Cxcl10 binding proteins and uses thereof

Abstract

The present invention relates to C-X-C motif chemokine ligand 10 (CXCL10) binding proteins and their use in methods of detecting and / or diagnosing a condition in a subject, including determining the level of CXCL10 in a subject. Specific antibodies that bind total CXCL10 (full length, N-terminally truncated and citrullinated) and antibodies that bind active CXCL10 (full length) are used to measure the level of total CXCL10 and active CXCL10 in ovarian cancer patient samples. The calculated ratio between active CXCL10 and total CXCL10 is lower in patients with malignant disease compared to patients with benign tumors or healthy individuals, and this calculated ratio is the basis for methods of diagnosing malignant disease, monitoring tumor burden and disease progression.

Description

[0001] Relevant application data

[0002] This application claims priority to Australian Patent Application No. 2019904859, filed on December 20, 2019, entitled “XCL10 binding protein and uses Its”, the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application is submitted electronically along with the sequence list. The entire contents of the sequence list are incorporated herein by reference. Technical Field

[0005] This disclosure relates to CXCL10 binding proteins and their uses. Background Technology

[0006] Chemokines interferon-γ-induced protein (also known as CXC motif chemokine ligand 10; CXCL10; interferon-induced protein 10 or IP-10) are members of the CXC chemokine family and play an important role in leukocyte transport by generating chemotactic activity in cells expressing corresponding chemokine receptors.

[0007] CXCL10 exhibits both agonist and antagonist activities and participates in chemotaxis, apoptosis induction, cell growth regulation, and angiogenesis mediation. CXCL10 exerts its biological effects by specifically activating its receptor CXCR3, a seven-transmembrane G protein-coupled receptor primarily expressed in activated T lymphocytes (Th1), natural killer (NK) cells, inflammatory dendritic cells, macrophages, and B cells. The proliferative or antiproliferative effects of CXCL10 appear to depend on cell type and / or the isotype of its receptor CXCR3. Furthermore, post-translational modifications such as deamination or citrullination of CXCL10 via peptidyl arginine deiminase (PAD) or truncation of the NH2 terminus by proteases such as dipeptidyl peptidase IV (DPP4) can facilitate its biological effects by generating a dominant-negative form capable of binding CXCR3 without inducing signal transduction.

[0008] CXCL10 is associated with a variety of human diseases, including infectious diseases, central nervous system diseases, chronic inflammation, immune dysfunction, and cancer.

[0009] Given its broad association with many human diseases, CXCL10 has been a highly attractive candidate for biomarkers and targeted therapies. However, the application of commercially available diagnostic agents is limited by the inability to distinguish between different biologically relevant forms of the protein.

[0010] Based on the foregoing, those skilled in the art will recognize that there is an urgent need in the art for compounds (e.g., antibodies and antibody-derived proteins) that can accurately target all forms of CXCL10 as potential biomarkers. Summary of the Invention

[0011] In developing this invention, the inventors sought to produce reagents that bind to biologically relevant forms of CXCL10. The inventors produced an antibody that binds to full-length (i.e., biologically active) CXCL10, and an antibody that binds to both full-length CXCL10 and N-terminal truncated and citrullinated (i.e., inactive) forms. Surprisingly, the inventors found that detecting different forms of CXCL10, particularly the ratio between different forms, could distinguish between benign and malignant conditions. The inventors also found that the ratio between different forms of CXCL10 could distinguish between the presence (benign or malignant) and absence (i.e., healthy individuals) of disease. The inventors also surprisingly found that detecting the ratio between different forms of CXCL10 and other biomarkers (e.g., DPP4, CA-125, GM-CSF, IL-6, TNF-RII, HE4, and / or IL-8) could distinguish between stage I (i.e., early-stage) cancer or precancerous lesions and benign conditions.

[0012] In one example, this disclosure provides a CXCL10 binding protein, wherein the binding protein binds to full-length human CXCL10, N-terminal truncated CXCL10, and citrullinated CXCL10.

[0013] In one embodiment of the above example, the CXCL10 binding protein binds to the epitope NH2-LSRTVRCTCISISNQPVNPRSLE-COOH (SEQ ID NO: 26) of full-length human CXCL10, N-terminal truncated CXCL10, and citrullinated CXCL10.

[0014] In one instance, CXCL10 binds to proteins: (i) at 50 nM or less K D Combined with full-length human CXCL10; and / or (ii) with 5 nM or less K D It binds to N-terminal truncated human CXCL10.

[0015] In one example, CXCL10 binds to the protein at 5 nM or less K. D It binds to or specifically binds to full-length human CXCL10. For example, CXCL10-binding proteins bind at approximately 50 nM, or approximately 40 nM, or approximately 30 nM, or approximately 20 nM, or approximately 10 nM of K. D It binds to or specifically binds to full-length human CXCL10. In one example, the CXCL10-binding protein binds at 49 nM K.D It binds to or specifically binds to full-length human CXCL10.

[0016] In one embodiment of the above example, the CXCL10 binding protein requires the N-terminal valine and / or proline of epitope NH2-VPLSRTVRCTCISISNQPVNPRSLE-COOH (SEQ ID NO: 25) to bind to full-length human CXCL10.

[0017] In one example, CXCL10 binds to the protein at 5 nM or less K. D It binds to or specifically binds to N-terminally truncated human CXCL10. For example, CXCL10-binding proteins bind to K at approximately 5 nM, or approximately 4 nM, or approximately 3 nM, or approximately 2 nM, or approximately 1 nM. D It binds to or specifically binds to N-terminally truncated human CXCL10. In one example, CXCL10 binds to the protein at 3 nM K. D It binds to or specifically binds to N-terminal truncated human CXCL10.

[0018] This disclosure also provides a CXCL10 binding protein, wherein the binding protein is at a concentration of 50 nM or less. D It binds to full-length human CXCL10, but not to N-terminal truncated CXCL10 or citrullinated CXCL10.

[0019] In one example, the CXCL10-binding protein neither detectably nor significantly binds to N-terminal truncated CXCL10 nor citrullinated CXCL10.

[0020] The methods used to determine the binding of CXCL10-binding proteins to peptides are readily apparent to those skilled in the art. For example, the peptide is immobilized on a solid or semi-solid surface and the CXCL10-binding protein contacts the immobilized peptide. The binding is then determined, for example, by surface plasmon resonance (SPR) imaging.

[0021] In one example, the levels (e.g., by K) are combined. D (Identified) Measurements were taken using surface plasmon resonance (SPR) imaging.

[0022] In one example, the CXCL10 binding protein of this disclosure includes a variable region or antigen-binding domain.

[0023] In one example, the binding protein is selected from the group consisting of:

[0024] (i)Fv;

[0025] (ii) Single-stranded Fv fragments (scFv);

[0026] (iii) Dimer scFv (di-scFv);

[0027] (iv) Single-domain antibodies;

[0028] (v) Microantibodies;

[0029] (vi) Double-chain antibodies;

[0030] (vii) Triple-chain antibody;

[0031] (viii) Quadruplex antibody;

[0032] (ix)Fab;

[0033] (x)F(ab')2;

[0034] (xi) antibody;

[0035] (xii) Antibody mimics;

[0036] (xiii) Heavy chain immunoglobulins only;

[0037] (xiv)T cell receptor;

[0038] (xv) adendidine protein;

[0039] (xvi) anti-carrier protein;

[0040] (xvii) Affinity form;

[0041] (xvii) Avimer;

[0042] (xix) designed ankyrin repeat protein (DARPin); or

[0043] (xx) Linked to the constant region, Fc, or heavy chain constant domain of the antibody (C H One of (i) to (xix) of CH2 and / or CH3.

[0044] In one example, the binding protein includes the antigen-binding domain of the antibody. For example, the binding protein includes at least V... H and V L V H and V L They combine to form an Fv containing an antigen-binding domain.

[0045] In one example, the binding protein is an antibody or an antigen-binding fragment thereof (e.g., an scFv containing an antibody variable region). Exemplary antibodies are full-length and / or naked (e.g., unconjugated) antibodies. In one example, the antibody disclosed herein is a full-length antibody.

[0046] In one example, the antibody is an IgG, IgE, IgM, IgD, IgA, or IgY antibody. For instance, the antibody is an IgG antibody.

[0047] In one example, the IgG antibody is IgG1, IgG2, IgG3, or IgG4. For example, the antibody is an IgG1 antibody. In another example, the antibody is an IgG4 antibody. In one example, the antibody is a stable IgG4 antibody.

[0048] In one example, the binding protein is recombinant, chimeric, CDR-transplanted, humanized, co-humanized, primate-derived, deimmunized, or a human antibody.

[0049] In one example, the antigen-binding fragment of this disclosure is a hapten. For example, the CXCL10 binding protein is a hapten comprising a heavy chain and a light chain.

[0050] In one example, the antigen-binding fragment of this disclosure contains an IgG4 constant region or a stable IgG4 constant region.

[0051] In one example, the binding protein is an antibody mimic. For instance, the binding protein may contain an antigen-binding domain of an immunoglobulin, such as IgNAR, a camel antibody, or a T-cell receptor.

[0052] In one example, the binding protein is a domain antibody (e.g., containing only the heavy chain variable region or only the light chain variable region) or a heavy chain-only antibody (e.g., a camel antibody or IgNAR) or its variable region.

[0053] In one example, the binding protein competitively inhibits the binding of an antibody or its antigen-binding fragment to CXCL10, said antibody or its antigen-binding fragment comprising:

[0054] (i) Heavy chain variable region (V) containing the amino acid sequence shown in SEQ ID NO: 3 H ) and the light chain variable region (V) containing the amino acid sequence shown in SEQ ID NO: 4 L ); and / or

[0055] (ii) V containing the amino acid sequence shown in SEQ ID NO: 11 H and V containing the amino acid sequence shown in SEQ ID NO: 12 L .

[0056] In one example, the binding protein competitively inhibits the binding of an antibody or its antigen-binding fragment to CXCL10, said antibody or its antigen-binding fragment comprising V, which contains the amino acid sequence shown in SEQ ID NO: 3. H and V containing the amino acid sequence shown in SEQ ID NO: 4L .

[0057] In another example, the binding protein competitively inhibits the binding of an antibody or its antigen-binding fragment to CXCL10, said antibody or its antigen-binding fragment comprising V, which contains the amino acid sequence shown in SEQ ID NO: 11. H and V containing the amino acid sequence shown in SEQ ID NO: 12 L .

[0058] In one example, the binding proteins include:

[0059] (i) V containing a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 3 H and V containing a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 4 L ;or

[0060] (ii) V containing a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 11 H and V containing a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 12 L .

[0061] In one example, the binding protein includes: V containing a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 3. H and V containing a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 4 L For example, binding proteins include V that have at least 90%, or 95%, or 97%, or 98%, or 99% identity with the sequences disclosed herein. H and / or V L .

[0062] In one example, the binding protein includes: V containing a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 11. H and V containing a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 12 L For example, binding proteins include V that have at least 90%, or 95%, or 97%, or 98%, or 99% identity with the sequences disclosed herein. H and / or V L .

[0063] In one example, the binding protein of this disclosure may optionally include one or more amino acid substitutions, deletions, or insertions of any sequence disclosed herein. Amino acid substitutions applicable to this disclosure will be apparent to those skilled in the art and include both naturally occurring and engineered substitutions.

[0064] In one example, the CXCL10 binding protein of this disclosure is an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising:

[0065] (i) V containing the amino acid sequence shown in SEQ ID NO: 3 H and V containing the amino acid sequence shown in SEQ ID NO: 4 L ;

[0066] (ii) V containing the amino acid sequence shown in SEQ ID NO: 11 H and V containing the amino acid sequence shown in SEQ ID NO: 12 L .

[0067] In one example, the CXCL10 binding protein of this disclosure is an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising V containing the amino acid sequence shown in SEQ ID NO: 3. H and V containing the amino acid sequence shown in SEQ ID NO: 4 L .

[0068] In another example, the CXCL10 binding protein of this disclosure is an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising V containing the amino acid sequence shown in SEQ ID NO: 11. H and V containing the amino acid sequence shown in SEQ ID NO: 12 L .

[0069] In one example, the CXCL10 binding protein of this disclosure is an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising:

[0070] (i)V H It includes:

[0071] a) A CDR1 containing the sequence of amino acids 25-34 in SEQ ID NO: 3;

[0072] b) CDR2 containing the sequence of amino acids 49-65 in SEQ ID NO: 3; and

[0073] c) CDR3 containing the sequence of amino acids 98-108 in SEQ ID NO: 3; and

[0074] V L It includes:

[0075] a) A CDR1 containing the sequence of amino acids 23-33 in SEQ ID NO: 4;

[0076] b) CDR2 containing the sequence of amino acids 49-55 in SEQ ID NO: 4; and

[0077] c) A CDR3 containing the sequence of amino acids 88-96 in SEQ ID NO: 4; or

[0078] (ii)V H It includes:

[0079] a) A CDR1 containing the sequence of amino acids 25-34 in SEQ ID NO: 11;

[0080] b) CDR2 containing the sequence of amino acids 49-65 in SEQ ID NO: 11; and

[0081] c) CDR3 containing the sequence of amino acids 98-108 in SEQ ID NO: 11; and

[0082] V L It includes:

[0083] a) A CDR1 containing the sequence of amino acids 23-33 in SEQ ID NO: 12;

[0084] b) CDR2 containing the sequence of amino acids 49-55 in SEQ ID NO: 12; and

[0085] c) CDR3 containing the sequence of amino acids 88-96 in SEQ ID NO: 12.

[0086] In one example, the CXCL10 binding protein of this disclosure is an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising:

[0087] (i)V H It includes:

[0088] a) A CDR1 containing the sequence of amino acids 25-34 in SEQ ID NO: 3;

[0089] b) CDR2 containing the sequence of amino acids 49-65 in SEQ ID NO: 3; and

[0090] c) CDR3 containing the sequence of amino acids 98-108 in SEQ ID NO: 3; and

[0091] (ii)V L It includes:

[0092] a) A CDR1 containing the sequence of amino acids 23-33 in SEQ ID NO: 4;

[0093] b) CDR2 containing the sequence of amino acids 49-55 in SEQ ID NO: 4; and

[0094] c) CDR3 containing the sequence of amino acids 88-96 in SEQ ID NO: 4.

[0095] In one example, the CXCL10 binding protein of this disclosure is an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising:

[0096] (i)V H It includes:

[0097] a) A CDR1 containing the sequence of amino acids 25-34 in SEQ ID NO: 11;

[0098] b) CDR2 containing the sequence of amino acids 49-65 in SEQ ID NO: 11; and

[0099] c) CDR3 containing the sequence of amino acids 98-108 in SEQ ID NO: 11; and

[0100] (ii)V L It includes:

[0101] a) A CDR1 containing the sequence of amino acids 23-33 in SEQ ID NO: 12;

[0102] b) CDR2 containing the sequence of amino acids 49-55 in SEQ ID NO: 12; and

[0103] c) CDR3 containing the sequence of amino acids 88-96 in SEQ ID NO: 12.

[0104] In one example, the CXCL10 binding protein of this disclosure is an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising:

[0105] (i)V H It includes:

[0106] a) A CDR1 containing the sequence shown in SEQ ID NO: 5;

[0107] b) CDR2 containing the sequence shown in SEQ ID NO: 6; and

[0108] c) CDR3 containing the sequence shown in SEQ ID NO: 7; and

[0109] V L It includes:

[0110] a) A CDR1 containing the sequence shown in SEQ ID NO: 8;

[0111] b) CDR2 containing the sequence shown in SEQ ID NO: 9; and

[0112] c) A CDR3 containing the sequence shown in SEQ ID NO: 10; or

[0113] (ii)V H It includes:

[0114] a) A CDR1 containing the sequence shown in SEQ ID NO: 13;

[0115] b) CDR2 containing the sequence shown in SEQ ID NO: 14; and

[0116] c) CDR3 containing the sequence shown in SEQ ID NO: 15; and

[0117] V L It includes:

[0118] a) A CDR1 containing the sequence shown in SEQ ID NO: 16;

[0119] b) CDR2 containing the sequence shown in SEQ ID NO: 17; and

[0120] c) CDR3 containing the sequence shown in SEQ ID NO: 18.

[0121] In one example, the CXCL10 binding protein of this disclosure is an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising:

[0122] (i)V H It includes:

[0123] a) A CDR1 containing the sequence shown in SEQ ID NO: 5;

[0124] b) CDR2 containing the sequence shown in SEQ ID NO: 6; and

[0125] c) CDR3 containing the sequence shown in SEQ ID NO: 7; and

[0126] (ii)V L It includes:

[0127] a) A CDR1 containing the sequence shown in SEQ ID NO: 8;

[0128] b) CDR2 containing the sequence shown in SEQ ID NO: 9; and

[0129] c) CDR3 containing the sequence shown in SEQ ID NO: 10.

[0130] In one example, the CXCL10 binding protein of this disclosure is an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising:

[0131] (i)V H It includes:

[0132] a) A CDR1 containing the sequence shown in SEQ ID NO: 13;

[0133] b) CDR2 containing the sequence shown in SEQ ID NO: 14; and

[0134] c) CDR3 containing the sequence shown in SEQ ID NO: 15; and

[0135] (ii)V L It includes:

[0136] a) A CDR1 containing the sequence shown in SEQ ID NO: 16;

[0137] b) CDR2 containing the sequence shown in SEQ ID NO: 17; and

[0138] c) CDR3 containing the sequence shown in SEQ ID NO: 18.

[0139] In one instance, a protein, antibody, or antigen-binding fragment thereof is any form of protein, antibody, or functional fragment thereof encoded by a nucleic acid that encodes any of the aforementioned proteins, antibodies, or functional fragments.

[0140] In one example, the CXCL10 binding protein is conjugated to a detectable label. Detectable labels suitable for use in this disclosure will be obvious to those skilled in the art and / or described herein. For example, the detectable labels are selected from the group consisting of: radioactive labels, enzymes, fluorescent labels, luminescent labels, bioluminescent labels, magnetic labels, prosthetic groups, and contrast agents.

[0141] In one example, the detectable marker is a radioactive marker. For example, the radioactive marker may be, but is not limited to, radioactive iodine (125I, 131I), technetium, yttrium, 35S, or 3H.

[0142] In one example, the detectable marker is an enzyme. For example, the enzyme could be, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase.

[0143] In one example, the detectable marker is a fluorescent marker. For example, the fluorescent marker may be, but is not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, or phycoerythrin.

[0144] In one example, the detectable marker is a glowing marker. For example, a glowing marker could be, but is not limited to, luminol.

[0145] In one instance, the detectable marker is a bioluminescent marker. For example, a bioluminescent marker may be, but is not limited to, luciferase, luciferin, or jellyfish luminescent protein.

[0146] In one example, the detectable marker is a magnetic marker. For example, the magnetic marker can be, but is not limited to, gadolinium or iron oxide chelates.

[0147] In one example, the detectable marker is a prosthetic group. For example, the prosthetic group can be, but is not limited to, streptavidin / biotin or avidin / biotin.

[0148] In one example, the detectable marker is a contrast agent.

[0149] The present invention also provides a composition comprising the binding protein of the present invention and a carrier. The carriers suitable for use in this disclosure will be obvious to those skilled in the art and / or described herein.

[0150] The present invention also provides a polynucleotide encoding the CXCL10 binding protein according to the invention.

[0151] The present invention also provides an expression vector comprising a polynucleotide encoding the CXCL10 binding protein of the present invention. Exemplary vectors suitable for this disclosure will be apparent to those skilled in the art and / or described herein.

[0152] The present invention also provides an in vitro cell comprising the expression vector of the present invention. Exemplary cells suitable for use with respect to this disclosure will be obvious to those skilled in the art and / or described herein. In one embodiment, the present invention provides the use of this cell for preparing the CXCL10 binding protein of the present invention. For example, this use includes culturing the cells of the present disclosure and generating the CXCL10 binding protein therefrom; and isolating and purifying the generated binding protein. Methods for isolating and purifying the generated binding protein will be obvious to those skilled in the art and / or described herein.

[0153] This disclosure provides a method for detecting and / or diagnosing malignant diseases in a subject, the method comprising:

[0154] a) Determine the active CXCL10 level and the total CXCL10 level of the subjects; and

[0155] b) Determine the CXCL10 ratio of active CXCL10 to total CXCL10 in the subjects.

[0156] In one example, determining the levels of active CXCL10 and total CXCL10 includes determining the amount of active CXCL10 protein and the amount of total CXCL10 protein in the subject.

[0157] In one example, the method further includes comparing the CXCL10 ratio in the subject with the CXCL10 ratio in at least one reference. The method for determining the reference will be obvious to those skilled in the art and / or described herein.

[0158] In one example, the method includes determining whether: (a) the CXCL10 ratio in the subject is higher than the CXCL10 ratio in the reference; or (b) whether the CXCL10 ratio in the subject is lower than the CXCL10 ratio in the reference.

[0159] In one example, (i) a lower CXCL10 ratio in the subjects compared to the CXCL10 ratio in the reference indicates a malignant condition; or (ii) a higher CXCL10 ratio in the subjects compared to the CXCL10 ratio in the reference indicates a benign condition.

[0160] In one example, the method includes using:

[0161] (i) CXCL10-binding proteins that specifically bind to full-length human CXCL10, N-terminal truncated CXCL10, and citrullinated CXCL10 to determine the level of total CXCL10 in the subjects; and

[0162] (ii) CXCL10 binding protein, which specifically binds to full-length human CXCL10 but not to N-terminal truncated CXCL10 and citrullinated CXCL10, to determine the level of total CXCL10 in the subject.

[0163] In one example of any method described herein, the method includes using at least one CXCL10 binding protein according to this disclosure.

[0164] In one example

[0165] (i) The total CXCL10 level in the subject was determined using an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising V containing the amino acid sequence shown in SEQ ID NO: 11. H and V containing the amino acid sequence shown in SEQ ID NO: 12 L ; and / or

[0166] (ii) The level of active CXCL10 in the subject was determined using an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising V containing the amino acid sequence shown in SEQ ID NO: 3. H and V containing the amino acid sequence shown in SEQ ID NO: 4 L .

[0167] In one example, the level of active CXCL10 in a subject is determined using an antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising V, which includes the amino acid sequence shown in SEQ ID NO: 11. H and V containing the amino acid sequence shown in SEQ ID NO: 12 L .

[0168] In another example, the level of active CXCL10 in a subject is determined using an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising V containing the amino acid sequence shown in SEQ ID NO: 3. H and V containing the amino acid sequence shown in SEQ ID NO: 4 L .

[0169] In one example, one or more CXCL10-binding proteins are conjugated to a detectable marker. Detectable markers suitable for use in this disclosure will be obvious to those skilled in the art and / or described herein. For example, the detectable markers are selected from the group consisting of: radioactive markers, enzymes, fluorescent markers, luminescent markers, bioluminescent markers, magnetic markers, prosthetic groups, and contrast agents.

[0170] In one example, the detectable marker is a radioactive marker. For example, the radioactive marker may be, but is not limited to, radioactive iodine (125I, 131I), technetium, yttrium, 35S, or 3H.

[0171] In one example, the detectable marker is an enzyme. For example, the enzyme could be, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase.

[0172] In one example, the detectable marker is a fluorescent marker. For example, the fluorescent marker may be, but is not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, or phycoerythrin.

[0173] In one example, the detectable marker is a glowing marker. For example, a glowing marker could be, but is not limited to, luminol.

[0174] In one instance, the detectable marker is a bioluminescent marker. For example, a bioluminescent marker may be, but is not limited to, luciferase, luciferin, or jellyfish luminescent protein.

[0175] In one example, the detectable marker is a magnetic marker. For example, the magnetic marker can be, but is not limited to, gadolinium or iron oxide chelates.

[0176] In one example, the detectable marker is a prosthetic group. For example, the prosthetic group can be, but is not limited to, streptavidin / biotin or avidin / biotin.

[0177] In one example, the detectable marker is a contrast agent.

[0178] Methods for detecting CXCL10 levels will be obvious to those skilled in the art and / or described herein. For example, methods may include flow cytometry, enzyme-linked immunosorbent assay (ELISA), or Western blotting.

[0179] In one example, the method includes performing flow cytometry.

[0180] In one example, the method includes performing an enzyme-linked immunosorbent assay (ELISA).

[0181] In one example, the method includes performing protein blotting.

[0182] In one example, the method is performed on the subject either in vitro or ex vivo. For example, the method is performed on the subject in vitro. In another example, the method is performed on the subject ex vivo.

[0183] In one example, the method is performed on at least one biological sample obtained from the subject. The biological samples used in this disclosure will be obvious to those skilled in the art and / or described herein. For example, the biological sample is selected from biopsy sections, fluid samples, plasma samples, or cell swabs.

[0184] In one example, the biological sample is a slice of living tissue.

[0185] In one example, the biological sample is a fluid sample. For example, a fluid sample is cervical fluid, vaginal discharge, or ascites. In one example, the biological sample is ascites.

[0186] In one example, the biological sample is a plasma sample.

[0187] In one example, the biological sample is a cell swab. For instance, a cell swab is a cervical swab. In another example, a cell swab is a cervicovaginal swab (CVS).

[0188] In one example, the method was performed on plasma samples and cervical vaginal swabs (CVS).

[0189] In one example of any of the methods described herein, the present invention provides a method for detecting and / or diagnosing a malignant condition in a subject. For example, the malignant condition is a reproductive cancer. In one example, the reproductive cancer is ovarian cancer. In one example, the ovarian cancer is stage I cancer. In another example, the ovarian cancer is a precancerous lesion, such as a lesion with a p53 gene mutation.

[0190] In one example, the method includes detecting and / or diagnosing malignant conditions from benign conditions.

[0191] In one example of any of the methods described herein, the method further includes measuring the levels of dipeptidyl peptidase-4 (DPP4) and / or cancer antigen 125 (CA-125) in the subject. In another example, the method further includes measuring the levels of dipeptidyl peptidase-4 (DPP4) and / or cancer antigen 125 (CA-125) in the subject. In yet another example, the method further includes measuring the levels of cancer antigen 125 (CA-125) in the subject.

[0192] In one example of any of the methods described herein, the method further includes: determining the levels of one or more of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), tumor necrosis factor receptor II (TNF-RII), human epididymal protein 4 (HE4), and interleukin-8 (IL-8). For example, the method further includes determining the levels of GM-CSF, IL-6, TNF-RII, HE4, and IL-8.

[0193] In one example of any of the methods described herein, the method further includes determining the levels of DPP4, GM-CSF, IL-6, TNF-RII, HE4, and IL-8.

[0194] In one example of any of the methods described herein, the method further includes determining the levels of CA-125, GM-CSF, IL-6, TNF-RII, HE4, and IL-8.

[0195] In one example of any of the methods described herein, the method further includes determining the levels of DPP4, CA-125, GM-CSF, IL-6, TNF-RII, HE4, and IL-8.

[0196] A method for detecting and / or diagnosing a condition in a subject is also provided, the method comprising determining the level of CXCL10 in the subject using at least one of the CXCL10 binding proteins of the present invention.

[0197] In this example, the condition is characterized by the levels and / or relative ratios of one or more full-length (i.e., biologically active) CXCL10, N-terminal truncated CXCL10, and citrullinated CXCL10. In one example, the condition is an inflammatory condition. For example, an inflammatory condition is arthritis, such as rheumatoid arthritis and / or psoriatic arthritis. In one example, the condition is rheumatoid arthritis. In another example, the condition is psoriatic arthritis. In one example, the condition is hepatitis C. In another example, the condition is heart failure.

[0198] This disclosure also provides a method for monitoring tumor burden in subjects with malignant conditions, the method comprising determining the CXCL10 ratio between active CXLC10 and total CXCL10 in the subject at one or more time points.

[0199] This disclosure also provides a method for monitoring the progression of malignant disease in a subject, the method comprising determining the CXCL10 ratio between active CXLC10 and total CXCL10 in the subject at one or more time points.

[0200] This disclosure also provides a method for monitoring tumor regression in a subject with a malignant condition, the method comprising determining the CXCL10 ratio between active CXLC10 and total CXCL10 in the subject at one or more time points.

[0201] This disclosure also provides a method for monitoring tumor recurrence in subjects with malignant conditions, the method comprising determining the CXCL10 ratio between active CXLC10 and total CXCL10 in the subject at one or more time points.

[0202] This disclosure also provides a method for determining the efficacy of a treatment for a subject suffering from a malignant condition, the method comprising determining the CXCL10 ratio between active CXLC10 and total CXCL10 in the subject at one or more time points.

[0203] In one example, the subject has been diagnosed with a malignant disease. For example, the subject has a malignant disease. For example, a malignant disease is a reproductive cancer, such as ovarian cancer.

[0204] In one example, the subject was asymptomatic.

[0205] In one example, the subject has not yet received treatment for the malignant condition. For example, the subject has never received treatment. In another example, the subject is receiving treatment for the malignant condition. In yet another example, the subject has already received treatment for the malignant condition. The appropriate treatment for the malignant condition will be obvious to a technician and / or described herein. For example, treatments include surgery, chemotherapy, radiation therapy, targeted drug therapy, immunotherapy, or combinations thereof.

[0206] In one example, the treatment includes surgery.

[0207] In one example, the treatment includes chemotherapy.

[0208] In one example, the treatment includes radiation therapy.

[0209] In one example, the treatment includes targeted drug therapy.

[0210] In one example, the treatment includes immunotherapy.

[0211] In one example, the method includes determining:

[0212] (a) Whether the subject's CXCL10 rate was lower at subsequent time points than at the first time point; or

[0213] (b) Whether the subject’s CXCL10 ratio at subsequent time points was higher than the subject’s CXCL10 ratio at the first time point.

[0214] In one example, a lower CXCL10 ratio in subjects at subsequent time points compared to the first time point indicates an increased risk of tumor burden and / or tumor progression and / or tumor recurrence in the subjects. For instance, a lower CXCL10 ratio in subjects after treatment (i.e., at subsequent time points) compared to before treatment (i.e., the first time point) indicates an increased risk of tumor burden and / or tumor progression and / or tumor recurrence in the subjects.

[0215] In one example, a higher CXCL10 ratio in subjects at subsequent time points compared to the first time point indicates a reduced risk of tumor burden and / or tumor progression and / or tumor recurrence. For instance, a higher CXCL10 ratio in subjects after treatment (i.e., at subsequent time points) compared to before treatment (i.e., the first time point) indicates a reduced risk of tumor burden and / or tumor progression and / or tumor recurrence.

[0216] In one example of any of the methods described herein, the method further includes administering treatment to reduce the tumor burden of the subject and / or slow the progression of the subject's tumor.

[0217] This disclosure also provides a method for treating a malignant condition in a subject, the method comprising: detecting and / or diagnosing a malignant condition in the subject according to this disclosure, and treating the subject.

[0218] The appropriate treatment for a malignant condition will be obvious to a technician and / or described herein. For example, treatments include surgery, chemotherapy, radiation therapy, targeted drug therapy, immunotherapy, or combinations thereof.

[0219] This disclosure also provides a panel or kit for detecting and / or diagnosing malignancies in subjects, the panel or kit containing one or more CXCL10 binding proteins of this disclosure.

[0220] This disclosure also provides combinations or kits for monitoring tumor burden, monitoring progression, determining tumor regression, determining tumor recurrence, and / or determining the efficacy of treatment for malignant conditions in subjects, the combinations or kits comprising one or more CXCL10 binding proteins of this disclosure.

[0221] Unless otherwise expressly stated, any implementation described herein should be considered applicable to any other implementation. For example, as those skilled in the art will understand, the examples outlined above with respect to one example of the invention are equally applicable to other examples of the invention.

[0222] The scope of this invention is not limited to the specific embodiments described herein, which are for illustrative purposes only. As stated herein, functionally equivalent products, compositions, and methods are obviously within the scope of this invention.

[0223] Throughout this specification, unless otherwise expressly stated or required by the context, when referring to a single step, a substance composition, a group of steps, or a group of substance compositions shall be understood to include one or more (i.e., one or more) of such steps, substance compositions, groups of steps, or groups of substance compositions. Attached Figure Description

[0224] Figure 1 This is a graphical representation showing the difference between active CXCL10 and total CXCL10. Representative standard curves for detecting CXCL10 using RA2 and RG2 are shown, respectively. 2 >0.99.

[0225] Figure 2(a) Comparison of total CXCL10 quantification in malignant ascites of ovarian cancer patients (n=212) between ART and commercial ELISA. No significant difference in total CXCL10 quantification in matched ascites was observed between ART and commercial ELISA. The overall mean total CXCL10 values ​​for ART and commercial ELISA were 1126.1 ± 2158.6 pg / mL and 1192.4 ± 1059.5 pg / mL, respectively. Figure 2 (b) shows the correlation between ART and commercial ELISA for quantitative total CXCL10; the two tests for benign and malignant ascites showed a moderate positive correlation, with r values ​​of 0.3084 and 0.2594, respectively. P ≤ 0.05.

[0226] Figure 3 (a) Western blots demonstrating the detection of recombinant full-length CXCL10 and citrullinated CXCL10 by mAb-RA2 and mAb-RG2 are shown. CXCL10 was treated with (+) or untreated (-) PAD2 to induce citrullination within 60 minutes, followed by separation by Western blot. Untreated recombinant full-length CXCL10 (n / t) was used as a positive control. The commercially available anti-CXCL10 antibody (ab9807) did not detect citrullinated CXCL10 after 15 minutes of PAD2 latency. Note: A significant decrease in the detection ability of mAb-RA2 for citrullinated CXCL10 was observed after 15 minutes, and no effect of citrullination on the detection of CXCL10 by mAb-RG2 was observed. Figure 3 (b) shows the ART detection of citrullinated CXCL10 by mAb-RA2 and mAb-RG2. The binding of mAb-RA2 to citrullinated CXCL10 was significantly reduced, while mAb-RG maintained a certain degree of binding to citrullinated CXCL10.

[0227] Figure 4 This study demonstrates how to differentiate between benign and malignant ascites in ovarian cancer patients by quantitative analysis of active CXCL10 and total CXCL10, and by the ratio of active CXCL10 to active CXCL10. Figure 4 (a) The concentrations of active CXCL10 in benign (n=51) and malignant ascites (n=208) are shown: 240.4 ± 410.5 pg / mL and 818.6 ± 1098.0 pg / mL, respectively; Figure 4 (b) The total CXCL10 concentrations in benign (n=51) and malignant ascites (n=212) are shown to be 160.8 ± 362.0 pg / mL and 1126.1 ± 2158.6 pg / mL, respectively. Figure 4(c) shows a significant difference in the activity ratio between benign ascites (n=51) and malignant ascites (n=226): 2.59±1.18 and 1.43±1.04, respectively. Figure 4 (d) shows the activity ratio based on ovarian cancer stage compared to benign stage. Benign stage: 2.59 ± 1.18; Stage 1: 1.07 ± 0.44; Stage 3: 1.55 ± 1.23. ****P ≤ 0.0001.

[0228] Figure 5 The study demonstrated the use of DPP4 and plasma CA125 to differentiate between benign and malignant cases. Figure 5 (a) The concentrations of DPP4 in benign ascites (n=48) and malignant ascites (n=148) were measured by anti-DPP4 ELISA: 184.8 ± 177.6 ng / mL and 203.5 ± 154.3 ng / mL, respectively. P = 0.1031. Figure 4 (b) The specific activity (U / ng) measurements between benign ascites (n=49) and malignant ascites (n=50) are shown: 2.49±3.83 and 1.65±2.13, respectively. P=0.4229. Figure 5 (c) shows the measurement of CA125 in matched patient plasma: 200.8 ± 368.7 U / mL for benign (n = 30) patients and 1697.0 ± 3409.0 U / mL for malignant (n = 188) patients. ****P ≤ 0.0001.

[0229] Figure 6 The correlation between the activity ratio in benign and malignant ascites and plasma CA125, DPP4 concentrations and DPP4 specific activity was shown. Figure 6 (a) shows that there was no significant correlation between the activity rate and plasma CA125 in benign or malignant samples. Figure 6 (b) shows that the activity ratio was moderately negatively correlated with DPP4 (ng / mL) in malignant ascites samples (P = 0.0002), while no significant correlation was found in benign samples. Figure 6 (c) shows that the activity ratio was moderately negatively correlated with the specific activity of DPP4 (U / ng) in benign ascites samples (P = 0.0895), while no significant correlation was found in malignant samples.

[0230] Figure 7 Receiver operating curve (ROC) analysis is shown, demonstrating that ART has a superior AUC compared to other biomarkers in patient ascites samples. Figure 7 (a) shows that the activity ratio achieved an AUC (0.8617) that was higher than the AUC of total CXCL10 and active CXCL10 quantification (AUCs were 0.8122 and 0.7872, respectively); Figure 7(b) The activity ratio achieved a higher AUC than DPP4 and plasma CA125 (AUC 0.5598 and AUC 0.8262, respectively). The highest AUC was demonstrated by combining the activity ratio, DPP4 (ng / mL), and plasma CA125 (U / mL).

[0231] Figure 8 Demonstrations of cervical vaginal swabs (CVS) and plasma are shown, which are well-suited for use as biomarker-based ART tests. Figure 8 (a) shows a significant difference in the CVS activity ratio between benign (n=50) and malignant (n=50) samples: the activity ratios of benign and malignant CVS were 4.39±4.52 and 1.14±0.62, respectively. Figure 8 (b) shows a significant difference in plasma activity ratios between benign (n=30) and malignant (n=30) plasma samples: the activity ratios for benign and malignant plasma were 3.18 ± 1.79 and 2.02 ± 1.05, respectively. ****P<0.0001, **P<0.01.

[0232] Figure 9 This demonstrates how ART can differentiate between cancer-free patients and those with benign or malignant ovarian cancer. Figure 9 (a) shows the active CXCL10 and total CXCL10 concentrations measured in plasma samples. Figure 9 (b) Shows the active and total CXCL10 concentrations in the three patient groups. The calculated activity ratios between cancer-free (healthy) samples and (c) benign and malignant samples detected in plasma or (d) CVS are also included. *p≤0.05; ****p≤0.0001.

[0233] Figure 10 (a) shows the abundance of DPP4; Figure 10 (b) shows the specific activity of DPP4 in the prospective collection cohort; Figure 10 (c)-(d) show their correlation with the activity ratio.

[0234] Figure 11 The use of CVS swabs in ART to differentiate between non-cancerous and benign and malignant conditions is illustrated. Figure 11 (a) shows that the total concentration of active CXCL10 is significantly higher than the total concentration of total CXCL10. Figure 11 (b) shows the calculated activity ratios between cancer-free (healthy) samples and benign and malignant samples. *p≤0.05; ****p≤0.0001.

[0235] Figure 12 (a) shows the abundance of DPP4 on CVS; Figure 12(b) shows the correlation with the calculated CVS activity ratio.

[0236] Figure 13 (a) shows plasma CA125 in a prospective collection cohort of patients with benign or malignant ovarian tumors. Figure 13 (b) shows the correlation between CA125 and the activity ratio.

[0237] Figure 14 (a) shows a combination of the activity ratios of plasma and CVS (a), plasma and DPP4 and CA125 (b), and CVS and DPP4 and CA125 (c), each of which can be used to distinguish between healthy women and patients with malignant ovarian tumors.

[0238] Figure 15 The ROC assessments show (a) the discriminative power of individual biomarkers compared to CA125 and (b) biomarker combinations (based on benign + healthy and malignant constructs).

[0239] Key sequence list

[0240] The amino acid sequence of SEQ ID NO: 1 human CXCL10, including the pre-sequence.

[0241] Amino acid sequence of mature human CXCL10 SEQ ID NO: 2

[0242] SEQ ID NO: 3 Heavy chain VH amino acid sequence of anti-CXCL10 antibody RA2

[0243] SEQ ID NO: 4 Light chain VL amino acid sequence of anti-CXCL10 antibody RA2

[0244] SEQ ID NO: 5 Heavy chain VHCDR1 amino acid sequence of anti-CXCL10 antibody RA2

[0245] SEQ ID NO: 6 Heavy chain VHCDR2 amino acid sequence of anti-CXCL10 antibody RA2

[0246] SEQ ID NO: 7 Heavy chain VHCDR3 amino acid sequence of anti-CXCL10 antibody RA2

[0247] SEQ ID NO: 8 Light chain VLCDR1 amino acid sequence of anti-CXCL10 antibody RA2

[0248] SEQ ID NO: 9 Light chain VLCDR2 amino acid sequence of anti-CXCL10 antibody RA2

[0249] SEQ ID NO: 10 Light chain VLCDR3 amino acid sequence of anti-CXCL10 antibody RA2

[0250] SEQ ID NO: 11 Heavy chain VH amino acid sequence of anti-CXCL10 antibody RG2

[0251] SEQ ID NO: 12 Light chain VL amino acid sequence of anti-CXCL10 antibody RG2

[0252] SEQ ID NO: 13 Heavy chain VHCDR1 amino acid sequence of anti-CXCL10 antibody RG2

[0253] SEQ ID NO: 14 Heavy chain VHCDR2 amino acid sequence of anti-CXCL10 antibody RG2

[0254] SEQ ID NO: 15 Heavy chain VHCDR3 amino acid sequence of anti-CXCL10 antibody RG2

[0255] SEQ ID NO: 16 Light chain VLCDR1 amino acid sequence of anti-CXCL10 antibody RG2

[0256] SEQ ID NO: 17 Light chain VLCDR2 amino acid sequence of anti-CXCL10 antibody RG2

[0257] SEQ ID NO: 18 Light chain VLCDR3 amino acid sequence of anti-CXCL10 antibody RG2

[0258] SEQ ID NO: 19 Heavy chain VH nucleotide sequence of anti-CXCL10 antibody RA2

[0259] SEQ ID NO: 20 Light chain VL nucleotide sequence of anti-CXCL10 antibody RA2

[0260] SEQ ID NO: 21 Heavy chain VH nucleotide sequence of anti-CXCL10 antibody RG2

[0261] SEQ ID NO: 22 Light chain VL nucleotide sequence of anti-CXCL10 antibody RG2

[0262] SEQ ID NO: 23 contains the complete N-terminal peptide sequence of human CXCL10.

[0263] SEQ ID NO: 24 contains an N-terminal truncated peptide sequence of human CXCL10.

[0264] SEQ ID NO: 25 complete N-terminal epitope of CXCL10

[0265] SEQ ID NO: N-terminal truncated epitope of CXCL10 from SEQ ID NO: 26 Detailed Implementation

[0266] Overview

[0267] Throughout this specification, unless otherwise expressly stated or required by the context, when referring to a single step, a substance composition, a group of steps, or a group of substance compositions shall be understood to include one or more (i.e., one or more) of such steps, substance compositions, groups of steps, or groups of substance compositions.

[0268] The scope of this disclosure is not limited to the specific examples described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are obviously within the scope of this disclosure.

[0269] Those skilled in the art will understand that various changes and / or modifications can be made to the invention illustrated in the specific embodiments without departing from the spirit or scope of the invention as described broadly. Therefore, these embodiments should be considered illustrative rather than restrictive in all respects.

[0270] All publications discussed and / or cited in this article are incorporated herein in their entirety.

[0271] Any discussion of documents, actions, materials, devices, articles, etc., included in this specification is merely to provide context for the invention. It should not be construed as an admission that any or all of these matters constitute part of the prior art or common general knowledge in the field relating to the invention that existed prior to the priority date of each claim of this application.

[0272] Unless otherwise expressly stated, any example in this disclosure should be considered as applicable to any other example of this disclosure. In other words, any particular example of this disclosure may be combined with any other particular example of this disclosure (unless mutually exclusive).

[0273] Any example of this disclosure that discloses a particular feature or set of features or methods or method steps will be used to provide explicit support for abandoning that particular feature or set of features or methods or method steps.

[0274] Unless otherwise expressly defined, all technical and scientific terms used herein should be considered to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, molecular biology, immunohistochemistry, protein chemistry, and biochemistry).

[0275] Unless otherwise stated, the recombinant proteins, cell culture, and immunological techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained in literature from sources such as Perbal, 1984; Sambrook et al., 1989; Brown, 1991; Glover et al., 1995 and 1996; Ausubel et al., 1988; Harlow et al., 1988; and Coligan et al., 1991.

[0276] The descriptions and definitions of variable regions and their portions, immunoglobulins, antibodies and their fragments are further clarified in the following discussion: Kabat et al., 1987 and 1991; Bork et al., 1994; Chothia and Lesk, 1987; Chothia et al., 1989 and / or Al-Lazikani et al., 1997.

[0277] The ranges mentioned herein (e.g., ranges of residues) will be understood to be inclusive. For example, the reference to “the region containing amino acids 56 to 65” will be understood in an inclusive manner, that is, the region contains the amino acid sequence numbered 56, 57, 58, 59, 60, 61, 62, 63, 64 and 65 in the specified order.

[0278] The term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y”, and should be regarded as providing explicit support for both meanings or either meaning.

[0279] Throughout this specification, the word “comprise” or variations thereof such as “comprises” or “comprising” shall be understood to imply inclusion of the stated element, integer or step, or group of elements, integers or steps, without excluding any other element, integer or step, or group of elements, integers or steps.

[0280] As used herein, the term "subject" should be understood to include any animal, such as a mammal, including humans. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, a subject is a human.

[0281] Selected definition

[0282] Human CXCL10 was expressed as a pre-sequence (SEQ ID NO: 1), whose N-terminal 22 amino acids were cleaved to produce mature “full-length” human CXCL10 (SEQ ID NO: 2). Therefore, as used herein, the term “full-length CXCL10” refers to CXCL10 without post-translational modifications, rather than the “mature” full-length CXCL10 produced by post-translational cleavage to remove the 22 N-terminal amino acids.

[0283] As used herein, the term “N-terminal truncated CXCL10” refers to “mature” full-length CXCL10 that has been truncated by dipeptidyl peptidase IV (DPP4) and consists of, for example, amino acids 3 to 77 as shown in SEQ ID NO: 2.

[0284] As used herein, the term "citrullinated CXCL10" refers to CXCL10 that has been post-translational modified by peptidyl arginine deiminase (PAD) at, for example, the arginine residue at position 5 of amino acid position shown in SEQ ID NO: 2, for example.

[0285] Sequences of CXCL10 from other species can be determined using the sequences provided herein and / or in publicly available databases and / or using standard techniques (e.g., Ausubel et al., 1988 (including all updates to date) or Sambrook et al., 1989).

[0286] The term "recombinant" should be understood as the product of artificial genetic recombination. Therefore, in the context of recombinant proteins containing variable regions or antigen-binding domains (e.g., antibody-antigen-binding domains), this term does not include proteins naturally present in the body of a subject that are products of natural recombination during B cell maturation. However, if such a protein is isolated, it is considered an isolated protein containing variable regions or antigen-binding domains. Similarly, if nucleic acids encoding proteins are isolated and expressed using a recombinant approach, the resulting protein is a recombinant protein containing variable regions or antigen-binding domains. Recombinant proteins also include proteins expressed through artificial recombination when expressed, for example, in cells, tissues, or a subject.

[0287] The term "protein" should be understood to include a single polypeptide chain, that is, a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains (i.e., polypeptide complexes) linked covalently or nonvalently to each other. For example, a series of polypeptide chains can be covalently linked using suitable chemical bonds or disulfide bonds. Examples of nonvalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0288] The term “polypeptide” or “polypeptide chain” will be understood, based on the preceding paragraph, as referring to a series of consecutive amino acids linked by peptide bonds.

[0289] As used herein, the term “binding protein” should be understood to mean a protein or a portion thereof or other region of a protein that is capable of interacting with or specifically binding to an antigen (e.g., a cellular component or molecule, such as a protein).

[0290] As used herein, the term "antigen-binding domain" should be understood as referring to the region of an antibody that can specifically bind to an antigen, i.e., V.H or V L Or simultaneously include V H and V L The antigen-binding domain does not necessarily have to be in the case of an intact antibody; for example, it can be isolated (e.g., domain antibody) or exist in another form (e.g., as described herein, such as scFv).

[0291] For the purposes of this disclosure, the term "antibody" includes a protein capable of specifically binding one or more closely related antigens (e.g., CXCL10) through an antigen-binding domain contained in an Fv. This term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-transplantation antibodies, primate antibodies, deimmunized antibodies, synthetic humanized antibodies, haptens, bispecific antibodies). Antibodies typically contain constant domains that may be arranged into constant regions or constant fragments or crystallizable fragments (Fc). Exemplary forms of antibodies contain a four-chain structure as their basic unit. Full-length antibodies contain two covalently linked heavy chains (about 50 to 70 kDa) and two light chains (each about 23 kDa). The light chains typically contain a variable region (if present) and a constant domain, and in mammals are κ or λ light chains. The heavy chains typically contain a variable region and one or two constant domains connected to another constant domain via hinge regions. Mammalian heavy chains belong to one of the following types: α, δ, ε, γ, or μ. Each light chain is also covalently linked to one heavy chain. For example, two heavy chains, as well as heavy and light chains, are linked together via interchain disulfide bonds and non-covalent interactions. The number of interchain disulfide bonds may vary between different types of antibodies. Each chain has an N-terminal variable region (V... H or V L Each of the light chains is approximately 110 amino acids long and consists of one or more constant structural domains located at the C-terminus. L The constant structural domain (approximately 110 amino acids in length) and the first constant structural domain of the heavy chain (C) H 1. The antibody heavy chain (330 to 440 amino acids in length) is aligned and bound by disulfide bonds. The variable region of the light chain is aligned with the variable region of the heavy chain. The antibody heavy chain may contain two or more additional C... H Structural domains (e.g., C) H 2. C H 3, etc.) and can be included in C H 1 and C H2. Hinge region between constant structural domains. Antibodies can belong to any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In one example, the antibody is a mouse (mouse or rat) antibody or a primate (e.g., human) antibody. In one example, the antibody heavy chain lacks a C-terminal lysine residue. In one example, the antibody is a humanized, synthetically humanized, chimeric, CDR-grafted, or deimmunized antibody.

[0292] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably and refer to an antibody in its essentially complete form, rather than an antigen-binding fragment of the antibody. Specifically, a whole antibody includes antibodies with both heavy and light chains, including the Fc region. The constant domain can be a wild-type sequence constant domain (e.g., a human wild-type sequence constant domain) or a variant of its amino acid sequence.

[0293] As used herein, a “variable region” refers to the light and / or heavy chain portion of an antibody as defined herein, which is capable of specifically binding to an antigen and includes complementarity-determining regions (CDRs), namely, the amino acid sequences of CDR1, CDR2, and CDR3; and frame regions (FRs). For example, a variable region may contain three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) and three CDRs. H This refers to the variable region of the heavy chain. V L It refers to the variable region of a light chain.

[0294] As used herein, the term "complementarity-determining region" (also known as CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues in the antibody variable region, which are primarily used for specific antigen binding. Each variable region domain (V H or V L Typically, it has three CDRs, identified as CDR1, CDR2, and CDR3. In one example, the amino acid positions assigned to the CDRs and FRs are defined by Kabat et al. (1987 and 1991) (also referred to herein as the "Kabat numbering system"). In another example, the amino acid positions assigned to the CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html). According to Kabat's numbering system, V HThe positions of FR and CDR are as follows: residues 1 to 30 (FR1), 31 to 35 (CDR1), 36 to 49 (FR2), 50 to 65 (CDR2), 66 to 94 (FR3), 95 to 102 (CDR3), and 103 to 113 (FR4). According to Kabat's numbering system, V L The positions of FR and CDR are as follows: residues 1 to 23 (FR1), 24 to 34 (CDR1), 35 to 49 (FR2), 50 to 56 (CDR2), 57 to 88 (FR3), 89 to 97 (CDR3), and 98 to 107 (FR4). This disclosure is not limited to the FR and CDR defined by the Kabat numbering system, but includes all numbering systems, including the canonical numbering system or the numbering systems described by Chothia and Lesk (1987), Chothia et al. (1989), and / or Al-Lazikani et al. (1997); the numbering system described by Honnegger and Plükthun (2001); or the IMGT system discussed by Giudicelli et al. (1997). In one example, CDR is defined according to the Kabat numbering system. Optionally, the heavy chain CDR2 according to the Kabat numbering system does not contain any of the five C-terminal amino acids listed herein, or any one or more of these amino acids are substituted by another naturally occurring amino acid. In this regard, Padlan et al. determined in 1995 that the five C-terminal amino acids of the heavy chain CDR2 do not usually participate in antigen binding.

[0295] The "frame region" (FR) consists of variable region residues other than CDR residues.

[0296] As used herein, the term "Fv" (or variable fragment) should be understood to refer to any protein, whether it consists of multiple polypeptides or a single polypeptide, where V L and V H They combine to form a complex having an antigen-binding domain, i.e., capable of specifically binding to an antigen. The VH and VL forming the antigen-binding domains may be located in a single polypeptide chain or in different polypeptide chains. Furthermore, the Fv of this disclosure (and any protein of this disclosure) may have multiple antigen-binding domains, which may bind to the same antigen or not to the same antigen-binding structure. This term should be understood to include fragments directly derived from antibodies and proteins corresponding to such fragments produced using recombinant methods. In some examples, V... H Not connected to the heavy chain constant domain (C H )1; and / or V L Not connected to the light chain constant domain (C LExemplary peptides or proteins containing Fv include Fab fragments, Fab' fragments, F(ab') fragments, scFv, double-chain antibodies, triple-chain antibodies, quadruple-chain antibodies, or higher-order complexes, or any of the aforementioned substances linked to a constant region or domain, such as C. H 2 or C H 3. Structural domains, such as microantibodies. The “antigen-binding fragment” or “Fab fragment” consists of a monovalent antigen-binding fragment of an immunoglobulin and can be produced by digesting the whole antibody with papain to generate a fragment consisting of a complete light chain and a portion of the heavy chain; alternatively, it can be produced using a recombinant method. The antibody’s “Fab” fragment can be obtained by treating the whole antibody with pepsin and then reducing it to produce a fragment consisting of a complete light chain and a portion of the heavy chain. H A molecule composed of a portion of a heavy chain and a single constant structural domain. Each antibody treated in this way yields two Fab' fragments. Fab' fragments can also be generated via recombination. The antibody's "F(ab')2 fragment" consists of a dimer of two Fab' fragments linked together by two disulfide bonds and is obtained by treating the entire antibody molecule with pepsin without subsequent reduction. The "Fab2" fragment contains, for example, a leucine zipper or C... H 3. Recombinant fragments of two Fab fragments linked by a structural domain. “Single-chain Fv” or “scFv” is a recombinant molecule containing an antibody-containing variable region fragment (Fv), in which the light chain variable region and the heavy chain variable region are covalently linked by a suitable flexible peptide linker.

[0297] As used herein, the term "binds" in relation to the interaction of CXCL10 binding proteins or their antigen-binding domains with antigens means that the interaction depends on the presence of a specific structure on the antigen (e.g., an antigenic determinant or epitope). For example, antibodies recognize and bind to specific protein (rather than protein in general) structures. If an antibody binds to epitope "A," then in a reaction involving labeled "A" and a protein, the presence of a molecule containing epitope "A" (or free, unlabeled "A") will reduce the amount of labeled "A" bound to the antibody.

[0298] As used herein, the terms “specific binding” or “specifically binding” should be understood to mean that the CXCL10 binding protein of this disclosure reacts more frequently, more rapidly, for a longer duration, and / or with greater affinity to alternative antigens or cells compared to specific antigens or cells expressing the same antigen or cell. For example, the affinity of the CXCL10 binding protein to CXCL10 is much greater than that to antigens typically recognized by other chemokine receptors or by multireactive natural antibodies (i.e., naturally occurring antibodies known to bind to a variety of naturally occurring human antigens) (e.g., 1.5-fold, or 2-fold, or 5-fold, or 10-fold, or 20-fold, or 40-fold, or 60-fold, or 80-fold to 100-fold, or 150-fold, or 200-fold). The reference to “binding” provides explicit support for the term “specific binding”, and vice versa.

[0299] As used herein, the term “does not bind to” should be understood to mean that the CXCL10 binding protein of this disclosure does not bind to a specific antigen or a cell expressing that antigen.

[0300] As used herein, the term "undetectable binding" should be understood to mean that CXCL10 binding proteins, such as antibodies, bind to candidate antigens in the aforementioned background at a level of less than 10%, or 8%, or 6%, or 5%. This background can be the level of binding signal detected in the absence of the protein and / or in the presence of a negative control protein (e.g., an allotype control antibody), and / or the level of binding detected in the presence of a negative control antigen. In one example, this binding level is detected using a biosensor assay (e.g., Biacore) in which the antigen (e.g., a peptide) is immobilized and contacted with the CXCL10 binding protein.

[0301] As used herein, the term "non-significant binding" should be understood to mean that the binding level of the CXCL10 binding protein of this disclosure to the peptide is not statistically significantly higher than the background level, for example, the binding signal level detected in the absence of the CXCL10 binding protein and / or in the presence of a negative control protein (e.g., an isotype control antibody), and / or the binding level detected in the presence of the negative control peptide. In one example, this binding level is detected using a biosensor assay (e.g., Biacore), in which the antigen (e.g., the peptide) is immobilized and contacted with the CXCL10 binding protein.

[0302] For purposes of illustrative purposes and based on the subject matter of the examples herein, it will be apparent to those skilled in the art that “affinity” as used herein refers to the K-axis of a protein or antibody. D .

[0303] For purposes of clarification and based on the description herein, it will be apparent to those skilled in the art that references to “at least about” affinity will be understood to mean affinity (or K).D The affinity is equal to or greater than the value listed (i.e., the value listed when the affinity is low), meaning an affinity of 2 nM is greater than an affinity of 3 nM. In other words, the term can be "X or less affinity," where X is the value listed here.

[0304] As used herein, the term "epitope" (also known as "antigenic determinant") should be understood to refer to the region where CXCL10 binds to a protein. The term is not necessarily limited to a specific residue or structure that contacts the CXCL10-binding protein. For example, the term includes a region spanning the amino acid that contacts the CXCL10-binding protein and 5-10 (or more), 2-5, or 1-3 amino acids beyond that region. In some examples, the epitope comprises a discontinuous series of amino acids that fold close together when the CXCL10 polypeptide folds, for example, when binding to another CXCL10 polypeptide; this is known as a "conformational epitope."

[0305] The term "competitive inhibition" should be understood to mean that the CXCL10 binding protein (or its antigen-binding domain) of this disclosure reduces or prevents the binding of the antibody or the CXCL10 binding protein to CXCL10. This may be due to the binding of the CXCL10 binding protein (or antigen-binding domain) and the antibody to the same or overlapping epitopes. It will be apparent from the foregoing that the CXCL10 binding protein does not need to completely inhibit antibody binding, but rather it only needs to reduce binding by a statistically significant amount, for example, at least about 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90%, or 95%. For example, the CXCL10 binding protein reduces antibody binding by at least about 30%, for example at least about 50%, for example at least about 70%, for example at least about 75%, and even more preferably, at least about 80% or 85%, for example at least about 90%. Methods for determining competitive inhibition of binding are known in the art and / or described herein. For example, antibodies are exposed to CXCL10 with or without the CXCL10-binding protein. If antibody binding is reduced in the presence of the CXCL10-binding protein compared to the absence of the CXCL10-binding protein, the protein is considered to competitively inhibit antibody binding. In one example, the competitive inhibition is not due to steric hindrance.

[0306] CXCL10 binding protein

[0307] As discussed herein, the binding proteins of this disclosure can take on various forms and bind to full-length human CXCL10, N-terminal truncated CXCL10, and / or citrullinated CXCL10.

[0308] In one example, this disclosure provides a CXCL10 binding protein, wherein the binding protein binds to full-length human CXCL10, N-terminal truncated CXCL10, and citrullinated CXCL10.

[0309] In another example, this disclosure provides a CXCL10 binding protein that binds to full-length human CXCL10 but not to N-terminal truncated CXCL10 or citrullinated CXCL10.

[0310] Antibody

[0311] In one example, the CXCL10 binding protein of this disclosure includes an antibody or an antigen-binding fragment thereof.

[0312] Immunological methods

[0313] Methods for generating antibodies are known in the art and / or described in Harlow et al. (1988). Typically, in such methods, a protein, or an immunogenic fragment or epitope thereof, formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient (i.e., an immunogen), is administered to a non-human animal, such as a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cattle, goat, or pig. The immunogen can be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or via other known routes.

[0314] Polyclonal antibody production can be monitored by sampling the blood of immunized animals at different time points following immunization. If necessary, one or more further immunizations can be performed to achieve the desired antibody titer. The booster and titration process is repeated until the appropriate titer is reached. Once the desired level of immunogenicity is obtained, blood is collected from the immunized animals, and the serum is separated and stored, and / or the animals are used to produce monoclonal antibodies (mAbs).

[0315] Monoclonal antibodies are one exemplary form of antibody contemplated in this disclosure. The term "monoclonal antibody" or "mAb" refers to a group of homogeneous antibodies capable of binding to the same antigen, for example, to the same epitope within the antigen. This term is not intended to limit the source of the antibody or the manner in which it is prepared.

[0316] For the production of mAb, any of a variety of known techniques can be used, such as those listed in US4196265 or by Harlow et al. (1988).

[0317] For example, an immunogen is used to immunize a suitable animal under conditions sufficient to stimulate antibody-producing cells. Rabbits and rodents such as mice and rats are exemplary animals. Mice that are genetically engineered to express human immunoglobulins and, for example, do not express mouse immunoglobulins can also be used to produce antibodies of this disclosure (e.g., as described in WO2002066630).

[0318] Following immunization, somatic cells with antibody-producing potential, such as B lymphocytes (B cells), are selected for use in the mAb generation protocol. These cells can be obtained from biopsies of the spleen, tonsils, or lymph nodes, or from peripheral blood samples. The B cells from the immunized animal are then fused with immortalized myeloma cells, typically derived from the same species as the animal immunized with the immunogen.

[0319] Hybrids are amplified by culturing in a selective medium containing reagents that block de novo nucleotide synthesis in tissue culture media. Exemplary reagents are aminopterin, methotrexate, and azoserine.

[0320] For example, functional selection of amplified hybridomas based on antibody specificity and / or titer can be performed using flow cytometry and / or immunohistochemistry and / or immunoassays (e.g., radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, spot immunoassay).

[0321] Alternatively, ABL-MYC technology (NeoClone, Madison WI 53713, USA) can be used to produce cell lines that secrete mAbs (e.g., as described in the literature by Largaespada et al. (1996)).

[0322] Library-based approach

[0323] This disclosure also includes a library of screening antibodies or their antigen-binding fragments (e.g., containing their variable regions).

[0324] Examples of libraries considered in this disclosure include initial libraries (from untreated subjects), immune libraries (from subjects immunized with antigens), or complex libraries. Nucleic acids encoding antibodies or regions thereof (e.g., variable regions) are cloned using conventional techniques (e.g., as disclosed by Sambrook et al., 2001) and used to encode and display proteins using methods known in the art. Other techniques for generating protein libraries are described, for example, in US6300064 (e.g., the HuCAL library from Morphosys AG), US5885793, US6204023, US6291158, or US6248516.

[0325] The antigen-binding fragments according to this disclosure may be soluble secreted proteins or may be presented as fusion proteins on the surface of cells or particles (e.g., bacteriophages or other viruses, ribosomes, or spores). Various display library formats are known in the art. For example, the library is an in vitro display library (e.g., a ribosome display library, a covalent display library, or an mRNA display library, as described in, for example, US7270969). In yet another example, the display library is a phage display library in which a protein containing an antigen-binding fragment of an antibody is expressed on a phage, as described in, for example, US6300064, US5885793, US6204023, US6291158, or US6248516. Other phage display methods are known in the art and are contemplated by this disclosure. Similarly, methods of cell display are contemplated by this disclosure, for example, by a bacterial display library as described in US5516637; by a yeast display library as described in US6423538; or by a mammalian display library.

[0326] Methods for screening display libraries are known in the art. In one example, affinity purification is used to screen display libraries disclosed herein, for example, as described by Scopes in 1994. Affinity purification methods typically involve contacting a protein containing an antigen-binding fragment displayed by the library with a target antigen and eluting those domains that remain bound to the antigen after washing.

[0327] If desired, any variable region or scFv identified through screening can be readily modified into a complete antibody. Exemplary methods for modifying or reformatting variable regions or scFvs into complete antibodies are described, for example, in the literature Jones et al. (2010), or Jostock et al. (2004), or WO2012040793. Alternatively, standard cloning methods, such as those described in Ausubel et al. (1987) and / or Sambrook et al. (2001), can also be used.

[0328] Deimmunized, chimeric, humanized, as-humanized, primate-derived, and human antibody or antigen-binding fragments. The antibody or antigen-binding fragment disclosed herein may be humanized.

[0329] The term "humanized antibody" should be understood to refer to a protein containing a human-like variable region, including the CDR of an antibody transplanted into or inserted into a human antibody's FR (this type of antibody is also called a "CDR transplanted antibody") from a non-human species (e.g., mouse or rat or non-human primate). Humanized antibodies also include antibodies in which one or more residues of a human protein are modified by substitution of one or more amino acids and / or one or more FR residues of a human antibody are substituted by corresponding non-human residues. Humanized antibodies may also contain residues not found in either human or non-human antibodies. Any other region of the antibody (e.g., the Fc region) is typically human. Humanization can be performed using methods known in the art, such as those described in US5225539, US6054297, US7566771, or US5585089. The term "humanized antibody" also includes hyperhumanized antibodies, for example, as described in US7732578. A similar meaning will also apply to the term "humanized antigen-binding fragment."

[0330] The antibodies or antigen-binding fragments thereof disclosed herein may be human antibodies or antigen-binding fragments thereof. As used herein, the term "human antibody" refers to an antibody found in humans that has variable and optional constant antibody regions, such as those in human germline or somatic cells or derived from libraries using these regions. "Human" antibodies may include amino acid residues not encoded by human sequences, such as mutations introduced through random or site-directed mutagenesis in vitro (particularly involving conserved substitutions or mutations in a few residues of a protein, such as mutations in residues 1, 2, 3, 4, or 5). These "human antibodies" do not necessarily need to be generated by a human immune response; instead, they may be generated using recombinant methods (e.g., screening phage display libraries) and / or by transgenic animals (e.g., mice) containing nucleic acids encoding the constant and / or variable regions of human antibodies and / or using guided selection (e.g., as described in US5565332). The term also includes affinity-matured forms of such antibodies. For the purposes of this disclosure, human antibodies will also be considered to include proteins comprising a FR derived from a human antibody or a sequence comprising a common sequence derived from human FRs, wherein one or more CDRs are random or semi-random FRs, for example as described in US6300064 and / or US6248516. A similar meaning will also apply to the term "human antigen-binding fragment".

[0331] The antibody or antigen-binding fragment thereof disclosed herein may be a synthetic humanized antibody or antigen-binding fragment thereof. The term "synthetic humanized antibody" refers to an antibody prepared by the method described in WO2007019620. A synthetic humanized antibody includes a variable region of the antibody, wherein the variable region comprises the FR from the variable region of a New World primate antibody and the CDR from the variable region of a non-New World primate antibody.

[0332] The antibody or antigen-binding fragment disclosed herein may be primate-derived. A “primate-derived antibody” comprises a variable region derived from an antibody produced after immunization of a non-human primate (e.g., a cynomolgus monkey). Optionally, the variable region of the non-human primate antibody is linked to a human constant region to produce a primate-derived antibody. Exemplary methods for producing primate-derived antibodies are described in US6113898.

[0333] In one example, the antibody or antigen-binding fragment thereof disclosed herein is a chimeric antibody or fragment. The terms "chimeric antibody" or "chimeric antigen-binding fragment" refer to an antibody or fragment in which one or more variable domains originate from a specific species (e.g., murine animals, such as mice or rats) or belong to a specific antibody class or subclass, while the remainder of the antibody or fragment originates from another species (e.g., human or non-human primates) or belongs to another antibody class or subclass. In one example, a V-cell containing a non-human antibody (e.g., a mouse antibody) is described. H and / or V L The chimeric antibody and the remaining region of the antibody are derived from human antibodies. The production of such chimeric antibodies and their antigen-binding fragments is known in the art and can be achieved by standard methods (such as those described in US6331415; US5807715; US4816567 and US4816397).

[0334] This disclosure also covers deimmunized antibodies or antigen-binding fragments thereof, such as those described in WO2000034317 and WO2004108158. Deimmunized antibodies and fragments have one or more epitopes, such as B-cell or T-cell epitopes, that have been removed (i.e., mutated), thereby reducing the likelihood that a subject will generate an immune response against the antibody or protein. For example, the antibodies of this disclosure are analyzed to identify one or more B-cell or T-cell epitopes, and one or more amino acid residues within the epitope are mutated, thereby reducing the immunogenicity of the antibody.

[0335] Antibody-binding domain containing protein

[0336] Single-domain antibodies;

[0337] In some examples, the CXCL10 binding protein of this disclosure is or contains a single-domain antibody (which may be used interchangeably with the terms "domain antibody" or "dAb"). A single-domain antibody is a single polypeptide chain containing all or part of the variable domain of the antibody heavy chain.

[0338] Double-chain antibodies, triple-chain antibodies, and quadruple-chain antibodies

[0339] In some examples, the CXCL10 binding protein of this disclosure is or includes a double-chain antibody, a triple-chain antibody, a quadruple-chain antibody, or a higher-order protein complex, such as those described in WO98 / 044001 and / or WO94 / 007921.

[0340] For example, a double-chain antibody is a protein consisting of two related polypeptide chains, each containing a structural V. L -XV H or V H -XV L Where X is a subset of V that does not contain enough to allow a single polypeptide chain to contain V. H and V L Associated (or formed) Fv residues or a linker that is not present; and the V of one of the polypeptide chains H With another polypeptide chain V L These molecules combine to form antigen-binding sites, i.e., Fv molecules capable of specifically binding to one or more antigens. Each polypeptide chain contains V... L and V H It can be the same, or V in each polypeptide chain L and V H They can be different, thus forming bispecific double-chain antibodies (i.e., comprising two Fvs with different specificities).

[0341] Single-stranded Fv (scFv) fragment

[0342] The CXCL10 binding protein disclosed herein may be scFv. Those skilled in the art will recognize that scFv contains V in a single polypeptide chain. H and V L District and V H and V L The polypeptide linker between them allows scFv to form the desired structure for antigen binding (i.e., for a single polypeptide chain V). H and V L They combine to form Fv). For example, this linker contains more than 12 amino acid residues, and (Gly4Ser)3 is one of the more favorable linkers for scFv.

[0343] This disclosure also considers Fv (or diFv or dsFv) stabilized by disulfide bonds, wherein a single cysteine ​​residue is introduced into V. H FR and V L In the FR, cysteine ​​residues are linked by disulfide bonds to produce a stable Fv.

[0344] Alternatively or otherwise, this disclosure covers dimer scFvs, namely proteins comprising two scFv molecules linked by non-covalent or covalent bonds, such as through a leucine zipper domain (e.g., derived from Fos or Jun). Alternatively, two scFvs may be linked by a peptide linker of sufficient length to allow the two scFvs to form and bind to an antigen, for example, as described in US20060263367.

[0345] Haptop

[0346] In some examples, the antigen-binding fragment of the present invention is a hapten or a hapten. Those skilled in the art will recognize that a hapten is a protein comprising a single heavy chain and a single light chain. The term "hapten" also encompasses proteins comprising an antibody light chain and an antibody heavy chain, wherein the antibody heavy chain has been mutated to prevent binding to another antibody heavy chain. In one example, a hapten is formed when an antibody dissociates to form two molecules, each containing one heavy chain and one light chain.

[0347] Methods for generating haptens are known in the art and exemplary methods are described herein.

[0348] In one example, a hapten can be secreted for expression by introducing genes for the single heavy and light chains that constitute the target IgG into cells. In one example, the constant region (e.g., the IgG4 constant region) contains a "bond or pore" (or "protrusion or pore") mutation to prevent heterodimer formation. In one example, the constant region (e.g., the IgG4 constant region) contains a T366W mutation (or protrusion). In another example, the constant region (e.g., the IgG4 constant region) contains T366S, L368A, and Y407V mutations (or pores). In another example, the constant region contains T350V, T366L, K392L, and T394W mutations (protrusions). In yet another example, the constant region contains T350V, L351Y, F405A, and Y407V mutations (pores). Exemplary constant region amino acid substitutions are numbered according to the EU numbering system.

[0349] Other antibodies and proteins containing their antigen-binding domains

[0350] This disclosure also considers other antibodies and proteins containing their antigen-binding domains, such as:

[0351] (i) Microantibodies, for example, as described in US5837821;

[0352] (ii) Conjugates and proteins, for example, as described in US4676980;

[0353] (iii) Modifiers and proteins produced using chemical cross-linking agents, such as those described in US4676980; and

[0354] (iv) Fab3, for example, as described in EP19930302894.

[0355] Immunoglobulins and immunoglobulin fragments

[0356] An example of the CXCL10 binding protein disclosed herein is a protein containing the variable region of an immunoglobulin, such as a T-cell receptor or a heavy chain immunoglobulin (e.g., IgNAR, camel antibody).

[0357] Heavy chain immunoglobulins

[0358] Heavy chain immunoglobulins differ structurally from many other forms of immunoglobulins (such as antibodies) because they contain heavy chains but not light chains. Therefore, these immunoglobulins are also known as "heavy chain-only antibodies." Heavy chain immunoglobulins are found in animals such as camels and cartilaginous fish (also known as IgNAR).

[0359] The variable region, which exists in naturally occurring heavy chain immunoglobulins, is usually referred to as "V" in camel Ig. HH The term "structural domain" is used in IgNAR as a way to distinguish them from the heavy chain variable region (called "V-NAR") present in conventional four-chain antibodies. H The structural domain) and the light chain variable region (called "V") present in conventional four-chain antibodies L Distinguish them from "structural domains".

[0360] Heavy chain immunoglobulins can bind to relevant antigens with high affinity and high specificity without the presence of light chains. This means that single-domain binding fragments can be derived from heavy chain immunoglobulins, which are readily expressed and are generally stable and soluble.

[0361] A general description of heavy chain immunoglobulins from camelids, their variable regions, and methods of their production and / or isolation and / or use can be found in the following references WO94 / 04678, WO97 / 49805 and WO97 / 49805.

[0362] For a general description of heavy chain immunoglobulins from cartilaginous fish, their variable regions, and methods of their production and / or isolation and / or use, see WO2005118629.

[0363] V-like protein

[0364] In one example, the CXCL10 binding protein of this disclosure comprises a T-cell receptor. The T-cell receptor has two V domains that bind together to form an antibody-like Fv module structure. Novotny et al. described in 1991 how the two V domains of the T-cell receptor (designated α and β) fuse and are expressed as a single-chain polypeptide, and how surface residues are altered to reduce the direct hydrophobicity similar to antibody scFv. Other publications describing the generation of single-chain or multimeric T-cell receptors comprising two V-α and V-β domains include WO1999045110 or WO2011107595.

[0365] Other non-antibody proteins containing antigen-binding domains include proteins with V-like domains, which are typically constructed as single molecules. Examples of proteins containing such V-like domains include CTLA-4, CD28, and ICOS. Further disclosures of proteins containing such V-like domains are included in WO1999045110.

[0366] Adnectins

[0367] In one example, the CXCL10 binding protein of this disclosure comprises adenettin protein. Adenettin protein is based on the tenth fibronectin type III in human fibronectin (…). 10 The Fn3) domain, in which the loop region is altered for antigen binding. For example, it can be used for... 10 The three loops at one end of the β-sandwich of the Fn3 domain are engineered to enable the adenodetin protein to specifically recognize antigens. See US20080139791 or WO2005056764 for more details.

[0368] Antiticalin

[0369] In yet another example, the CXCL10-binding protein of this disclosure comprises an anticarrier protein. Anticarrier proteins are derived from lipidcarrier proteins, a family of extracellular proteins that transport small hydrophobic molecules such as steroids, choline, retinoids, and lipids. Lipidcarrier proteins have a rigid β-sheet secondary structure with multiple loops at the open end of the cone structure, which can be engineered to bind to antigens. Such engineered lipidcarrier proteins are referred to as anticarrier proteins. Further descriptions of anticarrier proteins can be found in US7250297 or US20070224633.

[0370] Affinity

[0371] In another example, the CXCL10 binding protein of this disclosure comprises an affinity compound. The affinity compound is a scaffold of the Z-domain (antigen-binding domain) derived from Protein A of Staphylococcus aureus, which can be engineered to bind antigens. The Z-domain consists of a triple-helix bundle of approximately 58 amino acids. A library has been generated by randomization of surface residues. See EP1641818 for further details.

[0372] Avimer

[0373] In another example, the CXCL10-binding protein disclosed herein comprises avigmer. Avigmer is a multidomain protein derived from the A-domain scaffold family. The native domain, consisting of approximately 35 amino acids, employs a defined disulfide bond structure. Diversity arises from natural variations exhibited by reorganizing the A-domain family. See WO2002088171 for further details.

[0374] DARPin

[0375] In another example, the CXCL10 binding protein disclosed herein comprises a designed ankyrin repeat protein (DARPin). The designed ankyrin repeat is derived from ankyrins, a family of proteins that mediate the attachment of integrated membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two α-helices and one β-turn. They can be designed to bind different target antigens by randomizing the residues in the first α-helix and the β-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). See US20040132028 for further details.

[0376] Mutations in binding proteins

[0377] This disclosure also provides a CXCL10 binding protein or nucleic acid encoding the sequence disclosed herein that has at least 90% identity with the sequence disclosed herein. In one example, the CXCL10 binding protein or nucleic acid of this disclosure comprises a sequence that has at least about 90%, or 95%, or 97%, or 98%, or 99% identity with the sequence disclosed herein, wherein, according to any example, as described herein, the protein specifically binds to CXCL10.

[0378] Alternatively or otherwise, the CXCL10 binding protein includes V as described in any of the examples herein. H or V LThe CDRs must be at least about 90%, or 95%, or 97%, or 98%, or 99% identical (e.g., three CDRs), wherein the protein, according to any example as described herein, is capable of specifically binding to CXCL10. Methods for determining the binding between a protein and CXCL10 are described herein.

[0379] It is known in the art that the five C-terminal residues of the heavy chain CDR2 can be mutated to conserved or non-conserved amino acid substitutions (31% of the residues) (Padlan et al., 1995). Therefore, proteins can contain a CDR2 with at least about 35% identity to the heavy chain CDR2 sequence disclosed herein.

[0380] This disclosure also considers mutant forms of the CXCL10 binding protein of this disclosure that contain one or more conserved amino acid substitutions compared to the sequence described herein. In some examples, the CXCL10 binding protein contains 10 or fewer, such as 9, 8, 7, 6, 5, 4, 3, 2, or 1 conserved amino acid substitutions. "Conserved amino acid substitution" refers to the substitution of an amino acid residue with a similar side chain and / or hydrophilicity and / or hydrophilicity. Exemplary conserved amino acid substitutions are provided in Table 1.

[0381] Table 1: Exemplary Amino Acid Substitutions

[0382]

[0383]

[0384] Families of amino acid residues with similar side chains have been defined in this art, including: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Hydrophilicity indices are described, for example, by Kyte and Doolittle (1982), while hydrophilicity indices are described, for example, in US4554101.

[0385] This disclosure also considers non-conservative amino acid variations. For example, there is particular interest in replacing charged amino acids with another charged amino acid and replacing charged amino acids with neutral or positively charged amino acids. In some examples, CXCL10 binding proteins contain 10 or fewer, such as 9, 8, 7, 6, 5, 4, 3, 2, or 1, non-conservative amino acid substitutions.

[0386] In one example, the mutation occurs within the FR of the antigen-binding domain of the CXCL10 binding protein of this disclosure. In another example, the mutation occurs within the CDR of the CXCL10 binding protein of this disclosure.

[0387] Exemplary methods for generating mutant forms of the CXCL10 binding protein include:

[0388] • DNA mutagenesis (Thie et al., 2009) or RNA mutagenesis (Kopsidas et al., 2006; Kopsidas et al., 2007; and WO1999 / 058661);

[0389] • Introduce the nucleic acid encoding the polypeptide into mutant cells, such as XL-1Red, XL-mutS, and XL-mutS-Kanr bacterial cells (Stratagene);

[0390] • DNA restructuring (e.g., as disclosed by Stemmer (1994)); and

[0391] • Targeted mutagenesis (e.g., as described by Dieffenbach et al. (1995)).

[0392] Exemplary methods for determining the biological activity of the mutant CXCL10 binding protein of this disclosure are readily apparent to those skilled in the art and include, for example, antigen binding, competitive inhibition of binding, affinity, association, and dissociation.

[0393] In another example, the nucleic acid of this disclosure comprises a sequence having at least about 90%, or 95%, or 97%, or 98%, or 99% identity with the sequences disclosed herein, and encodes a CXCL10 binding protein having the function described in any example herein. This disclosure also includes nucleic acids encoding the CXCL10 binding protein of this disclosure, which differ from the sequences listed herein due to the degeneracy of the genetic code.

[0394] The percentage of identity between nucleic acids or peptides was determined by GAP (Needleman and Wunsch, 1970) analysis (GCG procedure), with a vacancy penalty of 5 and a vacancy extension penalty of 0.3. The query sequence was at least 50 residues long, and the GAP analysis aligned the two sequences over a region of at least 50 residues. For example, the query sequence was at least 100 residues long, and the GAP analysis aligned the two sequences over a region of at least 100 residues. Alternatively, the two sequences could be aligned over their entire length.

[0395] constant region

[0396] This disclosure covers the CXCL10 binding protein and / or antibody described herein, which contains a constant region of the antibody. This includes the antigen-binding fragment of an antibody fused to an Fc.

[0397] The constant region sequences that can be used to generate the proteins of this disclosure can be obtained from many different sources. In some examples, the constant region of a protein, or a portion thereof, is derived from a human antibody. The constant region, or a portion thereof, can be derived from any antibody class, including IgM, IgG, IgD, IgA, and IgE, and any antibody isotype, including IgG1, IgG2, IgG3, and IgG4. In one example, the constant region is human isotype IgG4 or the stable IgG4 constant region. In one embodiment, the constant region is the IgGκ constant region.

[0398] In one example, the Fc region of the constant region has the ability to reduce the inducible effect function compared to, for example, the Fc region of natural or wild-type human IgG1 or IgG3.

[0399] In the context of this disclosure, "effective function" refers to those biological activities mediated by cells or proteins that bind to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region), leading to cell death. Examples of antibody-induced effector functions include complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent phagocytosis (ADCP); and B cell activation. In one example, the effector function is ADCC and / or ADCP and / or CDC. Methods for assessing the level of effector function of proteins containing Fc regions are known in the art and / or described herein.

[0400] In one example, the Fc region is the IgG4 Fc region (i.e., derived from the IgG4 constant region), such as the human IgG4 Fc region. The sequence of a suitable IgG4 Fc region is obvious to a person skilled in the art and / or available in publicly available databases (e.g., from the National Center for Biotechnology Information).

[0401] In one example, the constant region is the stable IgG4 constant region. The term "stable IgG4 constant region" will be understood to refer to an IgG4 constant region that has been modified to reduce Fab arm exchange or the tendency to undergo Fab arm exchange or form a hapten. "Fab arm exchange" refers to a protein modification of human IgG4 in which the IgG4 heavy chain and its linked light chain (half-molecule) are exchanged for the heavy-light chain pair of another IgG4 molecule. Thus, the IgG4 molecule can acquire two distinct Fab arms to recognize two different antigens (producing a bispecific molecule). Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or reducing agents (such as reduced glutathione). When an IgG4 antibody dissociates to form two molecules, a "hapten" is formed, each molecule containing one heavy chain and one light chain.

[0402] In one example, according to the Kabat system (Kabat et al., 1987 and / or 1991), the stable IgG4 constant region contains proline at position 241 of the hinge region. According to the European Union numbering system (Kabat et al., 2001 and Edelman et al., 1969), this position corresponds to position 228 of the hinge region. In human IgG4, this residue is typically serine. After replacing proline with serine, the IgG4 hinge region contains the sequence CPPC. In this respect, those skilled in the art will recognize that the “hinge region” is the proline-rich portion of the antibody heavy chain constant region that connects the Fc and Fab regions, which confer mobility to the two Fab arms of the antibody. The hinge region includes cysteine ​​residues contained in the inter-heavy chain disulfide bonds. According to the Kabat numbering system, it is generally defined as extending from Glu226 to Pro243 in human IgG1. The hinge region of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the heavy chain disulfide (SS) bond in the same position (see, for example, WO2010 / 080538).

[0403] Other examples of stabilized IgG4 antibodies are those in which arginine at position 409 of the human IgG4 heavy chain constant region (according to the EU numbering system) is replaced by lysine, threonine, methionine, or leucine (e.g., as described in WO2006 / 033386). The Fc region of the constant region may additionally or optionally contain residues selected from the group consisting of alanine, valine, glycine, isoleucine, and leucine at position corresponding to 405 (according to the EU numbering system). Optionally, the hinge region contains proline at position 241 (i.e., the CPCC sequence) (as described above).

[0404] In another example, the Fc region is a region modified to have reduced effector function, i.e., a "non-immunostimulatory Fc region". For example, the Fc region contains a substituted IgG1 Fc region at one or more locations selected from the group consisting of 268, 309, 330, and 331. In another example, the Fc region is an IgG1 Fc region containing one or more of the following variations: E233P, L234V, L235A, and G236 deletion, and / or one or more of the variations: A327G, A330S, and P331S (Armour et al., 1999; Shields et al., 2001). Other examples of non-immunostimulatory Fc regions are described in the following literature (e.g., Dall'Acqua et al., 2006; and / or Hezareh, 2001).

[0405] In another example, the Fc region is a chimeric Fc region, for example, comprising at least one CH2 domain from an IgG4 antibody and at least one CH3 domain from an IgG1 antibody, wherein the Fc region contains substitutions (e.g., as described in WO2010 / 085682) at one or more amino acid positions selected from the group consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409, and 427 (EU numbers). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

[0406] Protein production

[0407] In one example, the CXCL10 binding protein described herein according to any example is produced by culturing cells of the present invention under conditions sufficient to produce the protein, for example, as described herein and / or as known in the art.

[0408] Recombination expression

[0409] In another example, the CXCL10 binding protein described in this paper according to any example is recombinant.

[0410] In the case of recombinant proteins, the nucleic acid encoding it can be cloned into an expression construct or vector, which is then transfected into host cells, such as *E. coli* cells, yeast cells, insect cells, or mammalian cells (e.g., simian COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells), or myeloma cells that do not produce the protein. Exemplary cells used for protein expression are CHO cells, myeloma cells, or HEK cells. Molecular cloning techniques for achieving these purposes are known in the art and described, for example, in Ausubel et al., 1988 (including all updates to date) or Sambrook et al., 1989. Various cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods for generating recombinant antibodies are also known in the art; see, for example, US4816567 or US5530101.

[0411] After isolation, the nucleic acid is inserted into the promoter of the expression construct or expression vector and operatively linked to further clone (DNA amplification) or express in a cell-free system or in a cell.

[0412] As used herein, the term "promoter" should be used in its broadest sense and includes transcriptional regulatory sequences of genomic genes, including TATA boxes or initiation elements required for accurate transcription initiation in the presence or absence of regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers, and silencers), which, for example, respond to developmental and / or external stimuli or alter nucleic acid expression in a tissue-specific manner. In this document, the term "promoter" is also used to describe recombinant, synthetic, or fusion nucleic acids or derivatives that confer, activate, or enhance the expression of nucleic acids operatively linked to them. Exemplary promoters may contain additional copies of one or more specific regulatory elements to further enhance the expression of said nucleic acid and / or alter spatial and / or temporal expression.

[0413] As used in this article, the term "operably linked" refers to locating the promoter relative to the nucleic acid so that the expression of the nucleic acid is controlled by the promoter.

[0414] Many vectors for expression in cells are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, a protein-coding sequence (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. Those skilled in the art will know suitable sequences for protein expression. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, α-factor leader, or acid phosphatase leader), or mammalian secretion signals (e.g., herpes simplex gD signal).

[0415] Exemplary promoters active in mammalian cells include: cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1-promoter (EF1), small nuclear RNA promoters (U1a and U1b), myosin heavy chain promoter, simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), adenovirus major late promoter, β-actin promoter; and hybrid regulatory elements containing CMV enhancer / β-actin promoter or immunoglobulin promoter or their active fragments. Examples of useful mammalian host cell lines include the monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); the human embryonic kidney cell line (293 or 293 cell subclones for growth in suspension culture); juvenile hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

[0416] Typical promoters suitable for expression in yeast cells, such as those selected from Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, include, but are not limited to: ADH1 promoter, GAL1 promoter, GAL4 promoter, CUP1 promoter, PHO5 promoter, nmt promoter, RPR1 promoter, or TEF1 promoter.

[0417] Methods for introducing isolated nucleic acids or expression constructs containing them into cells for expression are known to those skilled in the art. Techniques used for a given cell depend on known successful techniques. Methods for introducing recombinant DNA into cells include microinjection, DEAE-glucan-mediated transfection, liposome-mediated transfection such as using lipofectamine (Gibco, MD, USA) and / or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation, and particle bombardment such as using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA).

[0418] Host cells used for protein production can be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham's Fl0 (Sigma), Minimal Essential Medium (MEM, Sigma), RPM1-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.

[0419] Protein isolation

[0420] Methods for separating proteins are known in the art and / or described herein.

[0421] When the CXCL10-binding protein is secreted into the culture medium, the supernatant from this expression system can first be concentrated using a commercially available protein concentrator filter such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors (such as PMSF) can be used in any of the above steps to inhibit proteolysis, and antibiotics can be used to prevent the growth of foreign contaminants. Alternatively, the supernatant can be filtered and / or separated from the protein-expressing cells, for example, using continuous centrifugation.

[0422] CXCL10 binding proteins prepared from cells can be purified using methods such as ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination of the above methods. These methods are known in the art and described, for example, in WO99 / 57134 or Harlow et al. (1988).

[0423] Those skilled in the art will also recognize that proteins can be modified to include tags that facilitate purification or detection, such tags being, for example, polyhistidine tags like a hexahistine tag, or influenza virus hemagglutinin (HA) tags, or Simian Virus 5 (V5) tags, or FLAG tags, or glutathione S-transferase (GST) tags. The resulting protein is then purified using methods known in the art (e.g., affinity purification). For example, a protein containing a hexahistine tag can be purified by contacting a sample containing the protein with nickel-nitrotriacetic acid (Ni-NTA) specifically bound to a hexahistine tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and then eluting the bound protein. Alternatively or additionally, a ligand or antibody bound to the tag can be used in affinity purification methods.

[0424] Conjugate

[0425] In one example, the CXCL10 binding protein of this disclosure is conjugated with a detectable tag.

[0426] As used herein, the terms “conjugate” or “conjugated” should be understood to include both indirect and direct conjugations. For example, direct conjugation includes chemical conjugations, which can be non-covalent, covalent, or genetic conjugations (also known as “fusions”). In one example, the conjugation is covalent, for example, in the form of a disulfide bond.

[0427] As used herein, a “detectable label” is a molecular or atomic tag or label that produces or can be induced to produce an optical or other signal or product that is visually detectable or detectable by using a suitable detector. Detectable labels are well known in the art and include, for example, radioactive labels, enzyme labels, fluorescent labels, luminescent labels, bioluminescent labels, magnetic labels, cofactors, contrast agents, and sonicating agents.

[0428] Commonly used fluorescent labels include, but are not limited to, anthocyanins such as Alexa, Cy5, and Cy5.5, indocyanine, and fluorescein isothiocyanate (FITC). Fluorescent labels useful in the practice of this disclosure may include, but are not limited to: 1,5IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-carboxyfluorescein (pH 10); 5-carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-carboxyfluorescein); 5-HAT (hydroxytryptamine); 5-hydroxytryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA (5-carboxytetramethylrhodamine); 6-carboxyrhodamine 6C; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); Acridine Orange + DNA; Acridine Orange + RNA; Acridine Orange + DNA & RNA; Acridine Red; Acridine Yellow; Acridine Yellow Feulgen SITSA; Jellyfish Luminescent Protein (Luminescent Protein); Alexa Fluorescence Fluor 350; Alex fluorescence 430; Alex fluorescence 488; Alex fluorescence 532; Alex fluorescence 546; Alex fluorescence 568; Alex fluorescence 594; Alex fluorescence 633; ​​Alex fluorescence 647; Alex fluorescence 660; Alex fluorescence 680; Alizarin complexing agent; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); Aniline Blue; Anthracycline stearate; APC (Allophycocyanin); APC-Cy7; APTRA-BTC=Ratio dye, Zn 2+APTS; AstraZeneca Orange Brilliant Red 4G; AstraZeneca Orange R; AstraZeneca Red 6B; AstraZeneca Yellow 7GLL; Atabrine; ATTO-TAG.TM.CBQCA; ATTO-TAG.TM.FQ; Atabrine; Aurophosphine G; Aurophosphine; BAO 9 (bisaminophenyldiazole); BCECF (high pH); BCECF (low pH); Berberine sulfate; β-lactamase; BFP blue-shifted GFP (Y66H); Blue fluorescent protein; BFP / GFP FRET; Bimane; Bisbenzemide; Hoechst; BisBTC-Ratio dye, Zn 2+ Blancophor FFG; Blancophor SV; BOBO-1; BOBO-3; Bodipy 492 / 515; Bodipy 493 / 503; Bodipy 500 / 510; Bodipy 505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; Bodipy 558 / 568; Bodipy 564 / 570; Bodipy 576 / 589; Bodipy 581 / 591; Bodipy 630 / 650-X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy F1; Bodipy FL ATP; Bodipy F1-ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; BodipyTR-X SE; BO-PRO-1; BO-PRO-3; Bright Sulphoflavin FF; BTC-Ratio dye Ca 2+ BTC-5N-atio dye, Zn 2+ Calcein; Calcein Blue; Calcium Red; Calcium Green; Calcium Green-1Ca 2+ + dye; calcium green-2Ca 2+ ; Calcium Green-5N Ca 2 ; Calcium Green-C18 Ca 2+; Calcium Orange; Calcofluor White; Carboxy-X-Rhodamine (5-ROX); Cascade Blue; Cascade Yellow 399; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein); CFP / YF P FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF (Ratio Dye, pH); CMFDA; Coelenterazine; Coelenterazine cp (Ca 2+(Dye); Coelentrin f; Coelentrin fcp; Coelentrin h; Coelentrin hcp; Coelentrin ip; Coelentrin n; Coelentrin O; Coumarin Phalloidin; C-phycocyanin; CPM methylcoumarin; CTC; CTC formazan; Cy2; Cy3.18; Cy3.5; Cy3; Cy5.18; Cy5.5; Cy5; Cy7; Cyan GFP; Cyclic AMP Fluorosensor (FiCRhR); CyQuant Cell Proliferation Assay; Dabcyl; Danshenyl; Danshenamide; Danshenyl cadaverine; Danshenyl chloride; Danshenyl DHPE; Danshen fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (Dichlorodihydrofluorescein diacetate); DDAO; DHR (Dihydorhodamine 123); bis-4-ANEPPS; bis-8-ANEPPS (non-isotropic); DiA (4-Di-16-ASP); Dichlorodihydrofluorescein diacetate (DCFH); DiD-lipophilic tracer; DiD (DilC18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DilC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (Di1C18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; Red fluorescent protein; DTA F; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF97; Eosin; Phycoerythrone; Phycoerythrone ITC; Ethidium bromide; Ethylphenidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium chloride (111); EYFP; Speed ​​Blue; FDA; Fulgen (para-ferrochrome); FIF (formaldehyd-induced fluorescence); FITC; FITC antibody; Flazo orange; Fluo-3; Fluo-4; FITC; FITC diacetate; Fluorescent emerald; Fluoro-Gold (hydroxystilbene); Fluor-Ruby; FluorX; FM1-43; FM 4-46; Fura Red (high pH); Fura Red / Fluo-3; Fura-2, high calcium; Fura-2, low calcium; iFura-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF;Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); GFP (S65T); GFP Redshift (rsGFP); Non-UV Excited Wild-Type GFP (wtGFP); UV Excited Wild-Type GFP (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; Hematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbene (fluorescent gold); Serotonin; Indo-1 (high calcium); Indo-1 (low calcium); Indo Dicyanine (DiD); Indo Tricyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Rhodamine sulfadiazine; Rhodamine sulfadiazine B; LIVE / DEAD kit for animal cells; Calcein / ethindrone homodimer; LOLO-1; LO-PRO-1; Lucifer yellow; Lysosomal blue probe (Lyso Tracker Blue); Lysosomal blue-white probe; Lysosomal green probe; Lysosomal red probe; Lysosomal yellow probe; LysoSensor blue; LysoSensor green; LysoSensor yellow / blue; Mag green; Sudan Red (Magdala Red) (Root Bark Red B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; Magnesium green; Magnesium orange; Malachite green; Navy blue; Maxilon Brilliant Flavin 10GFF; Methionin 8GFF; Merocyanin; Methoxycoumarin; Mitotracker Greeu FM; Mitotracker Orange; Mitotracker Red; Procainamide; Monobromodiamine; mBBr-GSH; Monochlorobimane; MPS (Methyl-green pyronin stilbene); NBD; NBD amine; Nile Red; Nitrobenzoxedidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant Lavin E8G;Oregon Green; Oregon Green 488-X; Oregon Green 500; Oregon Green 514; Pacific Blue; Fulgen Red; PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-Texas Red [613 Red]; Phloxin B (Metalla Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; Photoresist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-IPRO-3; Primuline; Procion Yellow); Propidine Iodide (P1); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613 [PE-Texas Red]; Resorufin; RH414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B; Rhodamine B200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Phalloidine; Rhodamine Red; Rhodamine WT; Rose Red; R-phycocyanin; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Dark Blue GFP; SBFI; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFPTM (super glow BFP); sgGFP; sgGFP (super glow GFP); SITS; SITS (Primula Extract); SITS (Stilbene Isothiosulphonic Acid); SNAFL Calcein; SNAFL-1; SNAFL-2; SNAR Calcein; SNARF1; Sodium Green Green); SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red;SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium); stilbene; sulphorhodamine B and C; SulphoRhodamien Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN, Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; Tricolor (PE-Cy5); TRITC tetramethylrhodamine isothiocyanate; True Blue; True Red; Ultralite; Uranine B; Uvitex SFC; wtGFP; WW781; X-Rhodamine; XRITC; Xylene Organge; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; and YOYO-3.

[0429] In one example, the detectable marker is an enzyme. This enzyme can act on a suitable substrate to produce a detectable dye. Examples of enzymes that can be used in this disclosure include, but are not limited to, alkaline phosphatase and horseradish peroxidase. Alternatively, the enzyme may be, for example, luciferase. The enzyme can be conjugated to an antibody using conventional chemical methods or expressed as a fusion protein together with an antibody.

[0430] The radioactive isotopes used as detectable markers in this disclosure are well known in the art and may include 3 H, 11C 18 F, 35 S, 64 Cu、 67 Ga、 68 Ga、 99 mTc, 111 In、 123 I, 124 I, 125 I and 131 I. Any gamma-emitting radioactive material that can react with the carboxyl, amino, or thiol groups of compounds that bind to calcitonin receptors, such as... 99 mTc and 111 In attachment is suitable for detection methods using gamma scintillation scanning. Radioactive substances that can react with the carboxyl, amino, or thiol groups of a compound. 11 C 18 F, 64 Cu、 67 Ga、 68 Ga、 124 I and 131 The adhesion of compound I is suitable for detection methods using PET / SPECT imaging.

[0431] Detection of CXCL10 binding protein

[0432] CXCL10 Combinations and Modifications

[0433] As will be apparent to those skilled in the art from the disclosure herein, some of the CXCL10 binding proteins of this disclosure bind to full-length CXCL10 and / or to specific post-translational modifications of CXCL10 (e.g., N-terminal truncated CXCL10 and / or citrullinated CXCL10). Methods for assessing protein binding are known in the art, for example, as described in Scopes (1994). Such methods typically involve immobilizing the CXCL10 binding protein and contacting it with a labeled antigen. After washing to remove non-specific binding proteins, the amount of labeling is detected, thereby detecting the bound antigen. Of course, the CXCL10 binding protein can be labeled and the antigen immobilized. A panning assay can also be used. Alternatively, a surface plasmon resonance assay can also be used.

[0434] Determining affinity

[0435] Optionally, the dissociation constant (Kd), association constant (Ka), or equilibrium constant (K) of the binding protein can be determined. D In one example, these constants of the binding region (e.g., antibody or antigen-binding fragment) are measured using biosensor analysis employing surface plasmon resonance (SPR). An exemplary SPR method is described in US7229619.

[0436] Affinity measurements can be determined using standard methods of antibody reactions, such as immunoassays, surface plasmon resonance (SPR) (Rich and Myszka, 2000; Englebienne, 1998), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art.

[0437] Determine the competitive combination

[0438] The determination of antibodies or antigen-binding fragments thereof used to identify competitive inhibition of binding to the CXCL10 binding protein described herein will be obvious to those skilled in the art and / or to those described herein.

[0439] For example, the antibody or its antigen-binding fragment can be conjugated to a detectable label, such as a fluorescent or radioactive label. The labeled antibody and the test CXCL10 binding protein are then mixed and contacted with CXCL10 or its region or cells expressing it. The level of the labeled antibody is then measured and compared to the level measured when the labeled antibody is contacted with CXCL10, its region, or cells in the absence of the CXCL10 binding protein. If the level of the labeled antibody in the presence of the test CXCL10 binding protein is reduced compared to the absence of the CXCL10 binding protein, the CXCL10 binding protein is considered to competitively inhibit antibody binding to CXCL10.

[0440] Optionally, different label conjugations of the CXCL10-binding protein with the antibody can be tested. This alternative labeling allows for the detection of the binding level of the CXCL10-binding protein to CXCL10 or its region or cells.

[0441] In another example, the CXCL10 binding protein is allowed to bind to CXCL10 or its region or cells expressing it before contacting the CXCL10, region, or cell with the antibody. The reduced amount of antibody bound in the presence of the CXCL10 binding protein compared to its absence indicates that the protein competitively inhibits antibody binding to CXCL10. Alternatively, a labeled CXCL10 binding protein can be used, with the antibody first bound to CXCL10 for mutual analysis. In this case, the reduced amount of labeled CXCL10 binding protein binding to CXCL10 in the presence of the antibody compared to its absence indicates that the CXCL10 binding protein competitively inhibits antibody binding to CXCL10.

[0442] Determine the level of CXCL10

[0443] As discussed above, CXCL10 is associated with a variety of human diseases, including infectious diseases, central nervous system diseases, chronic inflammation, immune dysfunction, and cancer.

[0444] The inventors have developed a CXCL10 binding protein to detect different forms of the protein, namely full-length or mature CXCL10, N-terminal truncated CXCL10, and / or citrullinated CXCL10.

[0445] The inventors have discovered that different forms of proteins exist at different levels in benign and malignant diseases.

[0446] Therefore, any disclosed methods described herein include determining the level of CXCL10 in subjects.

[0447] As used in this article, the term “level” for CXCL10 should be understood to refer to the functional level of the protein (i.e., functional level). For example, level (or “expression level”) refers to a measure of the protein’s encoding.

[0448] In particular, the inventors have discovered that measuring the levels of active CXCL10 protein and total CXCL10 protein in subjects can differentiate between benign and malignant conditions. This procedure is referred to herein as the Active Ratio Test (ART).

[0449] As used herein, the term “active” in the context of CXCL10 levels refers to the biologically active form of CXCL10. For example, CXCL10-binding proteins that bind to active CXCL10 in this disclosure are binding proteins that bind to full-length or mature (i.e., N-terminally intact) CXCL10, but not to N-terminally truncated or citrullinated CXCL10.

[0450] As used herein, the term “total” in the context of CXCL10 levels refers to all forms of CXCL10. For example, CXCL10 binding proteins that bind to active CXCL10 in this disclosure refer to binding proteins that bind to both full-length or mature (i.e., N-terminally intact) CXCL10 and N-terminally truncated or citrullinated CXCL10.

[0451] In one example, determining the levels of active CXCL10 and total CXCL10 includes determining the amount of active CXCL10 protein and the amount of total CXCL10 protein in the subject.

[0452] As used herein, the term "quantity" regarding CXCL10 levels will be understood to refer to the amount of protein (i.e., active CXCL10 protein or total CXCL10 protein). Those skilled in the art can use various methods to assess protein quantity, and will recognize that specific values ​​or quantities will vary depending on the assessment method used. Clearly, the term includes both absolute and relative values. For example, the quantity may be relative to a reference or control sample. In another example, the quantity may be the absolute amount of protein present in the sample.

[0453] The inventors were surprised to find that they could distinguish between benign and malignant diseases by measuring the CXCL10 ratio in subjects.

[0454] As used in this article, the term "CXCL10 ratio" refers to the ratio of the level of active CXCL10 in a subject to the total level of CXCL10.

[0455] In one example, the method further includes comparing the CXCL10 ratio in the subject with the CXCL10 ratio in at least one reference.

[0456] In one example of any of the methods described herein, the method includes determining whether: (a) the CXCL10 ratio in the subject is higher than the CXCL10 ratio in the reference; or (b) whether the CXCL10 ratio in the subject is lower than the CXCL10 ratio in the reference.

[0457] The term "higher" in relation to the CXCL10 ratio refers to a larger or increased ratio in subjects compared to the control or reference level. Based on the foregoing, it will be clear that the CXCL10 ratio only needs to increase by a statistically significant amount, such as at least about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%.

[0458] The term "lower" in relation to the CXCL10 ratio refers to a reduction or decrease in the ratio among subjects compared to the control or reference level. Based on the above, it will be apparent that the CXCL10 ratio only needs to be reduced by a statistically significant amount, such as at least about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%.

[0459] Methods for determining CXCL10 levels

[0460] Methods for determining CXCL10 protein levels will be obvious to those skilled in the art and / or described herein. Examples include immunohistochemistry, immunofluorescence, Western blotting, protein blotting, dot blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay, fluorescence resonance energy transfer (FRET), matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF), electrospray ionization (ESI), mass spectrometry (including tandem mass spectrometry, such as LC MS / MS), biosensor technology, evanescent fiber optic technology, or protein chip technology. Suitable assays, for example, are semi-quantitative and / or quantitative assays.

[0461] In one embodiment, a method for determining CXCL10 levels in a sample includes: contacting a biological sample from a subject with a CXCL10-binding protein (as described herein) that specifically binds to a CXCL10 peptide or protein for a period of time, and then detecting the complex under conditions sufficient to form a complex between the binding protein and the peptide or protein. For example, in any of the methods described herein, the levels of active CXCL10 and total CXCL10 are measured and the ratio between active CXCL10 and total CXCL10 is determined.

[0462] Enzyme-linked immunosorbent assay (ELISA) and fluorescence-linked immunosorbent assay (FLISA)

[0463] Standard solid-phase ELISA or FLISA formats are particularly useful for determining protein concentrations in a variety of samples. In one form, this assay involves immobilizing a biological sample on a solid matrix, such as polystyrene or polycarbonate microwells or test paper, membrane, or glass support (e.g., a glass slide).

[0464] An antibody that specifically binds to a label within the CXCL10 peptide is directly contacted with an immobilized biological sample and forms a direct bond with any of its target proteins present in the sample. In the case of FLISA or enzymes (e.g., horseradish peroxidase (HRP), alkaline phosphatase (AP), or β-galactosidase), the antibody is typically labeled with a detectable reporter molecule label, such as a fluorescent label (e.g., FITC or Texas Red) or fluorescent semiconductor nanocrystals (as described in US 6,306,610), or in the case of ELISA, a second labeled antibody bound to a first antibody may be used. After washing to remove any unbound antibody, the label is detected directly, either in the case of fluorescent labeling or by adding a substrate, such as hydrogen peroxide, TMB, or toluidine, or 5-bromo-4-chloro-3-indole-β-D-galactoside (x-gal), in the case of enzyme labeling. Such ELISA or FLISA-based systems are suitable for quantifying the amount of protein in a sample by calibrating the detection system against a known amount of antibody-bound protein standards, such as isolated and / or recombinant CXCL10 peptides or their immunogenic fragments or epitopes. In another example, an ELISA consists of an antibody or ligand specifically binding to a disease or symptom marker within the CXCL10 peptide, immobilized on a solid matrix, such as a membrane, polystyrene or polycarbonate micropores, a polystyrene or polycarbonate micropore measuring tape, or a glass support. The sample is then physically contacted with the antibody, causing the marker within the sample to bind or be “captured.” The bound protein is then detected using a labeled antibody. Alternatively, a third labeled antibody bound to a second (detection) antibody can be used.

[0465] In one example, the fixed antibody is a polyclonal antibody.

[0466] It will be apparent to those skilled in the art that the assays described herein are suitable for high-throughput applications, such as automated screening processes or microarrays as described by Mendoza et al. (1999). Furthermore, variations of the aforementioned assays, such as competitive ELISAs, will be readily apparent to those skilled in the art.

[0467] In one example, the measurement form is a microfluidic device, such as a microfluidic chip or a droplet-based microfluidic device. Microfluidic chips (e.g., microelectromechanical systems (MEMS) devices) typically range in size from a few square millimeters to a few square centimeters. These microfluidic chips are designed to handle or manipulate small amounts of fluid to perform biological or medical procedures or tests. Fluids can be moved, mixed, or processed within a single microfluidic chip.

[0468] In another example, the test is performed on a test strip, such as a polycarbonate test strip.

[0469] SIMOA Measurement

[0470] Another assay that can be used in this invention is the single-molecule array (Simoa) assay, which is described in the literature of Kuhle et al. (2016) and Gisslen et al. (2016).

[0471] Protein blot

[0472] In another example, Western blotting is used to determine the level of a marker within the CXCL10 peptide in a sample. In such an assay, the protein is separated from the sample using techniques known in the art, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and described, for example, in Scopes (1994). The separated protein is then transferred to a solid support, such as a membrane (e.g., a PVDF membrane), using methods known in the art, such as electrotransfer. The membrane is then blocked with a labeled antibody or ligand that specifically binds to the marker within the CXCL10 peptide, and the membrane is probed. Alternatively, a labeled secondary or even triple antibody or ligand is used to detect the binding of a specific primary antibody. The labeling level is then determined using an assay appropriate to the label used.

[0473] Appropriate assays will be obvious to a skilled technician and include, for example, densitometric measurements. In one example, the intensity of a protein band or spot is normalized relative to the total amount of protein loaded onto an SDS-PAGE gel using methods known in the art. Alternatively, the level of the detected marker is normalized relative to the level of a control / reference protein. Such control proteins are known in the art and include, for example, actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β2-microglobulin, hydroxymethylcholesterol synthase, hypoxanthine phosphoribosyltransferase 1 (HPRT), ribosomal protein L13c, succinate dehydrogenase complex subunit A, and TATA box-binding protein (TBP).

[0474] Radioimmunoassay

[0475] Alternatively, radioimmunoassay (RIA) can be used to detect CXCL10 levels. The basic principle of this assay is to detect antibody-antigen interactions using radiolabeled antibodies or antigens. An antibody or ligand that specifically binds to a marker within the CXCL10 polypeptide is bound to a solid support, and the sample is in direct contact with the antibody. To detect the level of bound antigen, isolated and / or recombinant forms of the antigen are radiolabeled and contacted with the same antibody. After washing, the level of radioactivity after binding is detected. Since any antigen in a biological sample inhibits the binding of the radiolabeled antigen, the detected level of radioactivity is inversely proportional to the level of antigen in the sample. This assay can be quantified using a standard curve with increased known concentrations of the isolated antigen.

[0476] As is obvious to a technician, this assay can be modified to use any reporter molecule, such as an enzyme or a fluorescent molecule, instead of a radioactive label.

[0477] Biosensor or optical immunosensor system

[0478] Alternatively, biosensors or optical immunosensor systems can be used to determine the level of CXCL10 in a sample. Generally, an optical biosensor is a device that uses optical principles to quantitatively convert the binding of a ligand or antibody to a target peptide into an electrical signal. These systems can be categorized into four main types: reflectance technology; surface plasmon resonance; fiber optic technology; and integrated optics. Reflectance technology includes elliptic polarization, multiple integral reflectance spectroscopy, and fluorescent capillary-filled devices. Fiber optic technology includes evanescent field fluorescence, fiber optic capillaries, and fiber optic fluorescence sensors. Integrated optics include planar evanescent field fluorescence, input hierarchical coupler immunosensors, Mach-Zehnder interferometers, Hartman interferometers, and differential interferometer sensors. Examples of these optical immunosensors are generally described in the literature provided by Robins (1991). More specific descriptions of these devices can be found, for example, in U.S. patents such as Nos. 4,810,658; 4,978,503; 5,186,897; and Brady et al. (1987).

[0479] biological samples

[0480] As is obvious to technicians, the type and size of a biological sample will depend on the detection method used. For example, protein-based assays require a sufficient number of cells to provide enough protein for antigen-based assays.

[0481] As used herein, the term "sample" or "biological sample" refers to any type of suitable material obtained from a subject. This term includes clinical samples (e.g., cervical or cervicovaginal swabs), biological fluids (e.g., cervical fluid, vaginal secretions, plasma, ascites), tissue samples, live cells, and also includes cultured cells, cell supernatants, and cell lysates derived therefrom. Samples may be obtained directly from the source or used after at least one step (partial) purification. It will be apparent to those skilled in the art that samples can be prepared in any medium that does not interfere with the methods of this disclosure. Typically, samples contain cells or tissues and / or aqueous solutions or biological fluids containing cells or tissues. Those skilled in the art will understand the selection and pretreatment methods. Pretreatment may include, for example, diluting viscous fluids. Sample handling may involve filtration, distillation, separation, and concentration.

[0482] In one example, the biological sample has already been obtained from the subject. Therefore, in one example, the method as described herein according to any embodiment further includes providing a biological sample.

[0483] In one example, biological samples may be collected from subjects at more than one time point to, for example, monitor the progression of malignant disease, monitor recurrence, and / or assess the efficacy of treatment regimens. In one example, biological samples may be collected from subjects before, during, and / or after treatment for their malignant disease. Samples may be collected weekly, bi-weekly, monthly, bi-monthly ... or bi-monthly to monitor the progression of malignant disease or assess the efficacy of treatment regimens.

[0484] In one example, the method described herein, according to any implementation, is performed using an extract from a sample, such as a protein.

[0485] Reference Sample

[0486] As is apparent from the foregoing description, some of the determinations of this disclosure can be quantified using appropriate reference samples or controls.

[0487] Suitable reference samples used in the methods of this disclosure will be obvious to those skilled in the art and / or described herein. For example, a reference may be an internal reference (i.e., from the same subject), from normal individuals, or from an established dataset (e.g., matched for age, sample type, and / or period stage).

[0488] In one example, the reference is an internal reference or a sample. For example, the reference is a self-reference. In one example, the internal reference is obtained from the subject concurrently with the analysis of the sample. In another example, the internal reference is obtained from the subject at an earlier time point, serving as the sample being analyzed.

[0489] As used herein, the term "normal individual" should be understood as an individual selected based on the fact that they do not have malignant and / or benign conditions or that they are not suspected of having such conditions. For example, a normal individual is a healthy individual.

[0490] In one example, the reference is an established dataset. The established datasets applicable to this disclosure will be obvious to those skilled in the art, and include, for example:

[0491] • Data sets from normal subjects or normal subject groups matched by age and sample type;

[0492] • Datasets from another subject or subject group matched for age, sample type, and / or disease / condition;

[0493] • A dataset containing in vitro cells, wherein the cells have been treated to induce CXCL10 expression; and

[0494] • A dataset containing in vitro endometrial epithelial cells, in which the cells have been treated to suppress CXCL10 expression.

[0495] In one example, the reference is not included in the determination. Instead, a suitable reference is derived from a previously generated, established dataset. The data from the processed, analyzed, and / or determined test samples are then compared with the data obtained from the samples.

[0496] Detection and / or diagnosis of malignant diseases

[0497] As disclosed herein, the inventors have demonstrated the role of CXCL10 in the detection and / or diagnosis of malignant conditions. It will be apparent to those skilled in the art that the methods disclosed herein can be used to differentiate between malignant and benign conditions in a subject. For example, the methods disclosed herein can be used as a screening test for diagnosing malignant conditions in a subject.

[0498] Therefore, this disclosure provides, for example, a method for detecting and / or diagnosing malignant diseases in a subject, the method comprising:

[0499] (i) Determine the active CXCL10 level and the total CXCL10 level of the subject; and

[0500] (ii) Determine the ratio of active CXCL10 to total CXCL10 in the subjects.

[0501] As used in this article, the terms “detecting” or “diagnosis” refer to the identification of malignant conditions in a subject.

[0502] As used herein, the term "malignant disease" refers to a condition or disease that grows uncontrollably, invades normal tissue, and often metastasizes and grows in sites far from the tissue of origin. In one example, a malignant disease or condition is cancer or cancer-related. Those skilled in the art will understand that cancer can arise from virtually any tissue in the body, and as used herein, the term encompasses all forms of disease, including, for example, carcinoma, sarcoma, lymphoma, and leukemia (i.e., solid and non-solid forms of cancer).

[0503] In one example, this disclosure provides a method for distinguishing between malignant and benign conditions.

[0504] As used in this article, the term "benign disease" refers to a cluster of cells that lack the ability to invade adjacent tissues.

[0505] In one example, this disclosure provides a method for distinguishing precancerous lesions from benign conditions.

[0506] As used herein, the term "precancerous lesion" refers to an abnormally growing cluster of cells that differs in size, shape, or appearance from normal cells, but which have not yet become cancerous or malignant. In one example, a precancerous lesion is a p53 precancerous lesion. As used herein, the term p53 precancerous lesion refers to cells with a mutation in the p53 gene.

[0507] In one example, the subject has a malignant condition (i.e., cancer). For example, the cancer is a solid tumor, such as a sarcoma or carcinoma. For example, the cancer could be prostate cancer, ovarian cancer, breast cancer, lung cancer, liver cancer, kidney cancer, colon cancer, pancreatic cancer, or stomach cancer. For example, the subject has ovarian cancer. In one example, the cancer is a non-solid tumor, such as leukemia or lymphoma. In one example, the subject has stage 0 cancer. For example, the cancer is carcinoma in situ. In another example, the subject has stage I, II, or III cancer. For example, the cancer has spread to nearby lymph nodes outside the organ of origin and / or tissues or organs adjacent to the location of the primary tumor. In one example, the subject has stage IV cancer. For example, the cancer has spread to distant tissues and / or organs.

[0508] In one example, the subject had not yet received treatment for the malignant condition. For example, the subject had not received any treatment.

[0509] In one example, the subject is receiving treatment for a malignant condition. In another example, the subject has already received treatment for a malignant condition. The appropriate treatment for the malignant condition will be obvious to a technician and / or described herein. For example, treatments include surgery, chemotherapy, radiation therapy, targeted drug therapy, immunotherapy, or combinations thereof.

[0510] Ovarian cancer

[0511] In one example of any of the methods described herein, the method includes detecting and / or diagnosing ovarian cancer in the subject. For example, the subject has ovarian cancer.

[0512] As used in this article, the term "ovarian cancer" refers to any cancerous growth that begins in the ovary.

[0513] In one example, the method includes a way to distinguish a subject's ovarian cancer from a benign condition.

[0514] It will be apparent to those skilled in the art that the methods described herein are applicable to the detection and / or diagnosis of all subtypes of ovarian cancer, including, for example, epithelial, endometrioid, germ cell, clear cell, and mucinous adenocarcinoma.

[0515] In one example, this disclosure provides a method for detecting and / or diagnosing epithelial ovarian cancer in a subject.

[0516] Those skilled in the art will understand that ovarian cancer is staged using the FIGO staging system, as described in Table 2 below.

[0517] In one example of any of the methods described herein, this disclosure provides a method for detecting and / or diagnosing ovarian cancer in a subject, independent of cancer stage.

[0518] In one example of any of the methods described herein, this disclosure provides a method for detecting and / or diagnosing stage I ovarian cancer in a subject.

[0519] Those skilled in the art will also understand that ovarian cancer is classified based on cancer grades. For example, grade 1 tumors have well-differentiated cells; grade 2 tumors are moderately well-differentiated; and grade 3 tumors are poorly differentiated.

[0520] In one example of any of the methods described herein, this disclosure provides a method for detecting and / or diagnosing ovarian cancer in a subject, regardless of the cancer grade.

[0521] In another example, ovarian cancer is serous carcinoma, mucinous carcinoma, endometrioid carcinoma, clear cell carcinoma, GCT or a mixture thereof, or ovarian cancer. In one example, ovarian cancer is serous carcinoma.

[0522] In one example, the subject was at risk of developing ovarian cancer.

[0523] As used herein, a subject at "risk" for ovarian cancer may or may not have detectable ovarian cancer or symptoms of ovarian cancer. "At risk" means that the subject has one or more risk factors, which are measurable parameters associated with the development of the disease or condition, as known in the art and / or described herein. For example, a subject has a p53 gene mutation.

[0524] Table 2: FIGO stages of ovarian cancer

[0525]

[0526] Risk factors include, for example:

[0527] • Family history of ovarian cancer and / or breast cancer;

[0528] • Reproductive history, i.e., having given birth after age 35 or never having given birth, is associated with a higher risk;

[0529] • History of breast cancer;

[0530] Hormone therapy, such as postmenopausal hormone replacement therapy (HRT), is associated with an increased risk; and

[0531] • Obesity, such as a body mass index greater than 30.

[0532] A subject is considered at risk if their risk of developing ovarian cancer is higher than that of a control group. A control group may include one or more subjects randomly selected from the general population who have never had ovarian cancer or have a family history of ovarian cancer (e.g., matched for age, sex, race, and / or ethnicity). If a subject is found to have a “risk factor” associated with ovarian cancer, they may be considered at risk. For example, through statistical or epidemiological studies of a subject population, risk factors may include any activity, characteristic, event, or trait associated with a given disease. Therefore, a subject may be classified as at risk even if the study identifying potential risk factors did not specifically include them.

[0533] In one example, the method of this disclosure is performed before or after the onset of ovarian cancer symptoms. The symptoms of ovarian cancer are obvious to a person skilled in the art and include, for example:

[0534] • Abdominal swelling or distension;

[0535] • Abdominal bloating and pain;

[0536] Lower abdominal pain;

[0537] • Feeling full after eating very little;

[0538] ·tired;

[0539] • Changes in bowel or bladder habits;

[0540] The clothes don't fit properly;

[0541] • Leg swelling;

[0542] Rapid breathing;

[0543] Vaginal bleeding;

[0544] • Irregular menstrual cycle;

[0545] • Weight loss or gain; and

[0546] • Back pain of unknown cause.

[0547] The inventors have also discovered that the method disclosed herein can be combined with the detection of other biomarkers.

[0548] In one example, the method of this disclosure further includes measuring the levels of dipeptidyl peptidase-4 (DPP4) and / or cancer antigen 125 (CA-125) in a subject. In one example, the method also includes measuring the level of DPP4. In another example, the method also includes measuring the level of CA-125. In yet another example, the method also includes measuring the levels of both DPP4 and CA-125.

[0549] Methods for measuring DPP4 and / or CA-125 are known in the art (see, for example, US 5,356,817, Saho et al., 2019; Scholler et al., 2007 and Vento et al., 1997) and / or described herein.

[0550] In one example, the method further includes determining the levels of one or more of the following: granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), tumor necrosis factor receptor II (TNF-RII), human epididymal protein 4 (HE4), and interleukin-8 (IL-8).

[0551] In one example, the method also includes determining the level of GM-CSF.

[0552] In one example, the method also includes determining the level of IL-6.

[0553] In one example, the method also includes measuring the level of TNF-RII.

[0554] In one example, the method also includes determining the level of HE4.

[0555] In one example, the method also includes determining the level of IL-8.

[0556] In one example, the method further includes determining the levels of GM-CSF, IL-6, TNF-RII, HE4, and IL-8.

[0557] In one example of any of the methods described herein, the method further includes determining the levels of DPP4, GM-CSF, IL-6, TNF-RII, HE4, and IL-8.

[0558] In one example of any of the methods described herein, the method further includes determining the levels of CA-125, GM-CSF, IL-6, TNF-RII, HE4, and IL-8.

[0559] In one example of any of the methods described herein, the method further includes determining the levels of DPP4, CA-125, GM-CSF, IL-6, TNF-RII, HE4, and IL-8.

[0560] Methods of treating malignant diseases

[0561] In one example, the present invention provides a method for treating a subject's malignant condition, the method comprising performing the method described herein and treating the subject's malignant condition.

[0562] As used herein, the term "treatment" includes the surgical removal of all or part of a cancer or the administration of a therapeutically effective amount of a compound / molecule / radiation sufficient to alleviate or eliminate at least one symptom of a malignant disease. For example, an "effective amount" for therapeutic use is the amount of compound required to provide clinically significant relief of disease symptoms without excessive adverse side effects. Appropriate "effective amounts" for any individual case can be determined using techniques such as dose escalation studies. An "effective amount" of a compound is the amount that effectively achieves the desired pharmacological effect or therapeutic improvement without excessive adverse side effects. It should be understood that an "effective amount" or "therapeutic effective amount" can vary depending on the subject's age, weight, general condition, the condition being treated, the severity of the condition being treated, and the prescribing physician's judgment.

[0563] In one example, treatment includes surgery, chemotherapy, radiation therapy, targeted drug therapy, or a combination thereof.

[0564] In one example, the treatment includes surgery. For instance, the surgery is a tumor reduction procedure.

[0565] In another example, treatment includes chemotherapy. Exemplary chemotherapeutic agents include, for example, carboplatin, cytarabine, chlorambucil, cisplatin, cyclophosphamide, doxorubicin, docetaxel, erlotinib, etoposide, fluorouracil, fludarabine, idarubicin, irinotecan, liposomal doxorubicin, methotrexate, mitoxantrone, paclitaxel, topotecan, vincristine, and vinblastine.

[0566] In one example, the treatment includes radiation therapy. For example, radiation therapy is selected from the group consisting of: external beam radiation therapy (EBRT), three-dimensional conformal radiation therapy (3D-CRT), intensity modulated radiation therapy (IMRT), volume modulated arc therapy (VMAT), conformal proton beam radiation therapy, stereotactic radiosurgery (SRS) / stereotactic radiation therapy (SRT), image-guided radiation therapy (IGRT), brachytherapy (internal radiation therapy), and whole brain and spinal cord radiation therapy (craniospinal radiation therapy).

[0567] In one example, the treatment includes targeted drug therapy. For example, the targeted drug therapy is a therapeutic antibody. Exemplary therapeutic antibodies are known to those skilled in the art and include, but are not limited to: abagovomab, abciximab, abituzumab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afelimomab, afutuzumab, alacizumab pegol, alemtuzumab, aliromumab, alirocumab, altumomab pentetate, amatuximab, and anatumomab. Mafenatox, anetumabravtansine, anifrolumab, anrukinzumab, apolizumab, arcitumomab, ascrinvacumab, aselizumab, atezolizumab, atinumab, atlizumab (also known as tocilizumab), atorolimumab, bapineumab (List of names and related terms - likely related to specific abbreviations or specific types of abbreviations)Abvedotin, briakinumab, brodalumab, brolucizumab, brontictuzumab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, CBR96-doxorubicin immunoconjugate, cedelizumab, PEGylated certolizumabpegol, cetuximab, citatuzumab Bogatox, Cixutumab, Clavazizumab, Clenoliximab, Clivatuzumab-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid conjugate, Codrituzumab, Coltuximab-DM4 conjugate, Conatumumab, Concizumab, Cotara (iodine I-131 derlotuximab) Biotin), CR6261, Clenzamab, Dacetuzumab, Daclizumab, Dalotuzumab, PEGylated Dapirolizumab pegol, Daratumumab, Dectrekumab, Demcizumab, Denintuzumab-MMAF conjugate, Denosumab, Delotuximab biotin, Detumomab, Dinutuximab, Diridavumab, Dorlimomabaritox, drozitumab, duligotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enavatuzumab, enfortumab-vedotin conjugate vedotin, PEGylated enlimomab pegol, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomabcituxetan, epratuzumab, erlizumab, ertumaxomab, etaranitidine, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, felvizumab, fezakinumab, ficlatuzumab, figitumumab, futilumab, futilumab Monoclonal antibodies (firivumab), flanvotumab, fletikumab, fontolizumab, foralumab, foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab, gantemumab, gavilimomab, gemtuzumab-ozomicin conjugate, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, guselkumab, ibalizumab, ibritumomab tiuxetan, icrucumab, idarucizumab, igovomab, imalumab, imciromab, imgatuzumab, inclacumab, indatuximabravtansine, timidumab (USA)Vedotin, infliximab, inoliximab, and inotuzumab-ozomicin conjugate. ozogamicin, intetumumab, ipilimumab, iratumumab, isatuximab, itolizumab, ixekizumab, keliximab, labetuzumab, lambrolizumab, lampalizumab, lebrikizumab, lemalesomab, lenzilumab, lerdelimumab, lexatumumab, libivirumab, lidafuzumab vedotin, ligelizumab, lilotomab-setatan conjugate satetraxetan, lintuzumab, lirilumab, lodelcizumab, lokivetmab, lorvotuzumabMertansine, Lucatumumab, PEGylated Lulizumab, Lumiliximab, Lumretuzumab, Mapatumumab, Margetuximab, Maslimomab, Matuzumab, Mavrilimumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mivetuximab-Sorazosine conjugate, Mitumomab, Mogamulizumab, Morolimumab (Mab), Motavizumab, Moxetumomab-Padox conjugate, Muromonab-CD3, Nacolomab-Tafenatox conjugate, Namilumab, Naaptumab-Tafenatox conjugate, Narnatumab, Natalizumab, Nebacumab, Nemitumumab, Nemolizumab, Neelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab-Mepetan conjugate Merpentan, Obiltoxaximab, Obinutuzumab, Ocarutuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Ontuxizumab, Opicinumab, Atrazomab-Monatoxin ConjugateMonatox, oregovomab, orticumab, otelixizumab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, pankomab, panobacumab Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab, Vedotin, Pintumomab, Placulumab, Polatuzumab Vedotin, ponezumab, priliximab, pritoxaximab, pritumumab, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, revivalizumab Monoclonal antibodies (refanezumab), regavirumab, reslizumab, rilotumumab, rinucumab, rituximab, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, sacituzumab-gavita conjugategovitecan, samalizumab, sarilumab, satumomab-penditide conjugate, secukinumab, seribantumab, setoxaximab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab Vedotin, solanezumab, solitomab, soneccizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid conjugate, tadocizumab, talizumab, tanezumab, and taplitumomab-papretoxin conjugate.Paptox, Tarextumab, Tefibazumab, Telimomabaritox (atemoxetine conjugate), Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, Tesidolumab, Tetulomab, Tiglimumab, Tigatuzumab Tildrakizumab, tocilizumab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, tregalizumab, tremelimumab, trevogrumab, tucotuzumab-simmointerleukin conjugate celmoleukin, tuvirumab, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, vadastuximab-talirine conjugate, vandortuzumab The following are listed: vedotin, vantictumab, vanucizumab, vapaliximab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab-mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, and zolimomab-azolidin conjugate.aritox).

[0568] In one example, the treatment includes immunotherapy. For instance, immunotherapy could be selected from checkpoint inhibitors, oncolytic virus therapy, T-cell therapy, and cancer vaccines.

[0569] In one example, immunotherapy is a checkpoint inhibitor. Suitable checkpoint inhibitors include, for example, ipilimumab. nivolumab pembrolizumab atezolizumab avelumab durvalumab

[0570] In one example, immunotherapy is oncolytic virus therapy. For instance, oncolytic virus therapy is talimogenelaherparepvec. Or T-VEC.

[0571] In one example, immunotherapy is T-cell therapy. For instance, T-cell therapy is CAR T-cell therapy.

[0572] In one example, immunotherapy is a cancer vaccine.

[0573] Monitoring tumor burden, progression, recurrence, and regression

[0574] It will be apparent to those skilled in the art that this disclosure also provides methods for monitoring tumor burden, monitoring progression, monitoring recurrence, and / or determining tumor regression in subjects with malignant conditions, the methods comprising: (i) determining the level of active CXCL10 and the total CXCL10 level of the subject; and (ii) determining the CXCL10 ratio between active CXCL10 and total CXCL10 in the subject.

[0575] As used herein, the term “monitoring” may include determining prognosis, response to drug therapy, assessing ongoing drug therapy, predicting outcomes, determining response to treatment (including diagnosis of complications), progression of tumor volume, or selecting patients most likely to benefit from treatment.

[0576] As used in this article, the term “tumor burden” refers to the volume of tumor cells, excluding other changes such as inflammation, necrosis, or edema.

[0577] As used in this article, the term "progression" refers to the continued growth and invasiveness of a tumor.

[0578] As used in this article, the term "regression" refers to a reduction in the size or volume of a tumor.

[0579] As used in this article, the term "relapse" refers to a recurrence or relapse of malignancy after a period of time when the malignancy was undetectable.

[0580] In one example, methods for monitoring tumor burden, progression, recurrence, and / or determining tumor regression in subjects with malignant conditions include measuring the CXCL10 ratio in subjects at one or more time points. For example, miRNA expression levels are measured at time points 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0581] In one example, the CXCL10 ratio is measured in at least one biological sample obtained from the subject before, during, and / or after treatment. For example, the CXCL10 ratio is measured at one or more time points before treatment. In another example, the CXCL10 ratio is measured at one or more time points during treatment. In yet another example, the CXCL10 ratio is measured at one or more time points after treatment. In yet another example, the CXCL10 ratio is measured before and during treatment. In yet another example, the CXCL10 ratio is measured before and after treatment. In yet another example, the CXCL10 ratio is measured during and after treatment. In yet another example, the CXCL10 ratio is measured before and after treatment.

[0582] In one example, the method includes comparing the subject's CXCL10 ratio at a first time point with the subject's CXCL10 ratio at a subsequent time point.

[0583] As will be apparent to those skilled in the art from this disclosure, references to the first and subsequent time points are not intended to refer to defined or specific time points and are for comparative purposes only. The first, second (and any subsequent) time points can be separated by any time period during which malignancy in the subject is to be monitored. The monitoring methods of this disclosure can utilize additional samples at other time points (e.g., a third sample at a third time point in addition to the first and second samples).

[0584] As will be apparent to those skilled in the art, the ability to monitor the CXCL10 ratio of this disclosure during the course of disease will help in monitoring tumor burden and disease progression.

[0585] It is obvious to technicians that methods for monitoring tumor burden and disease progression in subjects can be used to determine tumor regression and / or recurrence in subjects.

[0586] Combinations and kits

[0587] This disclosure provides combinations or kits for detecting and / or diagnosing malignancies in subjects. This disclosure also provides combinations or kits for monitoring tumor burden, tumor progression, and / or tumor regression. The combinations or kits of the present invention will preferably contain one or more of the CXCL10 binding proteins described herein. Optionally, the combinations or kits include instructions for use in the methods described herein.

[0588] In one example, the combination or kit includes a reference sample.

[0589] In one example, the combination or kit described herein is used for in vitro analysis. In one example, the kit is suitable for whole blood, plasma, cervical-vaginal (CVS) swabs, and / or serum samples.

[0590] In one example, the combination or kit described herein is suitable for high-throughput screening. The term "high-throughput screening" refers to a screening method that can be used to test or evaluate multiple samples at once and can reduce the time required to test multiple samples. In one example, the method is suitable for testing or evaluating at least 5, at least 10, at least 20, at least 30, at least 50, at least 70, at least 90, at least 150, at least 200, or at least 300 samples at once. This high-throughput screening method can rapidly analyze multiple samples, for example, within at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, or at least 10 hours. High-throughput screening may also involve the use of liquid handling devices. In one example, high-throughput analysis can be automated.

[0591] This disclosure includes the following non-limiting examples.

[0592] Example

[0593] Example 1: Materials and Methods

[0594] reagents

[0595] Nunc-Immuno Microwell 96-well solid plates were purchased from Sigma-Aldrich (USA). TMB chromogen solution was from ThermoFisher (USA). Recombinant human CXCL10 protein (for immunoassay and assay standards) was from Genscript (Hong Kong, China) (SEQ ID NO: 2). Mimotopes (Australia) synthesized active or truncated N-terminal short peptides containing CXCL10. Biotin (Type A) rapid conjugation kit, anti-IP-10 antibody, streptavidin peroxidase, anti-human IP-10 ELISA kit, human dipeptidyl peptidase IV ELISA kit, and active peptidyl arginine deiminase (PAD) cocktail were from Abcam (UK). All other reagents were analytical grade.

[0596] Clinical samples

[0597] Clinical samples were taken from archived samples stored in the Ovarian Cancer Research Foundation Tissue Bank. These samples were prospectively collected from women who underwent surgery for suspected gynecological malignancies between 2007 and 2014. All samples were from anesthetized patients who had not undergone surgery or chemotherapy.

[0598] Histological assessment of tumor type, stage and grade, preoperative CA125 measurement, age, menopausal status, pre-existing conditions, and any history of previous malignancy was obtained from the identified patients' medical records. Serum CA125 measurements in samples were performed in the Diagnostic Pathology Laboratory at Monash Medical Centre, Melbourne, Australia. Ethical approval was obtained from the Southern Health Human Research Ethics Committee (HREC Certificates; #06032C, #02031B), and all participants provided prior informed written consent.

[0599] Patient samples were divided into two groups based on pathology: benign and malignant.

[0600] The median CA125 measurement and the interquartile range (IQR) were used for variability measurements in each group.

[0601] Both the benign and malignant groups involve premenopausal and postmenopausal (i.e., mixed) women.

[0602] The sample types tested in this study were ascites, plasma, and cervical vaginal swabs (CVS), as detailed in Table 3.

[0603] Table 3. Summary of patient samples (ascites, plasma, CVS) used to compare the active CXCL10 ratio between benign and malignant samples.

[0604]

[0605] Generate monoclonal antibodies against human CXCL10

[0606] Monoclonal antibodies were generated against the full-length recombinant CXCL10 protein (SEQ ID NO:2) at the Montash Antibody Technologies Facility. Short peptides containing the N-terminus of the complete (NH2-VPLSRTVRCTCISISNQPVNPRSLE-COOH) (SEQ ID NO:25) or truncated (NH2-LSRTVRCTCISISNQPVNPRSLE-COOH) (SEQ ID NO:26) human CXCL10 were used for screening.

[0607] Mice were intraperitoneally injected with 16 μg of adjuvanted (Sigma adjuvant) at three doses, once every two weeks. S6322) full-length CXCL10, co-injected with methylated CpG. Serum titers were measured by ELISA and compared with pre-immunized, non-immunized serum. Serum comparisons. Mice showing the highest titers were selected for hybridoma generation. Spleen cells were extracted and fused with SP2 / 0-Ag14 myeloma cells using polyethylene glycol. The resulting hybridoma cells were grown for 13 days in 96-well tissue culture plates containing diazoserine-hypoxanthine. The supernatant of each individual hybridoma was screened for reactivity to the full-length protein and each peptide antigen by microarray, with positive clones rescreened by ELISA.

[0608] Amplification and subcloning of the highest-responding clones, demonstrating appropriate antigen specificity, were performed to ensure the derivation of monoclonal hybridoma cell lines. Monoclonal antibodies were purified from the supernatant using Protein G Sepharose, and Ig isotypes were determined using a commercially available assay kit. The final monoclonal hybridomas of interest were grown to 80% confluence, flash-frozen with 10% DMSO as a cryoprotectant, and stored in liquid N2.

[0609] Surface plasmon resonance imaging

[0610] Two monoclonal antibodies, full-length, N-terminally intact CXCL10-specific mAb-RA2 and full-length, truncated CXCL10-specific mAb-RG2, were analyzed by surface plasmon resonance (SPR). Experiments were performed using a ProteOn XPR36 SPRi biosensor (BioRad) equipped with a GLC chip. The chip was conditioned with 0.5% SDS, 50 mM NaOH, and 100 mM HCl, and then the lanes were activated with aliquots of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and N-hydroxysuccinimide (NHS). The antibodies were immobilized in individual lanes at a concentration of 50 μg / mL in sodium acetate buffer (pH 4.5), and each lane was then inactivated using ethanolamine. The antigen was applied to each lane, and binding was recorded as RU. All lanes were regenerated with 0.85% H3PO3 between antigen applications. Interspots and control RUs were subtracted to give specific binding.

[0611] Biotinylation of RA2 and RG2 monoclonal antibodies

[0612] The mAb-RA2 and mAb-RG2 antibodies were biotinylated using a biotinylation kit (Abcam; ab201795) for use as detection antibodies in the activity ratio assay (ART) of this invention, in which streptavidin-peroxidase was used as the detection reagent. Biotinylation of the two monoclonal antibodies was performed according to the manufacturer's instructions; 10 μL of biotin-modifying reagent was added to 100 μL of each purified monoclonal antibody (2 mg mL⁻¹; PBS, pH 7.4), followed by gentle mixing. The mixture was then directly added to lyophilized biotin and gently mixed. They were incubated at room temperature for 15 minutes. After incubation, 10 μL of biotin quenching reagent was added to complete the biotinylation of both antibodies.

[0613] Quantification of activity and total CXCL10

[0614] Prewash ascites and plasma samples by centrifugation (18, 400x g, 4°C for 20 min) and transfer the clear supernatant to fresh tubes. Dilute the samples (1:5) with assay buffer (0.1% BSA / 0.05% Tween 20 / PBS, pH 7.4) and keep on ice before assay. Vortex the CVS samples for 30 seconds, sonicate in an ice bath for 15 min, and then vortex again for 30 seconds. Centrifuge the supernatant as above and keep on ice until assay.

[0615] For evaluation by ART, anti-human IP-10 polyclonal antibody (Abcam; #ab9807) prepared in coating buffer (15 mM NaCO3 and 35 mM NaHCO3, pH 9.6) was fixed at a coating concentration of 0.5 μg mL⁻¹ in 96-well plates (100 μL per well) for 2 hours at room temperature. The plates were washed once with 280 μL of wash solution (0.05% Tween 20 / Milli-QH₂O) and then incubated for 2 hours at room temperature with 300 μL of blocking solution (5% BSA / 0.05% Tween 20 / PBS, pH 7.4). The plates were washed four times in wash solution and then standards (recombinant CXCL10 (SEQ ID NO:2), 48.8 pg mL⁻¹ to 200,000 pg mL⁻¹) or samples were added in quadruplicate to the corresponding wells (100 μL per well). After 2 hours of incubation, each well was washed four times with wash buffer. Biotinylated detection antibodies mAb-RA2 and mAb-RG2 (1 μg mL⁻¹ in assay buffer) were added appropriately (100 μL per well), and the plate was incubated at room temperature for 1 hour. After incubation, the plate was washed five times with wash buffer, and then incubated for 45 minutes at room temperature with diluted (1:1000) streptavidin peroxidase (1 μg / mL / well). The plate was washed four more times with wash buffer, and then 100 μL of TMB chromophore solution was added to each well, and the plate was incubated in the dark at room temperature for 20 minutes. The reaction was stopped by adding stop solution (1 M hydrochloric acid, 50 μL per well). Absorbance was measured at 450 nm using a Cytation3 multitemplate reader (Biotek, La Jolla CA) equipped with Gen5 v3.08 analysis software. The comparison between ART and standard ELISA (detecting only total CXCL10) was performed using the colorimetric anti-human IP-10 ELISA kit (Abcam#ab100579) according to the manufacturer's instructions.

[0616] Quantitative analysis of DPP4 and analysis of DPP4 specific activity

[0617] DPP4 abundance was assessed in matched ascites samples (1:4 dilution) using a commercial anti-human DPP4 ELISA (Abcam#ab222872) according to the manufacturer's instructions. Detection and concentration analysis were performed using a Cytation multimodal plate reader (as described above). An asymmetric sigmoid log nonlinear regression curve was constructed for DPP4 concentration to quantify soluble DPP4 in the samples.

[0618] DPP4-specific activity (μmol / min / ng DPP4) was measured using Gly-Pro-7-acylamino-4-methylcoumarin hydrobromide (H-Gly-Pro-AMC) as a DPP4 substrate, as previously described (Sinnathurai et al., 2018). Full-length CXCL10 was citrullinated by incubation with human protein-arginine deiminase 2 (PAD2) at 37°C for up to 1 hour. Aliquots were taken every 15 minutes during incubation for time-course measurements. Time-course samples were then separated by SDS-PAGE and detected by Western blotting with mAb-RA2, mAb-RG2, a commercial anti-CXCL10 antibody, and an anti-citrullinated antibody, as previously described (Loos et al., 2008).

[0619] Statistical analysis

[0620] Statistical analysis was performed using GraphPad PRISM (GraphPad Software, La Jolla, CA), and all measured data were logarithmically transformed to approximate normality. The best-fit line was determined for the log total CXCL10 concentrations measured in mAb-RA2 and mAb-RG2 using asymmetric sigmoid (5PL) nonlinear regression curves to quantify active and truncated CXCL10, respectively. The concentrations of CXCL10 obtained from the measurements were in pg mL⁻¹. Significance was determined using one-way ANOVA and Bonferroni post-hoc tests, and pairwise comparisons were performed using Student's t-test. Welch correction was applied to groups with significantly different variances. Spearman's rank test was used for correlation analysis. A p-value < 0.05 was considered significant.

[0621] Example 2: Formulation of functional assays for differentiating active ingredients from total CXCL10

[0622] The inventors developed monoclonal antibodies capable of distinguishing between the intact N-terminus, chemically active form, and all other variants of CXCL10. Using the full-length CXCL10 protein as the antigen, hybridoma clones were isolated and screened for reactivity to (i) the full-length CXCL10 protein and (ii) a short synthetic peptide representing the intact or truncated N-terminus of CXCL10. Antibodies secreted by clone mAb-RA2 recognized both the full-length and intact N-terminus forms of CXCL10, but not the truncated N-terminus form (Table 4). Antibodies secreted by clone mAb-RG2 reacted with all tested CXCL10 protein forms. SPR analysis confirmed binding affinity in the nM to pM range in each case. Open reading frames encoding the variable regions of both antibodies were sequenced.

[0623] Using biotinylated mAb-RA2 and mAb-RG2 as detection antibodies, a sandwich ELISA was constructed to independently measure whole or total CXCL10. Specific parameters were evaluated, including limit of detection (LOD), limit of quantitation (LOQ), linearity, inter- and intra-assay precision, and dynamic range (Table 4). mAb-RA2 and mAb-RG2 each exhibited good affinity for full-length CXCL10, with an R-squared value of 0.996 and a dynamic range of 5 orders of magnitude (OOM). Figure 1 The detection limits for mAb-RA2 and mAb-RG2 were 95.9 pg mL⁻¹ and 116.3 pg mL⁻¹, respectively (Table 4). Inter-assay accuracy was determined using 10 replicates prepared independently with a single dose; and intra-assay variability was determined between 10 individual assay runs performed on different days. In each case, the coefficient of variation (CV) was less than 10%, indicating good reproducibility and assay accuracy (Table 4).

[0624] Table 4. Binding affinity of mAb-RA2 and mAb-RG2 determined by SPR.

[0625]

[0626] The inventors then attempted to validate the quantitative detection of CXCL10 using ART compared to commercially available ELISA kits. Ascites fluid from ovarian cancer patients is rich in CXCL10, which is involved in CXCR3-mediated cancer cell migration and specific T cell subsets (Rainzuk et al., 2012; Windmuller et al., 2017). Therefore, the inventors evaluated the detection of CXCL10 in ascites fluid from ovarian cancer patients (n=212) as a representative complex biological matrix. Although there was considerable bias in the concentration range within the sample groups (CV range 88.4%–225.1%), there was no significant difference in the quantification of total CXCL10 between ART and commercial ELISA. Figure 2 a). For benign and malignant ascites, a positive correlation was observed between ART and commercial ELISA for quantitative total CXCL10, resulting in r values ​​of 0.3084 and 0.2594, respectively. Figure 2 b).

[0627] Example 3: Functional differentiation of mAb-RA2 and mAb-RG2 in non-functional CXCL10

[0628] Besides the N-terminal truncation of CXCL10 due to proteolytic hydrolysis, other post-translational modifications can affect CXCL10 activity (Mortier et al., 2011). In particular, the deamination of citrulline (R5Cit) from arginine in the N-terminal region of CXCL10 is an established in vivo functional modification and must therefore be considered in any assay to assess CXCL10 function (Mortier et al., 2011). Therefore, the inventors investigated whether R5Cit modification could affect the detection of full-length CXCL10 via mAb-RA2 or mAb-RG2.

[0629] Recombinant full-length CXCL10 (SEQ ID NO:2) was incubated with protein-arginine deiminase 2 (PAD2) to induce deamination at R5, and the protein was separated by SDS-PAGE and analyzed by Western blotting. In vitro citrullination had no effect on the detection of CXCL10 using mAb-RG2 (total CXCL10 was detected), indicating that mAb-RG2 binds to CXCL10 regardless of post-translational modifications. Figure 3 a). In contrast, mAb-RA2 showed a substantial reduction in the detection of R5Cit-CXCL10 ( Figure 3 a) This is consistent with epitope modification following R5Cit modification at the N-terminus.

[0630] Similar results were obtained when R5Cit-CXCL10 was evaluated by ELISA. Citrullination significantly reduced mAb-RA2 binding, while mAb-RG2 maintained its ability to detect R5Cit-CXCL10. Figure 3 b). Overall, the data demonstrate that mAb-RA2 and mAb-RG2 are effective in distinguishing active ingredients from total CXCL10.

[0631] Example 4: Viability ratio test to distinguish between benign and malignant ovarian cancer samples

[0632] To establish the use of ART to differentiate between benign and malignant diseases, the inventors measured the activity and total CXCL10 concentration in ascites fluid harvested from patients with benign or malignant ovarian tumors (Table 3). Compared to benign (160.8 ± 362.0 pg mL⁻¹) ascites, malignant (853.1 ± 1574.0 pg mL⁻¹) ascites showed a significantly higher total CXCL10 concentration. Figure 4 b). Similarly, a wide range of measurements of active CXCL10 was also observed (240.4 ± 410.5 pg mL⁻¹ and 818.6 ± 1098.0 pg mL⁻¹, respectively). Figure 4a). This wide bias within the population (CV% ranging between 134.1% and 225.1%) leads to low sensitivity of the differences between benign and malignant samples (Table 5) and highlights the difficulties associated with direct CXCL10 measurement for diagnostic or prognostic purposes.

[0633] Since proportionally higher levels of active CXCL10 were observed in benign vs. malignant samples, function in each sample was examined as a mechanism for normalizing CXCL10 function among patients: the ratio of total CXCL10. Figure 4 c). This “activity ratio” measurement significantly improved the separation between groups, resulting in differences between benign and malignant samples, with cutoff values ​​of <1.43 and <1.25 for 90% and 95% specificity, respectively (Table 5). When malignant samples were compared according to disease stage (FIGO stage I versus stage III; Figure 4 d) This relationship remains valid even upon separation. Although plasma CA125 is significantly elevated in patients with malignant diseases compared to benign diseases ( Figure 5 c), but there was no significant correlation between the activity ratio measured in ascites and plasma CA125 levels. Figure 6 a). Therefore, the activity ratio provides a malignancy marker independent of plasma CA125 concentration.

[0634] Example 5: DPP4 abundance and activity are not correlated with functional CXCL10.

[0635] DPP4 catalyzes the removal of the Val-Pro or Ala-Pro dipeptide from the N-terminus of CXCL10, converting it into an antagonist recruited by T cells. Therefore, previous studies have sought to quantify the levels of DPP4-cleaved CXCL10 in biological fluids as an indicator of disease (Casrouge et al., 2011 and 2012). However, multiple modifications of CXCL10, both proteolytic and non-proteolytic, can lead to an expanded repertoire beyond simple DPP4-catalyzed N-terminal processing of CXCL10 (Loos et al., 2008; Mortier et al., 2011). Consequently, the differences in CXCL10 measured in clinical samples compared to the total amount of CXCL10 caused by these multiple variants have not been adequately accounted for, and the functional state of CXCL10 cannot be fully captured (Casrouge et al., 2012).

[0636] Table 5. Prognostic performance of ovarian cancer patients by activity ratios in ascites and other biomarkers distinguishing between benign and malignant diseases. Cutoff values ​​for each biomarker were obtained based on ROC analysis (90 and 95% specificity) to determine sensitivity, positive predictive value (PPV), and negative predictive value (NPV) accordingly.

[0637]

[0638]

[0639] Note: Consider the prevalence of EOC in the patient cohort to determine the positive predictive value (PPV) and negative predictive value (NPV) with 90% and 95% specificity.

[0640] To elucidate the relationship between DPP4 activity and CXCL10 function in clinical samples, we measured the abundance and specific activity of DPP4 in the same group of benign and malignant ascites.

[0641] First, receiver operating characteristic (ROC) curves (activity ratio, DPP4, CA125) for each marker were constructed based on the results from the patient samples to obtain the area under the curve (AUC). To obtain the combined ROC, binary logistic regression was performed; the measurement of each marker was used as the dependent variable for the benign and malignant groups to obtain the probability as the test variable, which was then used to construct the combined ROC.

[0642] No significant difference in abundance or activity was observed in either case. Figure 5 a and Figure 5 b). While DPP4 abundance correlated with the calculated activity ratio in malignant samples (p = 0.002), its specific activity did not; interestingly, benign samples showed an inverse (non-significant) trend, suggesting a correlation between specific activity and activity ratio measurements. Figure 6 b and Figure 6 c). These data suggest that while DPP4 activity may be associated with CXCL10 function in non-malignant diseases, multiple CXCL10 variants may be important in determining the overall functional status of CXCL10 in malignant tumors.

[0643] Example 6: Activity ratio provides prognostic differentiation between benign and malignant diseases.

[0644] To assess whether the activity ratio could provide useful clinical information, we used recipient operating characteristic (ROC) curves to evaluate its performance in ascites. Figure 7 Compared to measurements of activity or total CXCL10 alone, the activity ratio achieved a higher AUC and a significant improvement in sensitivity / specificity. Figure 7 a; Table 3) highlights its enhancing effect on these single measurements. The sensitivity, specificity, and predictive value of the activity ratio are also higher than those of plasma CA125 ( Figure 7b; Table 3). The activity ratio also achieved a larger effect size compared to individual measurements of DPP4, CA125, or CXCL10 (active or total) (Cohen's d) (Table 4). Therefore, the combination of the activity ratio, DPP4, and plasma CA125 measurements yielded an AUC >0.95, with good PPV (87-94%) and NPV (93-95%) for differentiating between benign and malignant diseases. Thus, the combination of ART with DPP4 and plasma CA125 provides a method for differentiating between benign and malignant diseases in patients with ascites.

[0645] Table 6. Effect sizes of biomarkers based on sample type. AUC, Cohen's d, and CV% values ​​were obtained, independent of cutoff values. P-values ​​were based on ROC analysis.

[0646]

[0647] Example 7: ART as a clinical diagnosis

[0648] The inventors explored whether cervical vaginal swabs (CVS), which represent the reproductive tract, could be used to measure the activity ratio.

[0649] Parallel to our findings in ascites, activity ratio measurements of CVS extracts (n = 50 / group) showed good differentiation between benign and malignant disease samples, with an AUC of 0.8 (p < 0.0001). Figure 8 a; Table 6). In matched plasma samples (n=30), ART was also able to distinguish between groups (AUC 0.8, p<0.001).

[0650] To explore diagnostic utility beyond ROC analysis, the inventors also examined the effect size (Cohen's d) for each biomarker and sample type (Table 6). The measurement of plasma CA125 returned an AUC of 0.83, second only to the ART measurement in ascites (AUC 0.86); however, the effect size was "moderate" (Cohen's d = 0.62). In contrast, the ART measurement achieved a "large" effect size in ascites and CVS (Cohen's d = 0.86 and 1.0, respectively), indicating a significant reduction in bias within the same population. Similarly, the ART measurement in plasma returned Cohen's d = 0.79 (Table 6).

[0651] These results demonstrate that ART is the preferred biomarker for achieving statistically robust measurements, and that analysis of it using CVS can provide robust and clinically useful measurements to differentiate between malignant and non-malignant diseases.

[0652] Example 8: Differentiating patients without cancer from those with benign conditions or malignant ovarian tumors using ART.

[0653] After determining the application of ART to differentiate between patients with benign and malignant ovarian tumors by activity rate, ART was applied to test a new patient cohort (i.e., a prospectively collected cohort)—patients who underwent prophylactic, risk-reducing salpingo-oophorectomy and were confirmed BRCA1 / 2 mutation carriers, or had a significant family history of breast and / or ovarian cancer. Therefore, these patients represented an ideal disease-free control cohort for validating ART designed to detect the presence of early-stage ovarian cancer.

[0654] The concentration of active CXCL10 in healthy women was significantly higher than that of total CXCL10. Figure 9 a) This suggests that patients without cancer may have higher levels of active, functional CXCL10. When the overall active CXCL10 and total CXCL10 concentrations in healthy women from a prospectively collected cohort were compared to their respective benign and malignant CXCL10 concentrations, they were significantly lower than those of benign and malignant CXCL10. Figure 9 b). Regarding the activity ratio, the activity ratio in healthy women was significantly higher than that in both the benign and malignant groups in both plasma and CVS. Figure 9 c and d).

[0655] Given that the levels and activity ratio of active CXCL10 in healthy women were significantly higher than those of total CXCL10 and the activity ratio in patients with benign and malignant tumors, the data suggest that the degree of functional CXCL10 cleavage initiated by DPP4 may be lower in patients without benign conditions or malignant ovarian tumors.

[0656] Example 9: DPP4 abundance and activity are independent of activity ratio.

[0657] To establish the relationship between DPP4 and CXCL10 in a prospectively collected cohort, the abundance and specific activity of DPP4 in plasma samples were measured and compared with results from patients with benign or malignant ovarian cancer.

[0658] The level of DPP4 in plasma samples from healthy patients was significantly higher than that in both benign and malignant patient groups. However, in these healthy female samples, DPP4 abundance was not correlated with the calculated activity ratio. Figure 10 a). Although DPP4 levels were significantly higher in healthy women than in the benign and malignant patient groups, its specific activity in the plasma of healthy women was significantly lower than in the benign and malignant groups (a). Figure 10 (b) Specific activity was also independent of the calculated activity ratio. These data suggest that DPP4 function in healthy women may be inhibited by unknown factors.

[0659] Example 10: Differentiating healthy women from patients with benign or malignant ovarian tumors using CVS

[0660] Since CVS is the preferred sample for biomarker-based testing, the levels of active CXCL10 and total CXCL10 concentrations in CVS were measured in a prospectively collected cohort to obtain the activity ratio. Furthermore, the activity ratio was compared between healthy patients and patients with benign or malignant ovarian tumors in the prospectively collected cohort.

[0661] As for healthy women, the overall level of active, functional CXCL10 was significantly higher than that of total CXCL10 ( Figure 11 a). Compared with patients with benign or malignant ovarian tumors, the calculated activity ratio of CVS in healthy patients was consistent with plasma results; the overall activity ratio in the healthy female sample was significantly higher than that in the benign and malignant patient groups ( Figure 11 b).

[0662] Example 11: DPP4 abundance on CVS and its correlation with CVS activity ratio

[0663] Due to the limited quantity of CVS extracts, DPP4 abundance on CVS was not measured in previous studies involving ovarian cancer biobanks. Because it is necessary to measure the activity ratios and DPP4 abundances from the same sample source and establish their correlation within the same sample type, CVS preparation was optimized to measure the activity ratios and DPP4 abundances of CVS, thereby correlating them. Figure 12 As shown, DPP4 in CVS can be quantified, and the calculated activity ratio is negatively correlated with DPP4 (p = 0.0094). This data demonstrates the feasibility of linking the activity ratio with DPP4 in CVS.

[0664] Example 12: Activity ratio and plasma CA125

[0665] CA125 levels in healthy women were significantly lower than in the benign and malignant groups. This data is consistent with the fact that CA125 levels are elevated in patients with malignant diseases. Figure 13 The calculated activity ratio was positively correlated with CA125 (p = 0.0015).

[0666] Example 13: Activity ratio and plasma CA125

[0667] To assess whether the activity ratio could provide useful clinical information, its performance in plasma and CVS was evaluated using receiver operating characteristic (ROC) curves. The ROC curves were constructed based on a comparison between healthy women and patients with malignant ovarian tumors in a prospective cohort.

[0668] Because previous ascites studies showed a significant improvement in sensitivity / specificity in the activity ratio compared to quantitative CXCL10 or total CXCL10 alone, the combination of plasma and CVS activity ratios also achieved a higher AUC ( ). Figure 14a). This validates the use of these two sample types to improve the prognostic effect of ART. Combined measurement of the activity ratios of plasma, DPP4, and plasma CA125 yielded an AUC > 0.98 ( Figure 14 (b) The combination of activity ratios of CVS, DPP4, and plasma CA125 yielded an AUC > 0.99, which was used to differentiate between healthy women and malignant tumors. These data suggest that the combination of activity ratios with DPP4 and plasma CA125, as well as the combination of the two sample types, can be used to differentiate between healthy women and malignant diseases.

[0669] Example: Differentiating malignant tumor patients with complex backgrounds using ART

[0670] To investigate the prognostic potential of ART in identifying malignant tumors in different contexts, women from healthy and benign cohorts were combined, and ROC analysis was performed on the malignant group. When applied to plasma samples, the AUC for ART alone was 0.79, and the AUC for CVS samples was 0.72. In contrast, the AUC for CA125 was 0.93. Figure 15 A). However, CA125 showed high variability in the cohort (CV 189.5%) and a small effect size (Cohen's d 0.097), indicating that it is not a reliable measure for distinguishing malignant samples from a cohort of healthy plus benign individuals (Table 7).

[0671] In contrast, the activity ratio measurement had a larger impact on CVFS (d = 0.64) and plasma (d = 1.01), indicating a significant reduction in bias within the same population (Table 7). This result demonstrates that ART can achieve statistically robust measurements and is superior to the current gold standard for differentiating malignant ovarian cancer in complex contexts, whether in healthy women or patients with benign disease.

[0672] Table 7 shows the effect sizes of individual biomarkers used to distinguish malignant diseases in a complex background cohort.

[0673]

[0674] The combination of ART (CVS and / or plasma), DPP4, and CA125 was also investigated as a mechanism to improve intergroup differentiation. Compared with CA125 (AUC 0.94), the combination of ART measured using CVS with all other markers measured in plasma yielded an AUC of 0.98 (Table 8).

[0675] The combination of all biomarkers (ART, DPP4, and CA125) achieved an AUC of 0.98 and a sensitivity of 93.1% in this cohort (Table 8). In contrast, CA125 alone had an AUC of 0.94 but a sensitivity of only 78.6% (Table 8).

[0676] Table 8 shows the prognostic outcomes of individual and combined biomarkers to differentiate malignant disease from a patient cohort consisting of healthy women and women with benign gynecological conditions.

[0677]

[0678] Therefore, the combination of ART, DPP4, and CA125 distinguishes malignant tumors from a complex background of women with both healthy and benign diseases; and is significantly superior to the current clinical gold standard, CA125.

[0679] Example 15: ART improves the identification of patients with early-stage (stage I) cancer.

[0680] Cancer stage at diagnosis is closely associated with clinical outcomes in patients with ovarian cancer. In particular, patients diagnosed with FIGO stage I (“early”) have significantly improved 5-year and overall survival (approximately 95%) compared to patients diagnosed with stage III-IV (<40%). Therefore, this study evaluated whether ART (alone or in combination) could successfully differentiate patients with early-stage cancer from healthy women or women with benign disease.

[0681] CA125 provided the largest single biomarker differentiation between patients with early-stage cancer and those with benign disease or healthy women (Table 9). However, the combination of ART (plasma) with DPP4 and CA125 significantly improved the differentiation between benign and early-stage malignant tumors (AUC 0.75 vs 0.63; Table 9).

[0682] Notably, the combination of three biomarkers (AUC = 0.99; sensitivity = 95.4%) demonstrated high precision in distinguishing patients with early-stage malignant tumors from healthy women compared to CA125 alone (AUC 0.88; sensitivity 81.8%).

[0683] Therefore, compared to the current CA125 gold standard, ART can be used to improve patient diagnosis or prognostic assessment, especially for detecting disease-specific changes associated with early-stage cancer.

[0684] Table 9 shows the prognostic performance of single biomarkers and combined biomarkers for identifying stage 1 cancer.

[0685]

[0686]

[0687] Example 16: Identifying patients with precancerous conditions in a prospectively collected cohort using a combination of ART groups and multiple 5-label groups The patient who changed

[0688] Although the causes of ovarian cancer remain unclear, it is now evident that many ovarian tumors begin with the presence of a precancerous "p53 signature" in the fallopian tubes. Similar to other cancer types, detecting lesions at a very early or precancerous stage will significantly improve outcomes.

[0689] The inventors previously identified several other biomarkers (GM-CSF, IL-6, TNF-RII, HE4, IL8 (PMID: https: / / doi.org / 10.1038 / s41598-020-59009-z) that are associated with diagnostic analysis of high-grade epithelial ovarian cancer. Therefore, the potential for improved diagnostic performance in combination with these biomarkers, along with existing ART, DPP4, and CA125, was evaluated.

[0690] For these studies, a combination of biomarkers was evaluated in a prospective cohort of women who underwent prophylactic risk-reducing surgery (typically bilateral salpingo-oophorectomy) and had a known genetic risk of developing ovarian cancer. Histological evaluation was used to classify all patients as “healthy” (i.e., confirmed to be disease-free) or “cancer” (presence of p53 lesions or early-stage occult tumors).

[0691] The results are shown in Table 10 (only the best-performing combinations are shown). The 5-biomarker group returned a combined AUC of 0.87 for detecting early / precancerous lesions; while the established ART group had an AUC of 0.97 (Table 10). These are significantly superior to CA125, which has an AUC of 0.77.

[0692] However, it is noteworthy that when the two groups were combined, a perfect AUC of 1.0 was achieved, suggesting that this particular combination of biomarkers has great potential in diagnosing early and precancerous lesions of the ovary.

[0693] Table 10 shows the prognostic performance of single and combined biomarkers to identify patients with stage 1 cancer or p53 precancerous lesions.

[0694]

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Claims

1. A CXC motif chemokine ligand 10 (CXCL10) binding protein, wherein the binding protein comprises a variable region of an immunoglobulin or an antigen-binding domain of an antibody, and binds to full-length human CXCL10, N-terminally truncated CXCL10, and citrullinated CXCL10, wherein the binding protein binds to the epitope NH2-LSRTVRCTCISISNQPVNPRSLE-COOH of full-length human CXCL10, N-terminally truncated CXCL10, and citrullinated CXCL10, wherein the binding protein is an antibody comprising: Heavy chain variable region (V H ), which includes: a) CDR1 shown in SEQ ID NO: 13; b) CDR2 shown in SEQ ID NO: 14; and c) CDR3 shown in SEQ ID NO: 15; and Light chain variable region (V L ), which includes: a) CDR1 shown in SEQ ID NO: 16; b) CDR2 shown in SEQ ID NO: 17; and c) CDR3 shown in SEQ ID NO:

18.

2. The CXCL10 binding protein according to claim 1, wherein the binding protein: (i) with K 50 nM or less D With full-length CXCL10; and / or (ii) with K 50 nM or less D It binds to N-terminal truncated human CXCL10.

3. The CXCL10 binding protein according to claim 1 or 2, wherein the binding protein is an antibody comprising the V shown in SEQ ID NO:

11. H and V shown in SEQ ID NO: 12 L .

4. A CXC motif chemokine ligand 10 (CXCL10) binding protein, wherein the binding protein comprises a variable region of an immunoglobulin or an antigen-binding domain of an antibody, and is expressed at a K+ level of 50 nM or less. D It binds to full-length human CXCL10, but not to N-terminal truncated CXCL10 or citrullinated CXCL10, wherein the CXCL10-binding protein requires the N-terminal valine and / or proline of the epitope NH2-VPLSRTVRCTCISISNQPVNPRSLE-COOH to bind to full-length human CXCL10, wherein the binding protein is an antibody comprising: V H It includes: a) CDR1 shown in SEQ ID NO: 5; b) CDR2 shown in SEQ ID NO: 6; and c) CDR3 shown in SEQ ID NO: 7; and V L It includes: a) CDR1 shown in SEQ ID NO: 8; b) CDR2 shown in SEQ ID NO: 9; and c) CDR3 shown in SEQ ID NO:

10.

5. The CXCL10 binding protein according to claim 4, wherein the binding protein is an antibody, comprising: The V shown in SEQ ID NO: 3 H And the V shown in SEQ ID NO:4 L .

6. The CXCL10 binding protein according to any one of claims 1 to 5, wherein the binding protein binds to a detectable marker.

7. A composition comprising a binding protein and a carrier according to any one of claims 1 to 6.

8. A polynucleotide encoding a CXCL10-binding protein according to any one of claims 1 to 6.

9. An expression vector comprising the polynucleotide according to claim 8.

10. An in vitro cell comprising the expression vector according to claim 9.

11. Use of the cells of claim 10 for the preparation of CXCL10 binding protein.

12. The use according to claim 11, comprising culturing the cells according to claim 10 and generating the CXCL10 binding protein therefrom.

13. The use according to claim 12 further includes isolating and purifying the generated CXCL10 binding protein.

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