US20140065164A1 - Ip-10 antibody dosage escalation regimens - Google Patents

Ip-10 antibody dosage escalation regimens Download PDF

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US20140065164A1
US20140065164A1 US14/114,652 US201214114652A US2014065164A1 US 20140065164 A1 US20140065164 A1 US 20140065164A1 US 201214114652 A US201214114652 A US 201214114652A US 2014065164 A1 US2014065164 A1 US 2014065164A1
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antibody
mdx
antibodies
seq
chain variable
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Allison Y. Luo
Wendy L. Trigona
Jinshan Shen
Li-An Xu
Yan Zhang
Bruce Stouffer
Haibin Chen
Haichun Huang
Xiaolu Tao
Catherine Brockus
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Bristol Myers Squibb Co
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Bristol Myers Squibb Co
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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/686—Anti-idiotype
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • C07K16/24—Immunoglobulins [IGs], 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 [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • 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
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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

Definitions

  • Interferon gamma inducible protein 10 (also known as CXCL10) is a 10 kDa chemokine that is secreted by a variety of cells, including endothelial cells, monocytes, fibroblasts, and keratinocytes, in response to IFN-gamma. IP-10 is also present in dermal macrophages and endothelial cells in delayed type hypersensitivity (DTH) responses in human. Although originally identified based on its being induced by IFN-gamma, IP-10 can also be induced by IFN-alpha, for example in dendritic cells. IP-10 expression can also be induced in cells of the central nervous system, such as astrocytes and microglia, by stimuli such as IFN-gamma, viruses and lipopolysaccharide.
  • CXCL10 Interferon gamma inducible protein 10
  • the receptor for IP-10 has been identified as CXCR3, a seven transmembrane receptor.
  • CXCR3 is expressed on activated T lymphocytes but not on resting T lymphocytes, nor on B lymphocytes, monocytes or granulocytes.
  • CXCR3 expression is upregulated on NK cells by stimulation with TGF-beta 1.
  • MIG and ITAC Two other ligands for CXCR3 are identified: MIG and ITAC. Binding of IP-10 to CXCR3 mediates calcium mobilization and chemotaxis in activated T cells. Chemotaxis and intracellular calcium mobilization are also induced by IP-10 binding to CXCR3 on activated NK cells.
  • IP-10 is a chemoattractant for TCR ⁇ + CD8 + T cells, TCR ⁇ + T cells and NK-type cells.
  • IP-10 or its receptor CXCR3 have been identified in a variety of different inflammatory and autoimmune conditions, including multiple sclerosis, rheumatoid arthritis, ulcerative colitis, hepatitis, spinal cord injury, systemic lupus erythematosus, transplant rejection, Sjögren's syndrome. Accordingly, there is a need for therapeutic agents (e.g., anti-IP10 antibodies) as well as methods for the treatment of IP-10 related diseases (e.g., inflammatory and autoimmune conditions).
  • therapeutic agents e.g., anti-IP10 antibodies
  • methods for the treatment of IP-10 related diseases e.g., inflammatory and autoimmune conditions.
  • the present invention provides a method of treating an IP10-related disease in a subject in need of treatment.
  • Such method comprises: (a) administering to the subject a predetermined dosage of an anti-IP10 antibody; (b) detecting the level of the anti-IP10 antibody in a sample of the subject; and (c) if the level of the anti-IP10 antibody from step (b) is below a threshold exposure level, increasing the dosage of the anti-IP10 antibody in the subject such that the IP-10 related disease in the subject is treated.
  • the anti-IP10 antibody used in the methods specifically binds to human IP-10 and does not cross-react with human MIG or human ITAC.
  • the anti-IP10 antibody is MDX-1100 (a fully human monoclonal antibody).
  • An exemplary anti-IP10 antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7.
  • Another exemplary anti-IP10 antibody comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 3; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 4; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 5; (d) a light chain variable region CDR1 comprising SEQ ID NO: 8; (e) a light chain variable region CDR2 comprising SEQ ID NO: 9; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 10.
  • step (b) of the above described method is performed by detecting the level of the anti-IP10 antibody via a method which comprises contacting said sample with an antibody which binds to the anti-IP10 antibody under conditions suitable for antibody-antigen complex formation, followed by the detection of the antibody-antigen complex formation.
  • the antibody which binds to the anti-IP10 antibody is an anti-idiotypic antibody.
  • the anti-idiotypic antibody binds to one or more CDRs of MDX-1100.
  • Exemplary anti-idiotypic antibodies include, but are not limited to, 10C8, 6C9, 2F5, and 23H10 as described in the working examples.
  • the detection method utilizes two anti-idiotypic antibodies, i.e., 10C8 and 23H10, as capture antibody and a detectable antibody (also referable to as “detection antibody”), respectively.
  • detection is accomplished by a means selected from the group consisting of EIA, ELISA, RIA, indirect competitive immunoassay, direct competitive immunoassay, non-competitive immunoassay, sandwich immunoassay, and agglutination assay.
  • the IP10-related disease of the above-described method is an inflammatory or autoimmune disease.
  • the inflammatory or autoimmune diseases include, but are not limited to, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), systemic lupus erythematosus, Type I diabetes, inflammatory skin disorders (e.g., psoriasis, lichen planus), autoimmune thyroid disease (e.g., Graves' disease, Hashimoto's thyroiditis), Sjögren's syndrome, pulmonary inflammation (e.g., asthma, chronic obstructive pulmonary disease, pulmonary sarcoidosis, lymphocytic alveolitis), transplant rejection, spinal cord injury, brain injury (e.g., stroke), neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease), gingivitis, gene therapy-induced inflammation, diseases of angiogenesis, inflammatory kidney disease (e.
  • the present invention provides an isolated monoclonal antibody (e.g., an anti-idiotypic antibody) or an antigen binding portion thereof, which specifically binds to an anti-IP10 antibody.
  • the anti-IP10 antibody is MDX-1100.
  • An exemplary anti-IP10 antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7.
  • Another exemplary anti-IP10 antibody comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 3; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 4; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 5; (d) a light chain variable region CDR1 comprising SEQ ID NO: 8; (e) a light chain variable region CDR2 comprising SEQ ID NO: 9; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 10.
  • Exemplary anti-idiotypic antibodies include, but are not limited to, 10C8, 6C9, 2F5, and 23H10 as described in the working examples.
  • the present invention provides a hybridoma cell line which produce a monoclonal antibody (e.g., an anti-idiotypic antibody) or an antigen binding portion thereof, which specifically binds to an anti-IP10 antibody (e.g., MDX-1100).
  • a monoclonal antibody e.g., an anti-idiotypic antibody
  • an antigen binding portion thereof which specifically binds to an anti-IP10 antibody (e.g., MDX-1100).
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising (1) a monoclonal antibody (e.g., an anti-idiotypic antibody) or an antigen binding portion thereof, which specifically binds to an anti-IP10 antibody (e.g., MDX-1100), and (2) a pharmaceutically acceptable carrier.
  • a monoclonal antibody e.g., an anti-idiotypic antibody
  • an anti-IP10 antibody e.g., MDX-1100
  • the present invention provides a method of detecting a therapeutic anti-IP 10 antibody (e.g., MDX-1100) in a sample.
  • a therapeutic anti-IP 10 antibody e.g., MDX-1100
  • Such method comprises contacting said sample with an antibody against the anti-IP10 antibody (e.g., an anti-idiotypic antibody), or antigen-binding portion thereof, under conditions suitable for antibody-antigen complex formation, followed by the detection of said complex formation.
  • the anti-idiotypic antibody binds to one or more CDRs of MDX-1100.
  • Exemplary anti-idiotypic antibodies include, but are not limited to, 10C8, 6C9, 2F5, and 23H10 as described in the working examples.
  • the detection method utilizes two anti-idiotypic antibodies, i.e., 10C8 and 23H10, as capture antibody and a detectable antibody (also referable to as “detection antibody”), respectively.
  • detection is accomplished by a means selected from the group consisting of EIA, ELISA, RIA, indirect competitive immunoassay, direct competitive immunoassay, non-competitive immunoassay, sandwich immunoassay, and agglutination assay.
  • the present invention provides a kit which comprises (1) a monoclonal antibody (e.g., an anti-idiotypic antibody) or an antigen binding portion thereof, which specifically binds to an anti-IP10 antibody (e.g., MDX-1100), and (2) reagents necessary for facilitating an antibody-antigen complex formation.
  • a monoclonal antibody e.g., an anti-idiotypic antibody
  • an anti-IP10 antibody e.g., MDX-1100
  • the present invention provides a method of treating an IP10-related disease in a subject in need of treatment, comprising: (a) administering to the subject an anti-IP10 antibody; (b) detecting the level of the anti-IP10 antibody in a sample of the subject by an immunoassay; and (b) increasing the dosage of the anti-IP10 antibody in the subject if the level of the anti-IP10 antibody is below a threshold exposure level; and not increasing the dosage of the anti-IP10 antibody in the subject if the level of the anti-IP10 antibody is at or above a threshold exposure level.
  • FIG. 1 shows Exposure-Response (E-R) relationship of clinical response, clinical remission and mucosal healing rates stratified by Cminss of MDX-1100 at Study Day 57.
  • FIG. 2 shows logistic regression analysis of clinical response rate vs. Cminss of MDX-1100 at Study Day 57.
  • FIG. 3 shows logistic regression analysis of clinical remission rate vs. Cminss of MDX-1100 at Study Day 57.
  • FIG. 4 shows logistic regression analysis of mucosal healing rate vs. Cminss of MDX-1100 at Study Day 57.
  • FIG. 5 shows the negative log 10 transformed p-values vs. different Cminss as possible target exposure.
  • FIGS. 6A and 6B show analysis of binding activities of two anti-idiotypic Clones 10C8 and 6C9.
  • FIG. 7 shows analysis of binding activities of two anti-idiotypic Clones 2F5 and 23H10.
  • FIG. 8 shows analysis of binding activities of Subclone of 10C8.
  • FIG. 9 shows analysis of binding activities of Subclones of 6C9, 2F5 and 23H10.
  • FIG. 10 shows analysis of binding activities of Clone 10C8 competition with IP 10.
  • FIG. 11 shows analysis of binding activities of Clone 2F5 and 23H10 competition with IP10.
  • FIG. 12A shows the nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of the heavy chain variable region of the 6A5 human monoclonal antibody.
  • the CDR1 (SEQ ID NO: 3), CDR2 (SEQ ID NO: 4) and CDR3 (SEQ ID NO: 5) regions are delineated and the V, D and J germline derivations are indicated.
  • FIG. 12B shows the nucleotide sequence (SEQ ID NO: 6) and amino acid sequence (SEQ ID NO: 7) of the light chain variable region of the 6A5 human monoclonal antibody.
  • the CDR1 (SEQ ID NO: 8), CDR2 (SEQ ID NO: 9) and CDR3 (SEQ ID NO: 10) regions are delineated and the V and J germline derivations are indicated.
  • the present invention relates to isolated monoclonal antibodies, particularly human monoclonal antibodies, which bind specifically to IP-10 (herein referred to as “IP10 antibodies” or “anti-IP10 antibodies”) and inhibit functional properties of IP-10.
  • IP10 antibodies human monoclonal antibodies
  • anti-IP10 antibodies anti-IP10 antibodies
  • the present invention provides anti-idiotypic antibodies which bind to an IP-10 antibody.
  • the present invention provides methods of detecting an IP10 antibody in a biologic sample using such anti-idiotypic antibodies.
  • the invention provides novel and effective methods of treating IP10-related diseases (e.g., inflammatory or autoimmune diseases), which comprise: (a) administering to the subject an anti-IP10 antibody; (b) detecting the level of the anti-IP10 antibody in a sample of the subject; and (c) increasing dosages of the anti-IP10 antibody to the subject if the level of the anti-IP10 antibody from step (b) is below a level, such that the IP-10 related disease in the subject is treated.
  • IP10-related diseases e.g., inflammatory or autoimmune diseases
  • the present invention provides a method of treating an IP10-related disease in a subject in need of treatment, comprising: (a) administering to the subject an anti-IP10 antibody; (b) detecting the level of the anti-IP10 antibody in a sample of the subject by an immunoassay; and (b) increasing the dosage of the anti-IP10 antibody in the subject if the level of the anti-IP10 antibody is below a threshold exposure level; and not increasing the dosage of the anti-IP10 antibody in the subject if the level of the anti-IP10 antibody is at or above a threshold exposure level.
  • IP-10 interferon gamma inducible protein 10
  • CXCL10 CXCL10
  • human IP10 antibodies of the invention may, in certain cases, cross-react with IP-10 from species other than human. In other cases, the antibodies may be completely specific for human IP-10 and may not exhibit species or other types of cross-reactivity.
  • the complete amino acid sequence of human IP-10 has GENBANK® accession number NP — 001556.
  • the complete amino acid sequence of rhesus monkey IP-10 has GENBANK® accession number AAK95955.
  • the complete amino acid sequence of mouse IP-10 has GENBANK® accession number NP — 067249.
  • CXCR3 refers to the receptor for IP-10 (CXCL10).
  • CXCL10 IP-10
  • the complete amino acid sequence of human CXCR3 has GENBANK® accession number NP — 001495.
  • MIG refers to a ligand for CXCR3, also know as monokine induced by gamma interferon, which is distinct from IP-10.
  • the complete amino acid sequence of human MIG has GENBANK® accession number NP — 002407.
  • ITAC refers to a ligand for CXCR3, also known as interferon-inducible T cell alpha chemoattractant, which is distinct from IP-10.
  • the complete amino acid sequence of human ITAC has GENBANK® accession number NP — 005400.
  • antibody as referred to herein includes whole antibodies and any antigen binding fragment (i.e., “antigen-binding portion”) or single chains thereof.
  • An “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, C H1 , C H2 and C H3 .
  • Each light chain is comprised of a light chain variable region (abbreviated herein as V L ) and a light chain constant region.
  • the light chain constant region is comprised of one domain, C L .
  • the V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • antibody portion refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., IP-10 or an IP10 antibody). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V H , V L , C L and C H1 domains; (ii) a F(ab′) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C H1 domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature, 341:544-546 (1989)), which consists of a V H domain; and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the V H , V L , C L and C H1 domains
  • F(ab′) 2 fragment a bivalent fragment comprising
  • the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., Science, 242:423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988)).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody.
  • an “isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds IP-10 is substantially free of antibodies that specifically bind antigens other than IP-10).
  • An isolated antibody that specifically binds IP-10 may, however, have cross-reactivity to other antigens, such as IP-10 molecules from other species.
  • an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • human antibody is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.
  • the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term “human antibody”, as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • human monoclonal antibody refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
  • recombinant human antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V H and V L regions of the recombinant antibodies are sequences that, while derived from and related to human germline V H and V L sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • the present invention provides monoclonal or polyclonal antibodies specific for an anti-IP10 antibody (e.g., MDX-1100).
  • an anti-IP10 antibody e.g., MDX-1100
  • an anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody.
  • An Id antibody can be prepared by immunizing an animal with an antibody to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody). See, e.g., U.S. Pat. No. 4,699,880. Exemplary techniques for the production of monoclonal antibodies which bind to an anti-IP10 antibody are provided below.
  • Monoclonal antibodies may be obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
  • the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
  • a mouse or other appropriate host animal such as a hamster
  • lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
  • lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press (1986)).
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • HAT medium hypoxanthine, aminopterin, and thymidine
  • Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • exemplary myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2, P3X63Ag.U.1, or X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Va. USA.
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , pp. 51-63, Marcel Dekker, Inc., New York (1987)).
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antibody of interest.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or ELISA.
  • RIA radioimmunoassay
  • ELISA ELISA-Linked Immunosorbent assay
  • Such clones are also screened for those that produce the least background noise in the assay when used as capture reagents and/or detectable antibodies.
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press (1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-SEPHAROSE® agarose chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • One specific preparation technique using hybridoma technology comprises immunizing mice such as CAF1 mice or Balb/c, for example, by injection in the footpads or spleen, with the antibody of interest in an adjuvant such as monophosphoryl lipid A/trehalose dicorynomycolate or as a conjugate of the antibody of interest with keyhole limpet haemocyanin (KLH) or with Limulus hemocyanin. Injections are done as many times as needed.
  • an adjuvant such as monophosphoryl lipid A/trehalose dicorynomycolate or as a conjugate of the antibody of interest with keyhole limpet haemocyanin (KLH) or with Limulus hemocyanin.
  • KLH keyhole limpet haemocyanin
  • Limulus hemocyanin Limulus hemocyanin
  • mice are sacrificed and popliteal lymph nodes or splenocytes obtained from the immunized mice, especially those with high titers, are fused with a murine myeloma cell line such as SP2/0 or P3X63Ag.U.1 (American Type Culture Collection (ATCC, Manassas, Va.)).
  • a murine myeloma cell line such as SP2/0 or P3X63Ag.U.1 (American Type Culture Collection (ATCC, Manassas, Va.)).
  • the resulting hybridomas are screened for antibodies with binding affinity for the antibody of interest but not other antibodies binding to a different antigen. This screening may take place by conventional ELISA for secretion of antibody that binds to immobilized antibody of interest or for production of IgG with an inhibition capacity of more than about 95% (inhibition of binding of the antibody of interest to the protein antigen).
  • This screen defines a population of antibodies with nominal or higher reactivity as well as selectivity for the antibody of interest. Further selection may be performed to identify those antibodies with properties especially preferred for ELISAs.
  • the criteria used for selecting a preferred anti-idiotypic antibody include that it binds to the antibody of interest with relatively high affinity (Kd ⁇ about 10 ⁇ 8 M), and that its binding to the antibody of interest should be mutually exclusive with binding to the analyte transmembrane protein. It should also provide the cleanest assay with the least background noise.
  • the positive clones may be re-screened using surface plasmon resonance using a BIACORE® instrument to measure the affinity of the anti-idiotypic antibody for the antibody of interest (as reflected in its off-rate) and the mutual exclusivity of binding.
  • Rabbit anti-mouse IgG(Fc) may be immobilized onto the biosensor surface and used to capture anti-idiotypic antibodies from hybridoma culture supernates.
  • the antibody of interest at 0.2 nM alone and in the presence of 0.9 nM C-reactive protein (CRP) may be injected over the surface of the immobilized anti-idiotypic antibody and the relative mass accumulation compared.
  • CRP C-reactive protein
  • the hybridoma cells that are selected are cloned as by limiting dilution to obtain the desired clones.
  • the anti-idiotypic antibody can then be purified and isolated from these clones. See, e.g., U.S. Publication Nos. 2002/0142356 and 2008/0176257 for examples of preparing an anti-idiotypic antibody, as well as Durrant et al., Int. J. Cancer, 1:92(3):414-420 (2001) and Bhattacharya-Chatterjee, Curr. Opin. Mol. Ther., 3(1):63-69 (2001).
  • the monoclonal antibodies may also be produced recombinantly.
  • DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (see, e.g., U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalently joining to the immunoglobulin-coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • the antibodies against an anti-IP10 antibody (e.g., MDX-1100) or antigen binding portion thereof, of the invention can be used in a method for the detection of a therapeutic anti-IP10 antibody (e.g., MDX-1100) and fragments and derivatives thereof in a subject.
  • a therapeutic anti-IP10 antibody e.g., MDX-1100
  • fragments and derivatives thereof e.g., fragments and derivatives thereof in a subject.
  • such antibody is an anti-idiotypic (anti-Id) antibody.
  • a body fluid e.g., blood, serum or plasma
  • tissue sample from the test subject is contacted with an anti-MDX-1100 monoclonal antibody, or antigen binding portion thereof, of the invention under conditions suitable for the formation of antibody-antigen complexes.
  • the presence or amount of such complexes can then be determined by methods described herein and otherwise known in the art (see, e.g., O'Connor et al., Cancer Res., 48:1361-1366 (1988)), in which the presence or amount of complexes found in the test sample is compared to the presence or amount of complexes found in a series of standards or control samples containing a known amount of antigen.
  • the present invention relates to methods for detecting an anti-IP10 antibody such as MDX-1100 (or a fragment and/or derivative thereof) in a biological sample (e.g., blood, serum, plasma, urine, cerebrospinal fluid, mucus, or saliva).
  • the method can employ an immunoassay, e.g., an enzyme immunoassay (EIA), enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), indirect competitive immunoassay, direct competitive immunoassay, non-competitive immunoassay, sandwich immunoassay, agglutination assay or other immunoassay describe herein and known in the art (see, e.g., Zola, Monoclonal Antibodies: A Manual of Techniques , pp. 147-158, CRC Press, Inc. (1987)) Immunoassays may be constructed in heterogeneous or homogeneous formats.
  • EIA enzyme immunoassay
  • ELISA enzyme-linked immunosorbant assay
  • RIA radioimmunoassay
  • indirect competitive immunoassay direct competitive immunoassay
  • non-competitive immunoassay non-competitive immunoassay
  • sandwich immunoassay sandwich immunoassay
  • Heterogeneous immunoassays are distinguished by incorporating a solid phase separation of bound analyte from free analyte or bound label from free label.
  • Solid phases can take a variety of forms well known in the art, including but not limited to tubes, plates, beads, and strips. One particular form is the microtiter plate.
  • the solid phase material may be comprised of a variety of glasses, polymers, plastics, papers, or membranes. Particularly desirable are plastics such as polystyrene.
  • Heterogeneous immunoassays may be competitive or non-competitive (i.e., sandwich formats) (see, e.g., U.S. Pat. No. 7,195,882).
  • the present invention provides a method of detecting MDX-1100 in a biologic sample from a subject, which comprises the following steps (see below).
  • the biological sample is contacted and incubated with an immobilized capture antibody such as an anti-idiotypic antibody directed against MDX-1100.
  • immobilized capture antibody such as an anti-idiotypic antibody directed against MDX-1100.
  • anti-idiotypic antibodies are preferably monoclonal antibodies, and may be from any species, but preferably they are rodent, more preferably murine (e.g., 10C8, 6C9, 2F5, and 23H10 as described in the working examples).
  • Immobilization conventionally is accomplished by insolubilizing the capture antibody either before the assay procedure, as by adsorption to a water-insoluble matrix or surface (U.S. Pat. No.
  • non-covalent or covalent coupling for example, using glutaraldehyde or carbodiimide cross-linking, with or without prior activation of the support with, e.g., nitric acid and a reducing agent as described in U.S. Pat. No. 3,645,852 or in Rotmans et al., J. Immunol. Methods, 57:87-98 (1983)), or afterward, e.g., by immunoprecipitation.
  • glutaraldehyde or carbodiimide cross-linking with or without prior activation of the support with, e.g., nitric acid and a reducing agent as described in U.S. Pat. No. 3,645,852 or in Rotmans et al., J. Immunol. Methods, 57:87-98 (1983)
  • afterward e.g., by immunoprecipitation.
  • the solid phase used for immobilization may be any inert support or carrier that is essentially water insoluble and useful in immunometric assays, including supports in the form of, e.g., surfaces, particles, porous matrices, etc.
  • supports in the form of, e.g., surfaces, particles, porous matrices, etc.
  • commonly used supports include small sheets, SEPHADEX® gels, polyvinyl chloride, plastic beads, and assay plates or test tubes manufactured from polyethylene, polypropylene, polystyrene, and the like, including 96-well microtiter plates, as well as particulate materials such as filter paper, agarose, cross-linked dextran, and other polysaccharides.
  • reactive water-insoluble matrices such as cyanogens-bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are suitably employed for capture-reagent immobilization.
  • the immobilized capture antibody is coated on a microtiter plate, and in particular the solid phase used is a multi-well microtiter plate that can be used to analyze several samples at one time. The most preferred is a MICROTEST® or MaxiSorp 96-well ELISA plate such as that sold as NUNC® MaxiSorb or IMMULON®.
  • the solid phase is coated with the capture antibody as defined above, which may be linked by a non-covalent or covalent interaction or physical linkage as desired. Techniques for attachment include those described in U.S. Pat. No. 4,376,110 and the references cited therein. If covalent, the plate or other solid phase is incubated with a cross-linking agent together with the capture antibody under conditions well known in the art such as for one hour at room temperature.
  • cross-linking agents for attaching the capture reagents to the solid-phase substrate include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane.
  • Derivatizing agents such as methyl-3-((p-azidophenyl)-dithio)propioimidate yield photoactivatable intermediates capable of forming cross-links in the presence of light.
  • the coated plates are then typically treated with a blocking agent that binds non-specifically to and saturates the binding sites to prevent unwanted binding of the free ligand to the excess sites on the wells of the plate.
  • a blocking agent that binds non-specifically to and saturates the binding sites to prevent unwanted binding of the free ligand to the excess sites on the wells of the plate.
  • appropriate blocking agents for this purpose include, e.g., gelatin, bovine serum albumin, egg albumin, casein, and non-fat milk.
  • the blocking treatment typically takes place under conditions of ambient temperatures for about 1-4 hours, preferably about 1.5 to 3 hours.
  • the conditions for incubation of sample and immobilized capture antibody are selected to maximize sensitivity of the assay and to minimize dissociation, and to ensure that any antibody of interest present in the sample binds to the immobilized capture antibody.
  • the incubation is accomplished at fairly constant temperatures, ranging from about 0° C. to about 40° C., preferably at or about room temperature.
  • the time for incubation is generally no greater than about 10 hours.
  • the incubation time is from about 0.5 to 3 hours, and more preferably about 1.5-3 hours at or about room temperature to maximize binding of the antibody of interest to the capture antibody.
  • the duration of incubation may be longer if a protease inhibitor is added to prevent proteases in the biological fluid from degrading the antibody of interest.
  • the biological sample is separated (preferably by washing) from the immobilized capture antibody to remove uncaptured antibody of interest (e.g., MDX-1100).
  • the washing may be done three or more times.
  • the temperature of washing is generally from refrigerator to moderate temperatures, with a constant temperature maintained during the assay period, typically from about 0-40° C., more preferably about 4-30° C.
  • a cross-linking agent or other suitable agent may also be added at this stage to allow the now-bound antibody of interest to be covalently attached to the capture reagents if there is any concern that the captured antibody of interest may dissociate to some extent in the subsequent steps.
  • the immobilized capture antibody with any bound antibody of interest are contacted with a detectable antibody, preferably at a temperature of about 20-40° C., more preferably about 36-38° C.
  • the detectable antibody may be a polyclonal or monoclonal antibody, preferably it is a monoclonal antibody, more preferably rodent, still more preferably murine.
  • a detectable antibody of the assay herein is an anti-idiotypic antibody against MDX-1100, such as 10C8, 6C9, 2F5, and 23H10 as described in the working examples.
  • the detectable antibody is directly detectable, and such as biotinylated.
  • the detection means for the biotinylated label is preferably avidin or streptavidin-HRP, and the readout of the detection means is preferably fluorimetric or colorimetric.
  • the same anti-idiotypic antibody can be used for coat (capture) and detection in the assay, or different antibodies can be used for coat (capture) and detection. They are preferably selected so that the background noise is minimized.
  • the level of any free antibody of interest (e.g., MDX-1100) from the sample that is now bound to the capture antibody is measured using a detection means for the detectable antibody.
  • the measuring step preferably comprises comparing the reaction that occurs as a result of the above three steps with a standard curve to determine the level of antibody of interest compared to the known amount.
  • the detectable antibody (herein referred to as the “first antibody”) will be either directly labeled, or detected indirectly by addition, after washing off of excess first antibody, of a molar excess of a second, labeled antibody directed against IgG of the animal species of the first antibody. In the latter, indirect assay, labeled antisera against the first antibody are added to the sample so as to produce the labeled antibody in situ.
  • the label used for either the first or second antibody is any detectable functionality that does not interfere with the binding of free antibody of interest to the anti-idiotypic antibodies.
  • suitable labels are those numerous labels known for use in immunoassay, including moieties that may be detected directly, such as fluorochrome, chemiluminscent, and radioactive labels, as well as moieties, such as enzymes, that must be reacted or derivatized to be detected.
  • suitable labels include the radioisotopes 32 P, 14 C, 125 I, 3 H, and 131 I, fluorophores such as rare-earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (see, e.g., U.S.
  • luciferin 2,3-dihydrophthalazinediones, HRP, alkaline phosphatase, galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin (detectable by, e.g., avidin, streptavidin, streptavidin-HRP, and streptavidin-( ⁇ -galactosidase with MUG), spin labels, bacteriophage labels, stable free radicals, and the like.
  • the label is biot
  • coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized benzidine, and the like may be used to tag the antibodies with the above-described fluorescent, chemiluminescent, and enzyme labels. See, e.g., U.S. Pat. No. 3,940,475 (fluorimetry) and U.S. Pat. No. 3,645,090 (enzymes); Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014-1021 (1974); Pain et al., J. Immunol.
  • An exemplary label is biotin using streptavidin-HRP for detection means.
  • the conjugation of such label, including the enzymes, to an antibody is a standard manipulative procedure for one of ordinary skill in immunoassay techniques. See, e.g., O'Sullivan et al. “Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay,” in Methods in Enzymology, Langone, J. J. and Van Vunakis, H., eds. Vol. 73, pp. 147-166, Academic Press, New York, N.Y. (1981).
  • the amount of bound antibody is determined by removing excess unbound labeled antibody through washing and then measuring the amount of the attached label using a detection method appropriate to the label, and correlating the measured amount with the amount of the antibody of interest in the biological sample.
  • the amount of color developed and measured will be a direct measurement of the amount of the antibody of interest present.
  • HRP is the label
  • the color is detected using the substrate OPD at 490-nm absorbance.
  • color or chemiluminiscence is developed and measured by incubating the immobilized capture reagent with a substrate of the enzyme. Then the concentration of the antibody of interest is calculated by comparing with the color or chemiluminescence generated by the standard antibody of interest run in parallel.
  • kits that can be used in the assays described above, which comprise one or more antibodies (monoclonal or polyclonal) against an anti-IP10 antibody of interest (e.g., MDX-1100), or an antigen binding portion thereof as well as reagents necessary for facilitating an antibody-antigen complex formation and/or detection.
  • an anti-IP10 antibody of interest e.g., MDX-1100
  • reagents necessary for facilitating an antibody-antigen complex formation and/or detection e.g., such antibodies of the kits are anti-idiotypic antibodies.
  • a kit of the present invention is a packaged combination including the basic elements of: (a) capture reagents comprising at least one anti-idiotypic antibody against MDX-1100 (herein referred to as a “capture antibody”); (b) at least one detectable (labeled or unlabeled) anti-idiotypic antibody that binds to a different epitope on MDX-1100; and (c) instructions on how to perform the assay method using these reagents.
  • the kit further comprises a solid support for the capture antibodies, which may be provided as a separate element or on which the capture antibodies are already immobilized.
  • the capture antibodies in the kit may be immobilized on a solid support, or they may be immobilized on such support that is included with the kit or provided separately from the kit.
  • the capture antibodies are coated on a microtiter plate.
  • the detectable antibodies may be labeled antibodies detected directly or unlabeled antibodies that are detected by labeled antibodies directed against the unlabeled antibodies raised in a different species.
  • the kit will ordinarily include substrates and cofactors required by the enzyme, where the label is a fluorophore, a dye precursor that provides the detectable chromophore, and where the label is biotin, an avidin such as avidin, streptavidin, or streptavidin conjugated to HRP or ⁇ -galactosidase with MUG.
  • the capture antibodies are anti-idiotypic antibodies selected from 10C8, 6C9, 2F5, and 23H10 as described in the working examples.
  • the detectable antibodies are anti-idiotypic selected from 10C8, 6C9, 2F5, and 23H10, wherein the capture antibody and the detectable antibody bind to different epitopes on MDX-1100.
  • the kit may further comprise, as a positive control, the antibody of interest (e.g., purified MDX-1100) or a fragment thereof which binds to the anti-idotypic antibody.
  • the kits may further comprise, as a negative control, an antibody which does not react with the anti-idiotypic antibody.
  • the kit may further comprise other additives such as stabilizers, washing and incubation buffers, and the like.
  • the components of the kit will be provided in predetermined ratios, with the relative amounts of the various reagents suitably varied to provide for concentrations in solution of the reagents that substantially maximize the sensitivity of the assay.
  • the reagents may be provided as dry powders, usually lyophilized, including excipients, which on dissolution will provide for a reagent solution having the appropriate concentration for combining with the sample to be tested.
  • the invention provides novel and effective methods of treating IP10-related diseases (e.g., inflammatory or autoimmune diseases), which comprise: (a) administering to the subject a predetermined dosage of an anti-IP10 antibody; (b) detecting the level of the anti-IP10 antibody in a sample of the subject; and (c) if the level of the anti-IP10 antibody from step (b) is below a threshold exposure level, increasing the dosage of the anti-IP10 antibody (e.g., to a therapeutically effective dosage) in the subject, such that the IP-10 related disease in the subject is treated; and if the level of the anti-IP10 antibody from step (b) is at or above a threshold exposure level, not increasing the dosage of the anti-IP10 antibody in the subject.
  • the level of the anti-IP10 antibody in a sample can be detected by any of assays as described above.
  • treating includes the administration of anti-IP10 antibodies to prevent or delay the onset of the symptoms, complications, or biochemical indicia of an IP10-related disease, alleviating the symptoms or arresting or inhibiting further development of the disease (e.g., an inflammatory or autoimmune disease).
  • Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.
  • dose refers to an amount of an anti-IP10 antibody which is administered to a subject.
  • terapéuticaally effective dosage refers to a dosage of an anti-IP-10 antibody which preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
  • One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • threshold exposure level refers to a minimum exposure level which allows for clinically meaningful induction and/or maintenance of disease remission after administering an anti-IP10 antibody in a subject during the induction phase and/or maintenance phase.
  • the threshold exposure level can be readily determined, such as by the exposure-response analyses as described in the working examples.
  • the threshold exposure level can be a trough concentration ranging from 40-150 ⁇ g/mL (e.g., 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 ⁇ g/mL).
  • the present invention is based, at least in part, on observations made during clinical testing of a human anti-IP10 antibody (i.e., MDX-1100) in treating an inflammatory bowel disease (IBD), as described below.
  • MDX-1100 a human anti-IP10 antibody
  • IBD inflammatory bowel disease
  • the tests demonstrate variability in MDX-1100's pharmacokinetic (PK) parameters in patients treated with MDX-1100. Meanwhile, the tests demonstrate very low immunogenicity of MDX-1100 in this study. Furthermore, the tests demonstrate a strong drug exposure/response relationship whereby trough drug levels were directly related to efficacy.
  • the present invention provides a method of treating an IP-10-related disorder, which comprises: (1) administering to a subject with a maintenance dosage (e.g., a predetermined dosage) of an anti-IP10 antibody; (2) if the subject fails to maintain response (also referred to as “lose response” or “relapse”), a diagnostic assay will be used to measure the exposure level (e.g., blood concentration) of the anti-IP10 antibody in the subject; (3)) if the exposure level of the anti-IP10 antibody is below a threshold exposure level, escalating the dosage in the subject, such that drug response is maintained in the subject.
  • a maintenance dosage e.g., a predetermined dosage
  • a diagnostic assay will be used to measure the exposure level (e.g., blood concentration) of the anti-IP10 antibody in the subject
  • the exposure level of the anti-IP10 antibody is below a threshold exposure level, escalating the dosage in the subject, such that drug response is maintained in the subject.
  • the present invention relates to use of anti-IP10 antibodies which are characterized by particular functional features or properties.
  • the antibodies bind specifically to human IP-10.
  • the antibodies may cross react with IP-10 from one or more non-human primates, such as rhesus monkey.
  • the antibodies do not cross react with mouse IP-10.
  • MIG and ITAC are also ligands for the CXCR3 receptor
  • the antibodies of the invention preferably do not cross react with human MIG or human ITAC.
  • the antibodies of the invention are capable of inhibiting one or more functional activities of IP-10.
  • the antibodies inhibit the binding of IP-10 to CXCR3.
  • the antibodies inhibit IP-10 induced calcium flux.
  • the antibodies inhibit IP-10 induced cell migration (chemotaxis).
  • Other functional features or properties of anti-IP10 antibodies are also described in U.S. Publication No. 2005/0191293, the content of which is expressly incorporated by reference.
  • Exemplary anti-IP-10 antibodies are monoclonal antibodies 1D4, 1E1, 2G1, 3C4, 6A5, 6A8, 6B10, 7C10, 8F6, 10A12 and 13C4 as described in U.S. Publication No. 2005/0191293, the content of which is expressly incorporated by reference.
  • an anti-IP10 antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7.
  • an anti-IP 10 antibody comprises: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 3; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 4; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 5; (d) a light chain variable region CDR1 comprising SEQ ID NO: 8; (e) a light chain variable region CDR2 comprising SEQ ID NO: 9; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 10.
  • anti-IP-10 antibodies of the invention include antibodies whose heavy and light chain variable regions comprising amino acid sequences that are homologous to the amino acid sequences of the preferred antibodies described herein, and wherein the antibodies retain the desired functional properties of the anti-IP-10 antibodies of the invention.
  • an anti-IP10 antibody includes an antibody comprising: (a) the heavy chain variable region comprises an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2; (b) the light chain variable region comprises an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7.
  • an anti-IP10 antibody includes an antibody comprising: (a) a heavy chain variable region CDR1 comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 3; (b) a heavy chain variable region CDR2 comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 4; (c) a heavy chain variable region CDR3 comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 5; (d) a light chain variable region CDR1 comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 8; (e) a light chain variable region CDR2 comprising an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 9; and (f) a light chain variable region CDR3 comprising: (
  • any of the homologous antibody specifically binds to IP-10, and exhibits at least one of the following functional properties: (i) the antibody inhibits binding of IP-10 to CXCR3; (ii) the antibody inhibits IP-10 induced calcium flux; (iii) the antibody inhibits IP-10 induced cell migration; (iv) the antibody cross-reacts with rhesus monkey IP-10; (v) the antibody does not cross-react with mouse IP-10; (vi) the antibody does not cross-react with human MIG; (vii) the antibody does not cross-react with human ITAC.
  • Homologous anti-IP10 antibodies are also described in U.S. Publication No. 2005/0191293, the content of which is expressly incorporated by reference.
  • anti-IP-10 antibodies of the invention also include an anti-IP-10 antibody or a fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin.
  • a therapeutic moiety such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin.
  • conjugates are referred to herein as “immunoconjugates”
  • Immunoconjugates that include one or more cytotoxins are referred to as “immunotoxins.”
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells.
  • Examples include TAXOL®, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vin
  • An example of a calicheamicin antibody conjugate is commercially available (MYLOTARG®; Wyeth-Ayerst) Immunoconjugates of anti-IP10 antibodies are also described in U.S. Publication No. 2005/0191293, the content of which is expressly incorporated by reference.
  • anti-IP-10 antibodies of the invention further include bispecific molecules comprising an anti-IP-10 antibody or a fragment thereof.
  • An anti-IP10 antibody or antigen-binding portions thereof can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules.
  • An anti-IP10 antibody or a fragment thereof may in fact be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be encompassed by the term “bispecific molecule” as used herein.
  • an anti-IP10 antibody can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule results.
  • Bispecific anti-IP10 antibodies are also described in U.S. Publication No. 2005/0191293, the content of which is expressly incorporated by reference.
  • the present invention relates to use of anti-IP10 antibodies (including immunoconjugates and bispecific molecules) for treating IP10-related disorders in subjects.
  • subject as used herein in intended to include human and non-human animals.
  • Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles.
  • the methods are particularly suitable for treating human patients having a disorder associated with aberrant IP-10 expression.
  • antibodies to IP-10 are administered together with another agent, the two can be administered in either order or simultaneously.
  • IP10-related disease or disorder refers to a local and/or systemic physiological disorder where IP10 is a primary mediator leading to the manifestation of the disorder.
  • An exemplary IP10-related disorder is an inflammatory or autoimmune disease, including but not limited to, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), systemic lupus erythematosus, Type I diabetes, inflammatory skin disorders (e.g., psoriasis, lichen planus), autoimmune thyroid disease (e.g., Graves' disease, Hashimoto's thyroiditis), Sjögren's syndrome, pulmonary inflammation (e.g., asthma, chronic obstructive pulmonary disease, pulmonary sarcoidosis, lymphocytic alveolitis), transplant rejection, spinal cord injury, brain injury (e.g., stroke), neurodegenerative diseases (e.g., Alzheimer's disease
  • the present invention relates to a composition (e.g., a pharmaceutical composition) containing one or a combination of anti-IP10 monoclonal antibodies or antigen-binding portion(s) thereof, formulated together with a pharmaceutically acceptable carrier.
  • a composition may include one or a combination of (e.g., two or more different) antibodies, or immunoconjugates or bispecific molecules of the invention.
  • a pharmaceutical composition can comprise a combination of antibodies (or immunoconjugates or bispecifics) that bind to different epitopes on the target antigen or that have complementary activities.
  • the present invention provides combination therapy by using a pharmaceutical composition which comprises an anti-IP10 antibody combined with other agents.
  • the combination therapy can include an anti-IP-10 antibody combined with at least one other anti-inflammatory or immunosuppressant agent.
  • therapeutic agents that can be used in combination therapy are described in greater detail in U.S. Publication No. 2005/0191293, the content of which is expressly incorporated by reference.
  • “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the active compound i.e., antibody, immunoconjuage, or bispecific molecule
  • the pharmaceutical compounds may include one or more pharmaceutically acceptable salts.
  • Other ingredients of the pharmaceutical compositions are described in greater detail in U.S. Publication No. 2005/0191293, the content of which is expressly incorporated by reference.
  • Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of an anti-IP 10 antibody calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the dosage ranges from about 1 to 50 mg/kg of the host body weight.
  • dosages can be 1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg or 50 mg/kg, of body weight.
  • An exemplary treatment regime entails administration once per day, three times per week, twice per week, once per week, once every two weeks, once every three weeks, once every four weeks, once per month, once every 3 months or once every three to 6 months.
  • dosage regimens for an anti-IP-10 antibody include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks.
  • administration of an anti-IP10 antibody is by intravenous infusion during the induction phase, and by subcutaneous injection during the maintenance phase.
  • the frequency of administration may vary from once per day to once per month. If a subject fails to maintain response (also referred to as “lose response” or “relapse”) during the maintenance phase, a diagnostic assay will be used to measure the subject's exposure level (e.g., blood level) of the anti-IP10 antibody; (3) if the exposure level of the anti-IP10 antibody is below a threshold exposure level, escalating the dosage in the subject.
  • the dosage can be escalated in the subject by increasing the frequency of administration (e.g., increasing the frequency from once a week to twice a week).
  • two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated.
  • An anti-IP antibody is usually administered on multiple occasions. Intervals between single dosages can be, for example, weekly, monthly, every three months or yearly. Intervals can also be irregular as indicated by measuring blood levels of an IP-10 antibody in the patient. In certain aspects, dosage is adjusted to achieve a plasma concentration of about 1-600 ⁇ g/ml and in some methods about 25-300 ⁇ g/ml.
  • an anti-IP10 antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
  • an anti-IP10 antibody in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the anti-IP10 antibody employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the anti-IP10 antibody being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • MDX-1100 was studied for the induction of clinical response in patients with moderate to severely active ulcerative colitis (UC) in comparison to placebo.
  • E-R Exposure-response
  • the study was conducted in accordance with Good Clinical Practice, as defined by the International Conference on Harmonization and in accordance with the ethical principles underlying European Union Directive 2001/20/EC and the United States Code of Federal Regulations, Title 21, Part 50 (21CFR50) and in accordance with the ethical principles that have their origin in the Declaration of Helsinki.
  • the study protocol, amendments, and subject informed consent received appropriate approval by the Institutional Review Board (IRB)/Independent Ethics Committee (IEC) prior to initiation of study at the site.
  • IRB Institutional Review Board
  • IEC Independent Ethics Committee
  • the investigator Prior to the beginning of the study, the investigator provided each subject with the IRB/IEC's written approval/favorable opinion of the written informed consent form and any other relevant information. Freely given written informed consent was obtained from each subject, or, in those situations where consent could not be given by the subject, their legally acceptable representatives, prior to study participation, including informed consent for any screening procedures conducted to establish subject eligibility in the study.
  • Serum samples for pharmacokinetic evaluation of MDX-1100 were taken from each patient up to 60 minutes prior to each dose on Study Days 1, 15, 29, and 43 and on Study Day 57. Serum concentrations of MDX-1100 were assayed using a validated ELISA assay method. Serum trough concentrations of MDX-1100 on Study Day 57 were derived as steady-state trough concentration (Cminss) for each patient received MDX-1100 treatment.
  • Cminss steady-state trough concentration
  • Clinical response which was defined as a decrease from baseline (screening) in the total Mayo score of at least 3 points and at least 30% in a patient at Study Day 57, with an accompanying decrease in the subscore for rectal bleeding of at least 1 point or an absolute subscore for rectal bleeding of 0 or 1 (the clinical response rate was defined as the proportion of patients in each treatment group who had a clinical response);
  • Clinical remission which was defined as a total Mayo score of ⁇ 2 points with no individual subscore exceeding 1 point and no blood in stool in a patient at Study Day 57 (the clinical remission rate was defined as the proportion of patients in each treatment group who were in clinical remission); and (3) Mucosal healing, which was defined as endoscopy subscore of 0 or 1 in a patient at Study Day 57 (the mucosal healing rate was defined as the proportion of patients
  • the optimal target exposure to MDX-1100 was chosen to give the maximum separation between the patients whose exposure is less than the target exposure and the patients whose exposure is greater than the target exposure in terms of overall efficacy.
  • the target exposure to MDX-1100 was determined based on the following algorithm—for any given exposure c, a Fisher's exact test was performed to determine if there is a difference between patients whose exposure is less than c and patients whose exposure is greater than c for clinical response, clinical remission and mucosal healing rate.
  • the exposure with the minimum corresponding p-value was selected as the target exposure.
  • Cmin trough concentrations
  • the safety profile of patients treated with MDX-1100 and achieved Cminss in the highest tertile subgroup was comparable to the overall MDX-1100 safety population (Table 5).
  • the number of patients whose Cminss of MDX-1100 were in the highest tertile subgroup and experiencing at least one AE was also comparable to that in the placebo group (6/16 [37.5%] vs. 17/52 [32.7%], respectively).
  • b SAEs include all Grade 3 (severe) and above events (or events with missing severity) considered serious by the investigator up to 70 days post study drug.
  • the Grade level is based on USA NCI AE clarification guideline.
  • c Possibly, probably, or definitely related to the study drug (missing relationships presumed as related) 4.
  • MDX-1100 also referred to herein as 6A5
  • 6A5 is a human anti-IP10 antibody (see, e.g., U.S. Publication No. 2005/0191293).
  • MDX-1100 (10 mg/ml) was used for Fab preparation.
  • Fab fragment of MDX-1100 (2.92 mg/ml) was used as an immunogen in antibody generation.
  • mice were used to generate a few hybridomas, for example, 10C8, 6C9, 2F5, and 23H10. Table 6 below summarizes the hybridomas and the corresponding mice used in their production.
  • Fab of MDX-1100 was used to immunize the mice through ip, sc, and foot pad injection. Such immunization was conducted on five separate days. Subsequently, spleen and lymph nodes were harvested.
  • the Sp2/0 myeloma cell line was used for the fusions. Cells were maintained in culture for 1 month, passed twice a week. Supernatant from P388D1 (ATCC, TIB-63 FL) cells was used as conditioned media for the hybridomas. Briefly, cells were grown and expanded to 200 mL. Stationary cultures were grown for about 7 days. The exhausted supernatant was spun down and filtered through a 0.2 ⁇ m sterile filter. This cell line was passed for 1 month and then a new vial was thawed.
  • DMEM Gibco#12382-024 containing 10% FBS (HYCLONE®, cat #SH30071.03; Lot# ASL31024) were used to culture the myeloma fusion partner and P388D1 cells. Additional media supplements were added to the Hybridoma growth media, which included 5% Origen—Hybridoma Cloning Factor (IGEN®, cat #210001), 15% P388D1 conditioned media, ⁇ -mercaptoethanol (Gibco cat #1019091), Hepes (CELLGRO® #25060037) and HAT (Sigma, H 0262; 1.0 ⁇ 10 ⁇ 4 M Hypoxanthine, 4.0 ⁇ 10 ⁇ 7 M Aminopterin, 1.6 ⁇ 10 ⁇ 5 M Thymidine), or HT (Sigma, H0137; 1.0 ⁇ 10 4 M Hypoxanthine, 1.6 ⁇ 10 ⁇ 5 M Thymidine).
  • the viable cells yielded from the spleens and lymph nodes of the three mice for fusions were listed in Table 7 below. Electrode fusion was performed on the splenocytes. The resulting hybridomas were plated out into 96-well COSTAR® tissue-culture treated plates seeded at 200 ⁇ l/well.
  • the fusions were screened for antibodies directly binding to MDX1100 6A5 (whole 6A5, 6A5-biotin, or 6A5 Fab) initially; then following up the specific binding vs. other anti-IP10 human antibodies (e.g., 1D4 and 10A12), and pooled human IgG.
  • MDX1100 6A5 whole 6A5, 6A5-biotin, or 6A5 Fab
  • FIG. 6A shows that clones 10C8 and 6C9 were positive when hybridoma supernatant was screened for MDX1100 6A5 specific antibodies by ELISA.
  • FIG. 6B shows that clones 10C8 and 6C9 bind to 6A5 specifically.
  • FIG. 7 shows that clones 2F5 and 23H10 bind to 6A5 specifically.
  • the antibody selection was based on direct binding to MDX1100 6A5, specific for 6A5 vs. irrelevant antibodies 1D4 and 10A12, and a pooled human IgG, and competition of anti-idiotypic antibodies with 6A5-IP 10 interaction.
  • 1 ⁇ g/ml, 50 ⁇ l of goat anti-mouse IgG gamma was coated on the plates.
  • 1 ⁇ g/ml, 50 ⁇ l of a diluted anti-idiotypic antibody was added as well.
  • goat anti-human IgG Fc gamma-HRP was added for detection.
  • FIG. 10 shows that Clone 10C8 competes with IP10 in the competitive binding assays.
  • FIG. 11 shows that Clones 2F5 and 23H10 do not compete with IP10 in the competition binding assays.
  • MSD Meso Scale Discovery
  • the standard curve prepared in 2% human serum, ranged from 5 ng/mL to 300 ng/mL (250 to 15,000 ng/mL in 100% human serum), and was fitted to a 4-parameter logistic regression model with 250 ng/mL as an anchor point.
  • the intra-assay precision for QCs was within 7.2% and inter-assay precision was within 10.9%.
  • the intra-assay precision for reference standards was within 6.7% and inter-assay precision was within 6.4%.
  • the QCs accuracy was within ⁇ 15.6% of the nominal values.
  • the reference standards accuracy was within ⁇ 5.4% of the nominal values.
  • Assay Buffer 1% BSA and 0.05% Tween-20 in DPBS
  • Blocking Buffer (5% BSA and 0.05% Tween-20 in DPBS):
  • PBS Wash Buffer PBS Dry Powder Packets, pH 7.4 ⁇ 0.2 containing 0.05% v/v Tween 20
  • the reagent was aliquoted, and stored at ⁇ 70° C. A final concentration of 25 ng/mL solution of the Sulfo-Tag labeled mouse anti-idiotypic IP10 6A5 MAb 23H10 in assay buffer was prepared for the assay. Each new lot of this reagent must be titrated against the previous lot and used in the assay at a dilution that gives results equivalent to those observed with the previous lot.
  • the reagent was stored in room temperature.
  • a final 2 ⁇ solution was prepared by diluting 5 mL of MSD Read Buffer T (4 ⁇ ) in 5 mL dIH2O before use.
  • the standard curve was prepared in assay buffer/2% human serum as described below.
  • volume is enough for one plate. Proportional volumes were prepared as needed.
  • a 1:50 dilution of each of the QC samples was prepared by adding 10 ⁇ L of the 100% human serum QC samples, prepared supra, to 490 ⁇ L of assay buffer to provide QC samples containing 15000, 10000, 6000, 1000 and 500 ng/mL in 2% human serum. These dilutions were prepared on the day of the analysis.
  • the accuracy of the method was assessed by calculating the deviation of the predicted concentrations from their nominal values.
  • the accuracy and precision information based on the three analytical reference standards and QCs, obtained using a one-way ANOVA in SAS are listed in Tables 10 and 11.
  • the intra-assay precision for QCs was within 7.2% and inter-assay precision was within 10.9%.
  • the intra-assay precision for reference standards was within 6.7% and inter-assay precision was within 6.4%.
  • the QCs accuracy was within ⁇ 15.6% of the nominal values.
  • the reference standards accuracy was within ⁇ 5.4% of the nominal values.
  • Dilutional linearity of MDX-1100 was assessed by diluting a 2.5 mg/mL QC sample at 50-fold with assay buffer first, then serially diluting 10-fold in assay buffer/2% human serum. These individual dilutions were analyzed with a standard curve and QC samples. The results, summarized in Table 13, indicated that the predicted concentrations of the individually diluted test samples were within ⁇ 10% of the nominal value. The data demonstrated that study samples could be diluted at least 50.000-fold in assay buffer/2% human serum without adversely affecting the accuracy and precision of the assay.
  • IP10 and Heparan Sulfate were combined at the following concentrations and used to test the potential inference with the quantitation of the low, mid and high QCs: (a) IP10 at 0, 10, 100, 1000 ng/ml; and (b) Heparan Sulfate at 0, 500, 5000, 50000 ng/ml.
  • MSD immunoassay for the quantitation of MDX-1100 in human serum has been developed over the standard curve concentration ranging from 250 ng/mL to 15,000 ng/mL in neat human serum.
  • This enzyme-linked immunosorbent assay is designed to detect MDX-1100 in human serum.
  • NeutrAvidin coated plates are coated with biotinylated anti-MDX-1100 mouse monoclonal antibody (clone 10C8) at a concentration of 1.5 ⁇ g/mL in Assay Buffer.
  • Calibrators, controls and samples are diluted to the assay MRD (1:1000) in Assay Buffer and incubated on the plate to capture MDX-1100.
  • the captured MDX-1100 is then detected using HRP-labeled anti-MDX-1100 mouse monoclonal antibody (clone 23H10) at a concentration of 0.25 ⁇ g/mL in Assay Buffer.
  • TMB is added as the HRP substrate. Plates are read on the Spectramax Plus plate reader after the addition of Stop Solution and the measured optical density (OD) is directly proportional to the concentration of the MDX-1100 on the plate.
  • Analyte concentrations are determined by interpolation from the standard curve, which has been fit using a five-parameter logistic regression model with a weighting factor of 1/response2.
  • the minimum required sample volume is 10.0 ⁇ L.
  • the MRD is 1:1000.
  • the calibration range is 1.25 to 320 ⁇ g/mL with a quantification range of 2.5 ⁇ g/mL to 320 ⁇ g/mL. Samples are stored at approximately ⁇ 80° C.
  • PCV Percent Coefficient of Variation
  • Intra-assay precision and accuracy analyses were performed in runs 1JHX2 (QCs 1-3), 2JHX2 (QCs 4-6) and 6JHX2 (QC 7).
  • the PCV values for the intra-assay samples at all QC levels were acceptable, ranging from 3% for QC 4 (LLOQ) to 14% for QC 5 (Back-up LLOQ).
  • the PDT values for the intra-assay precision and accuracy samples for all QC levels also fell within the acceptance criteria, ranging from ⁇ 8% (QC 7; ULOQ) to 13% (QC 1 and QC 2). These data are presented in Table 16.
  • Assay sensitivity was evaluated by assessing the accuracy and precision of QC 4 (LLOQ). It was expected that QC 4 would have PCV values less than 25% and PDT values within ⁇ 25%. Two replicates of QC 4 were plated in runs 1JHX2-8JHX2, and one of these runs (7JHX2) was rejected due to unacceptable QC quantitation of the mid QC (QC 2; 120 ⁇ g/mL). In the seven acceptable runs, QC 4 had an average PCV value of 13% and an average PDT value of 0% (Table 17), well within the acceptable limits. These data indicate that the assay sensitivity for ICD 426 is acceptable at the LLOQ.
  • QC 8 The ability to dilute samples originally above the upper limit of the standard curve was assessed by evaluating a series of dilutions that were prepared from an over-the-curve QC pool (QC 8).
  • QC 8 was prepared by diluting the MDX-1100 stock (10,200 ⁇ g/mL) 1:20 in pooled normal human serum to achieve a concentration of 510 ⁇ g/mL. Note that this concentration was the highest that could be achieved to maintain at least 95% serum for QC 8, in accordance with the guidelines of PPD's Standard Operating Procedure (SOP No.: LP-PAL-1013).
  • Samples with dilution factors ranging from Dil 3.75 to Dil 101.25 had PDT values ranging from ⁇ 16% to ⁇ 2%, and met the acceptance criteria that the PDT be within ⁇ 20%.
  • the prozone or hook effect was examined by the comparing responses of QC 8 (510 ⁇ g/mL) and the QC 8 Dil 1.25 sample (408 ⁇ g/mL) to the responses of CAL 10 (320 ⁇ g/mL; ULOQ). Both replicates of each dilution had O.D. values that were greater than the recorded values for CAL 10, but were not quantitated by extrapolating the calibration curve beyond the ULOQ. Therefore, there is no hook effect for MDX-1100 concentrations beyond the ULOQ of 320 ⁇ g/mL and including 510 ⁇ g/mL.
  • Hemolysis data for each of the spiked hemolysis samples were expected to have PCV values ⁇ 20% and the accuracy of the mean value was expected to be within ⁇ 20% of the theoretical value for that pool. Blank samples were expected to have a PCV less than 20% and quantitate lower than the LLOQ. At least 80% of hemolysis samples at each level were required to meet acceptance criteria.
  • the remaining nine individuals spiked at the High QC level had acceptable PCV values ranging from 0% to 5% and acceptable PDT values ranging from ⁇ 7% to 16%.
  • the acceptance criteria for samples exhibiting a low level of hemolysis were met at the blank (90% acceptable), low (100% acceptable), and high (90% acceptable) QC levels.
  • the stability of MDX-1100 in thawed matrix was evaluated at 2-8° C. to determine whether holding samples in a thawed state for a period of time adversely affected analyte stability in normal human serum.
  • the PCV of the replicate determinations was expected to not exceed 20% and accuracy of the mean value for each level was expected to be within 20% of the theoretical concentration.
  • the overall PCV for the low and high stability QCs was 4% and 5%, respectively. Percent difference from the theoretical for the low and high QCs was ⁇ 11% and 10%, respectively.
  • QCs 1-3 were also analyzed in 13JHX2 and 1JHX4.
  • QC 2 and QC 3 had % DIFF of 13% and 7%, respectively, and met the acceptance criteria.
  • QC 1 failed to meet the acceptance criteria with a % DIFF value of 35%.

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WO2012149320A1 (en) 2012-11-01
US20140127229A1 (en) 2014-05-08
EA201391582A1 (ru) 2014-03-31
CA2834552A1 (en) 2012-11-01
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