WO2009155962A1 - Use - Google Patents
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- WO2009155962A1 WO2009155962A1 PCT/EP2008/011135 EP2008011135W WO2009155962A1 WO 2009155962 A1 WO2009155962 A1 WO 2009155962A1 EP 2008011135 W EP2008011135 W EP 2008011135W WO 2009155962 A1 WO2009155962 A1 WO 2009155962A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/401—Proline; Derivatives thereof, e.g. captopril
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/72—Increased effector function due to an Fc-modification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- the present invention relates in general to the treatment and/or prevention of diseases which involve amyloid deposition.
- the invention relates to the treatment of amyloidosis.
- Amyloidosis is a serious and usually fatal disease caused by accumulation in the i tissues of abnormal insoluble protein fibres known as amyloid fibrils . These are derived from different proteins in different forms of the disease but all amyloid fibrils share a common cross- ⁇ core structure and all are derived by misfolding of normally 1 soluble precursor proteins . In addition to the amyloid fibrils themselves, amyloid deposits are always rich in proteoglycans, some of which are tightly bound to the
- SAP serum amyloid P component
- Human SAP is a constitutive protein in the plasma , at a concentration of around 20-40 mg/1 and with a total of about 50-100 mg of SAP in the combined plasma and extravascular compartments both of normal individuals and patients with diseases
- SAP is also specifically concentrated in the amyloid deposits and in an individual with extensive
- Amyloid deposits are extracellular and they cause disease by progressive accumulation until they damage the structure and thus the function of whatever tissue they occupy . There is very rarely any inflammatory or 'foreign body' response to amyloid deposition, either seen locally in the tissues or suggested by systemic markers of inflammation. In so called systemic amyloidosis the deposits can be present in any tissue or organ in the body but deposits are never seen within the brain substance in these forms of the disease. Systemic amyloidosis is the cause of about 1 per 1000 of all deaths in developed countries, and is always fatal unless the abundance of the protein which is the precursor of the amyloid fibrils can be sufficiently and persistently reduced.
- tissue or organ system also occur and may cause serious disease .
- amyloidosis the damage to the structure and function of tissues and organs which leads to clinical disease is unequivocally caused by the progressive accumulation of the amyloid deposits themselves.
- amyloid deposits are always present, most importantly Alzheimer's disease and type 2 diabetes mellitus, in which the contribution of the amyloid deposition to the pathogenesis of disease, specifically loss of cognitive and pancreatic islet function respectively, is not i known .
- amyloid deposits anywhere else in the body are demonstrably pathogenic and it is likely that the cerebral amyloid deposits of Alzheimer's disease and the islet amyloid deposits of type 2 diabetes are also harmful. Since treatment which clears amyloid deposits in systemic and local amyloidosis will certainly be i therapeutic , removal of the amyloid deposits in Alzheimer's disease and type 2 diabetes should also be clinically beneficial.
- AA amyloid A protein (AA) amyloidosis is readily induced in mice by chronic inflammation following intravenous injection of an extract of amyloidotic tissue
- amyloid enhancing factor 9 containing amyloid fibrils, and known as amyloid enhancing factor . This model very closely resembles human AA amyloidosis with major amyloid deposition in the spleen
- the AA protein which forms the amyloid fibrils is derived from its circulating precursor, serum amyloid A protein (SAA), which is an acute phase protein.
- SAA serum amyloid A protein
- the plasma concentration of SAA rises sharply from its normal trace value of less than 5 mg/1 in response to almost any form of inflammation and tissue damage and can persist at values up to 1000 mg/1, or even more, in the face of persistent stimulation. This increased production of SAA is a necessary precondition for development of AA amyloidosis, and both in humans and in mice, when the SAA concentration falls to
- European patent application EP 0 915 088 discloses compounds which are competitive inhibitors of binding of SAP to amyloid fibrils, as well as methods for their manufacture.
- a preferred compound disclosed in EP 0 915 088 is (R)- 1 -[6-[(R)-2-Carboxy-pyrrolidin- 1 -yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), however, any of the compounds described therein, or any other compound which depletes circulating SAP can be used in the practice of the present invention.
- International Patent Application WO 2004/099173 incorporated herein by reference, also describes palindromic compounds that could also be used in the practice of the present invention.
- (R)-I -[6-[(R)-2-Carboxy-pyrrolidin-l-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC) is specifically bound by human SAP in a complex composed of two 16 native pentameric SAP molecules and 5 CPHPC molecules . This complex is recognised as abnormal by the liver and is very rapidly taken up by the hepatocytes and
- the present invention is based, at least in part, on the surprising finding that this can now be achieved by treatment with a compound which effectively depletes human SAP from the circulation, and additionally treatment with an antibody specific for SAP.
- a pharmaceutical composition comprising a compound which depletes serum amyloid P component (SAP) from the circulation, in combination with an antibody specific for SAP.
- SAP serum amyloid P component
- the compound which depletes SAP from the circulation is an SAP cross linking agent. It has been found that compounds capable of cross linking a plurality of SAP molecules in the circulation cause the SAP to be rapidly eliminated from the circulation; see WO03/013508. Examples of such compounds include multivalent ligands specific for SAP, for example multivalent competitive inhibitors of SAP binding. Competitive inhibitors of binding of SAP to amyloid are set forth, for example in EP 0 915 088, the disclosure of which is incorporated herein by reference; the use of these, and other molecules to deplete SAP from the circulation is described,
- the compound which depletes SAP from the circulation is a
- D-proline preferred are D-prolines of the Formula:
- X-Y-X' is a linker having at least 4, advantageously at least 5, advantageously at least 6, up to 20 linear or straight-chain carbon atoms, wherein
- X is -(CH ) -; -CH(R )(CH ) -; CH 0(CH ) -; CH NH-; benzyl,
- -C(R ) CH-; -CH 2 CH(OH)-; or thiazol-2,5-diyl;
- X' is -(CH ) -; -(CH ) CH(R )-; -(CH ) OCH -; -NHCH -; benzyl,
- -CH C(R )-; CH(OH)CH 2 ; or thiazol-2,5-diyl;
- R is lower alkyl, lower alkoxy or benzyl and n is 0-3, or a pharmaceutically acceptable salt or mono- or diester thereof.
- SAP-depleting compound The compound which depletes SAP from the circulation, including the preferred embodiments mentioned above, is referred to hereinafter as an SAP-depleting compound.
- D-proline of formula I-A above can be written as
- Linker (X-Y-X') can be from 4 to 20 linear carbon atoms in length, including from 4-15 linear carbon atoms, 5-10 linear carbon atoms, and 6-8 linear carbon atoms in length.
- the linker can be a straight or branched chain, or can optionally form one or more ring structures, with the proviso that at least 4 linear or straight-chain carbon atoms are present in the linker.
- at least one of the linear or straight-chain C atoms can be optionally substituted by at least one hetero atom selected from N, O, or S, advantageously O or S, preferably O.
- the composition does not comprise ethanolamines and/or phosphoethanolamines and/or 4,6-pyruvate acetyl of ⁇ -D galactopyranose and/or calcium and/or IL-4 and/or IL-3.
- the composition is not indicated for enhancing f ⁇ brocyte formation.
- the composition does not enhance fibrocyte formation.
- said antibody specific for SAP does not target a portion of SAP asserted to be functional in inhibiting fibrocyte formation from monocytes.
- composition is indicated for use in the treatment of amyloid disease, and there is accordingly provided a pharmaceutical composition comprising an SAP-depleting compound and an antibody specific for SAP for use in the treatment of amyloid disease.
- Amyloid disease can be any disease which is associated with the extracellular deposition of amyloid fibrils in the tissues.
- amyloid disease is a disease selected from the group consisting of any form of systemic (visceral) or local amyloidosis, type 2 diabetes and Alzheimer's disease.
- SAP is human SAP
- references to anti-SAP antibodies and SAP-depleting compounds are preferably references to compounds which target and/or deplete human SAP.
- an SAP-depleting compound in combination with an antibody specific for SAP in the manufacture of a composition for the treatment or prophylaxis of amyloid disease.
- SAP is present in all amyloid deposits of all types in human diseases associated with amyloid deposition, including amyloidosis, Alzheimer's disease and type 2 diabetes, this approach to treatment is applicable in all such conditions.
- the invention is for the treatment of amyloidosis.
- a method for treating a subject suffering from or at risk from amyloid disease comprising administering to a subject in need thereof a composition comprising an SAP-depleting compound and an antibody specific for SAP.
- the SAP-depleting compound and the antibody may be administered simultaneously, for instance separately or in admixture, or sequentially.
- the treatment regime comprises administration of the SAP-depleting compound alone, followed by administration of the antibody.
- the SAP-depleting compound administration may be continued during administration of the antibody.
- kits for use in the treatment of amyloidosis comprising an SAP-depleting compound and an antibody specific for SAP.
- the kit components can be provided for simultaneous, simultaneous separate or sequential administration, or a combination thereof.
- the SAP-depleting compound and the anti-SAP antibody are administered sequentially, such that the SAP-depleting compound is administered before the antibody.
- Administration may be conducted over an extended period of time, by infusion, repeated bolus doses or in any other way; or single dose administration may be employed, in which the SAP-depleting compound and/or the antibody are administered once only.
- the SAP-depleting compound is administered over a prolonged period, but the antibody is administered in a single dose.
- a method for identifying an agent that can be used in combination with the SAP-depleting compound for the treatment of amyloidosis comprising the steps of: (a) contacting a non-human animal with transgenic expression of human SAP in which systemic AA amyloidosis has been induced, with the SAP-depleting compound thereby depleting the circulating SAP; (b) contacting said transgenic non-human animal with one or more agents; and (c) determining if said agent(s) promote substantial or complete regression of the amyloid deposits in the non-human animal, wherein an agent that causes substantial regression of the amyloid deposits in the non-human animal is indicative of an agent that can be used for the treatment of amyloidosis.
- the transgenic non-human animal is a mouse, suitably a C57BL/6 mouse with the mouse SAP gene deleted and which is transgenic for human SAP.
- a method for identifying an agent that can be used in combination with an SAP-depleting compound for the treatment of amyloidosis comprising the steps of: (a) administering to a non-human animal in which systemic
- AA amyloidosis has been induced, a dose of isolated pure human SAP, thereby loading the amyloid deposits with human SAP; (b) contacting said amyloidotic SAP laden non-human animal with one or more agents; and (c) determining if said agent(s) promote substantial or complete regression of the amyloid deposits in the non-human animal, wherein an agent that causes substantial regression of the amyloid deposits in the non-human animal is indicative of an agent that can be used for the treatment of amyloidosis.
- Amyloid is identified by its pathognomonic green birefringence which must be distinguished from the white or other bright birefringence of collagen in the tissues and artefacts of foreign bodies, dust, etc. Spleen amyloid scores: 7722, zero (no amyloid
- Liver amyloid scores 6980, zero (no green birefringent amyloid, only white-yellow collagen birefringence); 7482, 10 ; 8028, 10 ; 8156, 10 ; 8272, 10 .
- Figure 4 Time course of cellular infiltration and amyloid destruction after administration of anti-SAP antibody. Spleen sections stained with Congo red and viewed in polarised light (left) and stained with haematoxylin and eosin (right) from animals killed at the times shown after antibody treatment. On day 1 ( Figure 4A) there is abundant green birefringent amyloid in the typical perifollicular marginal zone but in contrast to its usual acellular appearance it is densely infiltrated with predominantly mononuclear inflammatory cells. On day 2 ( Figure 4B) macrophages surrounding the amyloid are already fusing to form multinucleate giant cells.
- Figure 5 Electron micrographs of spleen taken 1 day after anti-SAP antibody treatment.
- Figure 5A macrophages (top right) surrounding typical fibrillar amyloid deposit (centre and left); magnification x4,500.
- Figure 5B granulocytes (upper half of the image) and amyloid deposit (lower half of the image); neutrophils and macrophages have darker cytoplasm; one eosinophil is seen in the centre of the image; x3,000.
- Figure 5C magnified fragment of an amyloid deposit, granulocytes and macrophages (also seen in 5B); x 7,000.
- FIG. 6 Immunochemical identification of cells and proteins in anti-SAP antibody mediated clearance of AA amyloid deposits.
- Top two panels show the intense macrophage infiltration, identified by strong staining with anti-F4/80, in all congophilic amyloid deposits in spleen and liver. Such staining is completely absent in amyloid deposits of mice not treated with anti-SAP antibody (not shown).
- Third panel shows the co-localisation of AA amyloid, CD68 (a marker of phagocytic endocytotic activity), and mouse C3.
- Bottom panel shows phagocytically active macrophages surrounding and engulfing a fragment of mouse AA amyloid.
- Figure 6A is day 1 spleen with Congo red, left; anti-F4/80, right;
- Figure 6B is day 1 liver with Congo red, left; anti-F4/80, right;
- Figure 6C is day 4 spleen with anti-AA protein, left; anti-CD68, centre; anti-mouse C3, right;
- Figure 6D is day 4 confocal image with anti-CD68, red; anti-AA protein, green; nuclear conterstain, blue.
- Figure 7. Immunohistochemical staining with anti-human SAP antibody in spleen of an amyloidotic wild type mouse after injection of isolated pure human SAP. There is strong positive staining of all the amyloid deposits in their typical marginal zone distribution. This bound human SAP is the target of the therapeutic anti-SAP antibody according to the present invention.
- FIG 8. Three dimensional structure of the SAP molecule.
- the SAP molecule is composed of five identical non-covalently associated subunits arranged with cyclic pentameric symmetry in a disc-like configuration.
- On one planar face of the disc each protomer has a short ⁇ -helix shown in red and this face is designated the A face.
- On the opposite face, each protomer has the calcium dependent ligand binding pocket through which SAP recognizes and binds to its ligands, including amyloid fibrils, and is designated as the B face.
- the two calcium atoms in each binding pocket are shown as yellow spheres.
- Figure 9 Epitope mapping for monoclonal anti-human SAP antibody SAP-5.
- A complete amino acid sequence of human SAP showing the points at which it is cleaved by CNBr in 70%TFA (residue 159M, in blue) and by chymotrypsin, without reduction/carbamidomethylation, in ammonium bicarbonate in the absence of calcium, (residues 140Y and 144F, in red).
- B SDS-PAGE analysis of SAP cleaved with CNBr.
- D Sequence comparison between human SAP (h) and mouse SAP (m) for residues 136-147. Top panel, differences indicated above by residues shown in red in the murine sequence. Bottom panel, position of this extended loop with 140Y at its apex shown in red in the 3D structure of human SAP. The different residues in the murine sequence are shown in blue. The yellow spheres represent the calcium atoms bound in the ligand binding pocket of each protomer.
- FIG 10. The complex of SAP with CPHPC.
- the complex of SAP with CPHPC comprises two pentameric SAP molecules cross linked, B face to B face by 5 CPHPC molecules, the D-proline head groups at either end of which are bound in the calcium dependent ligand binding pockets of opposite pairs of SAP protomers.
- Figure 11. The complex of SAP and CPHPC cross linked with BS3.
- the complex of SAP and CPHPC was covalently cross linked using the bifunctional cross linker, BS3, to produce a stable complex in which most of the B face of SAP is occluded exactly as it must be when SAP is bound to amyloid fibrils in vivo.
- Anti-human SAP antibodies according to the present invention must be able to recognise and bind to SAP when it is in this ligand-bound form.
- FIG. 13 Immunoblot analysis of native human SAP and C-reactive protein (CRP). Isolated pure human SAP and CRP were electrophoresed in agarose gel, shown by Coomassie blue staining on the left of each panel, and replicate gels were blotted onto a membrane by standard techniques and then detected using either SAP-5 at 3 ⁇ g/ml or Abpl at 10 ⁇ g/ml. Bound mouse IgG was revealed using peroxidise labelled polyclonal rabbit anti-mouse IgG followed by peroxidise substrate and DAB, on the right in each panel. Both SAP-5 and Abpl bind to human SAP but neither binds to human CRP, the protein most closely related to human SAP with which it shares 55% strict residue-for-residue identity. DETAILED DESCRIPTION OF THE INVENTION
- aspects of the present invention relate to the treatment and/or prevention of disease caused by deposition of amyloid in the tissues, such disease being known as amyloidosis.
- prophylaxis refers to a course of action (such as administering one or more compounds or pharmaceutical compositions) initiated (e.g., prior to the onset of a clinical symptom of amyloidosis) so as to prevent, suppress or reduce, either temporarily or permanently, the onset of a clinical manifestation of amyloidosis.
- treatment refers to a course of action (such as administering one or more compounds or pharmaceutical compositions) initiated after the onset of clinical manifestations of amyloidosis so as to eliminate or reduce, either temporarily or permanently, a clinical manifestation or progression of amyloidosis.
- Amyloidosis is any disease characterized by the extracellular accumulation of amyloid in various organs and tissues of the body.
- amyloid refers to extracellular deposits in the tissues of insoluble protein fibres composed of fibrils with characteristic ultrastructural morphology, a cross- ⁇ sheet core structure and the pathognomonic histochemical tinctorial property of binding Congo red dye from alkaline alcoholic solution and then giving red-green dichroism when viewed microscopically in strong cross polarised light. About 25 different unrelated proteins are known to form amyloid fibrils which deposit in human tissues and share all these typical properties. Amyloid deposits in the brain substance, cerebral amyloid, differ somewhat from amyloid deposits elsewhere in the body in that they are always focal and microscopic in size, and are commonly referred to as amyloid plaques.
- Amyloidosis that is disease directly caused by deposition of amyloid in the tissues, comprises both local amyloidosis, in which the deposits are confined to one anatomical region and/or one tissue or organ system, and systemic amyloidosis in which the deposits can occur in any organ or tissue in the body, including blood vessels and connective tissues.
- the cause of amyloidosis can be either acquired or hereditary.
- amyloid A protein (AA) type is a complication of chronic active inflammatory diseases such as rheumatoid arthritis, juvenile rheumatoid arthritis,
- Dialysis related amyloidosis is caused by accumulation of ⁇ -microglobulin as a result of end stage renal failure.
- Monoclonal immunoglobulin light chain (AL) amyloidosis is a complication of multiple myeloma or otherwise benign monoclonal gammopathy (monoclonal gammopathy of uncertain significance,
- amyloidosis of transthyretin type can occur without any preceding illness and is merely a complication of old age.
- Hereditary amyloidosis is caused by mutations in the genes for various proteins which encode expression of variant proteins having an increased propensity to form amyloid fibrils, and includes disease caused by transthyretin, apolipoprotein AI, gelsolin, lysozyme, cystatin C and amyloid ⁇ -protein.
- Alzheimer's disease and type 2 diabetes in which amyloid deposits are always present but in which the precise mechanisms causing these respective diseases are not yet known. Nevertheless the local deposition of amyloid in the brain and cerebral blood vessels in Alzheimer's disease, and in the pancreatic islets in diabetes is very likely to exacerbate pathology and disease. Accordingly, in one embodiment, the invention relates to treatment of both Alzheimer's disease and type 2 diabetes, indeed to any condition associated with the presence of amyloid deposits in the tissues.
- transmissible spongiform encephalopathy are associated with amyloid deposits in the brain, and the present invention therefore relates to all these conditions, including variant Creutzfeldt- Jakob disease in humans, Creutzfeldt- Jakob disease itself, kuru and the various other forms of human prion disease, and also bovine spongiform encephalopathy, chronic wasting disease of mule-deer and elk, and transmissible encephalopathy of mink.
- animals including poultry such as chickens, ducks, turkeys and geese, and, preferably, mammals, including humans, as well as dogs, cats, horses, cows, sheep, pigs, guinea pigs, mice and rats is contemplated.
- mammals including humans, as well as dogs, cats, horses, cows, sheep, pigs, guinea pigs, mice and rats is contemplated.
- the treatment of humans is preferred.
- SAP-depleting compound in combination with an antibody specific for SAP for use in the treatment of amyloidosis.
- references herein to anti-SAP antibodies, SAP-binding antibodies and antibodies specific for SAP are coterminous and refer to antibodies, or binding fragments derived from antibodies, which bind to SAP in a specific manner and substantially do not cross-react with other molecules present in the circulation or the tissues.
- the antibodies according to the invention target SAP which is bound to amyloid fibrils in the tissue amyloid deposits.
- the SAP molecule is a pentamer composed of five identical non-covalently associated protomers arranged with cyclic pentameric symmetry in a disc-like configuration.
- each protomer On one face of the molecule each protomer has a short ⁇ -helix, designated herein as the A face.
- each protomer On the opposite face each protomer has the calcium dependent ligand binding pocket through which SAP recognizes and binds to its ligands, including amyloid fibrils, and designated herein as the B face.
- human SAP forms stable decarneric dimers, probably via A-face to A-face interactions.
- an “antibody” as used herein includes but is not limited to, polyclonal, monoclonal, recombinant, chimeric, complementarity determining region (CDR)-grafted, single chain, bi-specific, Fab fragments and fragments produced by a Fab expression library.
- Such fragments include fragments of whole anti-SAP antibodies which retain their binding activity for SAP, Fv, F(ab'), F(ab')2 fragments, and F(v) antibody fragments as well as fusion proteins and other synthetic proteins which comprise the antigen-binding site of the anti-SAP antibody.
- the antibodies and fragments thereof may be humanized antibodies, as described in further detail below.
- Variable regions and CDRs in an antibody sequence may be identified by aligning the sequences against a database of known variable regions. Methods for identifying these regions are described in, for example, Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001. Databases of antibody sequences are described in, for example, VBASE2 at www.vbase2.org, as described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005).
- Monoclonal anti-human SAP antibody produced in mouse is commercially available from various sources - such as Sigma-Aldrich, Gillingham, Dorset, UK (Catalogue # A9191); US Biological (catalogue # S 1003-3, 1003-4); Acris Antibodies (catalogue #
- Examples of monoclonal anti-human SAP antibodies include eight distinct monoclonal anti-human SAP IgG antibodies (SAP-I - SAP-7 and NH) generated using standard techniques and tested by immunoradiometric assays (IRMA) for their binding to human SAP (see Example 9 below). Two of these antibodies, SAP-5 and SAP-2, are of the IgG2a isotype while the others are of the IgGl isotype.
- murine IgG2a is an isotype known to activate mouse complement whereas murine IgGl antibodies do not and the amyloid clearance mechanism according to the present invention is complement dependent (see Examples 6 and 7, below).
- the hybridoma that secretes SAP-5 will be deposited.
- Examples of monoclonal anti-human SAP antibodies also include a set of six distinct monoclonal anti-SAP IgG2a antibodies generated by standard techniques (3H8.H3, 3H8.H6, 3H8.H8, 6Hl . G2, 6Hl . G3, 6Hl . Hl, one of which, 6Hl .
- G2 is designated here as Abpl and has been deposited with American Type Culture Collection (ATCC) and assigned accession number (To Be Assigned; US attorney docket number 674599-2007; PCT attorney docket number 305407-20073, both referenced in the deposit papers).
- ATCC American Type Culture Collection
- the epitope on human SAP that is recognized and bound by SAP-5 is part of the linear amino acid sequence of human SAP, as shown by the fact that this antibody binds equally well to completely denatured and to native human SAP (Example 9).
- the present application also shows that SAP-5 binds to a linear amino acid sequence in the large amino terminal fragment of human SAP, residues 1-159, after cleavage of SAP by cyanogen bromide at residue 159M ( Figure 9).
- Dig estion of human SAP with chymotrypsin cleaves SAP immediately after residues 140Y and 144F to give fragments 1-140 and 145-204 with the intermediate small fragment 140-144 being lost.
- Monoclonal antibody SAP-5 reacts very weakly with the 1-140 fragment but very strongly with the small amount of residual undigested intact SAP protomer, suggesting that the sequence recognized by SAP-5 may include residues 140-159 ( Figure 9). Sequence comparison between human and mouse SAP reveals 3 amino acid differences at residues 139, 143 and 145, consistent with potential immunogenicity of this part of the human SAP sequence in mice in which the monoclonal antibody was produced ( Figure 9). In contrast, antibody Abpl only binds to native and not to denatured or cleaved human SAP and thus recognises a discontinuous or conformational epitope (Example 9).
- Native human SAP a protein composed of 5 identical 204 residue glycosylated protomers each of mass 25,462 Da 7 , contains discrete and different epitopes. Two of these epitopes are: (1) a linear (contiguous) amino acid sequence found in the amino terminal 159 residues of the protein, and (2) a conformational epitope present only in the native molecule. Nevertheless, both antibodies described above bind optimally to SAP in its ligand-bound conformation (Example 9), which is essential for an effective anti-human SAP antibody according to the present invention in which the antibody must bind to, and activate complement on, human SAP bound to amyloid deposits in vivo.
- the present invention also relates to antibodies that bind to an epitope of human SAP that is recognized by either SAP-5 or Abpl, including antibodies which bind to the linear amino acid sequence in the amino terminal fragment of human SAP after cleavage of the protein by chymotrypsin and the conformational epitope described above.
- the term "monoclonal antibody” refers to an antibody obtained from a single clone of B lymphocyte derived plasma cells producing a homogeneous antibody of a single heavy and light chain class and epitope specificity.
- Monoclonal antibodies are typically highly specific, and are directed against a single antigenic site (epitope), in contrast to conventional antibodies within an antiserum induced in a whole animal by immunisation with a particular antigen.
- Such conventional antibodies are derived from many different clones of B lymphocytes which recognise either the same or different epitopes on the immunising antigen, and are known as polyclonal antibodies.
- monoclonal antibodies are readily produced in pure form uncontaminated by other immunoglobulins, whereas isolation of specific antibodies from a polyclonal antiserum requires demanding immunopurification procedures.
- Monoclonal antibodies may be prepared by the hybridoma method (see Kohler et al., Nature, 256:495-7, 1975), or by recombinant DNA methods. The monoclonal antibodies may even be isolated from phage antibody libraries using well known techniques.
- the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA,
- a host animal typically a mouse
- Lymphocytes harvested from the immunised animal are then fused in vitro with a continuous line of myeloma cells grown in vitro to form so-called hybridoma cells.
- myeloma cells include, but are not limited to, human myeloma and mouse-human heteromyeloma cell lines which have been described for the production of human monoclonal antibodies.
- the culture medium from the growing hybridoma cells may be assayed for monoclonal antibodies directed against the antigen.
- the binding specificity of the antibodies produced by the cells may be determined by various methods - such as immunoprecipitation or an in vitro binding assay - such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) or immunoradiometric assay (IRMA).
- RIA radioimmunoassay
- ELISA enzyme-linked immunosorbent assay
- IRMA immunoradiometric assay
- the clones may be subcloned by limiting dilution procedures and grown by standard methods.
- the monoclonal antibodies secreted by the subclones are separated from the culture medium or serum by well-known immunoglobulin purification procedures - such as protein A-Sepharose, gel electrophoresis, dialysis, hydroxylapatite chromatography or affinity chromatography.
- Polyclonal antibodies may be raised in animals by multiple subcutaneous, intramuscular or intraperitoneal injections of the relevant antigen and an adjuvant.
- An improved antibody response may be obtained by conjugating the relevant antigen to a protein that is immunogenic in the species to be immunized.
- Animals may be immunized against the antigen, immunogenic conjugates, or derivatives by combining, eg., 100 ⁇ g or 5 ⁇ g of the protein or conjugate (for rabbits or mice, respectively) with suitable adjuvants, including, but not limited to, Freund's complete adjuvant, aluminium hydroxide, Freund's incomplete adjuvant, L-tyrosine, nitrocellulose-absorbed protein, immune-stimulating complexes (ISCOMS, antigen-modified saponin/cholesterol micelles) or MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate), for instance, and injecting the solution intradermally at multiple sites.
- the animals may then be later boosted with 1/5 of the original amount of peptide or conjugate in adjuvant by subcutaneous injection at multiple sites. At 7-14 days post-booster injection, the animals can be bled and the serum is assayed for antibody titre.
- anti-SAP antibodies and fragments also encompass variants of the anti-SAP antibodies and fragments thereof.
- variantants include peptides and polypeptides comprising one or more amino acid sequence substitutions, deletions, and/or additions that have the same or substantially the same affinity and specificity of epitope binding as the anti-SAP antibody or fragments thereof.
- deletions, insertions or substitutions of amino acid residues may produce a silent change and result in a functionally equivalent substance.
- Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues.
- negatively charged amino acids include aspartic acid and glutamic acid
- positively charged amino acids include lysine and arginine
- amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
- Homologous substitution substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue
- substitution and replacement may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc.
- Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids - such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.
- Z ornithine
- B diaminobutyric acid ornithine
- O norleucine ornithine
- pyriylalanine pyriylalanine
- Replacements may also be made by unnatural amino acids include; alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, ⁇ -alanine*, L- ⁇ -amino butyric acid*, L- ⁇ -amino butyric acid*, L- ⁇ -amino isobutyric acid*, L- ⁇ -amino caproic
- natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, ⁇ -alanine*, L- ⁇ -amino butyric
- Phe phenylalanine
- Phe such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino) ,
- variants may include peptides and polypeptides comprising one or more amino acid sequence substitutions, deletions, and/or additions to the anti-SAP antibodies and fragments thereof wherein such substitutions, deletions and/or additions do not cause substantial changes in affinity and specificity of epitope binding.
- a variant of an anti-SAP antibody or fragment thereof may result from one or more changes to an anti-SAP antibody or fragment thereof, where the changed anti-SAP antibody or fragment thereof has the same or substantially the same affinity and specificity of epitope binding as the starting sequence.
- Variants may be naturally occurring, such as allelic or splice variants, or may be artificially constructed. Variants may be prepared from the corresponding nucleic acid molecules encoding said variants.
- Variants of the anti-SAP antibodies or fragments thereof may have changes in light and/or heavy chain amino acid sequences that are naturally occurring or are introduced by in vitro engineering of native sequences using recombinant DNA techniques.
- Naturally occurring variants include "somatic" variants which are generated in vivo in the corresponding germ line nucleotide sequences during the generation of an antibody response to a foreign antigen.
- Variants of SAP binding antibodies and binding fragments may also be prepared by mutagenesis techniques. For example, amino acid changes may be introduced at random throughout an antibody coding region and the resulting variants may be screened for binding affinity for SAP or for another property. Alternatively, amino acid changes may be introduced into selected regions of the anti-SAP antibody, such as in the light and/or heavy chain CDRs, and/or in the framework regions, and the resulting antibodies may be screened for binding to SAP or some other activity. Amino acid changes encompass one or more amino acid substitutions in a CDR, ranging from a single amino acid difference to the introduction of multiple permutations of amino acids within a given CDR. Also encompassed are variants generated by insertion of amino acids to increase the size of a CDR.
- Anti-SAP antibodies or fragments thereof may be provided with a modified Fc region where a naturally-occurring Fc region is modified to increase the half-life of the antibody or fragment in a biological environment, for example, the serum half-life or a half-life measured by an in vitro assay.
- Variants also include anti-SAP antibodies or fragments thereof comprising a modified Fc region, wherein the modified Fc region comprises at least one amino acid modification relative to a wild-type Fc region.
- the variant Fc region may be designed, relative to a comparable molecule comprising the wild-type Fc region, so as to bind Fc receptors with a greater or lesser affinity.
- the SAP binding antibodies and fragments thereof may comprise a modified Fc region.
- Fc region refers to naturally-occurring or synthetic polypeptides homologous to the IgG C-terminal domain that is produced upon papain digestion of IgG.
- IgG Fc has a molecular weight of approximately 50 kD.
- an entire Fc region can be used, or only a half-life enhancing portion.
- many modifications in amino acid sequence are acceptable, as native activity is not in all cases necessary or desired.
- SAP binding antibodies and fragments thereof also encompass derivatives of the antibodies, fragments and sequences disclosed herein.
- Derivatives include polypeptides or peptides, or variants, fragments or derivatives thereof, which have been chemically modified. Examples include covalent attachment of one or more polymers - such as water soluble polymers, N-linked, or O-linked carbohydrates, sugars, phosphates, and/or other such molecules.
- the derivatives are modified in a manner that is different from naturally occurring or starting peptide or polypeptides, either in the type or location of the molecules attached. Derivatives further include deletion of one or more chemical groups which are naturally present on the peptide or polypeptide.
- SAP binding antibodies that include two full length heavy chains and two full length light chains.
- the SAP binding antibodies may be constructs such as single chain antibodies or "mini" antibodies that retain binding activity to SAP. Such constructs may be prepared by methods well known in the art.
- SAP binding antibodies and fragments thereof Methods for creating recombinant DNA versions of the antigen-binding regions of antibody molecules which bypass the generation of monoclonal antibodies are contemplated for the SAP binding antibodies and fragments thereof.
- DNA is cloned into a bacterial expression system.
- One example of such a technique uses a bacteriophage lambda vector system having a leader sequence that causes the expressed Fab protein to migrate to the periplasmic space (between the bacterial cell membrane and the cell wall) or to be secreted.
- SAP binding agents Fab fragments with specificity for an SAP polypeptide
- SAP binding agents are specifically encompassed within the SAP binding antibodies and fragments thereof.
- the SAP binding antibodies and fragments thereof may be humanized or human engineered antibodies.
- a humanized antibody or antigen binding fragment thereof, is a recombinant polypeptide that comprises a portion of an antigen binding site from a non-human antibody and a portion of the framework and/or constant regions of a human antibody.
- a human engineered antibody or antibody fragment is a non-human (e.g., mouse) antibody that has been engineered by modifying (e.g., deleting, inserting, or substituting) amino acids at specific positions so as to reduce or eliminate any detectable immunogenicity of the modified antibody in a human.
- Humanized antibodies include chimeric antibodies and CDR-grafted antibodies.
- Chimeric antibodies are antibodies that include a non-human antibody variable region linked to a human constant region. Thus, in chimeric antibodies, the variable region is mostly non-human, and the constant region is human. Chimeric antibodies and methods for making them are described in, for example, Proc. Natl. Acad. ScL USA, 81 : 6841-6855 (1984). Although, they can be less immunogenic than a mouse monoclonal antibody, administrations of chimeric antibodies have been associated with human immune responses (HAMA) to the non-human portion of the antibodies.
- HAMA human immune responses
- Chimeric antibodies can also be produced by splicing the genes from a mouse antibody molecule of appropriate antigen-binding specificity together with genes from a human antibody molecule of appropriate biological activity, such as the ability to activate human complement and mediate antibody dependent cellular cytotoxicity (ADCC).
- ADCC antibody dependent cellular cytotoxicity
- One example is the replacement of a Fc region with that of a different isotype.
- CDR-grafted antibodies are antibodies that include the CDRs from a non-human "donor” antibody linked to the framework region from a human “recipient” antibody.
- CDR-grafted antibodies include more human antibody sequences than chimeric antibodies because they include both constant region sequences and variable region (framework) sequences from human antibodies.
- a variable region framework sequences from human antibodies.
- CDR-grafted humanized antibody of the invention can comprise a heavy chain that comprises a contiguous amino acid sequence (e.g., about 5 or more, 10 or more, or even 15 or more contiguous amino acid residues) from the framework region of a human antibody (e.g., FR-I, FR-2, or FR-3 of a human antibody) or, optionally, most or all of the entire framework region of a human antibody.
- a human antibody e.g., FR-I, FR-2, or FR-3 of a human antibody
- CDR-grafted antibodies and methods for making them are described in Nature, 321 : 522-525 (1986). Methods that can be used to produce humanized antibodies also are described in, for example, US 5,721,367 and 6,180,377.
- Veneered antibodies are non-human or humanized (e.g., chimeric or CDR-grafted antibodies) antibodies that have been engineered to replace certain solvent-exposed amino acid residues so as to reduce their immunogenicity or enhance their function. Veneering of a chimeric antibody may comprise identifying solvent-exposed residues in the non-human framework region of a chimeric antibody and replacing at least one of them with the corresponding surface residues from a human framework region. Veneering can be accomplished by any suitable engineering technique.
- humanized or human engineered antibodies are IgG, IgM, IgE, IgA, and IgD antibodies.
- the antibodies may be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa or lambda light chain.
- a human antibody may comprise an IgG heavy chain or defined fragment, such as at least one of isotypes, IgGl, IgG2, IgG3 or IgG4.
- the antibodies or fragments thereof can comprise an IgGl heavy chain and a kappa or lambda light chain.
- the anti-SAP antibodies and fragments thereof may be human antibodies - such as antibodies which bind the SAP polypeptides and are encoded by nucleic acid sequences which may be naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequence, and fragments, synthetic variants, derivatives and fusions thereof.
- Such antibodies may be produced by any method known in the art, such as through the use of transgenic mammals (such as transgenic mice) in which the native immunoglobulins have been replaced with human V-genes in the mammal chromosome.
- Human antibodies to target SAP can also be produced using transgenic animals that have no endogenous immunoglobulin production and are engineered to contain human immunoglobulin loci, as described in WO 98/24893 and WO 91/00906.
- an immune response can be produced to a selected antigenic molecule, and antibody producing cells can be removed from the animal and used to produce hybridomas that secrete human monoclonal antibodies.
- Immunization protocols, adjuvants, and the like are known in the art, and are used in immunization of, for example, a transgenic mouse.
- the antibodies produced by phage technology are produced as antigen binding fragments-usual Iy Fv or Fab fragments-in bacteria and thus lack effector functions.
- Effector functions can be introduced by one of two strategies: the fragments can be engineered either into complete antibodies for expression in mammalian cells, or into bispecific antibody fragments with a second binding site capable of triggering an effector function.
- Human antibodies may be generated through the in vitro screening of antibody display libraries (J. MoI. Biol. (1991) 227: 381). Various antibody-containing phage display libraries have been described and may be readily prepared. Libraries may contain a diversity of human antibody sequences, such as human Fab, Fv, and scFv fragments, that may be screened against an appropriate target. Phage display libraries may comprise peptides or proteins other than antibodies which may be screened to identify agents capable of selective binding to SAP.
- Phage-display processes mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice.
- One such method is described in WO 99/10494.
- Anti-SAP antibodies can be isolated by screening of a recombinant combinatorial antibody library, preferably a scFv phage display library, prepared using human V and V cDNAs prepared from mRNA derived from human lymphocytes.
- the antibodies of the invention include "cross-linked” antibodies.
- cross-linked refers to binding of at least two IgG molecules together to form one (or single) molecule.
- the SAP antibodies may be cross-linked using various linker molecules and optionally the antibodies are cross-linked using an anti-IgG molecule, complement, chemical modification or molecular engineering.
- IgM, IgG2a and IgG2b are known to fix complement.
- the antibodies of the invention advantageously comprise "complement fixing” antibodies and fragments thereof (Examples 6 & 7).
- a complement fixing antibody or fragment thereof is one which having bound to its specific epitopes on the target antigen then activates the complement cascade.
- IgG antibodies required for the present invention such activation takes place predominantly via the classical pathway via CIq although all complement activation is amplified by the feedback loop of the alternative pathway.
- the key effects of complement in relation to the present invention are then cleavage and binding of C3, the pivotal and most abundant complement component, and the subsequent activation and cleavage of C5.
- the small split fragments of C3 and C5, respectively C3a and C5a, are potent chemotactic attractants for phagocytic cells of all types, polymorphonuclear granulocytes and mononuclear phagocytes, monocytes and macrophages.
- the latter group are essential for amyloid clearance according to the present invention (Example 8).
- amyloid deposits of bound complement predominantly C3b and its further breakdown products, C3bi, C3c and C3d
- C3bi, C3c and C3d amyloid deposits of bound complement
- C3bi, C3c and C3d amyloid deposits of bound complement
- opsonises the deposits that is enhances their phagocytosis and destruction, leading to the desirable and clinically beneficial removal of amyloid which is the purpose of the present invention.
- Antibodies of the present invention are specific for SAP and do not recognise or bind to any cell surface to other cellular antigens. They are accordingly free of specific agonistic activity.
- antibodies of the invention may optionally comprise dimeric antibodies, as well as multivalent forms of antibodies.
- Those skilled in the art may construct such dimers or multivalent forms by techniques known in the art and using the anti-SAP antibodies herein.
- the antibodies of the invention may also comprise monovalent antibodies.
- Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain.
- the heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain cross linking.
- the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent cross linking.
- In vitro methods are also suitable for preparing monovalent antibodies.
- Digestion of antibodies to produce fragments thereof, particularly, Fab fragments can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566.
- Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields an F(ab').sub.2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.
- the SAP binding antibodies and fragments thereof may comprise one or more portions that do not bind SAP but instead are responsible for other functions, such as circulating half-life, direct cytotoxic effect, detectable labeling, or activation of the recipient's endogenous complement cascade or endogenous cellular cytotoxicity.
- the antibodies or fragments thereof may comprise all or a portion of the constant region and may be of any isotype, including IgA (e.g., IgAl or IgA2), IgD, IgE, IgG (e.g. IgGl, IgG2, IgG3 or IgG4), or IgM.
- antigen-binding compounds of the invention may include an epitope tag, a salvage receptor epitope, a label moiety for diagnostic or purification purposes, or a cytotoxic moiety such as a radionuclide or toxin.
- the anti-SAP antibody or fragment thereof may be modified in order to increase its serum half-life, for example, by adding molecules - such as PEG or other water soluble polymers, including polysaccharide polymers to increase the half-life.
- the SAP binding antibodies and fragments thereof may be bispecific.
- bispecific antibodies may resemble single antibodies (or antibody fragments) but have two different antigen binding sites (variable regions).
- Bispecific antibodies can be produced by various methods - such as chemical techniques, "polydoma” techniques or recombinant DNA techniques.
- Bispecific antibodies may have binding specificities for at least two different epitopes, at least one of which is an epitope of SAP.
- the SAP binding antibodies and fragments may be heteroantibodies. Heteroantibodies are two or more antibodies, or antibody binding fragments (Fab) linked together, each antibody or fragment having a different specificity.
- antibody fragments refers to portions of an intact full length antibody - such as an antigen binding or variable region of the intact antibody.
- antibody fragments include Fab, Fab', F(ab') , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibody fragments such as bispecific, trispecific, and multispecific antibodies (e.g., diabodies, triabodies, tetrabodies); binding-domain immunoglobulin fusion proteins; camelized antibodies; minibodies; chelating recombinant antibodies; tribodies or bibodies; intrabodies; nanobodies; small modular immunopharmaceuticals (SMIP), V containing antibodies; and any other polypeptides formed from antibody
- SMIP small modular immunopharmaceuticals
- anti-SAP antibody and SAP binding antibody encompass SAP binding antibody fragments comprising any part of the heavy or light chain sequences of the full length antibodies, and which bind SAP.
- fragments refers to fragments capable of binding SAP, for example any of at least 3 contiguous amino acids (e.g., at least 4, 5, 6, 7, 8, 9 or 10 or more contiguous amino acids, for example from a CDR) of the antibody involved in antigen binding, and encompasses Fab, Fab', F(ab') , and F(v) fragments, or the individual light or heavy chain variable regions or portion thereof.
- SAP binding fragments include, for example, Fab, Fab', F(ab') , Fv and scFv. These fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation, and can have less non-specific tissue binding than an intact antibody.
- fragments can be produced from intact antibodies using well known methods, for example by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') fragments).
- fragments of the present invention activate complement.
- SAP binding antibodies and fragments also encompass single-chain antibody fragments (scFv) that bind to SAP.
- An scFv comprises an antibody heavy chain variable region (V H ) operably linked to an antibody light chain variable region (V L ) wherein the heavy chain variable region and the light chain variable region, together or individually, form a binding site that binds SAP.
- An scFv may comprise a V region at the amino-terminal end and a V region at the carboxy-terminal end.
- scFv may comprise a V region at the amino-terminal end and a V region at the
- VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv).
- An scFv may optionally further comprise a polypeptide linker between the heavy chain variable region and the light chain variable region.
- SAP binding antibodies and fragments also encompass domain antibody (dAb) fragments as described in Nature 341 : 544-546 (1989) which consist of a V H domain.
- dAb domain antibody
- SAP binding antibodies and fragments also encompass heavy chain antibodies (HCAb). These antibodies can apparently form antigen-binding regions using only heavy chain variable region, in that these functional antibodies are dimers of heavy chains only (referred to as “heavy-chain antibodies” or “HCAbs”). Accordingly, SAP binding antibodies and fragments may be heavy chain antibodies (HCAb) that specifically bind to SAP.
- HCAb heavy chain antibodies
- SAP binding antibodies and fragments also encompass antibodies that are SMIPs or binding domain immunoglobulin fusion proteins specific for SAP protein. These constructs are single-chain polypeptides comprising antigen binding domains fused to immunoglobulin domains necessary to carry out antibody effector functions (see WO03/041600).
- the SAP binding antibodies and fragments also encompass diabodies. These are bivalent antibodies in which V H and V L domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain. This forces the domains to pair with complementary domains of another chain and thereby creates two antigen binding sites (see, for example, WO 93/1 1 161). Diabodies can be bispecific or monospecific.
- SAP binding antibodies and fragments thereof also encompass immunoadhesins.
- One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin.
- An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently.
- the CDRs permit the immunoadhesin to specifically bind to SAP.
- SAP binding antibodies and fragments thereof also encompass antibody mimics comprising one or more SAP binding portions built on an organic or molecular scaffold (such as a protein or carbohydrate scaffold).
- an organic or molecular scaffold such as a protein or carbohydrate scaffold.
- Proteins having relatively defined three-dimensional structures commonly referred to as protein scaffolds, may be used as reagents for the design of antibody mimics.
- These scaffolds typically contain one or more regions which are amenable to specific or random sequence variation, and such sequence randomization is often carried out to produce libraries of proteins from which desired products may be selected.
- an antibody mimic can comprise a chimeric non-immunoglobulin binding polypeptide having an immunoglobulin-like domain containing scaffold having two or more solvent exposed loops containing a different CDR from a parent antibody inserted into each of the loops and exhibiting selective binding activity toward a ligand bound by the parent antibody.
- Non-immunoglobulin protein scaffolds have been proposed for obtaining proteins with novel binding properties.
- Anti-SAP antibodies or antibody fragments thereof typically bind to human SAP with high affinity (e.g., as determined with BIACORE), such as for example with an equilibrium binding dissociation constant (K ) for SAP of about 15nM or less, 10 nM or less, about 5 nM or less, about 1 nM or less, about 500 pM or less, about 250 pM or less, about 100 pM or less, about 50 pM or less, or about 25 pM or less, about 10 pM or less, about 5 pM or less, about 3 pM or less about 1 pM or less, about 0.75 pM or less, or about 0.5 pM or less.
- K equilibrium binding dissociation constant
- the anti-SAP antibody or antibody fragment thereof does not cross-react with any target other than SAP.
- the antibodies and antibody fragments described herein can be prepared by any suitable method. Suitable methods for preparing such antibodies and antibody fragments are known in the art.
- the antibody or antibody fragment may be isolated or purified to any degree.
- an "isolated compound” is a compound that has been removed from its natural environment.
- a purified compound is a compound that has been increased in purity, such that it exists in a form that is more pure than it exists in its natural environment and/or when initially synthesized and/or amplified under laboratory conditions. Purity is a relative term and does not necessarily mean absolute purity.
- SAP-DEPLETING COMPOUND Compounds of the present invention include those compounds that result in the depletion of circulating SAP.
- Such compounds include those which are competitive inhibitors of binding of SAP to amyloid fibrils, as described in European patent application EP 0 915 088, including ( ⁇ -l-f ⁇ - ⁇ -Carboxy-pyrrolidin-l-ylJ- ⁇ -oxo-hexanoylJpyrrolidine ⁇ -carboxylic acid (CPHPC), however, any of the compounds described therein, or any other compound which depletes circulating SAP can be used in the practice of the present invention.
- CPHPC ⁇ -l-f ⁇ - ⁇ -Carboxy-pyrrolidin-l-ylJ- ⁇ -oxo-hexanoylJpyrrolidine ⁇ -carboxylic acid
- the SAP-depleting compound is a D-proline; preferred are D-prolines of the Formula:
- X-Y-X' is a linker having at least 4, advantageously at least 5, advantageously at least 6, up to 20 linear or straight-chain carbon atoms, wherein
- X is -(CH ) -; -CH(R )(CH ) -; CH O(CH ) -; CH NH-; benzyl,
- X' is -(CH ) -; -(CH ) CH(R )-; -(CH ) OCH -; -NHCH -; benzyl,
- -CH C(R )-; CH(OH)CH 2 ; or thiazol-2,5-diyl; O;
- R is lower alkyl, lower alkoxy or benzyl and n is 0-3, or a pharmaceutically acceptable salt or mono- or diester thereof.
- SAP-depleting compound D-proline of formula I-A above can be written as
- Ligand - linker - Ligand wherein the X-Y-X' moiety of formal I-A forms the linker.
- linker (X-Y-X') can be from 4 to
- the linker can be a straight or branched chain, or can optionally form one or more ring structures, with the proviso that at least 4 linear or straight-chain carbon atoms are present in the linker.
- at least one of the linear or straight-chain C atoms can be optionally substituted by at least one hetero atom selected from N, O, or S, advantageously O or S, preferably O.
- an "optionally substituted linker” can have one or more substitutions that lead to branching and /or one or more substitutions of carbon atom(s) of the linear or straight-chain carbon atoms of the linker, e.g. the linker can be an ether or a substituted ether.
- the complex of SAP with CPHPC comprises two pentameric SAP molecules cross linked, B face to B face by five CPHPC molecules, and the D-proline head groups at either end of which are bound in the calcium dependent ligand binding pockets of opposite pairs of SAP protomers (Figure 10).
- This complex may be covalently cross linked, using the bifunctional cross linker, BS3, to produce a stable complex in which the SAP molecules are fixed in the orientation and with the same stereochemical accessibility to binding by antibodies as SAP deposited on amyloid fibrils in vivo ( Figure 1 1).
- B inding of anti-human SAP antibodies to SAP immobilised on a solid surface, such as microtiter plates, can be accurately quantified. Comparison of such binding to SAP immobilised alone and in the stable complex with CPHPC after cross linking with BS3, and in the presence and absence of calcium, provides information about the position and nature of the epitope recognised by each antibody.
- the SAP-depleting compound as described herein and the anti-human SAP antibody or fragment thereof described herein will be administered as pharmaceutical compositions comprising therapeutically effective amounts.
- terapéuticaally effective amount refers to an amount of SAP-depleting compound and the anti- (human) SAP antibody or fragment thereof or as a part of a pharmaceutical composition, that is capable of having any detectable, positive effect on any symptom, aspect, or characteristic of amyloidosis when administered to a patient (e.g., as one or more doses).
- the term "patient”, includes a subject, advantageously an animal subject.
- Animal subjects of the present invention include mammals, i.e., humans, companion animals including felines and canines, as well as other animals including equines.
- the combination of an SAP-depleting compound and the anti-SAP antibody or fragment thereof may be administered separately, simultaneously, sequentially, concurrently or consecutively, or the combination may be presented in the form of one pharmaceutical formulation.
- the present invention also involves the SAP-depleting compound and the anti-SAP antibody or fragment thereof as a combined preparation for simultaneous, separate or sequential use in the therapeutic or prophylactic treatment of amyloidosis.
- compositions comprising the SAP-depleting compound may be administered separately from the anti-SAP antibodies or fragments thereof, and such separate administrations may be performed at the same point or different points in time, such as for example the same or different days. If the combination of SAP-depleting compound and the anti-SAP antibody or fragment thereof are to be administered sequentially then the SAP-depleting compound is administered first such that the SAP-depleting compound treatment can clear almost all of the circulating SAP. Since this leaves substantial amounts of SAP associated with the amyloid deposits in the tissues the sequential administration of the anti-SAP antibody or fragment thereof enables the localisation and specific binding to the amyloid deposits to promote their rapid and extensive regression.
- the anti-SAP antibody or fragment thereof may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or 25 or more days after the treatment(s) with the SAP-depleting compound.
- the sequential administration may involve two or more sequential treatments with SAP-depleting compound followed by two or more sequential treatments with the anti-SAP antibody or fragment thereof.
- the sequential administration may involve one treatment with SAP-depleting compound followed by one sequential treatment with the anti-SAP antibody or fragment thereof, which is then repeated one or more times.
- the sequential/subsequent dose may be an amount that is more than the initial/previous dose or less than the initial/previous dose.
- the administration of an initial dose of SAP-depleting compound and/or the anti-SAP antibody or fragment thereof may be followed by the administration of one or more sequential (eg. subsequent) doses of SAP-depleting compound and/or the anti-SAP antibody or fragment thereof, and wherein said one or more sequential doses may be in an amount that is approximately the same or less than the initial dose.
- the administration of an initial dose of SAP-depleting compound and/or the anti-SAP antibody or fragment thereof may be followed by the administration of one or more sequential (eg. subsequent) doses, and wherein at least one of the subsequent doses is in an amount that is more than the initial dose.
- the administration may use a pre-determined or routine schedule for administration, thereby resulting in a predetermined designated period of time between dose administrations.
- the schedule may encompass periods of time which are identical or which differ in length, as long as the schedule is predetermined. Any particular combination would be covered by the schedule as long as it is determined ahead of time that the appropriate schedule involves administration on a certain day.
- compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable compounds - such as carriers, excipients, diluents, antioxidants, preservatives, colouring, flavouring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, and surfactants.
- a pharmaceutically acceptable compounds such as carriers, excipients, diluents, antioxidants, preservatives, colouring, flavouring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, and surfactants.
- Acceptable compounds for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington
- the pharmaceutical compositions may include antioxidants - such as ascorbic acid; low molecular weight polypeptides; proteins - such as serum albumin and/or gelatin, hydrophilic polymers - such as polyvinylpyrrolidone; chelating agents - such as EDTA; sugar alcohols - such as mannitol and/or sorbitol; amino acids; monosaccharides, disaccharides, and other carbohydrates; salt-forming counterions - such as sodium; and/or nonionic surfactants - such as Tween, pluronics, or polyethylene glycol (PEG).
- antioxidants such as ascorbic acid
- proteins - such as serum albumin and/or gelatin
- hydrophilic polymers such as polyvinylpyrrolidone
- chelating agents - such as EDTA
- sugar alcohols - such as mannitol and/or sorbitol
- amino acids monosaccharides, disaccharides, and other carbohydrates
- suitable tonicity enhancing agents include alkali metal halides (preferably sodium or potassium chloride), mannitol, sorbitol, and the like.
- Suitable preservatives include benzalkonium chloride, thimerosal, methylparaben, propylparaben, chlorhexidine phenethyl alcohol and sorbic acid.
- Suitable cosolvents include glycerine and/or propylene glycol.
- Suitable complexing agents include caffeine and/or polyvinylpyrrolidone.
- Suitable surfactants or wetting agents include sorbitan esters and/or polysorbates.
- the buffers may be conventional buffers such as citrate, acetate, borate, bicarbonate, or Tris-HCl.
- Acetate buffer may be about pH 4-5.5, and Tris buffer may be about pH 7-8.5. Additional pharmaceutical agents are set forth in Remington 's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990.
- the pharmaceutical carrier, excipient or diluent etc. may be selected based upon the intended route of administration and standard pharmaceutical practice.
- composition/formulation requirements may differ dependent on the different delivery system(s).
- the composition may be in liquid, lyophilized or freeze-dried form and may include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and/or bulking agents.
- a lyoprotectant is included, which is a non-reducing sugar - such as sucrose and/or lactose.
- the amount of lyoprotectant generally included is such that, upon reconstitution, the resulting formulation will typically be isotonic.
- a surfactant may be included - such as nonionic surfactants and ionic surfactants. Exemplary amounts of surfactant that may be present in the pre-lyophilized formulation are from about 0.001-0.5%.
- High molecular weight structural additives ⁇ e.g.
- fillers may be included.
- binders may be included.
- concentrations of high molecular weight structural additives are from 0.1% to 10% by weight.
- a bulking agent ⁇ e.g., mannitol, glycine
- compositions may be suitable for parenteral administration.
- Compositions may be suitable for injection or infusion into an animal by any route available to the skilled person, such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intralesional, oral, and inhaled routes.
- a parenteral formulation typically will be a sterile, pyrogen-free, isotonic aqueous solution, optionally containing pharmaceutically acceptable preservatives.
- compositions may be formulated for controlled or sustained delivery in a manner that provides local concentration of the product (e.g., bolus, depot effect) sustained release and/or increased stability or half-life in a particular local environment.
- product e.g., bolus, depot effect
- such compositions may include a significantly larger amount of the SAP-depleting compound and/or the anti-SAP antibody or fragment thereof in the initial deposit, while the effective amount of antibody or fragment actually released and available at any point in time is in an amount much lower than the initial deposit.
- compositions can include the formulation with particulate preparations of polymeric compounds as well as agents such as a biodegradable matrix, injectable microspheres, microcapsular particles, microcapsules, bioerodible particles beads, liposomes, and implantable delivery devices that provide for the controlled or sustained release of the active agent which then can be delivered as a depot injection.
- agents such as a biodegradable matrix, injectable microspheres, microcapsular particles, microcapsules, bioerodible particles beads, liposomes, and implantable delivery devices that provide for the controlled or sustained release of the active agent which then can be delivered as a depot injection.
- Such sustained- or controlled-delivery means are known and a variety of polymers have been developed and used for the controlled release and delivery of drugs.
- Such polymers are typically biodegradable and biocompatible.
- Polymer hydrogels including those formed by complexation of enantiomeric polymer or polypeptide segments, and hydrogels with temperature or pH sensitive properties, may be desirable for providing drug depot effect because of the mild and aqueous conditions involved in trapping bioactive protein agents (e.g., antibodies).
- Bioadhesive polymers may also be present use in the compositions.
- Bioadhesives are synthetic and naturally occurring materials able to adhere to biological substrates for extended time periods.
- Carbopol and polycarbophil are both synthetic cross-linked derivatives of poly(acrylic acid).
- the pharmaceutical composition may be formulated for inhalation, such as for example, as a dry powder.
- Inhalation solutions also can be formulated in a liquefied propellant for aerosol delivery.
- solutions may be nebulized.
- Additional pharmaceutical composition for pulmonary administration include, those described, for example, in PCT Application Publication WO 94/20069, which discloses pulmonary delivery of chemically modified proteins.
- the particle size should be suitable for delivery to the distal lung.
- the particle size can be from 1 ⁇ m to 5 ⁇ m; however, larger particles may be used, for example, if each particle is fairly porous.
- Another preparation can involve a mixture with non-toxic excipients which are suitable for the manufacture of tablets.
- excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
- formulations may be administered orally.
- Formulations administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules.
- a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized.
- Additional agents can be included to facilitate absorption of a selective binding agent. Diluents, flavourings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders also can be employed.
- compositions used in the invention may comprise a therapeutically effective amount or a prophylactically effective amount of the SAP-depleting compound and/or the anti-SAP antibody or fragment thereof.
- a therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
- a therapeutically effective amount of the antibody or antibody portion may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual.
- a therapeutically effective amount is also one in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects.
- a prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
- a therapeutically or prophylactically effective amount of the pharmaceutical composition will depend, for example, upon the therapeutic objectives - such as the indication for which the composition is being used, the route of administration, and the condition of the subject.
- Pharmaceutical compositions are administered in a therapeutically or prophylactically effective amount to treat amyloidosis.
- a "therapeutically or prophylactically effective amount” is that amount which can treat or prevent one or more symptoms of amyloidosis in a subject.
- composition described herein may also be used in combination with conventional treatments for amyloidosis.
- SAP-depleting compound may be administered in the form of a pharmaceutically acceptable salt.
- Suitable acid addition salts are formed from acids which form non-toxic salts and include the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, hydrogenphosphate, acetate, trifluoroacetate, gluconate, lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate, gluconate, formate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate and p-toluenesulphonate salts.
- suitable pharmaceutically acceptable base addition salts can be formed from bases which form non-toxic salts and include the aluminium, calcium, lithium, magnesium, potassium, sodium, zinc, and pharmaceutically-active amines such as diethanolamine, salts.
- a pharmaceutically acceptable salt may be readily prepared by mixing together solutions of the SAP-depleting compound and the desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.
- the components may be administered alone but will generally be administered as a pharmaceutical composition - e.g. when the components are as an admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
- the components can be administered in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.
- the tablet may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
- excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine
- disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates
- Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
- Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.
- the agent may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
- the routes for administration may include, but are not limited to, one or more of oral (e.g. as a tablet, capsule, or as an ingestable solution), topical, mucosal
- nasal e.g. as a nasal spray or aerosol for inhalation
- parenteral e.g. by an injectable form
- gastrointestinal intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal, buccal, vaginal, epidural and sublingual.
- the mode of administration is intravenous infusion.
- DOSE LEVELS Suitable and/or preferred pharmaceutical formulations can be determined in view of the present disclosure and general knowledge of formulation technology, depending upon the intended route of administration, delivery format, and desired dosage.
- the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.
- the SAP-depleting compound may be administered at a dose of between 2mg/kg and O.lmg/kg, depending on its activity.
- the anti-SAP antibody or fragment thereof may be administered as a fixed dose, independent of a dose per subject weight ratio, or at an appropriate dose in mg/kg body weight with an approximate maximum of 200 mg/kg.
- the antibody or fragment thereof may be administered to a 70 kg individual in one or more separate, simultaneous or sequential doses of 14,000 mg or less of antibody or fragment thereof, 13,000 mg or less of antibody or fragment thereof, 12,000 mg or less of antibody or fragment thereof, 1 1 ,000 mg or less of antibody or fragment thereof, 10,000 mg or less of antibody or fragment thereof, 9000 mg or less of antibody or fragment thereof, 8000 mg or less of antibody or fragment thereof, 7000 mg or less of antibody or fragment thereof, 6000 mg or less of antibody or fragment thereof, 5000 mg or less of antibody or fragment thereof, 4500 mg or less of antibody or fragment thereof, 4000 mg or less of antibody or fragment thereof, 3700 mg or less of antibody or fragment thereof, 3500 mg or less of antibody or fragment thereof, mg or less of antibody or fragment thereof, 3200 mg or less of antibody or fragment thereof, 3
- the antibody or fragment is administered in one or more doses of at least 20 mg of antibody or fragment thereof. Since the total protein concentration in human plasma is 70,000 mg/1, and standard blood transfusion or plasma or albumin infusions routinely deliver tens or even hundreds of grams of protein intravenously, administration of the maximal doses of anti-SAP antibody are safe and acceptable.
- the SAP-depleting compound may be administered as a fixed dose, independent of a dose per subject weight ratio.
- the SAP-depleting compound may be administered in one or more separate, simultaneous or sequential parenteral doses of 100 mg or less, of 50 mg or less, 25 mg or less, or 10 mg or less.
- the SAP-depleting compound may be administered in a dose per subject weight ratio as easily determined by one of skill in the art.
- the component(s) may be formulated into a pharmaceutical composition, such as by mixing with one or more of a suitable carrier, diluent or excipient, by using techniques that are known in the art.
- EXPRESSION A wide variety of expression systems are available for the production of anti-SAP antibodies and antibody fragments including Fab fragments, scFv, and V HH s.
- expression systems of both prokaryotic and eukaryotic origin may be used for the large-scale production of antibody fragments and antibody fusion proteins.
- the gram-negative bacterium E. coli is widely used as a host for heterologous gene expression.
- large amounts of heterologous protein tend to accumulate inside the cell.
- Subsequent purification of the desired protein from the bulk of E. coli intracellular proteins can sometimes be difficult.
- bacteria from the genus Bacillus are very suitable as heterologous hosts because of their capability to secrete proteins into the culture medium.
- Other bacteria suitable as hosts are those from the genera Streptomyces and Pseudomonas.
- eukaryotic hosts such as yeasts or other fungi may be preferred.
- yeast cells are preferred over fungal cells because they are easier to manipulate.
- some proteins are either poorly secreted from the yeast cell, or in some cases are not processed properly (e.g. hyperglycosylation in yeast). In these instances, a different fungal host organism should be selected.
- fungi such as Aspergillus species (such as those described in EP-A-Ol 84438 and EP-A-0284603) and Trichoderma species; bacteria such as Bacillus species (such as those described in EP-A-Ol 34048 and EP-A-0253455), Streptomyces species and
- yeasts such as Kluyveromyces species (such as those described in EP-A-0096430 and EP-A-0301670) and Saccharomyces species.
- suitable host cells - such as yeast, fungal and plant host cells - may provide for post-translational modifications (e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products.
- post-translational modifications e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation
- an article of manufacture, or "kit”, containing materials useful for the treatment of the amyloidosis is provided.
- the kit is formulated for the separate or sequential administration of the D- proline and the anti-SAP antibody or a fragment thereof.
- the kit comprises a container comprising the SAP-depleting compound and the anti-SAP antibody or fragment thereof. In another embodiment, the kit comprises a first container comprising the SAP-depleting compound and a second container comprising the anti-SAP antibody or fragment thereof.
- the SAP-depleting compound and the anti-SAP antibody or fragment thereof are present in therapeutically or prophylactically effective amounts for the treatment and/or prevention of amyloidosis.
- Suitable containers include, for example, bottles, vials, syringes, blister pack, etc.
- the container may be formed from a variety of materials such as glass or plastic.
- the container holds the SAP-depleting compound or a pharmaceutical formulation thereof and/or the anti-SAP antibody or a fragment thereof, in an amount effective for treating amyloidosis, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
- the kit may further comprise a label or package insert on or associated with the container.
- the label or package insert may indicate that the composition(s) is used for treating amyloidosis.
- the kit may further comprise an additional container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection, phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
- a pharmaceutically acceptable buffer such as bacteriostatic water for injection, phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
- the kit may further comprise directions for the administration of the SAP-depleting compound and the anti-SAP antibody or fragment thereof for treating or preventing amyloidosis.
- the kit may further comprise directions for the simultaneous, sequential or separate administration of the first and second pharmaceutical compositions to a subject in need thereof.
- kits may be suitable for the delivery of solid oral forms of the compositions - such as tablets or capsules.
- a kit includes, for example, a number of unit dosages.
- Such kits can include a card having the dosages oriented in the order of their intended use.
- An example of such a kit is a "blister pack".
- Blister packs are well known in the packaging industry and are widely used for packaging pharmaceutical unit dosage forms.
- a memory aid can be provided, for example in the form of numbers, letters, or other markings or with a calendar insert, designating the days in the treatment schedule in which the dosages can be administered.
- the kit may comprise a separate container for containing the separate formulations, such as a divided bottle or a divided foil packet; however, the separate compositions may also be contained within a single, undivided container.
- the kit comprises directions for the administration of the separate components. This kit form is particularly advantageous when the separate components are administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician.
- the present invention provides an assay method for identifying one or more agents that can be used in combination with an SAP-depleting compound as described herein for the treatment of amyloidosis, in particular, for the essentially complete clearance of the amyloid deposits.
- the agent may be an organic compound or other chemical.
- the agent may be a compound, which is obtainable from or produced by any suitable source, whether natural or artificial.
- the agent may be an amino acid molecule, a polypeptide, or a chemical derivative thereof, or a combination thereof.
- the agent may even be a polynucleotide molecule - which may be a sense or an anti-sense molecule, or an antibody, for example, a polyclonal antibody, a monoclonal antibody or a monoclonal humanized antibody.
- the agent is an antibody - such as an antibody that is derived or derivable from an anti-SAP antibody.
- the agent may be prepared by chemical synthesis techniques.
- the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N. Y.); B. Roe, J. Crabtree, and A.
- control groups are essential to provide comparison with the known spontaneous regression of the amyloid deposits when inflammation ceases.
- the groups also need to be sufficiently large to compensate for the different rates of amyloid regression in different individual mice, even in these inbred pure line animals. Similarly the experiment cannot be conducted whilst amyloid induction is continuing because of the variable rates at which amyloid deposition occurs.
- a preliminary experiment on groups of 15 mice each, conducted according to the same protocol and with the same reagents as described here we obtained the same result as we show below. The present experiment was then performed with larger numbers in each group to confirm that the observed effect was reproducible and not due to a chance occurrence of accelerated amyloid regression in one of the groups unrelated to the treatment given.
- human SAP is present in both the circulation and the amyloid deposits.
- the drug CPHPC is specifically bound by human SAP in a complex composed of two native pentameric SAP molecules and 5 CPHPC
- This complex is recognised as abnormal by the liver and is very rapidly taken up by the hepatocytes and degraded, thus efficiently clearing SAP from the
- Plasma SAP concentrations remain very low for as long as the drug is
- CPHPC treatment clears almost all the circulating human SAP but leaves substantial amounts of SAP associated with the amyloid deposits in the tissues (unpublished observations).
- the greatest depletion of SAP from amyloid deposits which we have observed in human patients is about 90% after months of continuous CPHPC administration.
- Intravenous infusion of monospecific antibodies against human SAP into patients whose circulating SAP has been depleted enables the antibodies to locate and bind specifically to the amyloid deposits and promote their rapid and extensive regression, with corresponding clinical benefit. We show here the efficacy of this
- mice underwent whole body counting 24, 48, 72 96 and 168 h after the tracer injection to determine retention of radioactivity as an index of whole body amyloid load. There was consistently more retention in all the treated mice compared to the controls at all time points, indicating that they all had substantial
- mice 18 systemic amyloidosis deposits .
- the mice were then allocated to three closely matched groups with as close as possible to the same total numbers and sex
- mice 19 red staining . All mice were weighed at the time of assignment to the groups after
- mice were bled four times: (1) immediately before groups two and three were started on CPHPC; (2) the day before injection of IgG preparations in groups two and three; (3) 14 days after the IgG injections; (4) at the time of killing. Sections of spleen and liver of all animals stained with Congo red were independently examined by three different expert observers blinded to the treatment each mouse had received and scored for the amount 0 4 of amyloid present as previously reported.
- the scores of 10 -10 represent an o approximately log base 10 ranking scale from 10 , corresponding to one or two tiny
- SAP is not expressed in the spleen and there was no difference between the males and females with respect to either the quantity of spleen amyloid or the amount of human SAP within any of the three groups.
- SAP content of the spleens is therefore shown here for the whole of each group.
- amyloid deposits present in groups two and three, and absent in group one, there were modest changes consistent with chronic inflammation in many, though not all, of the tissues and these were not different between the groups.
- mice were all killed for estimation of their amyloid load at 60 days after the last casein injection and 28 days after treatment of the test group with anti-SAP antibodies. All mice had substantial amyloid deposits before anti-SAP or control treatments, as
- Example 2 Treatment of a patient with systemic amyloidosis using CPHPC and anti-SAP antibody
- a patient suffering from systemic amyloidosis is diagnosed by clinical examination and routine investigations leading to suspicion of amyloidosis, followed by specific confirmation by expert histochemical examination of biopsies of affected tissues.
- Radiolabeled SAP scintigraphy is performed in the UK NHS National Amyloidosis Centre at the Centre for Amyloidosis and Acute Phase Proteins in the Department of Medicine of University College London at the Royal Free Hospital.
- the tissue examination identifies the particular type of amyloid present and the scan, coupled with echocardiographic examination of the heart, shows where the amyloid is present and quantifies its extent.
- Routine clinical investigations of organ function establish the extent and severity of tissue and organ damage caused by the amyloid deposits, as well as the presence and severity of any underlying primary disease which may have led to amyloid deposition.
- the two essential first steps in treatment of such a patient consist of:
- organ function by all possible means, including drug therapy as appropriate and organ replacement if necessary comprising renal dialysis and organ transplantation, and (2) deployment of whatever therapy may be available to reduce the abundance of the precursor protein which is forming the amyloid fibrils.
- the latter may be very difficult to achieve and is sometimes impossible, so that organ damage progresses to organ failure, serious morbidity and usually leads to death.
- the patient is treated to arrest amyloid deposition and to clear away existing established amyloid deposits from the tissues, leading to clinical benefit.
- the patient is treated with the SAP depleting drug, CPHPC, administered by bolus intravenous injection of a dose of 100 mg.
- CPHPC the SAP depleting drug
- bolus intravenous injection of a dose of 100 mg The following day a blood sample is taken. This confirms by specific immunoassay that the SAP concentration in the serum has been reduced by 90%.
- An intravenous infusion of anti-SAP antibody is then commenced and over a period of several hours a dose of lOOOmg of antibody is administered sufficient to bind to SAP within the amyloid deposits throughout the body.
- CPHPC is administered parenterally twice daily for the next two weeks, to ensure that plasma SAP values remain suppressed. All aspects of the patient's clinical state and organ function are monitored closely on a daily basis throughout, and improvements in organ function are detected within days after the antibody infusion.
- mice In an experiment using the same protocol and reagents as described in Example 1 above, different groups of 5 mice each received the following doses of the same IgG fraction of sheep anti-human SAP antiserum as in Example 1 : 50 mg (same dose as in Example 1); 10 mg; 2 mg; 0.4 mg; none. The quantity of anti-SAP antibody in these doses was 7 mg, 1.4 mg, 0.28, 0.056 mg and zero respectively.
- the minimal effective dose of this particular sheep polyclonal anti-SAP antibody administered in a single dose in the protocol described in Example 1 was greater than 0.28 mg, and a dose of 1.4 mg showed maximal efficacy.
- Example 4 Time course, mechanism and clinical effects of amyloid clearance by anti-SAP antibody.
- mice In an experiment using the same protocol and reagents as described in Example 1 above, different groups of 5 mice each received the same treatments as in that experiment, including the 50 mg dose of IgG fraction containing 7 mg of anti-SAP antibody, and were then killed 1 , 2, 3, 4, 7, 10, 14, 21 and 25 days respectively after administration of the antibody. Plasma samples obtained from each animal at the time each group of mice was killed were stored frozen before biochemical analysis in a single batch at the end of the experiment. The spleen and liver were removed from each mouse at the time of death and processed for analysis as described in Example 1.
- C3 is the most abundant complement component and is responsible for the key chemotactic and opsonic activities of the complement system.
- any of the plasma analytes tested comprising: sodium, potassium, chloride, urea, creatinine, calcium, phosphate, alkaline phosphatase, alanine transaminase, aspartate transaminase, total protein, albumin, total cholesterol, triglycerides, glucose, total bilirubin, creatine kinase, lactate dehydrogenase.
- the predominant cell type persistently invading amyloid deposits was identified by hematoxylin and eosin staining as macrophages and this was confirmed by electron microscopy and immunocytochemistry, as was their active role in phagocytosis and destruction of the amyloid.
- the presence of human SAP, sheep IgG and mouse C3 on the AA amyloid deposits within and surrounded by macrophages is consistent with the following mechanism.
- Anti-human SAP antibody binds to human SAP associated with the amyloid deposits and activates complement, generating the potent chemotactic attractants, C3a and C5a.
- Phagocytic cells mostly macrophages, then surround and infiltrate the amyloid deposits and actively ingest the amyloid which has been opsonised by complement and IgG antibody, and macrophage mediated degradation rapidly clears the deposits.
- Example 1 The standard protocol described in detail in Example 1 uses mice in which the mouse SAP gene has been deleted and the human SAP transgene inserted to provide expression of human SAP. It also used administration of CPHPC in the drinking water starting from shortly before administration of the anti-SAP antibody and continuing through the rest of the experiment. In order to explore the need for such prolonged treatment with CPHPC, a single dose of parenteral CPHPC was used in mice which were human SAP transgenic on a wild type background, rather than mouse SAP knockouts as in the previous examples. In another approach, which is critical for studies in mice with genetic modifications unrelated to SAP, amyloidotic wild type non-transgenic mice had their amyloid deposits 'loaded' with human SAP by a single parenteral injection of isolated pure human SAP.
- each mouse received a single intraperitoneal injection of 50 mg of the IgG fraction of sheep anti-SAP antiserum and all were then killed 15 days after that for estimation of amyloid load by Congo red staining.
- Example 6 Complement dependence of amyloid clearance by anti-SAP antibody.
- mice lacking either CIq or C3 anti-SAP treatment did not clear amyloid deposits.
- the therapeutic efficacy of anti-SAP is thus very substantially complement dependent and is not mediated by IgG antibody alone which could, in theory, engage phagocytic cells via their Fc( ⁇ ) receptors. Nevertheless the more fragmented appearance of the persistent amyloid deposits in the complement deficient mice suggested at least some effect of antibody alone. Also the trend to more clearance in CIq deficient compared to C3 deficient animals suggested that C3 activation is critical and that even in the absence of CIq and the classical pathway, which is typically activated by IgG antibody, some complement activation may be taking place.
- Complement activation by IgG antibody requires the whole intact molecule, including the Fc region, and proceeds via the classical pathway initiated by binding of CIq. In some antibody-antigen systems less efficient complement activation via the alternative pathway can be mediated by the F(ab) 2 fragment. In order to confirm the complement dependence of amyloid clearing by anti-SAP antibody and to investigate the potential requirement for the Fc region of the antibody, the effect was tested of F(ab) 2 anti-SAP antibody.
- AA amyloidosis was induced and confirmed in wild type C57BL/6 mice as detailed in Example 1 above.
- the dose of anti-SAP antibody activity injected was 7.28 mg per mouse receiving F(ab) 2 and 7 mg (50 mg of total IgG as usual) per mouse receiving whole IgG. All mice were killed 14 days later for estimation of amyloid load by Congo red staining.
- mice receiving IgG anti-SAP antibody which had a median (range) spleen amyloid score of 10° (10°-10 3 ), compared to the massive amyloid deposits in the control mice with median (range) spleen amyloid score of 10 5 (10 4 -10 5 ).
- the mice receiving F(ab) 2 had less amyloid than untreated controls, median score 10 2 , range 10°-10 4 , but still substantially more than the mice treated with whole IgG anti-SAP antibody. Discussion
- the molar dose of F(ab) 2 anti-SAP antibody used in this study was about one third greater than that of IgG antibody, given the smaller molecular weight of the F(ab) 2 fragment compared to whole IgG.
- mice After induction and confirmation of AA amyloidosis in wild type mice, using the protocol detailed in Example 1 above, all animals received a single intraperitoneal dose of 10 mg of isolated pure human SAP to load their amyloid deposits with human SAP.
- mice which received the liposomal clodronate in a regime known to completely ablate macrophage function there was no clearance of amyloid deposits, the amyloid load score being 10 5 (10 2 -10 5 )
- Example 9 In vitro binding to SAP of mouse monoclonal antibodies against human SAP.
- the first set of antibodies comprised the antibodies from 7 hybridomas generated in a single conventional immunization and fusion protocol and designated SAP-I to SAP-7, and these were tested together with a separate monoclonal, designated NH.
- SAP-5 and SAP-2 are IgG2a isotype while the others are all IgGl isotype.
- the second set of antibodies comprised 6 different IgG2a monoclonals derived by standard techniques from immunization with pure human SAP and a conventional fusion to produce hybridomas which were cloned by routine methods.
- Supernatants from the fusion initially evaluated after the original fusion were using initially screened by standard enzyme-linked immunosorbent assay (ELISA) to identify the six mouse monoclonal anti-human SAP IgG antibodies that bound most abundantly to isolated pure human SAP which had been non-specifically non-covalently immobilised on microtiter plates. After cloning the supernatants and then the isolated antibodies were evaluated by IRMA as described below, and hybridomas clones producing IgG2a antibodies with maximal binding to SAP binding were selected.
- ELISA enzyme-linked immunosorbent assay
- Binding of antibodies to SAP was quantified by immunoradiometric assays performed in microtiter plates coated with immobilised pure human SAP. Bound mouse IgG was detected with radiolabeled sheep polyclonal antibody specific either for all mouse IgG classes or with isotype specific sheep antibodies against mouse IgG2a. Isolated pure human C-reactive protein (CRP) was used as a specificity control antigen. CRP is the protein most closely related to SAP and shares 55% strict residue for residue identity with it. None of the antibodies showed any reactivity at all against human CRP.
- CRP C-reactive protein
- Isolated pure human SAP was immobilised on the plates in several different formats: directly by non-specific, non-covalent binding to the plastic surface; by covalent attachment via its free amino groups to jV-hydroxysuccinimide activated plates; by capture on specific sheep anti-human SAP antibodies coated onto the plates; and in the form of complexes of SAP with CPHPC which had been further cross-linked by covalent interaction with the bifunctional reagent BS3.
- the SAP molecules are associated in pairs with their binding, B, faces, apposed and thereby occluded as they are when SAP is bound to amyloid fibrils.
- Use of SAP in this format therefore tests whether an anti-SAP antibody can access its specific epitope when the SAP molecule is bound to a macromolecular ligand.
- Binding of selected antibodies to SAP was further characterized qualitatively by double immunodiffusion in gel, and by blotting from both native agarose electrophoresis and SDS-PAGE (western blotting), all by routine Standard methods.. Quantitative analysis of the thermodynamic parameters of antibody binding was performed in the Biacore instrument, using isolated pure SAP alone immobilized on the chip, according to the manufacturer's instructions.
- the second set of anti-SAP antibodies were selected initially as those of IgG2a isotype with the most abundant binding in ELISA assays, and were then evaluated by IRMA using both polyvalent anti-mouse IgG and isotype specific anti-mouse IgG2a, a shown in Table 6. Table 6. Binding of mouse monoclonal anti-human SAP IgG2a antibodies to human SAP directly immobilized by non-covalent attachment and detected with radiolabeled sheep polyvalent anti-mouse IgG or sheep specific anti-mouse anti-IgG2a.
- Example 10 Efficacy of mouse monoclonal anti-human SAP antibody SAP-5 in clearing mouse systemic AA amyloid deposits.
- Amyloid clearance according to the present invention requires an anti-human SAP antibody which binds sufficiently avidly to the human SAP associated with amyloid deposits in the tissues and activates complement there in order to attract and activate macrophages to mediate destruction and clearance of the amyloid deposits.
- the capacity of various monoclonal antibodies to mediate this effect was sought in comparison with the standard sheep polyclonal anti-human SAP antibody as a positive control.
- mice received a single dose of 5 mg CPHPC by intraperitoneal injection, followed 5 hours later via the same route by the either the standard sheep polyclonal anti-human SAP IgG fraction (1 ml at 50 mg/ml total protein, 7 mg/ml anti-human SAP antibody) or 5 mg of each of the different monoclonal anti-human SAP antibodies. All mice were killed 21 days after the antibody injection and amyloid load determined by Congo red histology of their spleens.
- Table 9 The results shown in Table 9 are the scores for amyloid abundance in the spleen according to the logarithmic scale set out in Example 1.. Table 9. The presence of amyloid in spleen of mice with systemic AA amyloidosis after treatment with CPHPC and various anti-human SAP antibodies.
- SAP-5 is of the IgG2a isotype which is known to activate mouse complement while all the other monoclonals except SAP-2 were IgG 1 isotype which is not complement activating.
- SAP-2 is IgG2a isotype, its binding to SAP as quantified in vitro in Example 9, Tables 4 and 5, was notably less than that of SAP-5 and evidently was not sufficient in vivo to be effective.
- Example 11 Efficacy of Abpl monoclonal anti-human SAP antibody.
- mice were killed 17 days later for estimation of amyloid load by Congo red staining.
- mice treated with 5 mg of Abpl showed the same remarkable clearance of splenic and hepatic amyloid deposits as seen with the 7 mg dose of sheep polyclonal antibody. Only trace specks of amyloid remained in the spleens of the treated mice and none at all was detected in many of the livers, contrasting sharply with the extensive splenic and hepatic amyloid deposits in all animals which received the control antibody (Table 10).
- mice which were not treated because they had not developed sufficient amyloid load by the end of the induction period had massive amyloid deposits in all cases (Table 10), enhancing the contrast with the striking efficacy of amyloid clearance produced by both the polyclonal antibody, as usual, and by the conformation specific Abpl monoclonal.
- the lower dose of 1 mg of Abpl per mouse there was reduced amyloid in the liver but no significant effect in the spleens.
- monoclonal anti-human SAP antibodies for use according to the present invention can be directed at either sequence epitopes, such as antibody SAP-5, or at conformational epitopes, such as Abpl .
- Amyloid degradation is complete by 15 days and normal histology is restored by 21-25 days after administration of a single dose of anti-SAP antibody.
- Amyloid clearance requires an intact complement system and is absolutely dependent on macrophages.
- Patent No. US 7,045,499 B2 issued May 16, 2006.
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ES08874772T ES2729648T3 (en) | 2007-06-27 | 2008-12-30 | Combinations of agents that deplete SAP and anti-SAP antibodies |
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EP3025729A1 (en) * | 2010-03-03 | 2016-06-01 | Glaxo Group Limited | Antigen binding proteins specific for serum amyloid p component |
WO2011107480A1 (en) * | 2010-03-03 | 2011-09-09 | Glaxo Group Limited | Antigen binding proteins specific for serum amyloid p component |
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JP2013521252A (en) * | 2010-03-03 | 2013-06-10 | グラクソ グループ リミテッド | Antigen-binding protein specific for serum amyloid P component |
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