CXCL10-binding proteins and uses thereof

Abstract

The present invention relates to a C-X-C motif chemokine ligand 10 (CXCL10) binding protein and its use in a method for detecting and / or diagnosing a condition in a subject, the method comprising measuring the level of CXCL10 in the subject. Using specific antibodies that bind to total CXCL10 (full-length, N-terminal truncated, and citrullinated) and to active CXCL10 (full-length), the levels of total and active CXCL10 were measured in samples from ovarian cancer patients. The calculated ratio of active CXCL10 to total CXCL10 was lower in patients with malignant conditions compared to patients with benign tumors or healthy individuals. This provides the basis for methods of diagnosing malignant conditions and monitoring tumor burden and disease progression.

Description

[Technical Field]

[0001] Related application data This application claims priority from Australian Patent Application No. 2019904859 (entitled "CXCL10 binding proteins and uses thereof") (filed December 20, 2019), the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application is filed with a Sequence Listing in electronic form, the entire contents of which are incorporated herein by reference.

[0003] Technical Field The present disclosure relates to CXCL10 binding proteins and uses thereof. [Background technology]

[0004] The chemokine interferon-γ-inducible protein (CXC motif chemokine ligand 10; CXCL10; also known as interferon-inducible protein 10 or IP-10) is a member of the CXC chemokine family and plays an important role in leukocyte tracking by generating chemotactic activity in cells expressing the corresponding chemokine receptor.

[0005] CXCL10 possesses both agonist and antagonist activities and is involved in chemotaxis, induction of apoptosis, and regulation of cell proliferation and mediating antiangiogenic effects. CXCL10 exerts its biological effects by specifically activating its receptor CXCR3, a seven-transmembrane, G protein-coupled receptor preferentially expressed on activated T lymphocytes (Th1), natural killer (NK) cells, inflammatory dendritic cells, macrophages, and B cells. The proliferative or antiproliferative effects of CXCL10 appear to be cell type-dependent and / or dependent on the subtype of its receptor, CXCR3. Furthermore, post-translational modifications such as deamination or citrullination of CXCL10 by peptidylarginine deiminase (PAD) or NH2-terminal cleavage by proteases such as dipeptidyl peptidase IV (DPP4) contribute to its biological effects by generating dominant-negative forms that can bind to CXCR3 but do not induce signaling.

[0006] CXCL10 is associated with a variety of human diseases, including infectious diseases, central nervous system diseases, chronic inflammation, immune dysfunction, and cancer.

[0007] Given the widespread association of CXCL10 with numerous human diseases, it has become an attractive biomarker and candidate for targeted therapy, but commercially available diagnostic applications are limited by the inability to distinguish between biologically distinct forms of the protein.

[0008] Based on the foregoing, it will be apparent to those skilled in the art that there is a need in the art for compounds (e.g., antibodies and antibody-derived proteins) that can precisely target all forms of CXCL10 as potential biomarkers. Summary of the Invention

[0009] In producing the present invention, the inventors have identified biologically relevant forms of CXCL10. The inventors attempted to generate reagents that bind to CXCL10. The inventors generated antibodies that bind to full-length (i.e., biologically active) CXCL10, as well as antibodies that bind to full-length CXCL10 and further to N-terminally truncated and citrullinated (i.e., inactive) forms of CXCL10. Surprisingly, the inventors discovered that detecting different forms of CXCL10, and specifically the ratio of different forms of CXCL10, allows for the differentiation of benign and malignant conditions. The inventors also discovered that the ratio of different forms of CXCL10 allows for the differentiation of the presence of disease (either benign or malignant) from the absence of disease (i.e., healthy individuals). Surprisingly, the inventors have also discovered that by detecting the ratio of different forms of CXCL10 in combination with other biological markers (e.g., DPP4, CA-125, GM-CSF, IL-6, TNF-RII), HE4, and / or IL-8), it is possible to distinguish between stage I (i.e., early stage) cancer or precancerous lesions and benign conditions.

[0010] In one example, the disclosure provides a CXCL10 binding protein, wherein the binding protein binds to full-length human CXCL10, N-terminal truncated CXCL10, and citrullinated CXCL10.

[0011] In one embodiment of the above examples, the CXCL10 binding protein binds to the epitope NH2-LSRTVRCTCISISNQPVNPRSLE-COOH (SEQ ID NO: 26) of full-length human CXCL10, N-terminally truncated CXCL10, and citrullinated CXCL10.

[0012] In one example, the CXCL10 binding protein has (i) a K of 50 nM or less. D and / or (ii) binds to full-length human CXCL10 with a K of 5 nM or less. D It binds to N-terminally truncated human CXCL10 at 100 kJ / s.

[0013] In one embodiment, the CXCL10 binding protein has a K DFor example, the CXCL10 binding protein may bind to or specifically bind to full-length human CXCL10 with a K of about 50 nM, or about 40 nM, or about 30 nM, or about 20 nM, or about 10 nM. D In one example, the CXCL10 binding protein binds to or specifically binds to full-length human CXCL10 with a K of about 49 nM. D It binds to or specifically binds to full-length human CXCL10.

[0014] In one embodiment of the above examples, the CXCL10 binding protein requires an N-terminal valine and / or proline of the epitope NH2-VPLSRTVRCTCISISNQPVNPRSLE-COOH (SEQ ID NO: 25) to bind to full-length human CXCL10.

[0015] In one example, the CXCL10 binding protein is 5n M The following K D For example, the CXCL10 binding protein may bind to or specifically bind to N-terminally truncated human CXCL10 with a K of about 5 nM, or about 4 nM, or about 3 nM, or about 2 nM, or about 1 nM. D In one example, the CXCL10 binding protein binds to or specifically binds to N-terminal truncated human CXCL10 with a K of about 3 nM. D binds to or specifically binds to N-terminal truncated CXCL10 at

[0016] The present disclosure provides a CXCL10 binding protein, wherein the binding protein has a K D Also provided is a binding protein as described above that binds to full-length human CXCL10 at 200 ng / L, but not to N-terminal truncated CXCL10 or citrullinated CXCL10.

[0017] In one example, the CXCL10 binding protein does not detectably or significantly bind to N-terminal truncated CXCL10 and citrullinated CXCL10.

[0018] Methods for measuring the binding of a CXCL10-binding protein to a polypeptide will be apparent to those skilled in the art. For example, the polypeptide is immobilized on a solid or semi-solid surface, and the CXCL10-binding protein is contacted with the immobilized polypeptide. Binding is then measured, for example, by surface plasmon resonance (SPR) imaging.

[0019] In one embodiment, (e.g., K D The level of binding (as measured by surface plasmon resonance (SPR) imaging) is measured by surface plasmon resonance (SPR) imaging.

[0020] In one example, a CXCL10 binding protein of the disclosure comprises a variable region or antigen-binding domain.

[0021] In one embodiment, the binding protein is (i) Fv; (ii) single-chain Fv fragments (scFv); (iii) dimeric scF (di-scFv); (iv) single domain antibodies; (v) Minibody; (vi) diabodies; (vii) triabodies; (viii) tetrabodies; (ix)Fab; (x)F(ab')2; (xi) Antibodies; (xii) antibody mimetics; (xiii) heavy chain-only immunoglobulins; (xiv) T cell receptor; (xv) Adnectin; (xvi) anticalins; (xvii) Affibody; (xviii)Avimar; (xix) designed ankyrin repeat proteins (DARPins); and (xx) Antibody, (i) to (xix) bound to the Fc constant region or a heavy chain constant region (CH2) and / or CH3.

[0022] In one example, the binding protein comprises the antigen-binding domain of an antibody. For example, the binding protein may comprise at least one V H and V L Including V H and V L combine to form the antigen-binding domain, Fv.

[0023] In one example, the binding protein is an antibody or antigen-binding fragment thereof (e.g., an scFv comprising the variable region of an antibody). Exemplary antibodies are full-length and / or naked (e.g., unconjugated) antibodies. In one example, the antibodies of the present disclosure are full-length antibodies.

[0024] In one example, the antibody is an IgG, or IgE, or IgM, or IgD, or IgA, or IgY antibody. For example, the antibody is an IgG antibody.

[0025] In one embodiment, the IgG antibody is an IgG1, or an IgG2, or an IgG3, or an IgG4. For example, the antibody is an IgG1 antibody. In another embodiment, the antibody is an IgG4 antibody. In one embodiment, the antibody is a stabilized IgG4 antibody.

[0026] In one example, the binding protein is recombinant, chimeric, CDR-grafted, humanized, synthetic humanized, primatized, deimmunized, or human.

[0027] In one example, the antigen-binding fragment of the present disclosure is a half antibody, for example, a CXCL10 binding protein is a half antibody comprising one heavy chain and one light chain.

[0028] In one example, the antigen-binding fragment of the present disclosure comprises an IgG4 constant region, or a stabilized IgG4 constant region.

[0029] In one example, the binding protein is an antibody mimetic, e.g., the binding protein comprises the antigen-binding domain of an immunoglobulin, e.g., an IgNAR, a camelid antibody, or a T-cell receptor.

[0030] In one example, the binding protein is a domain antibody (e.g., comprising only a heavy chain variable region or only a light chain variable region), or a heavy chain-only antibody (e.g., a camelid antibody or IgNAR), or a variable region thereof.

[0031] In one example, the binding protein is an antibody or antigen-binding fragment thereof. (i) a heavy chain variable region (V) comprising the amino acid sequence set forth in SEQ ID NO: 3; H ), and a light chain variable region (V L ); and / or (ii) V comprising the amino acid sequence set forth in SEQ ID NO: 11 H and V comprising the amino acid sequence set forth in SEQ ID NO: 12. L It competitively inhibits the binding of CXCL10, including

[0032] In one example, the binding protein comprises a V domain of an antibody or antigen-binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:3. H and V comprising the amino acid sequence set forth in SEQ ID NO:4 L It competitively inhibits the binding of CXCL10, including

[0033] In another embodiment, the binding protein comprises a V domain of an antibody or antigen-binding fragment thereof comprising the amino acid sequence set forth in SEQ ID NO:11. H and V comprising the amino acid sequence set forth in SEQ ID NO: 12. L It competitively inhibits the binding of CXCL10, including

[0034] In one embodiment, the binding protein is (i) V comprising a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 3 H and V comprising a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:4. L ;or (ii) V comprising a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 11 Hand V comprising a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 12. L Includes.

[0035] In one example, the binding protein comprises a V sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:3. H and V comprising a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:4. L For example, the binding protein may include a V that is at least 90%, or 95%, or 97%, or 98%, or 99% identical to a sequence disclosed herein. H and / or V L Includes.

[0036] In one example, the binding protein comprises a V sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:11. H and V comprising a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 12. L For example, the binding protein may include a V that is at least 90%, or 95%, or 97%, or 98%, or 99% identical to a sequence disclosed herein. H and / or V L Includes.

[0037] In one example, the binding proteins of the present disclosure optionally contain one or more amino acid substitutions, deletions, or insertions of any of the sequences disclosed herein. Amino acid substitutions suitable for use in the present disclosure will be apparent to those of skill in the art and include naturally occurring substitutions and recombinant substitutions.

[0038] In one example, a CXCL10 binding protein of the disclosure comprises: (i) V comprising the amino acid sequence set forth in SEQ ID NO: 3 H and V comprising the amino acid sequence set forth in SEQ ID NO:4 L ;or (ii) V comprising the amino acid sequence set forth in SEQ ID NO: 11 H and V comprising the amino acid sequence set forth in SEQ ID NO: 12. L and wherein the antibody or antigen-binding fragment thereof comprises:

[0039] In one example, a CXCL10 binding protein of the disclosure comprises a V H and V comprising the amino acid sequence set forth in SEQ ID NO:4 L or an antigen-binding fragment thereof comprising:

[0040] In another example, a CXCL10 binding protein of the disclosure comprises a V comprising the amino acid sequence set forth in SEQ ID NO:11. H and V comprising the amino acid sequence set forth in SEQ ID NO: 12. L or an antigen-binding fragment thereof comprising:

[0041] In one example, a CXCL10 binding protein of the disclosure comprises: (i)V H And, a) CDR1 comprising the sequence set forth in amino acids 25 to 34 of SEQ ID NO: 3; b) CDR2 comprising the sequence set forth in amino acids 49 to 65 of SEQ ID NO: 3; and c) a CDR3 comprising the sequence set forth in amino acids 98 to 108 of SEQ ID NO: 3; and H , and V L And, a) CDR1 comprising the sequence set forth in amino acids 23 to 33 of SEQ ID NO: 4; and b) CDR2 comprising the sequence set forth in amino acids 49 to 55 of SEQ ID NO: 4; and c) a CDR3 comprising the sequence set forth in amino acids 88 to 96 of SEQ ID NO: 4; and L ,or (ii)V H And, a) CDR2 comprising the sequence set forth in amino acids 25 to 34 of SEQ ID NO: 11; and b) CDR2 comprising the sequence set forth in amino acids 49 to 65 of SEQ ID NO: 11; and c) a CDR3 comprising the sequence set forth in amino acids 98 to 108 of SEQ ID NO: 11; and H , and V LAnd, a) CDR1 comprising the sequence set forth in amino acids 23 to 33 of SEQ ID NO: 12; and b) CDR2 comprising the sequence set forth in amino acids 49 to 55 of SEQ ID NO: 12; and c) a CDR3 comprising the sequence set forth in amino acids 88 to 96 of SEQ ID NO: 12; and L and wherein the antibody or antigen-binding fragment thereof comprises:

[0042] In one example, a CXCL10 binding protein of the disclosure comprises: (i)V H And, a) CDR1 comprising the sequence set forth in amino acids 25 to 34 of SEQ ID NO: 3; b) CDR2 comprising the sequence set forth in amino acids 49 to 65 of SEQ ID NO: 3; and c) a CDR3 comprising the sequence set forth in amino acids 98 to 108 of SEQ ID NO: 3; and H , and (ii)V L And, a) CDR1 comprising the sequence set forth in amino acids 23 to 33 of SEQ ID NO: 4; and b) CDR2 comprising the sequence set forth in amino acids 49 to 55 of SEQ ID NO: 4; and c) a CDR3 comprising the sequence set forth in amino acids 88 to 96 of SEQ ID NO: 4; and L and wherein the antibody or antigen-binding fragment thereof comprises:

[0043] In one example, a CXCL10 binding protein of the disclosure comprises: (i)V H And, a) CDR1 comprising the sequence set forth in amino acids 25 to 34 of SEQ ID NO: 11; and b) CDR2 comprising the sequence set forth in amino acids 49 to 65 of SEQ ID NO: 11; and c) a CDR3 comprising the sequence set forth in amino acids 98 to 108 of SEQ ID NO: 11; and H , and (ii)V L And, a) CDR1 comprising the sequence set forth in amino acids 23 to 33 of SEQ ID NO: 12; and b) CDR2 comprising the sequence set forth in amino acids 49 to 55 of SEQ ID NO: 12; and c) a CDR3 comprising the sequence set forth in amino acids 88 to 96 of SEQ ID NO: 12; and L and wherein the antibody or antigen-binding fragment thereof comprises:

[0044] In one example, a CXCL10 binding protein of the disclosure comprises: (i)V H And, a) a CDR1 comprising the sequence set forth in SEQ ID NO: 5; b) a CDR2 comprising the sequence set forth in SEQ ID NO: 6; c) a CDR3 comprising the sequence set forth in SEQ ID NO: 7; and a V H , and V L And, a) a CDR1 comprising the sequence set forth in SEQ ID NO: 8; b) a CDR2 comprising the sequence set forth in SEQ ID NO: 9; and c) a CDR3 comprising the sequence set forth in SEQ ID NO: 10; and a V L ,or (ii)V H And, a) CDR1 comprising the sequence set forth in SEQ ID NO: 13; and b) a CDR2 comprising the sequence set forth in SEQ ID NO: 14; and c) a CDR3 comprising the sequence set forth in SEQ ID NO: 15; and a V comprising H , and V L And, a) CDR1 comprising the sequence set forth in SEQ ID NO: 16; b) a CDR2 comprising the sequence set forth in SEQ ID NO: 17; c) a CDR3 comprising the sequence set forth in SEQ ID NO: 18; and a V comprising L and wherein the antibody or antigen-binding fragment thereof comprises:

[0045] In one example, a CXCL10 binding protein of the disclosure comprises: (i)VH And, a) a CDR1 comprising the sequence set forth in SEQ ID NO: 5; b) a CDR2 comprising the sequence set forth in SEQ ID NO: 6; c) a CDR3 comprising the sequence set forth in SEQ ID NO: 7; and a V H , and (ii)V L And, a) a CDR1 comprising the sequence set forth in SEQ ID NO: 8; b) a CDR2 comprising the sequence set forth in SEQ ID NO: 9; and c) a CDR3 comprising the sequence set forth in SEQ ID NO: 10; and a V L and wherein the antibody or antigen-binding fragment thereof comprises:

[0046] In one example, a CXCL10 binding protein of the disclosure comprises: (i)V H And, a) CDR1 comprising the sequence set forth in SEQ ID NO: 13; and b) a CDR2 comprising the sequence set forth in SEQ ID NO: 14; and c) a CDR3 comprising the sequence set forth in SEQ ID NO: 15; and a V comprising H , and (ii)V L And, a) CDR1 comprising the sequence set forth in SEQ ID NO: 16; b) a CDR2 comprising the sequence set forth in SEQ ID NO: 17; c) a CDR3 comprising the sequence set forth in SEQ ID NO: 18; and a V comprising L and wherein the antibody or antigen-binding fragment thereof comprises:

[0047] In one example, the protein, antibody, or antigen-binding fragment thereof is in the form of a protein, antibody, or any functional fragment thereof encoded by a nucleic acid encoding any of the aforementioned proteins, antibodies, or functional fragments.

[0048] In one example, the CXCL10 binding protein is conjugated to a detectable label. Detectable labels suitable for use in the present disclosure will be apparent to one of skill in the art and / or are described herein. For example, the detectable label is selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, a linking group, and an imaging agent.

[0049] In one example, the detectable label is a radiolabel, for example, but not limited to, radioactive iodine (I, I), technetium, yttrium, S, or H.

[0050] In one example, the detectable label is an enzyme, for example, but not limited to, the enzyme can be horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase.

[0051] In one example, the detectable label is a fluorescent label, for example, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, fluorescein, dansyl chloride, or phycoerythrin.

[0052] In one example, the detectable label is a luminescent label, for example, but not limited to, luminol.

[0053] In one example, the detectable label is a bioluminescent label, for example, but not limited to, luciferase, luciferin, or aequorin.

[0054] In one example, the detectable label is a magnetic label, for example, but not limited to, a gadolinium or iron oxide chelate.

[0055] In one example, the detectable label is a prosthetic group. For example, but not limited to, the prosthetic group can be streptavidin / biotin or avidin / biotin.

[0056] In one embodiment, the detectable label is an imaging agent.

[0057] The present disclosure also provides compositions comprising a binding protein of the present disclosure and a carrier. Suitable carriers for use in the present disclosure will be apparent to those of skill in the art and / or are described herein.

[0058] The present disclosure also provides polynucleotides encoding CXCL10 binding proteins according to the present disclosure.

[0059] The present disclosure further provides expression vectors comprising a polynucleotide encoding a CXCL10 binding protein of the present disclosure. Exemplary vectors suitable for use in the present disclosure will be apparent to those of skill in the art and / or are described herein.

[0060] The present disclosure further provides cells comprising an expression vector of the present disclosure in vitro. Exemplary cells suitable for use in the present disclosure will be apparent to one of skill in the art and / or are described herein. In one example, the present disclosure provides a use of the cells for preparing a CXCL10 binding protein of the present disclosure. For example, the use includes culturing a cell of the present disclosure and producing a CXCL10 binding protein from the cell, and isolating and purifying the produced binding protein. Methods for isolating and purifying the produced binding protein will be apparent to one of skill in the art and / or are described herein.

[0061] The present disclosure provides a method for detecting and / or diagnosing a malignant condition in a subject, the method comprising: a) measuring the level of active CXCL10 in the subject and the level of total CXCL10 in the subject; b) measuring the CXCL10 ratio of active CXLC10 to total CXCL10 in the subject.

[0062] In one example, measuring the levels of active CXCL10 and total CXCL10 comprises measuring the amount of active CXCL10 protein and the amount of total CXCL10 protein in the subject.

[0063] In one embodiment, the method further comprises comparing the CXCL10 ratio in the subject to the CXCL10 ratio in at least one reference, the measurement of which will be apparent to one skilled in the art and / or described herein.

[0064] In one embodiment, the method includes determining (a) whether 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.

[0065] In one embodiment, (i) a lower CXCL10 ratio in the subject compared to the reference CXCL10 ratio indicates a malignant condition, or (ii) a higher CXCL10 ratio in the subject compared to the reference CXCL10 ratio indicates a benign condition.

[0066] In one embodiment, the method comprises: (i) measuring the level of total CXCL10 in a subject using a CXCL10 binding protein that specifically binds full-length human CXCL10, N-terminal truncated CXCL10, and citrullinated CXCL10; (ii) measuring the level of active CXCL10 in the subject using a CXCL10-binding protein that specifically binds to full-length human CXCL10, but not to N-terminal truncated CXCL10 and citrullinated CXCL10; Includes.

[0067] In one embodiment of any of the methods described herein, the method comprises using at least one CXCL10 binding protein according to the present disclosure.

[0068] In one embodiment, (i) The level of total CXCL10 in the subject is determined by measuring the level of a V H and V comprising the amino acid sequence set forth in SEQ ID NO: 12. L and / or (ii) The level of active CXCL10 in the subject is measured using a V H and V comprising the amino acid sequence set forth in SEQ ID NO:4 L The antibody or antigen-binding fragment thereof is measured using an antibody comprising:

[0069] In one example, the level of total CXCL10 in a subject is determined by measuring the level of a V CXCL10 comprising the amino acid sequence set forth in SEQ ID NO: 11. H and V comprising the amino acid sequence set forth in SEQ ID NO: 12. L The antibody or antigen-binding fragment thereof is measured using an antibody comprising:

[0070] In another embodiment, the level of active CXCL10 in a subject is determined by measuring the level of active CXCL10 in a V H and V comprising the amino acid sequence set forth in SEQ ID NO:4 L The antibody or antigen-binding fragment thereof is measured using an antibody comprising:

[0071] In one example, one or more of the CXCL10 binding proteins are conjugated to a detectable label. Detectable labels suitable for use in the present disclosure will be apparent to one of skill in the art and / or are described herein. For example, the detectable label is selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, a linking group, and an imaging agent.

[0072] In one example, the detectable label is a radiolabel, for example, but not limited to, radioactive iodine (I, I), technetium, yttrium, S, or H.

[0073] In one example, the detectable label is an enzyme, for example, but not limited to, the enzyme can be horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase.

[0074] In one example, the detectable label is a fluorescent label, for example, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, fluorescein, dansyl chloride, or phycoerythrin.

[0075] In one example, the detectable label is a luminescent label, for example, but not limited to, luminol.

[0076] In one example, the detectable label is a bioluminescent label, for example, but not limited to, luciferase, luciferin, or aequorin.

[0077] In one example, the detectable label is a magnetic label, for example, but not limited to, a gadolinium or iron oxide chelate.

[0078] In one example, the detectable label is a prosthetic group. For example, but not limited to, the prosthetic group can be streptavidin / biotin or avidin / biotin.

[0079] In one embodiment, the detectable label is an imaging agent.

[0080] Methods for detecting levels of CXCL10 will be apparent to those of skill in the art and / or are described herein, for example, methods include performing flow cytometry, enzyme-linked immunosorbent assay, or Western blot.

[0081] In one embodiment, the method includes performing flow cytometry.

[0082] In one embodiment, the method includes performing an enzyme-linked immunosorbent assay.

[0083] In one example, the method comprises performing a Western blot.

[0084] In one embodiment, the method is performed in vitro or ex vivo in a subject. For example, the method is performed in vitro in a subject. In another embodiment, the method is performed ex vivo in a subject.

[0085] In one embodiment, the method is performed on at least one biological sample obtained from a subject. Suitable biological samples for use in the present disclosure will be apparent to one of skill in the art and / or are described herein. For example, the biological sample is selected from the group consisting of a biopsy, a fluid sample, a plasma sample, or a cell swab.

[0086] In one embodiment, the biological sample is a biopsy.

[0087] In one embodiment, the biological sample is a fluid sample. For example, the fluid sample is cervical fluid, vaginal fluid, or peritoneal fluid. In one embodiment, the biological sample is peritoneal fluid.

[0088] In one embodiment, the biological sample is a plasma sample.

[0089] In one embodiment, the biological sample is a cytological swab. For example, the cytological swab is a cervical swab. In one embodiment, the cytological swab is a cervicovaginal swab (CVS).

[0090] In one embodiment, the method is performed on a plasma sample and a cervicovaginal swab (CVS).

[0091] In one embodiment of any of the methods described herein, the 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 embodiment, the reproductive cancer is ovarian cancer. In one embodiment, the ovarian cancer is stage I cancer. In another embodiment, the ovarian cancer is a precancerous lesion, e.g., a lesion with a p53 gene mutation.

[0092] In one embodiment, the method includes detecting and / or diagnosing a malignant condition from a benign condition.

[0093] In one example of any of the methods described herein, the method further comprises measuring the level of dipeptidyl peptidase-4 (DPP4) and / or cancer antigen 125 (CA-125) in the subject. In one example, the method further comprises measuring the level of dipeptidyl peptidase-4 (DPP4) and cancer antigen 125 (CA-125) in the subject. In another example, the method further comprises measuring the level of dipeptidyl peptidase-4 (DPP4) in the subject. In a further embodiment, the method further comprises measuring the level of cancer antigen 125 (CA-125) in the subject.

[0094] In one embodiment of any of the methods described herein, the method includes administering to a subject a therapeutically effective amount of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), tumor necrosis factor receptor 1 (TNF-α), or a combination ... For example, the method further comprises measuring the levels of GM-CSF, IL-6, TNF-RII, HE4, and IL-8.

[0095] In one embodiment of any of the methods described herein, the method further comprises measuring levels of DPP4, GM-CSF, IL-6, TNF-RII, HE4, and IL-8.

[0096] In one embodiment of any of the methods described herein, the method further comprises measuring levels of CA-125, GM-CSF, IL-6, TNF-RII, HE4, and IL-8.

[0097] In one embodiment of any of the methods described herein, the method further comprises measuring levels of DPP4, CA-125, GM-CSF, IL-6, TNF-RII, HE4, and IL-8.

[0098] Also provided is a method for detecting and / or diagnosing a condition in a subject, the method comprising measuring the level of CXCL10 in said subject using at least one CXCL10 binding protein of the invention.

[0099] In this example, the condition is characterized by the levels and / or relative proportions of one or more of 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, the inflammatory condition is arthritis, e.g., 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.

[0100] The present disclosure also provides a method for monitoring tumor burden in a subject suffering from a malignant condition, the method comprising measuring the CXCL10 ratio of active CXLC10 to total CXCL10 in the subject at one or more time points.

[0101] The present disclosure further provides a method for monitoring the progression of a malignant condition in a subject, the method comprising measuring the CXCL10 ratio of active CXLC10 to total CXCL10 in the subject at one or more time points.

[0102] The present disclosure also provides a method for measuring tumor regression in a subject suffering from a malignant condition, the method comprising measuring the CXCL10 ratio of active CXLC10 to total CXCL10 in the subject at one or more time points.

[0103] The present disclosure also provides a method for measuring tumor recurrence in a subject suffering from a malignant condition, the method comprising measuring the CXCL10 ratio of active CXLC10 to total CXCL10 in the subject at one or more time points.

[0104] The present disclosure also provides a method for measuring the effectiveness of a treatment for a malignant condition in a subject suffering from the malignant condition, the method comprising measuring the CXCL10 ratio of active CXLC10 to total CXCL10 in the subject at one or more time points.

[0105] In one embodiment, the subject has been diagnosed with a malignant condition. For example, the subject is For example, the malignant condition is a reproductive cancer, such as ovarian cancer.

[0106] In one embodiment, the subject is asymptomatic.

[0107] In one embodiment, the subject has not received treatment for the malignant condition. For example, the subject is treatment naive. In one embodiment, the subject is receiving treatment for the malignant condition. In another embodiment, the subject has been treated for the malignant condition. Suitable treatments for treating the malignant condition will be apparent to one of skill in the art and / or are described herein. For example, the treatment may include surgery, chemotherapy, radiation therapy, targeted drug therapy, immunotherapy, or a combination thereof.

[0108] In one embodiment, the treatment comprises surgery.

[0109] In one embodiment, the treatment comprises chemotherapy.

[0110] In one embodiment, the treatment comprises radiation therapy.

[0111] In one embodiment, the treatment comprises targeted drug therapy.

[0112] In one embodiment, the treatment comprises immunotherapy.

[0113] In one embodiment, the method comprises: (a) whether the CXCL10 ratio in the subject at the later time point is lower than the CXCL10 ratio in the subject at the first time point; or (b) determining whether the ratio of CXCL10 in the subject at the later time point is higher than the ratio of CXCL10 in the subject at the first time point.

[0114] In one example, a lower CXCL10 ratio in a subject at a later time point compared to a first time point indicates increased tumor burden and / or tumor progression and / or tumor recurrence in the subject. For example, a lower CXCL10 ratio after treatment (i.e., at a later time point) compared to before treatment (i.e., at a first time point) indicates increased tumor burden and / or tumor progression and / or tumor recurrence in the subject.

[0115] In one example, a higher CXCL10 ratio in a subject at a later time point compared to a first time point indicates a decrease in tumor burden and / or tumor regression and / or tumor recurrence in the subject. For example, a higher CXCL10 ratio after treatment (i.e., at a later time point) compared to before treatment (i.e., at a first time point) indicates a decrease in tumor burden and / or tumor regression and / or tumor recurrence in the subject.

[0116] In one embodiment of any of the methods described herein, the method further comprises administering a treatment to reduce tumor burden and / or tumor progression in the subject.

[0117] The present disclosure further provides a method of treating a malignant condition in a subject, the method comprising detecting and / or diagnosing a malignant condition in the subject in accordance with the present disclosure and administering a treatment to the subject.

[0118] Suitable therapies for treating malignant conditions will be apparent to those skilled in the art and / or are described herein, for example, the treatment may include surgery, chemotherapy, radiation therapy, targeted drug therapy, immunotherapy, or a combination thereof.

[0119] The present disclosure provides a panel or kit for detecting and / or diagnosing a malignant condition in a subject. Also provided is a panel or kit, wherein the panel or kit comprises one or more CXCL10 binding proteins of the disclosure.

[0120] The present disclosure further provides a panel or kit for monitoring tumor burden of a malignant condition, monitoring progression of a malignant condition, measuring tumor regression of a malignant condition, measuring tumor recurrence of a malignant condition, and / or measuring the effectiveness of a treatment for a malignant condition in a subject, wherein the panel or kit comprises one or more CXCL10 binding proteins of the present disclosure.

[0121] Any embodiment herein is deemed to apply mutatis mutandis to any other embodiment unless expressly stated otherwise. For example, one skilled in the art will appreciate that the examples outlined above with respect to one embodiment of the invention apply equally to other embodiments of the invention.

[0122] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions and methods as described herein are clearly within the scope of the invention.

[0123] Throughout this specification, unless otherwise specified or otherwise required by context, references to a single step, composition of matter, group of steps or group of compositions of matter shall be interpreted as encompassing one and more (i.e., one or more) of that step, composition of matter, group of steps or group of compositions of matter. [Brief explanation of the drawings]

[0124] [Figure 1] 1 is a graphical representation showing the difference between active and total CXCL10. Exemplary calibration curves using RA2 and RG2 for the detection of CXCL10. Each has an R2>0.99. [Figure 2] (A) Comparison of quantified total CXCL10 in malignant ascites fluids from ovarian cancer (n=212) between ART and commercial ELISA. No significant differences in quantified total CXCL10 were observed in matched ascites fluids between ART and commercial ELISA. The overall mean total CXCL10 values ​​by ART and commercial ELISA were 1126.1±2158.6 pg / mL and 1192.4±1059.5 pg / mL, respectively. (B) Correlation of quantified total CXCL10 between ART and commercial ELISA and a moderate positive correlation between the two tests for both benign and malignant ascites are shown, with r values ​​of 0.3084 and 0.2594, respectively. P≦0.05. [Figure 3](A) Western blots showing the detection of recombinant full-length CXCL10 and citrullinated CXCL10 by mAb-RA2 and mAb-RG2. CXCL10 was treated with (+) or without (-) PAD2 to induce citrullination for 60 minutes, followed by Western blot separation. Recombinant full-length CXCL10 without any treatment (n / t) was used as a positive control. A commercial anti-CXCL10 antibody (ab9807) did not detect citrullinated CXCL10 after 15 minutes of PAD2 incubation. Note that a substantial decrease in the detection of citrullinated CXCL10 by mAb-RA2 was observed after 15 minutes, indicating no effect of citrullination on CXCL10 detection by mAb-RG2. (B) Detection of citrullinated CXCL10 by ART by mAb-RA2 and mAb-RG2. A substantial decrease in the binding of citrullinated CXCL10 by mAb-RA2 was observed, while mAb-RG maintained constant binding of citrullinated CXCL10. [Figure 4] Quantification of active and total CXCL10 and the difference in active CXCL10 between benign and malignant ascites fluids from ovarian cancer are shown. (A) Active CXCL10 concentrations in benign (n=51) and malignant ascites (n=208): 240.4±410.5 pg / mL and 818.6±1098.0 pg / mL, respectively. (B) Total CXCL10 concentrations in benign (n=51) and malignant ascites (n=212): 160.8±362.0 pg / mL and 1126.1±2158.6 pg / mL, respectively. (C) Significant differences in activity ratios in benign (n=51) and malignant ascites (n=226): 2.59±1.18 and 1.43±1.04, respectively. (D) Activity ratios based on ovarian cancer stage compared to benign. Benign: 2.59 ± 1.18; Stage 1: 1.07 ± 0.44; and Stage 3: 1.55 ± 1.23. ****P ≤ 0.0001. [Figure 5]Figure 1 shows the effects of DPP4 and plasma CA125 on distinguishing benign from malignant cases. (A) Measurement of DPP4 concentrations in benign (n = 48) and malignant ascites (n = 148) by anti-DPP4 ELISA: 184.8 ± 177.6 ng / mL and 203.5 ± 154.3 ng / mL, respectively. P = 0.1031. (B) Measurement of DPP4 specific activity (U / ng) in benign (n = 49) and malignant (n = 50) ascites: 2.49 ± 3.83 and 1.65 ± 2.13, respectively. P = 0.4229. (C) Measurement of plasma CA125 in matched patients: 200.8 ± 368.7 U / mL and 1697.0 ± 3409.0 U / mL for benign (n = 30) and malignant (n = 188) patient samples, respectively. ****P ≤ 0.0001. [Figure 6] Correlations between activity ratio and plasma CA125, DPP4 concentrations, and DPP4 specific activity in benign and malignant ascites fluids are shown. (A) No significant correlation between activity ratio and plasma CA125 in either benign or malignant samples. (B) A moderate negative correlation between activity ratio and DPP4 (ng / mL) in malignant ascites samples (P=0.0002), while no significant correlation exists in benign samples. (C) A moderate negative correlation between activity ratio and DPP4 specific activity (U / ng) in benign samples, while no correlation exists in malignant samples. [Figure 7] Receiver operating curves (ROCs) showing superior AUCs for ART versus other markers in patient ascites samples are shown. (A) The active fraction achieves a higher AUC (0.8617) than the AUCs for quantification of total CXCL10 and active CXCL10 (0.8122 and 0.7872, respectively). (B) The active fraction achieves a higher AUC than the AUCs for DPP4 and plasma CA125 (0.5598 and 0.8262, respectively). Combining active fraction, DPP4 (ng / mL), and plasma CA125 (U / mL) demonstrates a high AUC. [Figure 8]Cervicovaginal swabs (CVS) and plasma samples are ideal for ART as biomarker-based tests. (A) Significant differences in the activity ratios in CVS between benign (n=50) and malignant (n=50) samples are shown. The activity ratios in benign and malignant CVS are 4.39±4.52 and 1.14±0.62, respectively. (B) Significant differences in the activity ratios in plasma between benign (n=30) and malignant (n=30) plasma samples are shown. The activity ratios in benign and malignant plasma are 3.18±1.79 and 2.02±1.05, respectively. ****P<0.0001, **P<0.01. [Figure 9] ART distinguishes cancer-free patients from those with benign or malignant ovarian cancer. A shows the concentrations of active and total CXCL10 measured in plasma samples. B shows the concentrations of active and total CXCL10 across the three patient groups. C and D show the calculated activity ratios between non-cancerous (healthy) samples and benign and malignant samples as detected in plasma (C) or CVS (D). *p≦0.05; ****p≦0.0001. [Figure 10] In the cohort recruited for prophylaxis, A shows the amount of DPP4, B shows the DPP4-specific activity, and C and D show the correlation between these and the activity percentage. [Figure 11] Figure 1 shows ART performed using CVS swabs to distinguish between non-cancerous conditions and benign and malignant diseases. A shows that the overall concentration of active CXCL10 was significantly higher than total CXCL10. B shows the calculated activity ratio between non-cancerous (healthy) samples and benign and malignant samples. *p≦0.05; ****p≦0.0001. [Figure 12] A shows the amount of DPP4 in the CVS, and B shows the correlation with the calculated activity rate of the CVS. [Figure 13] A shows plasma CA125 in a prophylactically recruited cohort of patients with either benign or malignant ovarian tumors. B shows the correlation between CA125 and percent activity. [Figure 14]A: Activity ratio of plasma and CVS, B: Activity ratio of plasma containing DPP4 and CA125, and C: Activity ratio of CVS containing DPP4 and CA125 combinations are shown, which distinguish between healthy women and patients with malignant ovarian tumors, respectively. [Figure 15] A: ROC for assessing the discriminatory power of individual markers compared to CA125 and B: combinations of markers (built on benign + healthy vs malignant). DETAILED DESCRIPTION OF THE INVENTION

[0125] Sequence Listing explanation SEQ ID NO: 1 Amino acid sequence of human CXCL10 including the presequence SEQ ID NO: 2: Amino acid sequence of mature human CXCL10 SEQ ID NO: 3 Heavy chain V of anti-CXCL10 antibody RA2 H Amino acid sequence SEQ ID NO: 4 Light chain V of anti-CXCL10 antibody RA2 L Amino acid sequence SEQ ID NO: 5 Heavy chain V of anti-CXCL10 antibody RA2 H CDR1 amino acid sequence SEQ ID NO: 6 Heavy chain V of anti-CXCL10 antibody RA2 H CDR2 amino acid sequence SEQ ID NO: 7 Heavy chain V of anti-CXCL10 antibody RA2 H CDR3 amino acid sequence SEQ ID NO: 8 Light chain V of anti-CXCL10 antibody RA2 L CDR1 amino acid sequence SEQ ID NO: 9 Light chain V of anti-CXCL10 antibody RA2 L CDR2 amino acid sequence SEQ ID NO: 10 Light chain V of anti-CXCL10 antibody RA2 L CDR3 amino acid sequence SEQ ID NO: 11 Heavy chain V of anti-CXCL10 antibody RG2 H Amino acid sequence SEQ ID NO: 12 Light chain V of anti-CXCL10 antibody RG2 L Amino acid sequence SEQ ID NO: 13 Heavy chain V of anti-CXCL10 antibody RG2H CDR1 amino acid sequence SEQ ID NO: 14 Anti-CXCL10 antibody R G Heavy chain V of 2 H CDR2 amino acid sequence SEQ ID NO: 15 Heavy chain V of anti-CXCL10 antibody RG2 H CDR3 amino acid sequence SEQ ID NO: 16 Light chain V of anti-CXCL10 antibody RG2 L CDR1 amino acid sequence SEQ ID NO: 17 Anti-CXCL10 antibody R G 2 light chain V L CDR2 amino acid sequence SEQ ID NO: 18 Light chain V of anti-CXCL10 antibody RG2 L CDR3 amino acid sequence SEQ ID NO: 19 Heavy chain V of anti-CXCL10 antibody RA2 H Nucleotide sequence SEQ ID NO: 20 Light chain V of anti-CXCL10 antibody RA2 L Nucleochi Do distribution column SEQ ID NO: 21 Heavy chain V of anti-CXCL10 antibody RG2 H Nucleotide sequence SEQ ID NO: 22 Light chain V of anti-CXCL10 antibody RG2 L Nucleochi Do distribution column SEQ ID NO: 23 Peptide sequence containing the intact N-terminus of human CXCL10 SEQ ID NO: 24 Peptide sequence containing the truncated N-terminus of human CXCL10 SEQ ID NO: 25 Intact N-terminal epitope of human CXCL10 SEQ ID NO: 26 Cleaved N-terminal epitope of human CXCL10

[0126] overview Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, configuration of events, group of steps or group of events shall be interpreted as encompassing one and more (i.e., one or more) of that step, configuration of events, group of steps or group of events.

[0127] The present disclosure is not intended to be limited in scope by the specific examples described herein, as such specific examples are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0128] Those skilled in the art will recognize that many variations and / or modifications may be made to the invention shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described, and the present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0129] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0130] Any discussion of documents, acts, materials, devices, articles or the like which has been included in this specification is solely for the purpose of providing a context for the present invention and is not to be considered an admission that any or all of such matters were part of the prior art or common general knowledge in the fields relevant to this invention as they existed prior to the priority date of each claim in this application.

[0131] Any example of the present disclosure shall be construed as applying mutatis mutandis to any other example of the present disclosure, unless expressly stated otherwise. In other words, any embodiment of the present disclosure can be combined with any other embodiment of the present disclosure (except those that are mutually exclusive).

[0132] Any example of this disclosure that discloses a particular trait, or group of traits, or method, or method step, will be used to provide explicit support for distinguishing that particular trait, or group of traits, or method, or method step.

[0133] Unless specifically defined otherwise, all technical and scientific terms used herein shall be construed 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).

[0134] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in this disclosure are standard procedures well known to those skilled in the art, and are described and explained throughout the literature in the following sources: Perbal, 1984; Sambrook et al., 1989; Brown, 1991; Glover et al., 1995 and 1996; Ausubel et al., 1988; Harlow et al., 1988; Coligan et al., 1991.

[0135] The descriptions and definitions of variable regions and portions thereof, immunoglobulins, antibodies, and fragments thereof herein are as set forth in Kabat et al., 1987 and 1991; Bork et al., 1994; Chothia and Lesk, 1987; Chothia et al., 1989, and / or the discussion in Al-Lazikani et al., 1997 may provide further clarity.

[0136] For example, references herein to ranges of residues will be understood to be inclusive, e.g., a reference to "the region comprising amino acids 56-65" will be understood in an inclusive manner, i.e., that region includes the sequence of numbered amino acids 56, 57, 58, 59, 60, 61, 62, 63, 64, and 65 in a particular sequence.

[0137] The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be deemed to provide clear support for both meanings or either meaning.

[0138] Throughout this specification, the words "comprise" or "comprises" It will be understood that variations such as "includes" or "comprising" imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0139] As used herein, the term "subject" should be understood to mean any animal, e.g., a mammal, including a human. Exemplary subjects include, but are not limited to, humans and non-human primates. In one example, the subject is a human. For example, the subject is a human.

[0140] Selected Definitions Human CXCL10 is represented as a presequence (SEQ ID NO: 1), which is truncated by 22 N-terminal amino acids to produce mature "full-length" human CXCL10 (SEQ ID NO: 2). Thus, as used herein, the term "full-length CXCL10" refers to CXCL10 that has not been post-translationally modified other than post-translational cleavage to remove the 22 N-terminal amino acids to generate "mature" full-length CXCL10.

[0141] As used herein, the term "N-terminally truncated CXCL10" refers to "mature" full-length CXCL10 that has been cleaved by dipeptidyl peptidase IV (DPP4), for example, consisting of amino acids 3 to 77 of SEQ ID NO:2.

[0142] As used herein, the term "citrullinated CXCL10" refers to CXCL10 that has been translationally modified by peptidylarginine deiminase (PAD) by deamination or citrullination, for example, at the arginine residue at amino acid position 5 of SEQ ID NO:2.

[0143] Sequences of CXCL10 from other species can be determined using the sequences provided herein and / or from publicly available databases and / or using standard techniques (e.g., as described in Ausubel et al., 1988 (including any updates to date), or Sambrook et al., 1989).

[0144] The term "recombinant" should be understood to mean a product of artificial genetic engineering. Thus, in the context of a recombinant protein containing a variable region or antigen-binding domain (e.g., an antibody antigen-binding domain), the term does not include proteins that occur naturally in a subject's body that are the product of natural recombination that occurs during B-cell maturation. However, if such a protein is isolated, the protein is considered an isolated protein that contains a variable region or antigen-binding domain. Similarly, if a nucleic acid encoding a protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein that contains a variable region or antigen-binding domain. Recombinant protein also includes proteins expressed by artificial recombinant means when the protein is present, for example, in the cell, tissue, or subject in which it is expressed.

[0145] The term "protein" should be understood to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains (i.e., a polypeptide complex) that are covalently or non-covalently bonded to one another. For example, a series of polypeptide chains can be covalently bonded using suitable chemical or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0146] The term "polypeptide" or "polypeptide chain" will be understood from the preceding paragraph to mean a series of consecutive amino acids linked by peptide bonds.

[0147] 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, e.g., a protein).

[0148] As used herein, the term "antigen binding domain" refers to a domain that binds to an antigen, i.e., a V H Or V L , or V H and V L The term "antigen-binding region" should be understood to mean the region of an antibody capable of specifically binding to an Fv, including both the Fv and the antibody. The antigen-binding region need not refer to the entire antibody, but can be, for example, isolated (e.g., domain antigen) or in another form, such as an scFv, as described herein.

[0149] For purposes of this disclosure, the term "antibody" includes proteins capable of specifically binding to one or a small number of closely related antigens (e.g., CXCL10) via an antigen-binding region contained within the 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-grafted antibodies, primatized antibodies, deimmunized antibodies, synthetic humanized antibodies, half antibodies, and bispecific antibodies). Antibodies typically contain a constant domain, which can be incorporated into a constant region or constant fragment or fragment crystallizable (Fc). Typical forms of antibodies contain a four-chain structure as a basic unit. Full-length antibodies contain two covalently linked heavy chains (approximately 50-70 kDa) and two light chains (approximately 23 kDa). The light chain generally consists of a variable region (if present) and a constant domain, and in mammals is either a kappa or lambda light chain. Heavy chains generally consist of a variable region and one or two constant domain(s) connected by a hinge region to additional constant domain(s). Mammalian heavy chains are of one of the following types: α, β, ε, γ, or μ. Each light chain is also covalently linked to one of the heavy chains. For example, two heavy chains, and heavy and light chains, are linked to each other by interchain disulfide bonds and by non-covalent interactions. The number of interchain disulfide bonds varies between different types of antibodies. Each chain contains an N-terminal variable region (VLs), each about 110 amino acids long. H or V L ), and one or more constant domains at the C-terminus. The light chain constant domain (C, which is about 110 amino acids long) L ) is the first constant domain of the heavy chain (C, which is 330-440 amino acids long). H 1) and are disulfide bonded. The light chain variable region is aligned with the heavy chain variable region. The antibody heavy chain consists of two or more additional C H Region (e.g., C H 2. C H 3, etc.), and C H 1 constant domain and C HA hinge region may be included between the two constant domains. The antibody can be of 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 murine (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 humanized, synthetically humanized, chimeric, CDR-grafted, or deimmunized.

[0150] The terms "full length antibody," "intact antibody," or "whole antibody" are used interchangeably and refer to an antibody in its substantially intact form, as opposed to an antigen-binding fragment thereof. In particular, whole antibodies include those having heavy and light chains, including the Fc region. The constant domains may be wild-type sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0151] As used herein, "variable region" refers to a portion of the light and / or heavy chain of an antibody, as defined herein, capable of specifically binding to an antigen, including the complementarity determining regions (CDRs); i.e. A variable region includes the amino acid sequences of CDR1, CDR2, and CDR3, as well as framework regions (FR). For example, a variable region includes three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) combined with three CDRs. H refers to the variable region of the heavy chain. L refers to the variable region of the light chain.

[0152] As used herein, the term "complementarity determining region" (synonym: CDR, i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues in the variable region of an antibody that are primarily responsible for specific binding to an antigen. H or V L) typically have three CDRs designated as CDR1, CDR2, and CDR3. In one example, the amino acid positions assigned to the CDRs and FRs are defined according to 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 the Kabat numbering system, V H The FRs and CDRs of V are located as follows: residues 1-30 (FR1), 31-35 (CDR1), 36-49 (FR2), 50-65 (CDR2), 66-94 (FR3), 95-102 (CDR3), and 103-113 (FR4). According to the Kabat numbering system, V L The FRs and CDRs of are located as follows: residues 1-23 (FR1), 24-34 (CDR1), 35-49 (FR2), 50-56 (CDR2), 57-88 (FR3), 89-97 (CDR3), and 98-107 (FR4). The present disclosure is not limited to FRs and CDRs defined by the Kabat numbering system, but includes any numbering system, including the canonical numbering systems of Chothia and Lesk, 1987; Chothia et al., 1989; and / or Al-Lazikani et al., 1997; the numbering system of Honnegher and Plukthun, 2001; or the TMGT system discussed in Giudicelli et al., 1997. In one embodiment, the CDRs are defined according to the Kabat numbering system. Optionally, the heavy chain CDR2 according to the Kabat numbering system does not include the five C-terminal amino acids listed herein, or one or more of these amino acids are substituted with other naturally occurring amino acids. In this regard, Padlan et al., 1995, established that the five C-terminal amino acids of heavy chain CDR2 are generally not involved in antigen binding.

[0153] "Framework regions" (FR) are those variable region residues other than the CDR residues.

[0154] As used herein, the term "Fv" (or a deformable fragment) refers to a fragment of an Fv polypeptide, whether composed of multiple polypeptides or a single polypeptide, in which the V L and V H The term "antigen-binding domain" should be understood to mean any protein that associates with a V that forms a complex having an antigen-binding domain that is capable of specifically binding to an antigen. H and V L The V can be present on a single polypeptide chain or on different polypeptide chains. Furthermore, an Fv of the disclosure (and similarly any protein of the disclosure) may have multiple antigen-binding domains that may or may not be capable of binding to the same antigen. The term should be understood to encompass fragments derived directly from antibodies, as well as proteins corresponding to such fragments produced using recombinant means. In some examples, the V H is the heavy chain constant domain (C H ) 1 and / or V L is the light chain constant domain (C L Exemplary Fv-containing polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFv, diabodies, triabodies, tetrabodies or higher order complexes, or constant regions or domains thereof, such as C H 2 or C H An "antigen-binding fragment" or "Fab fragment" consists of a monovalent antigen-binding fragment of an immunoglobulin, which is produced by cleavage of a whole antibody with the enzyme papain. An "Fab' fragment" of an antibody can be obtained by treating whole antibody with pepsin followed by reduction, resulting in an intact light chain and a fragment consisting of a portion of the heavy chain, or can be produced using recombinant means. HA molecule consisting of a portion of a heavy chain containing a single constant domain can be obtained. Two Fab' fragments can be obtained from one antibody treated in this manner. Fab' fragments can also be produced by recombinant means. An "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments linked by two disulfide bonds and can be obtained by treating a whole antibody molecule with the enzyme pepsin without subsequent reduction. "Fab2" fragments can be obtained by, for example, leucine zipper or C H A "single-chain Fv" or "scFv" is a recombinant molecule containing the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable flexible peptide linker.

[0155] As used herein, the term "bind" with respect to the interaction of a CXCL10-binding protein, or its antigen-binding domain, with an antigen means that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) in the antigen. For example, an antigen recognizes and binds to a specific protein structure, rather than all proteins. If an antibody binds to epitope "A," in a reaction containing 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.

[0156] As used herein, the term "specifically binds" should be understood to mean that a CXCL10-binding protein of the disclosure reacts with or associates with a particular antigen or cells expressing that antigen more frequently, faster, and for a longer period of time and / or with higher affinity than with alternative antigens or cells. For example, a CXCL10-binding protein binds to CXCL10 with substantially higher affinity (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) than it binds to other chemokine receptors or antigens commonly recognized by polyreactive natural antibodies (i.e., naturally occurring antibodies known to bind to a variety of antigens naturally found in humans). Reference to "binding" provides explicit support for the term "specifically binds," and vice versa.

[0157] As used herein, the term "does not bind" should be understood to mean that the CXCL10 binding proteins of the present disclosure do not bind to a particular antigen or to a cell expressing that antigen.

[0158] As used herein, the term "does not detectably bind" should be understood to mean that the CXCL10-binding protein, e.g., antibody, binds to the candidate antigen at a background level that is less than 10%, or 8%, or 6%, or greater than 5%. 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 isotype control antibody), and / or the level of binding detected in the presence of a negative control antigen. In one example, the level of binding is detected using a biosensor assay (e.g., Biacore) in which an antigen (e.g., a polypeptide) is immobilized and contacted with the CXCL10-binding protein.

[0159] As used herein, the term "does not specifically bind" refers to the level of binding of a CXCL10 binding protein of the disclosure to a polypeptide, e.g., the level of binding signal detected in the absence of a CXCL10 binding protein and / or in the presence of a negative control protein (e.g., an isotype control antibody) and / or in the presence of a negative control polypeptide. This should be understood to mean that the level of binding reported is not statistically significantly higher than background. In one example, the level of binding is detected using a biosensor assay (e.g., Biacore) in which an antigen (e.g., a polypeptide) is immobilized and contacted with a CXCL10-binding protein.

[0160] For clarity, and as will be apparent to one of skill in the art based on the subject matter exemplified herein, references herein to "affinity" refer to the K D This is a reference to.

[0161] Reference to "at least about affinity" refers to the affinity (or K D ) will be understood to mean equal to or greater than the cited value (i.e., the cited value for affinity is less), i.e., an affinity of 2 nM is greater than an affinity of 3 nM. Alternatively stated, the term can be "affinity less than or equal to X," where X is a value cited herein.

[0162] As used herein, the term "epitope" (synonymous with "antigenic determinant") should be understood to mean the region to which a CXCL10-binding protein binds. The term is not necessarily limited to the particular residues or structures that contact the CXCL10-binding protein. For example, the term includes a region spanning the amino acids that contact the CXCL10-binding protein, as well as 5-10 (or more), or 2-5, or 1-3 amino acids outside of that region. In some examples, an epitope includes a series of discontinuous amino acids that are located in close proximity to each other when a CXCL10 polypeptide is folded and, for example, associated with another CXCL10 polypeptide, i.e., a "conformational epitope."

[0163] The term "competitively inhibit" should be understood to mean that a CXCL10-binding protein (or antigen-binding domain thereof) of the present disclosure reduces or prevents the binding of the referenced antibody or CXCL10-binding protein to CXCL10. This may be due to the CXCL10-binding protein (or antigen-binding domain) and the antibody that binds to it, or to overlapping epitopes. From the foregoing, it will be apparent that a CXCL10-binding protein does not necessarily completely inhibit antibody binding; rather, it only needs to reduce binding by a statistically significant amount, e.g., at least 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90%, or 95%. For example, a CXCL10 binding protein reduces antibody binding by at least about 30%, e.g., at least about 50%, e.g., at least about 70%, e.g., at least about 75%, and more preferably, at least about 80% or 85%, e.g., at least about 90%. Methods for measuring competitive inhibition of binding are known in the art and / or described herein. For example, an antibody is exposed to CXCL10 in the presence or absence of a CXCL10 binding protein. If less antibody binds in the presence of the CXCL10 binding protein than in its absence, the protein is considered to competitively inhibit antibody binding. In one example, competitive inhibition is not due to steric hindrance.

[0164] CXCL10-binding protein As discussed herein, the binding proteins of the disclosure can take a variety of forms and can bind to full-length human CXCL10, N-terminally truncated CXCL10, and / or citrullinated CXCL10.

[0165] In one example, the disclosure provides a CXCL10 binding protein, wherein the binding protein binds to full-length human CXCL10, N-terminal truncated CXCL10, and citrullinated CXCL10.

[0166] In another embodiment, the disclosure provides a CXCL10 binding protein, wherein the binding protein binds to full-length human CXCL10 but does not bind to N-terminal truncated CXCL10 and citrullinated CXCL10.

[0167] antibody In one example, a CXCL10 binding protein of the disclosure comprises an antibody or antigen-binding fragment thereof.

[0168] Immunization-based methods Methods for generating antibodies are known in the art and / or described in Harlow et al., 1988. Generally, in such methods, a protein, or an immunogenic fragment or epitope thereof, or a cell expressing and displaying the same (i.e., immunogen), optionally formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, is administered to a non-human animal, such as a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat, or pig. The immunogen can be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known routes.

[0169] The production of polyclonal antibodies can be monitored by sampling the blood of the immunized animal at various time points after immunization. One or more further immunizations can be administered if necessary to achieve the desired antibody titer. The process of boosting and titering is repeated until a suitable titer is achieved. When the desired level of immunogenicity is obtained, the immunized animal is bled and the serum is isolated and stored, and / or the animal is used to generate monoclonal antibodies (mAbs).

[0170] Monoclonal antibodies are an exemplary form of antibody contemplated by the present disclosure. The term "monoclonal antibody" or "mAb" refers to a homogeneous antibody population capable of binding to the same antigen(s), e.g., the same epitope within the antigen. The term is not intended to be limited as to the source of the antibody or the manner in which it is made.

[0171] For the production of mAbs, any one of a number of known techniques can be used, such as the procedures exemplified in US Pat. No. 4,196,265 or Harlow et al., 1988.

[0172] For example, a suitable animal is immunized with an immunogen under conditions sufficient to stimulate antibody-producing cells. Rabbits and rodents (e.g., mice and rats) are exemplary animals. Mice that express human immunoglobulin proteins and, for example, that have been genetically engineered not to express murine immunoglobulin proteins can also be used to generate antibodies of the present disclosure (e.g., as described in WO2002066630).

[0173] After immunization, somatic cells with the potential to produce antibodies, such as B lymphocytes (B cells), are selected for use in mAb production protocols. Such 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 cells of immortalized myeloma cells. These myeloma cells are generally derived from the same species as the animal immunized with the immunogen.

[0174] The hybrids are amplified by culturing in selective medium containing drugs that block de novo synthesis of nucleotides in tissue culture medium. Exemplary drugs are aminopterin, methotrexate, and azaserine.

[0175] The expanded hybridomas are subjected to functional selection for antibody specificity and / or titer, for example, by flow cytometry and / or immunohistochemistry and / or immunoassays (e.g., radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunoassays, etc.).

[0176] Alternatively, ABL-MYC technology (NeoClone, Madison WI 53713, USA) is used to generate mAb-secreting cell lines (such as those described in Largaespada et al., 1996).

[0177] Library-based methods The present disclosure also encompasses the screening of libraries of antibodies or antigen-binding fragments thereof (eg, comprising the variable regions thereof).

[0178] Examples of libraries contemplated by the present disclosure include naive libraries (from subjects not administered an antigen), immunized libraries (from subjects immunized with an antigen), or synthetic libraries. Nucleic acids encoding antibodies or regions thereof (e.g., variable regions) can be cloned by conventional techniques (e.g., as disclosed in Sambrook et al., 2001) and used to encode and display proteins using methods known in the art. Other techniques for generating protein libraries are disclosed, for example, in US6300064 (e.g., the HuCAL library from Morphosys AG); US5885793; US6204023; US62911581 or US6248516.

[0179] Antigen-binding fragments according to the present disclosure can be soluble, secreted proteins, or displayed as fusion proteins on the surface of cells, or particles (e.g., phage 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, such as those disclosed in US 7,270,969). In yet another example, the display library is a phage display library, in which proteins comprising antigen-binding fragments of antibodies are expressed on phage, as disclosed in, for example, US 6,300,064; US 5,885,793; US 6,204,023; US 6,291,158; or US 6,248,516. Other phage display methods are known in the art and are contemplated by the present disclosure. Also contemplated are cell display methods according to the present disclosure, such as bacterial display libraries (e.g., those described in US5516637); yeast display libraries (e.g., those described in US6423538), or mammalian display libraries.

[0180] Methods for screening display libraries are known in the art. In one example, a display library of the present disclosure is screened using affinity purification, e.g., as described in Scopes, 1994. Affinity purification methods typically involve contacting proteins containing antigen-binding fragments displayed by the library with the target antigen, followed by washing and elution of those domains that remain bound to the antigen.

[0181] If desired, any variable regions or scFvs identified by screening can be rapidly modified into complete antibodies. Exemplary methods for modifying or reformatting variable regions or scFvs into complete antibodies are described, for example, in Jones et al., 201 or WO2012040793. Alternatively, or additionally, standard cloning methods may be used, e.g., as described in Ausubel et al., 1987, and / or Sambrook et al., 2001.

[0182] Deimmunized, chimeric, humanized, synthetic humanized, primatized, and human antibodies or antigen-binding fragments The antibodies or antigen-binding fragments of the present disclosure may be humanized.

[0183] The term "humanized antibody" should be understood to mean a protein comprising a human-like variable region, which comprises the CDRs of an antibody of a non-human species (e.g., mouse or rat, or non-human primate) grafted onto or inserted into the FRs of a human antibody (such antibodies are also called "CDR-grafted antibodies"). Humanized antibodies also include antibodies in which one or more residues of the human protein have been modified by one or more amino acid substitutions and / or one or more FR residues of the human antibody have been replaced by corresponding non-human residues. Humanized antibodies may further comprise residues that are not found in either human or non-human antibodies. Additional antibody regions (e.g., the Fc region) are typically human. Humanization can be performed using methods known in the art, for example, US Pat. No. 5,225,539, US Pat. No. 6,054,297, US Pat. No. 7,566,771, or US Pat. No. 5,585,089. The term "humanized antibody" also encompasses superhumanized antibodies, for example, as described in US Pat. No. 7,732,578. A similar meaning will apply to the term "humanized antigen-binding fragment."

[0184] The antibody or antigen-binding fragment thereof of the present disclosure can be a human antibody or antigen-binding fragment thereof. As used herein, the term "human antibody" refers to an antibody having variable antibody regions, and optionally constant antibody regions, found in a human, e.g., in a human germline or somatic cell, or from a library produced using such regions. A "human" antibody can include amino acid residues not encoded by human sequences, e.g., mutations introduced in vitro by random or site-specific mutagenesis (particularly mutations involving conservative substitutions or mutations of a small number of residues in the protein, e.g., 1, 2, 3, 4, or 5 residues in the protein). Such "human antibodies" need not necessarily be generated as the result of a human immune response; instead, they can be generated using recombinant means (e.g., screening of phage display libraries) and / or by transgenic animals (e.g., mice) containing nucleic acids encoding human antibody constant and / or variable regions, and / or by guided selection (e.g., as described in U.S. Pat. No. 5,565,332). The term also encompasses affinity-matured forms of such antibodies. For the purposes of this disclosure, a human antibody is also considered to include proteins comprising FRs of a human antibody, or sequences from a consensus sequence of human FRs, in which one or more CDRs comprise FRs that are random or semi-random, e.g., as described in US6300064 and / or US6248516. A similar meaning would apply to the term "human antigen-binding fragment."

[0185] The antibody or antigen-binding fragment thereof of the present disclosure can 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 comprises an antibody variable region comprising FRs from a New World primate antibody variable region and CDRs from a non-New World primate antibody variable region.

[0186] The antibodies or antigen-binding fragments thereof of the present disclosure may be primatized. A "primatized antibody" comprises variable region(s) from an antibody generated after immunization of a non-human primate (e.g., a cynomolgus macaque). Optionally, the variable region of the non-human primate antibody is linked to a human constant region to produce the primatized antibody. An exemplary method for producing a primatized antibody is: Described in US6113898.

[0187] In one example, an antibody or antigen-binding fragment thereof of the present disclosure is a chimeric antibody or fragment. The term "chimeric antibody" or "chimeric antigen-binding fragment" refers to an antibody or fragment in which one or more of the variable domains are derived from a particular species (e.g., murine, such as mouse or rat) or belong to a particular antibody class or subclass, while the remainder of the antibody or fragment is derived from another species (e.g., human or non-human primate) or belongs to another antibody class or subclass. In one example, a chimeric antibody is an antibody in which one or more of the variable domains are derived from a non-human antibody (e.g., murine antibody) or belong to a different antibody class or subclass. H and / or V L and the remaining regions of the antibody are derived from a human antibody. The production of such chimeric antibodies and antigen-binding fragments thereof is known in the art and can be achieved by standard means (e.g., as described in US6331415; US5807715; US4816567, and US4816397).

[0188] The present disclosure also contemplates deimmunized antibodies or antigen-binding fragments thereof, e.g., as described in WO2000034317 and WO2004108158. Deimmunized antibodies and fragments reduce the likelihood that a subject will mount an immune response to the antibody or protein by removing (i.e., mutating) one or more epitopes, e.g., B-cell or T-cell epitopes. For example, an antibody of the present disclosure can be analyzed to identify one or more B- or T-cell epitopes, and one or more amino acid residues within the epitope can be mutated to reduce the immunogenicity of the antibody.

[0189] Antibody-binding domain-containing proteins Single Domain Antibodies In some examples, a CXCL10 binding protein of the disclosure is or comprises a single domain antibody (used interchangeably with the term "domain antibody" or "dAb"). A single domain antibody is a single polypeptide chain that comprises all or part of the heavy chain variable domain of an antibody.

[0190] Diabodies, triabodies, and tetrabodies In some examples, a CXCL10 binding protein of the disclosure is or comprises a diabody, triabody, tetrabody, or higher order protein complex, such as those described in WO98 / 044001 and / or WO94 / 007921.

[0191] For example, a diabody is one in which each polypeptide chain has the structure V L -XV H or V H -XV L wherein X is a V in a single polypeptide chain. H and V L a linker that contains insufficient residues for the V of one polypeptide chain to associate (or form an Fv), or is absent, H is the V of the other polypeptide chain. L to form an antigen-binding site, i.e., an Fv molecule capable of specifically binding to one or more antigens. L and V H can be the same for each polypeptide chain, or V L and V H can be different on each polypeptide chain to form bispecific diabodies (i.e., diabodies comprising two Fvs with different specificities).

[0192] Single chain Fv (scFv) fragment The CXCL10 binding proteins of the present disclosure can be scFvs. Those skilled in the art will recognize that scFvs are composed of V in a single polypeptide chain. H and V L The V domains of a single polypeptide chain, as well as the scFv, enable the scFv to form the desired structure for antigen binding (i.e., the V domains of a single polypeptide chain to associate with each other to form the Fv). H and V L ), V H and V L Polypeptide phosphorus between For example, the linker contains an excess of 12 amino acid residues, and (Gly4Ser)3 is one of the more preferred linkers for scFvs.

[0193] The present disclosure also provides a method for treating a cysteine ​​residue comprising administering to a subject a subject therapies comprising administering to a subject a cysteine ​​residue comprising administering to a subject ... H FR and V L Disulfide-stabilized Fvs (or diFvs or dsFvs) are also contemplated, in which cysteine ​​residues are introduced into the FR of the Fv and linked by disulfide bonds to yield a stable Fv.

[0194] Alternatively, or in addition, the present disclosure encompasses dimeric scFs, i.e., proteins comprising two scFv molecules linked non-covalently or covalently, for example, by a leucine zipper domain (e.g., Fos or Jun). Alternatively, the two scFvs are linked by a peptide linker of sufficient length to allow both scFvs to be formed and capable of binding to antigen, e.g., as described in US20060263367.

[0195] half antibody In some embodiments, the antigen-binding fragments of the present disclosure are half antibodies or half molecules. Those skilled in the art will recognize that a half antibody refers to a protein comprising a single heavy chain and a single light chain. The term "half antibody" also encompasses proteins comprising an antibody light chain and an antibody heavy chain, where the antibody heavy chain has been mutated to prevent association with another antibody heavy chain. In one embodiment, a half antibody is formed when an antibody dissociates to form two molecules, each containing a single heavy chain and a single light chain.

[0196] Methods for generating half antibodies are known in the art, and exemplary methods are described herein.

[0197] In one example, half antibodies can be secreted by introducing into the cell genes for expression a single heavy chain and a single light chain that make up the IgG of interest. In one example, the constant region (e.g., an IgG4 constant region) contains a "lock or lock" (or "knob or hole") mutation to prevent heterodimer formation. In one example, the constant region (e.g., an IgG4 constant region) contains a T366W mutation (or knob). In another example, the constant region (e.g., an IgG4 constant region) contains T366S, L368A, and Y407V mutations (or holes). In another example, the constant region contains T350V, T366L, K392L, and T394W mutations (knobs). In another example, the constant region contains T350V, L351Y, F405A, and Y407V mutations (holes). Exemplary amino acid substitutions in constant regions are numbered according to the EU numbering system.

[0198] Other antibodies and proteins containing their antigen-binding domains The present disclosure also provides (i) Minibodies, e.g., as described in US Pat. No. 5,837,821; (ii) heteroconjugate proteins, e.g., as described in US4676980; (iii) heteroconjugate proteins produced using chemical cross-linkers, for example, as described in US 4,676,980; and (iv) Fab3 (e.g., as described in EP19930302894) Other antibodies, such as, and proteins comprising the antigen-binding domain thereof, are also contemplated.

[0199] Immunoglobulins and immunoglobulin fragments An example of a CXCL10 binding protein of the disclosure is a protein that comprises a variable region of an immunoglobulin, such as a T cell receptor or a heavy chain immunoglobulin (e.g., IgNAR, camelid antibody).

[0200] Heavy chain immunoglobulins Heavy chain immunoglobulins are structurally different from many other forms of immunoglobulins (e.g., antibodies) in that they contain heavy chains but no light chains. Thus, these immunoglobulins are also called "heavy chain-only antibodies." Heavy chain immunoglobulins are found, for example, in camelids and cartilaginous fish (also called IgNARs).

[0201] The variable regions present in naturally occurring heavy chain immunoglobulins generally correspond to the heavy chain variable regions ("V") present in conventional four-chain antibodies. H domain) and the light chain variable region (V L domain), and in camelid Ig, the domain is called "V HH domain" and in IgNARs, V-NAR.

[0202] Heavy chain immunoglobulins do not require the presence of light chains to bind relevant antigens with high affinity and specificity, meaning that single domain binding fragments can be derived from heavy chain immunoglobulins, which are easy to express and generally stable and soluble.

[0203] General descriptions of camelid-derived heavy chain immunoglobulins, and their variable regions, and methods for producing and / or isolating and / or using them can be found, inter alia, in the following references: WO94 / 04678, WO97 / 49805, and WO97 / 49805.

[0204] A general description of cartilaginous fish-derived heavy chain immunoglobulins, and their variable regions, and how to produce and / or isolate and / or use them, can be found, inter alia, in WO2005118629.

[0205] V-like protein In one example, a CXCL10-binding protein of the present disclosure comprises a T cell receptor. T cell receptors have two V domains that combine into a structure similar to the Fv module of an antibody. Novotny et al., 1991, describe how the two V domains (called alpha and beta) of a T cell receptor can be fused and expressed as a single polypeptide chain, and further describe how surface residues can be modified to directly reduce hydrophobicity, similar to an antibody scFv. Other publications describing the production of single-chain T cell receptors or multimeric T cell receptors comprising two V alpha and V beta domains include WO1999045110 or WO2011107595.

[0206] Other non-antibody proteins containing antigen-binding domains include proteins with V-like domains, which are generally monomeric. Examples of such V-like domain-containing proteins include CTLA-4, CD28, and ICOS. Further disclosure of such V-like domain-containing proteins is contained in WO1999045110.

[0207] Adnectins In one example, the CXCL10 binding protein of the disclosure comprises an Adnectin. Adnectins are a 10th fibronectin type III (F10) fragment of human fibronectin in which the loop region has been modified to confer antigen binding. 10 Fn3) domains, e.g. 10 Three loops at one end of the beta sandwich of the Fn3 domain can be engineered to enable Adnectins to specifically recognize antigens. For further details, see US20080139791 or WO2005056764.

[0208] Anticalin In further embodiments, the CXCL10-binding proteins of the present disclosure include anticalins. Anticalins are derived from lipocalins, a family of extracellular proteins that transport small hydrophobic molecules (e.g., steroids, bilins, retinoids, and lipids). Lipocalins have a rigid beta-sheet secondary structure with multiple loops at the open end of a conical structure, which can be modified to bind antigens. Such modified lipocalins are known as anticalins. For further description of anticalins, see US7250297 or US20070224633.

[0209] Affibody In further embodiments, the CXCL10-binding proteins of the present disclosure include affibodies. Affibodies are scaffolds derived from the Z domain (antigen-binding domain) of Staphylococcus aureus protein A, which can be engineered to bind to antigens. The Z domain consists of a three-helix bundle of approximately 58 amino acids. Libraries are generated by randomization of surface residues. For further details, see EP1641818.

[0210] Abima In a further embodiment, the CXCL10-binding protein of the present disclosure comprises an avimer. Avimers are multidomain proteins derived from the A-domain scaffold family. Native domains of approximately 35 amino acids conform to defined disulfide bond structures. Diversity is generated by shuffling the natural variations exhibited by the A-domain family. For further details, see WO2002088171.

[0211] DARPins In further examples, the CXCL10-binding proteins of the present disclosure comprise designed ankyrin repeat proteins (DARPins). DARPins are derived from ankyrins, a family of proteins that mediate the attachment of complex membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two α-helices and a β-turn. These can be modified to bind different target antigens by randomizing residues in the first α-helix and β-turn of each repeat. The binding interface can be increased by increasing the number of modules (affinity maturation method). For further details, see US20040132028.

[0212] Mutations to binding proteins The present disclosure also provides CXCL10-binding proteins or nucleic acids encoding CXCL10-binding proteins having at least 90% identity to a sequence disclosed herein. In one example, a CXCL10-binding protein or nucleic acid of the present disclosure comprises a sequence that is at least about 90%, or 95%, or 97%, or 98%, or 99% identical to a sequence disclosed herein, and the protein specifically binds to CXCL10 as described herein, according to any example.

[0213] Alternatively, or in addition, the CXCL10 binding protein may be a V-type CXCL10 binding protein as described herein, according to any of the examples. H or V L and the protein comprises CDRs (e.g., three CDRs) that are at least about 90%, or 95%, or 97%, or 98%, or 99% identical to the CDR(s) of, and the protein is capable of specifically binding to CXCL10 as described herein, according to any of the examples. Methods for measuring the binding of proteins to CXCL10 are described herein.

[0214] The five C-terminal residues of the heavy chain CDR2 were replaced with conservative or non-conservative amino acid substitutions (3 of the residues). It is known in the art that the heavy chain CDR2 sequences can be mutated to about 1% identity (Padlan et al., 1995). Thus, the protein can comprise a CDR2 having at least about 35% identity to the heavy chain CDR2 sequences disclosed herein.

[0215] The present disclosure also contemplates variants of the CXCL10 binding proteins of the present disclosure that contain one or more conservative amino acid substitutions compared to the sequences described herein. In some examples, the CXCL10 binding proteins contain 10 or fewer conservative amino acid substitutions, for example, 9, or 8, or 7, or 6, or 5, or 4, or 3, or 2, or 1. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain and / or hydrophobicity and / or hydrophilicity. Exemplary conservative amino acid substitutions are shown in Table 1. [Table 1]

[0216] Families of amino acid residues having similar side chains have been defined in the art and include 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). Hydropathic indexes are described, for example, in Kyte and Doolittle (1982), and hydrophilicity indices are described, for example, in US4554101.

[0217] The present disclosure also contemplates non-conservative amino acid changes. For example, of particular interest are substitutions of a charged amino acid with another charged amino acid and a neutral or positively charged amino acid. In some embodiments, the CXCL10 binding protein contains 10 or fewer non-conservative amino acid substitutions, e.g., 9, or 8, or 7, or 6, or 5, or 4, or 3, or 2, or 1.

[0218] In one example, the mutation(s) occur within a FR of the antigen-binding domain of a CXCL10 binding protein of the disclosure. In another example, the mutation(s) occur within a CDR of a CXCL10 binding protein of the disclosure.

[0219] Representative methods for generating mutant forms of CXCL10 binding proteins include: DNA (Thie et al., 2009) or RNA (Kopsidas et al. al., 2006; Kopsidas et al., 2007; and WO1999 / 058661); introducing a nucleic acid encoding the polypeptide into a mutator cell, such as XL-1Red, XL-mutS, and XL-mutS-Kanr bacterial cells (Stratagene); DNA shuffling, as disclosed, for example, in Stemmer, 1994; and Site-directed mutagenesis, as described, for example, in Dieffenbach et al., 1995.

[0220] Exemplary methods for measuring the biological activity of mutant CXCL10 binding proteins of the disclosure will be apparent to those of skill in the art and include, for example, antigen binding, competitive inhibition of binding, affinity, association, and dissociation.

[0221] In another example, a nucleic acid of the disclosure includes a sequence that is at least about 90%, or 95%, or 97%, or 98%, or 99% identical to a sequence described herein and encodes a CXCL10-binding protein having a function as described herein, according to any example. The disclosure also includes nucleic acids encoding CXCL10-binding proteins of the disclosure that differ from the sequences exemplified herein as a result of the degeneracy of the genetic code.

[0222] The percent identity of nucleic acids or polypeptides is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap opening penalty of 5 and a gap extension penalty of 0.3. The query sequence is at least 50 residues long, and the GAP analysis aligns the two sequences over a region of at least 50 residues. For example, the query sequence is at least 100 residues long, and the GAP analysis aligns the two sequences over a region of at least 100 residues. For example, the two sequences are aligned over their entire length.

[0223] constant region The present disclosure encompasses CXCL10 binding proteins and / or antibodies described herein that comprise the constant region of an antibody, including an antigen-binding fragment of an antibody fused to Fc.

[0224] Constant region sequences useful for producing the proteins of the present disclosure can be obtained from a number of different sources. In some examples, the constant region of the 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 derived from a human isotype. In one embodiment, the constant region is an IgGκ constant region.

[0225] In one example, the Fc region of the constant region has a reduced ability to induce effector function, for example, compared to a native or wild-type human IgG1 or IgG3 Fc region. In the context of the present disclosure, "effector function" refers to those biological activities mediated by cells or proteins that bind to the Fc region of an antibody (a native-sequence Fc region or an amino acid sequence variant Fc region) that result in cell killing. Examples of antibody-induced effector functions include complement-dependent cytotoxicity (CDC); antibody-dependent cellular cytotoxicity (ADCC); antibody-dependent cellular 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 an Fc region containing protein are known in the art and / or described herein.

[0226] In one example, the Fc region is an IgG4 Fc region (i.e., derived from an IgG4 constant region), e.g., a human IgG4 Fc region. The sequences of suitable IgG4 Fc regions will be apparent to those of skill in the art and / or are available from publicly available databases (e.g., available from the National Center for Biotechnology Information).

[0227] In one example, the constant region is a stabilized IgG4 constant region. The term "stabilized IgG4 constant region" will be understood to mean an IgG4 constant region that has been modified to reduce the tendency to undergo Fab arm exchange, or the formation of half antibodies, or to form half antibodies. "Fab arm exchange" refers to a type of protein modification to human IgG4 in which an IgG4 heavy chain and associated light chain (half molecule) are exchanged for a heavy / light chain pair from another IgG4 molecule. Thus, an IgG4 molecule can acquire two different Fab arms that recognize two different antigens (resulting in a bispecific molecule). Fab arm exchange occurs naturally in vivo and can be induced in vitro with purified blood or a reducing agent such as reduced glutathione. "Half antibodies" form when an IgG4 antibody dissociates to form two molecules, each containing a single heavy chain and a single light chain.

[0228] In one example, the stabilized IgG4 constant region comprises a proline at position 241 of the hinge region according to the Kabat system (Kabat et al., 1987 and / or 1991). This position corresponds to position 228 of the hinge region according to the EU numbering system (Kabat et al., 2001 and Edelman et al., 1969). In human IgG4, this residue is generally serine. After replacing the proline with serine, the IgG4 hinge region comprises the sequence CPPC. In this regard, those skilled in the art will recognize that the "hinge region" is the proline-rich part of the antibody heavy chain constant region that joins the Fc and Fab regions and confers mobility to the two Fab arms of the antibody. The hinge region contains the cysteine ​​residues involved in disulfide bonding between the heavy chains. According to the Kabat numbering system, the hinge region is generally defined as stretching from Glu226 to Pro243 of human IgG1. Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain disulfide S-S bonds in the same positions (see, for example, WO2010 / 080538).

[0229] Further examples of stabilized IgG4 antibodies are antibodies in which arginine at position 4 (according to the EU numbering system) of the heavy chain constant region of human IgG4 has been substituted with lysine, threonine, methionine, or leucine (e.g., those disclosed in WO2006 / 033386). The Fc region of the constant region may additionally or alternatively contain 405 (according to the EU numbering system) or 406 (according to the EU numbering system). Optionally, the hinge region comprises a residue selected from the group consisting of alanine, valine, glycine, isoleucine, and leucine at a position corresponding to (according to the sequence) 241. Optionally, the hinge region comprises a proline at position 241 (i.e., the CPPC sequence) (as described above).

[0230] In another embodiment, the Fc region is a region modified to reduce effector function, i.e., a "non-immunostimulatory Fc region." For example, the Fc region is an IgG1 Fc region comprising substitutions at one or more positions selected from the group consisting of 268, 309, 330, and 331. In another embodiment, the Fc region is an IgG1 Fc region comprising one or more of the following changes: E233P, L234V, L235A, and deletion of G236, and / or one or more of the following changes: A327G, A330S, and P331S (Armour et al., 1999; Shields et al., 2001). Further examples of non-immunostimulatory Fc regions are described, for example, in Dall'Acqua et al., 2006; and / or Hezareh, 2001.

[0231] In another embodiment, the Fc region comprises at least one C region, e.g., from an IgG4 antibody. H 2 domain and at least one C from an IgG1 antibody HA chimeric Fc region comprising three domains, wherein the Fc region comprises a substitution 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 numbering) (e.g., as described in WO2010 / 085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

[0232] Protein production The CXCL10 binding proteins described herein, according to any embodiment, are produced by culturing the cells of the invention under conditions sufficient to produce the protein, e.g., as described herein and / or known in the art.

[0233] Recombinant expression In another embodiment, the CXCL10 binding proteins described herein, according to any embodiment, are recombinant.

[0234] In the case of recombinant proteins, the nucleic acid encoding the recombinant protein can be cloned into an expression construct or vector, which is then transfected into host cells (e.g., E. coli cells, yeast cells, insect cells), or mammalian cells (e.g., monkey COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells), or myeloma cells that do not otherwise produce the protein. Exemplary cells used to express proteins are CHO cells, myeloma cells, or HEK cells. Molecular cloning techniques to achieve these ends are known in the art and are described, for example, in Ausubel et al., 1988 (including any updates to date) or Sambrook et al., 1989. A wide variety of cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art; see, for example, US4,816,567 or US5,530,101.

[0235] After isolation, the nucleic acid is inserted and operably linked to a promoter in an expression construct or expression vector for further cloning (amplification of the DNA) or for expression in a cell-free system or in a cell.

[0236] As used herein, the term "promoter" is to be interpreted in its broadest context. A "promoter" may comprise a transcriptional regulatory sequence of a genomic gene (including a TATA box or initiation element required for proper transcription initiation), with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers, and silencers) that modify expression of the nucleic acid, for example, in response to developmental and / or external stimuli or in a tissue-specific manner. In the context of the present invention, the term "promoter" is also used to describe a recombinant, synthetic, or fusion nucleic acid or derivative that confers, activates, or enhances expression of an operably linked nucleic acid. Exemplary promoters can include additional copies of one or more particular regulatory elements to further enhance expression of the nucleic acid and / or modify spatial and / or temporal expression.

[0237] As used herein, the term "operably linked to" means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.

[0238] Many vectors are available for intracellular expression. Generally, vector components include, but are not limited to, one or more of the following: a signal sequence (e.g., from the information provided herein), a protein-coding sequence, an enhancer element, a promoter, and a transcription termination sequence. Those skilled in the art will be aware of sequences suitable 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).

[0239] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate early promoter (CMV-IE), the human elongation factor 1-α promoter (EF1), the small nuclear RNA promoters (U1a and U1b), the α-myosin heavy chain promoter, the simian virus 40 promoter (SV40), the Rous sarcoma virus promoter (RSV), the adenovirus major late promoter, the β-actin promoter; hybrid control elements containing the CMV enhancer / β-actin promoter or the immunoglobulin promoter or active fragments thereof. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney lines (293 cells or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovarian (CHO) cells.

[0240] Exemplary promoters suitable for expression in yeast cells (e.g., yeast cells selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PHO5 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

[0241] Means for introducing isolated nucleic acids or expression constructs containing such nucleic acid molecules into cells are known to those of skill in the art. The technique used for a given cell will depend on the known techniques that have been used successfully. Means for introducing recombinant DNA into cells include microinjection, DEAE-dextran mediated transfection, liposome-mediated transfection using, for example, Lipofectamine (Gibco, MD, USA) and / or Cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation, and microparticle bombardment (e.g., DNA-coated tungsten or PEG-coated tungsten, among others). or fine particle bombardment using gold particles (Agracetus Inc., WI, USA).

[0242] Host cells used to produce proteins can be cultured in a variety of media depending on the cell type used. Commercially available media such as Ham's F10 (Sigma), Minimum 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 the other cell types discussed herein are known in the art.

[0243] Protein isolation Methods for isolating proteins are known in the art and / or described herein.

[0244] If the CXCL10-binding protein is secreted into the medium, the supernatant from such an expression system is first concentrated using a commercially available protein concentration filter (e.g., an Amicon or Millipore Pellicon ultrafiltration unit). A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants. Alternatively, or in addition, the supernatant can be filtered and / or isolated from the cells expressing the protein, for example, using sequential filtration.

[0245] The CXCL10-binding protein prepared from cells can be purified using, for example, 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 thereof. These methods are known in the art and are described, for example, in WO99 / 57134 or Harlow et al., 1988.

[0246] Those skilled in the art will also recognize that proteins can be modified to include tags to facilitate purification or detection, such as polyhistidine tags, e.g., hexahistidine tags, influenza virus hemagglutinin (HA) tags, simian virus type 5 (V5) tags, FLAG tags, or glutathione S-transferase (GST) tags. The resulting proteins are then purified using methods known in the art, such as affinity purification. For example, proteins containing hexa-His tags are purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA), which specifically binds to the hexa-His tag immobilized on a solid or semi-solid support, washing the sample to remove unbound proteins, and then eluting the bound proteins. Alternatively or additionally, a ligand or antibody that binds to the tag is used in affinity purification methods.

[0247] Conjugates In one example, a CXCL10 binding protein of the disclosure is conjugated to a detectable label.

[0248] As used herein, the term "conjugate" or "conjugated" should be understood to encompass both indirect and direct binding. For example, direct conjugation includes chemical conjugation, which can be non-covalent, covalent, or genetic conjugation (also called "fusion"). In one example, the conjugation is covalent, e.g., a disulfide bond.

[0249] As used herein, a "detectable label" is a molecular or atomic tag or marker that produces, or can be induced to produce, an optical or other signal or product that is detectable visually or by use of a suitable detector. Detectable labels are well known in the art and include, for example, radiolabels, enzymes, fluorescent labels, luminescent labels, bioluminescent labels, magnetic labels, prosthetic groups, contrast agents, and ultrasound agents.

[0250] Commonly used fluorescent labels include, but are not limited to, Alexa, cyanines (such as Cy5 and Cy5.5), indocyanines, and fluorescein isothiocyanate (FITC). Fluorescent labels useful in practicing the present disclosure include 1,5-IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-carboxynaphthofluorescein (pH10); 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 (both DNA and RNA); acridine red; acridine yellow; acriflavine; acriflavine feulgen SITSA; aequorin (photoprotein); Alexa Fluor 350; Alexa Fluor 430; Alexa Fluor 488; Alexa Fluor 532; Alexa Fluor 546; Alexa Fluor 568; Alexa Fluor 594; Alexa Fluor 633; ​​Alexa Fluor 647; Alexa Fluor 660; Alexa Fluor 680; Alizarin Complexone; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); Aniline Blue; Anthrosyl Stearate; APC (allophycocyanin); APC-Cy7; APTRA-BTC = Ratio Dye, Zn 2+;APTS;Astrazon Brilliant Red 4G;Astrazon Orange R;Astrazon Red 6B;Astrazon Yellow 7 GLL;Atabrine;ATTO-TAG CBQCA;ATTO-TAG FQ;Auramine;Aurophosphine G;Aurophosphine;BAO 9 (bisaminophenyloxazole);BCECF (high pH);BCECF (low pH);berberine sulfate;β-lactamase;BFP blue-shifted GFP (Y66H);blue fluorescent protein;BFP / GFP FRET bimane;bisbenzamide;bisbenzimide (Hoechst);bis-BTC = 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 Fl;Bodipy FL ATP;Bodipy Fl-Cer Amide;Bodipy R6G SE;Bodipy TMR;Bodipy TMR-X conjugate;Bodipy TMR-X, SE;Bodipy TR;Bodipy TR ATP;Bodipy TR-X SE;BO-PRO-1;BO-PRO-3;Brilliant Sulphoflavin FF;BTC-Ratio Dye Ca 2+ ;BTC-5N-atio Dye, Zn 2+ ;calcein;calcein Calcium Blue; Calcium Crimson; Calcium Green; Calcium Green 1 Ca 2+ Dye; Calcium Green-2 Ca 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; Catecholamines; CCF2 (GeneBlazer); CFDA; CFP--Cyan Fluorescent Protein; CFP / YFP; FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF (Ratio Dye, pH); CMFDA; Coelenterazine; Coelenterazine cp (Ca 2+Dye); Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hcp; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin phalloidin; C-psychocyanin; CPM I methylcoumarin; CTC; CTC formazan; Cy2; Cy3.1 8; Cy3.5; Cy3; Cy5.1 8; Cy5.5; Cy5; Cy7; Cyan GFP; cyclic AMP fluorosensor (FiCRhR); CyQuant cell proliferation assay; dabsyl; dansyl; dansylamine; dansylcadaverine; dansyl chloride; dansylDHPE; dansyl fluoride; DAPI; dapoxil; dapoxil 2; dapoxil 3; DCFDA; DCFH (dichlorodihydrofluorescein diacetate); DDAO; DHR (dihydrorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16-ASP); dichlorodihydrofluorescein diacetate (DCFH); DiD-lipophilic tracer; DiD (Di IC18(5)); DIDS; dihydrorhodamine 123 (DHR); DiI (DiIC18(3)); dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; red fluorescent protein; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF97; eosin; erythrosine; erythrosine ITC; ethidium bromide; ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; europium(III) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (formaldehyde-induced fluorescence); FITC; FITC antibody; Furazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein diacetate; Fluoro-Emerald; Fluoro-Gold (hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43; FM 4-46; Fura Red (high pH); Fura Red / Fluo-3; Fura-2, high calcium; Fura-2, low calcium; Fura-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); GFP (S65T); red-shifting GFP (rsGFP); GFP wild-type non-UV-excitable (wtGFP); GFP wild-type UV-excitable (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; Hematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-1, High Calcium; Indo-1, Low Calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; LIVE / DEAD Kit Animal Cells, Calcein / Ethidium Homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green;Lyso Tracker Red;Lyso Tracker Yellow;LysoSensor Blue, LysoSensor G green; LysoSensor Yellow / Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanine; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mithramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Proline Stilbene); NBD; NBD-amine; Nile Red; Nitrobenzoxazidol; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant Iavin E8G; Oregon Green; Oregon Green 488-X; Oregon Green; Oregon Green 488; Oregon Green 500; Oregon Green 514; Pacific Blue; Pararosanin (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-Texas Red [Red 613]; Phloxine B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; Photoresist; Phyllicoerythrin B [PE]; Phyllicoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primulin; Procion Yellow; Propidium iodide (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613 [PE-Texas Red]; Resorufin; RH 414; Rhodamine-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B200;Rhodamine B Extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine phallicidin; Rhodamine phalloidin; Rhodamine Red; Rhodamine WT; Rose Bengal; R-psychocyanin; R-phyllicoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP; sgBFP (Superglow BFP); sgGFP; sgGFP (Superglow GFP); SITS; SITS (Primuline); SITS (Stilbene isothiosulfonic acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; SpectrumAqua; Spectrum Green; Spectrum Orange; Spectrum Red; SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulforhodamine B can C; Sulforhodamine G Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYT; 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;thiolite;thiozole Orange;Tinopol CBS (Calcofluor White);TMR;TO-PRO-1;TO-PRO-3;TO-; Examples of fluorescent dyes include, but are not limited to, PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC (tetramethylrhodamine isothiocyanate); True Blue; TruRed; Ultralite; uranine B; Uvitex SFC; wt GFP; WW 781; X-rhodamine; XRITC; xylene orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; and YOYO-3.

[0251] In one example, the detectable label is an enzyme. Enzymes can act on appropriate substrates to produce a detectable dye. Examples of enzymes useful in the disclosure include, but are not limited to, alkaline phosphatase and horseradish peroxidase. Alternatively, or in addition, the enzyme can be, for example, luciferase. The enzyme can be bound to the antibody by conventional chemical methods or can be expressed together with the antibody as a fusion protein.

[0252] Radioisotopes useful as detectable labels in the present disclosure are well known in the art and include: 3 H, 11 C. 18 F, 35 S, 64 Cu, 67 Ga, 68 Ga, 99 mTc, 111 In, 123 I, 124 I, 125 I, and 131 Any gamma-emitting radioactive substance capable of reacting with a carboxyl, amino, or sulfhydryl group of a compound that binds to the calcitonin receptor, such as 99 mTc and 111 The In attachment is suitable for use in detection methods using gamma scintigraphy. 11 C.18 F, 64 Cu, 67 Ga, 68 Ga, 124 I, and 131 The I bond is suitable for use in detection methods using PET / SPECT imaging.

[0253] Assay for CXCL10-binding proteins Binding to CXCL10 and its modified forms It will be apparent to those skilled in the art from the disclosure herein that some CXCL10-binding proteins of the present disclosure bind to full-length CXCL10 and / or specific post-translationally modified forms of CXCL10 (e.g., N-terminally 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 generally involve immobilizing a CXCL10-binding protein and contacting the protein with a labeled antigen. After washing to remove nonspecifically bound proteins, the amount of label, and consequently, bound antigen, is detected. Of course, the CXCL10-binding protein can be detectable and the antigen can be immobilized. Panning-type assays can also be used. Alternatively, or in addition, surface plasmon resonance assays can be used.

[0254] Affinity measurement Optionally, the dissociation constant (Kd) or the association rate constant (Ka) or the equilibrium constant (K D ) These constants for a binding domain (e.g., an antibody or antigen-binding fragment) are measured, in one example, by biosensor analysis using a surface plasmon resonance assay. An exemplary SPR method is described in US7229619.

[0255] Affinity measurements can be measured by standard techniques for antibody reactions, such as immunoassays, surface plasmon resonance (SPR) (Rich and Myszka, 2000; Englebienne, 1998), isothermal titration calorimetry (ITC), or kinetic interaction assays known in the art.

[0256] Competitive binding assay An antibody or a compound that competitively inhibits the binding of a CXCL10-binding protein described herein. Assays for measuring such antigen-binding fragments will be apparent to those skilled in the art and / or are described herein.

[0257] For example, the antibody or antigen-binding fragment thereof is 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, a region thereof, or cells expressing CXCL10. The level of the labeled antibody is then measured and compared to the level measured when the labeled antibody contacts CXCL10, a region thereof, or cells in the absence of the CXCL10-binding protein. If the level of the labeled antibody is reduced in the presence of the test CXCL10-binding protein compared to the absence of the CXCL10-binding protein, the CXCL10-binding protein is considered to competitively inhibit the binding of the antibody to CXCL10.

[0258] Optionally, the test CXCL10 binding protein is conjugated to an antibody at different levels, and this surrogate labeling allows for detection of the level of binding of the test CXCL10 binding protein to CXCL10, or a region thereof, or a cell.

[0259] In another example, a CXCL10 binding protein is bound to CXCL10, a region thereof, or a cell expressing CXCL10, before contacting the CXCL10, region, or cell with the antibody. A decrease in the amount of bound antibody in the presence of the CXCL10 binding protein compared to its absence indicates that the protein competitively inhibits the binding of the antibody to CXCL10. Reciprocal assays can also be performed by first binding the antibody to CXCL10 using a labeled CXCL10 binding protein. In this case, a decrease in the amount of labeled CXCL10 binding protein bound to CXCL10 in the presence of the antibody compared to its absence indicates that the CXCL10 binding protein competitively inhibits the binding of the antibody to CXCL10.

[0260] Measurement of CXCL10 levels As mentioned above, CXCL10 is associated with a variety of human diseases, including infectious diseases, central nervous system diseases, chronic inflammation, immune dysfunction, and cancer.

[0261] The present inventors developed CXCL10 binding proteins to detect different forms of the protein, namely full-length or mature CXCL10, N-terminally truncated CXCL10, and / or citrullinated CXCL10.

[0262] The inventors have found that different forms of the protein are present at different levels in benign and malignant conditions.

[0263] Thus, any of the disclosed methods described herein include measuring the level of CXCL10 in a subject.

[0264] As used herein, the term "level" in reference to CXCL10 should be understood to refer to the level of functionality (i.e., functional level) of the protein. For example, level (or "degree of expression") refers to a measure of the encoded protein.

[0265] In particular, the inventors have discovered that measuring the levels of active and total CXCL10 protein in a subject can distinguish between benign and malignant conditions, a procedure referred to herein as the Activity Rate Test (ART).

[0266] As used herein, the term "active" in the context of levels of CXCL10 refers to a biologically active form of CXCL10. For example, "binding to active CXCL10" refers to binding to CXCL10. By "CXCL10 binding protein of the present disclosure" is meant a binding protein that binds to full-length or mature (i.e., N-terminally intact) CXCL10, but does not bind to N-terminally truncated or citrullinated CXLC10.

[0267] As used herein, "total" in the context of CXCL10 levels refers to all forms of CXCL10. For example, a "CXCL10 binding protein of the disclosure that binds to total CXCL10" refers to a binding protein that binds to full-length or mature (i.e., N-terminally intact) CXCL10, as well as N-terminally truncated and citrullinated CXCL10.

[0268] In one example, measuring the levels of active CXCL10 and total CXCL10 comprises measuring the amount of active CXCL10 protein and the amount of total CXCL10 protein in the subject.

[0269] As used herein, the term "amount" when referring to the level of CXCL10 will be understood to mean the amount of protein (i.e., either active or total CXCL10). Various methods for assessing protein amount are available to those skilled in the art, and those skilled in the art will recognize that the particular value or amount may vary depending on the assessment method used. It will also be clear that the term encompasses both absolute and relative values. For example, the amount may be relative to a reference or control sample. In another example, the amount may be an absolute value of the amount of protein present in a sample.

[0270] Surprisingly, the inventors have discovered that by measuring the CXCL10 ratio in a subject, it is possible to distinguish between benign and malignant conditions.

[0271] As used herein, the term "CXCL10 ratio" means the ratio of the level of active CXLC10 to the level of total CXCL10 in a subject.

[0272] In one example, the method further comprises comparing the CXCL10 ratio in the subject with the CXCL10 ratio in at least one reference.

[0273] In one embodiment of any of the methods described herein, the method includes determining (a) whether 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.

[0274] The term "higher" in reference to CXCL10 ratio means that the ratio in a subject is greater or increased compared to a control or reference level. From the foregoing, it will be apparent that the CXCL10 ratio need only be increased by a statistically significant amount, for example, by 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%.

[0275] The term "lower" in reference to CXCL10 ratio means that the ratio in a subject is lowered or decreased compared to a control or reference level. From the foregoing, it will be apparent that the CXCL10 ratio need only be decreased by a statistically significant amount, for example, by 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%.

[0276] How to measure CXCL10 levels Methods for measuring CXCL10 levels will be apparent to those skilled in the art and / or are described herein. For example, methods include immunohistochemistry, immunofluorescence, immunoblot, Western blot, dot blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay, fluorescence resonance energy transfer (FRET), matrix-assisted laser desorption / ionization (MALDI-TOF), electrospray ionization (ESI), mass spectrometry (including tandem mass spectrometry, e.g., LC MS / MS), biosensor technology, transient fiber optic technology, or protein chip technology. For example, suitable assays are semi-quantitative and / or quantitative assays.

[0277] In one example, a method for measuring the level of CXCL10 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 polypeptide or protein for a period of time and under conditions sufficient to form a complex between the binding protein and the polypeptide or protein, and detecting the complex. For example, in any of the methods described herein, the level of active CXCL10 is measured, the level of total CXCL10 is measured, or the ratio of active CXCL10 to total CXCL10 is measured.

[0278] Enzyme-linked immunosorbent assay (ELISA) and fluorescence-linked immunosorbent assay (FLISA) Standard solid-phase ELISA or FLISA formats are particularly useful for measuring protein concentrations from various samples. In one form, such assays involve immobilizing a biological sample on a solid matrix, such as a polystyrene or polycarbonate microwell or dipstick, a membrane, or a glass support (e.g., a glass slide).

[0279] An antibody that specifically binds to a marker in a CXCL10 polypeptide is directly contacted with a fixed biological sample and directly binds to any target protein present in the sample. The antibody is generally labeled with a detectable reporter molecule, such as a fluorescent label (e.g., FITC or Texas Red) or a fluorescent semiconductor nanocrystal (described in US Pat. No. 6,306,610) for FLISA, or an enzyme (e.g., horseradish peroxidase (HRP), alkaline phosphatase (AP), or β-galactosidase) for ELISA. Alternatively, a second labeled antibody that binds to the first antibody can be used. After washing to remove any unbound antibody, the label is detected directly in the case of a fluorescent label, or by adding a substrate such as hydrogen peroxide, TMB, toluidine, or 5-bromo-4-chloro-3-indole-β-D-galaotopyranoside (x-gel) in the case of an enzyme label.

[0280] Such ELISA or FLISA-based systems are suitable for quantifying the amount of protein in a sample by calibrating the detection system against a protein standard of known quantity to which an antibody binds, for example, to isolated and / or recombinant CXCL10 polypeptide, or an immunogenic fragment thereof, or an epitope thereof.

[0281] In another example, an ELISA involves immobilizing an antibody or ligand that specifically binds to a disease or disorder marker within a CXCL10 polypeptide on a solid matrix, such as a membrane, a polystyrene or polycarbonate microwell, a polystyrene or polycarbonate microwell dipstick, or a glass support. The sample is then physically associated with the antibody, which binds or "captures" the marker in the sample. Bound proteins are then detected using a labeled antibody. Alternatively, a third labeled antibody may be used that binds to the second (detection) antibody.

[0282] In one embodiment, the immobilized antibody is a polyclonal antibody.

[0283] It will be apparent to one of skill in the art that the assay formats described herein are amenable to high throughput formats, such as automation of the screening process or microarray formats, e.g., as described in Mendoza et al., 1999. Additionally, variations on the above-described assays, such as competitive ELISAs, will be apparent to one of skill in the art.

[0284] In one example, the assay format is a microfluidic device, e.g., 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 volumes of liquid to perform biological or medical processing or testing. Fluids may be moved, mixed, or processed in a single microfluidic chip.

[0285] In another embodiment, the assay format is a dipstick, for example a polycarbonate dipstick.

[0286] SIMOA assay Another assay that can be used in the present invention is the single molecule array (Simoa) assay, which is described in detail in Kuhle et al. (2016) and Gisslen et al. (2016).

[0287] Western blot In another example, Western blots are used to measure the level of a marker within a sample's CXCL10 polypeptide. In such assays, proteins are separated from the sample using techniques known in the art, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as described in Scopes, 1994, for example. The separated proteins are then transferred to a solid support, such as a membrane (e.g., a PVDF membrane), using methods known in the art, such as electron transfer. The membrane is then blocked and probed with a labeled antibody or ligand that specifically binds to a marker within the CXCL10 polypeptide. Alternatively, a labeled second or even third antibody or ligand is used to detect binding of the specific primary antibody. The level of label is then measured using an assay appropriate for the label used.

[0288] Suitable assays will be apparent to those skilled in the art and include, for example, densitometry. In one example, the intensity of a protein band or spot is normalized 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 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, hydroxy-methylbilane synthase, hypoxanthine phosphoribosyltransferase 1 (HPRT), ribosomal protein L13c, succinate dehydrogenase complex subunit A, and TATA box-binding protein (TBP).

[0289] Radioimmunoassay Alternatively, the level of CXCL10 is detected using radioimmunoassay (RIA). The basic principle of the 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 attached to a solid support, and the sample is brought into direct contact with the antibody. To detect the level of bound antigen, an isolated and / or recombinant form of the antigen is radiolabeled and contacted with the same antibody. After washing, the level of bound radioactivity is detected. Because any antigen in the biological sample will inhibit the binding of the radiolabeled antigen, the level of detected radioactivity is inversely proportional to the level of antigen in the sample. Such assays can be quantified using a calibration curve using increasing known concentrations of isolated antigen.

[0290] Such assays can be modified to substitute any reporter molecule, such as an enzyme or a fluorescent molecule, in place of the radiolabel.

[0291] Biosensor or Optical Immunosensor Systems Alternatively, the level of CXCL10 in a sample is measured using a biosensor or optical immunosensor system. Generally, optical biosensors are devices that use optical principles to quantitatively convert the binding of a ligand or antibody to a target polypeptide into an electrical signal. These systems can be classified into four major categories: reflectance technology; surface plasmon resonance; fiber optic technology; and integrated optical devices. Reflectance technologies include ellipsometry, multiple integrated reflectance spectroscopy, and fluorescent capillary-filled devices. Fiber optic technologies include evanescent field fluorescence, fiber optic capillaries, and fiber optic fluorescent sensors. Integrated optical devices include planar evanescent field fluorescence, input gradient coupler immunosensors, Mach-Zehnder interferometers, Hartmann interferometers, and differential interferometer sensors. Examples of these optical immunosensors are generally described in Robins, 1991. More specific descriptions of these devices can be found, for example, in US Pat. Nos. 4,810,658; 4,978,503; 5,186,897; and Brady et al., 1987.

[0292] biological samples As will be apparent to one of skill in the art, the type and size of the biological sample will vary depending on the detection method used, for example, protein-based assays require sufficient cells to provide sufficient protein like antigen-based assays.

[0293] As used herein, the term "sample" or "biological sample" refers to any type of suitable material obtained from a subject. This term encompasses clinical samples (e.g., cervical or cervicovaginal swabs), biological fluids (e.g., cervical fluid, vaginal fluid, plasma, ascites), tissue samples, and live cells, as well as cells in culture, cell supernatants, and cell lysates derived therefrom. Samples can be used directly from the source or after at least one (partial) purification step. 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 the present disclosure. Typically, samples contain cells or tissues and / or are aqueous solutions or biological fluids containing cells or tissues. Those skilled in the art will recognize selection and pretreatment methods. Pretreatment may involve, for example, dilution of viscous fluids. Sample processing may involve filtration, distillation, separation, and concentration.

[0294] In one example, the biological sample has previously been obtained from the subject. Thus, in one example, the methods described herein, according to any embodiment, further include providing a biological sample.

[0295] In one example, a biological sample can be collected from a subject at two or more time points, e.g., to monitor the progression of a malignant condition, to monitor recurrence, and / or to assess the effectiveness of a treatment protocol. In one example, a biological sample can be collected from a subject before, during, and / or after treating the subject for a malignant condition. Samples can be collected weekly, biweekly, monthly, bimonthly, trimonthly, quaternary, quintile, or quintile to monitor the progression of a malignant condition or assess the effectiveness of a treatment regimen.

[0296] In one example, the methods described herein, according to any embodiment, are performed using an extract from a sample, such as a protein.

[0297] Reference Sample As will become apparent from the foregoing, some assays of the present disclosure may use suitable reference samples or controls for quantification purposes.

[0298] Suitable reference samples for use in the methods of the present disclosure will be apparent to those skilled in the art and / or are described herein. For example, the reference may be an internal reference derived from a normal individual (i.e., derived from the same subject) or may be an established dataset (e.g., matched by age, sample type, and / or cycle stage).

[0299] In one embodiment, the reference is an internal reference or sample. For example, the reference is self-referenced. In one embodiment, the internal reference is obtained from the subject at the same time as the sample being analyzed. In another embodiment, the internal reference is obtained from the subject at an earlier time than the sample being analyzed.

[0300] As used herein, the term "normal individual" should be understood to mean that the subject is selected on the basis of not having, or not suspected of having, a malignant and / or benign condition. For example, a normal individual is a healthy individual.

[0301] In one embodiment, the reference is an established data set. Established data sets suitable for use in the present disclosure will be apparent to those skilled in the art, and may include, for example: · Age- or sample-type-matched normal subjects or datasets from a population of normal subjects; A dataset from another subject or population of subjects matched for age, sample type, and / or disease / condition; a dataset comprising cells in vitro, wherein the cells have been treated to induce CXCL10 expression; and An example would be a dataset containing endometrial epithelial cells in vitro, where the cells have been treated to inhibit CXCL10 expression.

[0302] In one embodiment, a reference is not included in the assay. Instead, a suitable reference is derived from a previously generated, established data set. Test samples are processed, analyzed, and assay-derived data is then compared to data obtained from the sample.

[0303] Detection and / or diagnosis of malignant conditions As disclosed herein, the inventors of the present disclosure have demonstrated a role for CXCL10 in the detection and / or diagnosis of malignant conditions. It will be apparent to one skilled in the art that the methods disclosed herein are useful for distinguishing malignant from benign conditions in a subject. For example, the methods of the present disclosure are useful as screening tests for diagnosing malignant conditions in a subject.

[0304] Thus, the present disclosure provides, for example, a method for detecting and / or diagnosing a malignant condition in a subject, the method comprising: (i) measuring the level of active CXCL10 in the subject and the level of total CXCL10 in the subject; (ii) measuring the CXCL10 ratio of active CXLC10 to total CXCL10 in the subject.

[0305] As used herein, the terms "detect," "detecting," "diagnosis," or "diagnosing" refer to the identification of a malignant condition in a subject.

[0306] As used herein, the term "malignant condition" refers to a condition or disease that grows in an uncontrolled manner, invades normal tissues, and often metastasizes, growing at sites distant from the tissue of origin. In one example, the malignant disease or condition is or is associated with cancer. Those skilled in the art will understand that cancer can occur in almost any tissue in the body, and that as used herein, the term encompasses all forms of the disease, including, for example, carcinoma, sarcoma, lymphoma, and leukemia (i.e., solid and non-solid cancers).

[0307] In one embodiment, the present disclosure provides a method for distinguishing malignant from benign conditions.

[0308] As used herein, the term "benign condition" refers to a mass of cells that lacks the ability to invade adjacent tissue.

[0309] In one embodiment, the present disclosure provides a method for distinguishing precancerous lesions from benign conditions.

[0310] The term "precancerous lesion" refers to a mass of cells that has grown abnormally and appears different in size, shape, or appearance from normal cells, but is not yet cancerous or malignant. In one example, the precancerous lesion is a p53 precancerous lesion. As used herein, the term "p53 precancerous lesion" refers to cells that have a p53 gene mutation.

[0311] In one embodiment, 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 carcinoma is a carcinoma of the prostate, ovary, breast, lung, liver, colon, pancreas, or stomach. For example, the subject has ovarian cancer. In one embodiment, the cancer is a non-solid tumor, e.g., a leukemia or lymphoma. In one embodiment, the subject has stage 0 cancer. For example, the carcinoma is in situ. In one embodiment, the subject has stage I, II, or III cancer. For example, the carcinoma has spread beyond the organ of origin to nearby lymph nodes and / or tissues, or to organs adjacent to the primary tumor site. In one embodiment, the subject has stage IV cancer. For example, the cancer has spread to distant tissues and / or organs.

[0312] In one example, the subject has not received treatment for the malignant condition, e.g., the subject is treatment naive.

[0313] In one embodiment, the subject is undergoing treatment for a malignant condition. In one embodiment, the subject has been treated for a malignant condition. Suitable treatments for treating a malignant condition will be apparent to one of skill in the art and / or are described herein. For example, the treatment may include surgery, chemotherapy, radiation therapy, targeted drug therapy, immunotherapy, or a combination thereof.

[0314] ovarian cancer In one embodiment of any of the methods described herein, the method includes detecting and / or administering to a subject ovarian cancer. For example, the subject has ovarian cancer.

[0315] As used herein, the term "ovarian cancer" means any cancerous growth that begins in the ovary.

[0316] In one embodiment, the method includes a method of distinguishing ovarian cancer from a benign condition in a subject.

[0317] It will be apparent to one skilled in the art that the methods described herein are applicable to detecting and / or diagnosing all subtypes of ovarian cancer, including, for example, epithelial, endometrioid, germ cell, clear cell, and mucinous adenocarcinomas.

[0318] In one example, the present disclosure provides a method for detecting and / or diagnosing epithelial ovarian cancer in a subject.

[0319] Those skilled in the art will appreciate that ovarian cancer is staged using the International Federation of Gynaecology and Obstetrics (FIGO) staging system, as set forth in Table 2 below.

[0320] In one embodiment of any of the methods described herein, the disclosure provides a method for detecting and / or diagnosing ovarian cancer in a subject, regardless of the stage of the cancer.

[0321] In one embodiment of any of the methods described herein, the disclosure provides a method of detecting and / or diagnosing stage I ovarian cancer in a subject.

[0322] Those skilled in the art will also appreciate that ovarian cancer is classified based on the grade of the cancer, for example, grade 1 tumors have well-differentiated cells, grade 2 tumors are moderately well-differentiated, and grade 3 tumors are poorly differentiated.

[0323] In one embodiment of any of the methods described herein, the disclosure provides a method for detecting and / or diagnosing ovarian cancer in a subject, regardless of the grade of the cancer.

[0324] In another embodiment, the ovarian cancer is serous, mucinous, endometrioid, clear cell, GCT, or a mixture thereof, hi one embodiment, the ovarian cancer is serous.

[0325] In one embodiment, the subject is at risk for ovarian cancer.

[0326] As used herein, a subject "at risk" of 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, measurable parameters known in the art and / or described herein, that correlate with progression of the disease or condition. For example, the subject has a p53 gene mutation. [Table 2]

[0327] Risk factors include: · Family history of ovarian and breast cancer; · Parity, i.e., pregnancy after age 35 or never having been pregnant, is associated with a higher risk; History of breast cancer Postmenopausal hormone therapy, such as hormone replacement therapy (HRT), is associated with an increased risk; and Obesity, e.g., a body mass index greater than 30.

[0328] A subject is at risk if the subject has a higher risk of developing ovarian cancer than a control population. The control population can include one or more subjects randomly selected from the entire population (e.g., matched by age, sex, race, and / or ethnicity) who do not have ovarian cancer or who have a family history of ovarian cancer. If a "risk factor" associated with ovarian cancer is found to be associated with the subject, the subject can be considered to be at risk. A risk factor can include, for example, any activity, trait, event, or property associated with a given disease through statistical or epidemiological studies in a population of subjects. Thus, a subject can be classified as at risk even if the study identifying the underlying risk factor did not specifically include the subject.

[0329] In one example, the methods of the present disclosure are performed before or after the onset of ovarian cancer symptoms, which will be apparent to one of skill in the art and include, for example: Abdominal bloating or swelling; Abdominal bloating and pain; Lower abdominal pain; Feeling full after eating only a small amount; · Fatigue; · Changes in bowel habits; · Clothing doesn't fit well; · Swelling of the legs; ·Difficulty breathing; · Vaginal bleeding; ·Irregular menstrual cycles; Weight loss or gain; and Unexplained back pain.

[0330] The inventors have also discovered that the methods of the present disclosure can be combined with the detection of other biological markers.

[0331] In one example, the methods of the present disclosure further comprise measuring dipeptidyl peptidase-4 (DPP4) and / or cancer antigen 125 (CA-125) in the subject. In one example, the method further comprises measuring the level of DPP4. In another example, the method further comprises measuring the level of CA-125. In a further example, the method further comprises measuring the levels of DPP4 and CA-125.

[0332] Methods for measuring DPP4 and / or CA-125 are known in the art (see, e.g., US 5,356,817, Saho et al., 2019; Scholler et al., 2007, and Vuento et al., 1997) and / or described herein.

[0333] In one example, the disclosed method further comprises measuring the levels of one or more, or all, of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 6 (IL-6), tumor necrosis factor receptor II (TNF-RII), human epididymis protein 4 (HE4), and interleukin 8 (IL-8).

[0334] In one embodiment, the method further comprises measuring the level of GM-CSF.

[0335] In one example, the method further comprises measuring the level of IL-6.

[0336] In one embodiment, the method further comprises measuring the level of TNF-RII.

[0337] In one example, the method further comprises measuring the level of HE4.

[0338] In one example, the method further comprises measuring the level of IL-8.

[0339] In one example, the method further comprises measuring levels of GM-CSF, IL-6, TNF-RII, HE4, and IL-8.

[0340] In one embodiment of any of the methods described herein, the method further comprises measuring levels of DPP4, GM-CSF, IL-6, TNF-RII, HE4, and IL-8.

[0341] In one embodiment of any of the methods described herein, the method further comprises measuring levels of CA-125, GM-CSF, IL-6, TNF-RII, HE4, and IL-8.

[0342] In one embodiment of any of the methods described herein, the method further comprises measuring levels of DPP4, CA-125, GM-CSF, IL-6, TNF-RII, HE4, and IL-8.

[0343] Methods for treating malignant conditions In one embodiment, the present invention provides a method of treating a malignant condition in a subject, the method comprising performing a method described herein and treating the malignant condition in the subject.

[0344] As used herein, the terms "treating," "treat," or "treatment" include surgically removing all or part of a cancer or administering a therapeutically effective amount of a compound / molecule / radiation sufficient to reduce or eliminate at least one symptom of a malignant condition. For example, an "effective amount" for therapeutic use is the amount of compound necessary to produce a clinically significant reduction in disease symptoms without undue side effects. An appropriate "effective amount" in any individual case can be determined using techniques such as dose escalation studies. An "effective amount" of a compound is an amount effective to achieve a desired pharmacological effect or therapeutic improvement without undue side effects. It is understood that an "effective amount" or "therapeutically effective amount" may vary from subject to subject, depending on variations in the metabolism of the compound due to the subject's age, weight, or general condition, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.

[0345] In one embodiment, the treatment comprises surgery, chemotherapy, radiation therapy, targeted drug therapy, or a combination thereof.

[0346] In one embodiment, the treatment comprises surgery, for example, the surgery is weight loss surgery.

[0347] In another embodiment, the treatment comprises chemotherapy. Exemplary chemotherapeutic agents include, for example, caboplatin, cytarabine, chlorambucil, cisplatin, cyclophosphamide, danorubicin, docetaxel, doxorubicin, erlotinib, etoposide, fluorouracil, fludarabine, idarubicin, irinotecan, liposomal doxorubicin, methotrexate, mitoxantrone, paclitaxel, topotecan, vincristine, and vinblastine.

[0348] In one embodiment, the treatment comprises radiation therapy, for example, the 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), intensity-modulated arc therapy (VMAT), conformal proton beam radiation therapy, stereotactic radiosurgery (SRS) / stereotactic radiotherapy (SRT), image-guided radiation therapy (IGRT), brachytherapy (internal radiation therapy), and whole brain and spinal radiation therapy (craniospinal irradiation).

[0349] For example, the targeted drug therapy is a therapeutic antibody. For example, the targeted drug therapy is a therapeutic antibody. Exemplary therapeutic antibodies are known to those skilled in the art and include abagovomab; abciximab; abituzumab; abrilumab; actoxumab; adalimumab; adecatumumab; aducanumab; afelimomab; afutuzumab; alacizumab pegol; alemtuzumab; alirocumab; altumomab pentetate; amatuximab; anatumomab mafenatox; anetumab ravtansine; anifrolumab; anrukinzumab; apolizumab; arcitumomab; asclinbatumab; acelizumab; atezolizumab; atinumab; atlizumab ( Tocilizumab; Atrolimumab; Bapineuzumab; Basiliximab; Bavituximab; Bectumomab; Begelomab; Belimumab; Benralizumab; Bertilimumab; Besilesomab; Bevacizumab; Bezlotoxumab; Biciromab; Bimagrumab; Bimekizumab; Bivatuzumab mertansine; Blinatumomab; Brosozumab; Bococizumab; Brentuximab vedotin; Briakinumab; Brodalumab; Brolucizumab; Brontizumab; Canakinumab; Cantuzumab mertansine; Cantuzumab lavtansine; Caplacizumab AB; capromab pendetide; carlumab; catumaxomab; cBR96-doxorubicin immunoconjugate; cedelizumab; certolizumab pegol; cetuximab; sitatuzumab bogatox; cixutumumab; clazakizumab; crenoliximab; clivatuzumab tetraxetan; codrituzumab; cortuximab ravtansine; conatumumab; concizumab; crenezumab; dacetuzumab; daclizumab; dalotuzumab; dapirolizumab pegol; daratumumab; dectrecumab; demcizumab; denintuzumab mafodoti N; denosumab; dellotuximab biotin; detumomab; dinutuximab; dilidabumab; dorimomab alitox; drozitumab; durigotumab; dupilumab; durvalumab; dusigitumab; ecromeximab; eculizumab; edovacomab; edrecolomab; efalizumab; efalizumab; eldelumab; ergemtumab; elotuzumab; elsilimomab; emactuzumab; emibetuzumab; enabatuzumab; enfortumab vedotin; enlimomab pegol; enoblitzumab; enokizumab; enoticumab;Ensituximab; epitumomab; cituxetan; epratuzumab; elizumab; ertumaxomab; etanercept; etaracizumab; etrolizumab; evinacumab; evolocumab; exbivirumab; fanolesomab; faralimomab; farletuzumab; fasinumab; felvizumab; fezakinumab; ficlatuzumab; figitumumab; filibumab; framvotumab; fretikumab; f Fontolizumab; Foralumab; Foravirumab; Fresolimumab; Furanumab; Futuximab; Galiximab; Ganitumab; Gantenerumab; Gavilimomab; Gemtuzumab ozogamicin; Gevokizumab; Dilentuximab; Glembatumumab vedotin; Golimumab; Gomiliximab; Guselkumab; Ibalizumab; Ibritumomab tiuxetan; Icrucumab; Idarucizumab; Igovomab; Imalumab Imalumab; Imgatuzumab; Inclacumab; Indatuximab ravtansine; Indusatumab vedotin; Infliximab; Inolimomab; Inotuzumab ozogamicin; Intetumumab; Ipilimumab; Iratumumab; Isatuximab; Itolizumab; Ixekizumab; Keliximab; Labetuzumab; Lambrolizumab; Lampalizumab; Lebrikizumab; Remaresomab; Lenzilumab; Le Ludelimumab;lexatumumab;ribivirumab;rifastuzumab vedotin;ligelizumab;rilotomab satetraxetan;lintuzumab;lirilumab;ridelucizumab;rokivetmab;lorvotuzumab mertansine;lucatumumab;lurizumab pegol;rumiliximab;lumuletuzumab;mapatumumab;margetuximab;maslimumab;matuzumab;mavrilimumab;mepolizumab;metelimumab; Milatuzumab; minletumomab; mirvetuximab soravtansine; mitumomab; mogamulizumab; morolimu- mab; motavizumab; moxetumomab pasudotox; muromonab-CD3; nacolomab butafenatox; namilumab; naptumomab estafenatox; narunatumab; natalizumab; nebacumab; necitumumab; nemolizumab; nerelimomab; nesbacumab; nimotuzumab; nivolumab; nofetumomab merpentan; obilutoxaximab; obinutuzumab; occaratuzumab; ocrelizumab; odulimomab; ofatumumab; olara tuzumab; olokizumab; omalizumab; onartuzumab; ontuxizumab; opicinumab; oportuzumab monatox; oregovomab; olticumab; otelixizumab; otreltuzumab; oxilumab; ozanezumab; ozoralizumab; pagibaximab; palivizumab; panitumumab; pancomab; panobacumab; palsatuzumab; pascolizumab; pasotuxizumab; pateclizumab; patritumab; pembrolizumab; pemtumomab; perakizumab; pertuzumab; pexelizumab; pidilizumab; pinatuzumab vedotin; pintumomab; Pratulumab; Polatuzumab vedotin; Ponezumab; Priliximab; Pritoxaximab; Pritumumab; Kirizumab; Racotumomab; Ladretumab; Rafivirumab; Ralpancizumab; Ramucirumab; Ranibizumab; Raxibacumab; Refanezumab; Regavirumab; Reslizumab; Rilotumumab; Linukumab; Rituximab; Lobatumumab; Lorezumab; Romosozumab; Rontalizumab; Rovelizumab; Ruplizumab; Sacituzumab govitecan; Samalizumab; Sarilumab; Satumomab pendetide; Secukinumab; Seribantumab; Cetoxaximab Mabs; Sevirumab; Sibrotuzumab; Sifalimumab; Siltuximab; Simtuzumab; Siplizumab; Sirukumab; Sofituzumab vedotin; Solanezumab; Solitomab; Sonepcizumab; Sontuzumab; Stamulumab; Sulesomab; Suvisumab; Tabalumab; Tacatuzumab tetraxetan; Tadocizumab; Talizumab; Tanezumab; Taplitumomab paptox; Talectumab; Tefibazumab; Terimomab alitox; Tenatumomab; Teneliximab; Teplizumab; Teprotumumab; Tesidolumab; Tetulomab; Ticilimumab; Tigatuzumab;Tildrakizumab; Tocilizumab; Tralizumab; Tosatoxumab; Tositumomab; Tobetumab; Tralokinumab; Trastuzumab; Tregalizumab; Tremelimumab; Trevoglumab; Tucotuzumab-celmoleukin; Tuvilumab; Ublituximab; Urotuplumab; Urelumab; Urtoxazumab; Ustekinumab; Vandrutuzumab vedotin; These include, but are not limited to, vanticutumab; vanucizumab; bapaliximab; varlilumab; batelizumab; vedolizumab; veltuzumab; bepalimomab; besentumab; visilizumab; volociximab; borsetuzumab mafodotin; votumumab; zalutumumab; zanolimumab; zatuximab; zoralimumab; zolimomab alitoxin;

[0350] In one embodiment, the treatment comprises immunotherapy, for example, the immunotherapy is selected from the group consisting of checkpoint inhibitors, oncolytic virus therapy, T cell therapy, and cancer vaccines.

[0351] In one embodiment, the immunotherapy is a checkpoint inhibitor. Suitable checkpoint inhibitors include, for example, ipilimumab (Yervoy®), nivolumab (Opdivo®), pembrolizumab (Keytruda®), atezolizumab (Tecentriq®), avelumab (Bavencio®), and durvalumab (Imfinzi®).

[0352] In one example, the immunotherapy is an oncolytic virotherapy. For example, the oncolytic virotherapy is talimogene laherparepvec (Imlygic®), or T-VEC.

[0353] In one embodiment, the immunotherapy is a T cell therapy, for example, the T cell therapy is a CAR T cell therapy.

[0354] In one embodiment, the immunotherapy is a cancer vaccine.

[0355] Monitoring tumor burden, progression, recurrence, and regression It will be apparent to those skilled in the art that the present disclosure also provides a method for monitoring tumor burden, monitoring progression, monitoring recurrence, and / or measuring tumor regression in a subject suffering from a malignant condition, the method comprising: (i) measuring the level of active CXCL10 in the subject and the level of total CXCL10 in the subject; and (ii) measuring the CXCL10 ratio of active CXCL10 to total CXCL10 in the subject.

[0356] As used herein, the term "monitoring" can include prognosis, response to drug therapy, evaluation of ongoing drug therapy, predicting outcome, measuring response to therapy (including diagnosing complications), measuring subsequent progression of tumor volume, or selecting patients most likely to benefit from therapy.

[0357] As used herein, the term "tumor burden" means the volume of tumor cells and does not include other changes such as inflammation, necrosis, or edema.

[0358] As used herein, the term "progression" refers to the continued growth and invasiveness of a tumor.

[0359] As used herein, the term "regression" means a decrease in size or volume of a tumor.

[0360] As used herein, the term "recurrence" refers to a malignant condition that has recurred or returned after a period of time during which the malignant condition was undetectable.

[0361] In one example, a method of monitoring tumor burden, progression, recurrence, and / or tumor regression in a subject suffering from a malignant condition comprises measuring CXCL10 ratio in the subject at one or more time points, e.g., miRNA expression is measured at 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 time points.

[0362] In one embodiment, 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 embodiment, the CXCL10 ratio is measured at one or more time points during treatment. In a further embodiment, the CXCL10 ratio is measured at one or more time points after treatment. In another embodiment, the CXCL10 ratio is measured before and during treatment. In a further embodiment, the CXCL10 ratio is measured before and after treatment. In another embodiment, the CXCL10 ratio is measured during and after treatment. In a further embodiment, the CXCL10 ratio is measured before, during, and after treatment.

[0363] In one example, the method comprises comparing the CXCL10 ratio in the subject at a first time point to the CXCL10 ratio in the subject at a later time point.

[0364] It will be apparent to those skilled in the art from the present disclosure that references to first and subsequent time points are not intended to refer to defined or specific time points, but are for comparison purposes only. The first, second (and any subsequent) time points are separated by any time point at which it is desired to monitor the malignant status of the subject. The monitoring methods of the present disclosure may include collecting additional samples at additional time points (e.g., a third time point in addition to the first and second samples) and / or a third time point. 3 samples) may be used.

[0365] As will be apparent to one of skill in the art, the ability to monitor CXCL10 ratios of the present disclosure over the course of disease will aid in monitoring tumor burden and disease progression.

[0366] It will be apparent to one skilled in the art that methods for monitoring tumor burden and disease progression in a subject are useful for monitoring tumor regression and / or recurrence in a subject.

[0367] Panels and Kits The present disclosure provides panels or kits for detecting and / or diagnosing a malignant condition in a subject. The present disclosure also provides panels or kits for monitoring tumor burden, tumor progression, and / or tumor regression. The panels or kits of the present invention preferably include one or more, or both, of the CXCL10 binding proteins described herein. Optionally, the panels or kits include instructions for use in the methods described herein.

[0368] In one embodiment, the panel or kit includes a reference sample.

[0369] In one example, the panels or kits described herein are for ex vivo analysis, hi one example, the kits are suitable for use with whole blood, plasma, cervicovaginal (CVS) swabs, and / or serum samples.

[0370] In one embodiment, the panels or kits described herein are suitable for high-throughput screening. The term "high-throughput screening" refers to a screening method that can be used to test or evaluate two or more samples at a time, reducing the time required to test multiple samples. In one embodiment, 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 a time. Such high-throughput screening methods can analyze two or more samples quickly, 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 can also involve the use of liquid handling devices. In one embodiment, high-throughput analysis can be automated.

[0371] The present disclosure includes the following non-limiting examples. [Example]

[0372] Example 1: Materials and Methods reagent Nunc-Immuno Microwell 96-well solid plates were purchased from SigmaAldrich (USA). TMB chromogen solution was purchased from ThermoFisher (USA). Recombinant human CXCL10 protein (used for immunization and assay standard) was purchased from Genscript (Hong Kong) (SEQ ID NO: 2). Short peptides containing either the active or truncated N-terminus of CXCL10 were synthesized by Mimotopes (Australia). 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 peptidase IV ELISA kit were purchased from Genscript (Hong Kong). Potassium arginine deiminase (PAD) cocktail was purchased from Abcam (UK). All other reagents were of analytical grade.

[0373] Clinical samples Clinical samples were obtained from archival samples stored at the Ovarian Cancer Research Foundation Tissue Bank and prospectively collected from women undergoing surgery for suspected gynecological malignancies between 2007 and 2014. All samples were obtained from patients who had not previously undergone surgical treatment, had received anesthesia, or received chemotherapy.

[0374] Tumor type, stage, and grade, preoperative CA125 measurements, age, menopausal status, pre-existing medical conditions, and histological evaluation of any previous history of malignancy were obtained from anonymized patient medical records. Serum CA125 measurements on samples were performed at the diagnostic pathology laboratory at Monash Medical Centre, Melbourne, Australia. Ethical approval was obtained from the Southern Health Human Research Ethics Committee (HREC approval numbers #06032C, #02031B), and all participants gave prior signed informed consent.

[0375] Patient samples were divided into two groups according to the disease state; benign and malignant.

[0376] Median CA125 measurements with interquartile ranges (IQR) for variability will be determined for each group.

[0377] Both the benign and malignant groups involved pre- and postmenopausal women (ie, mixed).

[0378] The sample types tested in this study were ascites, plasma, and cervicovaginal swabs (CVS) and are detailed in Table 3 . [Table 3]

[0379] Generation of monoclonal antibodies against human CXCL10 Monoclonal antibodies against full-length recombinant CXCL10 protein (SEQ ID NO: 2) were generated at the Monash Antibody Technologies Facility. Short peptides containing the intact (NH2-VPLSRTVRCTCISISNQPVNPRSLE-COOH) (SEQ ID NO: 25) or truncated (NH2-LSRTVRCTCISISNQPVNPRSLE-COOH) (SEQ ID NO: 26) N-terminus of human CXCL10 were used for screening.

[0380] Mice were inoculated intraperitoneally with 16 μg of adjuvanted Sigma adjuvant system® (S6322) full-length CXCL10 at three doses every other week, co-injected with methylated CpG. Serum titers were tested by ELISA and compared with naive serum collected before immunization. Mice exhibiting the highest titers were selected for hybridoma generation. Splenocytes were extracted and fused to SP2 / 0-Ag14 myeloma cells using polyethylene glycol. The resulting hybridoma cells were grown in azaserine-hypoxanthine-containing medium in 96-well tissue culture plates for 13 days. Each individual hybridoma Sera against were screened by microarray for reactivity to both the full-length protein and each peptide antigen, and positive clones were rescreened by ELISA.

[0381] The most highly responsive clones exhibiting the appropriate antigen specificity were expanded and subcloned to reliably derive monoclonal hybridoma lines. Monoclonal antibodies were purified from the supernatant using protein G Sepharose, and Ig isotypes were measured using commercially available assay kits. Final monoclonal hybridoma lines of interest were grown to 80% confluence, frozen using 10% DMSO as cryoprotectant, and stored in liquid N2.

[0382] Surface Plasmon Resonance Imaging The binding affinities of two monoclonal antibodies, mAb-RA2 specific for full-length, N-terminally intact CXCL10, and mAb-RG2 specific for both full-length and truncated CXCL10, to both forms of CXCL10 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 lanes were activated using equal amounts of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDAC) and N-hydroxysuccinimide (NHS). Antibodies were immobilized in separate lanes at a concentration of 50 μg / mL in sodium acetate buffer (pH 4.5), and each lane was deactivated using ethanolamine. Antigen was applied to each lane, and binding was recorded as RU. All channels were regenerated with 0.85% H3PO3 between antigen applications. Interspot and control RU were subtracted to obtain specific binding.

[0383] Biotinylation of RA2 and RG2 monoclonal antibodies The mAb-RA2 and mAb-RG2 antibodies were biotinylated using a biotinylation kit (Abcam; ab201795) and used as detection antibodies in the present invention's activity ratio test (ART). In this experiment, streptavidin-peroxidase was used as the detection reagent. 10 μL of the biotin-modifying reagent was added to 100 μL of each purified monoclonal antibody (2 mg mL; PBS, pH 7.4) and then gently mixed. The mixture was added directly to the lyophilized biotin and gently mixed. The mixture was then incubated for 15 minutes at room temperature. After incubation, 10 μL of biotin quencher reagent was added to terminate the biotinylation of the two antibodies.

[0384] Quantification of active and total CXCL10 Ascites fluid and plasma samples were precleared by centrifugation (18,400 x g, 4°C for 20 minutes), and the cleared supernatant was transferred to a clean tube. Samples were diluted (1:5) with assay buffer (0.1% BSA / 0.05% Tween 20 / PBS, pH 7.4) and kept on ice before assay. CVS samples were vortexed for 30 seconds, sonicated in an ice bath for 15 minutes, and then vortexed for an additional 30 seconds. Supernatants were centrifuged as above and kept on ice until assay.

[0385] For evaluation by ART, coating buffer (15 mM NaCl and 35 mM Anti-human IP-10 polyclonal antibody (Abcam; #ab9807) prepared in NaHCO3, pH 9.6 was immobilized on a 96-well microplate (100 μL per well) at a coating concentration of 0.5 μg mL-1 for 2 hours at room temperature. The plate was washed once with 280 μL of washing solution (0.05% Tween 20 / Milli-Q H2O) and then incubated with 300 μL of blocking solution (5% BSA / 0.05% Tween 20 / PBS, pH 7.4) for 2 hours at room temperature. After washing the plate four times with washing solution, 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 each well (100 μL per well). After a 2-hour incubation, each well was washed four times with washing buffer. Biotinylated detection antibodies, mAb-RA2 and mAb-RG2 (1 μg mL-1 in assay buffer), were added appropriately (100 μL per well) and incubated for 1 hour at room temperature. After incubation, the plate was washed five times with washing buffer and then incubated with diluted (1:1000) streptavidin peroxidase (1 μg / mL / well) for 45 minutes at room temperature. After an additional four washes with washing buffer, 100 μL of TMB chromogen solution was added to each well and incubated for 20 minutes in the dark at room temperature. The reaction was stopped by adding stop solution (1 M HCl, 50 μL per well). Absorbance was measured at 450 nm using a Cytation 3 multimode plate reader (Biotek, La Jolla, CA) equipped with Gen5 v3.08 analysis software. Comparison of ART with standard ELISA (detecting only total CXCL10) was performed using a colorimetric anti-human IP-10 ELISA kit (Abcam #ab100579) according to the manufacturer's instructions.

[0386] Quantification of DPP4 and analysis of DPP4-specific activity The amount of DPP4 in matched ascites samples (diluted 1:4) was assessed using a commercial anti-human DPP4 ELISA (Abcam #ab222872) according to the manufacturer's instructions. Detection and concentration analysis were performed using a Cytation multimode plate reader (described above). A nonlinear, asymmetric sigmoidal, logarithmic regression curve was constructed against DPP4 concentration to quantify soluble DPP4 in the samples.

[0387] DPP4 specific activity (μmol / min / ng DPP4) was measured using Gly-Pro-7-amido-4-methylcoumarin hydrobromide (H-Gly-Pro-AMC) as a DPP4 substrate as previously described (Sinnathurai et al., 2018). Citrullination of full-length CXCL10 was performed by incubation with human protein-arginine deiminase 2 (PAD2) at 37°C for up to 1 hour. For time course measurements, aliquots were taken every 15 minutes during incubation. Time course samples were then separated by SDS-PAGE and probed by Western blotting with mAb-RA2, mAb-RG2, a commercial anti-CXCL10 antibody, and an anti-citrulline antibody, as previously described (Loos et al., 2008).

[0388] statistical analysis Statistical analysis was performed using GraphPad Prism (GraphPad Software, La Jolla, CA), and all assay data were log-transformed to the appropriate normality. For the mAb-RA2 and mAb-RG2 assays, a best-fit line was determined by asymmetric sigmoidal nonlinear logarithmic regression curve fitting against total CXCL10 concentration to quantify active and active or cleaved CXCL10, respectively. CXCL10 concentrations obtained from the assays are in pg mL-1. Significance was determined using one-way ANOVA with Bonferroni post hoc test, and pairwise comparisons were performed using Student's t-test. For groups with significantly different variances, Welch's correction was applied. Spearman's rank test was used for the correction analysis. Results with P<0.05 were considered significant.

[0389] Example 2: Development of a functional assay that distinguishes activity from total CXCL10 We developed a monoclonal antibody that can distinguish the N-terminally intact, chemotactically active form of CXCL10 from all other variants. Using the full-length CXCL10 protein as an antigen, we isolated hybridoma clones and identified CXCL10. We screened for reactivity against (i) full-length CXCL10 protein and (ii) short synthetic peptides representing either the intact or truncated N-terminus of CXCL10. The antibody secreted by clone mAb-RA2 recognized both full-length and N-terminally intact CXCL10, but not the N-terminally truncated forms (Table 4). The antibody secreted by clone mAb-RG2 reacted with all proteoforms of CXCL10 tested. SPR analysis revealed binding affinities in the nM-pM range in each case. The open reading frames encoding the variable regions of both antibodies were sequenced.

[0390] A sandwich ELISA was constructed using biotinylated mAb-RA2 and mAb-RG2 as detection antibodies to independently measure intact or total CXCL10. Specific parameters, including limit of detection (LOD), limit of quantification (LOQ), linearity, inter- and intra-assay precision, and dynamic range, were evaluated (Table 4). mAb-RA2 and mAb-RG2 each showed good affinity for full-length CXCL10, with an R of 0.996 and a dynamic range of 5 orders of magnitude (OOM) (Figure 1). The limits of detection for mAb-RA2 and mAb-RG2 were 95.9 pg mL-1 and 116.3 pg mL-1, respectively (Table 4). Inter-assay precision was assessed using 10 independently prepared replicates as a single dose, and intra-assay variation was measured among 10 separate assays performed on different days. The coefficients of variation (CV) for both intra- and inter-assay were below 10% in each case, indicating good reproducibility and precision of the assay (Table 4). [Table 4]

[0391] We next sought to confirm the quantitative detection of CXCL10 using the ART compared with a commercially available ELISA kit. CXCL10 is abundant in ascites fluid from ovarian cancer patients, where it is involved in CXCR3-mediated migration of cancer cells and specific T cell subsets (Rainczuk et al., 2012; Windmuller et al., 2017). Therefore, we evaluated CXCL10 detection in ascites fluid from ovarian cancer patients (n = 212) as a representative complex biological matrix. While there was considerable variation in the concentration range among the sample groups (CVs ranging from 88.4% to 225.1%), there was no significant difference in the quantification of total CXCL10 between the ART and the commercial ELISA (Figure 2a). A positive correlation was observed for quantified total CXCL10 between ART and commercial ELISA for both benign and malignant ascites fluids, with r values ​​of 0.3084 and 0.2594, respectively (Fig. 2b).

[0392] Example 3: mAb-RA2 and mAb-RG2 distinguish functional from non-functional CXCL10 In addition to proteolytic N-terminal cleavage of CXCL10, other post-translational modifications may affect CXCL10 activity (Mortier et al., 2011). Specifically, arginine to citrulline deamination (R5Cit) in the N-terminal region of CXCL10 is a well-established functional modification that occurs in vivo and therefore must be considered in any assay evaluating CXCL10 function (Mortier et al., 2011). Therefore, we investigated whether the R5Cit modification could affect the detection of full-length CXCL10 by either mAb-RA2 or mAb-RG2.

[0393] Recombinant, full-length CXCL10 (SEQ ID NO: 2) was incubated with protein-arginine deiminase 2 (PAD2) to induce deamination at R5, and proteins were separated by SDS-PAGE and analyzed by Western blot. In vitro citrullination had no effect on CXCL10 detection (detection of total CXCL10) using mAb-RG2, suggesting that mAb-RG2 bound to CXCL10 regardless of post-translational modifications (Fig. 3a). In contrast, mAb-RA2 showed a substantial decrease in detection of R5Cit-CXCL10 (Fig. 3a), consistent with epitope modification following the N-terminal R5Cit modification.

[0394] Similar results were obtained when R5Cit-CXCL10 was assessed by ELISA. Citrullination substantially reduced the binding of mAb-RA2, whereas mAb-RG2 was still able to detect R5Cit-CXCL10 (Fig. 3b). Taken together, these data indicate that mAb-RA2 and mAb-RG2 can be used to effectively distinguish active CXCL10 from total CXCL10.

[0395] Example 4: Activity Ratio Test Distinguishes Benign from Malignant Ovarian Cancer Samples We measured active and total CXCL10 concentrations in ascites fluid collected from patients with either benign or malignant ovarian tumors (Figure 3). Total CXCL10 was assessed in malignant (853.1 ± 1574.0 pg mL-1) compared with benign (160.8 ± 362.0 pg mL-1) ascites fluid (Figure 4b). Similarly, a wide range of active CXCL10 measurements was also observed (240.4 ± 410.5 pg mL-1 and 818.6 ± 1098.0 pg mL-1, respectively) (Figure 4a). This wide variation within the population (CV% ranged between 134.1 and 225.1%) resulted in poor sensitivity for distinguishing benign from malignant samples (Table 5), highlighting the difficulties associated with direct CXCL10 measurement for diagnostic or prognostic purposes.

[0396] Because proportionally higher levels of active CXCL10 were observed in benign vs. malignant samples, the ratio of functional:total CXCL10 in each sample was investigated as a mechanism for normalizing CXCL10 function between patients (Fig. 4c). This "activity ratio" measure significantly improved separation between groups, resulting in the differentiation of benign and malignant samples at cutoff values ​​of <1.43 and <1.25 at 90% and 95% specificity, respectively (Table 5). This relationship also held true when malignant samples were separated according to disease stage (FIGO stage I vs. stage III, Fig. 4d). Plasma CA125 was significantly elevated in patients with malignant vs. benign disease (Fig. 5c), but there was no clear correlation between activity ratios measured in ascites fluid and plasma CA125 levels (Fig. 6a). Thus, activity ratios provide a marker of malignancy independent of plasma CA125 concentration.

[0397] Example 5: DPP4 amount and activity do not correlate with functional CXCL10 DPP4 catalyzes the removal of Val-Pro or Ala-Pro dipeptides from the N-terminus of CXCL10, converting it into an antagonist of T cell recruitment. Previous studies have attempted 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 to CXCL10—both proteolytic and nonproteolytic—can result in an expanded repertoire of CXCL10 variants beyond simple DPP4-catalyzed N-terminal processing (Loos et al., 2008; Mortier et al., 2011). Therefore, ignoring these multiple variants can lead to discrepancies between measured and total amounts of CXCL10 in clinical samples and inadequately capture the functional state of CXCL10 (Casrouge et al., 2012). [Table 5-1] [Table 5-2]

[0398] To clarify the relationship between DPP4 activity and CXCL10 function in clinical samples, the amount and specific activity of DPP4 were measured in the same set of benign and malignant ascites.

[0399] Based on the results of the patient samples, receiver operating characteristic (ROC) curves were first constructed for each marker (activity rate, DPP4, CA125) to obtain the area under the curve (AUC). A combined ROC was obtained and a binary logistic regression was performed. Using the measured values ​​of each marker as the dependent variable for both the benign and malignant groups, the probability was obtained as the test variable, which was then used to construct a combined ROC.

[0400] No significant differences in quantity or activity were observed in either case (Figures 5a and 5b). DPP4 quantity correlated with the calculated activity fraction in malignant samples (p = 0.002), but DPP4 specific activity did not. Interestingly, benign samples showed an inverse (non-significant) trend suggesting a correlation between specific activity and activity fraction measurements (Figures 6b and 6c). These data suggest that while DPP4 activity may be related to CXCL10 function in nonmalignant diseases, CXCL10 variants are likely important for determining the overall functional status of CXCL10 in malignant tumors.

[0401] Example 6: Activity ratio provides prognostic differentiation between benign and malignant disease To assess whether the activity ratio could provide useful clinical information, we evaluated the performance of the activity ratio in ascites fluid using receiver operating characteristic (ROC) curves (Figure 7). The activity ratio achieved a high AUC and substantial improvements in sensitivity / specificity compared with measurements of either active or total CXCL10 alone (Figure 7a; Table 3), highlighting the improved utility of these single measures. The sensitivity, specificity, and predictive value of the activity ratio were also higher than those of plasma CA125 (Figure 7b; Table 3). The activity ratio also achieved a larger effect size (Cohen's d) than either DPP4, CA125, or CXCL10 (active or total) measurements alone (Table 4). Thus, combining the activity ratio, DPP4, and plasma CA125 measurements yielded an AUC > 0.95 with good PPV (87-94%) and NPV (93-95%) for distinguishing benign from malignant disease. Thus, ART in combination with DPP4 and plasma CA125 provided utility in distinguishing benign from malignant disease in patients presenting with ascites fluid. [Table 6]

[0402] Example 7: ART as a clinical diagnostic We investigated whether a cervicovaginal swab (CVS) could be used to represent the genital tract and provide a rate of activity measurement.

[0403] Paralleling our findings in ascites fluid, fractional activity measurements performed on CVS extracts (n = 50 / group) showed good discrimination between benign and malignant disease samples, with an AUC of 0.8 (p < 0.0001) (Figure 8a; Table 6). In matched plasma samples (n = 30), ART was also able to differentiate between groups (AUC 0.8, p < 0.001).

[0404] We also examined the effect size (Cohen's d) for each biomarker and sample type (Table 6). Plasma CA125 measurements returned an AUC of 0.83, second only to ART measurements in ascites fluid (AUC 0.86). However, the effect size was "moderate" (Cohen's d=0.62). In contrast, ART measurements achieved "large" effect sizes in ascites fluid and CVS (Cohen's d=0.86 and 1.0, respectively), suggesting a significant reduction in variance in the same population. Similarly, ART measurements performed in plasma also ... It returned to 0.79 (Table 6).

[0405] The results indicate that ART is a preferred biomarker for achieving statistically robust measurements, and that analyzing ART using CVS provides a robust, clinically useful measurement that can distinguish between malignant and non-malignant disease.

[0406] Example 8: ART distinguishes cancer-free patients from patients with benign or malignant ovarian tumors ART was applied to a new patient cohort (i.e., a prophylactically recruited cohort)—patients who underwent prophylactic, risk-reducing salpingo-oophorectomy and either harbored BrCA1 / 2 mutations or had a strong family history of breast and / or ovarian cancer. These patients therefore represent an ideal uncontrolled cohort for the validation of ART designed to detect the presence of early-stage ovarian cancer.

[0407] The concentrations of active CXCL10 were significantly higher than total CXCL10 in healthy women (Fig. 9a), suggesting that patients without cancer may have high levels of active and functional CXCL10. When comparing the overall active and total CXCL10 concentrations in healthy women with those in benign and malignant tumors in the prophylactic cohort, these concentrations were significantly lower than those in both benign and malignant tumors (Fig. 9b). Regarding the activity rate, the activity rate in healthy women was significantly lower than that in the benign and malignant tumor groups in both plasma and CVS (Fig. 9c and d).

[0408] Given that the levels of active CXCL10 and activity fraction in healthy women are significantly higher than total CXCL10 and activity fraction in benign and malignant patients, respectively, the data suggest that the degree of DPP4-initiated cleavage of functional CXCL10 may be low in patients without benign conditions or malignant ovarian tumors.

[0409] Example 9: Amount and activity of DPP4 do not correlate with activity rate To establish the relationship between DPP4 and CXCL10 in a prophylactically recruited cohort, the amount of DPP4 and its specific activity were measured in plasma samples and compared with the results of patients with benign or malignant ovarian cancer.

[0410] The DPP4 levels in plasma samples from healthy patients were significantly higher than those in both benign and malignant patient groups. However, the amount of DPP4 did not correlate with the calculated activity ratio in healthy female samples (Fig. 10a). Although the amount of DPP4 in healthy females was significantly higher than that in benign and malignant patient groups, its specific activity in the plasma of healthy females was significantly lower in both benign and malignant patients (Fig. 10b). The specific activity did not correlate with any of the calculated activity ratios. These data suggest that DPP4 function in healthy females may be suppressed by unknown factors.

[0411] Example 10: CVS distinguishes healthy patients from those with benign or malignant ovarian tumors Because CVS is the optimal sample for biomarker-based testing, we measured the levels of active and total CXCL10 in the CVS of the prophylactic cohort to obtain activity rates. Furthermore, we compared the activity rates between healthy patients and patients with benign or malignant ovarian tumors in the prophylactic cohort.

[0412] For healthy women, the overall levels of active, functional CXCL10 were significantly higher than total CXCL10 (Fig. 11a). In contrast to patients with benign or malignant ovarian tumors, the calculated activity percentages of CVS from healthy patients were consistent with the plasma results and were significantly higher than those from healthy women. The overall activity rate in our sample was significantly higher than in both the benign and malignant patient groups (Fig. 11b).

[0413] Example 11: Correlation between DPP4 amount in CVS and activity rate of CVS Due to the limited amount of extract from CVS, the amount of DPP4 in CVS was not measured in previous studies involving the Ovarian Cancer Biobank. Because it was desirable to measure both the activity fraction and the amount of DPP4 from the same sample source and establish a correlation within the same sample type, the preparation of CVS was optimized to measure and correlate both the activity fraction and DPP4 in CVS. As shown in Figure 12, DPP4 in CVS could be quantified, and the calculated activity fraction was inversely correlated with DPP4 (p=0.0094). This data suggests that the activity fraction may be correlated with DPP4 in CVS.

[0414] Example 12: Activity rate and plasma CA125 The CA125 measurements in healthy women were significantly lower than those in both the benign and malignant groups. This data is consistent with the fact that CA125 levels are increased in patients with malignant disease (Figure 13). The calculated activity rate is positively correlated with CA125 (p=0.0015).

[0415] Example 13: Activity rate and plasma CA125 To assess whether the activity rate can provide useful clinical information, receiver operating characteristic (ROC) curves were used to evaluate its performance in plasma and CVS. ROC curves were constructed based on a comparison between healthy women in a prophylactic cohort and patients with malignant ovarian tumors.

[0416] Because the activity ratio achieved substantial improvements in sensitivity / specificity compared with quantifying either active or total CXCL10 alone in previous ascites studies, combining the activity ratios of plasma and CVS also achieved a high AUC (Figure 14a). This demonstrates that using both sample types increases the prognostic efficacy of ART. For the differentiation of healthy women from malignant disease, combining the activity ratios of plasma, DPP4, and plasma CA125 measurements yielded an AUC > 0.98 (Figure 14b), while combining the activity ratios of CVS, DPP4, and plasma CA125 yielded an AUC > 0.99. These data suggest that combining the two sample types, as well as the activity ratios of DPP4 and plasma CA125, will prove useful for the differentiation of healthy women from malignant disease.

[0417] Example 14: ART distinguishes patients with malignancies from complex backgrounds To investigate the prognostic ability of ART to distinguish malignant tumors from diverse backgrounds, we combined women from the healthy and benign cohorts and performed a receiver operating characteristic (ROC) analysis on the malignant group. When applied to plasma samples, ART alone returned an AUC of 0.79, and when applied to CVS samples, it returned an AUC of 0.72. By comparison, CA125 achieved an AUC of 0.93 (Figure 15A). However, CA125 exhibited high within-cohort variability (CV 189.5%) and a small effect size (Cohen's d 0.097), suggesting that it was not a robust measure for distinguishing malignant samples from the combined healthy + benign cohort (Table 7).

[0418] In contrast, the measure of activity fraction had a large effect size in both CVFS (d = 0.64) and plasma (d = 1.01), suggesting a significant reduction in variance in the same population (Table 7). ART can provide a statistically robust measure, allowing for the detection of malignant eggs against a complex background of healthy women or patients with benign disease. The results suggest that it may be possible to surpass the performance of the current CA125 criterion for distinguishing foveal carcinoma. [Table 7]

[0419] The combination of ART (CVS and / or plasma), DPP4, and CA125 was also investigated as a mechanism for improving discrimination between groups. Compared to CA125 (AUC 0.94), the combination of ART with all other markers measured in plasma, measured using CVS, returned an AUC of 0.98 (Table 8).

[0420] The combination of all biomarkers (ART, DPP4, and CA125) achieved an AUC of 0.98, with a sensitivity of 93.1% for detecting malignancies in this cohort (Table 8). In contrast, CA125 alone demonstrated an AUC of 0.94, but only a sensitivity of 78.6% (Table 8). [Table 8]

[0421] Thus, the combination of ART, DPP4, and CA125 distinguished malignancies from a complex background of healthy women and women with benign disease, significantly outperforming only CA125, the current clinical gold standard.

[0422] Example 15: ART improves identification of patients with early stage (Stage I) cancer Cancer stage at diagnosis is strongly associated with clinical outcomes for ovarian cancer patients. Specifically, patients diagnosed with FIGO stage I disease ("early stage") have substantially improved 5-year and overall survival rates (approximately 95%) compared with patients diagnosed with stages III-IV (<40%). Therefore, we evaluated whether ART (alone or in combination) could successfully distinguish early-stage cancer patients from either healthy women or women with benign disease.

[0423] CA125 provided the highest single-marker discrimination between patients with early-stage cancer and those with benign disease or healthy women (Table 9). However, combining ART (plasma) with DPP4 and CA125 substantially improved discrimination between benign and early-stage malignant tumors (AUC 0.75 vs 0.63; Table 9).

[0424] Remarkably, patients with early-stage malignancies could be distinguished from healthy women with high accuracy using the three-marker combination (AUC = 0.99; sensitivity 95.4%) compared with CA125 alone (AUC 0.88; sensitivity 81.8%).

[0425] Therefore, ART could be used to improve the diagnostic or prognostic assessment of patients compared with the current CA125 criteria, particularly with regard to the detection of disease-specific changes associated with early-stage cancer. [Table 9]

[0426] Example 16: Combining an ART panel with a multiplex five-marker panel identifies patients with precancerous lesions in a prospectively recruited cohort

[0427] Although the pathogenesis of ovarian cancer remains unclear, it is becoming clear that the fallopian tubes begin to carry a precancerous "p53 signature" as many ovarian tumors form. As with other types of cancer, detection of lesions at a very early, precancerous stage can result in substantially improved outcomes.

[0428] We have previously identified several additional relevant biomarkers (GM-CSF, IL-6, TNF-RII, HE4, IL-8) for diagnostic profiling of high-grade epithelial ovarian cancer (PMID: https: / / doi.org / 10.1038 / s41598-020-59009-z). Therefore, we evaluated their potentially improved diagnostic performance in combination with the existing panel of ART, DPP4, and CA125.

[0429] For these studies, combinations of markers were collected to predict the risk of developing ovarian cancer and preventative risk-reducing surgery (typically both ovaries). This study was evaluated in a cohort of women undergoing oophorectomy (tubeo-oophorectomy). Using histological evaluation, all patients were characterized as either "healthy" (i.e., confirmed free of disease) or "cancer" (present with p53 lesions or early occult tumors).

[0430] The results are shown in Table 10 (only the best-performing combination is shown). For early / precancerous lesion detection, the five-marker panel returned a combined AUC of 0.87, while the established ART panel achieved an AUC of 0.97 (Table 10). These were substantially better than CA125, returning an AUC of 0.77.

[0431] However, remarkably, when both panels were combined, a perfect AUC of 1.0 was achieved, indicating that this particular combination of markers has high potential for diagnosing early stage and precancerous ovarian lesions.

Table 10

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Claims

1. A C-X-C motif chemokine ligand 10 (CXCL10) binding protein, wherein the binding protein binds to full-length human CXCL10, N-terminal truncated CXCL10, and citrullinated CXCL10, the binding protein comprising: (i) heavy chain variable region (V Ｈ ) where a) a CDR1 comprising the sequence set forth in SEQ ID NO: 13; b) a CDR2 comprising the sequence set forth in SEQ ID NO: 14; and c) a CDR3 comprising the sequence set forth in SEQ ID NO: 15; and Ｈ , and (ii) a light chain variable region (V Ｌ ) where a) a CDR1 comprising the sequence set forth in SEQ ID NO: 16; b) a CDR2 comprising the sequence set forth in SEQ ID NO: 17; and c) a CDR3 comprising the sequence set forth in SEQ ID NO: 18; and Ｌ The binding protein is an antibody or an antigen-binding fragment thereof, comprising:

2. the binding protein (i) binds to the epitope NH2-LSRTVRCTCISISNQPVNPRSLE-COOH (SEQ ID NO: 26) of full-length human CXCL10, N-terminally truncated CXCL10, and citrullinated CXCL10; and / or (ii) a K of 50 nM or less Ｄ and / or binds to full-length human CXCL10 at (iii) a K of 5 nM or less Ｄ binds to N-terminally truncated human CXCL10 at The CXCL10 binding protein of claim 1.

3. The binding protein comprises the amino acid sequence set forth in SEQ ID NO:

11. Ｈ and V comprising the amino acid sequence set forth in SEQ ID NO:

12. Ｌ 3. The CXCL10 binding protein of claim 1 or 2, comprising:

4. CXC motif chemokine ligand 10 (CXCL10) binding protein, wherein the binding protein has a K Ｄ and binding to full-length human CXCL10 at the N-terminal valine and / or proline of the epitope NH2-VPLSRTVRCTCISISNQPVNPRSLE-COOH (SEQ ID NO: 25) but not to N-terminal truncated CXCL10 and citrullinated CXCL10. wherein the binding protein is (i) heavy chain variable region (V Ｈ ) where a) a CDR1 comprising the sequence set forth in SEQ ID NO: 5; b) a CDR2 comprising the sequence set forth in SEQ ID NO: 6; and c) a CDR3 comprising the sequence set forth in SEQ ID NO: 7; and Ｈ , and (ii) a light chain variable region (V Ｌ ) where a) a CDR1 comprising the sequence set forth in SEQ ID NO: 8; b) a CDR2 comprising the sequence set forth in SEQ ID NO: 9; and c) a CDR3 comprising the sequence set forth in SEQ ID NO: 10; and Ｌ , The binding protein is an antibody or an antigen-binding fragment thereof, comprising:

5. The binding protein comprises the amino acid sequence set forth in SEQ ID NO:3 Ｈ and V comprising the amino acid sequence set forth in SEQ ID NO:4 Ｌ The CXCL10 binding protein of claim 4, comprising:

6. A composition comprising the CXCL10 binding protein of any one of claims 1 to 5 and a carrier.

7. A polynucleotide encoding the CXCL10 binding protein according to any one of claims 1 to 5.

8. An expression vector comprising the polynucleotide of claim 7.

9. A cell comprising the expression vector of claim 8 in vitro.

10. 10. Use of the cells described in claim 9 for preparing a CXCL10 binding protein, the use comprising culturing the cells and producing the CXCL10 binding protein from the cells, and optionally isolating and purifying the produced binding protein.

11. Use of a CXCL10 binding protein according to any one of claims 1 to 5 in the manufacture of a panel or kit for detecting and / or diagnosing a malignant condition.

12. Use of a CXCL10 binding protein according to any one of claims 1 to 5 in the manufacture of a panel or kit for monitoring tumor burden and / or monitoring the progression of a malignant condition and / or measuring tumor regression in a subject suffering from a malignant condition.

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