Patent Description:
A major component of the tumor microenvironment is the extracellular matrix (ECM). Disruption of the healthy tissue homeostasis and altered turnover of the ECM are hallmarks of cancer that may lead to progression of the disease (<NUM>). The basement membrane (BM) is a specialized layer of ECM that underlies and surrounds epithelial cells, endothelial cells and mesenchymal cells and is essential for embryonic development as well as for maintaining tissue architecture and cellular polarization (<NUM>). The BM also serves as a barrier for cell invasion and breaching of the BM, and loss of BM integrity has been associated with an invasive phenotype in cancer (<NUM>).

Nidogens are highly conserved proteins that are found in almost all BMs (<NUM>). Two different nidogens exist: nidogen-<NUM> (entactin) and nidogen-<NUM>. Nidogen-<NUM> is the most widely expressed and comprises three globular domains (G1, G2 and G3) connected by two rod-shaped domains. Nidogen-<NUM> serves as the linker between other BM proteins, namely collagen type IV, perlecan and laminin (<NUM>). Nidogen-<NUM> also plays a key role in the BM assembly and stabilization (<NUM>) as is evident by the lung and heart abnormalities and perinatal death observed in nidogen-deficient mice as a direct result of BM changes (<NUM>).

It has also been shown that nidogen-<NUM> can regulate specific gene-expression in mammary epithelial cells (<NUM>). When the expression of β-casein was investigated as a function of nidogen quality/composition, a fragment of nidogen containing the laminin-<NUM> binding domain, but lacking the type IV collagen binding domain was able to reduce the expression of β-casein. In contrast, a fragment of nidogen containing the type IV collagen binding domain, but lacking the laminin-<NUM> binding domain was not. This indicates that in addition to maintaining BM integrity, nidogen has a physiological role in BM-induced gene expression and this is influenced by the quality/composition of nidogen. Additionally, Kalluri (<NUM>) discloses that intact Nidogen-<NUM> in the plasma can be used as a marker for ovarian serous cancer.

Lin Li et al. (<NUM>) discloses a general diagnostic link between intact Nidogen-<NUM> in the plasma and ovarian serous carcinoma. There is no discussion of cleavage products of Nidogen-<NUM>, in particular Cathepsin-S (Cats) cleavage of Nidogen-<NUM>. Lin Li et al. fails to disclose a method of evaluating the efficacy of an anti-cancer drug; there is only a brief speculative comment in the abstract regarding potential cancer therapies. Sage et al. (<NUM>) showed that Nidogen-<NUM> is a substrate for cathespin-S (CatS), and that CatS is found in both normal and tumor tissues. However, the physiological relevance of nidogen cleavage by CatS in normal and tumor tissues remained unknown. <CIT> discloses methods of diagnosis or of quantitation of pathological conditions which comprise conducting an immunoassay to measure neo-epitope containing protein fragments naturally present in a biofluid sample. The disclosed neo-epitopes are fragments of collagen type I, collagen type III, collagen type IV, collagen type V, collagen type VI, elastin, biglycan, decorin, lumican, versican, C-reactive protein, ApoE and laminins. Further methods of peptide identification are disclosed in <CIT> and <CIT>.

It is thought that cathepsins may play an important role in cancer (<NUM> and <NUM>). As noted in Sage et al. CatS is produced in normal tissues and tumour tissues, and it has also been found that CatS is produced by tumor-associated macrophages (TAMs) (<NUM>). CatS has been linked to growth, angiogenesis, migration, invasion and mestatasis in different cancer types (<NUM>-<NUM>). However, Kos et al (<NUM>) showed that the role of CatS in cancer progression is strongly associated with an immune response rather than with the remodelling of the extracellular matrix.

The applicant has found that nidogen-<NUM> degraded by CatS has biomarker potential in cancer. Without being bound by theory, it is hypothesized that this may be a reflection of a loss of BM integrity, a phenomenon which is known to be associated with pro-cancer events and an invasive phenotype. Accordingly, an aim of the present invention was to enable non-invasive assessment of cancer using biomarkers produced by CatS degradation of nidogen-<NUM>.

In a first aspect, the present invention relates to a method of diagnosis or of quantitation of cancer comprising conducting an immunoassay on a biofluid sample obtained from a patient to measure fragments of Nidogen-<NUM> having an N- or C-terminal neo-epitope formed by cleavage of Nidogen-<NUM> by Cathepsin-S, said fragments being naturally present in said sample, and associating an elevation of said measure in said patient above a normal level with the presence or extent of cancer, wherein said immunoassay is conducted by a method comprising:
contacting the fragments of Nidogen-<NUM> having said N- or C-terminal neo epitope that are naturally present in said sample with an immunological binding partner specifically reactive with the N- or C-terminal neo-epitope but not reactive with intact Nidogen-<NUM>, and measuring the extent of binding of the N- or C-terminal neo-epitope to said immunological binding partner to measure therein fragments comprising said neo-epitope.

The cancer may be breast cancer or non-small cell lung cancer (NSCLC).

Preferably, the immunological binding partner is specifically reactive with a C-terminal neo-epitope selected from the group consisting of:.

or is specifically reactive with an N-terminal neo-epitope selected from the group consisting of:.

Preferably, an immunological binding partner specifically reactive with a C-terminal neo-epitope does not react with a truncated and/or elongated C-terminal sequence at the C-terminus. Similarly, an immunological binding partner specifically reactive with an N-terminal neo-epitope preferably does not react with a truncated and/or elongated N-terminal sequence at the N-terminus. "Elongated" as used herein means the sequence is elongated by one or more amino acids. "Truncated" as used herein means the sequence is truncated by one or more amino acids. In a preferred embodiment of the invention, the immunological binding partner is specifically reactive with the C-terminal neo-epitope VEKTRCQHERE-COOH (SEQ ID NO: <NUM>). Preferably, the immunological binding partner does not react with a truncated C-terminal sequence VEKTRCQHER-COOH (SEQ ID NO: <NUM>) and/or wherein the immunological binding partner does not react with an elongated C-terminal sequence VEKTRCQHEREH-COOH (SEQ ID NO: <NUM>).

The immunological binding partner may be a polyclonal antibody or a monoclonal antibody.

The diagnostic method described above may be used to differentiate between a patient with NSCLC and a patient with another cancer type. In this regard the method preferably comprises performing the method as described above and associating an elevation of said measure in said patient of at least <NUM>% above a normal level with the presence of NSCLC.

In another aspect, the present invention relates to a method for evaluating the efficacy of an anti-cancer drug, wherein said method comprises.

Preferably, the immunological binding partner is as described supra.

In a further aspect the present invention relates to a diagnostic kit for use in the methods described supra, the kit comprising an immunological binding partner specifically reactive with an N- or C-terminal neo-epitope formed by cleavage of Nidogen-<NUM> by Cathepsin-S but not reactive with intact Nidogen-<NUM>, and at least one of the following:.

Preferably, the assay kit comprises a biotinylated peptide Biotin-L-VEKTRCQHERE-COOH (SEQ ID NO: <NUM>), wherein L is an optional linker, and a calibrator peptide comprising the C-terminal sequence VEKTRCQHERE-COOH (SEQ ID NO: <NUM>).

The term "antibody" is used according to the invention in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, and antibody fragments, so long as they exhibit the desired biological activity.

"Antibody fragments" used in the invention comprise a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')<NUM>, Fv and Fc fragments.

"Fragments of Nidogen-<NUM>" used in the invention means a peptide fragment produced by CatS cleavage of Nidogen-<NUM>. "N- or C-terminal neo-epitope" used in the invention means an N- or C-terminal epitope formed at a CatS cleavage site of Nidogen-<NUM>. For example, the following sequence of Nidogen-<NUM>.

would produce the N-terminal neo-epitope H<NUM>N-HILGAA. (SEQ ID NO: <NUM>) and the C-terminal neo-epitope. CQHERE-COOH (SEQ ID NO: <NUM>) when cleaved by CatS at the site between the E<NUM>-H<NUM> peptide bond, as denoted by the symbol "↓".

The term "a normal level" is used herein to mean a level/amount consistent with healthy individuals in a population. This generally corresponds to the median value in control samples ± standard deviation.

"NIC" as used herein refers to fragments of Nidogen-<NUM> comprising the C-terminal neo-epitope VEKTRCQHERE-COOH (SEQ ID NO: <NUM>).

The following three main CatS cleavage-sites (↓) on nidogen-<NUM> were previously identified by N-terminal sequencing (<NUM>):.

The proteolytic cleavage corresponding to the C-terminal neo-epitope. HERE<NUM> originating from outside the globular domains in the direction towards the N-terminus was selected for raising antibodies and development of immunoassays.

To generate an antibody specific for the selected CatS cleavage site on nidogen-<NUM> a sequence of <NUM> amino acids adjacent to the cleavage site was chosen as the target: VEKTRCQHERE↓ (SEQ ID NO: <NUM>). This amino acid sequence was used to design the selection peptide. The sequence was blasted for homology to other human proteins using NPS@: Network Protein Sequence Analysis with the UniprotKB/Swiss-prot database (<NUM>) and found to be unique to human nidogen-<NUM>.

A biotinylated screening peptide (Biotin-VEKTRCQHERE; SEQ ID NO: <NUM>) was included for coating streptavidin-coated plates. To test for specificity of the antibody a truncated selection peptide (EVEKTRCQHER; SEQ ID NO: <NUM>) missing the one amino acid closest to the cleavage site, and an elongated selection peptide (VEKTRCQHEREH; SEQ ID NO: <NUM>), with an additional amino acid added C-terminally to the cleavage site, as well as a non-sense selection peptide (APVGGIIGWM; SEQ ID NO: <NUM>) and a non-sense biotinylated screening peptide (Biotin-APVGGIIGWM; SEQ ID NO: <NUM>) were included in the assay development. The latter two correspond to the cleavage site located within the central part of the G2 domain (↓APVG. The immunogenic peptide (KLH-VEKTRCQHERE; SEQ ID NO: <NUM>) was generated by covalently cross-linking the selection peptide to Keyhole Limpet Hemocyanin (KLH) carrier protein using Succinimidyl <NUM>-(N-maleimidomethyl)cyclohexane-<NUM>-carboxylate, SMCC (Thermo Scientific, Waltham, MA, USA, cat. The synthetic peptides used for NIC are captured in Table <NUM>.

All synthetic peptides were purchased from the American Peptide Company, Vista, CA, USA.

Six week old BALB/c mice (n=<NUM>) were immunized subcutaneously with <NUM>µL emulsified antigen and <NUM>µg of the immunogenic peptide (KLH-VEKTRCQHERE; SEQ ID NO: <NUM>) using Freund's incomplete adjuvant (Sigma-Aldrich, St. Louis, MO, USA). Immunizations were repeated every second week and blood samples collected from the second immunization until stable serum antibody titer levels were reached. The mice with the highest antibody titer rested for a month and were then boosted intravenously with <NUM>µg immunogenic peptide in <NUM>µL <NUM>% sodium chloride solution. Three days later the spleen was isolated for cell fusion. The fusion procedure was performed as described elsewhere (<NUM>). Briefly, mouse spleen cells were fused with SP2/<NUM> myeloma cells to produce hybridoma cells. The hybridoma cells were cloned in culture dishes and limited-dilution was used to secure monoclonal growth. Native reactivity and peptide binding of the monoclonal antibodies were evaluated by displacement using human serum samples and the selection-peptide (VEKTRCQHERE; SEQ ID NO: <NUM>) in a preliminary ELISA using <NUM> ng/mL screening peptide on a streptavidin-coated microtiter plate (Roche, Basel, Switzerland, cat. #<NUM>) and the supernatant from the growing monoclonal hybridoma cells (containing the antibodies). The clones with best reactivity were purified using protein-G-columns according to the manufacturer's instructions (GE Healthcare Life Sciences, Little Chalfont, UK, cat. #<NUM>-<NUM>-<NUM>). Two hybridoma clones qualified to a screening for their ability to react competitively only with the selection peptide (VEKTRCQHERE; SEQ ID NO: <NUM>) and not with the truncated (EVEKTRCQHER; SEQ ID NO: <NUM>), elongated (VEKTRCQHEREH; SEQ ID NO: <NUM>) and non-sense (APVGGIIGWM; SEQ ID NO: <NUM>) selection peptides. One hybridoma clone (NB613-<NUM>) was selected for further assay development. Optimal incubation-buffer, incubation-time and incubation-temperature, as well the optimal concentrations of antibody and screening peptide, were determined from checkerboard analysis.

Human recombinant (hr-) nidogen-<NUM> (R&D Systems, Minneapolis, MN, USA, cat. #<NUM>-ND) was dissolved in CatS-buffer (<NUM> NaH<NUM>PO<NUM>(H<NUM>O)<NUM>, <NUM> DTT, <NUM>% Brij-<NUM>, pH <NUM>) or MMP-buffer (<NUM> Tris, <NUM> NaCl, <NUM> CaCl<NUM>, <NUM> ZnCl, pH <NUM>) to a final concentration of <NUM>µg/ml. The nidogen-<NUM> solutions incubated at <NUM> for <NUM> and <NUM> with, or without, the addition of hr-cathepsin S (Calbiochem, Whitehouse Station, NJ, USA, cat. #<NUM>), hr-MMP-<NUM> (R&D Systems, cat. #<NUM>-MP), hr-MMP-<NUM> (R&D Systems, cat. #<NUM>-MP) or hr-MMP-<NUM> (R&D Systems, cat. #<NUM>-MP) in final concentrations of <NUM>µg/ml, resulting in an enzyme-to-protein ratio of <NUM>:<NUM>. The chosen MMPs had previously been shown to degrade nidogen-<NUM>. (<NUM>,<NUM>) Enzymatic activity tests were performed in parallel with the actual cleavage of nidogen-<NUM>. The reaction was stopped by adding E-<NUM> or EDTA (final concentration of <NUM>) to CatS- and MMP-solutions, respectively. CatS- and MMP-buffer with relevant proteases were included as controls. The experiment was repeated twice. Samples were stored at -<NUM> until analysis. The cleavage of nidogen-<NUM> was confirmed by silverstaining according to the manufacturer's instructions (SilverXpress®, Invitrogen, cat. #LC6100) (data not shown).

The competitive ELISA procedure was as follows: a <NUM>-well streptavidin-coated microtiter plate, was coated with <NUM> ng/ml screening-peptide (Biotin-VEKTRCQHERE; SEQ ID NO: <NUM>) dissolved in assay buffer (<NUM> Tris-BTB, <NUM>/L NaCl, pH <NUM>). The plate was incubated for <NUM> at <NUM> in darkness and was subsequently washed five times in washing buffer (<NUM> Tris, <NUM> NaCl, pH <NUM>). <NUM>µl of selection-peptide (VEKTRCQHERE; SEQ ID NO: <NUM>) or sample (e.g. serum) was added to appropriate wells. This was followed by immediately adding <NUM>µl of monoclonal antibody dissolved in assay-buffer to a concentration of <NUM> ng/ml. The plate was incubated for <NUM> at <NUM> followed by x5 wash in washing buffer. Then, <NUM>µl of goat anti-mouse POD-conjugated IgG antibody (Thermo Scientific, Waltham, MA, USA, cat. #<NUM>) diluted <NUM>:<NUM> in assay buffer (final concentration of <NUM> ng/ml) was added to each well. The plate was incubated for <NUM> at <NUM> followed by five washes in washing buffer. Next, <NUM>µL tetra-methyl-benzinidine (Kem-En-Tec, Taastrup, Denmark, cat. #438OH) was added, the plate incubated for <NUM> at <NUM>, then <NUM>µl of stopping solution (<NUM>% H<NUM>SO<NUM>) was added. All incubation steps were performed while the plate was shaking with <NUM> rpm. Finally, the optical density was measured in a VersaMax ELISA microplate reader at <NUM> with <NUM> as reference. A <NUM>-parametric mathematical fit model was used to plot a calibration curve. Data were analyzed using the SoftMax Pro v. <NUM> software.

The lower limit of detection (LLOD) was determined from <NUM> measurements of the zero sample (assay buffer) and was calculated as the mean + three standard deviations (SD). The upper limit of detection (ULOD) was determined from ten independent runs of the highest selection peptide concentration employed in the standard curve and calculated as the mean back-calibration concentration + three SD. The lower limit of quantification (LLOQ) was calculated as the highest NIC2-levels quantifiable in serum with a coefficient of variation below <NUM>% reproduced from three independent runs of serum samples diluted stepwise. The intra-assay variation was calculated as the mean coefficient of variance (CV %) within plates, and the inter-assay variation was calculated as the mean CV % between plates. The inter- and intra-assay variations were determined by ten independent runs of eight quality control samples and two internal controls covering the detection range (LLOD-ULOD), with each run consisting of double-determinations of the samples. The eight samples included two human serum samples, two animal serum samples spiked with the selection peptide and four pools of human serum samples spiked with the selection peptide. Two-fold dilutions of human serum and plasma-EDTA samples were used to calculate linearity as a percentage of dilution-recovery of the undiluted sample. Accuracy was determined by spiking human serum and plasma-EDTA samples with two-fold dilutions of the selection peptide, and calculating the percentage spiking-recovery using the expected concentration (serum and peptide combined) as reference. The effect of repeated freezing and thawing of the samples was determined for three human serum and plasma-EDTA samples in four freeze/thaw cycles. The freeze-thaw recovery was calculated with the first cycle as reference. Analyte stability was determined for three human serum and plasma-EDTA samples after <NUM> of storage at either <NUM> or <NUM>. Recovery was calculated with samples stored at -<NUM> as reference. Interference was determined by adding a low/high content of hemoglobin (<NUM>/<NUM>), lipemia/lipids (<NUM>/<NUM>) and biotin (<NUM>/<NUM> ng/ml) to a serum sample of known concentration. Recovery percentage was calculated with the normal serum sample as reference.

Serum samples consisted of two cohorts. An overview is given in Table <NUM>.

Appropriate Institutional Review Board/Independent Ethical Committee approved sample collection and all the patients filed informed consent. The first cohort was acquired from the commercial vendor Proteogenex (Culver City, CA, USA). The second cohort was a combination of cancer patient samples acquired from the commercial vendor Asterand (Detroit, MI, USA) and healthy controls samples obtained from another study population (reg. KA99070gm). According to Danish law, it is not required to get additional ethical approval when measuring biomarkers in previously collected samples. All investigations were carried out in accordance with the Helsinki Declaration.

Serum levels of NIC were compared by the by one-way ANOVA adjusted for multiple comparisons with Holm-Sidak's test. The area under the receiver operating characteristics (AUROC) curves was used to assess the diagnostics power of NIC. Data were considered statistically significant when p<<NUM>. GraphPad Prism v6. <NUM> and MedCalc Statistical Software v14. <NUM> were used to perform statistical analysis.

The specificity of the competitive NIC ELISA was evaluated by assessing the percentage of inhibition induced by the selection peptide (VEKTRCQHERE; SEQ ID NO: <NUM>) as compared to an elongated selection peptide (VEKTRCQHEREH; SEQ ID NO: <NUM>), a truncated selection peptide (EVEKTRCQHER; SEQ ID NO: <NUM>), a non-sense selection peptide (APVGGIIGWM; SEQ ID NO: <NUM>) and a non-sense screening peptide (Biotin-APVGGIIGWM; SEQ ID NO: <NUM>) (the latter two corresponds to the cleavage site located within the central part of the G2 domain on nidogen-<NUM>). Results are shown in <FIG>. The selection peptide clearly inhibited the signal in a dose-dependent manner whereas the signal was only slightly inhibited by the truncated, elongated and non-sense peptides at the highest concentrations. No reactivity was detected towards the non-sense screening peptide. In addition, the levels of NIC generated by incubating nidogen-<NUM> with CatS and various MMPs were investigated. As shown in <FIG>, CatS generated NIC in a time-dependent manner whereas no NIC was generated neither without proteases, nor when incubating with MMP-<NUM>, MMP-<NUM> or MMP-<NUM>. Approximately <NUM>-fold higher levels of NIC were detected after incubating nidogen-<NUM> with CatS for <NUM>. Altogether, this indicates that the antibody is specific and that the assay accurately measures nidogen-<NUM> cleaved by CatS at amino acid position <NUM>.

The overall technical performance of the NIC assay is listed in Table <NUM>.

The assay had a LLOD of <NUM> and an ULOD of <NUM>. The LLOQ was <NUM>. Intra-assay variation was <NUM>% and the inter-assay variation was <NUM>%; below the acceptance level of <NUM>% and <NUM>%, respectively. All analyte recoveries were accepted if within <NUM>±<NUM>%. The dilution recovery was <NUM>% and <NUM>% for serum and plasma-EDTA, respectively, indicating good linearity when diluting the samples. Spiking recovery for the standard peptide was also acceptable in serum (<NUM>%) and plasma-EDTA (<NUM>%) indicating that these sample matrices do not affect assay response. The analyte was recovered in both serum and plasma-EDTA after four freeze-thaw cycles with <NUM>% and <NUM>% recoveries, respectively. The analyte was also recovered after storage at <NUM> and <NUM> for <NUM> resulting in <NUM>% and <NUM>% recoveries for serum and <NUM>% and <NUM>% recoveries for plasma-EDTA at <NUM> and <NUM>, respectively. Together this indicates that the analyte is relatively stable. No interference was detected from either low or high contents of biotin, lipids (lipemia) or hemoglobin, with recoveries ranging from <NUM>%-<NUM>%.

NIC levels were measured in serum from two different patient cohorts. As shown in <FIG>, NIC was significantly elevated (p><NUM>) in serum from NSCLC patients as compared to healthy controls and breast cancer patients. Median NIC in the NSCLC patients was <NUM> ranging from <NUM>-<NUM>. Median NIC in the breast cancer patients was <NUM> ranging from <NUM>-<NUM> and median NIC in the healthy controls was <NUM> ranging from <NUM>-<NUM>. As shown in <FIG>, NIC was significantly elevated in serum from NSCLC patients as compared to healthy controls (p<<NUM>), breast cancer patients (p<<NUM>) and patients with SCLC (p<<NUM>). In this cohort, median NIC in the NSCLC patients was <NUM> ranging from <NUM>-<NUM>. Median NIC in the SCLC patients was <NUM> ranging from <NUM>-<NUM>, median NIC in the breast cancer patients was <NUM> ranging from <NUM>-<NUM> and median NIC in the healthy controls was <NUM> ranging from <NUM>-<NUM>.

NIC levels were also assessed according to tumor stage, an important clinical parameter in cancer. Results are shown in <FIG> for all cancers combined. In detail, all stages of disease had elevated levels of NIC as compared to the healthy controls, whereas no difference could be detected between stages. This indicates that NIC is generated independent of tumor stage.

To analyze the diagnostic power of NIC with respect to NSCLC, both cohorts were pooled and grouped in 'NSCLC' vs. 'all others' and the AUROC calculated. The AUROC was <NUM> (<NUM>% confidence intervals: <NUM>-<NUM>), p<<NUM>. The ROC curve, as well as the sensitivity and specificity at estimated optimal cut-off value, is shown in <FIG>. These findings indicate that NIC are able to separate NSCLC patients from the other subjects combined. Altogether, the present findings suggest that NIC may be highly associated with NSCLC.

Described herein is the development and validation of a robust competitive NIC ELISA that enables non-invasive measurements of fragments of nidogen-<NUM> degraded specifically by CatS (NIC). Moreover, this is the first time it has been shows that nidogen-<NUM> degraded specifically by CatS has biomarker potential for cancer, in particular NSCLC.

CatS cleavage of nidogen-<NUM> resulted in the release of a specific fragment containing a neo-epitope with a C-terminal sequence VEKTRCQHERE (SEQ ID NO: <NUM>) (NIC).

Elevated NIC levels as measured by the NIC Assay described herein was detected in serum from patients with NSCLC as compared to patients with breast cancer, SCLC and healthy controls. This suggests that NSCLC patients have increased CatS mediated degradation of nidogen-<NUM>, which was further backed by the diagnostic accuracy of NIC (AUROC <NUM>) for NSCLC when compared to all other subjects. Furthermore, NIC levels may be indicative of a specific pathological event in a patient's tumor, and this pathological event seems to be more associated with NSCLC as compared to the other cancer types analyzed.

As NIC levels were elevated in all stages of disease, especially in NSCLC, this indicates that CatS degradation of nidogen-<NUM> is ongoing on both early and late stages of disease independent of tumor stage and that NIC may be applied in the early onset of disease.

Concerning the efficacy of anti-cancer drugs, ECM modifying drugs are currently being investigated as possible interventions for modulating the tumor microenvironment and obtain tumor shrinkage or more efficient delivery of chemotherapeutic drugs and targeted therapies. Interestingly, it has been shown that antibody-mediated blockage of CatS enhances the efficacy of chemotherapy (<NUM>). Moreover, this antibody has been suggested as a direct strategy for the treatment of solid tumors (<NUM>). Thus, the NIC assay could be used for predicting which patients are most likely to benefit from such treatment, or for monitoring early efficacy of treatment.

An important parameter of the NIC assay is related to robustness, i.e. technical and analytical evaluation. Technical evaluation of NIC included the establishment of detection range, sensitivity, inter- and intra-assay variation, linearity/precision (dilution-recovery), analyte stability, and interference, which all were within acceptable limits. Analytical evaluation of the NIC assay refers to the specificity of the assay and to whether the assay detects the analyte it was designed to detect in serum/plasma-EDTA. The specificity was evaluated by displacement analysis (<FIG>) and by testing reactivity towards intact and cleaved nidogen-<NUM> (<FIG>). Accuracy was evaluated by spiking the analyte into the relevant matrices (serum and plasma-EDTA) and calculating the percentage recovery (also within acceptable limits). Thus, the NIC assay was technically and analytically robust.

In conclusion, CatS degraded nidogen-<NUM> can be quantified in serum by the technically robust NIC assay. NIC levels were elevated in NSCLC patients as compared to breast cancer patients, SCLC patients and healthy controls. The NIC assay may therefore serve as a non-invasive biomarker for cancer and provide important insight into tumor biology.

Claim 1:
A method of diagnosis or of quantitation of cancer comprising conducting an immunoassay on a biofluid sample obtained from a patient to measure fragments of Nidogen-<NUM> having an N- or C-terminal neo-epitope formed by cleavage of Nidogen-<NUM> by Cathepsin-S, said fragments being naturally present in said sample, and associating an elevation of said measure in said patient above a normal level with the presence or extent of cancer, wherein said immunoassay is conducted by a method comprising:
contacting the fragments of Nidogen-<NUM> having said N- or C-terminal neo epitope that are naturally present in said sample with an immunological binding partner specifically reactive with the N- or C-terminal neo-epitope but not reactive with intact Nidogen-<NUM>, and measuring the extent of binding of the N- or C-terminal neo-epitope to said immunological binding partner to measure therein fragments comprising said neo-epitope.