Classifications

• • 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
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2884—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD44
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
  • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70585—CD44
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/10—Musculoskeletal or connective tissue disorders
  • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
  • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints

Definitions

• the invention in some embodiments thereof relates to an antibody which specifically binds the novel CD44vRA variant, polynucleotides encoding same and, methods and kits using same for diagnosing and treating diseases associated with expression of the novel CD44vRA variant such as inflammatory, autoimmune, rheumatic and malignant diseases.
• the cell surface adhesion molecule, designated CD44 has been implicated in cell-cell and cell-matrix interactions, as well as in cell traffic and cell transendothelial migration.
• CD44 is a single chain molecule comprising a conserved amino terminal extracellular domain, a nonconserved membrane proximal region, a variable region expressing various combinations of variant exons, a conserved transmembrane spanning domain and a conserved cytoplasmic tail.
• the genomic sequence of mouse and human CD44 includes 5 constant exons at the 5' terminus, and 5 constant exons at the 3' end.
• the mouse CD44 gene includes 10 variant exons in the middle of the molecule, designated V 1 -V 10 , resulting in a total of 20 exons.
• the human CD44 gene comprises only 9 of these 10 variant exons (V 2 -V 1O ) thus comprising a total of 19 exons (Screaton, G.R., et al., 1992).
• These molecules are designated CD44 variants (CD44v). To date, a few dozen isoforms of CD44 are known.
• CD44s In standard CD44 (CD44s, SEQ ID NO: 17), constant exon number 5 is spliced directly to constant exon number 16 and therefore this molecule lacks the entire variable region. The resulting protein product is expressed predominantly on hematopoietic cells and therefore, this product is also known as hematopoietic CD44 (CD44H) or standard CD44 product (CD44s product, SEQ ID NO: 18).
• CD44H hematopoietic CD44
• CD44s product SEQ ID NO: 18
• keratinocyte CD44 the longest CD44 identified so far, exon V3 to exon VlO are inserted in tandem between the two constant regions of the molecule.
• the CD44 N-terminus contains the ligand binding site of the molecule.
• Hyaluronic acid is the principal ligand of CD44, but other extracellular matrix (ECM) components (e.g. laminin, collagen, fibronectin and chondroitin sulfate) as well as non-ECM constituents (mucosal vascular addressin, serglycin, osteopontin and class II invariant chain) can also interact with the CD44 receptor. Marked accumulation of CD44, and sometimes hyaluronic acid, is detected in areas of intensive cell migration and cell proliferation, as in wound healing, tissue remodeling, inflammation (including auto inflammation), morphogenesis and carcinogenesis.
• ECM extracellular matrix
• CD44 protein and variants thereof in autoimmune diseases.
• anti-CD44 monoclonal antibodies mAbs
• these mAbs are directed against the constant region of CD44 (and are thus designated anti-pan CD44 mAbs shared by all CD44 isoforms). Therefore, such mAbs may also block CD44 expressed on normal cells, which is required for migratory activity of immune and inflammatory cells engaged in microorganism eradication.
• Monoclonal Abs directed against various variant regions of CD44 have also been suggested as potential agents for treatment of autoimmune diseases.
• Herrlich et al, 1991 describe mAbs directed against metastasis-specific variants of CD44v surface protein of a rat pancreatic adenocarcinoma.
• Anti-CD44-monoclonal antibodies, which inhibit T-cell proliferation, were also provided for treatment of various autoimmune diseases (Aune, T.M. et al., 1992).
• Monoclonal antibodies specific for forms of CD44 containing exon v6 were also reported as being useful for diagnosing inflammatory diseases (Jalkanen, S. et al., 1995).
• Anti-CD44 mAbs which were injected into mice, were shown to inhibit or prevent infiltration of various lymphoma and carcinoma cells into their target organs.
• transfection of a variant CD44 isoform into non metastatic rat pancreatic adenocarcinoma cells conferred metastatic potential to these cells.
• US Patent Application No. 11/130,206 to the present inventors discloses antibodies which comprise an antigen recognition domain capable of specifically binding the CD44vRA polypeptide (SEQ ID NO:22) found in rheumatoid arthritis patients but not the standard (hematopoietic) CD44 (SEQ ID NO:18) or the CD44v3- vlO (SEQ ID NO:20). Additional background art includes Golan I., et al., 2007.
• an isolated antibody comprising an antigen recognition domain which comprises complementarity determining region (CDR) amino acid sequences as set forth in SEQ ID NOs:8, 9, 10, 14, 15 and 16.
• CDR complementarity determining region
• an isolated polynucleotide comprising a nucleic acid sequence encoding the antibody of the invention.
• a nucleic acid construct comprising the isolated polynucleotide of the invention and a promoter for regulating expression of the isolated polynucleotide in a host cell.
• a pharmaceutical composition comprising as an active ingredient the antibody of the invention and a pharmaceutically effective carrier or diluent.
• a method of detecting a presence of a CD44vRA polypeptide in a biological sample comprising contacting the biological sample with the antibody of the invention under conditions which allow immunocomplex formation, wherein a presence and/or a level above a predetermined threshold of the immunocomplex is indicative of the presence of the CD44vRA polypeptide in the biological sample.
• a kit for detecting a presence of a CD44vRA polypeptide in a biological sample comprising the antibody of the invention and at least one reagent suitable for detection of an immunocomplex between the antibody and the CD44vRA polypeptide.
• a method of diagnosing a disease associated with expression of a CD44vRA polypeptide comprising contacting a biological sample of a subject with the antibody of the invention under conditions which allow immunocomplex formation, wherein a presence or a level above a predetermined threshold of the immunocomplex is indicative of the disease in the subject.
• kits for diagnosing a disease associated with expression of a CD44vRA polypeptide comprising the antibody of the invention, at least one reagent suitable for detection of an immunocomplex and instructions for use in diagnosing the disease associated with expression of the CD44vRA polypeptide.
• a method of treating a disease associated with expression of a CD44vRA polypeptide comprising administering to a subject the antibody of the invention or the pharmaceutical composition of the invention, thereby treating the disease associated with expression of the CD44vRA polypeptide.
• a heavy chain of the antibody is encoded by the nucleic acid sequence set forth by SEQ ID NO:1.
• a light chain of the antibody is encoded by the nucleic acid sequence set forth by SEQ ID NO:3. According to some embodiments of the invention, the antibody being conjugated to a therapeutic moiety.
• the antibody being conjugated to a detectable moiety.
• the biological sample is selected from the group consisting of a synovial fluid sample, a blood sample, a bone marrow sample, a tissue biopsy sample and a lymph fluid sample.
• the disease associated with expression of a CD44vRA polypeptide is selected from the group consisting of a rheumatoid disease, an inflammatory disease, an autoimmune disease and a cancerous disease.
• the CD44vRA polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22.
• the rheumatoid disease is selected from the group consisting of rheumatoid arthritis (RA), psoriatic arthritis (PSA), spondyloarthropathy (SPA), juvenile rheumatoid arthritis (JRA), chronic Reiter's syndrome (CRS), palindrome rheumatism (PR), inflammatory bowel disease (IBD), infected rheumatic disease (IRD), seronegative rheumatoid arthritis (SRA), Gout, and non-diagnosed arthritis.
• RA rheumatoid arthritis
• PSA psoriatic arthritis
• SPA spondyloarthropathy
• JRA juvenile rheumatoid arthritis
• CRS chronic Reiter's syndrome
• PR palindrome rheumatism
• IBD inflammatory bowel disease
• IBD infected rheumatic disease
• SRA seronegative rheumatoid arthritis
• FIGs. IA-D depict the nucleic acid ( Figures IA, C) and amino acid ( Figures IB, D) sequences of the anti CD44vRA antibody according to some embodiments of the invention.
• Figure IA - nucleic acid sequence of the antibody heavy chain SEQ ID NO:1.
• CDRs complementarity determining regions
• CDRl SEQ ID NO:5
• CDR2 SEQ ID NO:6
• CDR3 SEQ ID NO:7
• Figure IB amino acid sequence of the antibody heavy chain (SEQ ID NO:2), note CDRl (SEQ ID NO:8), CDR2 (SEQ ID NO:9) and CDR3 (SEQ ID NO:10) in red
• Figure 1C nucleic acid sequence of the antibody light chain (SEQ ID NO:3), note CDRl (SEQ ID NO: 11), CDR2 (SEQ ID NO: 12) and CDR3 (SEQ ID NO: 13) in red
• Figure ID - amino acid sequence of the antibody light chain SEQ ID NO:4, note CDRl (SEQ ID NO: 14), CDR2 (SEQ ID NO: 15) and CDR3 (SEQ ID NO: 16) in red
• CDR landmarks are shown in green.
• the invention in some embodiments thereof relates to an antibody which specifically binds the novel CD44vRA variant, polynucleotides encoding same and methods and kits using same for diagnosing and treating diseases associated with expression of the novel CD44vRA variant such as inflammatory, autoimmune, rheumatic and malignant diseases.
• diseases associated with expression of the novel CD44vRA variant such as inflammatory, autoimmune, rheumatic and malignant diseases.
• the present inventors have uncovered a novel antibody which specifically binds the CD44vRA variant (SEQ ID NO:22) present in synovial fluid cells of rheumatoid arthritis (RA) but not other CD44 variants such as the CD44 standard (hematopoietic form; SEQ ID NO: 18) and CD44v3- vlO variant (SEQ ID NO:20).
• the novel CD44vRA antibody identified herein comprises complementarity determining regions (CDRs) amino acid sequences as set forth in SEQ ID NOs: 8, 9 and 10 for the antibody heavy chain and SEQ ID NOs: 14, 15 and 16 for the antibody light chain ( Figures IA-D).
• CDRs complementarity determining regions
• Such an antibody can be used for diagnosing and treating various diseases associated with expression of the CD44vRA variant such as rheumatic, inflammatory, autoimmune and cancerous diseases.
• an isolated antibody comprising an antigen recognition domain which comprises complementarity determining region (CDR) amino acid sequences as set forth in SEQ ID NOs:8, 9 and 10 for an heavy chain of the antibody and SEQ ID NOs: 14, 15 and 16 for a light chain of the antibody.
• CDR complementarity determining region
• antibody as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of binding to macrophages.
• These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab")2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab")2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy
• Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
• Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
• antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab * )2.
• This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
• a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
• an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
• cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
• Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Natl Acad. Sci. USA 69:2659-62 (1972O]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker.
• sFv single-chain antigen binding proteins
• the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
• the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
• Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); and US Patent No. 4,946,778, which is hereby incorporated by reference in its entirety.
• CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
• Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
• Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
• CDR complementary determining region
• donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
• Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
• Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
• the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
• the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
• Fc immunoglobulin constant region
• a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
• humanized antibodies are chimeric antibodies (US Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
• humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
• Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. MoI. Biol., 227:381 (1991); Marks et al., J. MoI. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al.
• human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in US Patent Nos.
• expressible polynucleotides e.g., a polynucleotide which comprises the nucleic acid sequence set forth by SEQ ID NO: 3 encoding the antibody light chain; a polynucleotide which comprises the nucleic acid sequence set forth by SEQ ID NO:1 encoding the antibody heavy chain
• expressible polynucleotides e.g., a polynucleotide which comprises the nucleic acid sequence set forth by SEQ ID NO: 3 encoding the antibody light chain
• SEQ ID NO:1 a polynucleotide which comprises the nucleic acid sequence set forth by SEQ ID NO:1 encoding the antibody heavy chain
• the isolated polynucleotide(s) can be included in a nucleic acid construct which includes a promoter sequence for regulating expression of the isolated polynucleotide in a host cell.
• the promoter can direct transcription of the polynucleotide in a host cell in a constitutive or inducible manner.
• constitutive promoters include the cytomegalovirus (CMV) and Rous sarcoma virus (RSV).
• the nucleic acid construct may further include sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors); transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal; a signal sequence for secretion of the peptide from a host cell in which it is placed; enhancer elements; other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA; sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
• prokaryotes eukaryotes, or preferably both (e.g., shuttle vectors)
• transcription and translation initiation sequence e.g., transcription and translation terminator and a polyadenylation signal
• the recombinant antibody of the invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
• standard protein purification techniques such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
• the antibody of some embodiments of the present invention can be used in vitro, ex vivo and in vivo in various therapeutic or diagnostic applications.
• the human or humanized antibody or antibody fragment will generally tend to be better tolerated immunologically than one of non human origin since non variable portions of non human antibodies will tend to trigger xenogeneic immune responses more potent than the allogeneic immune responses triggered by human antibodies which will typically be allogeneic with the individual. It will be preferable to minimize such immune responses since these will tend to shorten the half- life, and hence the effectiveness, of the antibody of the invention in the individual. Furthermore, such immune responses may be pathogenic to the individual, for example by triggering harmful inflammatory reactions.
• an antibody or antibody fragment of human origin or a humanized antibody
• a functional physiological effect for example an immune response against a target cell, activated by a constant region of the antibody or antibody fragment in the individual is desired.
• a functional physiological effect for example an immune response against a target cell, activated by a constant region of the antibody or antibody fragment in the individual is desired.
• Such applications particularly include those in which the functional interaction between a functional portion of the antibody or antibody fragment, such as an Fc region, with a molecule such as an Fc receptor or an Fc-binding complement component, is optimal when such a functional portion is, similarly to the Fc region, of human origin.
• the antibody of the invention which includes a constant region, or a portion thereof, of any of various isotypes may be employed.
• the isotype is selected so as to enable or inhibit a desired physiological effect, or to inhibit an undesired specific binding of the antibody of the present invention via the constant region or portion thereof.
• ADCC antibody-dependent cell mediated cytotoxicity
• NK natural killer
• the isotype will preferably be IgG
• the isotype will preferably be IgE
• the isotype will preferably be IgE or IgA.
• the antibody or antibody fragment includes a constant region or portion thereof capable of initiating the cascade.
• the antibody or antibody fragment may advantageously comprise a Cgamma2 domain of IgG or Cmu3 domain of IgM to trigger a Clq-mediated complement cascade.
• the antibody of the present invention will preferably not comprise a constant region, or a portion thereof, of the relevant isotype.
• the antibody or antibody fragment of the invention may be attached to any of various functional moieties (e.g., therapeutic or diagnostic moieties).
• the functional moiety can be conjugated by translationally fusing a polynucleotide encoding the antibody with a polynucleotide encoding the functional moiety.
• the functional moiety can be chemically conjugated (coupled) to the antibody of the invention, using any conjugation method known to one skilled in the art.
• a peptide can be conjugated to an antibody of interest, using a 3-(2-pyridyldithio)propionic acid Nhydroxysuccinimide ester (also called N-succinimidyl 3-(2pyridyldithio) propionate) (“SDPD”) (Sigma, Cat. No. P-
• the functional moiety is a therapeutic moiety.
• the therapeutic moiety can be, for example, a cytotoxic moiety, a toxic moiety
• cytokine moiety e.g., interleukin 2 (GenBank Accession Nos. CAA00227 and A02159), interleukin 10
• an antibody or antibody fragment attached to a toxin may be referred to in the art as an "immunotoxin".
• the functional moiety is a detectable moiety which enables specific detection of an immunocomplex formed between the CD44vRA polypeptide and the antibody or antibody fragment of the invention.
• the detectable moiety can be a label which is directly visualized [e.g., radioactive isotopes (e.g., ⁇ 125 Hodine), phosphorescent chemicals, chemiluminescent chemicals, fluorescent chemicals].
• radioactive isotopes e.g., ⁇ 125 Hodine
• phosphorescent chemicals e.g., phosphorescent chemicals, chemiluminescent chemicals, fluorescent chemicals.
• fluorescent molecules include, but are not limited to, phycoerythrin (PE), fluorescein isothiocyanate (FITC; e.g., GenBank Accession Nos. AAF22695 and AF098239), Cy-chrome, rhodamine, green fluorescent protein (GFP; e.g., GenBank Accession Nos. AAL33912 and AF435427), blue fluorescent protein (BFP), orange fluorescent protein (; e.g., GenBank Accession Nos. AAL33917 and AF435432), Texas red, PE-Cy5, and the like.
• PE phycoerythrin
• FITC fluorescein isothiocyanate
• Cy-chrome Cy-chrome
• rhodamine Cy-chrome
• GFP green fluorescent protein
• BFP blue fluorescent protein
• orange fluorescent protein e.g., GenBank Accession Nos. AAL33917 and AF435432
• Texas red PE-Cy5, and the like
• fluorophore selection methods of linking fluorophores to various types of molecules, such as an antibody or antibody fragment of the invention, and methods of using such conjugates to detect molecules which are capable of being specifically bound by antibodies or antibody fragments comprised in such immunoconjugates is available in the literature of the art [for example, refer to: Richard P. Haugland, "Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals 1992-1994", 5th ed., Molecular Probes, Inc. (1994); US Patent. No. 6,037,137 to Oncoimmunin Inc.; Hermanson, "Bioco ⁇ jugate Techniques", Academic Press New York, N.Y. (1995); Kay M. et al, 1995.
• the detectable moiety can be a member of a binding (affinity) pair, which is identifiable via its interaction with an additional member of the binding pair. It should be noted that any of members of the binding pair can be labeled.
• the member of the binding pair is an antigen (e.g., a tag such as a Myc of Histidine tag) which is identified by a corresponding labeled antibody.
• the member of the binding pair is a biotin molecule which binds to streptavidin or avidin molecules.
• the label is a fluorescent protein or an enzyme producing a colorimetric reaction.
• suitable enzymes include horseradish peroxidase (HPR), peroxidase (e.g., GenBank Accession Nos.
• beta-galactosidase e.g., GenBank Accession Nos.
• a detectable moiety which comprises a fluorophore or results in activation of a fluorescent molecule can be used in numerous fluorescence detection methods such as fluorescence activated flow cytometry (FACS), immunofluorescence, fluorescence resonance energy transfer (FRET), and the like.
• FACS fluorescence activated flow cytometry
• FRET fluorescence resonance energy transfer
• detectable moieties e.g., radioactive molecules or chemiluminescent chemicals
• RIA radioimmuno assay
• Detection methods which utilize enzymes include, but are not limited to, enzyme linked immunosorbent assay (ELISA which can be performed to detect the CD44vRA polypeptide in a solution), enzyme-linked chemiluminescence assay (for example, to detect the CD44vRA polypeptide in an electrophoretically separated protein mixture), and enzyme-linked immunohistochemical assay (for example, to detect the CD44vRA polypeptide in a fixed tissue).
• ELISA enzyme linked immunosorbent assay
• enzyme-linked chemiluminescence assay for example, to detect the CD44vRA polypeptide in an electrophoretically separated protein mixture
• enzyme-linked immunohistochemical assay for example, to detect the CD44vRA polypeptide in a fixed tissue.
• the antibody of the invention can be used to detect the CD44vRA polypeptide in a biological sample.
• a method of detecting a presence of a CD44vRA polypeptide in a biological sample is effected by contacting the biological sample with the antibody of the invention under conditions which allow immunocomplex formation, wherein a presence or a level above a predetermined threshold of the immunocomplex is indicative of the presence of the
• the biological sample can be any sample comprising cells or portion thereof (e.g., a portion of a cell membrane) which is derived from a subject. Examples include, but are not limited to a synovial fluid sample, a blood sample, a bone marrow sample, lymph fluid, a tissue biopsy sample such as synovial membrane tissue (SMTs) and splenocytes. It should be noted that for in vivo detection the biological sample may not be physically removed from the subject.
• SMTs synovial membrane tissue
• Procedures for obtaining biological samples from individuals are well known in the art. Such procedures include, but are not limited to, drawing blood (e.g., of a vein, umbilical cord), bone biopsy, lymph node biopsy, pleural biopsy, skin biopsy, thyroid biopsy, CT-guided biopsy, joint biopsy (e.g., of the knee), needle aspiration biopsy and breast biopsy.
• drawing blood e.g., of a vein, umbilical cord
• lymph node biopsy e.g., pleural biopsy
• skin biopsy e.g., thyroid biopsy
• CT-guided biopsy e.g., of the knee
• needle aspiration biopsy e.g., of the knee
• a joint biopsy (a sample of the joint lining, synovial membrane or fluid) can be obtained by introducing an incision in the joint and withdrawing fluid using a needle attached to a syringe.
• contacting is performed under conditions sufficient to form an immunocomplex (e.g. a complex between the antibody of the invention and the
• CD44vRA polypeptide such conditions (e.g., appropriate concentrations, buffers, temperatures, reaction times) as well as methods of optimize same are known to those skilled in the art.
• the immunocomplex can be formed and detected on a cell surface, e.g., a cell membrane or a portion thereof (such as a ruptured membrane) which includes the CD44vRA polypeptide. Determining a presence or level of the immunocomplex is dependent on the detectable moiety attached to the antibody, essentially as described hereinabove.
• CD44vRA polypeptide SEQ ID NO:22
• a disease associated with CD44vRA expression such as an inflammatory disease, allergic disease, rheumatoid disease, autoimmune disease, graft rejection disease or a cancer disease.
• subject refers to a mammal, preferably, a male or female human subject.
• disease associated with expression of the CD44vRA polypeptide refers to any disease characterized by expression (Le., presence, e.g., above a predetermined threshold) of the CD44vRA polypeptide in cells of the affected subject.
• the predetermined threshold is determined in comparison to a level of the CD44vRA polypeptide in a control biological sample such as of an unaffected subject (Le., a healthy subject-free of the disease, same subject being in remission or prior to disease initiation) or based upon literature data.
• the inflammatory disease is associated with hypersensitivity.
• hypersensitivity examples include, but are not limited to, Type I hypersensitivity, Type II hypersensitivity, Type III hypersensitivity, Type IV hypersensitivity, immediate hypersensitivity, antibody mediated hypersensitivity, immune complex mediated hypersensitivity, T lymphocyte mediated hypersensitivity and DTH.
• a non-limiting example of Type I or immediate hypersensitivity, is asthma.
• Type II and type III hypersensitivity include, but are not limited to, rheumatoid diseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn V. et al, Histol
• paraneoplastic neurological diseases cerebellar atrophy, paraneoplastic cerebellar atrophy, non-paraneoplastic stiff man syndrome, cerebellar atrophies, progressive cerebellar atrophies, encephalitis, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydenham chorea, Gilles de Ia Tourette syndrome, polyendocrinopathies, autoimmune polyendocrinopathies (Antoine JC. and Honnorat J. Rev Neurol (Paris) 2000 Jan; 156 (1):23); neuropathies, dysimmune neuropathies (Nobile-Orazio E.
• vasculitises necrotizing small vessel vasculitises, microscopic polyangiitis, Churg and Strauss syndrome, glomerulonephritis, pauci-immune focal necrotizing glomerulonephritis, crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris). 2000 May; 151 (3): 178); antiphospholipid syndrome (Flamholz R. et al, J Clin Apheresis 1999;14 (4): 171); heart failure, agonist-like beta-adrenoceptor antibodies in heart failure (Wallukat G. et al, Am J Cardiol.
• Type IV or T cell mediated hypersensitivity include, but are not limited to, rheumatoid diseases, rheumatoid arthritis (Tisch R, McDevitt HO. Proc Natl Acad Sci U S A 1994 Jan 18; 91 (2):437), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Datta SK., Lupus 1998; 7 (9):591), glandular diseases, glandular autoimmune diseases, pancreatic diseases, pancreatic autoimmune diseases, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647); thyroid diseases, autoimmune thyroid diseases, Graves' disease (Sakata S.
• delayed type hypersensitivity examples include, but are not limited to, contact dermatitis and drug eruption.
• T lymphocyte mediating hypersensitivity examples include, but are not limited to, helper T lymphocytes and cytotoxic T lymphocytes.
• helper T lymphocyte-mediated hypersensitivity examples include, but are not limited to, T h I lymphocyte mediated hypersensitivity and T h 2 lymphocyte mediated hypersensitivity.
• the disease is rheumatoid disease, including, but not limited to, rheumatoid arthritis (RA), psoriatic arthritis (PSA), spondyloarthropathy (SPA), juvenile rheumatoid arthritis (JRA), chronic Reiter's syndrome (CRS), palindrome rheumatism (PR), inflammatory bowel disease (IBD), infected rheumatic disease (IRD), seronegative rheumatoid arthritis (SRA), Gout, and non-diagnosed arthritis.
• RA rheumatoid arthritis
• PSA psoriatic arthritis
• SPA spondyloarthropathy
• JRA juvenile rheumatoid arthritis
• CRS chronic Reiter's syndrome
• PR palindrome rheumatism
• IBD inflammatory bowel disease
• IBD infected rheumatic disease
• SRA seronegative rheumatoid arthritis
• Autoimmune diseases include, but are not limited to, cardiovascular diseases, rheumatoid diseases, glandular diseases, gastrointestinal diseases, cutaneous diseases, hepatic diseases, neurological diseases, muscular diseases, nephric diseases, diseases related to reproduction, connective tissue diseases and systemic diseases.
• autoimmune cardiovascular diseases include, but are not limited to atherosclerosis (Matsuura E. et al, Lupus. 1998; 7 Suppl 2:S135), myocardial infarction (Vaarala O. Lupus. 1998; 7 Suppl 2:S132), thrombosis (Tincani A. et al, Lupus 1998; 7 Suppl 2:S107-9), Wegener's granulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik S. et al, Wien Klin Klin Klin Klinschr 2000 Aug 25; 112 (15-16):660), anti-factor VIII autoimmune disease (Lacroix-Desmazes S.
• autoimmune rheumatoid diseases include, but are not limited to rheumatoid arthritis (Krenn V. et al, Histol Histopathol 2000 JuI; 15 (3):791; Tisch R, McDevitt HO. Proc Natl Acad Sci units S A 1994 Jan 18; 91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al, Arthritis Res 2001; 3 (3): 189).
• autoimmune glandular diseases include, but are not limited to, pancreatic disease, Type I diabetes, thyroid disease, Graves' disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmune prostatitis and Type I autoimmune polyglandular syndrome diseases include, but are not limited to autoimmune diseases of the pancreas, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res Clin Pract 1996 Oct;34 Suppl:S125), autoimmune thyroid diseases, Graves' disease (Orgiazzi J.
• autoimmune gastrointestinal diseases include, but are not limited to, chronic inflammatory intestinal diseases (Garcia Herola A. et al, Gastroenterol Hepatol. 2000 Jan; 23 (1):16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16; 138 (2):122), colitis, ileitis and Crohn's disease.
• autoimmune cutaneous diseases include, but are not limited to, autoimmune bullous skin diseases, such as, but are not limited to, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.
• autoimmune hepatic diseases include, but are not limited to, hepatitis, autoimmune chronic active hepatitis (Franco A. et al, Clin Immunol Immunopathol 1990 Mar; 54 (3):382), primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov; 91 (5):551; Strassburg CP. et al, Eur J Gastroenterol Hepatol. 1999 Jun; 11 (6):595) and autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug; 33 (2):326).
• autoimmune neurological diseases include, but are not limited to, multiple sclerosis (Cross AH. et al, J Neuroimmunol 2001 Jan 1; 112 (1-2):1), Alzheimer's disease (Oron L. et al, J Neural Transm Suppl. 1997; 49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999; 18 (l-2):83; Oshima M. et al, Eur J Immunol 1990 Dec; 20 (12):2563), neuropathies, motor neuropathies (Kornberg AJ. J Clin Neurosci.
• autoimmune muscular diseases include, but are not limited to, myositis, autoimmune myositis and primary Sjogren's syndrome (Feist E. et al, Int Arch Allergy Immunol 2000 Sep;123 (1):92) and smooth muscle autoimmune disease (Zauli D. et al, Biomed Pharmacother 1999 Jun;53 (5-6):234).
• autoimmune nephric diseases include, but are not limited to, nephritis and autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug;l (2):140).
• autoimmune diseases related to reproduction include, but are not limited to, repeated fetal loss (Tincani A. et al, Lupus 1998;7 Suppl 2:S107-9).
• autoimmune connective tissue diseases include, but are not limited to, ear diseases, autoimmune ear diseases (Yoo TJ. et al, Cell Immunol 1994 Aug;157 (1):249) and autoimmune diseases of the inner ear (Gloddek B. et al, Ann N Y Acad Sci 1997 Dec 29;830:266).
• autoimmune systemic diseases include, but are not limited to, systemic lupus erythematosus (Erikson J. et al, Immunol Res 1998; 17 (l-2):49) and systemic sclerosis (Renaudineau Y. et al, Clin Diagn Lab Immunol. 1999 Mar;6 (2): 156); Chan OT. et al, Immunol Rev 1999 Jun;169:107).
• Infectious diseases include, but are not limited to, chronic infectious diseases, sub-acute infectious diseases, acute infectious diseases, viral diseases, bacterial diseases, protozoan diseases, parasitic diseases, fungal diseases, mycoplasma diseases and prion diseases.
• Graft rejection diseases include, but are not limited to, graft rejection, chronic graft rejection, sub acute graft rejection, hyper acute graft rejection, acute graft rejection and graft versus host disease.
• Allergic diseases include, but are not limited to, asthma, hives, urticaria, pollen allergy, dust mite allergy, venom allergy, cosmetics allergy, latex allergy, chemical allergy, drug allergy, insect bite allergy, animal dander allergy, stinging plant allergy, poison ivy allergy and food allergy.
• Cancerous diseases include but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Particular examples of cancerous diseases but are not limited to: Myeloid leukemia such as Chronic myelogenous leukemia. Acute myelogenous leukemia with maturation. Acute promyelocytic leukemia, Acute nonlymphocytic leukemia with increased basophils, Acute monocytic leukemia. Acute myelomonocytic leukemia with eosinophilia; Malignant lymphoma, such as Burkitt's
• Non-Hodgkin's Lymphoctyic leukemia, such as Acute lymphoblastic leukemia,
• Benign Meningioma Mixed tumors of salivary gland, Colonic adenomas; Adenocarcinomas, such as Small cell lung cancer, Kidney, Uterus, Prostate, Bladder,
• Rhabdomyosarcoma (alveolar), Extraskeletal myxoid chondrosarcoma, Ewing's tumor; other include Testicular and ovarian dysgerminoma, Retinoblastoma, Wilms 1 tumor,
• Neuroblastoma Malignant melanoma, Mesothelioma, breast, skin, prostate, and ovarian.
• the antibody of the invention can be used to treat any of the above mentioned diseases which are associated with expression of the CD44vRA polypeptide.
• a method of treating a disease associated with expression of a CD44vRA polypeptide is effected by administering to a subject the antibody of the invention.
• treating refers to inhibiting or arresting the development of a disease, disorder or condition and/or causing the reduction, remission, or regression of a disease, disorder or condition.
• Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a disease, disorder or condition, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a disease, disorder or condition.
• the antibody of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
• a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
• the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
• active ingredient refers to the antibody of the invention accountable for the biological effect.
• physiologically acceptable carrier refers to the phrases "physiologically acceptable carrier" and
• pharmaceutically acceptable carrier refers to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
• An adjuvant is included under these phrases.
• excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in
• Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
• a tissue region of a patient e.g., joint tissue
• compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
• compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
• the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
• physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
• penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
• the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
• Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
• Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
• Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
• disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
• Dragee cores are provided with suitable coatings.
• suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
• compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
• the compositions may take the form of tablets or lozenges formulated in conventional manner.
• the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
• a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
• the dosage unit may be determined by providing a valve to deliver a metered amount.
• Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
• compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
• Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
• the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
• the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
• a suitable vehicle e.g., sterile, pyrogen-free water based solution
• the pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
• compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (Le., the antibody of the invention) effective to prevent, alleviate or ameliorate symptoms of a disease (e.g., inflammatory or rheumatoid disease) or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
• the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
• a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
• Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
• the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
• the dosage may vary depending upon the dosage form employed and the route of administration utilized.
• the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
• Dosage amount and interval may be adjusted individually to provide levels of the active ingredient which are sufficient to induce or suppress the biological effect
• MEC minimum effective concentration
• dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
• compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
• the active ingredient is provided to the individual with additional active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself.
• measures e.g., dosing and selection of the complementary agent
• Administration of such combination therapy can be simultaneous, such as in a single capsule having a fixed ratio of these active agents, or in multiple capsules for each agent.
• the active ingredient e.g., the antibody of the invention
• NSAID nonsteroidal anti-inflammatory drugs
• DARDS disease- modifying antirheumatic drugs
• corticosteroids analgesics
• Fibromyalgia medications chemotherapeutic agents and others.
• compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
• the pack may, for example, comprise metal or plastic foil, such as a blister pack.
• the pack or dispenser device may be accompanied by instructions for administration.
• the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
• Such notice for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
• compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
• the antibody of the invention which is described hereinabove for detecting the CD44vRA polypeptide may be included in a diagnostic kit/article of manufacture preferably along with appropriate instructions for use in diagnosing and/or assessing diseases associated with expression of the CD44vRA polypeptide and optionally labels indicating FDA approval for use in diagnosing and/or assessing diseases associated with expression of the CD44vRA polypeptide.
• kit can include, for example, at least one container including at least one of the above described diagnostic agents (e.g., the antibody of the invention, or an immunoconjugate thereof) and an imaging reagent packed in another container (e.g., enzymes, secondary antibodies, buffers, chromogenic substrates, fluorogenic material).
• the kit may also include appropriate buffers and preservatives for improving the shelf- life of the kit.
• CD44vRA peptide SNPEVLLQTTTRMTADVDRNGTTAYEGNWN
• SEQ ID NO:23 obtained from Corixa (Seattle, WA), and 100 ⁇ g/ml soluble CD44vRA (SEQ ID NO:22) produced as described in U.S. Pat. Appl. No. 11/130,206, both emulsified in complete Freund's adjuvant (CFA) (Sigma) and were used to immunize subcutaneously or intramuscularly 8-week-old female C75BL/6 mice. The immunization was repeated on days 14 and 28 and two weeks later the mice were bled and their sera were tested by flow cytometry for their ability to bind to Namalwa cells expressing CD44.
• CFA complete Freund's adjuvant
• mice with highest polyclonal anti-CD44 antibody titers were selected and boosted intraperitoneally (i.p.) with 10 8 Namalwa-CD44vRA cells. After 72 hours, spleen cells from the mice were harvested and fused with SP 2/0 myeloma cells according to Kohler and Milstein [Kohler, G., and Milstein C. Nature 256:495-497 (1975)].
• the supernatants from isolated hybridoma clones were screened by flow cytometry for their ability to bind to Namalwa-Neo, Namalwa-CD44v3-10 or Namalwa-CD44vRA cells.
• Hybridoma whose supernatants bound selectively or preferentially to Namalwa-CD44vRA were cloned by limiting dilution and then re-cloned for additional three cycles.
• the isolated hybridoma were maintained in enriched RPMI 1640 containing HAT media supplement and 20 % FBS.
• the isotype of the CD44vRA-positive hybridoma supernatant was determined by ELISA using Clonotype System-HRP (Southern Biotechnology Associates, Inc.). Determination of antibody heavy (VH) and light (VL) chain CDRs -
• Hybridoma cells were centrifuged (30 ml of hybridoma cells centrifuged for 10 minutes at 1200 rpm at 4 0 C).
• RNA was extracted using TRiReagent (Sigma) according to manufacturer instructions.
• TRiReagent Sigma
• RNA was quantified by nanodrop and integrity was checked by applying 3 ⁇ l from each sample on agarose gel.
• the isolated RNA was subjected to DNase I treatment [4 ⁇ g of RNA was taken for DNase I (Sigma) treatment according to the manufacturer instructions], followed by reverse transcription (using M-MLV, Reverse Transcriptase and random primers).
• PCR amplification of IgG was performed using Ig-Primer Sets kit (Novagen) according to manufacturer protocol.
• PCR conditions were denaturation at 94 °C for 5 minutes, 35 cycles, each including a denaturation at 94 0 C for 1 minute, annealing at 53 °C for 1 minute and elongation at 72 0 C for 1 minute, followed by 72 0 C for 5 minutes.
• Cloning of the PCR products of the heavy (HC) and light (LC) chains into pGEM-luc vector (Promega) was into the pGEM (ampR) vector (Promega).
• the plasmid DNA was purified (Pure YieldTM Plasmid Midiprep, Promega) from transformed bacteria and concentration determined by UV spectroscopy.
• the constructs (each containing 10 ⁇ g DNA) were sequenced using ABI37000 cycle sequencing.
• CD44vRA Production of anti-CD44vRA mAb - CD44vRA, CD44v3-vlO (derived from human keratinocytes) and CD44s (derived from HeLa cells) cDNAs were transfected into CD44-negative Namalwa Burkett lymphoma cell line.
• CD44 transfectants expressing high levels of CD44s, as well as CD44v3-vlO and CD44vRA transfectants expressing equal levels of v6-containing CD44 variant were selected.
• the transfectants were designated Namalwa-CD44s, Namalwa-CD44v3-vlO and Namalwa-CD44vRA, respectively.
• Namalwa cells transfected with an empty vector were designated Namalwa-Neo.
• C57BL/6 mice were immunized with soluble CD44vRA, incorporated into CFA and challenged with Namalwa-CD44vRA cells as described in Materials and Methods. Splenocytes from mice showing polyclonal anti-CD4vRA antibodies in their serum were fused with SP2/0 myeloma cells.
• Hybridoma cell clones were selected according to the ability of their supernatants to bind to Namalwa-CD44vRA (expressing the polypeptide set forth by SEQ ID NO:22), but not to Namalwa-CD44v3-vlO (expressing the polypeptide set forth by SEQ ID NO:20), Namalwa-CD44s (expressing the polypeptide set forth by SEQ ID NO: 18) or to Namalwa-Neo, as indicated by flow cytometry.
• Clones and sub-clones were established from positive hybridoma cell colonies, and they were stable in culture for over 8 months.
• Anti-CD44vRA mAbs from supernatants of positive hybridomas were purified on G protein column.

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