 (ATCC Accession Number PTA-9468) or antigen binding fragment thereof; (b) histidine; (c) trehalose; and (d) polysorbate 20. Lyophilized formulations can have a pH of between about 6 to about 7 when reconstituted, such as pH 6.5 when reconstituted. Lyophilized formulations typically comprise about 100 mg to about 1000 mg of the antibody. Lyophilized formulations typically comprise polysorbate 20 at a concentration within the range from about 0.005% to about 0.05% by weight. Following reconstitution, the lyophilized formulations yield an aqueous solution, for example, an aqueous solution comprising: (a) an antibody comprising a light chain comprising an amino acid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising an amino acid sequence set forth as any one of SEQ ID NOs: 14-16, and which is present at a concentration of about 10 mg/mL; (b) a histidine buffer present at a concentration of about 25 mM; (c) trehalose present at a concentration of about 230 mM; (d) polysorbate 20 present at a concentration of about 0.2 g/L; and (e) a pH of about 6.5. A representative lyophilized formulation comprises about 100 mg of the antibody following reconstitution with sterile water.

[0019] Also provided are nucleic acids encoding antibodies used to prepare the disclosed formulations. For example, such nucleic acids include nucleic acids comprising nucleotide sequences encoding an antibody light chain of SEQ ID NO: 13 and nucleic acids comprising nucleotide sequences encoding an antibody heavy chain of any one of SEQ ID NOs: 14-16. For example, the nucleotide sequences set forth as SEQ ID NO: 19 and SEQ ID NO: 20 (which is identical to SEQ ID NO: 19 and further includes a sequence encoding a signal peptide) each encode the humanized 2A4 light chain of SEQ ID NO: 13. As another example, the nucleotide sequences set forth as SEQ ID NO: 22 and SEQ ID NO: 23 (which is identical to SEQ ID NO: 22 and further includes a sequence encoding a signal peptide) each encode the humanized 2A4 heavy chain of SEQ ID NO: 15.

[0020] For the production of antibodies, the disclosed nucleic acids may be included in a vector, either singly or in combination (e.g., a combination of a nucleic acid encoding a humanized 2A4 light chain and a nucleic acid encoding a humanized 2A4 heavy chain). For example, a vector can comprise a nucleic acid comprising a nucleotide sequence encoding any one of SEQ ID NOs: 13-16, 21, and 24; a nucleic acid comprising the nucleotide sequence of any one of SEQ ID NOs: 19-20 and 22-23, or combinations thereof. Representative vectors of the invention include (a) a vector comprising a nucleic acid sequence encoding a humanized 2A4 light chain set forth as SEQ ID NO: 13 or 21 and a humanized 2A heavy chain set forth as SEQ ID NO: 15 or 24; (b) a vector comprising a nucleic acid having the nucleotide sequence of SEQ ID NO: 19 and a nucleic acid having the nucleotide sequence of SEQ ID NO: 22; and (c) a vector comprising a nucleic acid having the nucleotide sequence of SEQ ID NO: 20 and a nucleic acid having the nucleotide sequence of SEQ ID NO: 23.

[0021] Also provided are host cells (e.g., CHO cells) having stably incorporated into their genomes one or more of the nucleic acids disclosed herein. For example, a host cell can comprise in its genome a stably integrated nucleic acid comprising a nucleotide sequence encoding any one of SEQ ID NOs: 13-16, 21, and 24; a stably integrated nucleic acid comprising the nucleotide sequence of any one of SEQ ID NOs: 19-20 and 22-23, or combinations thereof. Representative host cells of the invention include (a) host cells comprising a nucleic acid sequence encoding a humanized 2A4 light chain set forth as SEQ ID NO: 13 or 21 and a humanized 2A heavy chain set forth as SEQ ID NO: 15 or 24; (b) host cells comprising a nucleic acid having the nucleotide sequence of SEQ ID NO: 19 and a nucleic acid having the nucleotide sequence of SEQ ID NO: 22; and (c) host cells comprising a nucleic acid having the nucleotide sequence of SEQ ID NO: 20 and a nucleic acid having the nucleotide sequence of SEQ ID NO: 23.

[0022] The present invention also provides methods of preparing pharmaceutical formulations. In one aspect of the invention, such a method comprises (a) culturing mammalian cells having stably incorporated into their genome nucleic acids encoding the light and heavy chains of a murine, chimeric or humanized 2A4 antibody or of a murine, chimeric or humanized 7D8 antibody so that the cells secrete the antibody into the cell culture media, and purifying the antibody from the cell culture media; (b) and preparing a formulation comprising (i) a chimeric or humanized version of antibody 2A4 (ATCC Accession Number PTA-9662) or of antibody 7D8 (ATCC Accession Number PTA-9468), or fragment thereof, that specifically competes for binding to antigen with 2A4 or 7D8, wherein the antibody is present at a concentration within the range from about 1 mg/mL to about 100 mg/mL; (ii) histidine buffer present at a concentration within the range from about 20 mM to about 30 mM; (iii) trehalose present at a concentration within the range from about 210 mM to about 250 mM; and (iv) polysorbate 20 present at a concentration within the range from about 0.005% to about 0.05% by weight; wherein the formulation is characterized by a pH within the range from about 6 to about 7. For example, in one aspect of the invention, mammalian cells having stably incorporated into their genomes nucleic acids encoding the light and heavy chains of a humanized 2A4 antibody are cultured. Mammalian cells useful for this purpose include (a) host cells having stably incorporated into their genomes a nucleic acid sequence encoding a humanized 2A4 light chain set forth as SEQ ID NO: 13 or 21 and a humanized 2A heavy chain set forth as SEQ ID NO: 15 or 24; (b) host cells having stably incorporated into their genomes a nucleic acid having the nucleotide sequence of SEQ ID NO: 19 and a nucleic acid having the nucleotide sequence of SEQ ID NO: 22; and (c) host cells having stably incorporated into their genomes a nucleic acid having the nucleotide sequence of SEQ ID NO: 20 and a nucleic acid having the nucleotide sequence of SEQ ID NO: 23. In some aspects of the invention, the disclosed methods of preparing a pharmaceutical formulation include the additional step of evaluating at least one property of antibody in the formulation, such as physical stability, chemical stability, and/or biological activity.

[0023] Still further provided are methods of therapeutically or prophylactically treating a human patient having or at risk of having amyloidosis characterized by the presence of amyloid protein fibrils, the method comprising administering to the patient an effective dosage of a formulation of the invention. Patients amenable to treatment have an amyloid disease such as amyloid A amyloidosis, which is characterized by the presence of amyloid A protein fibrils, or AL amyloidosis, which is characterized by the presence of amyloid light chain-type protein fibrils. Patients having AL amyloidosis may also suffer from an associated dyscrasis of the B lymphocyte lineage, for example a malignancy such as multiple myeloma.

[0024] The disclosed therapeutic and prophylactic treatment methods include combination therapies (i.e., administration of the disclosed antibody formulations with one or more additional drug substances) to thereby elicit synergistic results. The two or more drug substances are administered simultaneously or sequentially in any order, i.e., a formulation of the invention is administered prior to administration of a second drug substance, concurrently with a second drug substance, or subsequent to administration of a second drug substance. For example, a formulation of the invention can be administered concurrently or consecutively in combination with melphalan. As another example, a formulation of the invention can be administered concurrently or consecutively in combination with one or more of bortezomib, melphalan, lenalidomide and carfilzomib.

[0025] In accordance with the disclosed therapeutic and prophylactic treatment methods, formulations of the invention can be administered in multiple dosages, for example, at a frequency in a range of about daily to about annually, such as at a frequency in a range of about every other week to about every three months, or such as once a month. In one aspect, an antibody formulation of the invention is administered intravenously at a dose in a range from about 10 mg to about 5000 mg drug substance. For example, a formulation can be administered at a dose in a range from about 30 mg to about 2500 mg humanized 2A4 drug substance at a frequency in a range of about every other week to about every other month. Representative dosages used in the disclosed methods include 30 mg, 100 mg, 300 mg, 1000 mg, 2000 mg, and 2500 mg of humanized 2A4 drug substance.

[0026] In one aspect of the invention, a method of therapeutically or prophylactically treating a human patient having or at risk for having light chain (AL) amyloidosis characterized by the presence of amyloid fibrils, deposits or prefibrillar aggregates, comprises administering to the patient an effective dosage of a pharmaceutical formulation comprising: (a) an antibody comprising a light chain comprising an amino acid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising an amino acid sequence set forth as any one of SEQ ID NOs: 14-16, and which is present at a concentration of about 10 mg/mL; (b) a histidine buffer present at a concentration of about 25 mM; (c) trehalose present at a concentration of about 230 mM; (d) polysorbate 20 present at a concentration of about 0.2 g/L; and (e) a pH of about 6.5. In such a method, the dosage is typically from about 0.5 mg/kg to about 30 mg/kg of the antibody (e.g., about 0.5 mg/kg to about 8 mg/kg, or about 8 mg/kg to about 30 mg/kg) administered intravenously or subcutaneously at a frequency of from about weekly to about quarterly (e.g., once every 28 days).

[0027] The present invention further provides a pharmaceutical product comprising: (a) a vial comprising about 100 mg antibody in powder form; (b) instructions for reconstitution of the antibody; and (c) instructions for preparing the reconstituted antibody for infusion, wherein (i) the antibody comprises a light chain comprising an amino acid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising an amino acid sequence set forth as any one of SEQ ID NOs: 14-16; and (ii) the reconstitution instructions require reconstitution with water for injection to an extractable volume of 10 mL.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIGS. 1A-1B show various humanized 2A4 antibody light chain and heavy chain sequences. Bold and underlining, consensus sequence for N-linked glycosylation.

[0029] FIG. 2 shows murine 2A4 and 7D8 light chain variable region (VL) and heavy chain variable region (VH) sequences. Double underlining, leader sequence; underlining, complementarity determining region (CDR) sequences.

[0030] FIG. 3 shows humanized 2A4 version 3 light chain variable region (VL) and heavy chain variable region (VH) sequences. Lower case, back mutations.

[0031] FIGS. 4A-4B show nucleic acid sequences encoding humanized 2A4 version 3 heavy chain (FIG. 4A) and heavy chain (FIG. 4B) sequences. Single underline, leader sequence; no underline, variable region; double underline, constant region.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention provides antibody formulations useful for prophylaxis and treatment of amyloid disease. In one aspect of the invention, a pharmaceutical formulation comprises (a) a chimeric or humanized version of antibody 2A4 (ATCC Accession Number PTA-9662) or of antibody 7D8 (ATCC Accession Number PTA-9468), or fragment thereof, which specifically competes for binding to antigen with 2A4 or 7D8, and/or which is directed to an epitope comprising AEDS (SEQ ID NO: 18), wherein the antibody is present at a concentration within the range from about 1 mg/mL to about 100 mg/mL; (b) histidine buffer present at a concentration within the range from about 20 mM to about 30 mM; (c) trehalose present at a concentration within the range from about 210 mM to about 250 mM; and (d) polysorbate 20 present at a concentration within the range from about 0.005% to about 0.05% by weight; wherein the formulation is characterized by a pH within the range from about 6 to about 7.

[0033] In one aspect of the invention described herein, humanized 2A4 is an IgG1, kappa isotype version of murine 2A4. In the course of specificity characterization of humanized 2A4, the antibody was found to also react with high affinity and in a conformation-dependent manner with light chain in light chain amyloid fibrils, but not with free light chain in circulation.

[0034] The present invention provides methods for intravenous infusion of humanized 2A4 and/or humanized 7D8 antibodies to target misfolded amyloid protein in patients with AA amyloidosis and AL amyloidosis. Some humanized 2A4 antibodies specifically bind to pathologic amyloid forms of AL and SAA but do not bind to the parent molecules from which these pathologic forms are derived (SAA, native immunoglobulin light chain [LC], intact immunoglobulin [Ig]).

I. Pharmaceutical Formulations and Products

[0035] I.A. Characteristics

[0036] Provided herein are pharmaceutical formulations comprising a chimeric or humanized version of antibody 2A4 (ATCC Accession Number PTA-9662) or of antibody 7D8 (ATCC Accession Number PTA-9468), or fragment thereof, that specifically competes for binding to antigen (i.e., human AA or AL protein) with 2A4 or 7D8, respectively, and/or that is directed to the epitope AEDS (SEQ ID NO: 18). Also provided are pharmaceutical formulations comprising murine antibody 2A4 or murine antibody 7D8, or fragments thereof. The antibody is present at a concentration within the range from about 1 mg/mL to about 100 mg/mL. The formulation is characterized by a pH within the range from about 6 to about 7 and comprises a histidine buffer at a concentration within the range from about 20 mM to about 30 mM, trehalose at a concentration within the range from about 210 mM to about 250 mM; and polysorbate 20 at a concentration within the range from about 0.005% to about 0.05% by weight.

[0037] The term "humanized immunoglobulin" or "humanized antibody" refers to an immunoglobulin or antibody that includes at least one humanized immunoglobulin or antibody chain (i.e., at least one humanized light or heavy chain). The term "humanized immunoglobulin chain" or "humanized antibody chain" (i.e., a "humanized immunoglobulin light chain" or "humanized immunoglobulin heavy chain") refers to an immunoglobulin or antibody chain (i.e., a light or heavy chain, respectively) having a variable region that includes a variable framework region substantially from a human immunoglobulin or antibody and complementarity determining regions (CDRs) (e.g., at least one CDR, preferably two CDRs, more preferably three CDRs) substantially from a non-human immunoglobulin or antibody, and further includes constant regions (e.g., at least one constant region or portion thereof, in the case of a light chain, and preferably three constant regions in the case of a heavy chain). The term "humanized variable region" (e.g., "humanized light chain variable region" or "humanized heavy chain variable region") refers to a variable region that includes a variable framework region substantially from a human immunoglobulin or antibody and complementarity determining regions (CDRs) substantially from a non-human immunoglobulin or antibody.

[0038] The phrase "substantially from a human immunoglobulin or antibody" or "substantially human" means that, when aligned to a human immunoglobulin or antibody amino sequence for comparison purposes, the region shares at least 80-90%, preferably 90-95%, more preferably 95-99% identity (i.e., local sequence identity) with the human framework or constant region sequence, allowing, for example, for conservative substitutions, consensus sequence substitutions, germline substitutions, backmutations, and the like. The introduction of conservative substitutions, consensus sequence substitutions, germline substitutions, backmutations, and the like, is often referred to as "optimization" of a humanized antibody or chain. The phrase "substantially from a non-human immunoglobulin or antibody" or "substantially non-human" means having an immunoglobulin or antibody sequence at least 80-95%, preferably 90-95%, more preferably, 96%, 97%, 98%, or 99% identical to that of a non-human organism, e.g., a non-human mammal.

[0039] Accordingly, all regions or residues of a humanized immunoglobulin or antibody, or of a humanized immunoglobulin or antibody chain, except possibly the CDRs, are substantially identical to the corresponding regions or residues of one or more native human immunoglobulin sequences. The term "corresponding region" or "corresponding residue" refers to a region or residue on a second amino acid or nucleotide sequence which occupies the same (i.e., equivalent) position as a region or residue on a first amino acid or nucleotide sequence, when the first and second sequences are optimally aligned for comparison purposes.

[0040] In some formulations, the antibody comprises a light chain variable region comprising an amino acid sequence set forth as any one of SEQ ID NOs: 1, 2, or 4. In some formulations, the antibody comprises a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 3 or 5. In some formulations, the antibody comprises a light chain variable region comprising an amino acid sequence set forth as any one of SEQ ID NOs: 1, 2, or 4 and a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 3 or 5. In some formulations, the antibody comprises a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 1 and a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 3. In some formulations, the antibody comprises a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 4 and a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 5. In some formulations, the antibody comprises a light chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 2 and a heavy chain variable region comprising an amino acid sequence set forth as SEQ ID NO: 3.

[0041] In some formulations, the antibody comprises a light chain variable region comprising three complementarity determining regions set forth as SEQ ID NOs: 6, 7, and 8, and a heavy chain variable region comprising three complementarity regions set forth as SEQ ID NOs: 9, 10, and 11. In other formulations, the antibody comprises a light chain variable region comprising three complementarity determining regions set forth as SEQ ID NOs: 12, 7, and 8, and a heavy chain variable region comprising three complementarity regions set forth as SEQ ID NOs: 9, 10, and 11.

[0042] In other formulations of the present invention, the antibody comprises light chain and heavy chain variable regions of a murine, chimeric, or humanized 2A4 antibody, or of a murine, chimeric, or humanized 7D8 antibody, as described in U.S. Pat. No. 7,928,203 and PCT International Publication No. WO 2009/086539, each of which is incorporated herein by reference in its entirety, and the light chain and heavy chain variable region sequences described in the referenced patent and publication are specifically incorporated by reference herein.

[0043] In some formulations, the antibody comprises a light chain comprising an amino acid sequence set forth as SEQ ID NO: 13 or 21 and a heavy chain comprising an amino acid sequence set forth as any one of SEQ ID NOs: 14-16 and 24. The antibody can include, or not include, the leader sequences of the above-noted light chain and heavy chain amino acid sequences.

[0044] In other formulations, the antibody is a fragment of a 2A4 or 7D8 antibody, including chimeric and humanized versions thereof, such as a Fab fragment, a Fab' fragment, a F(ab').sub.2 fragment, a Fv fragment or a ScFv fragment.

[0045] In some aspects of the invention, the antibody specifically binds to aggregated amyloid protein without specifically binding to monomeric amyloid protein (e.g., at least a 10-fold and usually at least 100-fold lower specific binding affinity for monomeric forms of the amyloid protein).

[0046] In some formulations, the antibody is present at a concentration within the range from about 5 mg/mL to about 100 mg/mL. In some formulations, the antibody is present at a concentration within the range from about 5 mg/mL to about 15 mg/mL. In some formulations, the antibody is present at a concentration within the range from about 25 mg/mL to about 75 mg/mL. For example, the antibody may be present at a concentration of about 10 mg/mL, or present at a concentration of about 50 mg/mL. The antibody may be present in a sterile liquid dosage form of about 50 mg/vial to about 500 mg/vial, or greater. For example, the antibody may be present in a sterile liquid dosage form of about 100 mg/vial.

[0047] Antibodies used in the disclosed formulations can be coupled with a therapeutic moiety, such as a cytotoxic agent, a radiotherapeutic agent, an immunomodulator, a second antibody (e.g., to form an antibody heteroconjugate), or any other biologically active agent that facilitates or enhances the activity of a chimeric or humanized 2A4 or a chimeric or humanized 7D8 antibody. Representative therapeutic moieties include agent known to be useful for treatment, management, or amelioration of amyloid disease or symptoms of amyloid disease.

[0048] Antibodies used in the disclosed formulations can also be coupled with a detectable label, for example, as useful for diagnosing an amyloid disorder, for monitoring progression of amyloid disease, and/or for assessing efficacy of treatment. Antibodies formulated as described are particularly useful for performing such determinations in subjects having or being susceptible to AA amyloidosis or AL amyloidosis, or in appropriate biological samples obtained from such subjects. Representative detectable labels that may be coupled or linked to a humanized 2A4 antibody or humanized 7D8 antibody include various enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such streptavidinlbiotin and avidin/biotin; fluorescent materials, such as but umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as luminol; bioluminescent materials, such as luciferase, luciferin, and aequorin; radioactive materials, such as but not limited to iodine (.sup.131I, .sup.125I, .sup.123I, .sup.121I,), carbon (.sup.14C), sulfur (.sup.5S), tritium (.sup.3H), indium (.sup.115In, .sup.113In, .sup.112In, .sup.111In,), and technetium (.sup.99Tc), thallium (.sup.201Ti), gallium (.sup.68Ga, .sup.67Ga), palladium (.sup.103Pd), molybdenum (.sup.99Mo), xenon (.sup.133Xe), fluorine (.sup.18F), .sup.153Sm, .sup.177Lu, .sup.159Gd, .sup.149Pm, .sup.140La, .sup.175Y, .sup.166Ho, .sup.90Y, .sup.47Sc, .sup.186Re, .sup.188Re, .sup.142Pr, .sup.105Rh, .sup.97Ru, .sup.68Ge, .sup.57Co, .sup.65Zn, .sup.85Sr, .sup.32P, .sup.153Gd, .sup.169Y, .sup.51Cr, .sup.54Mn, .sup.75Se, .sup.113Sn, and .sup.117Tin; positron emitting metals using various positron emission tomographies, nonradioactive paramagnetic metal ions, and molecules that are radiolabelled or conjugated to specific radioisotopes.

[0049] Therapeutic moieties and/or detectable substances may be coupled or conjugated directly to a murine, chimeric or humanized 2A4 antibody or a murine, chimeric or humanized 7D8 antibody, or indirectly, through an intermediate (e.g., a linker) using techniques known in the art. See e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., Immunol. Rev., 1982, 62:119-58.

[0050] Antibodies used in the disclosed formulations also include modified forms of murine, chimeric or humanized 2A4 antibodies, or murine, chimeric or humanized 7D8 antibodies, which have increased in vivo half-lives relative to the corresponding unmodified antibodies. Such modified forms may be prepared, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. As one example, representative methods for antibody half-life extension are described in PCT International Publication No. WO 02/060919.

[0051] The present invention encompasses antibody formulations having stability at 38.degree. C.-42.degree. C. as assessed by high performance size exclusion chromatography (HPSEC) for at least about 30 days, formulations having stability at 20.degree. C.-24.degree. C. for at least about 1 year, and formulations having stability at 2.degree. C.-4.degree. C. for at least about 3 years. More particularly, the disclosed formulations exhibit low to undetectable levels of antibody aggregation and/or fragmentation, or a low or undetectable increase of antibody fragmentation and/or aggregation above an initial level (e.g., less than about 10% aggregation. A formulation having low to undetectable levels of fragmentation contains at least about 80%, 85%, 90%, 95%, 98%, or 99%, of the total protein, for example, in a single peak as determined by high performance size exclusion chromatography (HPSEC), or in two (2) peaks (one corresponding to each of the antibody heavy chains and antibody light chains) by reduced Capillary Gel Electrophoresis (rCGE), representing the non-degraded antibody, and containing no other single peaks having more than 5%, more than 4%, more than 3%, more than 2%, more than 1%, or more than 0.5% of the total protein each. A formulation having low to undetectable levels of aggregation contains no more than about 15%, no more than about 10%, no more that about 5%, no more than about 4%, no more than about 3%, no more than about 2%, no more than about 1%, or no more than about 0.5% aggregation by weight protein as measured by high performance size exclusion chromatography (HPSEC). For example, in some formulations, less than about 10% of the anti-amyloid antibody is present as an aggregate. Stable formulations of the invention also show little or no loss of biological activity(ies) of a chimeric or humanized 2A4 or chimeric or humanized 7D8, for example binding affinity measurable by ELISAs and/or additional functional assays, such as at least about at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of an initial measurable value of a given activity.

[0052] The histidine buffer may be present in some formulations at a concentration of about 25 mM. In some formulations, the histidine buffer comprises L-histidine and L-histidine HCl monohydrate. For example, in some formulations, L-histidine is present at a concentration within the range from about 16 mM to about 22 mM and L-histidine HCl monohydrate is present at a concentration within the range from about 4 mM to about 8 mM.

[0053] In some formulations, trehalose is present at a concentration from about 210 mM to about 250 mM, for example, about 230 mM. In some formulations, a different non-reducing sugar is used, such as sucrose, mannitol, or sorbitol.

[0054] In some formulations, polysorbate 20 is present at a concentration within the range of about from about 0.005% to about 0.05% by weight, for example, 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, or 0.05%. Alternatively, in some formulations, polysorbate 20 is present at a concentration within the range of about from about 0.05 g/L, 0.1 g/L, 0.15 g/L, 0.2 g/L, 0.25 g/L, 0.3 g/L, 0.35 g/L, 0.4 g/L, 0.45 g/L, or 0.5 g/L. Some formulations include polysorbate 20 at a concentration of 0.2 g/L.

[0055] Some formulations are characterized by a pH within the range of about 6-7, for example, a pH of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. Some formulations have a pH of about 6.5.

[0056] Some formulations are characterized by an osmolality of about 300 mOsm/kg.

[0057] A bulking agent may also be included some formulations.

[0058] Typically, the formulations are sterile, for example, as accomplished by sterile filtration using a 0.2 m or a 0.22 m filter. The formulations of the invention are also generally stable upon freezing and thawing.

[0059] Optionally, formulations of the invention may further comprise other excipients, such as saccharides, polyols, and amino acids (e.g., arginine, lysine, and methionine). In other aspects, the present invention also provides formulations substantially free of surfactant, inorganic salts, additional sugars, and/or other excipients, i.e., less than about less than 0.0005%, less than 0.0003%, or less than 0.0001% of such compounds.

[0060] An exemplary formulation comprises an antibody comprising a light chain comprising an amino acid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising an amino acid sequence set forth as any one of SEQ ID NOs: 14, 15, or 16, which is present at a concentration of about 10 mg/mL, a histidine buffer present at a concentration of about 25 mM, trehalose present at a concentration of about 230 mM; polysorbate 20 present at a concentration of about 0.2 g/L, and a pH of about 6.5.

[0061] I.B. Preparation of Pharmaceutical Formulations

[0062] The present invention also provides methods of preparing pharmaceutical formulations. In one aspect of the invention, such a method comprises (a) culturing mammalian cells having stably incorporated into their genome nucleic acids encoding the light and heavy chains of murine antibody 2A4 (ATCC Accession Number PTA-9662) or of antibody 7D8 (ATCC Accession Number PTA-9468), or of chimeric or humanized versions thereof, so that the cells secrete the antibody into the cell culture media, and purifying the antibody from the cell culture media; (b) and preparing a formulation comprising (i) the purified antibody present at a concentration within the range from about 1 mg/mL to about 100 mg/mL; (ii) histidine buffer present at a concentration within the range from about 20 mM to about 30 mM; (iii) trehalose present at a concentration within the range from about 210 mM to about 250 mM; and (iv) polysorbate 20 present at a concentration within the range from about 0.005% to about 0.05% by weight; wherein the formulation is characterized by a pH within the range from about 6 to about 7.

[0063] In some aspects of the invention, the disclosed methods of preparing a pharmaceutical formulation include the additional step of evaluating at least one property of antibody in the formulation selected from the group consisting of the physical stability, chemical stability and biological activity.

[0064] For example, in one aspect of the invention, mammalian cells are cultured for the production of antibodies, wherein the mammalian cells have stably incorporated into their genomes nucleic acids encoding the light and heavy chains of a humanized 2A4 antibody. Mammalian cells useful for this purpose include (a) host cells having stably incorporated into their genomes a nucleic acid sequence encoding a humanized 2A4 light chain set forth as SEQ ID NO: 13 or 21 and a humanized 2A heavy chain set forth as SEQ ID NO: 15 or 24; (b) host cells having stably incorporated into their genomes a nucleic acid having the nucleotide sequence of SEQ ID NO: 19 and a nucleic acid having the nucleotide sequence of SEQ ID NO: 22; and (c) host cells having stably incorporated into their genomes a nucleic acid having the nucleotide sequence of SEQ ID NO: 20 and a nucleic acid having the nucleotide sequence of SEQ ID NO: 23.

[0065] For the production of antibodies, the disclosed nucleic acids are included in a vector. In some examples, the vector contains the nucleic acid encoding murine 2A4 of 7D8 antibody, or a chimeric or humanized version thereof, operably linked to a suitable control sequence capable of effecting the expression of the DNA in a host cell. Such control sequences include a promoter to effect transcription (e.g., a constitutive promoter or inducible promoter as known in the art), an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, enhancers, polyadenylation signals, and sequences to control the termination of transcription and translation. The vector may be a plasmid, a phage particle (e.g., a viral vector such as adenovirus, adeno-associated-virus, retrovirus, herpes virus, vaccinia virus, lentivirus, poxvirus and cytomegalovirus vectors), or simply a genomic insert. Once transformed into a suitable host, the antibody nucleic acids may integrate into the genome of the host, or the vector may replicate and function independently of the host genome.

[0066] The disclosed nucleic acids are included in a vector either singly or in combination (e.g., a combination of a nucleic acid encoding an antibody light chain and a nucleic acid encoding an antibody heavy chain). For example, a vector can comprise a nucleic acid comprising a nucleotide sequence encoding any one of SEQ ID NOs: 13-16, 21, or 24; a nucleic acid comprising the nucleotide sequence of any one of SEQ ID NOs: 19-20 and 22-23, or combinations thereof. Representative vectors of the invention include (a) a vector comprising a nucleic acid sequence encoding a humanized 2A4 light chain set forth as SEQ ID NO: 13 and a humanized 2A heavy chain set forth as SEQ ID NO: 15; (b) a vector comprising a nucleic acid having the nucleotide sequence of SEQ ID NO: 19 and a nucleic acid having the nucleotide sequence of SEQ ID NO: 22; and (c) a vector comprising a nucleic acid having the nucleotide sequence of SEQ ID NO: 20 and a nucleic acid having the nucleotide sequence of SEQ ID NO: 23.

[0067] Host cells useful for preparing antibody formulations of the invention include mammalian cells, including cells of human origin, such as monkey kidney cells, human embryonic kidney cells, baby hamster kidney (BHK) cells, Chinese hamster ovary cells (CHO) cells, mouse sertoli cells, human cervical carcinoma (HeLa) cells, canine kidney cells, human lung cells, human liver cells, mouse mammary tumor cells, and NS0 cells. For example, a host cell can comprise in its genome a stably integrated nucleic acid comprising a nucleotide sequence encoding any one of SEQ ID NOs: 13-16, 21, and 24; a stably integrated nucleic acid comprising the nucleotide sequence of any one of SEQ ID NOs: 19-20 and 22-23, or combinations thereof. Representative host cells of the invention include (a) host cells comprising a nucleic acid sequence encoding a humanized 2A4 light chain set forth as SEQ ID NO: 13 or 21 and a humanized 2A heavy chain set forth as SEQ ID NO: 15 or 24; (b) host cells comprising a nucleic acid having the nucleotide sequence of SEQ ID NO: 19 and a nucleic acid having the nucleotide sequence of SEQ ID NO: 22; and (c) host cells comprising a nucleic acid having the nucleotide sequence of SEQ ID NO: 20 and a nucleic acid having the nucleotide sequence of SEQ ID NO: 23.

[0068] In another aspect of the invention, a chimeric or humanized 2A4 antibody or a chimeric or humanized 7D8 antibody is prepared by chemical synthesis and then used in the disclosed formulations.

[0069] Antibodies used to prepare the disclosed formulations are typically isolated or purified, i.e., substantially free of cellular material or other contaminating proteins from the cells in which they are produced, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, an antibody that is substantially free of cellular material includes preparations of the antibody having less than about 30%, 25%, 20%, 15%, 10%, 8%, 5%, 2%, 1%, 0.5%, 0.1%, or less (by dry weight) of contaminating protein. When an antibody is recombinantly produced, it is also substantially free of culture medium such that culture medium represents less than about 30%, 25%, 20%, 15%, 10%, 8%, 5%, 2%, 1%, 0.5%, 0.1%, or less, of the volume of the protein preparation. When an antibody is produced by chemical synthesis, it is preferably substantially free of or separated from chemical precursors or other chemicals involved in the synthesis of the protein. Accordingly, such antibody preparations have less than about 30%, 25%, 20%, 15%, 10%, 8%, 5%, 2%, 1%, 0.5%, 0.1%, or less (by dry weight) of chemical precursors or compounds other than the antibody drug substance. Purification of recombinantly expressed antibody can utilize any of a number of methods known in the art, such as, for example, affinity chromatography, acid treatment, depth filtration, anion exchange chromatography, cation exchange chromatography, nanofiltration, ultrafiltration, dialysis and diafiltration.

[0070] The purified antibody drug substance can be adjusted to a solution comprising any of the formulations described herein, diluted to the desired concentration and stored until ready for use. Optionally, the formulation can be stored in concentrated form until ready for use. Liquid formulations can be stored in frozen form, under refrigeration or at room temperature, depending upon their stability profile, which can be determined empirically. In some instances a further filtration step is applied. Some of the formulations described herein may be lyophilized and stored in powder form. Lyophilized formulations can be stored in frozen form, under refrigeration or at room temperature, depending upon their stability profile, which can be determined empirically. For example, the lyophilized formulations can be stored at a temperature of about 2.degree. C. to 8.degree. C. In such cases, the formulation would be reconstituted prior to administration to a patient to yield a liquid formulation having the antibody and excipients present in the concentrations described herein. In some cases, the formulation is reconstituted in sterile water. In some cases, the formulation is reconstituted and added to an infusion bag for administration to the patient. The reconstituted formulation can be stored under refrigeration or at room temperature prior to administration to a patient for a time consistent with the stability profile. Lyophilization and reconstitution techniques are known in the art and described in the Examples.

[0071] Thus, the present invention also encompasses pharmaceutical products comprising lyophilized antibody drug substance and instructions for reconstitution and use. For example, a representative pharmaceutical product can comprise: (a) a vial comprising about 100 mg antibody in powder form; (b) instructions for reconstitution of the antibody; and (c) instructions for preparing the reconstituted antibody for infusion, wherein (i) the antibody comprises a light chain comprising an amino acid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising an amino acid sequence set forth as any one of SEQ ID NOs: 14-16; and (ii) the reconstitution instructions require reconstitution with water for injection to an extractable volume of 10 mL.

II. Methods of Diagnosis and Treatment

[0072] Also provided are methods of therapeutically or prophylactically treating a human patient having or at risk of having amyloidosis characterized by the presence of amyloid protein fibrils, the method comprising administering to the patient an effective dosage of any of the formulations described herein.

[0073] II.A. Subjects Amenable to Diagnosis and Treatment

[0074] Humanized 2A4 drug substance is to be used for the treatment of systemic amyloidosis, such as amyloidoses involving either amyloid light chain AL or amyloid A (AA) proteins. Systemic amyloidoses are a complex group of diseases caused by tissue deposition of misfolded proteins that result in progressive organ damage. The most common type, AL amyloidosis or primary amyloidosis, involves a hematological disorder caused by clonal plasma cells that produce misfolded immunoglobulin light chains. Overproduction of misfolded light chain by plasma cells results in deposits of abnormal AL protein (amyloid), in the tissues and organs of individuals with AL amyloidosis. Clinical features of AL amyloidosis include a constellation of symptoms and organ dysfunction that can include cardiac, renal, and hepatic dysfunction, GI involvement, neuropathy's and macroglossia. A different form of systemic amyloidosis, AA amyloidosis or secondary amyloidosis, occurs "secondarily" as a result of other illness, such as chronic inflammatory diseases (for example, rheumatoid arthritis and ankylosing spondylitis) or chronic infections (for example, tuberculosis or osteomyelitis). In secondary amyloidosis, the depositing amyloid protein is amyloid A protein, derived from an acute-phase protein serum amyloid A.

[0075] Peripheral amyloidosis is be amenable to this type of amyloid-specific immunotherapy through antibody targeting of a neo-epitope that has been identified in AA amyloid, as well as in AL amyloid. Studies in animal models of both AA and AL have demonstrated that significant positive therapeutic effects may be possible at reasonable doses of antibody.

[0076] Subjects or patients amenable to treatment using the disclosed antibody formulations include individuals at risk of disease but not showing symptoms, as well as patients presently showing symptoms of amyloid disease. Therefore, the present methods can be administered prophylactically to the general population without the need for any assessment of the risk of the subject patient. For example, the present methods are especially useful for individuals who do have a known genetic risk autoimmune disorders. Such individuals include those having relatives who have experienced this disease and those whose risk is determined by analysis of genetic or biochemical markers. As another example, patients suffering from AA amyloidosis can be asymptomatic for a prolonged period of time, such that clinical diagnosis of AA amyloidosis is often delayed or missed until the amyloid deposits are extensive. For those patients who are symptomatic, it is estimated that only 53% of the cases are diagnosed. See e.g., L.E.K. Consulting, Independent Market Research (2003). Prophylactic administration disclosed antibody formulations may reduce the incidence of AA amyloidosis.

[0077] The present methods are especially useful for individuals who do have a known risk of, are suspected to have, or have been diagnosed with AA amyloidosis or AL amyloidosis. Such individuals include but are not limited to those having chronic inflammatory diseases, inherited inflammatory diseases, and chronic microbial infections, such as rheumatoid arthritis, juvenile chronic arthritis, ankylosing spondylitis, psoriasis, psoriatic arthropathy, Reiter's syndrome, Adult Still's disease, Behcet's syndrome, Crohn's disease, Familial Mediterranean Fever, leprosy, tuberculosis, bronchiectasis, decubitus ulcers, chronic pyelonephritis, osteomyelitis, Whipple's disease, myeloma, macroglobulinemia, immunocyte dyscrasia, monoclonal gammopathy, occult dyscrasia. Chronic inflammatory and infectious conditions are prerequisite to the development of AA amyloidosis and AL amyloidosis manifested by local nodular amyloidosis can be associated with chronic inflammatory diseases. Individuals who do have known risk of AA amyloidosis also include but are not limited to those having malignant neoplasms as Hodgkin's lymphoma, renal carcinoma, carcinomas of gut, lung and urogenital tract, basal cell carcinoma, and hairy cell leukemia. Additionally, individuals with known risk of AA amyloidosis also include but are not limited to those having lymphoproliferative disorders such as Castleman's Disease. Some of such patients have AA amyloidosis characterized by the presence of amyloid A protein fibrils. Some of such patients have AL amyloidosis characterized by the presence of amyloid light chain-type protein fibrils. Some patients have systemic organ dysfunction attributed to AL amyloidosis, including dysfunction of the heart, kidney, liver, peripheral nervous system, gastrointestinal system, autonomic nervous system, lung, and/or soft tissue or lymphatic system.

[0078] Patients amenable to treatment also include those patients who have received, are currently receiving, or will later receive an alternate therapy, for treatment of amyloid disease or an associated condition, such as, inflammatory diseases, chronic microbial infections, malignant neoplasms, inherited inflammatory diseases, and lymphoproliferative disorders. For example, patients may also receive or have received one or more of the therapeutic agents identified herein with respect to combination therapies. As a particular example, patients suffering from AL may also receive or have received bortezomib, melphalan, lenalidomide and/or carfilzomib. For those patients who have previously received alternate therapies for the treatment of amyloid disease, such therapies may or may not have been successful by the relevant clinical measures.

[0079] II.B. Treatment Regimes

[0080] As used herein, the terms "treat" and "treatment" refer to the alleviation or amelioration of one or more symptoms or effects associated with the disease, prevention, inhibition or delay of the onset of one or more symptoms or effects of the disease, lessening of the severity or frequency of one or more symptoms or effects of the disease, and/or increasing or trending toward desired outcomes as described herein.

[0081] Desired outcomes of the treatments disclosed herein vary according to the amyloid disease and patient profile and are readily determinable to those skilled in the art. Generally, desired outcomes include measurable indices such as reduction or clearance of pathologic amyloid fibrils, decreased or inhibited amyloid aggregation and/or deposition of amyloid fibrils, and increased immune response to pathologic and/or aggregated amyloid fibrils. Desired outcomes also include amelioration of amyloid disease-specific symptoms. For example, desired outcomes for the treatment of AL amyloidosis include a decrease in the incidence or severity of known symptoms, including organ dysfunction, peripheral and autonomic neuropathy, carpal tunnel syndrome, macroglossia, restrictive cardiomyopathy, arthropathy of large joints, immune dyscrasias, myelomas, as well as occult dyscrasias. As another example, desired outcomes for the treatment of AA include a decrease in associated inflammation, arthritis, psoriasis, microbial infection, malignancy, or symptoms of other preexisting or coexisting disease to which the AA amyloidosis is secondary.

[0082] Desired outcomes of the disclosed therapies are generally quantifiable measures as compared to a control or baseline measurement. As used herein, relative terms such as "improve," "increase," or "reduce" indicate values relative to a control, such as a measurement in the same individual prior to initiation of treatment described herein, or a measurement in a control individual or group. A control individual is an individual afflicted with the same amyloid disease as the individual being treated, who is about the same age as the individual being treated (to ensure that the stages of the disease in the treated individual and the control individual are comparable), but who has not received treatment using the disclosed antibody formulations. In this case, efficacy of the disclosed antibody formulations is assessed by a shift or trend away from measurable indices in the untreated control. Alternatively, a control individual is a healthy individual, who is about the same age as the individual being treated. In this case, efficacy of the disclosed antibody formulations is assessed by a shift or trend toward from measurable indices in the healthy control. Changes or improvements in response to therapy are generally statistically significant and described by a p-value less than or equal to 0.1, less than 0.05, less than 0.01, less than 0.005, or less than 0.001 may be regarded as significant.

[0083] In both asymptomatic and symptomatic patients, treatment according to the disclosed methods can begin at any time before or after the diagnosis of the underlying AA or AL amyloid diseases. Treatment typically entails multiple dosages over a period of time. Treatment can be monitored by assaying antibody, or employing radiolabeled SAP Scintigraphy over time. If the response falls, a booster dosage may be indicated. The response of patients with AL amyloidosis to treatment can be monitored by assessing cardiac markers, such as NT-proBNP and/or troponin, serum creatine, and/or alkaline phosphatase; by performing serum free light chain (SFLC) assays, quantitative immunoglobulin assays, biopsies, serum protein electrophoresis (SPEP), urine protein electrophoresis (UPEP), serum, urine immunofixation electrophoresis (IFE), and/or organ imaging techniques. An exemplary complete response (CR) can be determined from response criteria including negative IFE of serum and urine, normal K/k ration and/or <5% plasma cells in bone marrow. An exemplary very good partial response (VGPR) can be determined from a dFLC of <40 mg/L. An exemplary partial response (PR) can be determined from a dFLC decrease of .gtoreq.50%. In the kidney, a response to treatment can be determined, for example, from a .gtoreq.50% reduction (e.g., >0.5 g/24 hours) in 24 hour urine protein excretion in the absence of either a reduction in eGFR of .gtoreq.25% or an increase in serum creatine of .gtoreq.0.5 mg/dL. In the liver, a response to treatment can be determined, for example, from a .gtoreq.50% reduction in initially elevated alkaline phosphatase or a .gtoreq.2 cm reduction in liver size on CT scan or MRI. In the heart, a response to treatment can be determined, for example, from a .gtoreq.30% and 300 ng/L reduction in NT-proBNP in patients with baseline of NT-proBNP of >650 ng/L.

[0084] The antibody formulation can be administered intravenously in dosage ranges from about 10 mg to about 5000 mg for the patient in question, such as, for example, about 10 mg, about 30 mg, about 100 mg, about 300 mg, about 1000 mg, about 2000 mg, or about 2500 mg. The antibody formulation can also be administered intravenously in dosage ranges from about 0.1 mg/kg to about 50 mg/kg, or from about 0.5 mg/kg to about 30 mg/kg, of the host body weight. For example, dosages can be about 0.5 mg/kg body weight, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 4.0 mg/kg, about 5.0 mg/kg, about 8.0 mg/kg, about 10 mg/kg, about 15 mg/kg, about 16 mg/kg, about 20 mg/kg, about 25 mg/kg, or about 30 mg/kg body weight. Dose escalation for an individual patient can occur at the discretion of the prescriber in the absence of any clinically significant occurrence that the prescriber might reasonably believe would present an undue safety risk for the patient, such as, for example, Grade .gtoreq.3 non-hematologic toxicity, Grade .gtoreq.3 nausea, vomiting or diarrhea uncontrolled by maximum antiemetic/anti-diarrhea therapy, Grade 4 neutropenia lasting >7 days in the absence of growth factor support, Grade 3 or 4 neutropenia of any duration accompanied with fever .gtoreq.38.5.degree. C. and/or systemic infection, or other Grade .gtoreq.4 hematologic toxicity.

[0085] Antibody is usually administered on multiple occasions. An exemplary treatment regime entails administration once per every two weeks, once a month, or once every 3 to 6 months. For example, patients can receive the antibody formulation once every four weeks as a cycle, for example every twenty-eight days. The dosing frequency can be adjusted depending on the pharmacokinetic profile of the antibody formulation in the patient. For example, the half-life of the antibody may warrant a two week frequency of dosing. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of antibody to amyloid protein (e.g., AA) in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 .mu.g/ml or about 25-300 .mu.g/ml. Alternatively, antibody can be administered as a sustained release formulation, in which case less frequent administration is required.

[0086] Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, until a partial or complete response is achieved, and/or until the patient shows lessening or amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.

[0087] The formulations disclosed herein may be provided in a dosage form that is suitable for parenteral (e.g., intravenous, intramuscular, subcutaneous) administration. As appropriate for particular applications, the formulation may be alternately provided in a dosage suitable for rectal, transdermal, nasal, vaginal, inhalant, ocular or other administration. The pharmaceutical formulations are typically prepared according to conventional pharmaceutical practice. See e.g., Remington: The Science and Practice of Pharmacy, (19th ed.) ed. A. R. Gennaro, 1995, Mack Publishing Company, Easton, Pa. and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, N.Y.

[0088] In one aspect of the invention, a method of therapeutically or prophylactically treating a human patient having or at risk for having light chain (AL) amyloidosis characterized by the presence of amyloid fibrils, deposits or prefibrillar aggregates, comprises administering to the patient an effective dosage of a pharmaceutical formulation comprising: (a) an antibody comprising a light chain comprising an amino acid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising an amino acid sequence set forth as any one of SEQ ID NOs: 14-16, and which is present at a concentration of about 10 mg/mL; (b) a histidine buffer present at a concentration of about 25 mM; (c) trehalose present at a concentration of about 230 mM; (d) polysorbate 20 present at a concentration of about 0.2 g/L; and (e) a pH of about 6.5. In such a method, the dosage is typically from about 0.5 mg/kg to about 30 mg/kg of the antibody (e.g., about 0.5 mg/kg to about 8 mg/kg, or about 8 mg/kg to about 30 mg/kg) administered intravenously or subcutaneously at a frequency of from about weekly to about quarterly (e.g., once every 28 days).

[0089] II.C. Combinational Drug Therapy Treatment Regimes

[0090] The present invention also encompasses combination therapies for treatment or prophylaxis of amyloid disease, particularly AA amyloidosis and AL amyloidosis. Such combination therapies are performed by administering an antibody formulation of the invention in conjunction with one or more second therapeutic agents, such as another therapy to treat or effect prophylaxis of AA amyloidosis or AL amyloidosis, as the case may be. Combination therapy according to the invention may also be performed in conjunction with a second therapy is used to treat or effect prophylaxis of a disease or condition associated with amyloid disease, such as an inflammatory disease, a chronic microbial infection, a neoplasm (including malignant neoplasms), an inherited inflammatory disease, and/or a lymphoproliferative disorder. Numerous treatments are available in commercial use, in clinical evaluation, and in pre-clinical development, any of which could be selected for use in combination with the disclosed antibody formulations. Such treatments can be one or more compounds or treatments selected from, but not limited to several major categories, namely, (i) non-steroidal anti-inflammatory drugs (NSAIDs; e.g., detoprofen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenameate, mefenamic acid, meloxicam, nabumeone, naproxen sodium, oxaprozin, piroxicam, sulindac, tolmetin, celecoxib, rofecoxib, aspirin, choline salicylate, salsalte, and sodium and magnesium salicylate); (ii) steroids (e.g., cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone); (iii) DMARDs, i.e., disease modifying antirheumatic drugs (e.g., cyclosporine, azathioprine, methotrexate, leflunomide, cyclophosphamide, hydroxychloroquine, sulfasalazine, D-penicillamine, minocycline, and gold); (iv) recombinant proteins (e.g., ENBREL.RTM. (etanercept, a soluble TNF receptor) and REMICADE.RTM. (infliximab) a chimeric monoclonal anti-TNF antibody); (v) stem cell transplantation; and/or (vi) chemotherapy. Patients with AL amyloidosis may also receive treatment regimes that include drugs or combinations of drugs often used to treat hematological malignancies, such as melphalan, prednisone, dexamethasone, lenalidomide (REVLIMID) and proteosome inhibitors such as bortezomib (VELCADE.RTM.), and carfilzomib (KYPROLIS.TM.), at dosages in the range of the standard of care.

[0091] The duration of the combination therapy depends on the type of amyloid disease being treated, any underlying disease associated with the amyloid disease, the age and condition of the patient, the stage and type of the patient's disease, how the patient responds to the treatment, etc. A medical doctor can observe the therapy's effects closely and make any adjustments as needed. Additionally, a person having a greater risk of developing AA amyloidosis (e.g., a person who is genetically predisposed or previously had an inflammatory disorder or other underlying diseases) or AL amyloidosis may receive prophylactic combination treatments to inhibit or delay the development of AA AL aggregates such as fibrils, or as maintenance therapy post-treatment.

[0092] When performing a combination therapy, the two or more drug substances are administered simultaneously or sequentially in any order, i.e., a formulation of the invention is administered prior to administering a second drug substance, concurrently with a second drug substance, or subsequent to administration of a second drug substance. For example, a combination therapy may be performed by administering a first therapy prior to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administering a second agent/therapy.

[0093] The dosage, frequency and mode of administration of each component of the combination can be controlled independently. For example, one therapeutic agent/therapy may be administered orally three times per day, while the second therapeutic agent/therapy may be administered intramuscularly once per day. Combination therapy may be given in on-and-off cycles that include rest periods. The compounds may also be admixed or otherwise formulated together such that one administration delivers both compounds. In this case, each therapeutic agent is generally present in an amount of 1-95% by weight of the total weight of the composition. Alternatively, an antibody formulation of the invention and a second therapeutic agent can be formulated separately and in individual dosage amounts. Drug combinations for treatment can be provided as components of a pharmaceutical pack.

[0094] Preferably, the disclosed combination therapies elicit a synergistic therapeutic effect, i.e., an effect greater than the sum of their individual effects or therapeutic outcomes. Measurable therapeutic outcomes are described herein. For example, a synergistic therapeutic effect may be an effect of at least about two-fold greater than sum of the therapeutic effects elicited by the single agents of a given combination, or at least about five-fold greater, or at least about ten-fold greater, or at least about twenty-fold greater, or at least about fifty-fold greater, or at least about one hundred-fold greater. A synergistic therapeutic effect may also be observed as an increase in therapeutic effect of at least 10% compared to the sum of the therapeutic effects elicited by the single agents of a given combination, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or more. A synergistic effect is also an effect that permits reduced dosing of therapeutic agents when they are used in combination.

EXAMPLES

[0095] The following examples have been included to illustrate modes of the invention. Certain aspects of the following examples are described in terms of techniques and procedures found or contemplated by the present co-inventors to work well in the practice of the invention. In light of the present disclosure and the general level of skill in the art, those of skill appreciate that the following examples are intended to be exemplary only and that numerous changes, modifications, and alterations may be employed without departing from the scope of the invention.

Example 1. Selection of Humanized 2A4 for the Treatment of AL Amyloidosis

[0096] An IgG1, kappa isotype antibody was prepared, which is a humanized version of murine antibody 2A4. The light chain and heavy chain sequences of representative humanized 2A4 antibodies are set forth in FIGS. 1A-1B and 3. Nucleic acids encoding the particular humanized 2A4 antibody version 3, which amino acid sequences are shown in FIG. 3, are depicted in FIGS. 4A-4B.

[0097] The parent monoclonal 2A4 antibody is directed against a neo-carboxy terminal epitope of human serum Amyloid A (sAA), resulting from cleavage of the native sAA molecule at amino acid residue 76. The murine antibody does not cross-react with IgGs or free light chain (LC) and it has shown broad isotype recognition of patient derived AL amyloid samples examined to date. 2A4 recognizes multiple forms of AL light chain amyloid including soluble multimer and insoluble deposits. In addition, the antibody has been shown to promote regression of amyloidoma in a mouse xenograft model. The light chain and heavy chain sequences of murine 2A4 antibody are set forth in FIG. 2.

Example 2. Dose Determination for Humanized 2A4 Antibody

[0098] Nonclinical studies in the TRIAD mouse model and the cynomolgus monkey have utilized doses of 4 and 40 mg/kg in the mouse and 10, 50, and 100 mg/kg in the monkey. Conversion to the Human Equivalent Dose (HED) on a mg/kg basis (most appropriate conversion for monoclonal antibodies due to their restriction to the vascular space) gives HEDs of 0.32 and 3.2 for the mouse and 3.2, 16, and 32 for the monkey. Based on currently available data, the NOAEL in both species is expected to be the highest dose administered. Using a mouse HED (most sensitive species due to dosing limitations) of 3.2 and a 10.times. safety factor, the MRSD for first in man dosing would be approximately 0.32 mg/kg. Based upon animal studies, administration to humans is begun with a dose of 0.5 mg/kg.

Example 3. Preparation of the Expression Vector

[0099] For generation of the final h2A4 IgG1 HC vector the variable region of the heavy chain was isolated by PCR using the plasmid CET1019AS-hygro-h2A4VH3-Sce 4.23.07 as template. Primers used for the amplification introduced at the 5' end of the fragments an MfeI restriction site and at the 3' end a BlpI restriction site for subcloning. The variable region was cloned into the MfeI and BamHI digested eukaryotic expression vector pBI-61, which contains the genomic constant regions of human IgG1 of Glm(3) allotype. The resulting recombinant expression vector pBI-61/2A4 IgG1-REM is 9,015 base pairs in size and carries the selectable marker dihydrofolate reductase (DHFR) from hamster under the control of the DHFR promoter and polyadenylation signal. This vector also contains the beta-lactamase gene for selection in E. coli as well as origins of replication for E. coli (ColE1 ori), SV40 (SV40 ori) and filamentous phage f1 (f1 ori). Expression of the HC is driven by the immediate early promoter/enhancer region from human cytomegalovirus (CMV) combined with a transcription enhancing element (TE) derived from the hamster genome. For transcript termination and stabilization the polyadenylation signal from hamster growth hormone is used and for enhancement of transcription a non-coding sequence derived from the hamster genome (TE).

[0100] Using the plasmid CET1019AS-hu2A4VL3-hck-puro-Sce 4.19.07 as template the variable region of the h2A4 LC was isolated by PCR introducing at the 5' end of the fragments an SgrAI restriction site and at the 3' end a KpnI restriction site for subcloning into the final eukaryotic expression vector pBI-60 digested with the same restriction enzymes. This vector contains the genomic constant region of a human kappa chain. The resulting recombinant expression vector pBI-60/2A4 LC is 7,144 base pairs in size and contains the selectable marker neomycin phosphotransferase mutant, which confers resistance to geneticin, under the control of the SV40 promoter. For transcript termination the polyadenylation signal from Herpes simplex thymidine kinase is used. This vector also contains the beta-lactamase gene for selection in E. coli as well as origins of replication for E. coli (ColE1 ori) and filamentous phage f1 (f1 ori). Expression of the LC is driven by the immediate early promoter/enhancer region from human cytomegalovirus (CMV) combined with a transcription enhancing element (TE) derived from the hamster genome. For transcript termination and stabilization the polyadenylation signal from hamster growth hormone is used and for enhancement of transcription a non-coding sequence derived from the hamster genome (TE).

Example 4. Production of Humanized 2A4 Antibody (Pool-Derived Material)

[0101] Humanized 2A4 was produced in Chinese Hamster Ovary (CHO) cells, grown in chemically defined media without any bovine-derived components. Antibody was pooled from stable transfected cells from which the production cell line was ultimately derived. The pool-derived material was purified by protein A-affinity chromatography. This material was used for human tissue cross-reactivity studies and for a single dose pharmacokinetic (PK) study in cynomolgus monkeys. The formulation of the humanized 2A4 antibody is 10 mg/mL antibody, 25 mM L-Histidine/L-Histidine HCl monohydrate, 230 mM Trehalose dehydrate, 0.02% (w/v) Polysorbate (TWEEN.RTM.) 20, pH=6.5.

Example 5. Production of Humanized 2A4 Antibody (Clone-Derived Material)

[0102] A single CHO cell clone was isolated from cell pools as described in Example 3, and was used to establish the Master Cell Bank (MCB) without any bovine materials. Humanized 2A4 for nonclinical studies was manufactured at 80 L scale using the same cell cultivation and purification processes (except scale-up modifications) as the GMP clinical version of humanized 2A4 (2,000 L scale). Material from the 2,000 L scale production may also be used in nonclinical studies.

Example 6. Process of Manufacturing Humanized 2A4 Antibody

[0103] Vial Thaw & Inoculum Expansion. Cells from the MCB are thawed and transferred into an appropriate cell culture flask. The cells are incubated at approximately 37.degree. C. The thawed culture is propagated for one to four days (first passage after cell thaw). For sub-cultivation, an aliquot of a grown cell culture (and a defined volume of pre-warmed, 0.22 .mu.m or less filtered inoculum medium) is used to reach a seed density of approximately 0.1-0.5.times.10.sup.6 cells/mL in standard cell culture vessels of approximately 0.02 L to 1 L working volume. As an example, the first passages can be done in 0.125 L or 0.25 L or 0.5 L vessels, followed by passages in 1 L vessels. A stock culture can be initiated at this cultivation stage. For preparation of inoculum cultures for individual production fermenters, aliquots of the stock cultures are expanded to generate cultures with up to 25 L volume. Typically, the cell culture is scaled up from 1 L cultures to 2 or more 1 L or 2 L cultures, then to 2 or more 2 L or 3 L cultures and finally to 2 or more cultures with up to 25 L culture volume per vessel. Grown cell suspensions from several vessels can be pooled and used to inoculate the 80 L bioreactor. Shake flasks, T-flasks, spinner flasks and bags can be used as standard cell culture vessels for the above cultivation steps.

[0104] Seed Cultures in Bioreactors. Before inoculation with cells, 0.22 .mu.m or less filtered growth medium is added to the bioreactors. The content of the filled bioreactors is warmed to approximately 37.degree. C. and maintained at this temperature throughout incubation of the cells. Cells from the inoculum cultures are transferred into the pre-warmed medium. The initial cell density is targeted within the range of 0.1-0.5.times.10.sup.6 cells/mL. The cells are grown in an 80 L bioreactor and subsequently in a 400 L bioreactor. Cells are subcultivated approximately every two to four days. At this stage, cells may be transferred to another vessel of the same or larger volume. Typically, the cell culture is scaled up from 1.times.80 L bioreactor culture to 1.times.400 L culture. To initiate the production phase, the cells are transferred from the grown 400 L cell suspension to the production bioreactor of approximately 2,000 L working volume.

[0105] Production culture in 2,000 L Bioreactor. Before inoculation with cells, 0.22 .mu.m or less filtered production medium is added to the production bioreactor. The content of the filled production bioreactor is warmed to approximately 37.degree. C. and maintained at this temperature throughout incubation of the cells. The initial cell density in the production phase is targeted within the range of 0.1-0.5.times.10.sup.6 cells/mL. The production bioreactor is run in a fed batch mode. To support the production of antibody and to prolong culture duration, a nutrient feed medium is added during the production stage. The point at which to start feeding is determined either by culture time or by cell density. As needed, a glucose solution and/or glutamine solution can be added during the production stage to avoid depletion of these substances during the production period. The run time of the 2,000 L production bioreactor is typically 8 to 14 days. Pre-harvest samples are tested for sterility, mycoplasma, and adventitious virus in vitro.

[0106] Harvest and Clarification. After 8 to 14 days of cultivation in the production phase, the cell culture fluid is separated from the cells. After pre-harvest sampling and prior to harvest, the pH and the temperature of the culture can be adjusted to facilitate removal of cells, debris and particles during harvest. To remove the cells, the culture is passed through a centrifugation plus dead-end filtration unit. The cells are centrifuged and/or retained by the membranes. The harvested culture fluid is passed through filters of 0.22 .mu.m pore size or less and collected in an appropriate container. Residual culture fluid can be removed from the harvest system by flushing with Phosphate Buffered Saline (PBS) to recover residual product from the harvest system. The resulting recovered product amount is collected together with the harvested culture fluid to form the harvest pool, also called harvested cell-free culture fluid (HCCF). The pH and temperature of the HCCF can be adjusted to facilitate the subsequent downstream processing steps.

[0107] Purification. The antibody is purified from the HCCF by a series of steps involving affinity chromatography, acid treatment, depth filtration, anion exchange chromatography, cation exchange chromatography, nanofiltration and ultra-/diafiltration, several of which may be performed in several cycles. To remove contaminants the affinity chromatography process step specifically binds the antibody product. The HCCF is applied to the chromatography column packed with the MabSelect matrix. The matrix binds antibody at neutral pH, while contaminants appear in the flow through and are removed. The column is eluted in a step elution with a 100 mM acetic acid/sodium acetate solution at pH 3.5. To inactivate potential viral contaminants, the antibody solution is incubated at room temperature for a minimum of 60 minutes at pH 3.5.+-.0.1. After incubation the acid treated pool is adjusted to pH 7.2 using a 2 M Trometamol solution and subjected to depth filtration for clarification. For anion exchange chromatography, the depth filtered product pool is adjusted to a conductivity .ltoreq.7 mS/cm with Water for Injection (WFI). The adjusted pool is applied to a chromatography column packed with Q Sepharose FF resin. The antibody passes through the anion exchange matrix unbound. The flow through is monitored and the antibody containing fraction is collected based on absorbance measurement. For cation exchange chromatography, the product pool is adjusted to a pH of 5.5.+-.0.1 by addition of acetic acid up to a conductivity of .ltoreq.7.5 mS/cm with WFI. The adjusted product pool is applied onto a chromatography column packed with SP Sepharose FF cation exchange resin. This chromatography step is performed in a bind-elute mode. The antibody binds to the cation exchange matrix. The column is eluted in a step elution with a 100 mM acetic acid/sodium acetate and 138.5 mM sodium chloride solution at pH 5.5. Potential viral contaminants are removed by passing the antibody solution through a 0.1 .mu.m prefilter and a Planova 20N nanofilter at a maximum pressure of 1 bar differential pressure of the Planova 20N nanofilter. During ultrafiltration/diafiltration (UF/DF), the product is concentrated to the target concentration, and the buffer is exchanged with the formulation buffer. Concentration and diafiltration is performed using ultrafiltration membranes having a cut-off of approximately 30 kD. The material is processed by concentrating the product to 30-100 mg/mL. The 30 kD pool is then diafiltered with a solution of 25 mM L-Histidine, pH 6.5 and is flushed to a concentration of about 60-70 mg/mL. The 30 kD pool intermediate may be stored at -40.degree. C. until formulation is performed. For formulation, the 30 kD product pool is adjusted to a solution containing 17.5 mM L-Histidine/7.5 mM L-Histidine Hydrochloride, 230 mM Trehalose, and 0.02% (w/v) Polysorbate20, pH=6.5. The antibody is finally diluted with formulation buffer to the desired target concentration of 10 mg/mL. The resulting drug substance is filtered through a 0.22 .mu.m filter to remove any potential adventitious microbial contaminants and particulate material. The drug substance can be stored frozen at -40.degree. C. until filling.

Example 7. Characterization of Drug Substance Containing Humanized 2A4 Antibody

[0108] Humanized 2A4 used for formulation is composed of two heterodimers. Each of the heterodimers is composed of a heavy polypeptide chain of 50 kDa (449 amino acids) and a kappa light polypeptide chain of 24 kDa (219 amino acids). The antibody protein has a humanized amino acid sequence with a total molecular mass of approximately 147 kDa. The four polypeptide chains of the antibody molecule are linked together by disulfide bonds. Each heavy polypeptide chain contains one consensus sequence for N-linked glycosylation, which is occupied (positions 299 to 301, highlighted in bold and underlining in FIG. 1A). There are two binding sites for the serum amyloid A epitope per antibody molecule.

[0109] A competitive binding ELISA has been established to measure binding of humanized 2A4 to its antigen (CGGHEDT (SEQ ID NO: 17) when conjugated to Ovalbumin) compared to the reference standard.

Example 8. Humanized 2A4 Drug Substance Components and Composition

[0110] The humanized 2A4 drug substance (100 mg/vial) for clinical use is a sterile liquid dosage form consisting of a 10 mL fill in a 25 mL vial (20R). The nonclinical humanized 2A4 drug substance (200 mg/vial) is 20 mL fill in a 25 mL vial (20R). The nonclinical and clinical formulations of humanized 2A4 are provided in Table 1. The final formulation of the humanized 2A4 drug substance has a density of 1.034 g/mL at 20.degree. C. and a pH of 6.5.

TABLE-US-00001 TABLE 1 Composition of Nonclinical and Clinical Humanized 2A4 Drug Substance Nominal amount (mg/vial) Nonclinical Vial Clinical Vial Concentration Size = 25 mL Size = 25 mL Component Function (g/L) (20 R) (20 R) Humanized 2A4 Active 10 200 100 drug substance Substance L-Histidine Buffer 2.72 54.4 27.2 component L-Histidine HCl Buffer 1.57 31.4 15.7 monohydrate component Trehalose Tonicity 87.02 1,740.4 870.2 dihydrate agent Polysorbate Surfactant 0.20 4.0 2.0 (TWEEN .RTM.) 20 Water for Solvent -- Add WFI to a total Add WFI to a total Injection (WFI) volume of 20 mL volume of 10 mL

Example 9. Batch Formula for Drug Product (100 mg/ml Vial)

[0111] A formula was designed for a 2,600 vial batch of drug product as provided in Table 2.

TABLE-US-00002 TABLE 2 Batch Formula for 2,600 Vials Quantity Ingredient Grade per Batch Humanized 2A4 antibody -- 260.0 g L-Histidine USP, Ph. Eur. 70.72 g L-Histidine HCl monohydrate Ph. Eur. 40.82 g Trehalose dehydrate USP/NF, Ph. Eur. 2,262.52 g Polysorbate 20 USP/NF, Ph. Eur. 5.20 g

Example 10. Lyophilization

[0112] A Hof Com 26041 freeze dryer was used to lyophilize the formulated humanized 2A4 drug substance over a period of approximately 86 hours with the pressure regulated by an MKS control system (MKS Instruments) with N.sub.2 injection according to the program set forth in Table 3. The endpoint was detected by Pirani signal. During the drying mode, the vials stand directly on the shelves without lyo plates. The nitrogen backfill is at approximately 600 mbar with pharma grade, sterile N.sub.2. The vials were then closed and sorted at 5.degree. C. within the freeze dryer. The final drug product is stored at 2-8.degree. C., protected from light. The process should yield a white to yellowish lyo cake.

[0113] Table 3 summarizes the program for the lyophilization of humanized 2A4 drug substance.

TABLE-US-00003 TABLE 3 Lyophilization Steps Shelf Vacuum Step Time temperature MKS Step No. [hh:mm] [.degree. C.] [mbar] Loading 01 00:01 5 off Freezing 02 00:15 5 off 03 00:05 2 off 04 02:00 2 off 05 01:05 -50 off 06 02:30 -50 off Primary 07 00:05 -50 0.10 Drying 08 00:40 -10 0.10 09 55:00 -10 0.10 Secondary 10 04:30 30 0.10 Drying 11 20:00 30 0.10 Total Time 86:11

Example 11. Reconstitution of Lyophilized Drug Product

[0114] Prior to application, the lyophilisate has to be reconstituted with sterilized water for injection. The reconstitution of h2A4 vials has been performed according to the following procedure under laminar air-flow. The complete flip-off-cap of the respective product vial was removed. The rubber-stopper was also removed. The solvent was added by pipetting the necessary volume (2.times.5 mL WFI using a piston pipette). When performing this action, it was ensured that the solvent was added slowly to the lyophilized product. The vials were carefully swirled (not shaken), until the lyophilized product was completely dissolved. The solution was made homogenous by carefully rotating the vial end-over-end. The dissolved material was aliquoted according to table 1 and stored at -70.degree. C. until analysis

Example 12. Clinical Assessment of Humanized 2A4 Drug Substance

[0115] A clinical trial is designed to determine a maximum tolerated dose (MTD) and/or the Phase 2 recommended dose (P2RD) of humanized 2A4 drug substance in subjects with AL amyloidosis. Dosing will begin at 0.5 mg/kg and escalate to a high of 30 mg/kg or 2500 mg total (whichever is lower). Initially, humanized 2A4 drug substance will be given intravenously as a single agent every 28 days until progression of organ function or unacceptable treatment related toxicity or withdraw of consent. If the half-life (ti/2) of humanized 2A4 drug substance from the initial doses suggests that a different dosing schedule would be more appropriate (e.g., every two weeks or an alternate, less frequent schedule than once every 28 days), dosing in subsequent cohorts may be modified using an alternative dosing schedule.

Sequence CWU 1

1

241131PRTMus musculusMISC_FEATURE(1)..(19)LEADER SEQUENCEmat_peptide(20)..(131)MISC_FEATURE(43)..(58)CDR 1MISC_FEATURE(74)..(80)CDR 2MISC_FEATURE(113)..(121)CDR 3 1Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala -15 -10 -5Ser Ser Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val -1 1 5 10Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu 15 20 25Val His Ser Thr Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro30 35 40 45Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser 50 55 60Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Tyr Phe Thr 65 70 75Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys 80 85 90Ser Gln Ser Thr His Val Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu 95 100 105Glu Ile Lys1102131PRTMus musculusMISC_FEATURE(1)..(19)LEADER SEQUENCEmat_peptide(20)..(131)MISC_FEATURE(43)..(58)CDR1MISC_FEATURE(74).- .(80)CDR2MISC_FEATURE(113)..(121)CDR3 2Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala -15 -10 -5Ser Ser Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val -1 1 5 10Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Leu Ser Leu 15 20 25Val His Ser Thr Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro30 35 40 45Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser 50 55 60Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Tyr Phe Thr 65 70 75Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys 80 85 90Ser Gln Ser Thr His Val Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu 95 100 105Glu Ile Lys1103138PRTMus musculusMISC_FEATURE(1)..(19)LEADER SEQUENCEmat_peptide(20)..(138)MISC_FEATURE(45)..(54)CDR 1MISC_FEATURE(69)..(87)CDR 2MISC_FEATURE(120)..(127)CDR 3 3Met Val Leu Gly Leu Lys Trp Val Phe Phe Val Val Phe Tyr Gln Gly -15 -10 -5Val His Cys Glu Val Gln Leu Val Glu Ser Gly Gly Arg Leu Val Gln -1 1 5 10Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 15 20 25Asn Thr Tyr Ala Met Tyr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu30 35 40 45Glu Trp Val Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Ile Tyr 50 55 60Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Phe Arg Asp Asp Ser 65 70 75Gln Ser Met Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr 80 85 90Ala Met Tyr Tyr Cys Val Arg Pro Tyr Ser Asp Ser Phe Ala Tyr Trp 95 100 105Gly Gln Gly Thr Leu Val Thr Val Ser Ala110 1154112PRTArtificial SequenceHumanized antibody sequence containing murine and human residues (humanized 2A4 light chain variable region version 3) 4Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Thr Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95Thr His Val Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 1105119PRTArtificial SequenceHumanized antibody sequence containing murine and human residues (humanized 2A4 heavy chain variable region version 3) 5Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Tyr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Ile Tyr Tyr Ala Asp 50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Arg Pro Tyr Ser Asp Ser Phe Ala Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 115616PRTMus musculusMISC_FEATURE(1)..(16)2A4 VL CDR1 6Arg Ser Ser Gln Ser Leu Val His Ser Thr Gly Asn Thr Tyr Leu His1 5 10 1577PRTMus musculusMISC_FEATURE(1)..(7)2A4 VL CDR2 7Lys Val Ser Asn Arg Phe Ser1 589PRTMus musculusMISC_FEATURE(1)..(9)2A4 VL CDR3 8Ser Gln Ser Thr His Val Pro Phe Thr1 5910PRTMus musculusMISC_FEATURE(1)..(10)2A4 VH CDR1 9Gly Phe Thr Phe Asn Thr Tyr Ala Met Tyr1 5 101019PRTMus musculusMISC_FEATURE(1)..(19)2A4 VH CDR2 10Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Ile Tyr Tyr Ala Asp Ser1 5 10 15Val Lys Asp118PRTMus musculusMISC_FEATURE(1)..(8)2A4 VH CDR3 11Pro Tyr Ser Asp Ser Phe Ala Tyr1 51216PRTMus musculusMISC_FEATURE(1)..(16)7D8 VL CDR1 12Arg Ser Ser Leu Ser Leu Val His Ser Thr Gly Asn Thr Tyr Leu His1 5 10 1513219PRTArtificial SequenceHumanized antibody sequence containing murine and human residues (humanized 2A4 kappa light chain) 13Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Thr Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95Thr His Val Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 21514449PRTArtificial SequenceHumanized antibody sequence containing murine and human residues (humanized 2A4 IgG1 heavy chain variant 1 (G1m1 allotype)) 14Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Tyr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Ile Tyr Tyr Ala Asp 50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Arg Pro Tyr Ser Asp Ser Phe Ala Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445Lys15449PRTArtificial SequenceHumanized antibody sequence containing murine and human residues (humanized 2A4 IgG1 heavy chain variant 2 (G1m3 allotype))MISC_FEATURE(299)..(301)glycosylation site 15Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Tyr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Ile Tyr Tyr Ala Asp 50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Arg Pro Tyr Ser Asp Ser Phe Ala Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445Lys16445PRTArtificial SequenceHumanized antibody sequence containing murine and human residues (humanized 2A4 IgG2 heavy chain) 16Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Tyr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Ile Tyr Tyr Ala Asp 50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Arg Pro Tyr Ser Asp Ser Phe Ala Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu 210 215 220Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly385 390

395 400Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445177PRTHomo sapiens 17Cys Gly Gly His Glu Asp Thr1 5184PRTHomo sapiens 18Ala Glu Asp Ser119660DNAArtificial SequenceHumanized antibody sequence containing murine and human residues (Hu2A4 VH3VL3 hcg1,k cDNA sequence - light chain without signal sequence) 19gacgtggtga tgacccagtc ccctctgtcc ctgcctgtga cccctggcga gcctgcctcc 60atctcctgcc ggtcctccca gtccctggtg cactccaccg gcaacaccta tctgcactgg 120tatctgcaga agcctggcca gtctcctcag ctgctgatct acaaggtgtc caaccggttc 180tccggcgtgc ctgaccggtt ctctggctcc ggctccggca ccgacttcac cctgaagatc 240tcccgggtgg aggccgagga cgtgggcgtg tactactgct cccagtccac ccacgtgcct 300ttcaccttcg gcggaggcac caaggtggag atcaagcgaa ctgtggctgc accatctgtc 360ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 420ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 480tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc 540agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 600gtcacccatc agggcctgag ctcgcccgtc acaaagagct tcaacagggg agagtgttag 66020726DNAArtificial SequenceHumanized antibody sequence containing murine and human residues (Hu2A4 VH3VL3 hcg1,k cDNA sequence - light chain)sig_peptide(1)..(66)CDS(1)..(726)V_region(67)..(402)C_region(403)..- (726) 20atg gac atg cgg gtg ccc gca cag ctg ctg ggc ctg ctg atg ctg tgg 48Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp1 5 10 15gtg tcc ggc tcc tcc ggc gac gtg gtg atg acc cag tcc cct ctg tcc 96Val Ser Gly Ser Ser Gly Asp Val Val Met Thr Gln Ser Pro Leu Ser 20 25 30ctg cct gtg acc cct ggc gag cct gcc tcc atc tcc tgc cgg tcc tcc 144Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser 35 40 45cag tcc ctg gtg cac tcc acc ggc aac acc tat ctg cac tgg tat ctg 192Gln Ser Leu Val His Ser Thr Gly Asn Thr Tyr Leu His Trp Tyr Leu 50 55 60cag aag cct ggc cag tct cct cag ctg ctg atc tac aag gtg tcc aac 240Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn65 70 75 80cgg ttc tcc ggc gtg cct gac cgg ttc tct ggc tcc ggc tcc ggc acc 288Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 85 90 95gac ttc acc ctg aag atc tcc cgg gtg gag gcc gag gac gtg ggc gtg 336Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val 100 105 110tac tac tgc tcc cag tcc acc cac gtg cct ttc acc ttc ggc gga ggc 384Tyr Tyr Cys Ser Gln Ser Thr His Val Pro Phe Thr Phe Gly Gly Gly 115 120 125acc aag gtg gag atc aag cga act gtg gct gca cca tct gtc ttc atc 432Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile 130 135 140ttc ccg cca tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg 480Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val145 150 155 160tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag 528Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys 165 170 175gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag 576Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 180 185 190cag gac agc aag gac agc acc tac agc ctc agc agc acc ctg acg ctg 624Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 195 200 205agc aaa gca gac tac gag aaa cac aaa gtc tac gcc tgc gaa gtc acc 672Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 210 215 220cat cag ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg gga gag 720His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu225 230 235 240tgt tag 726Cys21241PRTArtificial SequenceSynthetic Construct 21Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp1 5 10 15Val Ser Gly Ser Ser Gly Asp Val Val Met Thr Gln Ser Pro Leu Ser 20 25 30Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser 35 40 45Gln Ser Leu Val His Ser Thr Gly Asn Thr Tyr Leu His Trp Tyr Leu 50 55 60Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn65 70 75 80Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 85 90 95Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val 100 105 110Tyr Tyr Cys Ser Gln Ser Thr His Val Pro Phe Thr Phe Gly Gly Gly 115 120 125Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile 130 135 140Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val145 150 155 160Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys 165 170 175Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 180 185 190Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 195 200 205Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 210 215 220His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu225 230 235 240Cys221350DNAArtificial SequenceHumanized antibody sequence containing murine and human residues (Hu2A4 VH3VL3 hcg1,k cDNA sequence - heavy chain without signal sequence) 22gaggtgcagc tggtcgagtc cggcggaggc ctggtgcagc ctggcggctc cctgagactg 60tcctgcgccg cctccggctt caccttcaac acctacgcca tgtactggat caggcaggct 120cctggcaagg gactggagtg ggtggcccgg atcaggtcca agtccaacaa ctacgctatc 180tactacgccg actccgtgaa ggaccggttc accatctccc gggacgactc caagaactcc 240ctgtatctgc agatgaactc cctgaaaacc gaggacaccg ccgtgtacta ctgcgctcgg 300ccttactccg actccttcgc ctactggggc cagggcaccc tggtgaccgt gtccagcgcc 360tccaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 600atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagagagt tgagcccaaa 660tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 720tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 840gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1020gccaaagggc agccccgaga accacaggtg tacacgctgc ccccatcccg ggaggagatg 1080accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1140gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag caggtggcag 1260caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1320aagagcctct ccctgtcccc gggtaaatga 1350231407DNAArtificial SequenceHumanized antibody sequence containing murine and human residues (Hu2A4 VH3VL3 hcg1,k cDNA sequence - heavy chain)sig_peptide(1)..(57)CDS(1)..(1407)V_region(58)..(414)C_region(415).- .(1407) 23atg gag ttc ggc ctg tcc tgg ctg ttc ctg gtg gcc atc ctg aag ggc 48Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly1 5 10 15gtg cag tgc gag gtg cag ctg gtc gag tcc ggc gga ggc ctg gtg cag 96Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30cct ggc ggc tcc ctg aga ctg tcc tgc gcc gcc tcc ggc ttc acc ttc 144Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45aac acc tac gcc atg tac tgg atc agg cag gct cct ggc aag gga ctg 192Asn Thr Tyr Ala Met Tyr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60gag tgg gtg gcc cgg atc agg tcc aag tcc aac aac tac gct atc tac 240Glu Trp Val Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Ile Tyr65 70 75 80tac gcc gac tcc gtg aag gac cgg ttc acc atc tcc cgg gac gac tcc 288Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser 85 90 95aag aac tcc ctg tat ctg cag atg aac tcc ctg aaa acc gag gac acc 336Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr 100 105 110gcc gtg tac tac tgc gct cgg cct tac tcc gac tcc ttc gcc tac tgg 384Ala Val Tyr Tyr Cys Ala Arg Pro Tyr Ser Asp Ser Phe Ala Tyr Trp 115 120 125ggc cag ggc acc ctg gtg acc gtg tcc agc gcc tcc acc aag ggc cca 432Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca 480Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr145 150 155 160gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg acg 528Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg 576Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc 624Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat 672Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc aaa tct 720His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser225 230 235 240tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg 768Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc 816Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc 864Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag 912His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg 960Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr305 310 315 320tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat 1008Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc 1056Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag 1104Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365gtg tac acg ctg ccc cca tcc cgg gag gag atg acc aag aac cag gtc 1152Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg 1200Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val385 390 395 400gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct 1248Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tat agc aag ctc acc 1296Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg 1344Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg 1392Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460tcc ccg ggt aaa tga 1407Ser Pro Gly Lys46524468PRTArtificial SequenceSynthetic Construct 24Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45Asn Thr Tyr Ala Met Tyr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Ile Tyr65 70 75 80Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser 85 90 95Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr 100 105 110Ala Val Tyr Tyr Cys Ala Arg Pro Tyr Ser Asp Ser Phe Ala Tyr Trp 115 120 125Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr145 150 155 160Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser225 230 235 240Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr305 310 315 320Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val385 390 395 400Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460Ser Pro Gly Lys465

Claims

1. A pharmaceutical formulation comprising: (a) a chimeric or humanized version of antibody 2A4 (ATCC Accession Number PTA-9662) or of antibody 7D8 (ATCC Accession Number PTA-9468), or fragment thereof, that specifically competes for binding to antigen with 2A4 or 7D8, wherein the antibody is present at a concentration within the range from about 1 mg/mL to about 100 mg/mL; (b) histidine buffer present at a concentration within the range from about 20 mM to about 30 mM; (c) trehalose present at a concentration within the range from about 210 mM to about 250 mM; and (d) polysorbate 20 present at a concentration within the range from about 0.005% to about 0.05% by weight; wherein the formulation is characterized by a pH within the range from about 6 to about 7.

2.-5. (canceled)

6. The formulation of claim 1, wherein the antibody comprises a light chain comprising an amino acid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising an amino acid sequence set forth as any one of SEQ ID NO: 14-16.

7. The formulation of claim 6, wherein the antibody comprises a light chain comprising an amino acid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising an amino acid sequence set forth as SEQ ID NO: 15.

8. The formulation of claim 1, wherein the antibody comprises a light chain variable region comprising three complementarity determining regions set forth as SEQ ID NOs: 6, 7, and 8, and a heavy chain variable region comprising three complementarity regions set forth as SEQ ID NOs: 9, 10, and 11.

9. The formulation of claim 1, wherein the antibody is a humanized or chimeric version of antibody 7D8 produced by ATCC Accession Number PTA-9468.

10. The formulation of claim 1, wherein the antibody comprises a light chain variable region comprising three complementarity determining regions set forth as SEQ ID NOs: 12, 7, and 8, and a heavy chain variable region comprising three complementarity regions set forth as SEQ ID NOs: 9, 10, and 11.

11.-12. (canceled)

13. The formulation of claim 1, wherein the antibody is present at a concentration within the range from about 25-75 mg/mL.

14. The formulation of claim 13, wherein the antibody is present at a concentration of about 50 mg/mL.

15.-26. (canceled)

27. A lyophilized formulation of an antibody, comprising (a) a humanized version of antibody 2A4 (ATCC Accession Number PTA-9662) or antibody 7D8 (ATCC Accession Number PTA-9468) or antigen binding fragment thereof, (b) histidine; (c) trehalose; and (d) polysorbate 20.

28. (canceled)

29. The lyophilized formulation of claim 27, wherein the formulation has a pH of about 6.5 when reconstituted.

30. The lyophilized formulation of claim 27, comprising about 100 mg to about 1000 mg of the antibody.

31. The lyophilized formulation of claim 27, wherein the polysorbate 20 is present at a concentration within the range from about 0.005% to about 0.05% by weight.

32. The lyophilized formulation of claim 27, that enables reconstitution to yield an aqueous solution comprising: (a) an antibody comprising a light chain comprising an amino acid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising an amino acid sequence set forth as any one of SEQ ID NOs: 14-16, and which is present at a concentration of about 10 mg/mL; (b) a histidine buffer present at a concentration of about 25 mM; (c) trehalose present at a concentration of about 230 mM; (d) polysorbate 20 present at a concentration of about 0.2 g/L; and (e) a pH of about 6.5.

33.-80. (canceled)

81. A method of therapeutically or prophylactically treating a human patient having or at risk for having light chain (AL) amyloidosis characterized by the presence of amyloid fibrils, deposits or prefibrillar aggregates, comprising administering to the patient an effective dosage of a pharmaceutical formulation comprising: (a) an antibody comprising a light chain comprising an amino acid sequence set forth as SEQ ID NO: 13 and a heavy chain comprising an amino acid sequence set forth as any one of SEQ ID NOs: 14-16, and which is present at a concentration of about 10 mg/mL; (b) a histidine buffer present at a concentration of about 25 mM; (c) trehalose present at a concentration of about 230 mM; (d) polysorbate 20 present at a concentration of about 0.2 g/L; and (e) a pH of about 6.5.

82. The method of claim 81, wherein the dosage is from about 0.5 mg/kg to about 30 mg/kg of the antibody administered intravenously or subcutaneously at a frequency of from about weekly to about quarterly.

83. The method of claim 82, wherein the frequency of administration is once every 28 days.

84. The method of claim 82, wherein the dosage is about 0.5 mg/kg to about 8 mg/kg.

85. The method of claim 82, wherein the dosage is about 8 mg/kg to about 30 mg/kg.

86. (canceled)

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