Methods of detecting and / or diagnosing a malignant condition

Abstract

The present disclosure relates to a method for assessing the risk that a subject suffers from a malignant condition and / or detecting and / or diagnosing a malignant condition comprising determining a CXCL10 ratio of active CXLC10 to total CXCL10 in the subject; and the level of cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) in the subject.

Description

[0001] METHODS OF DETECTING AND / OR DIAGNOSING A MALIGNANT CONDITION

[0002] RELATED APPLICATION DATA

[0003] The present application claims priority from Australian Patent Application No. 2023903261 entitled “Methods of detecting and / or diagnosing a malignant condition” filed on 11 October 2024. The entire contents of which is hereby incorporated by reference.

[0004] SEQUENCE LISTING

[0005] The present application is filed with a Sequence Listing in electronic form. The entire contents of the Sequence Listing are hereby incorporated by reference.

[0006] FIELD OF THE INVENTION

[0007] The present disclosure relates to a method for assessing the risk that a subject suffers from a malignant condition and / or detecting and / or diagnosing a malignant condition comprising determining a CXCL10 ratio of active CXLC10 to total CXCL10 in the subject; and the level of cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) in the subject.

[0008] BACKGROUND OF THE INVENTION

[0009] Ovarian cancer remains one of the most lethal gynaecological malignancies globally, with 314,000 new cases and 207,000 deaths in 2020. Appropriate surgical referral and initial management strategy is a key indicator of outcome for ovarian cancer patients; in particular, 5-year survival for patients diagnosed with early (FIGO stage I) disease is >90%; and recurrence rates for these patients are below 20%.

[0010] Whilst there are no universally adopted guidelines, clinical work-up for a suspected ovarian malignancy typically involves physical examination, transvaginal ultrasound (TVU) and measurement of serum biomarkers including cancer antigen 125 (CA125) and Human Epididymal Protein 4 (HE4) to direct referral for surgery. These measurements are commonly used in the calculation of Risk of Malignancy Index (RMI) or Risk of Malignancy Algorithm (ROMA) scores, used to indicate likelihood of malignancy when an ovarian mass is present. However, these modalities often lack sensitivity and specificity for the accurate identification of malignancy - particularly for early stage, low volume disease - against the background of other benign gynaecological conditions. Whilst RMI can achieve specificity up to -95% (i.e. the ability to correctly differentiate between benign and malignant disease), its sensitivity is generally lower at 71-80% (i.e. the ability to correctly identify the presence of disease). For the detection of Stage I- II ovarian cancers, sensitivity is further reduced to -54%. RMI is also dependent on the quality of ultrasound imaging. ROMA typically exhibits higher sensitivity but lower specificity than RMI.

[0011] Several biomarker-based tests have been introduced for pre-surgical triage, and include ROMATM (Fujirebio), 0VA1TM and OVERATM (Aspira Women’s Health) which are FDA-approved as aids to assess whether a pre- or post-menopausal woman who presents with a suspicious adnexal mass is at a low or high likelihood of finding malignancy on surgery.

[0012] It will be apparent to the skilled person from the foregoing that there is a need in the art for an alternative, biomarker-based approach for improved identification of ovarian malignancy with high specificity and sensitivity. In particular, there is a need in the art for the development of a biomarker approach for discrimination of early- stage cancers from benign gynaecological diseases.

[0013] SUMMARY OF THE INVENTION

[0014] In producing the present invention, the inventors sought to produce a panel of biomarkers that have high specificity and sensitivity for determining the presence of, or assessing the risk of a subject having, a malignant condition. The inventors identified a panel of biomarkers consisting of CXCL10, cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) that was able to distinguish between malignant ovarian cancer and benign ovarian disease with a high degree of specificity and sensitivity, compared to detecting the biomarkers alone and as compared to current standard of care. In particular, the inventors found that determining a CXCL10 ratio of active CXLC10 to total CXCL10 together with the level of CA-125, HE4 and IL-6 in the subject, they were able to accurately identify malignant disease with a high positive and negative predictive value. Furthermore, they were able to discriminate between stage I (i.e., early stage) cancers or pre-cancerous lesions and benign conditions. The inventors further found that the panel of biomarkers had even higher specificity and sensitivity in post-menopausal women, than detecting each of the biomarkers alone.

[0015] Accordingly, the present disclosure provides a method for assessing the risk that a subject suffers from a malignant condition, the method comprising: (i) determining a level of active C-X-C motif chemokine ligand 10 (CXCL10) in the subject and a level of total CXCL10 in the subject; and determining a CXCL10 ratio of active CXLC10 to total CXCL10 in the subject; and

[0016] (ii) determining the level of cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) in the subject.

[0017] The present disclosure also provides a method of detecting and / or diagnosing a malignant condition in a subject, the method comprising:

[0018] (i) determining a level of active C-X-C motif chemokine ligand 10 (CXCL10) in the subject and a level of total CXCL10 in the subject; and determining a CXCL10 ratio of active CXLC10 to total CXCL10 in the subject; and

[0019] (ii) determining the level of cancer antigen 125 (CA-125), human epididymis protein

[0020] 4 (HE4) and interleukin 6 (IL-6) in the subject.

[0021] In one example, the method comprises:

[0022] (i) determining the level of active CXCL10 and total CXCL10 comprises determining the amount of active CXCL10 protein and the amount of total CXCL10 protein in the subject; and / or

[0023] (ii) determining the level of CA-125, HE4 and / or IL-6 comprises determining the amount of CA-125, HE4 and / or IL-6 protein in the subject.

[0024] In one example, the method comprises determining the level of active CXCL10 and total CXCL10 comprises determining the amount of active CXCL10 protein and the amount of total CXCL10 protein in the subject.

[0025] In one example, the method comprises determining the level of CA-125, HE4 and IL-6 comprises determining the amount of CA-125, HE4 and IL-6 protein in the subject.

[0026] In one example, the method comprises:

[0027] (i) determining the level of active CXCL10 and total CXCL10 comprises determining the amount of active CXCL10 protein and the amount of total CXCL10 protein in the subject; and

[0028] (ii) determining the level of CA-125, HE4 and IL-6 comprises determining the amount of CA-125, HE4 and IL-6 protein in the subject.

[0029] In one example, the method further comprises comparing the CXCL10 ratio and the level of CA-125, HE4 and IL-6 in the subject to a CXCL10 ratio and a level of CA- 125, HE4 and IL-6 in at least one reference. Methods of determining a reference will be apparent to the skilled person and / or are described herein.

[0030] In one example, the method comprises determining: (i) if the CXCL10 ratio and the level of CA-125, HE4 and IL-6 in the subject are higher than the CXCL10 ratio and the level of CA-125, HE4 and LL6 in the reference; or

[0031] (ii) if the CXCL10 ratio and the level of CA-125, HE4 and IL-6 in the subject are lower than the CXCL10 ratio and the level of CA-125, HE4 and IL-6 in the reference.

[0032] In one example, the method comprises determining if the CXCL10 ratio and the level of CA-125, HE4 and IL-6 in the subject are higher than the CXCL10 ratio and the level of CA-125, HE4 and LL6 in the reference.

[0033] In one example, the method comprises if the CXCL10 ratio and the level of CA- 125, HE4 and IL-6 in the subject are lower than the CXCL10 ratio and the level of CA- 125, HE4 and IL-6 in the reference.

[0034] In one example, (i) a lower CXCL10 ratio and a lower level of CA-125, HE4 and IL-6 in the subject compared to the reference is indicative of an increased probability of the subject suffering from the malignant condition or is indicative of a malignant condition; or (ii) a higher CXCL10 ratio and a higher level of CA-125, HE4 and IL-6 in the subject compared to the reference is indicative of an increased probability of the subject suffering from a benign condition or is indicative of a benign condition.

[0035] In one example, a lower CXCL10 ratio and a lower level of CA-125, HE4 and IL- 6 in the subject compared to the reference is indicative of an increased probability of the subject suffering from the malignant condition or is indicative of a malignant condition.

[0036] In one example, a higher CXCL10 ratio and a higher level of CA-125, HE4 and IL-6 in the subject compared to the reference is indicative of an increased probability of the subject suffering from a benign condition or is indicative of a benign condition.

[0037] In one example, the method comprises determining a diagnostic score comprising the CXCL10 ratio and level of CA-125, HE4 and IL-6 in the subject.

[0038] In one example, the diagnostic score is obtained by transforming the data to approximate a standard normal distribution using Yeo Johnson transformation. Methods of using Yeo Johnson transformation will be apparent to the skilled person, described herein or as described for example, in Yeo and Johnson 2000. In one example, a transformation parameter is determined for each biomarker. For example, the transform for any individual marker Xi (i.e., CXCL10, CA-125, HE4 and IL-6) is defined by the formula:

[0039] In one example, transformed biomarker values are standardised according to the formula: where x(S)is the standardised individual measurement in each case.

[0040] In one example, model selection for combined biomarker analyses is estimated using one or more model types. For example, the model type is selected from the group consisting of Support Vector Classifier, Decision Tree, Naive Bayes, Logistic Regression and combinations thereof. In one example, performance is assessed by repeated stratified k-fold cross-validation (4 folds x 5 repeats).

[0041] In one example, a multivariate Logistic Regression model is defined according to the following formula: where p(x) indicates probability of malignancy.

[0042] In one example, the model is fit to a transformed and scaled dataset using Maximum Likelihood Estimation. In one example, a set of coefficients P is determined.

[0043] In one example, a Prediction of Malignancy for any given observation is determined. For example, the Prediction of Malignancy is determined by applying the cutoff to the Risk Score S (or diagnostic score), calculated according to the formula S=10p(X). For example, according to the formula:

[0044] In one example, the optimal risk score or diagnostic score is determined using Youden’s J index. Methods of using Youden’s J index will be apparent to the skilled person, described herein and / or, for example, in Watrowski et al., 2002.

[0045] In one example, the optimal risk score or diagnostic score cut-point is 3.6. For example, the cut-point is 3.684.

[0046] In one example, the method comprises determining the diagnostic score at one or more time points. In one example, (i) a score of >3.6 in the subject is indicative of an increased probability of the subject suffering from the malignant condition or is indicative of a malignant condition; or (ii) a score of <3.6 in the subject is indicative of an increased probability of the subject suffering from a benign condition or is indicative of a benign condition.

[0047] In one example, a score of >3.6 in the subject is indicative of an increased probability of the subject suffering from the malignant condition or is indicative of a malignant condition.

[0048] In one example, a score of <3.6 in the subject is indicative of an increased probability of the subject suffering from a benign condition or is indicative of a benign condition.

[0049] In one example, the methods as described herein can assess the risk and / or detect and / or diagnose a malignant condition in a subject with high specificity and sensitivity and / or high positive and negative predictive value. For example, the specificity and sensitivity is assessed by Receiver Operative Characteristic (ROC) analysis as area under the curve (AUC).

[0050] In one example, the method has:

[0051] (i) a sensitivity of at least 0.80;

[0052] (ii) a specificity of at least 0.80;

[0053] (iii) a positive predictive value of at least 0.90; and / or

[0054] (iv) a negative predictive value of at least 0.91.

[0055] In one example, the method has a sensitivity of at least 0.80. For example, the method has a sensitivity of 0.80, or 0.81, or 0.82, or 0.83, or 0.84, or 0.85. In one example, the method has a sensitivity of 0.86, or 0.87, or 0.88, or 0.89. In one example, the method has a sensitivity of at least 0.90. For example, the method has a sensitivity of 0.90, or 0.91, or 0.92, or 0.93, or 0.94, or 0.95. In one example, the method has a sensitivity of 0.96, or 0.97, or 0.98, or 0.99, or 1.00. For example, the method has a sensitivity of between 0.91 and 0.98. In one example, the method has a sensitivity of between 0.93 and 0.97. For example, the method has a sensitivity of 0.95. In one example, the method has a sensitivity of between 0.95 and 1.00. For example, the method has a sensitivity of between 0.98 and 1.00. In one example, the method has a sensitivity of 0.99.

[0056] In one example, the method has a sensitivity of 0.95 (95% confidence interval (CI) 0.91-0.98). In another example, the method has a sensitivity of 0.81 (95% CI 0.64- 0.93). In a further example, the method has a sensitivity of 0.99 (95% CI 0.95-1.00).

[0057] In one example, the method has a specificity of at least 0.80. For example, the method has a specificity of 0.80, or 0.81, or 0.82, or 0.83, or 0.84, or 0.85. In one example, the method has a specificity of 0.86, or 0.87, or 0.88, or 0.89. In one example, the method has a specificity of at least 0.90. For example, the method has a specificity of 0.90, or 0.91, or 0.92, or 0.93, or 0.94, or 0.95. In one example, the method has a specificity of 0.96, or 0.97, or 0.98, or 0.99, or 1.00. In one example, the method has a specificity of between about 0.90 and 0.98. For example, the method has a specificity of between about 0.93 and 0.97. For example, method has a specificity of 0.95. In one example, the method has a specificity of between 0.92 and 1.00. For example, the method has a specificity of 0.96 and 1.00. In one example, the method has a specificity of 0.98. In one example, the method has a specificity of between 0.84 and 0.97. In one example, the method has a specificity of between 0.90 and 0.94. For example, the method has a specificity of 0.92.

[0058] In one example, the method has a specificity of 0.95 (95% CI 0.90-0.98). In another example, the method has a specificity of 0.98 (95% CI 0.92-1.00). In a further example, the method has a specificity of 0.92 (95% CI 0.84-0.97).

[0059] In one example, the method has a positive predictive value of at least 0.75. For example, the method has a positive predictive value of 0.75, or 0.76, or 0.77, or 0.78, or 0.79, or 0.80. In one example, the method has a positive predictive value of at least 0.80. For example, the method has a positive predictive value of 0.81, or 0.82, or 0.83, or 0.84 or 0.85. In one example, the method has a positive predictive value of at least 0.85. For example, the method has a positive predictive value of 0.86, or 0.87, or 0.88, or 0.89, or 0.90. In one example, the method has a positive predictive value of at least 0.90. For example, the method has a positive predictive value of 0.91, or 0.92, or 0.93, or 0.94, or 0.95. In one example, the method has a positive predictive value of at least 0.95. For example, the method has a positive predictive value of 0.96, or 0.97, or 0.98, or 0.99, or 1.00. In one example, the method has a positive predictive value of between 0.90 and 0.98. For example, the method has a positive predictive value of between 0.93 and 0.97. For example, the method has a positive predictive value of 0.95. In one example, the method has a positive predictive value of between 0.77 and 0.99. For example, the method has a positive predictive value between 0.85 and 0.95. In one example, the method has a positive predictive value of between 0.90 and 0.95. For example, the method has a positive predictive value of 0.93.

[0060] In one example, the method has a positive predictive value of 0.95 (95% CI 0.90- 0.98). In another example, the method has a positive predictive value of 0.93 (95% CI 0.77-0.99). In a further example, the method has a positive predictive value of 0.95 (95% CI 0.90-0.98). In one example, the method has a negative predictive value of at least 0.85. For example, the method has a negative predictive value of 0.86, or 0.87, or 0.88, or 0.89, or 0.90. In one example, the method has a negative predictive value of at least 0.90. For example, the method has a negative predictive value of 0.91, or 0.92, or 0.93, or 0.94, or 0.95. In one example, the method has a negative predictive value of at least 0.95. For example, the method has a negative predictive value of 0.96, or 0.97, or 0.98, or 0.99, or 1.00. In one example, the method has a negative predictive value of between 0.90 and 0.98. For example, the method has a negative predictive value of between 0.91 and 0.98. In one example, the method has a negative predictive value of between 0.93 and 0.97. For example, the method has a negative predictive value of 0.95. In one example, the method has a negative predictive value of between 0.86 and 0.97. For example, the method has a negative predictive value between 0.90 and 0.95. For example, the method has a negative predictive value of 0.93. In one example, the method has a negative predictive value of between 0.92 and 1.00. In one example, the method has a negative predictive value of between 0.96 and 1.00. For example, the method has a negative predictive value of 0.98.

[0061] In one example, the method has a negative predictive value of 0.95 (95% CI 0.91- 0.98). In another example, the method has a negative predictive value of 0.93 (95% CI 0.86-0.97). In a further example, the method has a negative predictive value of 0.98 (95% CI 0.92-1.00).

[0062] In one example, the accuracy of a diagnostic test is characterized by a Receiver Operating Characteristic curve (“ROC curve”). The skilled person will understand that a ROC is a plot of the true positive rate against the false positive rate for the different possible points of a diagnostic test. A ROC curve shows the relationship between sensitivity and specificity. That is, an increase in sensitivity will be accompanied by a decrease in specificity. The closer the curve follows the left axis and then the top edge of the ROC space, the more accurate the test. Conversely, the closer the curve comes to the 45-degree diagonal of the ROC graph, the less accurate the test. The area under the ROC is a measure of test accuracy. The accuracy of the test depends on how well the test separates the group being tested into those with and without the disease in question. An area under the curve (AUC) of 1 represents a perfect test, while an area of 0.5 represents a less useful test.

[0063] In one example, the method has an AUC of at least 0.90. For example, the method has an AUC of between about 0.91 and 1.00. In one example, the method has an AUC of at least 0.95. For example, the method has an AUC of between about 0.97 and 1.00. For example, the method has an AUC of about 0.98. In one example, the method has an AUC of between about 0.98 and 1.00. For example, the method has an AUC of about 0.99.

[0064] In one example, the method has an AUC of 0.98 (95% CI 0.97-1.00). In another example, the method has an AUC of 0.95 (95% CI 0.91-1.00). In a further example, the method has an AUC of 0.99 (95% CI 0.98-1.00).

[0065] It will be apparent to the skilled person from the disclosure herein that reference to the AUC of the method refers to the combined AUC of the CXCL10 ratio and CA- 125, HE4 and IL-6.

[0066] In one example of any method described herein, the disclosure provides a method of assessing the risk of, and / or detecting and / or diagnosing a malignant condition in a subject. For example, the malignant condition is a reproductive cancer. In one example, the reproductive cancer is ovarian cancer. In one example, the ovarian cancer is a stage I cancer. In another example, the ovarian cancer is a pre-cancerous lesion, for example a lesion with a p53 gene mutation.

[0067] In one example, the method comprises detecting and / or diagnosing a malignant condition from a benign condition.

[0068] In one example, the method comprises assessing the risk that a subject suffers from a malignant condition and not a benign condition.

[0069] The present disclosure also provides a method for discriminating a malignant condition from a benign condition in a subject, the method comprising:

[0070] (i) determining a level of active C-X-C motif chemokine ligand 10 (CXCL10) in the subject and a level of total CXCL10 in the subject; and determining a CXCL10 ratio of active CXLC10 to total CXCL10 in the subject; and

[0071] (ii) determining the level of cancer antigen 125 (CA-125), human epididymis protein

[0072] 4 (HE4) and interleukin 6 (IL-6) in the subject.

[0073] The present disclosure further provides a method for identifying an early stage a malignant condition from a benign condition in a subject, the method comprising:

[0074] (i) determining a level of active C-X-C motif chemokine ligand 10 (CXCL10) in the subject and a level of total CXCL10 in the subject; and determining a CXCL10 ratio of active CXLC10 to total CXCL10 in the subject; and

[0075] (ii) determining the level of cancer antigen 125 (CA-125), human epididymis protein

[0076] 4 (HE4) and interleukin 6 (IL-6) in the subject.

[0077] In one example, the subject is pre-menopausal or post-menopausal. For example, the subject is pre-menopausal. In one example, the subject is post-menopausal. For example, the post-menopausal is peri-menopausal, menopausal or post-menopausal. Methods of determining menopausal status will be apparent to the skilled person and / or described herein. In on example, the subject is pre-menopausal. For example, the pre-menopausal subject has menstruated in the last 3 months and no change in menstrual frequency in the last 12 months. In one example, the subject is peri-menopausal. For example, the subject has changes in menstrual frequency or 3-11 months of amenorrhea. In one example, the subject is post-menopausal. For example, the subject has amenorrhea for 12 consecutive months or more. In one example, the post-menopausal subject is 52 years of age or older.

[0078] In one example, menopausal status is determined using follicle stimulating hormone (FSH) levels. For example, the FSH levels are determined using the Abbott ARCHITECT i2000 platform. In one example, the subject is pre-menopausal if the FSH levels are < 22 mIU / mL (< 22 IU / L). In one example, the subject is post-menopausal if FSH levels are > 22 mIU / mL (> 22 IU / L).

[0079] In one example, the subject is post-menopausal and the method has:

[0080] (i) a sensitivity of at least 0.95;

[0081] (ii) a specificity of at least 0.80;

[0082] (iii) a positive predictive value of at least 0.90; and / or

[0083] (iv) a negative predictive value of at least 0.90.

[0084] In one example, the subject is post-menopausal and the method has:

[0085] (i) a sensitivity of between about 0.95 and 1.00;

[0086] (ii) a specificity of between about 0.84 and 0.97 ;

[0087] (iii) a positive predictive value of between about 0.90 and 0.98; and / or

[0088] (iv) a negative predictive value of between about 0.92 and 1.00.

[0089] In one example, the subject is post-menopausal and the method has:

[0090] (i) a sensitivity of about 0.99;

[0091] (ii) a specificity of about 0.92;

[0092] (iii) a positive predictive value of about 0.95; and / or

[0093] (iv) a negative predictive value of about 0.98.

[0094] In one example, the method comprises using:

[0095] (i) a CXCL 10 binding protein that specifically binds full-length human CXCL 10 and N-terminally truncated CXCL 10 and citrullinated CXCL 10 to determine the level of total CXCL10 in the subject; and

[0096] (ii) a CXCL10 binding protein that specifically binds full-length human CXCL10, but does not bind N-terminally truncated CXCL 10 and citrullinated CXCL 10 to determine the level of active CXCL10 in the subject. In one example, the CXCL10 binding protein that binds full-length human CXCL10 and N-terminally truncated CXCL10 and citrullinated CXCL10:

[0097] (i) binds the epitope NH2-LSRTVRCTCISISNQPVNPRSLE-COOH (SEQ ID NO: 26) of full-length human CXCL10, N-terminally truncated CXCL10 and citrullinated CXCL10;

[0098] (ii) binds to full-length human CXCL10 with a KD of 50 nM or less; and / or

[0099] (iii) binds to N-terminally truncated human CXCL10 with a KD of 5 nM or less.

[0100] In one example, the CXCL10 binding protein binds the epitope NH2- LSRTVRCTCISISNQPVNPRSLE-COOH (SEQ ID NO: 26) of full-length human CXCL10, N-terminally truncated CXCL10 and citrullinated CXCL10.

[0101] In one example, the CXCL10 binding protein binds to full-length human CXCL10 with a KD of 50 nM or less. For example, the CXCL10 binding protein binds to or specifically binds to full-length human CXCL10 with a KD of about 50 nM, or about 40 nM, or about 30 nM, or about 20 nM, or about 10 nM. In one example, the CXCL10 binding protein binds to or specifically binds to full-length human CXCL10 with a KD of about 49 nM.

[0102] In one example, the CXCL10 binding protein binds to or specifically binds to N- terminally truncated human CXCL10 with a KD of 5 nM or less. For example, the CXCL10 binding protein binds to or specifically binds to N-terminally truncated human CXCL10 with a KD of about 5 nM, or about 4 nM, or about 3 nM, or about 2 nM, or about 1 nM. In one example, the CXCL10 binding protein binds to or specifically binds to N-terminally truncated CXCL10 with a KD of about 3 nM.

[0103] In one example, the CXCL10 binding protein that binds full-length human CXCL10, but does not bind N-terminally truncated CXCL10 and citrullinated CXCL10:

[0104] (i) binds to full-length human CXCL10 with a KD of 50 nM or less; and / or

[0105] (ii) requires the N-terminal valine and / or proline of the epitope NH2- VPLSRTVRCTCISISNQPVNPRSLE-COOH) (SEQ ID NO: 25) to bind to full- length human CXCL10.

[0106] In one example, the CXCL10 binding protein requires the N-terminal valine and / or proline of the epitope NH2-VPLSRTVRCTCISISNQPVNPRSLE-COOH (SEQ ID NO: 25) to bind to full-length human CXCL10.

[0107] Methods for determining binding of a CXCL10 binding protein to a polypeptide will be apparent to the skilled artisan. For example, the polypeptide is immobilized on a solid or semi- solid surface and the CXCL10 binding protein is contacted to the immobilized polypeptide. Binding is then determined, e.g., by surface plasmon resonance (SPR) imaging. In one example, the level of binding (e.g., as determined by KD) is measured by surface plasmon resonance (SPR) imaging.

[0108] In one example, the method comprises using a CA-125 binding protein, a HE4 binding protein and / or an IL-6 binding protein to determine the level of CA-125, HE4 and IL-6 in the subject.

[0109] In one example, the method comprises using a CA-125 binding protein to determine the level of CA-125 in the subject.

[0110] In one example, the method comprises using a HE4 binding protein to determine the level of HE4 in the subject.

[0111] In one example, the method comprises using an IL-6 binding protein to determine the level of IL- 6 in the subject.

[0112] In one example, of any method described herein:

[0113] (i) the CA-125 binding protein binds to one or more or all epitopes selected from the group consisting of LYSGCRLTLLRSEKDGAATGVDAICTHR (SEQ ID NO: 27), ERVLQSLLGPMFKNTSVGPLYS (SEQ ID NO: 28) and VDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTH (SEQ ID NO: 29); and / or

[0114] (ii) the HE4 binding protein binds to one or more or all epitopes selected from the group consisting of VNINFPQLGLCR (SEQ ID NO: 30) and CRDQCQVDSQCPGQMKCCRNGCGK (SEQ ID NO: 31); and / or

[0115] (iii) the IL-6 binding protein binds to one or more or all epitopes selected from the group consisting of

[0116] VPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRK (SEQ ID NO: 32), ETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEE (SEQ ID NO: 33), VYLEYLQNRFESSEEQARAVQM (SEQ ID NO: 34) and QKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLI (SEQ ID NO: 35).

[0117] In one example, the CA-125 binding protein binds to one or more or all epitopes selected from the group consisting of LYSGCRLTLLRSEKDGAATGVDAICTHR (SEQ ID NO: 27), ERVLQSLLGPMFKNTSVGPLYS (SEQ ID NO: 28) and VDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTH (SEQ ID NO: 29). For example, the CA-125 binding protein binds to the epitope LYSGCRLTLLRSEKDGAATGVDAICTHR (SEQ ID NO: 27). In one example, the CA-125 binding protein binds to the epitope ERVLQSLLGPMFKNTSVGPLYS (SEQ ID NO: 28). In one example, the CA-125 binding protein binds to the epitope VDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTH (SEQ ID NO: 29). In one example, the CA-125 binding protein binds to the epitope LYSGCRLTLLRSEKDGAATGVDAICTHR (SEQ ID NO: 27) and ERVLQSLLGPMFKNTSVGPLYS (SEQ ID NO: 28). In another example, the CA-125 binding protein binds to the epitope LYSGCRLTLLRSEKDGAATGVDAICTHR (SEQ ID NO: 27) and VDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTH (SEQ ID NO: 29). In a further example, the CA-125 binding protein binds to the epitope ERVLQSLLGPMFKNTSVGPLYS (SEQ ID NO: 28) and

[0118] VDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTH (SEQ ID NO: 29). In another example, the CA-125 binding protein binds to the epitope LYSGCRLTLLRSEKDGAATGVDAICTHR (SEQ ID NO: 27) and ERVLQSLLGPMFKNTSVGPLYS (SEQ ID NO: 28) and

[0119] VDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTH (SEQ ID NO: 29).

[0120] In one example, the HE4 binding protein binds to one or more or all epitopes selected from the group consisting of VNINFPQLGLCR (SEQ ID NO: 30) and CRDQCQVDSQCPGQMKCCRNGCGK (SEQ ID NO: 31). For example, the HE4 binding protein binds to the epitope VNINFPQLGLCR (SEQ ID NO: 30). In another example, the HE4 binding protein binds to the epitope CRDQCQVDSQCPGQMKCCRNGCGK (SEQ ID NO: 31). In a further example, the HE4 binding protein binds to the epitopes VNINFPQLGLCR (SEQ ID NO: 30) and CRDQCQVDSQCPGQMKCCRNGCGK (SEQ ID NO: 31).

[0121] In one example, the IL-6 binding protein binds to one or more or all epitopes selected from the group consisting of

[0122] VPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRK (SEQ ID NO: 32), ETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEE (SEQ ID NO: 33), VYLEYLQNRFESSEEQARAVQM (SEQ ID NO: 34) and

[0123] QKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLI (SEQ ID NO: 35). In one example, the IL-6 binding protein binds to the epitope VPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRK (SEQ ID NO: 32). In another example, the IL-6 binding protein binds to the epitope ETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEE (SEQ ID NO: 33). In a further example, the IL-6 binding protein binds to the epitope VYLEYLQNRFESSEEQARAVQM (SEQ ID NO: 34). In one example, the IL-6 binding protein binds to the epitope

[0124] QKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLI (SEQ ID NO: 35). In another example, the IL-6 binding protein binds to the epitopes

[0125] VPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRK (SEQ ID NO: 32), ETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEE (SEQ ID NO: 33), VYLEYLQNRFESSEEQARAVQM (SEQ ID NO: 34) and

[0126] QKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLI (SEQ ID NO: 35).

[0127] In one example, the binding protein of the present disclosure comprises a variable region or an antigen binding domain.

[0128] In one example, the binding protein is selected from the group consisting of:

[0129] (i) a Fv;

[0130] (ii) a single chain Fv fragment (scFv);

[0131] (iii) a dimeric scFv (di-scFv);

[0132] (iv) a single-domain antibody;

[0133] (v) a minibody;

[0134] (vi) a diabody;

[0135] (vii) a triabody;

[0136] (viii) a tetrabody;

[0137] (ix) a Fab;

[0138] (x) a F(ab’)2;

[0139] (xi) an antibody;

[0140] (xii) an antibody mimetic;

[0141] (xiii) a heavy chain only immunoglobulin;

[0142] (xiv) a T-cell receptor;

[0143] (xv) an adnectin;

[0144] (xvi) an anticalin;

[0145] (xvii) an affibody;

[0146] (xviii) an avimer;

[0147] (xix) a designed ankyrin-repeat protein (DARPin); and

[0148] (xx) one of (i) to (xix) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and / or CH3.

[0149] In one example, the binding protein comprises an antigen binding domain of an antibody. For example, the binding protein comprises at least a VH and a VL, wherein the VH and VL bind to form a Fv comprising an antigen binding domain.

[0150] In one example, the CXCL10 binding protein comprises an antigen binding domain of an antibody. For example, the CXCL10 binding protein comprises at least a VH and a VL, wherein the VH and VL bind to form a Fv comprising an antigen binding domain. In one example, the CA-125 binding protein comprises an antigen binding domain of an antibody. For example, the CA-125 binding protein comprises at least a VH and a VL, wherein the VH and VL bind to form a Fv comprising an antigen binding domain.

[0151] In one example, the HE4 binding protein comprises an antigen binding domain of an antibody. For example, the HE4 binding protein comprises at least a VH and a VL, wherein the VH and VL bind to form a Fv comprising an antigen binding domain.

[0152] In one example, the IL-6 binding protein comprises an antigen binding domain of an antibody. For example, the IL-6 binding protein comprises at least a VH and a VL, wherein the VH and VL bind to form a Fv comprising an antigen binding domain.

[0153] In one example, the binding protein is an antibody or antigen binding fragment thereof (e.g., a scFv comprising the variable regions of the antibody). Exemplary antibodies are full-length and / or naked (e.g., unconjugated) antibodies. In one example, an antibody of the present disclosure is a full length antibody.

[0154] In one example, the antibody is an IgG or an IgE or an IgM or an IgD or an IgA or an IgY antibody. For example, the antibody is an IgG antibody.

[0155] In one example, the IgG antibody is an IgGi or an IgGi or an IgGs or an IgG4. For example, the antibody is an IgGi antibody. In another example, the antibody is an IgG4antibody. In one example, the antibody is a stabilized IgG4antibody.

[0156] In one example, the binding protein is recombinant, chimeric, CDR grafted, humanized, synhumanized, primatized, deimmunized or human.

[0157] In one example, the antigen binding fragment of the present disclosure is a half antibody. For example, the binding protein is a half antibody comprising a single heavy chain and a single light chain.

[0158] In one example, the antigen binding fragment of the present disclosure comprises an IgG4constant region or a stabilised IgG4constant region.

[0159] In one example, the binding protein is an antibody mimetic. For example, the binding protein comprises an antigen binding domain of an immunoglobulin, e.g., an IgNAR, a camelid antibody or a T cell receptor.

[0160] 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 variable region thereof.

[0161] In one example, the CXCL10 binding protein competitively inhibits the binding of an antibody or antigen binding fragment thereof to CXCL10 comprising:

[0162] (i) a heavy chain variable region (VH) comprising an amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region (VL) comprising an amino acid sequence set forth in SEQ ID NO: 4; and / or (ii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 11 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 12.

[0163] In one example, the CXCL10 binding protein competitively inhibits the binding of an antibody or antigen binding fragment thereof to CXCL10 comprising a VH comprising an amino acid sequence set forth in SEQ ID NO: 3 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 4.

[0164] In another example, the CXCL10 binding protein competitively inhibits the binding of an antibody or antigen binding fragment thereof to CXCL10 comprising a VH comprising an amino acid sequence set forth in SEQ ID NO: 11 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 12.

[0165] In one example, the CXCL10 binding protein comprises:

[0166] (i) a VH comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 3 and a VL comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 4; or

[0167] (ii) a VH comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 11 and a VL comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 12.

[0168] In one example, the CXCL10 binding protein comprises a VH comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 3 and a VL comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 4. For example, the CXCL10 binding protein comprises a VH and / or a VL which is at least 90% or 95% or 97% or 98% or 99% identical to a sequence disclosed herein.

[0169] In one example, the CXCL10 binding protein comprises a VH comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 11 and a VL comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 12. For example, the CXCL10 binding protein comprises a VH and / or a VL which is at least 90% or 95% or 97% or 98% or 99% identical to a sequence disclosed herein.

[0170] In one example, the binding proteins of the present disclosure optionally comprises one or more amino acid substitutions, deletions or insertions of any sequence disclosed herein. Amino acid substitutions suitable for use in the present disclosure will be apparent to the skilled person and include naturally-occurring substitutions and engineered substitutions.

[0171] In one example, the CXCL10 binding protein of the present disclosure is an antibody or antigen binding fragment thereof comprising:

[0172] (i) a VH comprising an amino acid sequence set forth in SEQ ID NO: 3 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 4; or (ii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 11 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 12.

[0173] In one example, the CXCL10 binding protein of the present disclosure is an antibody or antigen binding fragment thereof comprising a VH comprising an amino acid sequence set forth in SEQ ID NO: 3 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 4.

[0174] In another example, the CXCL10 binding protein of the present disclosure is an antibody or antigen binding fragment thereof comprising a VH comprising an amino acid sequence set forth in SEQ ID NO: 11 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 12.

[0175] In one example, the CXCL10 binding protein of the present disclosure is an antibody or antigen binding fragment thereof comprising:

[0176] (i) a VH comprising: a) a CDR1 comprising a sequence set forth in amino acids 25-34 of SEQ ID NO: 3; and b) a CDR2 comprising a sequence set forth in amino acids 49-65 of SEQ ID NO: 3; and c) a CDR3 comprising a sequence set forth in amino acids 98-108 of SEQ ID NO: 3; and a VL comprising: a) a CDR1 comprising a sequence set forth in amino acids 23-33 of SEQ ID NO: 4; and b) a CDR2 comprising a sequence set forth in amino acids 49-55 of SEQ ID NO: 4; and c) a CDR3 comprising a sequence set forth in amino acids 88-96 of SEQ ID NO: 4; or

[0177] (ii) a VH comprising: a) a CDR1 comprising a sequence set forth in amino acids 25-34 of SEQ ID NO: 11 ; and b) a CDR2 comprising a sequence set forth in amino acids 49-65 of SEQ ID NO: 11 ; and c) a CDR3 comprising a sequence set forth in amino acids 98-108 of SEQ ID NO: 11; and a VL comprising: a) a CDR1 comprising a sequence set forth in amino acids 23-33 of SEQ ID NO: 12; and b) a CDR2 comprising a sequence set forth in amino acids 49-55 of SEQ ID NO: 12; and c) a CDR3 comprising a sequence set forth in amino acids 88-96 of SEQ ID NO: 12.

[0178] In one example, the CXCL10 binding protein of the present disclosure is an antibody or antigen binding fragment thereof comprising:

[0179] (i) a VH comprising: a) a CDR1 comprising a sequence set forth in amino acids 25-34 of SEQ ID NO: 3; and b) a CDR2 comprising a sequence set forth in amino acids 49-65 of SEQ ID NO: 3; and c) a CDR3 comprising a sequence set forth in amino acids 98-108 of SEQ ID NO: 3; and

[0180] (ii) a VL comprising: a) a CDR1 comprising a sequence set forth in amino acids 23-33 of SEQ ID NO: 4; and b) a CDR2 comprising a sequence set forth in amino acids 49-55 of SEQ ID NO: 4; and c) a CDR3 comprising a sequence set forth in amino acids 88-96 of SEQ ID NO: 4.

[0181] In one example, the CXCL10 binding protein of the present disclosure is an antibody or antigen binding fragment thereof comprising:

[0182] (i) a VH comprising: a) a CDR1 comprising a sequence set forth in amino acids 25-34 of SEQ ID NO: 11 ; and b) a CDR2 comprising a sequence set forth in amino acids 49-65 of SEQ ID NO: 11 ; and c) a CDR3 comprising a sequence set forth in amino acids 98-108 of SEQ ID NO: 11; and

[0183] (ii) a VL comprising: a) a CDR1 comprising a sequence set forth in amino acids 23-33 of SEQ ID NO: 12; and b) a CDR2 comprising a sequence set forth in amino acids 49-55 of SEQ ID NO: 12; and c) a CDR3 comprising a sequence set forth in amino acids 88-96 of SEQ ID NO: 12. In one example, the CXCL10 binding protein of the present disclosure is an antibody or antigen binding fragment thereof comprising:

[0184] (i) a VH comprising a) a CDR1 comprising a sequence set forth in SEQ ID NO: 5; and b) a CDR2 comprising a sequence set forth in SEQ ID NO: 6; and c) a CDR3 comprising a sequence set forth in SEQ ID NO: 7; and a VL comprising: a) a CDR1 comprising a sequence set forth in SEQ ID NO: 8; and b) a CDR2 comprising a sequence set forth in SEQ ID NO: 9; and c) a CDR3 comprising a sequence set forth in SEQ ID NO: 10; or

[0185] (ii) a VH comprising a) a CDR1 comprising a sequence set forth in SEQ ID NO: 13; and b) a CDR2 comprising a sequence set forth in SEQ ID NO: 14; and c) a CDR3 comprising a sequence set forth in SEQ ID NO: 15; and a VL comprising: a) a CDR1 comprising a sequence set forth in SEQ ID NO: 16; and b) a CDR2 comprising a sequence set forth in SEQ ID NO: 17; and c) a CDR3 comprising a sequence set forth in SEQ ID NO: 18.

[0186] In one example, the CXCL10 binding protein of the present disclosure is an antibody or antigen binding fragment thereof comprising:

[0187] (i) a VH comprising a) a CDR1 comprising a sequence set forth in SEQ ID NO: 5; and b) a CDR2 comprising a sequence set forth in SEQ ID NO: 6; and c) a CDR3 comprising a sequence set forth in SEQ ID NO: 7; and

[0188] (ii) a VL comprising: a) a CDR1 comprising a sequence set forth in SEQ ID NO: 8; and b) a CDR2 comprising a sequence set forth in SEQ ID NO: 9; and c) a CDR3 comprising a sequence set forth in SEQ ID NO: 10.

[0189] In one example, the CXCL10 binding protein of the present disclosure is an antibody or antigen binding fragment thereof comprising:

[0190] (i) a VH comprising a) a CDR1 comprising a sequence set forth in SEQ ID NO: 13; and b) a CDR2 comprising a sequence set forth in SEQ ID NO: 14; and c) a CDR3 comprising a sequence set forth in SEQ ID NO: 15; and

[0191] (ii) a VL comprising: a) a CDR1 comprising a sequence set forth in SEQ ID NO: 16; and b) a CDR2 comprising a sequence set forth in SEQ ID NO: 17; and c) a CDR3 comprising a sequence set forth in SEQ ID NO: 18.

[0192] In one example, the protein, antibody or antigen binding fragment thereof is any form of the protein, antibody or functional fragment thereof encoded by a nucleic acid encoding any of the foregoing proteins, antibodies or functional fragments.

[0193] In one example, the binding protein is conjugated to a detectable label. For example, the CXCL10, CA-125, HE4 and / or IL-6 binding protein is conjugated to a detectable label. Detectable labels suitable for use in the present disclosure will be apparent to the skilled person 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 biolumine scent label, a magnetic label, a prosthetic group and a contrast agent.

[0194] In one example, the detectable label is a radiolabel. For example, the radiolabel can be, but is not limited to, radioiodine (1251, 1311); technetium; yttrium; 35S or 3H.

[0195] In one example, the detectable label is an enzyme. For example, the enzyme can be, but is not limited to, horseradish peroxidase, alkaline phosphatase, P-galactosidase, or acetylcholinesterase.

[0196] In one example, the detectable label is a fluorescent label. For example, the fluorescent label can be, but is not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.

[0197] In one example, the detectable label is a luminescent label. For example, the luminescent label can be, but is not limited to, luminol.

[0198] In one example, the detectable label is a bioluminescent label. For example, the bioluminescent label can be, but is not limited to, luciferase, luciferin or aequorin.

[0199] In one example, the detectable label is a magnetic label. For example, the magnetic label can be, but is not limited to, gadolinium or iron-oxide chelate.

[0200] In one example, the detectable label is a prosthetic group. For example, the prosthetic group can be, but is not limited to, streptavidin / biotin or avidin / biotin.

[0201] In one example, the detectable label is a contrast agent.

[0202] The present disclosure also provides a cancer antigen 125 (CA-125) binding protein, wherein the CA-125 binding protein binds to one or more or all epitopes selected from the group consisting of LYSGCRLTLLRSEKDGAATGVDAICTHR (SEQ ID NO: 27), ERVLQSLLGPMFKNTSVGPLYS (SEQ ID NO: 28) and VDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTH (SEQ ID NO: 29). The present disclosure provides a CA-125 binding protein, wherein the CA-125 binding protein binds to the epitope LYSGCRLTLLRSEKDGAATGVDAICTHR (SEQ ID NO: 27).

[0203] The present disclosure also provides a CA-125 binding protein, wherein the CA- 125 binding protein binds to the epitope ERVLQSLLGPMFKNTSVGPLYS (SEQ ID NO: 28).

[0204] The present disclosure further provides a CA-125 binding protein, wherein the CA-125 binding protein binds to the epitope VDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTH (SEQ ID NO: 29).

[0205] The present disclosure provides a CA-125 binding protein, wherein the CA-125 binding protein binds to the epitope LYSGCRLTLLRSEKDGAATGVDAICTHR (SEQ ID NO: 27) and ERVLQSLLGPMFKNTSVGPLYS (SEQ ID NO: 28).

[0206] The present disclosure also provides a CA-125 binding protein, wherein the CA- 125 binding protein binds to the epitope LYSGCRLTLLRSEKDGAATGVDAICTHR (SEQ ID NO: 27) and VDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTH (SEQ ID NO: 29).

[0207] The present disclosure further provides a CA-125 binding protein, wherein the CA-125 binding protein binds to the epitope ERVLQSLLGPMFKNTSVGPLYS (SEQ ID NO: 28) and VDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTH (SEQ ID NO: 29).

[0208] The present disclosure provides a CA-125 binding protein, wherein the CA-125 binding protein binds to the epitope LYSGCRLTLLRSEKDGAATGVDAICTHR (SEQ ID NO: 27) and ERVLQSLLGPMFKNTSVGPLYS (SEQ ID NO: 28) and VDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTH (SEQ ID NO: 29).

[0209] The present disclosure further provides a human epididymis protein 4 (HE4) binding protein, wherein the HE4 binding protein binds to one or more or all epitopes selected from the group consisting of VNINFPQLGLCR (SEQ ID NO: 30) and CRDQCQVDSQCPGQMKCCRNGCGK (SEQ ID NO: 31).

[0210] The present disclosure also provides a HE4 binding protein, wherein the HE4 binding protein binds to the epitope VNINFPQLGLCR (SEQ ID NO: 30).

[0211] The present disclosure provides a HE4 binding protein, wherein the HE4 binding protein binds to the epitope CRDQCQVDSQCPGQMKCCRNGCGK (SEQ ID NO: 31).

[0212] The present disclosure also provides a HE4 binding protein, wherein the HE4 binding protein binds to the epitopes VNINFPQLGLCR (SEQ ID NO: 30) and CRDQCQVDSQCPGQMKCCRNGCGK (SEQ ID NO: 31). The present disclosure also provides an interleukin 6 (IL-6) binding protein, wherein the IL-6 binding protein binds to one or more or all epitopes selected from the group consisting of VPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRK (SEQ ID NO: 32), ETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEE (SEQ ID NO: 33), VYLEYLQNRFESSEEQARAVQM (SEQ ID NO: 34) and QKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLI (SEQ ID NO: 35).

[0213] The present disclosure provides an IL-6 binding protein, wherein the IL-6 binding protein binds to one or more or all epitopes selected from the group consisting of VPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRK (SEQ ID NO: 32), ETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEE (SEQ ID NO: 33), VYLEYLQNRFESSEEQARAVQM (SEQ ID NO: 34) and

[0214] QKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLI (SEQ ID NO: 35).

[0215] The present disclosure provides an IL-6 binding protein, wherein the IL-6 binding protein binds to the epitope

[0216] VPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRK (SEQ ID NO: 32).

[0217] The present disclosure provides an IL-6 binding protein, wherein the IL-6 binding protein binds to the epitope

[0218] ETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEE (SEQ ID NO: 33).

[0219] The present disclosure provides an IL-6 binding protein, wherein the IL-6 binding protein binds to the epitope VYLEYLQNRFESSEEQARAVQM (SEQ ID NO: 34).

[0220] The present disclosure provides an IL-6 binding protein, wherein the IL-6 binding protein binds to the epitope

[0221] QKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLI (SEQ ID NO: 35).

[0222] The present disclosure provides an IL-6 binding protein, wherein the IL-6 binding protein binds to the epitopes

[0223] VPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRK (SEQ ID NO: 32), ETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEE (SEQ ID NO: 33), VYLEYLQNRFESSEEQARAVQM (SEQ ID NO: 34) and

[0224] QKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLI (SEQ ID NO: 35).

[0225] The present disclosure also provides a composition comprising the CA-125 binding protein of the present disclosure and a carrier.

[0226] The present disclosure also provides a composition comprising the HE4 binding protein of the present disclosure and a carrier.

[0227] The present disclosure also provides a composition comprising the IL-6 binding protein of the present disclosure and a carrier. Carriers suitable for use in the present disclosure will be apparent to the skilled person and / or are described herein.

[0228] The present disclosure also provides a polynucleotide encoding the CA-125 binding protein according to the present disclosure.

[0229] The present disclosure also provides a polynucleotide encoding the HE4 binding protein according to the present disclosure.

[0230] The present disclosure also provides a polynucleotide encoding the IL-6 binding protein according to the present disclosure.

[0231] The present disclosure further provides an expression vector comprising the polynucleotide encoding the CA-125 binding protein of the present disclosure.

[0232] The present disclosure further provides an expression vector comprising the polynucleotide encoding the HE4 binding protein of the present disclosure.

[0233] The present disclosure further provides an expression vector comprising the polynucleotide encoding the IL-6 binding protein of the present disclosure.

[0234] Exemplary vectors suitable for use in the present disclosure will be apparent to the skilled person and / or are described herein.

[0235] The present disclosure further provides a cell comprising the expression vector of the present disclosure in vitro. Exemplary cells suitable for use in the present disclosure will be apparent to the skilled person and / or are described herein.

[0236] In one example, the present disclosure provides use of the cell for preparing a CA- 125 binding protein of the present disclosure. For example, the use comprises culturing the cell of the disclosure and producing the CA-125 binding protein therefrom; and isolating and purifying the produced binding protein.

[0237] In one example, the present disclosure provides use of the cell for preparing a HE4 binding protein of the present disclosure. For example, the use comprises culturing the cell of the disclosure and producing the HE4 binding protein therefrom; and isolating and purifying the produced binding protein.

[0238] In one example, the present disclosure provides use of the cell for preparing an IL-6 binding protein of the present disclosure. For example, the use comprises culturing the cell of the disclosure and producing the IL-6 binding protein therefrom; and isolating and purifying the produced binding protein.

[0239] Methods of isolating and purifying the produced binding protein will be apparent to the skilled person and / or are described herein.

[0240] Methods of detecting the level of CXCL10 will be apparent to the skilled person and / or described herein. For example, the method comprises performing flow cytometry, an enzyme-linked immunosorbent assay or western blot. In one example, the method comprises performing flow cytometry.

[0241] In one example, the method comprises performing an enzyme-linked immunosorbent assay.

[0242] In one example, the method comprises performing western blot.

[0243] In one example, the method is performed on the subject in vitro or ex vivo. For example, the method is performed on the subject in vitro. In another example, the method is performed on the subject ex vivo.

[0244] In one example, the method is performed on at least one biological sample obtained from the subject. Suitable biological samples for use in the present disclosure will be apparent to the skilled person 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 cellular swab.

[0245] In one example, the biological sample is a biopsy.

[0246] In one example, the biological sample is a fluid sample. For example, the fluid sample is cervical fluid, vaginal fluid or ascites. In one example, the biological sample is ascites fluid.

[0247] In one example, the biological sample is a plasma sample.

[0248] In one example, the biological sample is a cellular swab. For example, the cellular swab is a cervical swab. In one example, the cellular swab is a cervicovaginal swab (CVS).

[0249] In one example, the method is performed on a plasma sample and a cervicovaginal swab (CVS).

[0250] The present disclosure also provides a method of monitoring tumour burden, monitoring progression and / or determining tumour regression in a subject suffering from a malignant condition, the method comprising:

[0251] (i) determining a C-X-C motif chemokine ligand 10 (CXCL10) ratio of active CXLC10 to total CXCL10 in the subject at one or more time points; and

[0252] (ii) determining the level of cancer antigen 125 (CA-125), human epididymis protein

[0253] 4 (HE4) and interleukin 6 (IL-6) in the subject at one or more time points.

[0254] The present disclosure provides a method of monitoring tumour burden in a subject suffering from a malignant condition, the method comprising:

[0255] (i) determining a C-X-C motif chemokine ligand 10 (CXCL10) ratio of active CXLC10 to total CXCL10 in the subject at one or more time points; and

[0256] (ii) determining the level of cancer antigen 125 (CA-125), human epididymis protein

[0257] 4 (HE4) and interleukin 6 (IL-6) in the subject at one or more time points. The present disclosure also provides a method of monitoring tumour progression in a subject suffering from a malignant condition, the method comprising:

[0258] (i) determining a C-X-C motif chemokine ligand 10 (CXCL10) ratio of active CXLC10 to total CXCL10 in the subject at one or more time points; and

[0259] (ii) determining the level of cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) in the subject at one or more time points.

[0260] The present disclosure also provides a method of determining tumour regression in a subject suffering from a malignant condition, the method comprising:

[0261] (i) determining a C-X-C motif chemokine ligand 10 (CXCL10) ratio of active CXLC10 to total CXCL10 in the subject at one or more time points; and

[0262] (ii) determining the level of cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) in the subject at one or more time points.

[0263] The present disclosure also provides a method of determining efficacy of a treatment for a malignant condition in a subject suffering from a malignant condition, the method comprising:

[0264] (i) determining a C-X-C motif chemokine ligand 10 (CXCL10) ratio of active CXLC10 to total CXCL10 in the subject at one or more time points; and

[0265] (ii) determining the level of cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) in the subject at one or more time points.

[0266] In one example, the subject has been diagnosed as having a malignant condition. For example, the subject is suffering from a malignant condition. For example, the malignant condition is a reproductive cancer, such as ovarian cancer.

[0267] In one example, the subject is asymptomatic.

[0268] In one example, the subject has not received treatment for the malignant condition. For example, the subject is treatment naive. In one example, the subject is receiving treatment for the malignant condition. In another example, the subject has received treatment for the malignant condition. Suitable therapies for the treatment of the malignant condition will be apparent to the skilled person and / or described herein. For example, the treatment comprises surgery, chemotherapy, radiation therapy, targeted drug therapy, immunotherapy or a combination thereof.

[0269] In one example, the treatment comprises surgery.

[0270] In one example, the treatment comprises chemotherapy.

[0271] In one example, the treatment comprises radiation therapy.

[0272] In one example, the treatment comprises targeted drug therapy.

[0273] In one example, the treatment comprises immunotherapy. In one example, the method comprises determining the diagnostic score at one or more time points.

[0274] In one example, a score of >3.6 in the subject is indicative of increased tumour burden and / or tumour progression and / or tumour recurrence in the subject. For example, a score of >3.6 in the subject after treatment (i.e., at the subsequence time point) compared to before treatment (i.e., the first time point) is indicative of increased tumour burden and / or tumour progression and / or tumour recurrence in the subject.

[0275] In one example, a score of <3.6 in the subject is indicative of decreased tumour burden and / or tumour regression and / or tumour recurrence in the subject. For example, a score of <3.6 in the subject after treatment (i.e., at the subsequence time point) compared to before treatment (i.e., the first time point) is indicative of decreased tumour burden and / or tumour regression and / or tumour recurrence in the subject.

[0276] In one example of any method described herein, the method further comprises administering a treatment to reduce the tumour burden and / or tumour progression in the subject.

[0277] The present disclosure also provides a method of detecting and / or diagnosing malignant ovarian cancer in a subject, the method comprising:

[0278] (i) determining a C-X-C motif chemokine ligand 10 (CXCL10) ratio of active CXLC10 to total CXCL10 in at least one biological sample obtained from the subject; and

[0279] (ii) determining the level of cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) in at least one biological sample obtained from the subject, and

[0280] (iii) detecting and / or diagnosing malignant ovarian cancer in the subject based on the level determined in (i) and (ii); wherein the method further comprises administering or having administered a therapeutically effective amount of a treatment to the subject detected and / or diagnosed as having malignant ovarian cancer.

[0281] 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 according to the disclosure, and administering or having administered a treatment to the subject.

[0282] The present disclosure also provides a method of treating a subject identified as suffering from malignant ovarian cancer, the method comprising administering a therapeutically effective amount of a treatment to a subject, wherein the subject was identified as suffering from malignant ovarian cancer based on a C-X-C motif chemokine ligand 10 (CXCL10) ratio of active CXLC10 to total CXCL10 and a level of cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) in at least one biological sample obtained from the subject.

[0283] In one example, the treatment comprises surgery, chemotherapy, targeted drug therapy or a combination thereof.

[0284] The present disclosure also provides a kit or panel for assessing the risk of a subject suffering from malignant ovarian cancer, the kit or panel comprising one or more binding proteins for detecting a level of active C-X-C motif chemokine ligand 10 (CXCL10), total CXCL10, cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) in the subject.

[0285] The present disclosure also provides a kit or panel for detecting and / or diagnosing malignant ovarian cancer in a subject, the kit or panel comprising one or more binding proteins for detecting a level of active C-X-C motif chemokine ligand 10 (CXCL10), total CXCL10, cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) in the subject.

[0286] The present disclosure also provides a kit or panel for monitoring tumour burden, monitoring progression, determining tumour regression, determining tumour recurrence and / or determining efficacy of a treatment of a malignant ovarian cancer in a subject, the kit or panel comprising one or more binding proteins for detecting a level of active C-X- C motif chemokine ligand 10 (CXCL10), total CXCL10, cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) in the subject.

[0287] The present disclosure provides a panel of biomarkers for the diagnosis of malignant ovarian cancer consisting of active C-X-C motif chemokine ligand 10 (CXCL10), total CXCL10, cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6), wherein the biomarkers are bound to capture reagents attached to a substrate.

[0288] The present disclosure also provides a method for determining ovarian cancer status, the method comprising measuring the level of the panel of biomarkers of the disclosure using an immunoassay or mass spectroscopy. For example, the panel of biomarkers consists of active CXCL10, total CXCL10, CA-125, HE4 and IL-6.

[0289] The present disclosure further provides a method of detecting a panel of biomarkers in a sample obtained from a subject, the method comprising:

[0290] (i) contacting the sample from the subject to a substrate;

[0291] (ii) binding a panel of biomarkers in the sample to capture reagents attached to the substrate; wherein the panel of biomarkers consist of active C-X-C motif chemokine ligand 10 (CXCL10), total CXCL10, cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6), thereby detecting the panel of biomarkers in the sample from the subject.

[0292] In one example, the capture reagents are binding proteins against active CXCL10, total CXCL10, CA-125, HE4 and IL-6. For example, the capture reagents are binding proteins, wherein each binding protein comprises an antigen binding domain of an antibody against active CXCL10, total CXCL10, CA-125, HE4 or IL-6. In one example, the capture reagents are antibodies against active CXCL10, total CXCL10, CA-125, HE4 and IL-6 as described herein.

[0293] The present disclosure also provides a CXCL10 binding protein, wherein the CXCL10 binding protein binds to the epitope EIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP (SEQ ID NO: 36).

[0294] The present disclosure further provides a method for determining the CXCL10 active ratio in a sample, the method comprising:

[0295] (i) contacting a sample to a capture protein comprising an antibody variable region immobilized on a solid surface, wherein the immobilized capture protein binds to a CXCL10 protein to thereby form a protein complex;

[0296] (ii) contacting the protein complex with a first detection protein comprising an antibody variable region, wherein the first detection protein binds to full-length human CXCL10, N-terminally truncated CXCL10 and citrullinated CXCL10 and wherein the first detection protein comprises a first detectable label;

[0297] (iii)contacting the protein complex with a second detection protein comprising an antibody variable region, wherein the second detection protein binds to full- length human CXCL10 with a KD of 50 nM or less, but does not bind N- terminally truncated CXCL10 and citrullinated CXCL10 and wherein the second detection protein comprises a second detectable label;

[0298] (iv) detecting the first detectable label, wherein the presence of the first detectable label is indicative of the presence of full-length human CXCL10, N-terminally truncated CXCL10 and citrullinated CXCL10 in the sample; and

[0299] (v) detecting the second detectable label, wherein the presence of the second detectable label is indicative of the presence of full-length human CXCL10 only in the sample.

[0300] In one example, the detectable label is a prosthetic group. For example, the prosthetic group can be, but is not limited to, streptavidin / biotin or avidin / biotin. In one example, the detectable label is streptavidin / biotin. In another example, the detectable label is avidin / biotin.

[0301] In one example, the method further comprises immobilizing the capture protein onto the solid surface.

[0302] The present disclosure further provides a method for determining the CXCL10 active ratio in a sample, the method comprising:

[0303] (i) immobilising a capture protein comprising an antibody variable region onto a solid surface;

[0304] (ii) contacting a sample to a capture protein comprising an antibody variable region immobilized on a solid surface, wherein the immobilized capture protein binds to a CXCL10 protein to thereby form a protein complex;

[0305] (iii)contacting the protein complex with a first detection protein comprising an antibody variable region, wherein the first detection protein binds to full-length human CXCL10, N-terminally truncated CXCL10 and citrullinated CXCL10 and wherein the first detection protein comprises a first detectable label;

[0306] (iv) contacting the protein complex with a second detection protein comprising an antibody variable region, wherein the second detection protein binds to full- length human CXCL10 with a KD of 50 nM or less, but does not bind N- terminally truncated CXCL10 and citrullinated CXCL10 and wherein the second detection protein comprises a second detectable label;

[0307] (v) detecting the first detectable label, wherein the presence of the first detectable label is indicative of the presence of full-length human CXCL10, N-terminally truncated CXCL10 and citrullinated CXCL10 in the sample; and

[0308] (vi) detecting the second detectable label, wherein the presence of the second detectable label is indicative of the presence of full-length human CXCL10 only in the sample.

[0309] In one example, the method additionally comprises providing the sample.

[0310] In one example, the method additionally provides after step (ii) the solid surface is washed to remove unbound protein. For example, after contacting the sample to the capture protein comprising an antibody variable region immobilized on the solid surface, the solid surface is washed to remove unbound protein.

[0311] In one example, the method additionally provides after step (iii) and step (iv) the solid surface is washed to remove unbound protein. For example, after contacting the protein complex with a detection protein comprising an antibody variable region, the solid surface is washed to remove unbound protein. In one example, the capture protein is a CXCL10 binding protein, wherein the CXCL10 binding protein binds to the epitope EIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP (SEQ ID NO: 36).

[0312] In one example, the first detection protein binds the epitope NH2- LSRTVRCTCISISNQPVNPRSLE-COOH (SEQ ID NO: 26) of full-length human CXCL10, N-terminally truncated CXCL10 and citrullinated CXCL10.

[0313] In one example, the first detection protein is an antibody or antigen binding fragment thereof comprising a VH comprising an amino acid sequence set forth in SEQ ID NO: 11 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 12.

[0314] In one example, the second detection protein requires the N-terminal valine and / or proline of the epitope NH2-VPLSRTVRCTCISISNQPVNPRSLE-COOH) (SEQ ID NO: 25) to bind to full-length human CXCL10.

[0315] In one example, the second detection protein is an antibody or antigen binding fragment thereof comprising a VH comprising an amino acid sequence set forth in SEQ ID NO: 3 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 4.

[0316] The present disclosure further provides a method for determining the CXCL10 active ratio in a sample, the method comprising:

[0317] (i) contacting a sample to a capture protein comprising a CXCL10 binding protein that binds to the epitope

[0318] EIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP (SEQ ID NO: 36) immobilized on a solid surface, wherein the immobilized capture protein binds to a CXCL10 protein to thereby form a protein complex;

[0319] (ii) contacting the protein complex with a first detection protein comprising an antibody variable region, wherein the first detection protein binds the epitope NH2-LSRTVRCTCISISNQPVNPRSLE-COOH (SEQ ID NO: 26) of full-length human CXCL10, N-terminally truncated CXCL10 and citrullinated CXCLIOand wherein the first detection protein comprises a first detectable label;

[0320] (iii)contacting the protein complex with a second detection protein comprising an antibody variable region, wherein the second detection protein requires the N- terminal valine and / or proline of the epitope NH2- VPLSRTVRCTCISISNQPVNPRSLE-COOH) (SEQ ID NO: 25) to bind to full- length human CXCL10 but does not bind N-terminally truncated CXCL10 and citrullinated CXCL10 and wherein the second detection protein comprises a second detectable label; (iv) detecting the first detectable label, wherein the presence of the first detectable label is indicative of the presence of full-length human CXCL10, N-terminally truncated CXCL10 and citrullinated CXCL10 in the sample; and

[0321] (v) detecting the second detectable label, wherein the presence of the second detectable label is indicative of the presence of full-length human CXCL10 only in the sample.

[0322] The present disclosure further provides a method for determining the CXCL10 active ratio in a sample, the method comprising:

[0323] (i) contacting a sample to a capture protein comprising a CXCL10 binding protein that binds to the epitope

[0324] EIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP (SEQ ID NO: 36) immobilized on a solid surface, wherein the immobilized capture protein binds to a CXCL10 protein to thereby form a protein complex;

[0325] (ii) contacting the protein complex with a first detection protein comprising a VH comprising an amino acid sequence set forth in SEQ ID NO: 11 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 12 and wherein the first detection protein comprises a first detectable label;

[0326] (iii)contacting the protein complex with a second detection protein comprising a VH comprising an amino acid sequence set forth in SEQ ID NO: 3 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 4 and wherein the second detection protein comprises a second detectable label;

[0327] (iv) detecting the first detectable label, wherein the presence of the first detectable label is indicative of the presence of full-length human CXCL10, N-terminally truncated CXCL10 and citrullinated CXCL10 in the sample; and

[0328] (v) detecting the second detectable label, wherein the presence of the second detectable label is indicative of the presence of full-length human CXCL10 only in the sample.

[0329] BRIEF DESCRIPTION OF THE DRAWINGS

[0330] Figure 1 is a graphical representation showing median marker values and linear discriminants for individual parameters used in scoring. (A) Individual analytes and / or calculated scores (ROMA, RMI2) within the cohort, according to disease type and stage. Sample numbers are provided in Tables 3 and 4. *p<0.01; **p<0.01; ***p<0.001; ****p<0.0001. (B) Linear discriminant coefficients between groups for each parameter evaluated. Figure 2 is a graphical representation showing performance of individual scoring systems for discrimination between benign and malignant disease. (A) ROC curves were constructed to assess each scoring system (multi-marker panel, CA125, RMI2 and ROMA). (B) Violin plots demonstrating comparative scoring across all samples for each scoring system. Sample numbers are as indicated in Tables 3 and 4.

[0331] KEY TO SEQUENCE LISTING

[0332] SEQ ID NO: 1 amino acid sequence of human CXCL10 including pre- sequence

[0333] SEQ ID NO: 2 amino acid sequence of mature human CXCL10

[0334] SEQ ID NO: 3 heavy chain VH amino acid sequence of anti-CXCLIO antibody

[0335] RA2

[0336] SEQ ID NO: 4 light chain VL amino acid sequence of anti-CXCLIO antibody

[0337] RA2

[0338] SEQ ID NO: 5 heavy chain VH CDR1 amino acid sequence of anti-CXCLIO antibody RA2

[0339] SEQ ID NO: 6 heavy chain VH CDR2 amino acid sequence of anti-CXCLIO antibody RA2

[0340] SEQ ID NO: 7 heavy chain VH CDR3 amino acid sequence of anti-CXCLIO antibody RA2

[0341] SEQ ID NO: 8 light chain VL CDR1 amino acid sequence of anti-CXCLIO antibody RA2

[0342] SEQ ID NO: 9 light chain VL CDR2 amino acid sequence of anti-CXCLIO antibody RA2

[0343] SEQ ID NO: 10 light chain VL CDR3 amino acid sequence of anti-CXCLIO antibody RA2

[0344] SEQ ID NO: 11 heavy chain VH amino acid sequence of anti-CXCLIO antibody RG2

[0345] SEQ ID NO: 12 light chain VL amino acid sequence of anti-CXCLIO antibody

[0346] RG2

[0347] SEQ ID NO: 13 heavy chain VH CDR1 amino acid sequence of anti-CXCLIO antibody RG2

[0348] SEQ ID NO: 14 heavy chain VH CDR2 amino acid sequence of anti-CXCLIO antibody RG2

[0349] SEQ ID NO: 15 heavy chain VH CDR3 amino acid sequence of anti-CXCLIO antibody RG2 SEQ ID NO: 16 light chain VL CDR1 amino acid sequence of anti-CXCLIO antibody RG2

[0350] SEQ ID NO: 17 light chain VL CDR2 amino acid sequence of anti-CXCLIO antibody RG2

[0351] SEQ ID NO: 18 light chain VL CDR3 amino acid sequence of anti-CXCLIO antibody RG2

[0352] SEQ ID NO: 19 heavy chain VH nucleotide sequence of anti-CXCLIO antibody

[0353] RA2

[0354] SEQ ID NO: 20 light chain VL nucleotide acid sequence of anti-CXCLIO antibody

[0355] RA2

[0356] SEQ ID NO: 21 heavy chain VH nucleotide sequence of anti-CXCLIO antibody

[0357] RG2

[0358] SEQ ID NO: 22 light chain VL nucleotide acid sequence of anti-CXCLIO antibody

[0359] RG2

[0360] SEQ ID NO: 23 peptide sequence comprising the intact N-terminus of human

[0361] CXCL10

[0362] SEQ ID NO: 24 peptide sequence comprising the truncated N-terminus of human

[0363] CXCL10

[0364] SEQ ID NO: 25 Intact N-terminus epitope of human CXCL10

[0365] SEQ ID NO: 26 Truncated N-terminus epitope of human CXCL10

[0366] SEQ ID NO: 27 Epitope of human CA-125

[0367] SEQ ID NO: 28 Epitope of human CA-125

[0368] SEQ ID NO: 29 Epitope of human CA-125

[0369] SEQ ID NO: 30 Epitope of human HE4

[0370] SEQ ID NO: 31 Epitope of human HE4

[0371] SEQ ID NO: 32 Epitope of human IL-6

[0372] SEQ ID NO: 33 Epitope of human IL-6

[0373] SEQ ID NO: 34 Epitope of human IL-6

[0374] SEQ ID NO: 35 Epitope of human IL-6

[0375] SEQ ID NO: 36 Epitope of human CXCL10 DETAILED DESCRIPTION OF THE INVENTION

[0376] General

[0377] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. For example, reference to “a” includes a single as well as two or more; reference to "an" includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth.

[0378] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0379] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0380] Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise. Stated another way, any specific example of the present disclosure may be combined with any other specific example of the disclosure (except where mutually exclusive).

[0381] Any example of the present disclosure disclosing a specific feature or group of features or method or method steps will be taken to provide explicit support for disclaiming the specific feature or group of features or method or method steps.

[0382] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, molecular biology, immunohistochemistry, protein chemistry, and biochemistry).

[0383] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, Perbal, 1984; Sambrook et al., 1989; Brown, 1991; Glover et al., 1995 and 1996; Ausubel et al., 1988; Harlow et al., 1988; Coligan et al., 1991.

[0384] The description and definitions of variable regions and parts thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by the discussion in Kabat et al., 1987 and 1991; Bork et al., 1994; Chothia and Lesk, 1987; Chothia et al., 1989 and / or Al-Lazikani et al., 1997.

[0385] Reference herein to a range of, e.g., residues, will be understood to be inclusive. For example, reference to “a region comprising amino acids 56 to 65” will be understood in an inclusive manner, i.e., the region comprises a sequence of amino acids as numbered 56, 57, 58, 59, 60, 61, 62, 63, 64 and 65 in a specified sequence.

[0386] 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 taken to provide explicit support for both meanings or for either meaning.

[0387] Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to 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.

[0388] All publications cited herein are hereby incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0389] Any discussion of documents, acts, materials, devices, articles or the like that has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0390] Selected Definitions

[0391] The term “recombinant” shall be understood to mean the product of artificial genetic recombination. Accordingly, in the context of a recombinant protein comprising a variable region or an antigen binding domain (e.g., an antibody antigen binding domain), this term does not encompass naturally -occurring protein within a subject’s body that is the product of natural recombination that occurs during B cell maturation. However, if such a protein is isolated, it is to be considered an isolated protein comprising a variable region or an antigen binding domain. Similarly, if nucleic acid encoding the protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein comprising a variable region or an antigen binding domain. A recombinant protein also encompasses a protein expressed by artificial recombinant means when it is within a cell, tissue or subject, e.g., in which it is expressed.

[0392] The term “protein” shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

[0393] The term “polypeptide” or “polypeptide chain” will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.

[0394] As used herein, the term “binding protein” shall be understood to refer to a protein or part thereof or other region of the protein that is capable of interacting with or specifically binding to an antigen (e.g., a cell component or molecule, such as a protein).

[0395] As used herein, the term “antigen binding domain” shall be taken to mean a region of an antibody that is capable of specifically binding to an antigen, i.e., a VH or a VL or an Fv comprising both a VH and a VL. The antigen binding domain need not be in the context of an entire antibody, e.g., it can be in isolation (e.g., a domain antibody) or in another form, e.g., as described herein, such as a scFv.

[0396] For the purposes for the present disclosure, the term “antibody” includes a protein capable of specifically binding to one or a few closely related antigens (e.g., CXCE10) by virtue of an antigen binding domain contained within a 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, de-immunized antibodies, synhumanized antibodies, half-antibodies, bispecific antibodies). An antibody generally comprises constant domains, which can be arranged into a constant region or constant fragment or fragment crystallizable (Fc). Exemplary forms of antibodies comprise a four-chain structure as their basic unit. Full-length antibodies comprise two heavy chains (~50 to 70 kDa) covalently linked and two light chains (~23 kDa each). A light chain generally comprises a variable region (if present) and a constant domain and in mammals is either a K light chain or a light chain. A heavy chain generally comprises a variable region and one or two constant domain(s) linked by a hinge region to additional constant domain(s). Heavy chains of mammals are of one of the following types: a, 5, a, y, or p. Each light chain is also covalently linked to one of the heavy chains. For example, the two heavy chains and the heavy and light chains are held together by inter-chain disulfide bonds and by non-covalent interactions. The number of inter-chain disulfide bonds can vary among different types of antibodies. Each chain has an N-terminal variable region (VH or VL wherein each are -110 amino acids in length) and one or more constant domains at the C- terminus. The constant domain of the light chain (CL which is -110 amino acids in length) is aligned with and disulphide -bonded to the first constant domain of the heavy chain (CHI which is 330 to 440 amino acids in length). The light chain variable region is aligned with the variable region of the heavy chain. The antibody heavy chain can comprise 2 or more additional CH domains (such as, CH2, CH3 and the like) and can comprise a hinge region between the CHI and CH2 constant domains. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGi, IgGi. IgGs, IgG4, IgAi and IgAi) or subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (such as, human) antibody. In one example the antibody heavy chain is missing a C-terminal lysine residue. In one example, the antibody is humanized, synhumanized, chimeric, CDR-grafted or deimmunized.

[0397] The terms "full-length antibody," "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen binding fragment of an antibody. Specifically, whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be wildtype sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

[0398] As used herein, “variable region” refers to the portions of the light and / or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and includes amino acid sequences of complementarity-determining regions (CDRs); i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, the variable region comprises three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0399] As used herein, the term “complementarity determining regions” (syn. CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable region the presence of which are major contributors to specific antigen binding. Each variable region domain (VH or VL) typically has three CDRs identified as CDR1, CDR2 and CDR3. In one example, the amino acid positions assigned to 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 CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html). According to the numbering system of Kabat, VH FRS and CDRs are positioned as follows: residues 1 to 30 (FR1), 31 to 35 (CDR1), 36 to 49 (FR2), 50 to 65 (CDR2), 66 to 94 (FR3), 95 to 102 (CDR3) and 103 to 113 (FR4). According to the numbering system of Kabat, VL FRS and CDRs are positioned as follows: residues 1 to 23 (FR1), 24 to 34 (CDR1), 35 to 49 (FR2), 50 to 56 (CDR2), 57 to 88 (FR3), 89 to 97 (CDR3) and 98 to 107 (FR4). The present disclosure is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including the canonical numbering system or of Chothia and Lesk, 1987; Chothia et al., 1989; and / or Al-Lazikani et al., 1997; the numbering system of Honnegher and Pliikthun, 2001; or the IMGT system discussed in Giudicelli et al., 1997. In one example, the CDRs are defined according to the Kabat numbering system. Optionally, heavy chain CDR2 according to the Kabat numbering system does not comprise the five C-terminal amino acids listed herein or any one or more of those amino acids are substituted with another naturally -occurring amino acid. In this regard, Padlan et al., 1995 established that the five C-terminal amino acids of heavy chain CDR2 are not generally involved in antigen binding.

[0400] "Framework regions" (FRs) are those variable region residues other than the CDR residues.

[0401] As used herein, the term “Fv” (or variable fragment) shall be taken to mean any protein, whether comprised of multiple polypeptides or a single polypeptide, in which a VL and a VH associate and form a complex having an antigen binding domain, i.e., capable of specifically binding to an antigen. The VH and the VL which form the antigen binding domain can be in a single polypeptide chain or in different polypeptide chains. Furthermore, an Fv of the disclosure (as well as any protein of the disclosure) may have multiple antigen binding domains which may or may not bind the same antigen. This term shall be understood to encompass fragments directly derived from an antibody as well as proteins corresponding to such a fragment produced using recombinant means. In some examples, the VH is not linked to a heavy chain constant domain (CH) 1 and / or the VL is not linked to a light chain constant domain (CL). Exemplary Fv containing polypeptides or proteins include a Fab fragment, a Fab’ fragment, a F(ab’) fragment, a scFv, a diabody, a triabody, a tetrabody or higher order complex, or any of the foregoing linked to a constant region or domain thereof, e.g., CH2 or CH3 domain, e.g., a minibody. An “antigen binding fragment” or "Fab fragment" consists of a monovalent antigenbinding fragment of an immunoglobulin, and can be produced by digestion of a whole antibody with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain or can be produced using recombinant means. A "Fab' fragment" of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain comprising a VH and a single constant domain. Two Fab' fragments are obtained per antibody treated in this manner. A Fab’ fragment can also be produced by recombinant means. A "F(ab')2 fragment” of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A “Fabi” fragment is a recombinant fragment comprising two Fab fragments linked using, for example a leucine zipper or a CH3 domain. 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 polypeptide linker.

[0402] As used herein, the term “binds” in reference to the interaction of a CXCL10 binding protein or an antigen binding domain thereof with an antigen means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody binds to epitope "A", the presence of a molecule containing epitope “A” (or free, unlabeled “A”), in a reaction containing labeled “A” and the protein, will reduce the amount of labeled “A” bound to the antibody.

[0403] As used herein, the term “specifically binds” or “binds specifically” shall be taken to mean that a CXCL10 binding protein of the disclosure reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with a particular antigen or cell expressing same than it does with alternative antigens or cells. For example, a CXCL10 binding protein binds to CXCL10 with materially greater 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 does to other chemokine receptors or to antigens commonly recognized by polyreactive natural antibodies (i.e., by naturally occurring antibodies known to bind a variety of antigens naturally found in humans). Reference to “binding” provides explicit support for the term “specific binding” and vice versa.

[0404] As used herein, the term “does not bind” shall be understood to mean that a CXCL10 binding protein of the present disclosure does not bind to a particular antigen or cell expressing same.

[0405] As used herein, the term “does not detectably bind” shall be understood to mean that a CXCL10 binding protein, e.g., an antibody, binds to a candidate antigen at a level less than 10%, or 8% or 6% or 5% above background. The 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 biosensor analysis (e.g. Biacore) in which the antigen (e.g., a polypeptide) is immobilized and contacted with a CXCL10 binding protein.

[0406] As used herein, the term “does not significantly bind” shall be understood to mean that the level of binding of a CXCL10 binding protein of the disclosure to a polypeptide is not statistically significantly higher than background, e.g., the level of binding signal detected in the absence of the CXCL10 binding protein and / or in the presence of a negative control protein (e.g., an isotype control antibody) and / or the level of binding detected in the presence of a negative control polypeptide. In one example, the level of binding is detected using biosensor analysis (e.g. Biacore) in which the antigen (e.g., a polypeptide) is immobilized and contacted with a CXCL10 binding protein.

[0407] For the purposes of clarification and as will be apparent to the skilled artisan based on the exemplified subject matter herein, reference to “affinity” in this specification is a reference to KD of a protein or antibody.

[0408] For the purposes of clarification and as will be apparent to the skilled artisan based on the description herein, reference to an “affinity of at least about” will be understood to mean that the affinity (or KD) is equal to the recited value or higher (i.e., the value recited as the affinity is lower), i.e., an affinity of 2nM is greater than an affinity of 3nM. Stated another way, this term could be “an affinity of X or less”, wherein X is a value recited herein.

[0409] As used herein, the term “epitope” (syn. “antigenic determinant”) shall be understood to mean a region to which a CXCL10 binding protein binds. This term is not necessarily limited to the specific residues or structure to which the CXCL10 binding protein makes contact. For example, this term includes a region spanning amino acids contacted by the CXCL10 binding protein and 5-10 (or more) or 2-5 or 1-3 amino acids outside of this region. In some examples, the epitope comprises a series of discontinuous amino acids that are positioned close to one another when a CXCL10 polypeptide is folded and, for example, associated with another CXCL10 polypeptide, i.e., a “conformational epitope”.

[0410] The term “competitively inhibits” shall be understood to mean that a CXCL10 binding protein of the disclosure (or an antigen binding domain thereof) reduces or prevents binding of a recited antibody or CXCL10 binding protein to CXCL10. This may be due to the CXCL10 binding protein (or antigen binding domain) and antibody binding to the same or an overlapping epitope. It will be apparent from the foregoing that the CXCL10 binding protein need not completely inhibit binding of the antibody, rather it need only reduce binding by a statistically significant amount, for example, by at least about 10% or 20% or 30% or 40% or 50% or 60% or 70% or 80% or 90% or 95%. For example, the CXCL10 binding protein reduces binding of the antibody by at least about 30%, for example by at least about 50%, such as, by at least about 70%, for example by at least about 75%, even more preferably, by at least about 80% or 85% e.g., by at least about 90%. Methods for determining competitive inhibition of binding are known in the art and / or described herein. For example, the antibody is exposed to CXCL10 either in the presence or absence of the CXCL10 binding protein. If less antibody binds in the presence of the CXCL10 binding protein than in the absence of the CXCL10 binding protein, the protein is considered to competitively inhibit binding of the antibody. In one example, the competitive inhibition is not due to steric hindrance.

[0411] As used herein, the term “subject” shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and non-human primates. For example, the subject is a human.

[0412] Binding Proteins

[0413] As discussed herein, binding proteins of the present disclosure can take various forms and bind to full-length human CXCL10, N-terminally truncated CXCL10 and / or citrullinated CXCL10. Binding proteins of the present disclosure also bind to CA-125, HE4 and IL-6.

[0414] Human CXCL10 is expressed as a pre-sequence (SEQ ID NO: 1), with the N- terminal 22 amino acids cleaved to produce mature “full length” human CXCL10 (SEQ ID NO: 2). Thus, as used herein, the term “full-length CXCL10” refers to CXCL10 which has not been modified post-translationally other than the post-translational cleavage to remove the 22 N-terminal amino acids to produce “mature” full-length CXCL10.

[0415] As used herein, the term “N-terminally truncated CXCL10” refers to “mature” full-length CXCL10 which has been truncated by dipeptidylpeptidase IV (DPP4) and consists of e.g., amino acids 3 to 77 of SEQ ID NO: 2.

[0416] As used herein, the term “citrullinated CXCL10” refers to CXCL10 which has been post-translationally modified by deamination or citrullination at an arginine residue at e.g., amino acid position 5 of SEQ ID NO: 2, by peptidylarginine deiminases (PAD).

[0417] In one example, the present disclosure provides a CXCL10 binding protein, wherein the binding protein binds to full-length human CXCL10, N-terminally truncated CXCL10 and citrullinated CXCL10. In another example, the present disclosure provides a CXCL10 binding protein, wherein the binding protein binds to full-length human CXCL10, but does not bind N- terminally truncated CXCL10 and citrullinated CXCL10.

[0418] Exemplary CXCL10 binding proteins for use in the present disclosure are provided in WO 2021 / 119761, the entire contents of which is incorporated by reference.

[0419] CA-125 (also known as Mucin-16; MUC-16) is a protein that is encoded by the MUC16 gene. CA-125 is a membrane associated mucin that possesses a single transmembrane domain and contains about 22,000 amino acids. CA-125 comprises an N-terminal domain and a tandem repeat domain that are both entirely extracellular and highly O-glycosylated. The C-terminal domain contains multiple extracellular SEQ (sea urchin sperm protein, enterokinase and agrin) molecules, the transmembrane domain and a cytoplasmic tail. The extracellular region of CA-125 can be released from the cell surface by undergoing proteolytic cleavage. For the purposes of nomenclature only and not limitation, an exemplary sequence of CA-125 is set out in NCBI Reference Sequence NP_001388430.1.

[0420] The present disclosure provides a cancer antigen 125 (CA-125) binding protein, wherein the CA-125 binding protein binds to one or more or all epitopes selected from the group consisting of LYSGCRLTLLRSEKDGAATGVDAICTHR (SEQ ID NO: 27), ERVLQSLLGPMFKNTSVGPLYS (SEQ ID NO: 28) and

[0421] VDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTH (SEQ ID NO: 29).

[0422] HE4 is a secreted glycoprotein and is also designated WFDC2 because it contains two whey acidic protein (WAP) domains and a “four disulfide core” made up of eight cysteine residues. The HE4 gene resides on human chromosome 20ql2-13.1, a region that harbors a locus of 14 genes encoding protein domains that have homology with whey acidic protein (WAP). For the purposes of nomenclature only and not limitation, an exemplary sequence of human CA-125 is set out in NCBI Reference Sequence NP-006094.3.

[0423] The present disclosure provides a human epididymis protein 4 (HE4) binding protein, wherein the HE4 binding protein binds to one or more or all epitopes selected from the group consisting of VNINFPQLGLCR (SEQ ID NO: 30) and CRDQCQVDSQCPGQMKCCRNGCGK (SEQ ID NO: 31).

[0424] IL-6 is an interleukin that acts both as a pro-inflammatory cytokine and an antiinflammatory myokine. The IL6 gene resides on human chromosome 7pl5.3 and is also known as B cell stimulating factor 2 (BSF2), hybridoma growth factor (HGF) and interferon beta 2 (IFN P2). For the purposes of nomenclature only and not limitation, an exemplary sequence of human IL-6 is set out in NCBI Reference Sequence NG_011640.1.

[0425] The present disclosure provides an interleukin 6 (IL-6) binding protein, wherein the IL-6 binding protein binds to one or more or all epitopes selected from the group consisting of VPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRK (SEQ ID NO: 32), ETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEE (SEQ ID NO: 33), VYLEYLQNRFESSEEQARAVQM (SEQ ID NO: 34) and QKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLI (SEQ ID NO: 35).

[0426] The sequences of CXCL10, CA-125, HE4 and IL-6 from other species can be determined using sequences provided herein and / or in publicly available databases and / or determined using standard techniques (e.g., as described in Ausubel et al., 1988 (including all updates until present) or Sambrook et al., 1989).

[0427] Antibodies

[0428] In one example, a CXCL10, CA-125, HE4 and / or IL-6 binding protein of the present disclosure comprises an antibody or antigen binding fragment thereof. For example, a binding protein of the disclosure comprises an antigen binding domain of an antibody.

[0429] Immunization-based. Methods

[0430] Methods for generating antibodies are known in the art and / or described in Harlow et al., 1988. Generally, in such methods a protein or immunogenic fragment or epitope thereof or a cell expressing and displaying same (i.e., an immunogen), optionally formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, is administered to a non-human animal, for example, a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat or pig. The immunogen may be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known route.

[0431] The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. One or more further immunizations may be given, if required to achieve a desired antibody titer. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal is bled and the serum isolated and stored, and / or the animal is used to generate monoclonal antibodies (mAbs).

[0432] Monoclonal antibodies are one 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), for example, to the same epitope within the antigen. This term is not intended to be limited as regards to the source of the antibody or the manner in which it is made.

[0433] For the production of mAbs any one of a number of known techniques may be used, such as, for example, the procedure exemplified in US4196265 or Harlow et al., 1988.

[0434] For example, a suitable animal is immunized with an immunogen under conditions sufficient to stimulate antibody producing cells. Rabbits and rodents such as mice and rats are exemplary animals. Mice genetically engineered to express human immunoglobulin proteins and, for example, do not express murine immunoglobulin proteins, can also be used to generate an antibody of the present disclosure (e.g., as described in W02002066630).

[0435] Following immunization, somatic cells with the potential for producing antibodies, e.g., B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsies of spleens, tonsils or lymph nodes, or from a peripheral blood sample. The B cells from the immunized animal are then fused with cells of an immortal myeloma cell, generally derived from the same species as the animal that was immunized with the immunogen.

[0436] Hybrids are amplified by culture in a selective medium comprising an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary agents are aminopterin, methotrexate and azaserine.

[0437] The amplified hybridomas are subjected to a functional selection for antibody specificity and / or titer, such as, for example, by flow cytometry and / or immunohistochemstry and / or immunoassay (e.g. radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, and the like).

[0438] Alternatively, ABL-MYC technology (NeoClone, Madison WI 53713, USA) is used to produce cell lines secreting mAbs (e.g., as described in Largaespada et al., 1996).

[0439] Library-Based Methods

[0440] The present disclosure also encompasses screening of libraries of antibodies or antigen binding fragments thereof (e.g., comprising variable regions thereof).

[0441] Examples of libraries contemplated by this disclosure include naive libraries (from unchallenged subjects), immunized libraries (from subjects immunized with an antigen) or synthetic libraries. Nucleic acid encoding antibodies or regions thereof (e.g., variable regions) are cloned by conventional techniques (e.g., as disclosed in Sambrook et al., 2001) and used to encode and display proteins using a method known in the art. Other techniques for producing libraries of proteins are described in, for example in US6300064 (e.g., a HuCAL library of Morphosys AG); US5885793; US6204023; US6291158; or US6248516.

[0442] The antigen binding fragments according to the disclosure may be soluble secreted proteins or may be presented as a fusion protein on the surface of a cell, or particle (e.g., a phage or other virus, a ribosome or a spore). 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 a mRNA display library, e.g., as described in US7270969). In yet another example, the display library is a phage display library wherein proteins comprising antigen binding fragments of antibodies are expressed on phage, e.g., as described in US6300064; US5885793; US6204023; US6291158; or US6248516. Other phage display methods are known in the art and are contemplated by the present disclosure. Similarly, methods of cell display are contemplated by the disclosure, e.g., bacterial display libraries, e.g., as described in US5516637; yeast display libraries, e.g., as described in US6423538 or a mammalian display library.

[0443] 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. Methods of affinity purification typically involve contacting proteins comprising antigen binding fragments displayed by the library with a target antigen and, following washing, eluting those domains that remain bound to the antigen.

[0444] Any variable regions or scFvs identified by screening are readily modified into a complete antibody, if desired. Exemplary methods for modifying or reformatting variable regions or scFvs into a complete antibody are described, for example, in Jones et al., 2010; or Jostock et al., 2004; or W02012040793. Alternatively, or additionally, standard cloning methods are used, e.g., as described in Ausubel et al., 1987, and / or Sambrook et al., 2001.

[0445] Deimmunized, Chimeric, Humanized, Synhumanized, Primatized and Human Antibodies or Antigen Binding Fragments

[0446] The antibodies or antigen binding fragments of the present disclosure may be humanized.

[0447] The term “humanized antibody” shall be understood to refer to a protein comprising a human-like variable region, which includes CDRs from an antibody from a non-human species (e.g., mouse or rat or non-human primate) grafted onto or inserted into FRs from a human antibody (this type of antibody is also referred to a “CDR-grafted antibody”). Humanized antibodies also include antibodies in which one or more residues of the human protein are modified by one or more amino acid substitutions and / or one or more FR residues of the human antibody are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found in neither the human antibody or in the non-human antibody. Any additional regions of the antibody (e.g., Fc region) are generally human. Humanization can be performed using a method known in the art, e.g., US5225539, US6054297, US7566771 or US5585089. The term “humanized antibody” also encompasses a super-humanized antibody, e.g., as described in US7732578. A similar meaning will be taken to apply to the term “humanized antigen binding fragment”.

[0448] The antibodies or antigen binding fragments thereof of the present disclosure may be human antibodies or antigen binding fragments thereof. The term “human antibody” as used herein refers to antibodies having variable and, optionally, constant antibody regions found in humans, e.g. in the human germline or somatic cells or from libraries produced using such regions. The “human” antibodies can include amino acid residues not encoded by human sequences, e.g. mutations introduced by random or site directed mutations in vitro (in particular mutations which involve conservative substitutions or mutations in a small number of residues of the protein, e.g. in 1, 2, 3, 4 or 5 of the residues of the protein). These “human antibodies” do not necessarily need to be generated as a result of an immune response of a human, rather, they can be generated using recombinant means (e.g., screening a phage display library) and / or by a transgenic animal (e.g., a mouse) comprising nucleic acid encoding human antibody constant and / or variable regions and / or using guided selection (e.g., as described in or US5565332). This term also encompasses affinity matured forms of such antibodies. For the purposes of the present disclosure, a human antibody will also be considered to include a protein comprising FRs from a human antibody or FRs comprising sequences from a consensus sequence of human FRs and in which one or more of the CDRs are random or semirandom, e.g., as described in US6300064 and / or US6248516. A similar meaning will be taken to apply to the term “human antigen binding fragment”.

[0449] The antibodies or antigen binding fragments thereof of the present disclosure may be synhumanized antibodies or antigen binding fragments thereof. The term “synhumanized antibody” refers to an antibody prepared by a method described in W02007019620. A synhumanized antibody includes a variable region of an antibody, wherein the variable region comprises FRs from a New World primate antibody variable region and CDRs from a non-New World primate antibody variable region.

[0450] The antibody or antigen binding fragment thereof of the present disclosure may be primatized. A “primatized antibody” comprises variable region(s) from an antibody generated following immunization of a non-human primate (e.g., a cynomolgus macaque). Optionally, the variable regions of the non-human primate antibody are linked to human constant regions to produce a primatized antibody. Exemplary methods for producing primatized antibodies are described in US6113898.

[0451] In one example an antibody or antigen binding fragment thereof of the 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 is from a particular species (e.g., murine, such as mouse or rat) or belonging to a particular antibody class or subclass, while the remainder of the antibody or fragment is from another species (such as, for example, human or non-human primate) or belonging to another antibody class or subclass. In one example, a chimeric antibody comprising a VH and / or a VL from a non-human antibody (e.g., a murine antibody) and the remaining regions of the antibody are from a human antibody. The production of such chimeric antibodies and antigen binding fragments thereof is known in the art, and may be achieved by standard means (as described, e.g., in US6331415; US5807715; US4816567 and US4816397).

[0452] The present disclosure also contemplates a deimmunized antibody or antigen binding fragment thereof, e.g., as described in W02000034317 and W02004108158. De-immunized antibodies and fragments have one or more epitopes, e.g., B cell epitopes or T cell epitopes removed (i.e., mutated) to thereby reduce the likelihood that a subject will raise an immune response against the antibody or protein. For example, an antibody of the disclosure is analyzed to identify one or more B or T cell epitopes and one or more amino acid residues within the epitope is mutated to thereby reduce the immunogenicity of the antibody.

[0453] Antibody Binding Domain Containing Proteins

[0454] Single-Domain Antibodies

[0455] In some examples, a binding protein of the disclosure is or comprises a singledomain antibody (which is used interchangeably with the term “domain antibody” or “dAb”). A single-domain antibody is a single polypeptide chain comprising all or a portion of the heavy chain variable domain of an antibody.

[0456] Diabodies, Triabodies, Tetrabodies

[0457] In some examples, a binding protein of the disclosure is or comprises a diabody, triabody, tetrabody or higher order protein complex such as those described in W098 / 044001 and / or W094 / 007921.

[0458] For example, a diabody is a protein comprising two associated polypeptide chains, each polypeptide chain comprising the structure VL-X-VH or VH-X-VL, wherein X is a linker comprising insufficient residues to permit the VH and VL in a single polypeptide chain to associate (or form an Fv) or is absent, and wherein the VH of one polypeptide chain binds to a VL of the other polypeptide chain to form an antigen binding site, i.e., to form a Fv molecule capable of specifically binding to one or more antigens. The VL and VH can be the same in each polypeptide chain or the VL and VH can be different in each polypeptide chain so as to form a bispecific diabody (i.e., comprising two Fvs having different specificity).

[0459] Single Chain Fv (scFv) Fragments

[0460] The binding protein of the disclosure can be a scFv. The skilled artisan will be aware that scFvs comprise VH and VL regions in a single polypeptide chain and a polypeptide linker between the VH and VL which enables the scFv to form the desired structure for antigen binding (i.e., for the VH and VL of the single polypeptide chain to associate with one another to form a Fv). For example, the linker comprises in excess of 12 amino acid residues with (Gly4Ser)3 being one of the more favored linkers for a scFv.

[0461] The present disclosure also contemplates a disulfide stabilized Fv (or diFv or dsFv), in which a single cysteine residue is introduced into a FR of VH and a FR of VL and the cysteine residues linked by a disulfide bond to yield a stable Fv.

[0462] Alternatively, or in addition, the present disclosure encompasses a dimeric scFv, i.e., a protein comprising two scFv molecules linked by a non-covalent or covalent linkage, e.g., by a leucine zipper domain (e.g., derived from Fos or Jun). Alternatively, two scFvs are linked by a peptide linker of sufficient length to permit both scFvs to form and to bind to an antigen, e.g., as described in US20060263367.

[0463] Half-antibodies

[0464] In some examples, the antigen binding fragment of the present disclosure is a halfantibody or a half-molecule. The skilled artisan will be aware that a half-antibody refers to a protein comprising a single heavy chain and a single light chain. The term “halfantibody” also encompasses a protein comprising an antibody light chain and an antibody heavy chain, wherein the antibody heavy chain has been mutated to prevent association with another antibody heavy chain. In one example, a half-antibody forms when an antibody dissociates to form two molecules each containing a single heavy chain and a single light chain.

[0465] Methods for generating half-antibodies are known in the art and exemplary methods are described herein.

[0466] In one example, the half-antibody can be secreted by introducing into cells genes of the single heavy chain and single light chain that constitute the IgG of interest for expression. In one example, a constant region (e.g., an IgG4 constant region) comprises a “key or hole” (or “knob or hole”) mutation to prevent heterodimer formation. In one example, a constant region (e.g., an IgG4 constant region) comprises a T366W mutation (or knob). In another example, a constant region (e.g., an IgG4 constant region) comprises a T366S, L368A and Y407V mutation (or hole). In another example, the constant region comprises T350V, T366L, K392L and T394W mutations (knob). In another example, the constant region comprises T350V, L351Y, F405A and Y407V mutations (hole). Exemplary constant region amino acid substitutions are numbered according to the EU numbering system.

[0467] Other Antibodies and Proteins Comprising Antigen Binding Domains Thereof

[0468] The present disclosure also contemplates other antibodies and proteins comprising antigen-binding domains thereof, such as:

[0469] (i) minibodies, e.g., as described in US5837821;

[0470] (ii) heteroconjugate proteins, e.g., as described in US4676980;

[0471] (iii) heteroconjugate proteins produced using a chemical cross-linker, e.g., as described in US4676980; and

[0472] (iv) Fabs (e.g., as described in EP19930302894).

[0473] Immunoglobulins and Immunoglobulin Fragments

[0474] An example of a binding protein of the present disclosure is a protein comprising a variable region of an immunoglobulin, such as a T cell receptor or a heavy chain immunoglobulin (e.g., an IgNAR, a camelid antibody).

[0475] Heavy Chain Immunoglobulins

[0476] Heavy chain immunoglobulins differ structurally from many other forms of immunoglobulin (e.g., antibodies), in so far as they comprise a heavy chain, but do not comprise a light chain. Accordingly, these immunoglobulins are also referred to as “heavy chain only antibodies”. Heavy chain immunoglobulins are found in, for example, camelids and cartilaginous fish (also called IgNAR).

[0477] The variable regions present in naturally occurring heavy chain immunoglobulins are generally referred to as "VHH domains" in camelid Ig and V-NAR in IgNAR, in order to distinguish them from the heavy chain variable regions that are present in conventional 4-chain antibodies (which are referred to as "VH domains") and from the light chain variable regions that are present in conventional 4-chain antibodies (which are referred to as "VL domains"). Heavy chain immunoglobulins do not require the presence of light chains to bind with high affinity and with high specificity to a relevant antigen. This means that single domain binding fragments can be derived from heavy chain immunoglobulins, which are easy to express and are generally stable and soluble.

[0478] A general description of heavy chain immunoglobulins from camelids and the variable regions thereof and methods for their production and / or isolation and / or use is found inter alia in the following references WO94 / 04678, WO97 / 49805 and WO 97 / 49805.

[0479] A general description of heavy chain immunoglobulins from cartilaginous fish and the variable regions thereof and methods for their production and / or isolation and / or use is found inter alia in W02005118629.

[0480] V-Like Proteins

[0481] In one example, a 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 describes how the two V-domains of the T-cell receptor (termed alpha and beta) can be fused and expressed as a single chain polypeptide and, further, how to alter surface residues to reduce the hydrophobicity directly analogous to an antibody scFv. Other publications describing production of single-chain T-cell receptors or multimeric T cell receptors comprising two V-alpha and V-beta domains include WO 1999045110 or WO2011107595.

[0482] Other non-antibody proteins comprising antigen binding domains include proteins with V-like domains, which are generally monomeric. Examples of proteins comprising such V-like domains include CTLA-4, CD28 and ICOS. Further disclosure of proteins comprising such V-like domains is included in W01999045110.

[0483] Adnectins

[0484] In one example, a binding protein of the present disclosure comprises an adnectin. Adnectins are based on the tenth fibronectin type III (10Fn3) domain of human fibronectin in which the loop regions are altered to confer antigen binding. For example, three loops at one end of the P-sandwich of the10Fn3 domain can be engineered to enable an Adnectin to specifically recognize an antigen. For further details see US20080139791 or W02005056764. Anticalins

[0485] In a further example, a binding protein of the disclosure comprises an anticalin. Anticalins are derived from lipocalins, which are a family of extracellular proteins which transport small hydrophobic molecules such as steroids, bilins, retinoids and lipids. Lipocalins have a rigid P-sheet secondary structure with a plurality of loops at the open end of the conical structure which can be engineered to bind to an antigen. Such engineered lipocalins are known as anticalins. For further description of anticalins see US7250297 or US20070224633.

[0486] Affibodies

[0487] In a further example, a binding protein of the disclosure comprises an affibody. An affibody is a scaffold derived from the Z domain (antigen binding domain) of Protein A of Staphylococcus aureus which can be engineered to bind to antigen. The Z domain consists of a three-helical bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. For further details see EP1641818.

[0488] Avimers

[0489] In a further example, a binding protein of the disclosure comprises an Avimer. Avimers are multidomain proteins derived from the A -domain scaffold family. The native domains of approximately 35 amino acids adopt a defined disulphide bonded structure. Diversity is generated by shuffling of the natural variation exhibited by the family of A-domains. For further details see W02002088171.

[0490] DARPins

[0491] In a further example, a binding protein of the disclosure comprises a Designed Ankyrin Repeat Protein (DARPin). DARPins are derived from Ankyrin which is a family of proteins that mediate attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33 residue motif consisting of two a-helices and a P-tum. They can be engineered to bind different target antigens by randomizing residues in the first a-helix and a P-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details see US20040132028. Mutations to Binding Proteins

[0492] The present disclosure also provides a binding protein or a nucleic acid encoding same having at least 90% identity to a sequence disclosed herein.

[0493] In one example, a binding protein or nucleic acid of the disclosure comprises sequence at least about 90% or 95% or 97% or 98% or 99% identical to a sequence disclosed herein, wherein the protein specifically binds to CXCL10, CA- 125, HE4 and / or IL-6 as described herein according to any example.

[0494] Alternatively, or additionally, the binding protein comprises a CDR (e.g., three CDRs) at least about 90% or 95% or 97% or 98% or 99% identical to CDR(s) of a VH or VL as described herein according to any example, wherein the protein is capable of specifically binding to CXCL10, CA- 125, HE4 and / or IL-6 as described herein according to any example. Methods for determining binding of a protein to CXCL10, CA-125, HE4 and / or IL-6 are described herein.

[0495] It is known in the art that the five C -terminal residues of heavy chain CDR2 can be mutated to conservative or non-conservative amino acid substitutions (31% of residues) (Padlan et al., 1995). Thus, a protein can comprise a CDR2 having at least about 35% identity to a heavy chain CDR2 sequence disclosed herein.

[0496] The present disclosure also contemplates mutant forms of a CXCL10, CA-125, HE4 and / or IL-6 binding protein of the disclosure comprising one or more conservative amino acid substitutions compared to a sequence set forth herein. In some examples, the CXCL10, CA-125, HE4 and / or IL-6 binding protein comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 conservative amino acid substitutions. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain and / or hydropathicity and / or hydrophilicity. Exemplary conservative amino acid substitutions are provided in Table 1.

[0497] Table 1: Exemplary amino acid substitutions.

[0498] Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ^-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Hydropathic indices are described, for example in Kyte and Doolittle, 1982 and hydrophylic indices are described in, e.g., US4554101.

[0499] The present disclosure also contemplates non-conservative amino acid changes. For example, of particular interest are substitutions of charged amino acids with another charged amino acid and with neutral or positively charged amino acids. In some examples, the binding protein comprises 10 or fewer, e.g., 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 non-conservative amino acid substitutions.

[0500] In one example, the mutation(s) occur within a FR of an antigen binding domain of a binding protein of the disclosure. In another example, the mutation(s) occur within a CDR of a binding protein of the disclosure.

[0501] Exemplary methods for producing mutant forms of a binding protein include:

[0502] • mutagenesis of DNA (Thie et al., 2009) or RNA (Kopsidas et al., 2006; Kopsidas et al., 2007; and WO1999 / 058661); • introducing a nucleic acid encoding the polypeptide into a mutator cell, e.g., XL-

[0503] IRed, XL-mutS and XL-mutS-Kanr bacterial cells (Stratagene);

[0504] • DNA shuffling, e.g., as disclosed in Stemmer, 1994; and

[0505] • site directed mutagenesis, e.g., as described in Dieffenbach et al., 1995).

[0506] Exemplary methods for determining biological activity of the mutant binding proteins of the disclosure will be apparent to the skilled artisan and include, for example antigen binding, competitive inhibition of binding, affinity, association and dissociation.

[0507] In another example, a nucleic acid of the disclosure comprises a sequence at least about 90% or 95% or 97% or 98% or 99% identical to a sequence set forth herein and encoding a CXCL10, CA-125, HE4 and / or IL-6 binding protein having a function as described herein according to any example. The present disclosure also encompasses nucleic acids encoding a CXCL10, CA-125, HE4 and / or IL-6 binding protein of the disclosure, which differs from a sequence exemplified herein as a result of degeneracy of the genetic code.

[0508] The % identity of a nucleic acid or polypeptide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 50 residues in length, 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 in length 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.

[0509] Constant Regions

[0510] The present disclosure encompasses binding proteins and / or antibodies described herein comprising a constant region of an antibody. This includes antigen binding fragments of an antibody fused to a Fc.

[0511] Sequences of constant regions useful for producing the proteins of the present disclosure may be obtained from a number of different sources. In some examples, the constant region or portion thereof of the protein is derived from a human antibody. The constant region or portion thereof may be derived from any antibody class, including IgM, IgG, IgD, IgA and IgE, and any antibody isotype, including IgGl, IgG2, IgG3 and IgG4. In one example, the constant region is human isotype IgG4 or a stabilized IgG4 constant region. In an embodiment, the constant region is an IgG kappa constant region.

[0512] In one example, the Fc region of the constant region has a reduced ability to induce effector function, e.g., compared to a native or wild-type human IgGl or IgG3 Fc region. In the context of the present disclosure, “effector functions” refer to those biological activities mediated by cells or proteins that bind to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody that result in killing of a cell. Examples of effector functions induced by antibodies include: complement dependent cytotoxicity (CDC); antibody-dependent-cell-mediated cytotoxicity (ADCC); antibody-dependent-cell-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.

[0513] In one example, the Fc region is an IgG4 Fc region (i.e., from an IgG4 constant region), e.g., a human IgG4 Fc region. Sequences of suitable IgG4 Fc regions will be apparent to the skilled person and / or available in publically available databases (e.g., available from National Center for Biotechnology Information).

[0514] 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 Fab arm exchange or the propensity to undergo Fab arm exchange or formation of a half-antibody or a propensity to form a half antibody. “Fab arm exchange" refers to a type of protein modification for human IgG4, in which an IgG4 heavy chain and attached light chain (half-molecule) is swapped for a heavy-light chain pair from another IgG4 molecule. Thus, IgG4 molecules may acquire two distinct Fab arms recognizing two distinct antigens (resulting in bispecific molecules). Fab arm exchange occurs naturally in vivo and can be induced in vitro by purified blood cells or reducing agents such as reduced glutathione. A “half antibody” forms when an IgG4 antibody dissociates to form two molecules each containing a single heavy chain and a single light chain.

[0515] In one example, a stabilized IgG4 constant region comprises a proline at position 241 of the hinge region according to the systemof Kabat (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 a serine. Following substitution of the serine for proline, the IgG4 hinge region comprises a sequence CPPC. In this regard, the skilled person will be aware that the “hinge region” is a proline -rich portion of an antibody heavy chain constant region that links the Fc and Fab regions that confers mobility on the two Fab arms of an antibody. The hinge region includes cysteine residues which are involved in inter-heavy chain disulfide bonds. It is generally defined as stretching from Glu226 to Pro243 of human IgGl according to the numbering system of Kabat. Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain disulfide (S-S) bonds in the same positions (see for example W02010 / 080538).

[0516] Additional examples of stabilized IgG4 antibodies are antibodies in which arginine at position 409 in a heavy chain constant region of human IgG4 (according to the EU numbering system) is substituted with lysine, threonine, methionine, or leucine (e.g., as described in W02006 / 033386). The Fc region of the constant region may additionally or alternatively comprise a residue selected from the group consisting of: alanine, valine, glycine, isoleucine and leucine at the position corresponding to 405 (according to the EU numbering system). Optionally, the hinge region comprises a proline at position 241 (i.e., a CPPC sequence) (as described above).

[0517] In another example, the Fc region is a region modified to have reduced effector function, i.e., a “non-immunostimulatory Fc region”. For example, the Fc region is an IgGl Fc region comprising a substitution at one or more positions selected from the group consisting of 268, 309, 330 and 331. In another example, the Fc region is an IgGl Fc region comprising one or more of the following changes E233P, E234V, E235A and deletion of G236 and / or one or more of the following changes A327G, A33OS and P33 IS (Armour et al., 1999; Shields et al., 2001). Additional examples of non- immuno stimulatory Fc regions are described, for example, in Dall'Acqua et al., 2006; and / or Hezareh, 2001).

[0518] In another example, the Fc region is a chimeric Fc region, e.g., comprising at least one CH2 domain from an IgG4 antibody and at least one CH3 domain from an IgGl antibody, 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 W02010 / 085682). Exemplary substitutions include 240F, 262E, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

[0519] Protein Production

[0520] In one example, a binding protein described herein according to any example is produced by culturing a cell of the invention under conditions sufficient to produce the protein, e.g., as described herein and / or as is known in the art.

[0521] Recombinant Expression

[0522] In another example, a binding protein described herein according to any example is recombinant. In the case of a recombinant protein, nucleic acid encoding same can be cloned into expression constructs or vectors, which are then transfected into host cells, such as E. coli cells, yeast cells, insect cells, or mammalian cells, such as simian 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 for expressing a protein are CHO cells, myeloma cells or HEK cells. Molecular cloning techniques to achieve these ends are known in the art and described, for example in Ausubel et al., 1988 (including all updates until present) or Sambrook et al., 1989. A wide variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids. Methods of producing recombinant antibodies are also known in the art, see, e.g., US4816567 or US5530101.

[0523] Following isolation, the nucleic acid is inserted 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 cells.

[0524] As used herein, the term “promoter” is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter expression of a nucleic acid, e.g., in response to a developmental and / or external stimulus, or in a tissue specific manner. In the present context, the term “promoter” is also used to describe a recombinant, synthetic or fusion nucleic acid, or derivative which confers, activates or enhances the expression of a nucleic acid to which it is operably linked. Exemplary promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and / or alter the spatial expression and / or temporal expression of said nucleic acid.

[0525] 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.

[0526] Many vectors for expression in cells are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding a protein (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. The skilled artisan will be aware of suitable sequences for expression of a protein. 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, a factor leader, or acid phosphatase leader) or mammalian secretion signals (e.g., herpes simplex gD signal).

[0527] Exemplary promoters active in mammalian cells include cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1-a promoter (EFl), small nuclear RNA promoters (Ula and Ulb), a-myosin heavy chain promoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus major late promoter, P-actin promoter; hybrid regulatory element comprising a CMV enhancer / P- actin promoter or an immunoglobulin promoter or active fragment thereof. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

[0528] Typical promoters suitable for expression in yeast cells such as for example a yeast cell selected from the group comprising Pichia pastoris, Saccharomyces cerevisiae and Schizosaccharomyces pombe, include, but are not limited to, the ADH1 promoter, the GALI promoter, the GAL4 promoter, the CUP1 promoter, the PHO 5 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

[0529] Means for introducing the isolated nucleic acid or expression construct comprising same into a cell for expression are known to those skilled in the art. The technique used for a given cell depends on the known successful techniques. Means for introducing recombinant DNA into cells include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and / or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongst others.

[0530] The host cells used to produce the protein may be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPM1-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.

[0531] Isolation of Proteins

[0532] Methods for isolating a protein are known in the art and / or described herein.

[0533] Where a binding protein is secreted into culture medium, supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, for example, 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 additionally, supernatants can be filtered and / or separated from cells expressing the protein, e.g., using continuous centrifugation.

[0534] The CXCL10 binding protein prepared from the 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 of the foregoing. These methods are known in the art and described, for example in WO99 / 57134 or Harlow et al., 1988.

[0535] The skilled artisan will also be aware that a protein can be modified to include a tag to facilitate purification or detection, e.g., a poly-histidine tag, e.g., a hexa-histidine tag, or an influenza virus hemagglutinin (HA) tag, or a Simian Virus 5 (V5) tag, or a FLAG tag, or a glutathione S -transferase (GST) tag. The resulting protein is then purified using methods known in the art, such as, affinity purification. For example, a protein comprising a hexa-his tag is purified by contacting a sample comprising the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds a hexa-his tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and subsequently eluting the bound protein. Alternatively, or in addition a ligand or antibody that binds to a tag is used in an affinity purification method.

[0536] In one example, a binding protein of the present disclosure is conjugated to a detectable label.

[0537] As used herein, the term “conjugate” or “conjugated” shall be understood to encompass both indirect and direct binding. For example, direct conjugation includes chemical conjugation, which can be non-covalent or covalent or genetic conjugation (also referred to as “fusion”). In one example, the conjugation is covalent, e.g., a disulphide bond.

[0538] As used herein, a “detectable label” is a molecular or atomic tag or marker that generates or can be induced to generate an optical or other signal or product that can be detected visually or by using a suitable detector. Detectable labels are well known in the art and include, for example, a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, a prosthetic group, a contrast agent and an ultrasound agent. Fluorescent labels commonly used include Alexa, cyanine such as Cy5 and Cy5.5, and indocyanine, and fluorescein isothiocyanate (FITC), but they are not so limited. Fluorescent labels useful in the practice of the present disclosure can include, also without limitation, 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7- dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein (pH 10); 5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein); 5- HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X- rhodamine); 5-TAMRA (5 -Carboxy tetramethylrhodamine); 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 & RNA; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin 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 Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTRA-BTC=Ratio Dye, Zn2+; 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 (Bisamninophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein; BFP / GFP FRET Bimane; Bisbenzamnide; Bisbenzimide (Hoechst); bis-BTC=Ratio Dye, Zn2+; 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-Ceramide; 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 Ca2+; BTC-5N-atio Dye, Zn2+; Calcein; Calcein Blue; Calcium Crimson; Calcium Green; Calcium Green- 1 Ca2+Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White; Carboxy -X-rhodamine (5-ROX); Cascade Blue; Cascade Yellow 399; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP— Cyan Fluorescent Protein; CFP / YFP; FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF (Ratio Dye, pH); CMFDA; Coelenterazine; Coelenterazine cp (Ca2+Dye); Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM 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; Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4- ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16-ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD-Lipophilic Tracer; DiD (DiIC18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (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; ELF 97; Eosin; Erythrosin; Erythrosin 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; Flazo 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); GFP red shifted (rsGFP), GFP wild type, non- UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxy coumarin; Hydroxystilbamidine (FluoroGold); Hydroxy tryptamine; Indo-1, high calcium; Indo-1, low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intra white 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 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; Maxiion Brilliant Flavin 10 GFF; Maxiion Brilliant Flavin 8 GFF; Merocyanin; Methoxy coumarin; Mito tracker Green FM; Mito tracker Orange; Mito tracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green; Oregon Green 488-X; Oregon Green; Oregon Green 488; Oregon Green 500; Oregon Greene 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium Iodide (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613 [PE-TexasRed]; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (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 (super glow BFP); sgGFP; sgGFP (super glow GFP); SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine 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; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE- Cy5); TRITC (TetramethylRodamine-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.

[0539] In one example, a detectable label is an enzyme. The enzyme can act on an appropriate substrate to result in production of a detectable dye. Examples of enzymes useful in the disclosure include, without limitation, alkaline phosphatase and horseradish peroxidase. Alternatively or in addition, the enzyme can be, for example, luciferase. The enzyme can be linked to the antibody by conventional chemical methods, or it can be expressed together with the antibody as a fusion protein.

[0540] Radioisotopes useful as detectable labels in the disclosure are well known in the art and can include3H,nC,18F,35S, ^Cu,67Ga,68Ga, "mTc,i nIn,1231,1241,125I, and131I. Attachment of any gamma emitting radioactive materials, e.g., "mTc andi nIn, which can react with carboxyl, amino, or sulfhydryl groups of a compound that binds calcitonin receptor is suitable for use in detection methods using gamma scintigraphy. Attachment of radioactivenC,18F,64Cu,67Ga,68Ga,124I, and131I compounds which can react with carboxyl, amino, or sulfhydryl groups of a compound is suitable for use in detection methods using PET / SPECT imaging.

[0541] It will be apparent to the skilled artisan from the disclosure herein that some

[0542] CXCL10 binding proteins of the present disclosure bind to full-length CXCL10 and / or to specific post-translationally modified forms of CXCL10 (e.g., N-terminally truncated

[0543] CXCL10 and / or citrullinated CXCL10).

[0544] Binding proteins of the disclosure also bind CA-125, HE4 and / or IL-6.

[0545] Methods for assessing binding to a protein are known in the art, e.g., as described in Scopes, 1994. Such a method generally involves immobilizing the binding protein and contacting it with labeled antigen. Following washing to remove non-specific bound protein, the amount of label and, as a consequence, bound antigen is detected. Of course, the binding protein can be labeled and the antigen immobilized. Panning-type assays can also be used. Alternatively, or additionally, surface plasmon resonance assays can be used.

[0546] Determining Affinity

[0547] Optionally, the dissociation constant (Kd) or association constant (Ka) or equilibrium constant (KD) of a binding protein is determined. These constants for a binding region (e.g., an antibody or antigen binding fragment) are, in one example, measured by biosensor analysis using surface plasmon resonance assays. Exemplary SPR methods are described in US7229619.

[0548] Affinity measurements can be determined by standard methodology for antibody reactions, for example, immunoassays, surface plasmon resonance (SPR) (Rich and Myszka, 2000; Englebienne, 1998), isothermal titration calorimetry (ITC) or other kinetic interaction assays known in the art.

[0549] Assays for determining an antibody or antigen binding fragment thereof that competitively inhibits binding of a binding protein described herein will be apparent to the skilled artisan and / or described herein.

[0550] For example, the antibody or antigen binding fragment thereof is conjugated to a detectable label, e.g., a fluorescent label or a radioactive label. The labeled antibody and the test binding protein are then mixed and contacted with the antigen (e.g., CXCL10, CA-125, HE4 or IL-6) or a region thereof or a cell expressing same. The level of labeled antibody is then determined and compared to the level determined when the labeled antibody is contacted with the antigen, region or cells in the absence of the binding protein. If the level of labeled antibody is reduced in the presence of the test binding protein compared to the absence of the binding protein, the binding protein is considered to competitively inhibit binding of the antibody to the antigen.

[0551] Optionally, the test binding protein is conjugated to different label to the antibody. This alternate labeling permits detection of the level of binding of the test binding protein to the antigen or the region thereof or the cell.

[0552] In another example, the binding protein is permitted to bind to the antigen (e.g., CXCL10, CA-125, HE4 or IL-6) or a region thereof or a cell expressing same prior to contacting the antigen, region or cell with the antibody. A reduction in the amount of bound antibody in the presence of the binding protein compared to in the absence of the binding protein indicates that the protein competitively inhibits binding of the antibody to the antigen. A reciprocal assay can also be performed using labeled binding protein and first allowing the antibody to bind to the antigen. In this case, a reduced amount of labeled binding protein bound to the antigen in the presence of the antibody compared to in the absence of the antibody indicates that the binding protein competitively inhibits binding of the antibody to antigen. the Level of CXCL10, CA-125, HE4 and IL-6

[0553] As discussed herein, the present disclosure provides methods for detecting a panel of biomarkers (i.e., CXCL10 active ratio, CA-125, HE4 and IL-6) in a subject for diagnosis of a malignant condition. The present inventors have developed CXCL10 binding proteins to detect different forms of the protein, i.e., full-length or mature CXCL10, N-terminally truncated CXCL10 and / or citrullinated CXCL10.

[0554] The present inventors have found that different forms of the proteins are present at different levels in benign and malignant conditions.

[0555] In particular, the inventors have previously found that determining the level of active CXCL10 protein and total CXCL10 protein in a subject can distinguish benign and malignant conditions. This procedure is referred to herein as the active ratio test (ART).

[0556] As used herein, the term “active” in the context of the level of CXCL10 refers to biologically active forms of CXCL10. For example, a CXCL10 binding protein of the present disclosure that binds active CXCL10 refers to 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.

[0557] As used herein, the term “total” in the context of the level of CXCL10 refers to all forms of CXCL10. For example, a CXCL10 binding protein of the present disclosure that binds total CXCL10 refers to a binding protein that binds to full-length or mature (i.e., N-terminally intact) CXCL10 as well as to N-terminally truncated and citrullinated CXLC10.

[0558] Accordingly, the methods of any disclosure described herein comprise determining an active CXCL10 ratio.

[0559] As used herein, the term “CXCL10 ratio” refers to the ratio of the level of active CXLC10 to the level of total CXCL10 in the subject.

[0560] The inventors have presently found that determining the combined level of active CXCL10, CA-125, HE4 and IL-6 in a subject distinguishes a malignant condition from a benign condition with increased sensitivity and specificity in a subject, compared to the determining the level of active CXCL10, CA-125, HE4 and / or IL-6 alone.

[0561] Accordingly, the methods of any disclosure described herein comprise determining a level of CXCL10, CA-125, HE4 and IL-6 in a subject.

[0562] As used herein, the term “level” in reference to CXCL10, CA-125, HE4 and IL- 6 shall be understood to refer to the level of functionality of the protein (i.e., the functional level). For example, the level (or “level of expression”) refers to a measure of encoded protein.

[0563] In one example, determining the level of active CXCL10 and total CXCL10 comprises determining the amount of active CXCL10 protein and the amount of total CXCL10 protein in the subject; and / or determining the level of CA-125, HE4 and / or IL- 6 comprises determining the amount of CA-125, HE4 and / or IL-6 protein in the subject.

[0564] As used herein, the term “amount” with reference to the level of CXCL10, CA- 125, HE4 and IL-6 will be understood to refer to a quantity of protein. Various methods of assessing the quantity of protein are available to the skilled person and the skilled person will recognise that the specific value or amount will vary depending on the method of assessment used. It will also be apparent that this term encompasses both an absolute and relative value. 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 the sample.

[0565] The inventors have surprisingly found that determining the CXCL10 ratio in combination with the level of CA-125, HE4 and IL-6 in a subject can distinguish benign and malignant conditions. In particular, the inventors have found that determining the combination of the CXCL10 ratio with CA-125, HE4 and IL-6 in a subject can distinguish benign and malignant conditions with high specificity and sensitivity and a high positive and negative predictive value, when compared to determining the CXCL10 ratio, or level of CA-125, HE4 and IL-6 alone.

[0566] In one example, the method further comprises comparing the CXCL10 ratio and / or the level of CA-125, HE4 and / or IL-6 in the subject to a CXCL10 ratio and / or level of -125, HE4 and / or IL-6 in at least one reference.

[0567] In one example of any method described herein, the method comprises determining:

[0568] (i) if the CXCL10 ratio and the level of CA-125, HE4 and IL-6 in the subject are higher than the CXCL10 ratio and the level of CA-125, HE4 and LL6 in the reference; or

[0569] (ii) if the CXCL10 ratio and the level of CA-125, HE4 and IL-6 in the subject are lower than the CXCL10 ratio and the level of CA-125, HE4 and IL-6 in the reference.

[0570] The term “higher” in reference to the CXCL10 ratio and level of CA-125, HE4 and IL-6 means that the ratio or level in the subject is greater or increased, compared to a control or reference level. It will be apparent from the foregoing that the CXCL10 ratio or level of CA-125, HE4 and IL-6 needs 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%.

[0571] The term “lower” in reference to the CXCL10 ratio and level of CA-125, HE4 and IL-6means that the ratio or level in the subject is reduced or decreased, compared to a control or reference level. It will be apparent from the foregoing that the CXCL10 ratio or level of CA-125, HE4 and IL-6 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%.

[0572] In one example, the method comprises determining a diagnostic score comprising the CXCL10 ratio and level of CA-125, HE4 and IL-6 in the subject.

[0573] In one example, the diagnostic score uses Youden’s statistics to calculate a threshold or cut-off score to differentiate between malignant and benign conditions. For example, the threshold or cut-off score is 3.684 (e.g., 3.6).

[0574] The skilled person will understand Youden's J statistic (or Youden's index) is a characteristic parameter used to summarize the performance of a classifier (Youden, William J, Index for rating diagnostic tests, Cancer 3, 1, 32-35, 1950). Youden’s J index was applied as previously described (see Watrowski, R., et al., Biomarker-Based Models for Preoperative Assessment of Adnexal Mass: A Multicenter Validation Study. Cancers (Basel), 2022. 14(7)).

[0575] Methods of Determining the Level of CXCL10, CA-125, HE4 and IL-6

[0576] Methods of determining the level of CXCL10, CA-125, HE4 and IL-6 protein will be apparent to the skilled person and / or are described herein. For example, methods include immunohistochemistry, immunofluorescence, an immunoblot, a western blot, a dot blot, an enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay, fluorescence resonance energy transfer (FRET), matrix-assisted laser desorption / ionization time of flight (MALDI-TOF), electrospray ionization (ESI), mass spectrometry (including tandem mass spectrometry, e.g. LC MS / MS), biosensor technology, evanescent fiber-optics technology or protein chip technology. For example, a suitable assay is a semi-quantitative assay and / or a quantitative assay.

[0577] In one example, the method for determining the level of CXCL10, CA-125, HE4 and / or IL-6 in a sample comprises contacting a biological sample from a subject with a binding protein (as described herein) that specifically binds to the antigen (i.e., the CXCL10, CA-125, HE4 and / or IL-6 polypeptide or protein) for a time and under conditions sufficient for a complex between the binding protein and the polypeptide or protein to form and then detecting the complex.

[0578] In one example, in any method described herein the level of active CXCL10 is measured and the level of total CXCL10 is measured and the ratio of active CXCL10 to total CXCL10 determined. Enzyme Linked Immunosorbent Assay (ELISA) and Fluorescence Linked Immunosorbent Assay (FLISA)

[0579] Standard solid-phase ELISA or FLISA formats are particularly useful in determining the concentration of a protein from a variety of samples. In one form such an assay involves immobilizing a biological sample onto a solid matrix, such as, for example a polystyrene or polycarbonate microwell or dipstick, a membrane, or a glass support (e.g. a glass slide).

[0580] An antibody that specifically binds to a marker within an antigen of interest (i.e., the CXCL10, CA-125, HE4 and / or IL-6 polypeptide or protein) is brought into direct contact with the immobilized biological sample, and forms a direct bond with any of its target protein present in said sample. This antibody is generally labeled with a detectable reporter molecule, such as for example, a fluorescent label (e.g. FITC or Texas Red) or a fluorescent semiconductor nanocrystal (as described in US 6,306,610) in the case of a FLISA or an enzyme (e.g. horseradish peroxidase (HRP), alkaline phosphatase (AP) or P-galactosidase) in the case of an ELISA, or alternatively a second labeled antibody can be used that binds to the first antibody. Following washing to remove any unbound antibody the label is detected either directly, in the case of a fluorescent label, or through the addition of a substrate, such as for example hydrogen peroxide, TMB, or toluidine, or 5-bromo-4-chloro-3-indol-beta-D-galaotopyranoside (x-gal) in the case of an enzymatic label.

[0581] Such ELISA or FLISA based systems are suitable for quantification of the amount of a protein in a sample, by calibrating the detection system against known amounts of a protein standard to which the antibody binds, such as for example, an isolated and / or recombinant polypeptide or immunogenic fragment thereof or epitope thereof.

[0582] In another example, an ELISA consists of immobilizing an antibody or ligand that specifically binds a marker of a disease or disorder within a polypeptide of interest (i.e., the CXCL10, CA-125, HE4 and / or IL-6 polypeptide or protein) on a solid matrix, such as, for example, a membrane, a polystyrene or polycarbonate microwell, a polystyrene or polycarbonate microwell dipstick or a glass support. A sample is then brought into physical relation with said antibody, and said marker within the sample is bound or ‘captured’. The bound protein is then detected using a labeled antibody. Alternatively, a third labeled antibody can be used that binds the second (detecting) antibody.

[0583] In one example, the immobilized antibody is a polyclonal antibody.

[0584] It will be apparent to the skilled person that the assay formats described herein are amenable to high throughput formats, such as, for example automation of screening processes or a microarray format as described in Mendoza et al., 1999. Furthermore, variations of the above-described assay will be apparent to those skilled in the art, such as, for example, a competitive ELISA.

[0585] In one example, the assay format is a microfluidics device, for example, a microfluidic chip or a droplet-based microfluidics device. Microfluidic chips (e.g., a microelectromechanical systems (MEMS) device), typically range in size from a few square millimeters to a few square centimeters. These microfluidic chips are designed to handle or manipulate small fluid volumes in order to perform biological or medical processing or testing. The fluids may be moved, mixed, or processed in a single microfluidic chip.

[0586] In another example, the assay format is a dipstick, for example, a polycarbonate dipstick.

[0587] SIMOA assav

[0588] Another assay that can be used in the present invention is a single-molecule array (Simoa) assay, which is described in detail in Kuhle et al. (2016) and Gisslen et al. (2016).

[0589] Western blotting

[0590] In another example, western blotting is used to determine the level of a marker within a polypeptide of interest (i.e., the CXCL10, CA-125, HE4 and / or IL-6 polypeptide or protein) in a sample. In such an assay protein from a sample is separated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using techniques known in the art and described in, for example, Scopes, 1994. Separated proteins are then transferred to a solid support, such as, for example, a membrane (e.g., a PVDF membrane), using methods known in the art, for example, electrotransfer. This membrane is then blocked and probed with a labeled antibody or ligand that specifically binds to a marker within the polypeptide (i.e., the CXCL10, CA-125, HE4 and / or IL-6 polypeptide or protein). Alternatively, a labeled secondary, or even tertiary, antibody or ligand is used to detect the binding of a specific primary antibody. The level of label is then determined using an assay appropriate for the label used.

[0591] An appropriate assay will be apparent to the skilled artisan and include, for example, densitometry. In one example, the intensity of a protein band or spot is normalized against the total amount of protein loaded on a SDS-PAGE gel using methods known in the art. Alternatively, the level of the marker detected is normalized against 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), P2 microglobulin, hydroxy -methylbilane synthase, hypoxanthine phosphoribosyltransferase 1 (HPRT), ribosomal protein L13c, succinate dehydrogenase complex subunit A and TATA box binding protein (TBP).

[0592] Radioimmunoas s ay

[0593] Alternatively, the level of CXCL10, CA-125, HE4 and / or IL-6 polypeptide or protein is detected using a radioimmunoassay (RIA). The basic principle of the assay is the use of a radiolabeled antibody or antigen to detect antibody-antigen interactions. An antibody or ligand that specifically binds to the marker within a polypeptide of interest (i.e., the CXCL10, CA-125, HE4 and / or IL-6 polypeptide or protein) is bound to a solid support and a sample brought into direct contact with said antibody. To detect the level of bound antigen, an isolated and / or recombinant form of the antigen is radiolabeled and brought into contact with the same antibody. Following washing, the level of bound radioactivity is detected. As any antigen in the biological sample inhibits binding of the radiolabeled antigen the level of radioactivity detected is inversely proportional to the level of antigen in the sample. Such an assay may be quantitated by using a standard curve using increasing known concentrations of the isolated antigen.

[0594] As will be apparent to the skilled person, such an assay may be modified to use any reporter molecule, such as, for example, an enzyme or a fluorescent molecule, in place of a radioactive label.

[0595] Biosensor or optical immunosensor system

[0596] Alternatively, the level of a CXCL10, CA-125, HE4 and / or IL-6 polypeptide or protein in a sample is determined using a biosensor or optical immunosensor system. In general, an optical biosensor is a device that uses optical principles to quantitatively convert the binding of a ligand or antibody to a target polypeptide into electrical signals. These systems can be grouped into four major categories: reflection techniques; surface plasmon resonance; fibre optic techniques and integrated optic devices. Reflection techniques include ellipsometry, multiple integral reflection spectroscopy, and fluorescent capillary fill devices. Fibre-optic techniques include evanescent field fluorescence, optical fibre capillary tube, and fibre optic fluorescence sensors. Integrated optic devices include planer evanescent field fluorescence, input grading coupler immunosensor, Mach-Zehnder interferometer, Hartman interferometer and difference interfermoter sensors. These examples of optical immunosensors are described in general by Robins, 1991. More specific description of these devices are found for example in U.S. Patent. Nos. 4,810,658; 4,978,503; 5,186,897; and Brady et al., 1987.

[0597] As will be apparent to the skilled person, the type and size of the biological sample will depend upon the detection means used. For example, protein-based assays require sufficient cells to provide sufficient protein for an antigen based assay.

[0598] As used herein, the term “sample” or “biological sample” refers to any type of suitable material obtained from the subject. The term encompasses a clinical sample (e.g., cervical, or cervicovaginal, swab), biological fluid (e.g., cervical fluid, vaginal fluid, plasma, ascites), tissue samples, live cells and also includes cells in culture, cell supernatants, cell lysates derived therefrom. The sample can be used as obtained directly from the source or following at least one- step of (partial) purification. It will be apparent to the skilled person that the sample can be prepared in any medium which does not interfere with the method of the disclosure. Typically, the sample comprises cells or tissues and / or is an aqueous solution or biological fluid comprising cells or tissues. The skilled person will be aware of selection and pre-treatment methods. Pre-treatment may involve, for example, diluting viscous fluids. Treatment of a sample may involve filtration, distillation, separation, concentration.

[0599] In one example, the biological sample has been derived previously from the subject. Accordingly, in one example, a method as described herein according to any embodiment additionally comprises providing the biological sample.

[0600] In one example, biological samples may be collected from a subject at more than one time points to e.g. monitor progression of a malignant condition, to monitor for recurrence, and / or to assess the efficacy of a treatment protocol. In one example, the biological sample may be collected from a subject before, during and / or after treatment of a subject for a malignant condition. Samples may be collected, weekly, fortnightly, monthly, every two months, every three months, every four months, every five months or every six months to monitor progression of a malignant condition or assess the efficacy of a treatment regimen.

[0601] In one example, a method as described herein according to any embodiment is performed using an extract from a sample, such as, for example, protein.

[0602] Reference samples

[0603] As will be apparent from the preceding description, some assays of the present disclosure may utilize a suitable reference sample or control for quantification.

[0604] Suitable reference samples for use in the methods of the present disclosure will be apparent to the skilled person and / or described herein. For example, the reference may be an internal reference (i.e., from the same subject), from a normal individual or an established data set (e.g., matched by age, sample type and / or stage of cycle).

[0605] In one example, the reference is an internal reference or sample. For example, the reference is an autologous reference. In one example, the internal reference is obtained from the subject at the same time as the sample under analysis. In another example, the internal reference is obtained from the subject at an earlier time point as the sample under analysis.

[0606] As used herein, the term “normal individual” shall be taken to mean that the subject is selected on the basis that they do not have a malignant and / or a benign condition, or that they are not suspected of having such condition. For example, the normal individual is a healthy individual.

[0607] In one example, the reference is an established data set. Established data sets suitable for use in the present disclosure will be apparent to the skilled person and include, for example:

[0608] • A data set from a normal subject or a population of normal subjects matched by age and sample type;

[0609] • A data set from another subject or a population of subjects matched by age, sample type and / or disease / condition;

[0610] • A data set comprising cells in vitro, wherein the cells have been treated to induce CXCL10, CA-125, HE4 and IL-6 expression; and

[0611] • A data set comprising endometrial epithelial cells in vitro, wherein the cells have been treated to inhibit CXCL10, CA-125, HE4 and IL-6 expression.

[0612] In one example, a reference is not included in an assay. Instead, a suitable reference is derived from an established data set previously generated. Data derived from processing, analyzing and / or assaying a test sample is then compared to data obtained for the sample.

[0613] Assessing Risk and / or Detecting and / or Diagnosing a Malignant Condition

[0614] As disclosed herein, the inventors of the present disclosure have demonstrated a role of CXCL10, CA-125, HE4 and IL-6 in assessing the risk that a subject suffers from a malignant condition and / or detecting and / or diagnosing a malignant condition. In particular, the inventors of the present disclosure have demonstrated a role of the combined detection of CXCL10, CA-125, HE4 and IL-6 in assessing the risk that a subject suffers from a malignant condition and / or detecting and / or diagnosing a malignant condition compared to the detection of CXCL10, CA-125, HE4 and IL-6 alone. It will be apparent to the skilled person that the methods disclosed herein will be useful in distinguishing a malignant condition from a benign condition in a subject. For example, the methods of the present disclosure are useful as a screening test for the assessing the risk and / or for the diagnosis of a malignant condition in a subject.

[0615] Accordingly, the present disclosure provides, for example, a method of detecting and / or diagnosing a malignant condition in a subject, the method comprising:

[0616] (i) determining a level of active C-X-C motif chemokine ligand 10 (CXCL10) in the subject and a level of total CXCL10 in the subject; and determining a CXCL10 ratio of active CXLC10 to total CXCL10 in the subject; and

[0617] (ii) determining the level of cancer antigen 125 (CA-125), human epididymis protein

[0618] 4 (HE4) and interleukin 6 (IL-6) in the subject.

[0619] As used herein, the term “detect”, “detecting”, “diagnosis” or “diagnosing” refers to the identification of a malignant condition in a subject.

[0620] 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 and grows at sites distant from the tissue of origin. In one example, a malignant disease or condition is cancer or is related to cancer. The skilled person will understand that cancers can arise from almost any tissue in the body and as used herein the term encompasses all forms of the disease, including e.g., carcinomas, sarcomas, lymphomas, and leukemias (i.e., solid and non-solid forms of cancer).

[0621] In one example, the present disclosure provides a method of distinguishing a malignant condition from a benign condition.

[0622] As used herein, the term “benign condition” refers to a mass of cells that lacks the ability to invade neighbouring tissue.

[0623] In one example, the present disclosure provides a method of distinguishing pre- cancerous lesions from a benign condition.

[0624] As used herein, the term “pre-cancerous lesion” refers to a mass of cells that have grown abnormally, causing their size, shape or appearance to look different than normal cells, however they are not yet cancerous or malignant. In one example, the pre- cancerous lesion are p53 pre-cancerous lesions. As used herein, the term p53 pre- cancerous lesions refers to cells that have p53 gene mutations.

[0625] In one example, the subject suffers from 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, kidney, colon, pancreas or stomach. For example, the subject suffers from ovarian cancer. In one example, the cancer is a non-solid tumor, for example leukemia or lymphoma. In one example, the subject suffers from a stage 0 cancer. For example, the carcinoma is in situ. In another example, the subject suffers from a 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 organs adjacent to the location of the primary tumor. In one example, the subject suffers from a stage IV cancer. For example, the cancer has spread to distant tissues and / or organs.

[0626] In one example, the subject has not received treatment for the malignant condition. For example, the subject is treatment naive.

[0627] In one example, the subject is receiving treatment for the malignant condition. In one example, the subject has received treatment for the malignant condition. Suitable therapies for the treatment of the malignant condition will be apparent to the skilled person and / or described herein. For example, the treatment comprises surgery, chemotherapy, radiation therapy, targeted drug therapy, immunotherapy or a combination thereof.

[0628] Ovarian Cancer

[0629] In one example of any method described herein, the method comprises assessing the risk that a subject suffers from ovarian cancer (i.e., malignant ovarian cancer) and / or detecting and / or diagnosing ovarian cancer in a subject. For example, the subject suffers from ovarian cancer.

[0630] As used herein, the term “ovarian cancer” refers to any cancerous growth that begins in the ovary.

[0631] In one example, the method comprises a method of differentiating ovarian cancer in a subject from a benign condition.

[0632] It will be apparent to the skilled person that the methods described herein are applicable for detecting and / or diagnosing all subtypes of ovarian cancer including, for example, epithelial, endometrioid tumours, germ-cell tumours, clear cell and mucinous adenocarcinomas.

[0633] In one example, the present disclosure provides a method for assessing the risk that a subject suffers from epithelial ovarian cancer.

[0634] In one example, the present disclosure provides a method for detecting and / or diagnosing epithelial ovarian cancer in a subject.

[0635] The skilled person will understand that ovarian cancer is staged using the International Federation of Gynaecology and Obstetrics (FIGO) staging system, as described below in Table 2. In one example of any method described herein, the present disclosure provides a method for detecting and / or diagnosing ovarian cancer in a subject irrespective of stage of the cancer.

[0636] In one example, the present disclosure provides a method for assessing the risk of stage I ovarian cancer. For example, a method for assessing the risk of early stage ovarian cancer.

[0637] In one example of any method described herein, the present disclosure provides a method for detecting and / or diagnosing stage I ovarian cancer in a subject. For example, a method for detecting and / or diagnosing early stage ovarian cancer.

[0638] The skilled person will also understand that ovarian cancer is classified based on the grade of the cancer. For example, grade 1 tumours have well differentiated cells; grade 2 tumours are moderately well-differentiated; and grade 3 tumours are poorly differentiated.

[0639] In one example of any method described herein, the present disclosure provides a method for detecting and / or diagnosing ovarian cancer in a subject irrespective of grade of the cancer.

[0640] In another example, the ovarian cancer is serous, mucinous, endometrioid, clear cell, GCT or a mixture thereof, ovarian cancer. In an example, the ovarian cancer is serous.

[0641] In one example, the subject is at risk of ovarian cancer.

[0642] 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” denotes that a subject has one or more risk factors, which are measurable parameters that correlate with development of the disease or condition, as known in the art and / or described herein. For example, the subject has a p53 gene mutation and / or a BRCA gene mutation.

[0643] Table 2: FIGO stages of ovarian cancer

[0644] Risk factors include, for example:

[0645] • A family history of ovarian and / or breast cancer;

[0646] • Reproductive history, i.e., having children after the age of 35 or never having children are associated with a higher risk;

[0647] • History of breast cancer;

[0648] • Hormonal therapy, e.g., hormone replacement therapy (HRT) after menopause is associated with an increased risk; and

[0649] • Obesity, e.g., a body mass index of greater than 30. A subject is at risk if she has a higher risk of developing ovarian cancer than a control population. The control population may include one or more subjects selected at random from the general population (e.g., matched by age, gender, race and / or ethnicity) who have not suffered from or have a family history of ovarian cancer. A subject can be considered at risk if a "risk factor" associated with ovarian cancer is found to be associated with that subject. A risk factor can include any activity, trait, event or property associated with a given disorder, for example, through statistical or epidemiological studies on a population of subjects. A subject can thus be classified as being at risk even if studies identifying the underlying risk factors did not include the subject specifically.

[0650] In one example, the method of the present disclosure is performed before or after the onset of symptoms of ovarian cancer. Symptoms of ovarian cancer will be apparent to the skilled person and include, for example:

[0651] • Abdominal enlargement or swelling;

[0652] • Abdominal fullness and pain;

[0653] • Pain in lower abdomen;

[0654] • Feeling full after eating very little;

[0655] • Tiredness;

[0656] • Changes in bowel or bladder habits;

[0657] • Clothes not fitting well;

[0658] • Swelling of legs;

[0659] • Shortness of breath;

[0660] • Bleeding from vagina;

[0661] • Abnormal menstrual cycles;

[0662] • Weight loss or gain; and

[0663] • Unexplained back pain.

[0664] In one example, the subject is pre-menopausal.

[0665] In one example, the subject is menopausal. For example, the subject is postmenopausal. In one example, the subject is peri-menopausal.

[0666] The skilled person will understand that menopause is the period after the cessation of normal ovulation cycles, during which normal menstruation ceases. A decrease in estradiol (E2) production accompanies menopause, as the ovaries cease manufacture of E2. This decrease in E2 production results in a shift in hormone balance in the body, which often gives rise to a variety of symptoms associated with menopause.

[0667] Peri-menopause, which is also known as pre-menopause or the climacteric, is that period prior to menopause during which normal ovulation cycles gradually give way to cessation of menses. As the ovulatory cycles lengthen and become more irregular, the level of E2 may initially increase, but will eventually drop with the onset of menopause. Menopausal symptoms often accompany the drop in E2 levels. Symptoms of peri-menopause, menopause and post-menopause will be apparent to the skilled person and include physical symptoms such as hot flashes and sweating secondary to vasomotor instability. Additionally, psychological and emotional symptoms may accompany onset of climacteric, such as fatigue, irritability, insomnia, inability to concentrate, depression, memory loss, headache, anxiety and nervousness. Additional symptoms can include intermittent dizziness, paresthesias, palpitations and tachycardia as well as nausea, constipation, diarrhea, arthralgia, myalgia, cold hands and feet and weight gain. In addition, changes to the genitals, urinary incontinence, vaginal dryness, loss of pelvic muscle tone, increased risk of cardiovascular disease and osteoporosis increase with onset of menopause.

[0668] Methods of Treating a Malignant Condition

[0669] In one example, the present invention provides a method of treating a malignant condition in a subject, the method comprising performing the method as described herein and treating the subject for the malignant condition.

[0670] As used herein, the terms “treating”, “treat” or “treatment” includes surgically removing all or part of the cancer or administering a therapeutically effective amount of a compound / molecule / radiation sufficient to reduce or eliminate at least one symptom of the malignant condition. For example, an "effective amount" for therapeutic uses is the amount of the compound required to provide a clinically significant decrease in disease symptoms without undue adverse side effects. An appropriate "effective amount" in any individual case may be determined using techniques, such as a dose escalation study. An "effective amount" of a compound is an amount effective to achieve a desired pharmacologic effect or therapeutic improvement without undue adverse side effects. It is understood that "an effective amount" or "a therapeutically effective amount" can vary from subject to subject, due to variation in metabolism of the compound of any of age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.

[0671] In one example, treatment comprises surgery, chemotherapy, radiation therapy, targeted drug therapy or a combination thereof.

[0672] In one example, the treatment comprises surgery. For example, the surgery is debulking surgery.

[0673] In another example, the treatment comprises chemotherapy. Exemplary chemotherapy agents include, for example, caboplatin, cytarabine, chlorambucil, cisplatin, cyclophosphamide, danorubicin, docetaxal, doxorubicin, erlotinib, etoposide, fluorouracil, fludarabine, idarubicin, irinotecan, liposomal doxorubicin, methotrexate, mitoxantrone, paclitaxel, topotecan, vincristine and vinblastine.

[0674] In one example, 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), volumetric 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 cord radiation therapy (craniospinal radiation).

[0675] In one example, the treatment comprises targeted drug therapy. For example, the targeted drug therapy is a therapeutic antibody. Exemplary therapeutic antibodies are known to the skilled person and include, but are not limited to, 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; Ascrinvacumab; Aselizumab; Atezolizumab; Atinumab; Atlizumab (tocilizumab); Atorolimumab; Bapineuzumab; Basiliximab; Bavituximab; Bectumomab; Begelomab; Belimumab; Benralizumab; Bertilimumab; Besilesomab; Bevacizumab; Bezlotoxumab; Biciromab; Bimagrumab; Bimekizumab; Bivatuzumab mertansine; Blinatumomab; Blosozumab; Bococizumab; Brentuxim abvedotin; Briakinumab; Brodalumab; Brolucizumab; Brontictuzumab; Canakinumab; Cantuzumab mertansine; Cantuzumab ravtansine; Caplacizumab; Capromab pendetide; Carlumab; Catumaxomab; cBR96-doxorubicin immunoconjugate; Cedelizumab; Certolizumab pegol; Cetuximab; Citatuzumab bogatox; Cixutumumab; Clazakizumab; Clenoliximab; Clivatuzumab tetraxetan; Codrituzumab; Coltuximab ravtansine; Conatumumab; Concizumab; Crenezumab; Dacetuzumab; Daclizumab; Dalotuzumab; Dapirolizumab pegol; Daratumumab; Dectrekumab; Demcizumab; Denintuzumab mafodotin; Denosumab; Derlotuximab biotin; Detumomab; Dinutuximab; Diridavumab; Dorlimomab aritox; Drozitumab; Duligotumab; Dupilumab; Durvalumab; Dusigitumab; Ecromeximab; Eculizumab; Edobacomab; Edrecolomab; Efalizumab; Efungumab; Eldelumab; Elgemtumab; Elotuzumab; Elsilimomab; Emactuzumab; Emibetuzumab; Enavatuzumab; Enfortumab vedotin; Enlimomab pegol; Enoblituzumab; Enokizumab; Enoticumab; Ensituximab; Epitumomab cituxetan; Epratuzumab; Erlizumab; Ertumaxomab; Etanercept; Etaracizumab; Etrolizumab; Evinacumab; Evolocumab; Exbivirumab; Fanolesomab; Faralimomab; Farletuzumab; Fasinumab; Felvizumab; Fezakinumab; Ficlatuzumab; Figitumumab; Firivumab; Flanvotumab; Fletikumab; Fontolizumab; Foralumab; Foravirumab; Fresolimumab; Fulranumab; Futuximab; Galiximab; Ganitumab; Gantenerumab; Gavilimomab; Gemtuzumab ozogamicin; Gevokizumab; Girentuximab; Glembatumumab vedotin; Golimumab; Gomiliximab; Guselkumab; Ibalizumab; Ibritumomab tiuxetan; Icrucumab; Idarucizumab; Igovomab; Imalumab; Imciromab; Imgatuzumab; Inclacumab; Indatuximab ravtansine; Indusatumab vedotin; Infliximab; Inolimomab; Inotuzumab ozogamicin; Intetumumab; Ipilimumab; Iratumumab; Isatuximab; Itolizumab; Ixekizumab; Keliximab; Labetuzumab; Lambrolizumab; Lampalizumab; Lebrikizumab; Lemalesomab; Lenzilumab; Lerdelimumab; Lexatumumab; Libivirumab; Lifastuzumab vedotin; Ligelizumab; Lilotomab satetraxetan; Lintuzumab; Lirilumab; Lodelcizumab; Lokivetmab; Lorvotuzumab mertansine; Lucatumumab; Lulizumab pegol; Lumiliximab; Lumretuzumab; Mapatumumab; Margetuximab; Maslimomab; Matuzumab; Mavrilimumab; Mepolizumab; Metelimumab; Milatuzumab; Minretumomab; Mirvetuximab soravtansine; Mitumomab; Mogamulizumab; Morolimumab; Motavizumab; Moxetumomab pasudotox; Muromonab-CD3; Nacolomab tafenatox; Namilumab; Naptumomab estafenatox; Narnatumab; Natalizumab; Nebacumab; Necitumumab; Nemolizumab; Nerelimomab; Nesvacumab; Nimotuzumab; Nivolumab; Nofetumomab merpentan; Obiltoxaximab; Obinutuzumab; Ocaratuzumab; Ocrelizumab; Odulimomab; Ofatumumab; Olaratumab; Olokizumab; Omalizumab; Onartuzumab; Ontuxizumab; Opicinumab; Oportuzumab monatox; Grego vomab; Orticumab; Otelixizumab; Otlertuzumab; Oxelumab; Ozanezumab; Ozoralizumab; Pagibaximab; Palivizumab; Panitumumab; Pankomab; Panobacumab; Parsatuzumab; Pascolizumab; Pasotuxizumab; Pateclizumab; Patritumab; Pembrolizumab; Pemtumomab; Perakizumab; Pertuzumab; Pexelizumab; Pidilizumab; Pinatuzumab vedotin; Pintumomab; Placulumab; Polatuzumab vedotin; Ponezumab; Priliximab; Pritoxaximab; Pritumumab; Quilizumab; Racotumomab; Radretumab; Rafivirumab; Ralpancizumab; Ramucirumab; Ranibizumab; Raxibacumab; Refanezumab; Regavirumab; Reslizumab; Rilotumumab; Rinucumab; Rituximab; Robatumumab; Roledumab; Romosozumab; Rontalizumab; Rovelizumab; Ruplizumab; Sacituzumab govitecan; Samalizumab; Sarilumab; Satumomab pendetide; Secukinumab; Seribantumab; Setoxaximab; Sevirumab; Sibrotuzumab; Sifalimumab; Siltuximab; Simtuzumab; Siplizumab; Sirukumab; Sofituzumab vedotin; Solanezumab; Solitomab; Sonepcizumab; Sontuzumab; Stamulumab; Sulesomab; Suvizumab; Tabalumab; Tacatuzumab tetraxetan; Tadocizumab; Talizumab; Tanezumab; Taplitumomab paptox; Tarextumab; Tefibazumab; Telimomab aritox; Tenatumomab; Teneliximab; Teplizumab; Teprotumumab; Tesidolumab; Tetulomab; Ticilimumab; Tigatuzumab; Tildrakizumab; Tocilizumab; Toralizumab; Tosatoxumab; Tositumomab; Tovetumab; Tralokinumab; Trastuzumab; Tregalizumab; Tremelimumab; Trevogrumab; Tucotuzumab celmoleukin; Tuvirumab; Ublituximab; Ulocuplumab; Urelumab; Urtoxazumab; Ustekinumab; Vandortuzumab vedotin; Vantictumab; Vanucizumab; Vapaliximab; Varlilumab; Vatelizumab; Vedolizumab; Veltuzumab; Vepalimomab; Vesencumab; Visilizumab; Volociximab; Vorsetuzumab mafodotin; Votumumab; Zalutumumab; Zanolimumab; Zatuximab; Ziralimumab; Zolimomab aritox.

[0676] In one example, 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.

[0677] In one example, the immunotherapy is a checkpoint inhibitor. Suitable checkpoint inhibitors include, for example, ipilimumab (Yervoy®), nivolumab (Opdivo®), pembrolizumab (Keytruda®), atezolizumab (Tecentriq®), avelumab (Bavencio®), durvalumab (Imfinzi®).

[0678] In one example, the immunotherapy is an oncolytic virus therapy. For example, the oncolytic virus therapy is talimogene laherparepvec (Imlygic®), or T-VEC.

[0679] In one example, the immunotherapy is a T-cell therapy. For example, the T-cell therapy is chimeric antigen receptor (CAR) T-cell therapy.

[0680] In one example, the immunotherapy is a cancer vaccine.

[0681] Monitoring Tumour Burden, Progression, Recurrence and Regression

[0682] It will be apparent to the skilled person that the present disclosure also provides a method of monitoring tumour burden, monitoring progression, monitoring recurrence and / or determining tumour regression in a subject suffering from a malignant condition, the method comprising (i) determining a C-X-C motif chemokine ligand 10 (CXCL10) ratio of active CXLC10 to total CXCL10 in the subject at one or more time points; and (ii) determining the level of cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) in the subject at one or more time points.

[0683] As used herein, the term "monitoring" can include, determination of prognosis, response to drug therapy, assessment of ongoing drug therapy, prediction of outcomes, determining response to therapy (including diagnosis of a complication), following progression of tumour volume, or selecting patients most likely to benefit from a therapy.

[0684] As used herein, the term “tumour burden” refers to the volume of tumour cells and does not include other changes such as inflammation, necrosis or edema. As used herein, the term “progression” refers to continued growth and invasiveness of the tumour.

[0685] As used herein, the term “regression” refers to a decrease in the size or volume of the tumour.

[0686] As used herein, the term “recurrence” refers to a malignant condition that has returned or come back after a period of time in which the malignant condition could not be detected.

[0687] In one example, the method of monitoring tumour burden, monitoring progression, monitoring recurrence and / or determining tumour regression in a subject suffering from a malignant condition comprises determining a CXCL10 ratio and determining the level of CA-125, HE4 and IL-6 in the subject at one or more time points. For example, the ratio and level is determined at 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 time points.

[0688] In one example, the CXCL10 ratio and level of CA-125, HE4 and IL-6 are determined in at least one biological sample obtained from the subject prior to treatment, during treatment and / or after treatment. For example, the CXCL10 ratio and level of CA-125, HE4 and IL-6 are determined at one or more time points prior to treatment. In another example, the CXCL10 ratio and level of CA-125, HE4 and IL-6 are determined at one or more time points during treatment. In a further example, the CXCL10 ratio and level of CA-125, HE4 and IL-6 are determined at one or more time points after treatment. In another example, the CXCL10 ratio and level of CA-125, HE4 and IL-6 are determined prior to and during treatment. In a further example, the CXCL10 ratio and level of CA-125, HE4 and IL-6 are determined prior to and after treatment. In another example, the CXCL10 ratio and level of CA-125, HE4 and IL-6 are determined during and after treatment. In a further example, the CXCL10 ratio and level of CA-125, HE4 and IL- 6 are determined prior to, during and after treatment.

[0689] In one example, the method comprises comparing the CXCL10 ratio and level of CA-125, HE4 and IL-6 in the subject at a first time point to a CXCL10 ratio and level of CA-125, HE4 and IL-6 in the subject at a subsequent time point.

[0690] It will be apparent to the skilled person from the disclosure that reference to a first and subsequent time point is not reference to a defined or specific time point and is for the purposes of comparison only. The first, second (and any subsequent) time points may be separated by any period of time during which it is wished to monitor the subject’s malignant condition. The monitoring methods of the disclosure may make use of further samples at further time points (for example a third sample at a third time point, in addition to the first and second samples). As will be apparent to the skilled person, the ability to monitor the CXCL10 ratio and level of CA-125, HE4 and IL-6 of the present disclosure over the course of the disease will assist in monitoring tumour burden and disease progression.

[0691] It will be apparent to the skilled person, that methods of monitoring tumour burden and disease progression in a subject will be useful for determining tumour regression and / or recurrence in a subject.

[0692] Panels and Kits

[0693] The present disclosure provides panels or kits for assessing the risk of a malignant condition and / or detecting and / or diagnosing a malignant condition in a subject. The present disclosure also provides panels or kits for monitoring tumour burden, tumour progression and / or tumour regression. The panels or kits of the invention will preferably comprise one or more or both CXCL10 binding proteins and / or CA-125, HE4 and / or IL- 6 binding proteins described herein. Optionally, the panel or kit comprises instructions for use in a method described herein.

[0694] In one example, the panel or kit comprises a reference sample.

[0695] In one example, the panel or kit as described herein is for ex vivo analysis. In one example, the kit is suitable for use with whole blood, plasma, cervico vaginal (CVS) swabs and / or serum samples.

[0696] In one example, the panel or kit as described herein is suitable for high-throughput screening. The term “high-throughput screening” refers to screening methods that can be used to test or assess more than one sample at a time and that can reduce the time for testing multiple samples. In one example, the methods are suitable for testing or assessing at least 5 samples, at least 10, at least 20, at least 30, at least 50, at least 70, at least 90, at least 150, at least 200, at least 300 samples at a time. Such high-throughput screening methods can analyse more than one sample rapidly e.g. in at least 30 minutes, in at least 1 hour, in at least 2 hours, in at least 3 hours, in at least 4 hours, in at least 5 hours, in at least 6 hours, in at least 7 hours, in at least 8 hours, in at least 9 hours or in at least 10 hours. High-throughput screening may also involve the use of liquid handling devices. In one example, high-throughput analysis may be automated.

[0697] The present disclosure also provides a panel of biomarkers for the diagnosis of malignant ovarian cancer consisting of active C-X-C motif chemokine ligand 10 (CXCL10), total CXCL10, cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6), wherein the biomarkers are bound to capture reagents attached to a substrate. In one example, the capture reagents are antibodies against active CXCL10, total CXCL10, CA-125, HE4 and IL-6.

[0698] In one example, the substrate is a solid phase selected from the group consisting of a bead, a plate, a membrane, an array, a biochip and combinations thereof.

[0699] The present disclosure includes the following non-limiting examples. LES

[0700] 1: Materials and Methods

[0701] Antibodies against intact and total CXCL10, full-length CXCL10 protein standard and all reagents were as previously described (Kang et al., 2021). Luminex magnetic bead assay kits (IL-6, HE4) were from Thermo Fisher (cat# RDSLXSAHM05). All other reagents were of analytical grade.

[0702] Clinical Samples and Patient Characteristics

[0703] Assays were performed on retrospectively collected EDTA-chelated plasma samples accessed from the OCRF-sponsored Ovarian Cancer Tissue Bank, housed at the Hudson Institute of Medical Research, Australia. Ethical approval was obtained from the Southern Health Human Research Ethics Committee, with all participants providing prior informed written consent. All samples were collected from anaesthetised, chemo - naive patients who underwent surgery for suspected gynaecological malignancies. Patients were excluded if they were <18 years at the time of surgery; had a recent (<2 years) history of breast, ovarian, uterine or other gynaecological cancer; had undergone chemo, radio- or immune-therapy within the preceding 12 months; or were immunocompromised at the time of diagnosis. Histological assessment of tumour type, stage and grade, pre-surgical clinical markers (CA125, CEA, CA15.3 and CA19.9), pre- surgical pelvic imaging, age, self-reported menopausal status, pre-existing medical conditions and any prior history of malignancy were obtained from de-identified patient medical records. Imaging data was reviewed and scored according to the RMI2 schedule (Karimi-Zarchi et al., 2015) by a gynaecological oncology specialist.

[0704] The Risk of Malignancy Algorithm (ROMA) was calculated according to (Ortiz- Munoz et al., 2014) using the formula:

[0705] Pre-menopausal Predictive Index (PI): -12.0 + 2.38 x Ln(HE4) + 0.0626 x Ln(CA125) Post-menopausal Predictive index (PI): -8.09 + 1.04 x Ln(HE4) + 0.732 x Ln(CA125) Predicted Probability (PP): ROMA % = exp(PI) / [l+exp(PI)] x 100

[0706] The Risk of Malignancy Index (RMI) was calculated according to (Moore et al., 2009) using the formula;

[0707] RMI = ultrasound score x menopausal status x serum CA125 where ultrasound score is 1 or 4, menopausal status is 1 (pre) or 4 (post) and serum CA125 is in units / ml. The RMI2 scoring system was used as recommended (Karimi- Zarchi et al., 2015).

[0708] The characteristics of the retrospective cohort used for testing are provided in Table 3. A total of 334 patient samples met the requirements of the study and were included for analysis. All patients were recruited following referral to a gynaecological oncologist for exploratory surgery. In total there were 164 ovarian malignancies (49%) and 170 benign (51%) cases included, with 34% or 66% from pre- or post-menopausal women, respectively. Malignancy was more common in post-menopausal women (-60% of samples) compared to pre-menopausal (-28% of samples), and diagnosed malignancies were almost exclusively high grade (grade 2-3) ovarian cancers of serous epithelial pathology. Amongst the samples included were 17 (-10%) stage I ovarian cancers, of which 14 (82%) were grade 2-3. Approximately 37% of patients (123 / 334) had known genetic abnormalities at the time of diagnosis, with relatively even distributions between pre-menopausal (-54%) and post-menopausal (-47%) cohorts. Approximately 50% of the cohort had unknown mutational status.

[0709] Table 3. Cohort characteristics of patient samples included in this study.

[0710] ELISA Biomarker measurements by ELISA were performed using EDTA-chelated plasma samples recovered from previously bio-banked specimens. Plasma samples were thawed on ice and clarified by centrifugation (16,000g, 10 mins at room temp) prior to use. The CXCL10 active ratio ELISA was carried out as previously described (Kang et al. (2021)) using mAb-RA2 and mAb-RG2 antibodies as detection antibodies in the active ratio test (ART) and either anti -human IP- 10 polyclonal antibody (Abeam; #ab9807) or an in-house derived antibody against EIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP (SEQ ID NO: 36) as the capture antibody. Magnetic bead immunoassay for IL6 and HE4 was carried out as previously described (Kampan et al., 2023). A total of 100 beads per analyte were counted and the median fluorescence intensity was determined. Quantitation was performed against a standard curve for each analyte using five- parameter logistic curve fitting.

[0711] Statistical analyses

[0712] Statistical analyses were performed in Python v3.9.77 (https: / / www.python.org / ), with Confidence Intervals (Cis) calculated using R v4.3.1 (https: / / www.r-project.org / ). Cis for ROC curves were calculated as described (DeLong et al., 1988) using the ‘pROC’ package (version 1.18.2) (Robin et al., 2011), with all other Cis using the Exact Binomial method via the built-in ‘stats’ library. Plots were generated using GraphPad PRISM (GraphPad Software, La Jolla CA).

[0713] Exploration & estimation of diagnostic performance for the multi-marker panel (combining CXCL10 active ratio, IL-6, CA125, and HE4) was as follows. Missing values for HE4 and IL-6 in a single sample were assigned as their respective data median in each case. Biomarker measurements were transformed using the Yeo-Johnson method (Yeo and Johnson, 2011), and analytes contributing the greatest linear separation between groups identified by Linear Discriminant Analysis. A classification model was then defined by fitting a multivariate logistic regression model to Yeo-Johnson transformed data. Model performance was estimated using repeated stratified K-fold cross-validation (4 folds x 5 repeats), with performance estimated using the mean values across all sub-models. The model was then refit to the entire dataset of n=334 cases, and its performance re-evaluated. A scoring cut point was chosen according to Youden’s J index (Watrowski et al., 2022). Final estimates for the full-dataset model were compared to the cross-validation estimates to assess potential overfit in the model.

[0714] For the other classifiers, samples with missing values were first removed from the dataset prior to estimation of performance (total numbers remaining CA125 n=334; RMI n=169; ROMA n=333). For the calculation of RMI only 169 samples could be used as ultrasound information was not recovered for the remainder of the cohort. Defined cutoff values for CA125 (>35U / ml (Bast et al., 1983)), RMI (>200 (Jacobs et al., 1990)) and ROMA (pre-menopausal >13.1%; post-menopausal >27.7% (Moore et al., 2009)) were from published literature.

[0715] Example 2: Individual marker performance

[0716] Median values for each biomarker according to disease (benign or malignant) are provided in Table 4. Significant differences were observed between benign and malignant samples for patient age at diagnosis and all individual markers measured (Figure 1). Each of CA125, HE4 and IL-6 increased in a stage- specific manner and were highest in late-stage (stages 2-4) disease. As previously reported (Kang et al., 2021) the CXCL10 active ratio was independent of cancer stage (Figure 1A) suggesting it may assist in the differentiation of benign disease from early-stage malignancy. RMI2 score also appeared independent of stage, however only a limited number of stage I samples (n=7) could be included for analysis due to the absence of imaging data.

[0717] Linear discriminant analysis (LDA) was used to identify which individual markers contributed the greatest individual separation between benign and malignant samples. Amongst all biomarkers evaluated, discriminant coefficients with the largest magnitude were in descending order: HE4, CA125, CXCL10 active ratio, and IL-6 (Figure IB). Total CXCL10 also provided discrimination, but was excluded due to collinearity with CXCL10 active ratio. These four biomarkers were therefore evaluated further for their combined ability to differentiate benign from malignant disease, compared to currently used clinical approaches.

[0718] Table 4. Individual marker concentrations and calculations. CEA 1.0 (0.6, 1.9) n= 82 / 87 0.8 (0.5, 1.6) n=31 / 121

[0719] CA15.3 10.8 (7.1, n= 82 / 87 36.7 (17.3, n=31 / 121

[0720] 14.0) 89.5)

[0721] CA19.9 8.6 (5, 14.6) n= 82 / 87 9.8 (3.9, 23.1) n=31 / 121

[0722] Example 3: A multi-marker panel out-performs CA125, RMI and ROMA for the differentiation of benign from malignant disease

[0723] Commencing with individual biomarker measurements for HE4, CA125, IL-6 and CXCL10 active ratio, we evaluated the combined performance of the multi marker panel as follows. Data was first normalised using Yeo Johnson transformation (Yeo and Johnson, 2011) and a transformation parameter A. determined for each biomarker. The transform for any individual marker Xi was defined by the following; ed biomarker values were standardised according to x(S)= (x(x)- n) / o, tandardised individual measurement in each case.

[0724] A multivariate Logistic Regression model was then defined as below, and fit to the transformed and scaled dataset using Maximum Likelihood Estimation. This results in the determination of a set of coefficients p.

[0725] Where p(x) indicates probability of malignancy.

[0726] Prediction of Malignancy for any given observation was then obtained by applying the cutoff to the Risk Score S, calculated according to S=10p(X);

[0727] Using Youden’s J index (Watrowski et al., 2022) an optimal risk score cut-point of 3.684 was determined.

[0728] Potential overfit was assessed by comparison between cross-validation and full model performance estimates. Good agreement was observed for all metrics (within < 1% variation in every case - Table 5) indicating an acceptably low level of overfit and suggesting that performance estimates from the final model were reliable. Table 5. Performance and cross-validation estimates for the multi-marker model.

[0729] AUC Sensitivity Specificity PPV NPV

[0730] Cross- V alidation 0.981 0.930 0.952 0.950 0.935

[0731] Performance on Full Dataset 0.984 0.939 0.953 0.951 0.942

[0732] Subtractive Difference % -0.26% -0.85% -0.13% -0.09% -0.64%

[0733] Model performance for discrimination between benign and malignant samples was then assessed, with comparisons of the multi-marker panel score made against standard cut-off values for (>35U / ml (Bast et al., 1983)), RMI (>200 (Jacobs et al., 1990)) and ROMA (pre-menopausal >13.1%; post-menopausal >27.7% (Moore et al., 2009)). Metrics for comparison included the area under the curve (AUC), sensitivity / specificity and negative / positive predictive values (Table 6 and Figure 2). Receiver operator characteristic (ROC) curves were generated for each of the multi-marker panel, CA125, RMI and ROMA (Figure 2A). The multi-marker panel achieved a clear increase in overall efficacy, providing improved sensitivity / specificity characteristics for differentiation of between benign from malignant samples compared to CA125, RMI2 or ROMA (Figure 2A).

[0734] Table 6. Overall performance metrics for classification of benign vs malignant disease.

[0735] The AUC, sensitivity / specificity, PPV / NPV and overall accuracy for each test were determined for the combined cohort, as well as pre- and post-menopausal samples separately (Table 3B). Overall AUC in the combined cohort was above 0.95 in every case, with the highest AUC (0.98) achieved using the multi-marker panel (Table 6). Sensitivities were similar in each case, however the specificities of CA125 (0.82) and RMI2 (0.75) were reduced compared to the multi-marker panel and ROMA (Table 6). Corresponding PPV was also comparatively lower for each of CA125 (0.83) and RMI2 (0.66).

[0736] When post-menopausal samples were considered separately a similar pattern was observed; sensitivities ranged from 0.95 (CA125) to 0.99 (multi-marker), whilst specificity again was lower for CA125 (0.84) and RMI2 (0.64). Lowest PPV was again observed for RMI2 (0.70). For pre-menopausal samples considered separately both multi-marker panel and ROMA had reduced sensitivity (0.81 in each case) compared to CA125 and RMI2 (0.91 and 0.89 respectively); however, their specificities substantially exceeded those achieved using either CA125 (0.80) or RMI2 (0.50).

[0737] Overall, whilst NPV was similar across each marker score and group (combined, pre- or post-menopausal; between 0.93 - 0.98) the highest PPV was achieved by the multi-marker panel in each test (Table 6). The multi-marker panel was the only test to maintain specificity and PPV values above 90% in every case, demonstrating an overall performance that exceeded that of the other tests. Thus, within this cohort, the multimarker panel provided improved capability to differentiate benign from malignant disease.

[0738] The data demonstrates high accuracy using a multi-marker panel incorporating the CXCL10 “active ratio” as a method to discriminate benign from malignant disease. By comparison to common clinically used methods including CA125, RMI and ROMA, our multi-marker panel achieved superior sensitivity and specificity for the classification of benign from malignant samples in this dataset. Whilst all tests examined achieved the 75% specificity / 80% sensitivity suggested as a minimum requirement for clinical use (Moore et al., 2009), the multi-marker panel out-performed the other modalities (Figure 2 and Table 6). At a comparative specificity of 95%, the sensitivities of each of CA125 and RMI remained below 80%; whilst ROMA remained under 90% sensitivity at the same threshold. Only the multi-marker panel achieved a 95% sensitivity at this level, highlighting its superior ability to differentiate benign from malignant disease. Moreover, the multi-marker panel operated independently of ultrasound scoring suggesting that a two stage clinical workup - as is currently recommended under current ACOG guidelines (ACOG Practice Bulletin No. 83, 2007) - should provide a practical improvement in the pre-surgical classification of adnexal masses. The regression model also did not appear overly affected by menopausal status, suggesting this model may provide broad applicability pre-surgical discrimination of benign from malignant disease.

[0739] Example 4: A multi-marker panel assists in the identification of early-stage cancers

[0740] Differentiation of early-stage cancers (stages I and II) from non-malignant growths is particularly challenging, especially in the case of low-volume cancers where CA125 is can be below the >35U / ml threshold (Liest et al., 2019). Amongst 17 stage I cancers in the dataset, the multi-marker panel correctly classified 13 (81%) as cancer compared to ROMA (12 correct; 76%). Amongst these were four stage I cancer samples (2 pre- and 2 post-menopausal) with CA125 <35U / ml; none of these were detected using CA125 or ROMA index, whilst one additional sample (post-menopausal, CA125 = 9U / ml) was correctly identified by the multi-marker panel. A further 4 stage II cancers were correctly classified by all scoring systems. RMI2 could not be compared as ultrasound information was not recovered for the majority of these samples.

[0741] The multi-marker test correctly identified over 75% of FIGO stage I tumours within the dataset, compared to -59% using ROMA or -35% using RMI. Accordingly, this multi-marker panel may enhance the rapid and effective triage of patients with early- stage tumours to maximise treatment outcomes for these patients.

[0742] The multi-marker panel achieved a sensitivity of 98.8% sensitivity at specificity of 75% in this cohort, as well as correctly identifying over 75% of all stage I cancers present, indicating the potential of these markers to contribute to early-stage detection of ovarian cancers.

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Claims

CLAIMS1. A method for assessing the risk that a subject suffers from a malignant condition, the method comprising:(i) determining a level of active C-X-C motif chemokine ligand 10 (CXCL10) in the subject and a level of total CXCL10 in the subject; and determining a CXCL10 ratio of active CXLC10 to total CXCL10 in the subject; and(ii) determining the level of cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) in the subject.

2. A method of detecting and / or diagnosing a malignant condition in a subject, the method comprising:(i) determining a level of active C-X-C motif chemokine ligand 10 (CXCL10) in the subject and a level of total CXCL10 in the subject; and determining a CXCL10 ratio of active CXLC10 to total CXCL10 in the subject; and(ii) determining the level of cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) in the subject.

3. The method of claim 1 or 2, wherein:(i) determining the level of active CXCL10 and total CXCL10 comprises determining the amount of active CXCL10 protein and the amount of total CXCL10 protein in the subject; and / or(ii) determining the level of CA-125, HE4 and / or IL-6 comprises determining the amount of CA-125, HE4 and / or IL-6 protein in the subject.

4. The method of any one of claims 1 to 3, wherein the method further comprises:(i) comparing the CXCL10 ratio in the subject to a CXCL10 ratio in at least one reference; and / or(ii) comparing the level of CA-125, HE4 and / or IL-6 in the subject to a level of CA- 125, HE4 and / or IL-6 in at least one reference.

5. The method of claim 4, wherein the method comprises determining:(i) if the CXCL10 ratio and the level of CA-125, HE4 and IL-6 in the subject are higher than the CXCL10 ratio and the level of CA-125, HE4 and I-L6 in the reference; or(ii) if the CXCL10 ratio and the level of CA-125, HE4 and IL-6 in the subject are lower than the CXCL10 ratio and the level of CA-125, HE4 and IL-6 in the reference.

6. The method of claim 5, wherein:(i) a lower CXCL10 ratio and a lower level of CA-125, HE4 and IL-6 in the subject compared to the reference is indicative of an increased probability of the subject suffering from the malignant condition or is indicative of a malignant condition; or(ii) a higher CXCL10 ratio and a higher level of CA-125, HE4 and IL-6 in the subject compared to the reference is indicative of an increased probability of the subject suffering from a benign condition or is indicative of a benign condition.

7. The method of any one of claims 1 to 3, wherein the method comprises determining a diagnostic score comprising the CXCL10 ratio and level of CA-125, HE4 and IL-6 in the subject.

8. The method of claim 7, wherein the method comprises determining the diagnostic score at one or more time points.

9. The method of claims 7 or 8, wherein:(i) a score of >3.6 in the subject is indicative of an increased probability of the subject suffering from the malignant condition or is indicative of a malignant condition; or(ii) a score of <3.6 in the subject is indicative of an increased probability of the subject suffering from a benign condition or is indicative of a benign condition.

10. The method of any one of claims 1 to 9, wherein the method has:(i) a sensitivity of at least 0.80;(ii) a specificity of at least 0.80;(iii) a positive predictive value of at least 0.75; and / or(iv) a negative predictive value of at least 0.85.

11. The method of any one of claims 1 to 10, wherein the malignant condition is a reproductive cancer.

12. The method of claim 11, wherein the reproductive cancer is ovarian cancer.

13. The method of any one of claims 1 to 12, wherein the subject is pre-menopausal or post-menopausal.

14. The method of claim 13, wherein the subject is post-menopausal and the method has:(i) a sensitivity of at least 0.95;(ii) a specificity of at least 0.80;(iii) a positive predictive value of at least 0.90; and / or(iv) a negative predictive value of at least 0.90.

15. The method of any one of claims 1 to 14, wherein the method comprises using:(i) a CXCL 10 binding protein that specifically binds full-length human CXCL 10 and N-terminally truncated CXCL 10 and citrullinated CXCL 10 to determine the level of total CXCL10 in the subject; and(ii) a CXCL10 binding protein that specifically binds full-length human CXCL10, but does not bind N-terminally truncated CXCL 10 and citrullinated CXCL 10 to determine the level of active CXCL10 in the subject.

16. The method of any one of claims 1 to 15, wherein the CXCL10 binding protein that binds full-length human CXCL 10 and N-terminally truncated CXCL 10 and citrullinated CXCL 10:(i) binds the epitope NH2-LSRTVRCTCISISNQPVNPRSLE-COOH (SEQ ID NO: 26) of full-length human CXCL 10, N-terminally truncated CXCL 10 and citrullinated CXCL 10;(ii) binds to full-length human CXCL10 with a KD of 50 nM or less; and / or(iii) binds to N-terminally truncated human CXCL10 with a KD of 5 nM or less.

17. The method of any one of claims 1 to 15, wherein the CXCL 10 binding protein that binds full-length human CXCL 10, but does not bind N-terminally truncated CXCL 10 and citrullinated CXCL10:(i) binds to full-length human CXCL10 with a KD of 50 nM or less; and / or(ii) requires the N-terminal valine and / or proline of the epitope NH2- VPLSRTVRCTCISISNQPVNPRSLE-COOH) (SEQ ID NO: 25) to bind to full- length human CXCL 10.

18. The method of any one of claims 1 to 17, wherein the method comprises using a CA-125 binding protein, a HE4 binding protein and / or an IL-6 binding protein to determine the level of CA-125, HE4 and IL-6 in the subject.

19. The method of claim 18, wherein:(i) the CA-125 binding protein binds to one or more or all epitopes selected from the group consisting of LYSGCRLTLLRSEKDGAATGVDAICTHR (SEQ ID NO: 27), ERVLQSLLGPMFKNTSVGPLYS (SEQ ID NO: 28) and VDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTH (SEQ ID NO: 29); and / or(ii) the HE4 binding protein binds to one or more or all epitopes selected from the group consisting of VNINFPQLGLCR (SEQ ID NO: 30) and CRDQCQVDSQCPGQMKCCRNGCGK (SEQ ID NO: 31); and / or(iii) the IL-6 binding protein binds to one or more or all epitopes selected from the group consisting ofVPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRK (SEQ ID NO: 32), ETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEE (SEQ ID NO: 33), VYLEYLQNRFESSEEQARAVQM (SEQ ID NO: 34) and QKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLI (SEQ ID NO: 35).

20. The method of any one of claims 15 to 19, wherein the binding protein comprises a variable region or an antigen binding domain of an antibody.

21. The method of any one of claims 15 to 20, wherein the binding protein is selected from the group consisting of:(i) a Fv;(ii) a single chain Fv fragment (scFv);(iii) a dimeric scFv (di-scFv);(iv) a single-domain antibody;(v) a minibody;(vi) a diabody;(vii) a triabody;(viii) a tetrabody;(ix) a Fab;(x) a F(ab’)2;(xi) an antibody;(xii) an antibody mimetic;(xiii) a heavy chain only immunoglobulin;(xiv) a T-cell receptor;(xv) an adnectin;(xvi) an anticalin;(xvii) an affibody;(xviii) an avimer;(xix) a designed ankyrin-repeat protein (DARPin); and(xx) one of (i) to (xix) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH) 2 and / or CH3.

22. The method of any one of claims 15 to 21, wherein the CXCL10 binding protein comprises:(i) a VH comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 3 and a VL comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 4; or(ii) a VH comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 11 and a VL comprising a sequence which is at least 90% identical to a sequence set forth in SEQ ID NO: 12.

23. The method of any one of claims 15 to 22, wherein the CXCL10 binding protein is an antibody or antigen binding fragment thereof comprising:(i) a VH comprising an amino acid sequence set forth in SEQ ID NO: 3 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 4; or(ii) a VH comprising an amino acid sequence set forth in SEQ ID NO: 11 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 12.

24. The method of any one of claims 15 to 23, wherein the CXCL10 binding protein is an antibody or antigen binding fragment thereof comprising:(i) a VH comprising: a) a CDR1 comprising a sequence set forth in amino acids 25-34 of SEQ ID NO: 3; and b) a CDR2 comprising a sequence set forth in amino acids 49-65 of SEQ ID NO: 3; andc) a CDR3 comprising a sequence set forth in amino acids 98-108 of SEQ ID NO: 3; and a VL comprising: a) a CDR1 comprising a sequence set forth in amino acids 23-33 of SEQ ID NO: 4; and b) a CDR2 comprising a sequence set forth in amino acids 49-55 of SEQ ID NO: 4; and c) a CDR3 comprising a sequence set forth in amino acids 88-96 of SEQ ID NO: 4; or(ii) a VH comprising: a) a CDR1 comprising a sequence set forth in amino acids 25-34 of SEQ ID NO: 11; and b) a CDR2 comprising a sequence set forth in amino acids 49-65 of SEQ ID NO: 11; and c) a CDR3 comprising a sequence set forth in amino acids 98-108 of SEQ ID NO: 11; and a VL comprising: a) a CDR1 comprising a sequence set forth in amino acids 23-33 of SEQ ID NO: 12; and b) a CDR2 comprising a sequence set forth in amino acids 49-55 of SEQ ID NO: 12; and c) a CDR3 comprising a sequence set forth in amino acids 88-96 of SEQ ID NO: 12.

25. The method of any one of claims 15 to 24, wherein the CXCL10 binding protein is an antibody or antigen binding fragment thereof comprising:(i) a VH comprising: a) a CDR1 comprising a sequence set forth in SEQ ID NO: 5; and b) a CDR2 comprising a sequence set forth in SEQ ID NO: 6; and c) a CDR3 comprising a sequence set forth in SEQ ID NO: 7; and a VL comprising: a) a CDR1 comprising a sequence set forth in SEQ ID NO: 8; and b) a CDR2 comprising a sequence set forth in SEQ ID NO: 9; and c) a CDR3 comprising a sequence set forth in SEQ ID NO: 10; or(ii) a VH comprising: a) a CDR1 comprising a sequence set forth in SEQ ID NO: 13; andb) a CDR2 comprising a sequence set forth in SEQ ID NO: 14; and c) a CDR3 comprising a sequence set forth in SEQ ID NO: 15; and a VL comprising: a) a CDR1 comprising a sequence set forth in SEQ ID NO: 16; and b) a CDR2 comprising a sequence set forth in SEQ ID NO: 17; and c) a CDR3 comprising a sequence set forth in SEQ ID NO: 18.

26. The method of any one of claims 15 to 25, wherein:(i) the level of total CXCL10 in the subject is determined using an antibody or antigen binding fragment thereof comprising a VH comprising an amino acid sequence set forth in SEQ ID NO: 11 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 12; and / or(ii) the level of active CXCL10 in the subject is determined using an antibody or antigen binding fragment thereof comprising a VH comprising an amino acid sequence set forth in SEQ ID NO: 3 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 4.

27. The method of any one of claims 15 to 26, wherein one or more of the CXCL10 binding proteins, the CA-125 binding protein, the HE4 binding protein and / or the IL-6 binding protein is conjugated to a detectable label.

28. The method of claim 27, wherein 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 prosthetic group and a contrast agent.

29. The method of any one of claims 1 to 26, wherein the method comprises performing flow cytometry, an enzyme-linked immunosorbent assay or a western blot.

30. The method of any one of claims 1 to 29, wherein the method is performed on at least one biological sample obtained from the subject.

31. The method of claim 30, wherein the biological sample is a biopsy, a fluid sample, a plasma sample or cellular swab.

32. The method of any one of claims 1 to 31, wherein the method comprises determining if the subject has an increased probability of suffering from a malignantcondition or if the subject has a malignant condition and selecting an appropriate therapy for the subject based.

33. A method of monitoring tumour burden, monitoring progression and / or determining tumour regression in a subject suffering from a malignant condition, the method comprising:(i) determining a C-X-C motif chemokine ligand 10 (CXCL10) ratio of active CXLC10 to total CXCL10 in the subject at one or more time points; and(ii) determining the level of cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) in the subject at one or more time points.

34. A method of detecting and / or diagnosing malignant ovarian cancer in a subject, the method comprising:(i) determining a C-X-C motif chemokine ligand 10 (CXCL10) ratio of active CXLC10 to total CXCL10 in at least one biological sample obtained from the subject; and(ii) determining the level of cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) in at least one biological sample obtained from the subject, and(iii) detecting and / or diagnosing malignant ovarian cancer in the subject based on the level determined in (i) and (ii); wherein the method further comprises administering or having administered a therapeutically effective amount of a treatment to the subject detected and / or diagnosed as having malignant ovarian cancer.

35. A method of treating a malignant condition in a subject, the method comprising detecting and / or diagnosing the malignant condition in the subject according to any one of claims 2 to 32 and administering or having administered a treatment to the subject.

36. A method of treating a subject identified as suffering from malignant ovarian cancer, the method comprising administering a therapeutically effective amount of a treatment to a subject, wherein the subject was identified as suffering from malignant ovarian cancer based on a C-X-C motif chemokine ligand 10 (CXCL10) ratio of active CXLC10 to total CXCL10 and a level of cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) in at least one biological sample obtained from the subject.

37. The method of any one of claims 34 to 36, wherein the treatment comprises surgery, chemotherapy, targeted drug therapy or a combination thereof.

38. A kit or panel for detecting and / or diagnosing malignant ovarian cancer in a subject, the kit or panel comprising one or more binding proteins for detecting a level of active C-X-C motif chemokine ligand 10 (CXCL10), total CXCL10, cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6) in the subject.

39. A panel of biomarkers for the diagnosis of malignant ovarian cancer consisting of active C-X-C motif chemokine ligand 10 (CXCL10), total CXCL10, cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6), wherein the biomarkers are bound to capture reagents attached to a substrate.

40. A method for determining ovarian cancer status, the method comprising measuring the level of the panel of biomarkers of claim 39 using an immunoassay or mass spectroscopy.

41. A method for characterizing a biological sample, the method comprising contacting the biological sample with the panel of biomarkers of claim 39 and detecting binding.

42. A method of detecting a panel of biomarkers in a sample obtained from a subject, the method comprising:(i) contacting the sample from the subject to a substrate;(ii) binding a panel of biomarkers in the sample to capture reagents attached to the substrate; wherein the panel of biomarkers consist of active C-X-C motif chemokine ligand 10 (CXCL10), total CXCL10, cancer antigen 125 (CA-125), human epididymis protein 4 (HE4) and interleukin 6 (IL-6), thereby detecting the panel of biomarkers in the sample from the subject.

43. The panel of claim 39 or the method of any one of claims 40 to 42, wherein the capture reagents are antibodies against active CXCL10, total CXCL10, CA-125, HE4 and IL- 6.

44. The panel or method of claim 43, wherein the substrate is a solid phase selected from the group consisting of a bead, a plate, a membrane, an array, a biochip and combinations thereof.

45. A cancer antigen 125 (CA-125) binding protein, wherein the CA-125 binding protein binds to one or more or all epitopes selected from the group consisting of LYSGCRLTLLRSEKDGAATGVDAICTHR (SEQ ID NO: 27),ERVLQSLLGPMFKNTSVGPLYS (SEQ ID NO: 28) andVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTH (SEQ ID NO: 29).

46. A human epididymis protein 4 (HE4) binding protein, wherein the HE4 binding protein binds to one or more or all epitopes selected from the group consisting of VNINFPQLGLCR (SEQ ID NO: 30) and CRDQCQVDSQCPGQMKCCRNGCGK (SEQ ID NO: 31).

47. An interleukin 6 (IL-6) binding protein, wherein the IL-6 binding protein binds to one or more or all epitopes selected from the group consisting of VPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRK (SEQ ID NO: 32), ETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEE (SEQ ID NO: 33), VYLEYLQNRFESSEEQARAVQM (SEQ ID NO: 34) andQKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLI (SEQ ID NO: 35).

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