WO2004101511A2 - Anticorps anti-ip-10 et methodes d'utilisation pour le traitement d'affections intestinales inflammatoires - Google Patents

Anticorps anti-ip-10 et methodes d'utilisation pour le traitement d'affections intestinales inflammatoires Download PDF

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WO2004101511A2
WO2004101511A2 PCT/US2004/014507 US2004014507W WO2004101511A2 WO 2004101511 A2 WO2004101511 A2 WO 2004101511A2 US 2004014507 W US2004014507 W US 2004014507W WO 2004101511 A2 WO2004101511 A2 WO 2004101511A2
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antibody
antibodies
seq
amino acid
acid sequence
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WO2004101511A3 (fr
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Balaji Balasa
Naoya Tsurushita
Nicolas Landolfi
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PDL Biopharma Inc
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Protein Design Labs Inc
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Priority to DE602004031772T priority Critical patent/DE602004031772D1/de
Priority to EP04800979A priority patent/EP1708961B1/fr
Priority to AT04800979T priority patent/ATE501173T1/de
Priority to PCT/US2004/037600 priority patent/WO2005060457A2/fr
Publication of WO2004101511A2 publication Critical patent/WO2004101511A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/461—Igs containing Ig-regions, -domains or -residues form different species
    • C07K16/464—Igs containing CDR-residues from one specie grafted between FR-residues from another
    • C07K16/465—Igs containing CDR-residues from one specie grafted between FR-residues from another with additional modified FR-residues
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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 applies to the technical fields of immunology and disease treatment.
  • it concerns antibodies against chemokines and methods of using thereof for the treatment of inflammatory bowel diseases.
  • chemoattractant cytokines also known as chemokines, comprises the most diverse and largest subset of cytokines identified to date. Chemokines are characterized by their capacity to induce the directional migration and activation of leukocytes, as well as other somatic cell types, and thus play major roles in acute and chronic inflammation (William, P. et al., Fundamental Immunology, 4 th Edition, Lippincott-Raven pp. 791-794 (1990)).
  • Chemokines also promote humoral and cell-mediated immune reactions; regulate cell adhesion, angiogenesis, leukocyte trafficking, and homing; and contribute to lymphopoiesis and hematopoiesis.
  • Chemokines are produced by a wide variety of leukocytes and other cell types in response to irritants, polyclonal stimulants, antigens, and endogenous cytokines.
  • a variety of chemokines have been detected at local inflammatory sites in a great number of disease states. Chemokines play a central role in host defense against infectious organisms, including HIV-1.
  • chemokines participate in the pathogenesis of diverse conditions such as reperfusion injuries, including strokes, acute respiratory distress syndrome (ARDS), immune complex-induced glomerulonephritis, atherosclerosis, and autoimmune reactions (William, P. et al., Fundamental Immunology, 4 Edition, Lippincott-Raven pp. 791-794 (1990)).
  • ARDS acute respiratory distress syndrome
  • autoimmune reactions William, P. et al., Fundamental Immunology, 4 Edition, Lippincott-Raven pp. 791-794 (1990)).
  • IP-10 (CXCLIO) is a chemokine that is induced by interferon ⁇ and is the ligand for CXCR3.
  • CXCR3 is expressed on human activated/memory T cells, which are a subset of peripheral blood B cells, and activated B cells (Qin et al., J. Clin. Invest. 101:746 (1998)).
  • Over-expression of IP-10 at the site of inflammation potently attracts CDCR3+ activated T cells and antibody secreting plasma cells.
  • elevated levels of IP-10 have been found in a number of autoimmune diseases, including multiple sclerosis (MS) (Balashov et al., Proc. Natl. Acad.
  • the present invention provides for potent neutralizing anti-IP-10 antibodies comprising the disclosed amino acid sequences and the methods of using an antagonist of IP-10 for the treatment of inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis.
  • IBD inflammatory bowel diseases
  • the present invention is directed to an antibody that binds to a human IP-10 and neutralizes at least one biological activity of a human IP-10.
  • said antibody is capable of preventing or reducing severity of at least one symptom of an inflammatory bowel disease.
  • the antibody is a humanized antibody.
  • the present invention is also directed to an antibody or an antigen-binding fragment thereof, wherein said antibody comprises an amino acid sequence of any one of SEQ ID NOs: 3-10.
  • the present invention is also directed to an antibody or antigen-binding fragment thereof, wherein said antibody comprises an amino acid sequence sharing at least 85% identity with any one of SEQ ID NOs: 3-10.
  • the present invention is also directed to a humanized antibody or an antigen- binding fragment thereof, wherein said antibody comprises an amino acid sequence of any one of SEQ ID NOs: 13-14.
  • the present invention is also directed to a humanized antibody or antigen- binding fragment thereof, wherein said antibody comprises an amino acid sequence sharing at least 85% identity with any one of SEQ ID NOs: 13-14.
  • the present invention is also directed to a heavy chain complementarity determining region (CDR) of an antibody comprising an amino acid sequence of SEQ ID NOs: 5, 6, or 7.
  • CDR complementarity determining region
  • the antibody comprising the amino acid sequence of SEQ ID NOs: 5, 6, or 7 is a humanized antibody.
  • the present invention is also directed to a light chain complementarity determining region (CDR) of an antibody comprising an amino acid sequence of SEQ ID NOs: 8, 9, or 10.
  • CDR complementarity determining region
  • the antibody comprising the amino acid sequence of SEQ ID NOs: 8, 9, or 10 is a humanized antibody.
  • the present invention is also directed to a humanized antibody or chimeric antibody comprising the heavy chain CDR and/or a light chain CDR described herein.
  • the present invention is also directed to a polypeptide comprising any one of
  • the present invention is also directed to a polynucleotide molecule comprising anucleotide acid sequence of SEQ ID NOs: 11, 12, 51 or 53.
  • the present invention is also directed to a method of inhibiting chemotaxis of lymphocyte cells comprising contacting said cells with antibodies or antigen-binding fragments of the antibodies described herein.
  • the present invention is also directed to a method of reducing severity of at least one symptom of inflammatory bowel disease in a subject in need thereof comprising administering to said subject an effective amount of an antagonist of IP- 10, preferably, the antagonists are the antibodies described herein.
  • the present invention is also directed to a method of detecting an inflammatory bowel disease in a patient comprising: a) isolating a colon sample from the patient; b) contacting cells of said colon sample with an anti-IP-10 antibody; c) contacting normal colon cells with an anti-IP-10 antibody; and d) detecting and comparing the difference of the expression of IP-10 in said colon sample cells with said normal colon cells.
  • Figure 1 A depicts the amino acid sequences of the mature heavy chain variable region of ADP 13 (SEQ ID NO: 3) and the mature light chain variable region of AIP 13 (SEQ ID NO: 4).
  • the CDR's are underlined and in bold.
  • Figure IB depicts the nucleotide sequences encoding the mature heavy chain variable region of AIP 13 (SEQ ID NO: 11) and the mature light chain variable region of AIP 13 (SEQ ID NO: 12).
  • Figure 2A depicts the over-expression of IP-10 in colons of IBD mouse models.
  • Figure 2B depicts the over-expression of IP-10 in IBD patients (confirmed by Real-Time PCR)
  • Figure 3 A depicts reactivity of the mouse anti-IP-10 monoclonal antibodies against hIP-10.
  • Figure 3B depicts anti-IP-10 antibody mediated inhibition of IP-10 binding to CXCR3.
  • Figure 4 depicts the inhibition of chemotaxis of PHA blasts to IP-10 by the AIP antibodies in chemotaxis assays.
  • Figure 5 depicts the construction and amplification of heavy and light chain variable regions.
  • Figure 6 depicts vector pHuAIP13-IgGl.
  • Figure 7 depicts the comparison of the amino acid sequence of the heavy chain of MuAlP13 (SEQ ID No:3), HuAIP13 (SEQ ID No:13) and DP-3/JH6 (SEQ ID No: 15).
  • Figure 8 depicts the comparison of the amino acid sequence of the light chain of MuAIP13 (SEQ ID No:4), HuAEP13 (SEQ ID No:14) and DPK1/JK6 (SEQ ID No: 16).
  • Figure 9 depicts the DNA sequence (SEQ ID No: 17) and deduced amino acid sequence (SEQ ID No: 18) of the humanized NL mini-exon of HuAIP13.
  • Figure 10 depicts the D ⁇ A sequence (SEQ ID No: 19) and deduced amino acid sequence (SEQ ID No:20) of the humanized NH mini-exon of HuAIP13.
  • Figure 11 depicts competition ELISA results that show the affinity of
  • Figure 12 depicts Primers Hl-10 for the synthesis of the humanized heavy chain variable region (SEQ ID ⁇ Os: 21-30, respectively).
  • Figure 13 depicts Primers Ll-10 for the synthesis of the humanized light chain variable region (SEQ ID ⁇ Os:31-40, respectively).
  • Figure 14A depicts the amino acid sequences of the mature heavy chain variable region of AIP 12 (SEQ ID NO: 41) and the mature light chain variable region of AIP 12 (SEQ ID NO: 42). The CDR's are underlined and in bold.
  • Figure 14B depicts the nucleotide sequences encoding the mature heavy chain variable region of AIP 12 (SEQ ID NO: 43) and the mature light chain variable region of AIP 12 (SEQ ID NO: 44).
  • Figure 15 depicts vector pHuAIP12-IgGl.
  • Figure 16 depicts the comparison of the amino acid sequence of the heavy chain of MuAIP12 (SEQ ID No:41), HuAIP12 (SEQ ID No:45) and DP-3/JH6 (SEQ ID No:15).
  • Figure 17 depicts the comparison of the amino acid sequence of the light chain of MuAIP12 (SEQ ID No:42), HuAIP12 (SEQ ID No:46) and DPK1/JK6 (SEQ ID No:16).
  • Figure 18 depicts the DNA sequence (SEQ ID No:47) and deduced amino acid sequence (SEQ ID No:48) of the humanized NL mini-exon of HuAIP12.
  • Figure 19 depicts the D ⁇ A sequence (SEQ ID ⁇ o:49) and deduced amino acid sequence (SEQ ID No: 50) of the humanized NH mini-exon of HuAIP12.
  • Figure 20 depicts competition ELISA results that show the affinity of HuAIP12 to human IP-10.
  • Figure 21 depicts Primers 12H1-10 for the synthesis of the humanized heavy chain variable region (SEQ ID ⁇ Os: 51-60, respectively).
  • Figure 22 depicts the Primers 12L1-10 for the synthesis of the humanized light chain variable region (SEQ ID ⁇ Os:61-70, respectively).
  • Figure 23 A depicts the binding of murine AIP 13 and AIP 12 to human recombinant IP-10.
  • Figure 23B depicts the binding of HuAIP13 and HuATP12 to human recombinant IP-10.
  • Figure 24 depicts the AIP12, AIP13, HuAff 12 and HuAIP13 mediated inhibition of IP-10 binding to CXCR3.
  • Figure 25A depicts the AIP12, ATP13, HuAIP12 and HuAIP13 inhibition of recombinant human IP-10 mediated chemotaxis.
  • Figure 25B depicts the AIP 12, AIP13, HuAJP12 and HuAIP13 inhibition of native human IP-10 mediated chemotaxis.
  • Figure 25C depicts the AIP12, AIP13, HuAIP12 and HuAIP13 inhibition of cynomolgous IP-10 mediated chemotaxis.
  • Figure 26 A depicts the competition of AIP 12, HuAEP12 and HuAIP13 with biotinylated AJP13.
  • Figure 26B depicts the competition of AIP12, ATP13, HuAJP12 and HuAIP13 with biotinylated ATP 12. Both assays indicate that HuAIP12 exhibits higher relative affinity in IP-10 binding than HuATP13.
  • Figure 27 depicts that the disease results in weight loss in a mouse model of
  • antibody refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma (IgG- . , IgG 2 , IgG 3 , IgG 4 ), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable N region genes (as indicated below, there are V genes for both H -heavy- and L -light- chains).
  • Full-length immunoglobulin "light chains” (about 25 Kd or 214 amino acids) are encoded by a variable region gene, N- kappa or N-lambda, at the ⁇ H2-terminus (about 110 amino acids) and, respectively, a kappa or lambda constant region gene at the COOH-terminus.
  • Full-length immunoglobulin "heavy chains” (about 50 Kd or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).
  • immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions.
  • immunoglobulins may exist in a variety of other forms including, for example, Fv, Fab, and (Fab') 2 , as well as bifunctional hybrid antibodies (e.g., Lanzavecchia et al, Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g., Huston et al., Proc. Natl.
  • humanized antibody or “humanized immunoglobulin” refers to an immunoglobulin comprising a human framework, at least one and preferably all complementarity determining regions (CDRs) from a non-human antibody, and in which any constant region present is substantially identical to a human immunoglobulin constant region, i.e., at least about 85-90%, preferably at least 95% identical.
  • CDRs complementarity determining regions
  • all parts of a humanized immunoglobulin, except possibly the CDRs are substantially identical to corresponding parts of one or more native human immunoglobulin sequences. See, e.g. Queen et al., U.S. Patent Nos. 5,5301,101; 5,585,089; 5,693,762; and 6,180,370 (each of which is incorporated by reference in its entirety).
  • chimeric antibody refers to an antibody in which the constant region comes from an antibody of one species (typically human) and the variable region comes from an antibody of another species (typically rodent).
  • an effective amount of an antagonist, an antibody, a drug or pharmacologically active agent or pharmaceutical formulation is meant a sufficient amount of the antagonist, antibody, drug, agent or formulation to provide the desired effect.
  • a “subject,” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human.
  • differentiated means that a polypeptide or a mRNA encoding such a polypeptide is expressed in a type of cells or tissues that are in a diseased stage at a higher level than in the same type of cells that are in a non- diseased stage, for example, 30% more, 50% more, 100% more, 200% more, 300% more, or 400% more.
  • IP-10 is differentially expressed in diseased colon tissue of a patient suffering from an inflammatory bowel disease
  • the protein or mRNA of IP-10 will express at a higher level in the diseased colon cells of such a patient compared to the normal colon cells of the same patient.
  • the protein expression level can be measured by the standard technology known in the art, such as western blot, ELISA, immunohistological staining, or FACS analysis.
  • the mRNA expression level can be measured by the standard technology known in the art, such as, Real Time PCR, RT-PCR, Northern blot, RNA protection assay, in situ hybridization, or Taqman ® expression assay.
  • epitope determinants usually consist of active surface groupings of molecules such as amino acids or GAG side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • Two antibodies are said to bind to substantially the same epitope of a protein if amino acid mutations in the protein that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody, and/or if the antibodies compete for binding to the protein, i.e., binding of one antibody to the protein reduces or eliminates binding of the other antibody.
  • the determination of whether two antibodies bind substantially to the same epitope is accomplished by the methods known in the art, such as a competition assay.
  • a control antibody for example, one of the anti-IP-10 antibodies described herein
  • any test antibody one may first label the control antibody with a detectable label, such as biotin, enzymatic, radioactive label or fluorescence label, to enable the subsequent identification.
  • a detectable label such as biotin, enzymatic, radioactive label or fluorescence label
  • the antagonists of IP-10 include any molecules that directly or indirectly counteract, reduce, or inhibit IP-10 biological activities.
  • the antagonists of IP- 10 compete or even block the binding of IP-10 to their receptors, such as CXCR3.
  • the antagonists should counteract, reduce, or inhibit the chemoattractive activities of 3P-10, for example, the recruitment of lymphocytes to the inflammatory sites.
  • the antagonists directly interact with IP-10.
  • the antagonists are polypeptides. More preferably, the polypeptides bind to IP-10, and even more preferably, the antagonists are antibodies or antibody fragments that bind to IP-10 and inhibit at least one biological activity of IP- 10.
  • the antagonists can also be any polypeptides or peptides that inhibit IP-10 activities but do not directly interact with IP-10.
  • these antagonists block the binding of IP-10 to its receptors such as CXCR3.
  • the antagonists can be mutated IP-10 molecules, such as dominant-negative mutants derived from a wild-type IP-10 by terminal truncations or amino acid substitutions.
  • mutated IP-10 molecules retain the binding ability to the signaling molecules of IP-10 but lose the ability of triggering the downstream signaling transduction of IP-10. Therefore, the mutated IP-10 molecules can compete with the wild-type IP-10 and thus block the activities of the wild-type IP-10.
  • the terminal truncations and amino acid substitutions can be made by the standard mutagenesis and molecular cloning techniques.
  • the mutated IP-10 molecules can be administered into the target cells by standard delivery means known in the art, such as, lipid or viral transfections.
  • the antagonists interact with and regulate the up-stream or down-stream components of the IP-10 signaling pathway and indirectly reduce or enhance the activities of IP-10. It is known that IP-10 activities are induced by interferon ⁇ and act through receptors which belong to a superfamily of seven transmembrane spanning G-protein coupled receptors (Murphy, P. M., Annu. Rev. Immunol., 12: 593-633 (1994); Gerard, C. and N. P. Gerard, Curr.
  • G-protein coupled (serpentine) receptors comprises a large group of integral membrane proteins, containing seven transmembrane-spanning regions.
  • the receptors are coupled to G proteins, which are heterotrimeric regulatory proteins capable of binding GTP and mediating signal transduction from coupled receptors, for example, by the production of intracellular mediators.
  • G proteins which are heterotrimeric regulatory proteins capable of binding GTP and mediating signal transduction from coupled receptors, for example, by the production of intracellular mediators.
  • any molecules capable of regulating this pathway can be candidate antagonists, including, but not limited to, G-protein antagonists known in the art.
  • Yeast two-hybrid and variant screens offer preferred methods for identifying endogenous additional IP-10 interacting proteins (Finley, R. L. et al. in DNA Cloning-Expression Systems: A Practical Approach, eds.
  • Mass spectrometry is an alternative method for the elucidation of protein complexes (reviewed in, e. g., Pandley, A. and Mann, M., Nature 405: 837-846 (2000); Yates, J.R. 3rd, Trends Genet. 16: 5-8 (2000)).
  • the antagonists can inhibit the mRNA and/or protein expression of IP-10.
  • the IP- 10 expression can be regulated at the level of transcription, such as by regulators of transcription factors of IP-10, or at the level of mRNA splicing, or at translation or post-translation level.
  • the antagonists can also be nucleic acids, including, but not limited to, anti- sense nucleic acids of the nucleic acid sequence encoding part or full of IP-10 or having substantial sequence similarity of IP-10.
  • the DNA sequence of IP-10 is known in the art and is disclosed herein.
  • anti-sense nucleic acid probes of DNA of IP-10, and the optimal conditions of the anti-sense blocking can be developed by using the related techniques known to a skilled artisan in the field of molecular biology.
  • the nucleic acid reagent may belong to the class of short interfering RNA or siRNA.
  • the antagonists of the present invention also include small molecules, which often modulate the functions of proteins with enzymatic functions and/or proteins containing protein interaction domains.
  • Small molecule compounds are typically organic, non-peptide molecules, having a molecular weight less than 10,000, preferably less than 5,000, more preferably less than 1,000, and most preferably less than 500 daltons.
  • This class of antagonists includes chemically synthesized molecules, for instance, compounds from combinatorial chemical libraries. Synthetic compounds may be rationally designed or identified based on known or inferred properties of the IP-10 protein or may be identified by screening compound libraries. Alternative appropriate antagonists of this class are natural products, particularly secondary metabolites from organisms such as plants or fungi, which can also be identified by screening compound libraries for IP-10-modulating activities.
  • the antibodies against JP-10 of the present invention may be in a polyclonal or monoclonal form and should bind to at least one epitope of IP-10 derived from any species, such as human, rat, mouse, chicken, rabbit, preferably a human IP-10.
  • the antibodies should bind to a) a wild-type full-length IP-10 protein, or b) a functionally active fragment or derivative thereof.
  • the amino acid sequence of the full-length wild-type human JJP-10 is presented in SEQ ID NO: 1 (MNQTAILICC LIFLTLSGIQ GVPLSRTVRC TCISISNQPV NPRSLEKLEI IPASQFCPRV EIIATMKKKG EKRCLNPESK AK LLKANS KERSKRSP).
  • a "functionally active" IP-10 fragment or derivative exhibits one or more functional activities associated with the full-length, wild-type IP- 10 protein, such as antigenic or immunogenic activity, ability to bind natural cellular substrates, etc.
  • JJP-10 proteins, derivatives and fragments can be assayed by various methods known to one skilled in the art (Coligan et al., eds., Current Protocols in Protein Science, John Wiley & Sons, Inc., Somerset, New Jersey (1998)).
  • functionally active fragments also include those fragments that comprise one or more structural domains of an IP-10 polypeptide, such as a binding domain. Protein domains can be identified using the PFAM program (Bateman A., et al., Nucleic Acids Res. 27: 260-2 (1999); http://pfam.wustl.edu).
  • IP-10 polypeptide derivatives typically share a certain degree of sequence identity or sequence similarity with SEQ ID NO: 1 or a fragment thereof.
  • IP-10 derivatives can be produced by various methods known in the art. The manipulations that result in their production can occur at the gene or protein level. For example, a cloned IP-10 gene sequence can be cleaved at appropriate sites with restriction endonuclease(s) (Wells et al., Philos. Trans. R. Sot. London SerA 317: 415 (1986)) followed by further enzymatic modification, if desired, then isolated, and ligated in vitro, and expressed to produce the desired derivative.
  • restriction endonuclease(s) Wells et al., Philos. Trans. R. Sot. London SerA 317: 415 (1986)
  • an IP- 10 gene can be mutated in vitro or in vivo to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or to form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • a variety of mutagenesis techniques are known in the art such as chemical mutagenesis, in vitro site-directed mutagenesis (Carter et al., Nucl. Acids Res. 13: 4331(1986)), use of TAB ® linkers (available from Pharmacia and Upjohn, Kalamazoo, MI), etc.
  • the anti-IP-10 antibodies of the present invention include antibodies having all types of constant regions, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGl, IgG2a, IgG2b, IgG3 and IgG4.
  • the light chains of the antibodies can either be kappa light chains or lambda light chains.
  • anti-IP antibodies preferably bind to an IP-10 epitope at a binding affinity of at least 10 6 M _1 ,
  • the antibodies of the present invention neutralize at least one biological activity of IP-10, such as receptor binding activities, signaling transductions, and cellular responses induced by IP-10.
  • IP-10 such as receptor binding activities, signaling transductions, and cellular responses induced by IP-10.
  • These neutralizing antibodies are capable of competing with the binding of IP-10 to its receptors, such as CXCR3, or even block the binding completely.
  • These antibodies should inhibit or completely neutralize signaling activities, and/or induction of cellular responses, for example, calcium influx or chemotaxis of lymphocytes.
  • the antibodies are capable of inhibiting the interactions between IP- 10 and CXCR3 and/or IP-10 mediated chemotaxis activities.
  • a concentration of 0.005, 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2, 5, 10, or 100 ⁇ g/ml of the antibodies will block at least 10%, 25%, 50%, 90%, 95%, 99% or essentially 100% of the binding of IP-10 to CXCR3 or IP-10-mediated chemotaxis, especially when the IP-10 is also used at one of these concentrations or at a molar concentration that is 0.005, 0.01, 0.05, 0.1, 0.25, 0.5, or 1.0 of the concentration of the antibody.
  • Exemplary neutralizing antibodies are the monoclonal antibodies: AIP 13, AIP12, A1P8, AJP14, and A1P21 (AIP are PDL internal antibody names).
  • An exemplary neutralizing humanized antibody is the monoclonal antibody HuAJP13. Almost all of them inhibit, or even completely block the JJ?-10-mediated chemotaxis.
  • AIP 13 and AIP 12 have the most potent neutralizing capability. As little as 1.3 ⁇ g/ml of AIP13 or 1.6 ⁇ g/ml of AIP12 inhibit IP-10-mediated chemotaxis by about 50%.
  • SEQ ID NOs: 3 and 4 The amino acid sequences of the mature heavy chain variable region and the mature light chain variable region of AIP 13 are depicted in SEQ ID NOs: 3 and 4, respectively ( Figure 1A).
  • the amino acid sequences of the mature heavy chain variable region and the mature light chain variable region of HuAIP13 are depicted in SEQ ID NOs: 13 and 14, respectively ( Figures 7 and 8).
  • SEQ ID NOs: 5, 6, and 7 depict the amino acid sequences of the heavy chain CDR1 (DYSMH), CDR2 (WINTEIGEPTYADDFKG), and CDR3 (NYDYDAYFDV), respectively.
  • SEQ ID NOs: 8, 9, and 10 depict the amino acid sequences of the light chain CDR1
  • the amino acid sequences of the mature heavy chain variable region and the mature light chain variable region of AIP12 are depicted in SEQ ID NOs: 41 and 42, respectively ( Figure 14A).
  • the amino acid sequences of the mature heavy chain variable region and the mature light chain variable region of HuAIP13 are depicted in SEQ ID NOs: 45 and 46, respectively ( Figures 16 and 17).
  • SEQ ID NOs: 5, 73, and 74 depict the amino acid sequences of the heavy chain CDR1 (DYSMH), CDR2 (WINTETGEPTYADDFKG), and CDR3 (NYDYDGYFDV), respectively.
  • SEQ J_D NOs: 75, 76, and 77 depict the amino acid sequences of the light chain CDR1 (KASQDINKYIA), CDR2 (YTSTLQP), and CDR3 (LQYDNLLFT), respectively.
  • the present invention includes the analogs of the antibodies or antibody fragments describes herein. These analogs should retain the antigen-binding utility.
  • Preferred analogs include a) the CDRs comprising an amino acid sequences sharing at least 60%, 80% or 90-95% amino acid sequence identity with SEQ ID NOs: 5, 6, 7, 8, 9, or 10; b) the CDRs comprising an amino acid sequences sharing at least 60%, 80% or 90-95% amino acid sequence identity with SEQ ID NOs: 5, 73, 74, 75, 76, or 77; c) a mature heavy chain variable region comprising an amino acid sequences sharing at least 60%, 80% or 90-95% amino acid sequence identity with SEQ ID NO: 3 or 13; and/or a mature light chain variable region comprising an amino acid sequences sharing at least 60%, 80% or 90-95% amino acid sequence identity with SEQ ID NO: 4 or 14; d) a mature heavy chain variable region comprising an amino acid sequences sharing at least 60%, 80% or 90-95% amino acid sequence identity with SEQ ID NO: 41 or 45; and/or a mature light chain variable region comprising an amino acid sequences sharing at least 60%, 80% or 90-9
  • amino acids may be grouped as follows: Group I (hydrophobic sidechains): met, ala, val, leu, ile; Group JJ (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IN (basic side chains): asn, gin, his, lys, arg; Group N (residues influencing chain orientation): gly, pro; and Group NI (aromatic side chains): trp, tyr, phe.
  • Conservative substitutions involve substitutions between amino acids in the same class. ⁇ on-conservative substitutions constitute exchanging a member of one of these classes for a member of another.
  • the analogs of the present invention can be made by amino acid substitutions via mutagenesis methods known in the art.
  • the present invention provides for the polynucleotide molecules encoding the antibodies and antibody fragments and their analogs described herein. Because of the degeneracy of the genetic code, a variety of nucleic acid sequences encode each immunoglobulin amino acid sequence.
  • the desired nucleic acid sequences can be produced by de novo solid-phase D ⁇ A synthesis or by PCR mutagenesis of an earlier prepared variant of the desired polynucleotide.
  • the codons that are used comprise those that are typical for human or mouse (see, e.g., ⁇ akamura, Y., Nucleic Acids Res. 28: 292 (2000)).
  • An exemplary polynucleotide sequence encoding the mature anti-IP-10 antibody heavy chain variable region of SEQ ID NO: 3 is provided in SEQ ID NO: 11.
  • An exemplary polynucleotide sequence encoding the mature anti-IP-10 antibody light chain variable region of SEQ ID NO: 4 is provided in SEQ ID NO: 12.
  • An exemplary polynucleotide sequence encoding the mature anti-IP-10 antibody heavy chain variable region of SEQ ID NO: 41 is provided in SEQ ID NO: 43.
  • An exemplary polynucleotide sequence encoding the mature anti-IP-10 antibody light chain variable region of SEQ ID NO: 42 is provided in SEQ ID NO: 44.
  • Percent (%) sequence identity with respect to a specified subject sequence, or a specified portion thereof, may be defined as the percentage of nucleotides or amino acids in the candidate derivative sequence identical with the nucleotides or amino acids in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2.0al9 (Altschul et al, J. Mol. Biol. 215:403- 410 (1997); http: ⁇ last.wustl.edu/blast/README.html) with search parameters set to default values.
  • the HSPS and HSPS2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. Any two antibody sequences can only be aligned in one way, by using the numbering scheme in Kabat (Kabat, E. et al., "Sequences of Proteins of Immunological Interest", U.S. Department of Health and Human Service (1983)). Therefore, for antibodies, percent identity has a unique and well-defined meaning. A "% identity value" is determined by the number of matching identical nucleotides or amino acids divided by the sequence length for which the percent identity is being reported.
  • the present invention includes the monoclonal antibodies that bind to substantially the same epitope as any one of these above-exemplified antibodies.
  • Non-limiting exemplary natural antibodies include antibodies derived from human, chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits), including transgenic rodents genetically engineered to produce human antibodies (see, e.g., Lonberg et al., WO93/12227; U.S. Patent No. 5,545,806; and Kucherlapati, et al., WO91/10741; U.S. Patent No. 6,150,584, which are herein incorporated by reference in their entirety).
  • Natural antibodies are the antibodies produced by a host animal. In a preferred embodiment, the antibody is an isolated antibody that binds to or neutralizes IP-10.
  • the monoclonal antibodies are produced by conventional hybridoma methodology known in the art, as described originally by Kohler and Milstein, Nature 256: 495-7 (1975); Eur. J. Immunol. 6: 511 (1976)). More experimental details of producing the monoclonal antibodies described herein are disclosed in the Examples.
  • the polyclonal forms of the anti-IP-10 antibodies are also included in the present invention. Preferably, these antibodies neutralize at least one activity of IP- 10, or bind to the epitopes that the described monoclonal antibodies bind to in the present invention.
  • the polyclonal antibody can be produced by immunization of host animals by IP-10 or the fragments thereof. The polyclonal antibodies are secreted into the bloodstream and can be recovered with known techniques.
  • Purified forms of these antibodies can, of course, be readily prepared by standard purification techniques, preferably including affinity chromatography with Protein A, anti- immunoglobulin, or the antigen itself. In any case, in order to monitor the success of immunization, the antibody levels with respect to the antigen in serum will be monitored using standard techniques such as ELISA, RIA, and the like.
  • Recombinant antibodies against IP-10 are also included in the present invention. These recombinant antibodies have the same amino acid sequence as the natural antibodies or have altered amino acid sequences as compared with the natural antibodies in the present invention.
  • the genetically altered anti-IP-10 antibodies should be functionally equivalent to the above-mentioned natural antibodies and recombinant antibodies.
  • Modified antibodies providing improved stability and/or therapeutic efficacy are preferred.
  • modified antibodies include those with conservative substitutions of amino acid residues, and one or more deletions or additions of amino acids that do not significantly deleteriously alter the antigen binding utility. Substitutions can range from changing or modifying one or more amino acid residues to complete redesign of a region as long as the therapeutic utility is maintained.
  • Antibodies of this invention can be modified post-translationally (e.g., acetylation, and phosphorylation) or can be modified synthetically (e.g., the attachment of a labeling group or cytotoxic agent).
  • Preferred genetically altered antibodies are chimeric antibodies and humanized antibodies.
  • the chimeric antibody is an antibody having a variable region and a constant region derived from two different antibodies, typically derived from separate species.
  • the variable region of the chimeric antibody is derived from murine and the constant region is derived from human, so that the chimeric antibody has a longer half-life and is less immunogenic when administered to a human subject.
  • the murine variable regions are derived from any one of the monoclonal antibodies described herein, including the non-limiting examples: a) the monoclonal antibodies comprising a mature heavy chain variable region comprising amino acid sequence of SEQ ID NO: 3 and/or a mature light chain variable region comprising amino acid sequence of SEQ ID NO: 4; b) the antibodies that substantially bind to the same epitope of any antibodies of a); or c) the analogs of any antibodies of a).
  • the murine variable regions are derived from any one of the monoclonal antibodies described herein, including the non-limiting examples: a) the monoclonal antibodies comprising a mature heavy chain variable region comprising amino acid sequence of SEQ ID NO: 41 and/or a mature light chain variable region comprising amino acid sequence of SEQ ID NO: 42; b) the antibodies that substantially bind to the same epitope of any antibodies of a); or c) the analogs of any antibodies of a).
  • the portions derived from two different species can be joined together chemically by conventional techniques or can be prepared as single contiguous proteins with genetic engineering techniques.
  • the DNA molecules encoding the proteins of both the light chain and heavy chain portions of the chimeric antibody can be expressed as contiguous proteins.
  • the method of making the chimeric antibody is disclosed in U.S. Patent Nos. 5,677,427; 6,120,767; and 6,329,508, each of which is incorporated by reference in its entirety.
  • the genetically altered antibodies used in the present invention include humanized antibodies that bind to or neutralize JJM0.
  • said humanized antibody comprising CDRs of a mouse donor immunoglobulin and heavy chain and light chain frameworks of a human acceptor immunoglobulin.
  • the humanized antibodies are the humanized versions of a) the monoclonal antibodies comprising a mature heavy chain variable region comprising amino acid sequence of SEQ ID NO: 3 and/or a mature light chain variable region comprising amino acid sequence of SEQ ID NO: 4; b) the antibodies that bind to the same epitope of any antibodies of a); or c) the analogs of any antibodies of a).
  • the humanized antibodies are the humanized versions of a) the monoclonal antibodies comprising a mature heavy chain variable region comprising amino acid sequence of SEQ ID NO: 41 and/or a mature light chain variable region comprising amino acid sequence of SEQ ID NO: 42; b) the antibodies that bind to the same epitope of any antibodies of a); or c) the analogs of any antibodies of a).
  • the method of making humanized antibody is disclosed in U.S. Patent Nos: 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370, each of which is incorporated by reference in its entirety.
  • Anti-IP-10 fully human antibodies are also included in the present invention, hi a preferred embodiment of the present invention, said fully human antibodies are isolated human antibodies and neutralize the activities of IP-10 described herein.
  • HuAIF13 is an exemplification of humanized antibody that binds to IP-10.
  • the amino acid sequences of the HuAJF13 heavy chain variable region and light chain variable region are SEQ ID No.: 13 and 14, respectively.
  • HuAIF12 is another exemplification of humanized antibody that binds to IP-10.
  • the amino acid sequences of the HuAIF13 heavy chain variable region and light chain variable region are SEQ ID No.:45 and 46, respectively.
  • Fully human antibodies against IP-10 are produced by a variety of techniques.
  • trioma methodology The basic approach and an exemplary cell fusion partner, SPAZ-4, for use in this approach have been described by Oestberg et al, Hybridoma 2:361-367 (1983); Oestberg, U.S. Patent No. 4,634,664; and Engleman et al., U.S. Patent No. 4,634,666 (each of which is incorporated by reference in its entirety)
  • Human antibodies against IP-10 can also be produced from non-human transgenic animals having transgenes encoding at least a segment of the human immunoglobulin locus.
  • the production and properties of animals having these properties are described in detail by e.g., Lonberg et al., WO93/12227; U.S. Patent No. 5,545,806; and Kucherlapati, et al., WO91/10741; U.S. Patent No. 6,150,584, which are herein incorporated by reference in their entirety.
  • Various recombinant antibody library technologies may also be utilized to produce fully human antibodies.
  • one approach is to screen a DNA library from human B cells according to the general protocol outlined by Huse et al., Science 246:1275-1281 (1989). Antibodies binding IP-10 or a fragment thereof are selected. Sequences encoding such antibodies (or binding fragments) are then cloned and amplified. The protocol described by Huse is rendered more efficient in combination with phage-display technology. See, e.g., Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047; U.S. Patent No.
  • yeast system is also suitable for screening mammalian cell-surface or secreted proteins, such as antibodies.
  • Antibody libraries may be displayed on the surface of yeast cells for the purpose of obtaining the human antibodies against a target antigen. This approach is described by Yeung, et al., Biotechnol. Prog. 18(2):212-20 (2002); Boeder, E. T., et al., Nat.
  • human antibody libraries may be expressed intracellularly and screened via yeast two-hybrid system (WO 02/00729 A2, which is incorporated by reference in its entirety).
  • Antigen-binding fragments of the anti-IP-10 antibodies which retain the binding specificity to IP-10, are also included in the present invention. Examples include, but are not limited to, partial or full heavy chains or light chains, variable regions, or CDR regions of any anti-IP-10 antibodies described herein.
  • the antibody fragments are truncated chains (truncated at the carboxyl end). Preferably, these truncated chains possess one or more immunoglobulin activities (e.g., complement fixation activity).
  • truncated chains include, but are not limited to, Fab fragments (consisting of the NL, NH, CL and CHI domains); Fd fragments (consisting of the NH and CHI domains); Fv fragments (consisting of NL and NH domains of a single chain of an antibody); dab fragments (consisting of a NH domain); isolated CDR regions; (Fab') 2 fragments, bivalent fragments (comprising two Fab fragments linked by a disulphide bridge at the hinge region).
  • the truncated chains can be produced by conventional biochemistry techniques, such as enzyme cleavage, or recombinant D ⁇ A techniques, each of which is known in the art.
  • polypeptide fragments may be produced by proteolytic cleavage of intact antibodies by methods well known in the art, or by inserting stop codons at the desired locations in the vectors using site- directed mutagenesis, such as after CHI to produce Fab fragments or after the hinge region to produce (Fab') 2 fragments.
  • Site- directed mutagenesis such as after CHI to produce Fab fragments or after the hinge region to produce (Fab') 2 fragments.
  • Single chain antibodies may be produced by joining N L and N ⁇ -coding regions with a D ⁇ A that encodes a peptide linker connecting the NL and NH protein fragments
  • the genes of the antibody fragments may be fused to functional regions from other genes (e.g., enzymes, U.S. Patent No. 5,004,692, which is incorporated by reference in its entirety) to produce fusion proteins (e.g., immunotoxins) or conjugates having novel properties.
  • the present invention comprises the use of anti-IP-10 antibodies in immunotoxins. Conjugates that are immunotoxins including antibodies have been widely described in the art. The toxins may be coupled to the antibodies by conventional coupling techniques or immunotoxins containing protein toxin portions can be produced as fusion proteins. The conjugates of the present invention can be used in a corresponding way to obtain such immunotoxins.
  • immunotoxins those described by Byers, B. S. et al., Seminars Cell Biol. 2:59-70 (1991) and by Fanger, M. W. et al, Immunol Today 12:51-54 (1991).
  • Recombinant DNA techniques can be used to produce the recombinant anti- IP- 10 antibodies, as well as the chimeric or humanized anti-IP-10 antibodies or any other anti-IP-10 genetically-altered antibodies and the fragments or conjugate thereof in any expression systems including both prokaryotic and eukaryotic expression systems, such as bacteria, yeast, insect cells, plant cells, and mammalian cells (for example, NS0 cells).
  • prokaryotic and eukaryotic expression systems such as bacteria, yeast, insect cells, plant cells, and mammalian cells (for example, NS0 cells).
  • the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention can be isolated and purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like (see, generally, Scopes, R., Protein Purification (Springer-Verlag, ⁇ .Y., 1982)).
  • Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses.
  • polypeptides may then be used therapeutically (including extra corporeally) or in developing and performing assay procedures, immunofluorescent stainings, and the like. (See, generally, Immunological Methods, Nols. I and II, Lefkovits and Pernis, eds., Academic Press, ⁇ Y, 1979 and 1981).
  • the antibodies of the present invention should typically be antagonistic. However, agonistic antibodies that trigger or enhance the IP-10 ligand or receptor functions are also included in the present invention. These antibodies will find use in the treatment of disorders caused by the hypo-activities of IP- 10.
  • Antibodies of the present invention can be used to inhibit chemotaxis or migration of lymphocytes, such as T cells.
  • the present invention includes a method of inhibiting chemotaxis of lymphocyte cells comprising contacting said lymphocyte cells with any of the antibodies or antibody fragments described herein.
  • the antibodies can contact with the lymphocyte cells in vitro (such as in a cell culture environment) or in vivo (such as in a subject). When the antibodies contact the lymphocytes in vitro, the antibodies can be added to the cell culture where the lymphocytes are cultivated. When the antibodies contact with the lymphocyte cells in vivo, the antibodies can be administered in a subject via the various routes described below.
  • Antibodies of the present invention are also useful for the prevention and/or treatment of autoimmune diseases, preferably inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis.
  • IBD inflammatory bowel diseases
  • the present invention provides for a method of reducing the severity of at least one symptom of an inflammatory bowel disease comprising administering to a subject in need thereof an effective amoimt of an antagonist of IP-10, including all the antagonists described herein.
  • said antagonists are antibodies that bind to or neutralize human IP- 10, and more preferably, the antibodies or antibody fragments or conjugates described herein.
  • the symptoms of inflammatory bowel diseases include, but are not limited to, colon inflammatory lesions, body weight loss, diarrhea, and rectal prolapse.
  • subjects may be scored for CDAI.
  • subjects may be scored for MTWSI and MAYO.
  • colon biopsy materials may be evaluated for inflammatory activities. Therapeutic methods are usually applied to human patients but may be applied to other mammals.
  • the antagonists may be administered to a patient intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, inhalation routes, or other delivery means known to those skilled in the art.
  • compositions of the present invention commonly comprise a solution of antagonist, such as antibodies, or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • aqueous carriers can be used, e.g., water for injection (WFI), or water buffered with phosphate, citrate, acetate, etc. to a pH typically of 5.0 to 8.0, most often 6.0 to 7.0, and/or containing salts such as sodium chloride, potassium chloride, etc. to make isotonic.
  • WFI water for injection
  • the carrier can also contain excipients such as human serum albumin, polysorbate 80, sugars or amino acids to protect the active protein.
  • the concentration of an antibody in these formulations varies widely from about 0.1 to 100 mg/ml but is often in the range 1 to 10 mg/ml.
  • the formulated antagonists such as monoclonal antibodies are particularly suitable for parenteral administration, and can be administered as an intravenous infusion or by subcutaneous, intramuscular or intravenous injection.
  • Actual methods for preparing parentally administrable compositions are known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science (15th Ed., Mack Publishing Company, Easton, Pa., 1980), which is incorporated herein by reference.
  • the present invention provides for a pharmaceutical composition comprising any one of the antibodies described herein.
  • compositions can be administered for prophylactic and/or therapeutic treatments, comprising inhibiting the interactions between IP-10 and its receptors, inhibiting the IP-10 induced chemotaxis of lymphocytes, reducing the inflammatory responses, or reducing the clinical symptoms of IBD.
  • An amount adequate to accomplish the desired effect is defined as an "effective amount”.
  • the antibodies can be delivered into a patient by single or multiple administrations.
  • the appropriate dosage of the antibodies will depend on the severity and course of disease, the patient's clinical history and response, the toxicity of the antibodies, and the discretion of the attending physician.
  • the antibodies are suitably administered to the patient at one time or over a series of treatments.
  • the initial candidate dosage may be administered to a patient.
  • the proper dosage and treatment regimen can be established by monitoring the progress of therapy using conventional techniques known to the people skilled in the art.
  • the antagonist (such as antibodies) can be utilized alone in substantially pure form, or together with therapeutic agents for autoimmune diseases known to those of skill in the art.
  • Other therapies that may be used in conjunction with treatment with antibodies include administration of anti-sense nucleic acid molecules or biologicals, such as additional therapeutic antibodies.
  • the antibody will often be administered after or in combination with one or more other immunosuppressive drugs or other therapies, for example, corticosteroids, cyclosporine, methotrexate, phototherapy.
  • the treatment of the present invention is formulated in a manner allowing it to be administered serially or in combination with another agent for the treatment of inflammatory bowel diseases. IN. Diagnostic uses
  • Antibodies disclosed herein are useful in diagnostic and prognostic evaluation of diseases and disorders, particularly IBD (Crohn's disease and/or ulcerative colitis) associated with IP-10 expression. At each stage of disease, monoclonal antibodies may be used to improve diagnostic accuracy and facilitate treatment decisions. Methods of diagnosis can be performed in vitro using a cellular sample (e.g., blood sample, lymph node biopsy, or tissue) from a patient or can be performed by in vivo imaging.
  • a cellular sample e.g., blood sample, lymph node biopsy, or tissue
  • the present invention provides an antibody conjugate wherein the antibodies of the present invention are conjugated to a diagnostic imaging agent.
  • Compositions comprising the antibodies of the present invention can be used to detect IP-10, for example, by radioimmunoassay, ELISA, FACS, etc.
  • One or more labeling moieties can be attached to the antibodies.
  • Exemplary labeling moieties include radiopaque dyes, radiocontrast agents, fluorescent molecules, spin-labeled molecules, enzymes, or other labeling moieties of diagnostic value, particularly in radiologic or magnetic resonance imaging techniques.
  • the present invention provides for a method of detecting IBD comprising detecting the differential expression of mR ⁇ A or protein of IP- 10 in colon cells in a subject in need of such detection.
  • the method of diagnosing Crohn's disease or ulcerative colitis in a patient in need of such a diagnosis comprises: a) isolating a colon sample from said patient; b) contacting cells of said colon sample with the antibodies of the present invention; c) contacting normal colon cells with the antibodies of the present invention; and d) detecting and comparing the difference of the expression of IP-10 in said colon sample cells with the said normal colon cells.
  • the normal cells can be the colon cells isolated from a healthy subject or a non- diseased portion of the colon of the same patient.
  • the method of diagnosing Crohn's disease or ulcerative colitis in a patient in need of such a diagnosis comprises detecting elevated serum level of IP-10 in the blood of the patient as compared with that of a healthy subject, for example, including the step of a) isolating a blood sample from a patient; b) contacting said blood sample with the antibodies of the present invention; c) contacting a blood sample from a healthy subject with the antibodies of the present invention; and d) detecting and comparing the serum IP-10 level of said patient with said healthy subject.
  • the antibodies of the present invention can also be used to evaluate the treatment efficacy of a therapeutic approach, such as a method of treating IBD.
  • Antibodies are utilized to detect the expression level of IP-10 before and after certain treatment. Reduction in IP-10 expression level may be an indicator of the effectiveness of the treatment.
  • the antibodies of the present invention can also be used as detecting agents in in vitro assays for research purposes.
  • the antibodies can be used to identify or isolate novel receptors or other binding proteins for IP-10 via the methods known in the art, such as by screening protein expression libraries.
  • the present invention also provides for a diagnostic kit comprising anti-IP-10 antibodies described herein.
  • a diagnostic kit further comprises a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay.
  • the kit will include substrates and co-factors required by the enzyme.
  • other additives may be included such as stabilizers, buffers and the like.
  • the relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents that substantially optimize the sensitivity of the assay.
  • the reagents may be provided as dry powders, usually lyophilized, including excipients that, on dissolution, will provide a reagent solution having the appropriate concentration.
  • RNA samples from the diseased or control group mice were isolated, flushed with PBS, and snap-frozen in liquid nitrogen until use.
  • To isolate RNA the frozen tissues were ground into a fine powder using a mortar and pestle.
  • Total RNA was extracted from the homogenized tissues using a standard Trizol method. All reagents and equipments used for isolation purposes were kept sterile.
  • Total RNA was then treated with DNase I enzyme from Ambion (Austin, Texas) according to the manufacturer's protocol and stored at -80°C. 1 st strand cDNA synthesis The DNase treated RNA was then subjected to the 1 st strand cDNA synthesis using the SuperscriptTM reverse transcriptase purchased from Invitrogen (Carlsbad, California).
  • RNA was incubated with a random hexamer mix for 10 minutes at 70°C and then cooled on ice for 5 minutes.
  • the SuperscriptTM reverse transcriptase, dNTP mix, DTT, RNasin, and the SuperscriptTM buffer were then mixed together and allowed to be incubated at room temperature for 10 minutes.
  • the reaction was carried out at 40°C for 60 minutes.
  • An equal volume of water was added to the reaction and allowed to be incubated at 95°C for 10 minutes.
  • the reaction was then cooled on ice for 5 min and stored at -20°C.
  • SYBR Green Real-Time PCR All primers used for Real-Time PCR were designed using Primer Express
  • the reaction was set up in a 96-well Optical ReactionTM plate (ABI-Prism part #4306737) and sealed with OpticalTM caps (8caps/strip) (ABI-Prism part #4323032).
  • the master mix used for the reaction was SYBR Green PCR Master Mix (Applied Biosystems, Foster City, California part #4309155), supplied at 2x concentration.
  • the reaction was carried out in the GeneAmp 5700TM Sequence Detection System instrument.
  • the 1 st strand cDNA template was used at a 1/250 dilution, in an amount of 2 ⁇ l per well in a 25 ⁇ l total reaction volume.
  • a no-template control was set for each sample analyzed.
  • ⁇ -Actin was used as an endogenous control for normalizing samples and sample variations.
  • a standard curve was generated for each individual amplicon.
  • Normalized expression values for a gene was calculated by the following equation: Mean Qty Sample A Gene of interest/ Mean Qty of Sample A p-Actm- The fold change in expression of a gene of interest was obtained by using the equation: Normalized Expression Sample A/Normalized Expression Sample B.
  • cDNA microarrays cDNA microarray technique was performed according to the manufacturer's instructions (Incyte Genomics and Agilent Technologies, Palo Alto, California).
  • AgilentTM cDNA microarray analysis we have identified the mRNA for IP-10 elevated in colonic tissue isolated from the mice suffering from either of two distinct models of the inflammatory bowel disease, as compared to the levels of IP-10 mRNA present in colons from the nondiseased mice. RT-PCR analysis confirmed this difference.
  • RT- PCR analysis of mRNA isolated from the diseased tissues obtained from both Crohn's Disease (CD) and ulcerative colitis (UC) patients revealed elevated levels of IP-10 mRNA, as compared to the level present in the nondiseased colonic tissues.
  • CD Crohn's Disease
  • UC ulcerative colitis
  • This example describes the production of anti-IP-10 (AIP) monoclonal antibodies.
  • mice were immunized intraperitoneally or in the hind footpads with 5-20 ⁇ g of protein with an equal volume of Ribi adjuvant in a total final volume of 20 ⁇ l. Footpad immunizations were performed 4 times at 4- or 5-day interval. Intraperitoneal immunization consisted of 4 immunizations at two- week intervals.
  • ELISA Pre- and Post-immunization
  • Serum titers were determined by ELISA against human recombinant IP-10 with a standard antibody-capture ELISA assay and peroxidase-mediated detection. Serial dilutions of both pre-immune and post-immune serum were incubated with the capture protein and antigen-antibody complexes were detected with a horseradish peroxidase-conjugated anti-mouse-IgG secondary antibody and chromogenic reagent. Quantification of protein-specific serum titer was determined spectrophotometrically
  • mice with highest sera-titers against the IP-10 protein were sacrificed.
  • lymphocytes were isolated from the tissues and hybridomas were generated by standard procedures. Briefly, hybridomas were generated by polyethylene glycol (PEG) 1500 mediated fusion between lymphocytes and a murine myeloma cell line (NSO cells). Fused cells were plated into 96-well plates at a density of 10 5 cells per plate. Selection of fused cells was accomplished with HAT (hypoxanthine, aminopterin, and thymidine) media.
  • PEG polyethylene glycol
  • NSO cells murine myeloma cell line
  • Example 3 This example describes the blocking and neutralizing activities of the anti-IP-
  • Example 4 This example describes the chemotaxis assay for identifying neutralizing anti- human IP-10 monoclonal antibodies.
  • Peripheral blood mononuclear cells were purified on Ficoll-Paque PLUSTM and washed thoroughly with RPMI+, which is RPMI-1640 medium plus 10% FBS plus L-Glutamine plus Antibiotic- Antimycotic. Cells were adjusted to 1 to 5 x 10 /ml in RPMI+ and stimulated with PHA at 37°C/95% humidity/7.2% CO 2 for 3 days. Then the cells were split at 1 :3 in RPMI+ and cultured in the presence of rIL-2 at 10 ng/ml for 3 more days. Alternatively, PHA blasts were used that were frozen but thawed and cultured overnight in RPMI+ with 10 ng/ml rIL-2.
  • rhIP-10 R&D SystemsTM
  • rhJJP-10 mice were washed twice in a Chemotaxis buffer, which is RPMI-1640 + 0.5% BSA + 20 mM HEPES. Cells were adjusted to 1 x 10 6 /ml in the Chemotaxis buffer.
  • rhIP-10 R&D SystemsTM
  • rhIP-10 at certain doses (500, 50 and 25 ng/ml) were mixed with the graded doses of AIP monoclonal antibodies for 30 to 60 minutes at room temperature. Control wells with rhJJP-10 alone or without rhIP-10 were set up.
  • test reagent or control 30 ⁇ l of the test reagent or control were added to the bottom chamber of the ChemoTxTM plate (Neuro Probe, Gaithersburg, Maryland) and then placed the filter frame on the plate so that the fluid made contact with the filter.
  • the amount of 60 ⁇ l of cell suspension was placed on the filter top wells, covered, and incubated for 90 minutes at 37°C/95% humidity/7.2% CO 2 .
  • the non-migrating cells were removed by wiping the top of the filter with KimwipesTM. After centrifuge, cells were transferred from the microplates after assembly to an opaque- walled plate (DynexTM) and washed with 30 ⁇ l of buffer.
  • DynexTM opaque- walled plate
  • a panel of mouse anti-human-IP- 10 monoclonal antibodies that binds to recombinant hIP-10 was generated. These antibodies also bind to supernatants from the PHA blasts. Several of the monoclonal antibodies against hJP-10 neutralize the chemotaxis of PHA and LPS blasts in a dose-dependent manner. All of these monoclonal antibodies also bind to the mammalian-cell-expressed cynomolgus JJP-10 (Table 1).
  • Exemplary neutralizing antibodies are the monoclonal antibodies: AIP 13, AJP12, ATJ?8, AJP14, AIP6, AJJP21, AIP5, AJJ?6, AJP17, and AJJP18 ( Figure 4). All of these antibodies bind to IP-10 in ELISA and most of them block the binding of JJP-10 to CXCR3 expressed on CHO/CXCR3 transfectants. All of these antibodies inhibit or completely block the IP-10-induced chemotaxis of PHA and LPS blasts.
  • AIP5 As shown in Table 1, as little as 80 ng/ml of AEP13, 100 ng/ml of AIP12, 127ng/ml of AIP8, or 119 ng/ml of AIP14 inhibit the IP-10 (50 ng/ml)-induced chemotaxis of PHA blasts by 50%>.
  • AIP5, 6, 21, 32, and 36 are also potent neutralizing antibodies.
  • the amino acid sequences of the mature heavy-chain and light-chain variable regions of AIP 13 are presented here in SEQ ID NOs: 3 and 4, respectively.
  • the amino acid sequences of the mature heavy-chain and light-chain variable regions of AIP 12 are presented here in SEQ ED NOs: 41 and 42, respectively.
  • Table 1 ANTI-HUMAN IP-10 MONOCLONAL ANTIBODY PANEL
  • This example describes the humanization of anti-EP-10 antibodies.
  • Humanization of the murine monoclonal antibody AIP 13 was carried out essentially according to the procedure of Queen, C, et al., Proc. Natl. Acad. Sci. USA 86: 10029-10033 (1989).
  • Human V segments with high homology to the MuAJJP13 VH or NL amino acid sequences were identified.
  • the CDR sequences together with framework amino acids important for maintaining the structures of the CDRs were grafted into the selected human framework sequences.
  • human framework amino acids that were found to be rare in the corresponding V region subgroup were substituted with consensus amino acids to reduce potential immunogenicity.
  • the resulting humanized monoclonal antibody (HuAJJP13) was expressed in the mouse myeloma cell line NS0.
  • the affinity of HuAJJP13 to human IP- 10 was shown to be approximately 2.8-fold lower than that of MuAIP13.
  • variable region cDNAs for the heavy and light chains were amplified by polymerase chain reaction (PCR) using 3' primers that anneal respectively to the mouse gamma and kappa chain C regions, and a 5' universal primer provided in the SMART RACE cDNA Amplification Kit.
  • PCR polymerase chain reaction
  • the 3' primer has the sequence 5'- GCCAGTGGATAGACTGATGG-3' (SEQ ID NO: 71).
  • NL PCR the 3' primer has the sequence 5'-GATGGATACAGTTGGTGCAGC-3' (SEQ ID NO: 72).
  • VH and VL cDNAs were subcloned into the pCR4Blunt-TOPO vector (Invitrogen Corporation, Carlsbad, CA) for sequence determination.
  • DNA sequencing was carried out by PCR cycle sequencing reactions with fluorescent dideoxy chain terminators (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. The sequencing reactions were analyzed on a Model 377 DNA Sequencer (Applied Biosystems).
  • the heavy and light chain variable region genes were constructed and amplified using eight overlapping synthetic oligonucleotides ranging in length from approximately 65 to 80 bases as illustrated in Fig. 5 (He, X.-Y., et al., J. Immunol. 160: 1029-1035 (1998)).
  • the oligonucleotides were annealed pairwise and extended with the Klenow fragment of DNA polymerase I, yielding four double-stranded segments.
  • the resulting segments were denatured, annealed pairwise, and extended with Klenow fragment, yielding two segments. These segments were denatured, annealed pairwise, and extended once again, yielding a full-length gene.
  • the resulting product was amplified by PCR using the Expand High Fidelity PCR System (Roche Diagnostics Corporation, Indianapolis, IN).
  • the PCR-amplified fragments were gel-purified, digested with Mlul and Xbal, gel-purified, and subcloned respectively into pVgl.D.Tt (Cole, M.S., et al., J. Immunol. 159: 3613-3621 (1997)) and pHuCkappa.rgpt.dE (Kostelny, S.A., et al., Int. J. Cancer 93: 556-565 (2001)).
  • the EcoRI fragment containing the entire heavy chain transcription unit was subcloned into the unique EcoRI site in the light chain expression vector, resulting in a single vector for expression of heavy and light chains.
  • Stable transfection Mouse myeloma cell line NSO was obtained from the European Collection of Animal Cell Cultures (Salisbury, Wiltshire, UK), preadapted to Protein Free Basal Medium-1 (PFBM-1) (Protein Design Labs, Inc.), and maintained in PFBM-1 at 37°C in a 7.5% CO 2 incubator. Stable transfection into NSO was carried out by electroporation essentially as described in Bebbington, C.R., et al., Bio/Technology 10: 169-175 (1992). Before transfection, the expression vector was linearized using Fspl. Approximately 10 7 cells were transfected with 20 ⁇ g of linearized plasmid.
  • the transfected cells were suspended in DME medium (BioWhittaker, Inc., Walkersville, MD) containing 10% FBS (HyClone, Logan, UT) and plated into several 96-well plates. After 48 hr, selection media (DME medium containing 10% FBS, HT media supplement, 0.5 mg/ml xanthine and 2.4 ⁇ g/ml mycophenolic acid) was applied. Approximately 10 days after the initiation of selection, culture supernatants were assayed for antibody production by ELISA.
  • DME medium BioWhittaker, Inc., Walkersville, MD
  • FBS HyClone, Logan, UT
  • selection media DME medium containing 10% FBS, HT media supplement, 0.5 mg/ml xanthine and 2.4 ⁇ g/ml mycophenolic acid
  • ELISA plates (Nunc Nalge International, Rochester, NY) were coated overnight at 4°C with 100 ⁇ l/well of a 1:1000 dilution of AffiniPure goat anti-human IgG Fc ⁇ - chain specific polyclonal antibodies (Jackson JmmunoResearch Laboratories, Inc., West Grove, PA) in 0.2 M sodium carbonate-bicarbonate buffer, pH 9.4, washed with Wash Buffer (PBS containing 0.1% Tween 20), and blocked for 30 min at room temperature with 300 ⁇ l/well of SuperBlock Blocking Buffer in TBS (Pierce Chemical Company, Rockford, IL).
  • Wash Buffer PBS containing 0.1% Tween 20
  • the MuAIP13 heavy and light chain N-region cD ⁇ As were cloned from the hybridoma cells as described in Materials and Methods. Several heavy and light chain clones were sequenced from two independent PCR reactions. Unique sequences homologous to typical mouse heavy and light chain variable regions were identified. The predicted amino acid sequences of the mature heavy and light chain variable regions are shown in Fig. 1 A. The cD ⁇ A sequences encoding the heavy and light chain N-regions are shown in Fig. IB.
  • the J segment JH6 (Ravetch, J.V., et al., Cell 27: 583-591 (1981)) were selected to provide the frameworks for the HuAIP13 heavy chain variable region.
  • the NL segment DPK1 Cox, J.P.L., et al., Eur. J. Immunol. 24: 827-836 (1994)
  • the J segment JK2 Hieter, P.A., et al., J. Biol. Chem. 257: 1516- 1522 (1982) were used.
  • Genes encoding humanized NH or NL were designed as mini-exons including signal peptides, splice donor signals, and appropriate restriction enzyme sites for subsequent cloning into a mammalian expression vector.
  • the splice donor signals in the NH and NL mini-exons were derived from the corresponding human germline JH and JK sequences, respectively.
  • the signal peptide sequence in the humanized NH mini-exon was derived from the MuEP5C7 NH (He, X.-Y., et al., supra); the signal peptide sequence in the humanized NL mini-exon was derived from the corresponding MuAIP 13 NL.
  • the HuAIP13 NH and NL genes were constructed by extension of eight overlapping synthetic oligonucleotides and PCR amplification as illustrated in Fig. 5. A series of 8 overlapping oligonucleotides (1-8) was used.
  • Oligonucleotides 1 and 2, 3 and 4, 5 and 6, and 7 and 8 were separately annealed and extended with the Klenow fragment of D ⁇ A polymerase I.
  • the resulting double- stranded D ⁇ A segments, A and B, and C and D were separately mixed, denatured, annealed and extended to yield the D ⁇ A segments E and F, respectively, which were then mixed to generate the entire mini-exon (G) in the third annealing-and-extension step.
  • the mini-exon was amplified by PCR with primers 9 and 10 to incorporate the flanking Mlul and Xbal sites.
  • Primers Hl-10 for the synthesis of the humanized heavy chain variable region are presented in SEQ ID ⁇ Os: 21-30, respectively (Fig. 12).
  • Primers Ll-10 for the synthesis of the humanized light chain variable region are presented in SEQ ID ⁇ Os: 31 -40, respectively (Fig. 13).
  • the resulting N gene fragments were cloned into mammalian expression vectors pHuCkappa.rgpt.dE and pNgl.D.Tt, and then combined to generate a single expression vector (Fig. 6) for co-expression of the light and heavy chains.
  • the D ⁇ A sequences (SEQ ID ⁇ os: 17 and 19) and deduced amino acid sequences (SEQ ID ⁇ os: 18 and 20) of the humanized NL and NH mini-exons are shown in Figs. 9 and 10, respectively.
  • the single expression vector was introduced into the chromosome of mouse myeloma cell line ⁇ S0 by electroporation. Stable transfectants were selected for gpt expression as described in Materials and Methods. Culture supernatants of NSO stable transfectants were analyzed by ELISA for production of HuAIP13.
  • One of the high producing cell lines, clone DB2 was adapted to and expanded in PFBM-1 supplemented with PFFM-2.
  • HuAIP13 IgGl ⁇ monoclonal antibody was purified from spent culture supernatant with a protein-A Sepharose column as described in Materials and Methods.
  • HuAIP13 has a molecular weight of about 150-160 kD.
  • Analysis under reducing conditions indicated that HuAJJPl 3 is comprised of a heavy chain with a molecular weight of about 50 kD and a light chain with a molecular weight of about 25 kD. The purity of the antibody appeared to be more than 95%.
  • HuAJJPl 3 also blocks the binding of IP-10 to CXCR3 and exhibits nanomolar affinity for IP-10 (Table 5).
  • HuAIP13 also inhibits IP-10 mediated chemotaxis with a comparable IC 50 (-100 ng/ml) and inhibits 100%) of IP-10 mediated chemotaxis at ⁇ 1 ⁇ g/ml.
  • Table 2 AFFINITY OF HuAIP13 TO HUMAN IP-10
  • This example describes the humanization of anti-IP-10 antibodies.
  • Humanization of the murine monoclonal antibody AIP 12 (MuAJJP12) was carried out essentially according to the procedure of Queen, C, et al., Proc. Natl. Acad. Sci. USA 86: 10029-10033 (1989).
  • Human V segments with high homology to the MuAJJP12 NH or NL amino acid sequences were identified.
  • the CDR sequences together with framework amino acids important for maintaining the structures of the CDRs were grafted into the selected human framework sequences.
  • human framework amino acids that were found to be rare in the corresponding V region subgroup were substituted with consensus amino acids to reduce potential immunogenicity.
  • the resulting humanized monoclonal antibody (HuAIP12) was expressed in the mouse myeloma cell line NSO.
  • HuAJJPl 2 was expressed in the mouse myeloma cell line NSO.
  • affinity of HuAJJPl 2 to human EP- 10 was shown to be approximately 2.5-fold lower than that of MuAIP12.
  • variable region cDNAs for the heavy and light chains were amplified by polymerase chain reaction (PCR) using 3' primers that anneal respectively to the mouse gamma and kappa chain C regions, and a 5' universal primer provided in the SMART RACE cDNA Amplification Kit.
  • PCR polymerase chain reaction
  • the 3' primer has the sequence 5'- GCCAGTGGATAGACTGATGG-3' (SEQ ID NO: 71).
  • NL PCR the 3' primer has the sequence 5'-GATGGATACAGTTGGTGCAGC-3' (SEQ ID NO: 72).
  • the NH and NL cD ⁇ As were subcloned into the pCR4Blunt-TOPO vector (hivitrogen Corporation, Carlsbad, CA) for sequence determination.
  • D ⁇ A sequencing was carried out by PCR cycle sequencing reactions with fluorescent dideoxy chain terminators (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. The sequencing reactions were analyzed on a Model 377 D ⁇ A Sequencer (Applied Biosystems).
  • the heavy and light chain variable region genes were constructed and amplified using eight overlapping synthetic oligonucleotides ranging in length from approximately 65 to 80 bases as illustrated in Fig. 5 (He, X.-Y., et al., J. Immunol. 160: 1029-1035 (1998)).
  • the oligonucleotides were annealed pairwise and extended with the Klenow fragment of DNA polymerase I, yielding four double-stranded segments.
  • the resulting segments were denatured, annealed pairwise, and extended with Klenow fragment, yielding two segments. These segments were denatured, annealed pairwise, and extended once again, yielding a full-length gene.
  • the resulting product was amplified by PCR using the Expand High Fidelity PCR System (Roche Diagnostics Corporation, Indianapolis, IN).
  • the PCR-amplified fragments were gel-purified, digested with Mlul and Xbal, gel-purified, and subcloned respectively into pVgl.D.Tt (Cole, M.S., et al., J. Immunol. 159: 3613-3621 (1997)) and pHuCkappa.rgpt.dE (Kostelny, S.A., et al., h t. J. Cancer 93: 556-565 (2001)).
  • the EcoRI fragment containing the entire heavy chain transcription unit was subcloned into the unique EcoRI site in the light chain expression vector, resulting in a single vector for expression of heavy and light chains.
  • Mouse myeloma cell line NSO was obtained from the European Collection of Animal Cell Cultures (Salisbury, Wiltshire, UK), preadapted to Protein Free Basal Medium-1 (PFBM-1) (Protein Design Labs, Inc.), and maintained in PFBM-1 at 37°C in a 7.5% CO 2 incubator.
  • PFBM-1 Protein Free Basal Medium-1
  • Stable transfection into NSO was carried out by electroporation essentially as described in Bebbington, C.R., et al., Bio/Technology 10: 169-175 (1992). Before transfection, the expression vector was linearized using Fspl. Approximately 10 7 cells were transfected with 20 ⁇ g of linearized plasmid. The transfected cells were suspended in DME medium (BioWhittaker, Inc., WalkersviUe, MD) containing 10% FBS (HyClone, Logan, UT) and plated into several 96-well plates.
  • DME medium BioWhittaker, Inc., WalkersviUe, MD
  • FBS HyClone, Logan, UT
  • selection media DME medium containing 10% FBS, HT media supplement, 0.5 mg/ml xanthine and 2.4 ⁇ g/ml mycophenolic acid
  • DME medium containing 10% FBS, HT media supplement, 0.5 mg/ml xanthine and 2.4 ⁇ g/ml mycophenolic acid
  • PFFM-2 Protein-Free Feed Medium-2
  • ELISA plates (Nunc Nalge International, Rochester, NY) were coated overnight at 4°C with 100 ⁇ l/well of a 1:1000 dilution of AffiniPure goat anti-human IgG Fc ⁇ - chain specific polyclonal antibodies (Jackson hnmunoResearch Laboratories, Inc., West Grove, PA) in 0.2 M sodium carbonate-bicarbonate buffer, pH 9.4, washed with Wash Buffer (PBS containing 0.1 % Tween 20), and blocked for 30 min at room temperature with 300 ⁇ l/well of SuperBlock Blocking Buffer in TBS (Pierce Chemical Company, Rockford, IL).
  • Wash Buffer PBS containing 0.1 % Tween 20
  • MuAJP12 heavy and light chain N-region cD ⁇ As were cloned from the hybridoma cells as described in Materials and Methods. Several heavy and light chain clones were sequenced from two independent PCR reactions. Unique sequences homologous to typical mouse heavy and light chain variable regions were identified. The predicted amino acid sequences of the mature heavy and light chain variable regions are shown in Fig. 14 A. The cD ⁇ A sequences encoding the heavy and light chain N-regions are shown in Fig. 14B.
  • Genes encoding humanized NH or NL were designed as mini-exons including signal peptides, splice donor signals, and appropriate restriction enzyme sites for subsequent cloning into a mammalian expression vector.
  • the splice donor signals in the NH and NL mini-exons were derived from the corresponding human germline JH and JK sequences, respectively.
  • the signal peptide sequence in the humanized NH mini-exon was derived from the MuEP5C7 NH (He, X.-Y., et al., supra); the signal peptide sequence in the humanized NL mini-exon was derived from the corresponding MuAIP 12 NL.
  • the HuAJJPl 2 NH and NL genes were constructed by extension of eight overlapping synthetic oligonucleotides and PCR amplification as illustrated in Fig. 5. A series of 8 overlapping oligonucleotides (1-8) was used. Oligonucleotides 1 and 2, 3 and 4, 5 and 6, and 7 and 8 were separately annealed and extended with the Klenow fragment of D ⁇ A polymerase I. The resulting double- stranded D ⁇ A segments, A and B, and C and D, were separately mixed, denatured, annealed and extended to yield the D ⁇ A segments E and F, respectively, which were then mixed to generate the entire mini-exon (G) in the third annealing-and-extension step.
  • the mini-exon was amplified by PCR with primers 9 and 10 to incorporate the flanking Mlul and Xbal sites.
  • Primers 12H1-10 for the synthesis of the humanized heavy chain variable region are presented in SEQ ID ⁇ Os: 51-60, respectively (Fig. 21).
  • Primers 12L1-10 for the synthesis of the humanized light chain variable region are presented in SEQ ID ⁇ Os: 61-70, respectively (Fig. 22).
  • the resulting N gene fragments were cloned into mammalian expression vectors pHuCkappa.rgpt.dE and pNgl.D.Tt, and then combined to generate a single expression vector (Fig. 15) for co-expression of the light and heavy chains.
  • the D ⁇ A sequences (SEQ ID ⁇ os: 47 and 49) and deduced amino acid sequences (SEQ ID ⁇ os: 48 and 50) of the humanized NL and NH mini-exons are shown in Figs. 18 and 19, respectively.
  • the single expression vector was introduced into the chromosome of mouse myeloma cell line ⁇ SO by electroporation. Stable transfectants were selected for gpt expression as described in Materials and Methods. Culture supernatants of ⁇ SO stable transfectants were analyzed by ELISA for production of HuAIP12.
  • HuAIP12 IgGl/i monoclonal antibody was purified from spent culture supernatant with a protein-A Sepharose column as described in Materials and Methods.
  • HuAIP12 has a molecular weight of about 150-160 kD.
  • Analysis under reducing conditions indicated that HuAIP12 is comprised of a heavy chain with a molecular weight of about 50 kD and a light chain with a molecular weight of about 25 kD. The purity of the antibody appeared to be more than 95%.
  • HuAIP12 also blocks the binding of IP-10 to CXCR3 and exhibits nanomolar affinity for IP-10 (Table 5). HuAIP12 also inhibits IP-10 mediated chemotaxis with a comparable IC 5 o (-100 ng/ml) and inhibits 100% of IP-10 mediated chemotaxis at ⁇ 1 ⁇ g/ml.
  • This example describes the treatment of IBD with humanized anti-IP-10 antibodies in one or two different IBD mouse models.
  • Two mouse models of IBD used herein are the spontaneous colitis model and the adoptive transfer model.
  • IL-10-deficient NOD mice (Jackson Laboratories, Bar Harbor, Maine) are used.
  • the age-matched NOD mice are used as the control group mice.
  • the IL-10-deficient NOD mice develop IBD between 8-12 weeks. This IBD has been characterized as Thl mediated and it resembles Crohn's disease in humans.
  • the I1-10-KO (knockout) mice exhibit an increased expression of IP-10 as compared to the control mice.
  • CD4/CD45RB T cells from naive syngeneic BALB/c mice are isolated and injected at 3 to 4 x 10 5 /mouse intravenously into the CB17.SCID mice.
  • the recipient mice develop the inflammatory bowel diseases. This IBD has been characterized as Thl mediated and it resembles Crohn's disease in humans.
  • the age-matched non-diseased CB17.SCID mice are used as the control group mice. Mice are considered positive for the disease based on the development of one or more of the following clinical symptoms such as body weight loss, diarrhea, and rectal prolapse.
  • the CD4/CD45RB mice exhibit an increased expression of IP-10 as compared to the control mice.
  • the CD4/CD45RB hl mice experience weight loss (between 10- 20%) at least 2 weeks post transfer as compared to control mice ( Figure 27).
  • the recipient mice are treated at weekly intervals with humanized anti-mouse-IP- 10 polyclonal antibodies or monoclonal antibodies (0.5 to 1 mg mg/dose in PBS, intraperitoneal route).
  • the control group mice receive isotype-matched IgG or pre- immune polyclonal IgG.
  • the therapeutic efficacy of the neutralizing anti-IP-10 antibody is tested once the recipient mice develop the disease (based on their body weight loss with respect to the initial body weight). The mice are monitored for clinical signs of disease such as loss of body weight, diarrhea, and rectal prolapse. Colon samples are harvested from the mice for histological examination by H&E staining.

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Abstract

La présente invention concerne des anticorps anti-humains IP-10 ou des fragments de ces anticorps se liant à l'antigène, dont des anticorps humanisés, des anticorps anti-humains IP-10 ou des fragments de ces anticorps se liant à l'antigène. L'invention concerne également une méthode propre à réduire la gravité d'au moins un symptôme d'une affection intestinale inflammatoire chez un sujet, consistant à administrer audit sujet une dose efficace d'un antagoniste de IP-10. De plus, la présente invention concerne une méthode permettant de diagnostiquer une affection intestinale inflammatoire chez un patient, consistant é détecter un niveau sérique élevé de IP-10 chez ce patient par rapport à un sujet sain.
PCT/US2004/014507 2003-05-09 2004-05-07 Anticorps anti-ip-10 et methodes d'utilisation pour le traitement d'affections intestinales inflammatoires Ceased WO2004101511A2 (fr)

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DE602004031772T DE602004031772D1 (de) 2003-12-04 2004-11-10 Anti-ip-10-antikörper
EP04800979A EP1708961B1 (fr) 2003-12-04 2004-11-10 Anticorps anti-ip10
AT04800979T ATE501173T1 (de) 2003-12-04 2004-11-10 Anti-ip-10-antikörper
PCT/US2004/037600 WO2005060457A2 (fr) 2003-12-04 2004-11-10 Traitement de maladies intestinales inflammatoires avec des anticorps anti-ip10

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WO2006121207A1 (fr) * 2005-05-12 2006-11-16 Oncotherapy Science, Inc. Procedes pour endommager des cellules en utilisant une fonction effectrice d’un anticorps anti-dsc2
EP1708961A4 (fr) * 2003-12-04 2008-05-28 Pdl Biopharma Inc Traitement de maladies intestinales inflammatoires avec des anticorps anti-ip10
WO2009026117A3 (fr) * 2007-08-16 2009-12-30 Glaxo Group Limited Nouveaux composés
US7786268B2 (en) 2007-02-28 2010-08-31 Novimmune Sa Anti-IP-10 antibodies and methods of use thereof
AU2004298492B2 (en) * 2003-12-10 2011-08-18 E. R. Squibb & Sons, L.L.C. IP-10 antibodies and their uses
WO2012024242A1 (fr) * 2010-08-16 2012-02-23 Amgen Inc. Anticorps se liant à la myostatine, compositions et procédés
US8629257B2 (en) 2005-06-30 2014-01-14 Abbvie Inc. IL-12/p40 binding proteins
US9429581B2 (en) 2011-04-29 2016-08-30 Bristol-Myers Squibb Company IP-10 antibody dosage escalation regimens
WO2017220989A1 (fr) 2016-06-20 2017-12-28 Kymab Limited Anti-pd-l1 et cytokines il-2
US10556948B2 (en) 2015-11-30 2020-02-11 Bristol-Myers Squibb Company IP-10 antibodies and their uses
CN120737199A (zh) * 2025-09-03 2025-10-03 内蒙古博约微宏生物科技有限公司 一种抗ip10重组兔单克隆抗体对及其应用

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US6140064A (en) * 1996-09-10 2000-10-31 Theodor-Kocher Institute Method of detecting or identifying ligands, inhibitors or promoters of CXC chemokine receptor 3

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EP1708961A4 (fr) * 2003-12-04 2008-05-28 Pdl Biopharma Inc Traitement de maladies intestinales inflammatoires avec des anticorps anti-ip10
US8258266B2 (en) 2003-12-10 2012-09-04 Medarex, Inc. IP-10 antibodies and their uses
US9994633B2 (en) 2003-12-10 2018-06-12 Bristol-Myers Squibb Company IP-10 antibodies and their uses
US8945546B2 (en) 2003-12-10 2015-02-03 Medarex, L.L.C. IP-10 antibodies and their uses
AU2004298492B2 (en) * 2003-12-10 2011-08-18 E. R. Squibb & Sons, L.L.C. IP-10 antibodies and their uses
US7786266B2 (en) 2005-05-12 2010-08-31 Oncotherapy Science, Inc. Methods for damaging cells using effector function of anti-DSC2 antibody
WO2006121207A1 (fr) * 2005-05-12 2006-11-16 Oncotherapy Science, Inc. Procedes pour endommager des cellules en utilisant une fonction effectrice d’un anticorps anti-dsc2
US8629257B2 (en) 2005-06-30 2014-01-14 Abbvie Inc. IL-12/p40 binding proteins
US7786268B2 (en) 2007-02-28 2010-08-31 Novimmune Sa Anti-IP-10 antibodies and methods of use thereof
US8258267B2 (en) 2007-02-28 2012-09-04 Novimmune S.A. Human anti-IP-10 antibodies uses thereof
US8110661B2 (en) 2007-02-28 2012-02-07 Novlmmune S.A. Anti-IP-10 antibodies and methods of use thereof
WO2009026117A3 (fr) * 2007-08-16 2009-12-30 Glaxo Group Limited Nouveaux composés
US8999343B2 (en) 2010-08-16 2015-04-07 Amgen Inc. Antibodies that bind myostatin, compositions and methods
WO2012024242A1 (fr) * 2010-08-16 2012-02-23 Amgen Inc. Anticorps se liant à la myostatine, compositions et procédés
US9429581B2 (en) 2011-04-29 2016-08-30 Bristol-Myers Squibb Company IP-10 antibody dosage escalation regimens
US10556948B2 (en) 2015-11-30 2020-02-11 Bristol-Myers Squibb Company IP-10 antibodies and their uses
US11708405B2 (en) 2015-11-30 2023-07-25 Bristol-Myers Quibb Company IP-10 antibodies and their uses
WO2017220989A1 (fr) 2016-06-20 2017-12-28 Kymab Limited Anti-pd-l1 et cytokines il-2
WO2017220988A1 (fr) 2016-06-20 2017-12-28 Kymab Limited Anticorps multispécifiques pour l'immuno-oncologie
WO2017220990A1 (fr) 2016-06-20 2017-12-28 Kymab Limited Anticorps anti-pd-l1
CN120737199A (zh) * 2025-09-03 2025-10-03 内蒙古博约微宏生物科技有限公司 一种抗ip10重组兔单克隆抗体对及其应用
CN120737199B (zh) * 2025-09-03 2025-12-02 内蒙古博约微宏生物科技有限公司 一种抗ip10重组兔单克隆抗体对及其应用

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