Neo-Epitope Specific Assay Measuring Protease Mediated Degradation of Type IV Collagen
20230030529 · 2023-02-02
Assignee
Inventors
- Nicholas Willumsen (Dyssegard, DK)
- Christine Jensen (Kobenhavn N, DK)
- Morten A. Karsdal (Kobenhavn Ø, DK)
Cpc classification
C07K2317/34
CHEMISTRY; METALLURGY
International classification
Abstract
An assay measuring protease mediated degradation of type IV collagen and its biomarker potential for identifying cancer patients with a T-cell permissive tumor microenvironment is described.
Claims
1: A monoclonal antibody that specifically recognises and binds to a peptide having the N-terminus amino acid sequence MGNTGPTGAV (SEQ ID No. 1).
2: The monoclonal antibody of claim 1, wherein the monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having the N-terminus amino acid sequence MGNTGPTGAV (SEQ ID No. 1).
3: The monoclonal antibody of claim 1, wherein the monoclonal antibody does not specifically recognise or bind to a peptide having the N-terminus amino acid sequence selected from the group consisting of XMGNTGPTGAV (SEQ ID No. 2), wherein X represents any amino acid, GNTGPTGAV (SEQ ID NO. 3), MGQTGPTGAV (SEQ ID NO. 4), MGNSGPTGAV (SEQ ID NO. 5), and QGNTGPTGAV (SEQ ID NO. 6).
4-7. (canceled)
8: A method for identifying if a subject with cancer will respond to immunotherapy, said method comprising detecting the presence of a peptide having the N-terminus amino acid sequence MGNTGPTGAV (SEQ ID No. 1).
9: The method of claim 8, further comprising predicting survival outcome of the subject with cancer.
10: The method of claim 8, wherein said method is an immunoassay and comprises contacting a biofluid sample obtained from the subject with a monoclonal antibody that specifically recognises and binds to a peptide having the N-terminus amino acid sequence MGNTGPTGAV (SEQ ID No. 1), and detecting binding between the monoclonal antibody and peptides in the sample.
11: The method of claim 10, wherein the detection is quantitative.
12: The method of claim 10, wherein the immunoassay is a competitive immunoassay.
13: The method of claim 10, wherein the monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having the N-terminus amino acid sequence MGNTGPTGAV (SEQ ID No. 1).
14: The method of claim 10 wherein the monoclonal antibody does not specifically recognise or bind to a peptide having the N-terminus amino acid sequence selected from the group consisting of XMGNTGPTGAV (SEQ ID No. 2), wherein X represents any amino acid, GNTGPTGAV (SEQ ID NO. 3), MGQTGPTGAV (SEQ ID NO. 4), MGNSGPTGAV (SEQ ID NO. 5), and QGNTGPTGAV (SEQ ID NO. 6).
15-18. (canceled)
19: The method of claim 8, wherein the subject has been diagnosed with a cancer selected from the group consisting of melanoma, breast, colorectal, gastric, non-small cell lung cancer, small cell lung cancer, ovarian, prostate and pancreatic cancer.
20: The method of claim 19, wherein the subject has been diagnosed with a melanoma, preferably metastatic melanoma.
21: The method of claim 8 wherein the method further comprises correlating the amount of said peptide with values associated with normal healthy subjects and/or values obtained from cancer patients who have responded to immunotherapy.
22: The method of claim 21, wherein the method further comprises administering immunotherapy to the subject that has an elevated level of the peptide present.
23: The method of claim 8, wherein said immunotherapy comprises at least one immune checkpoint inhibitor.
24: The method of claim 23, wherein the immune checkpoint inhibitor is Ipilimumab.
25: An assay kit comprising a monoclonal antibody that specifically recognises and binds to a peptide having the N-terminus amino acid sequence MGNTGPTGAV (SEQ ID No. 1), and at least one of: a streptavidin coated well plate; a C-terminal biotinylated peptide having the N-terminus amino acid sequence MGNTGPTGAV (SEQ ID No. 1); a calibrator peptide having the N-terminus amino acid sequence MGNTGPTGAV (SEQ ID No. 1), an antibody biotinylation kit; an antibody HRP labeling kit; an antibody radiolabeling kit; or an assay visualization kit.
26: The assay kit of claim 25, wherein the monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having the N-terminus amino acid sequence MGNTGPTGAV (SEQ ID No. 1).
27: The assay kit of claim 25, wherein the monoclonal antibody does not specifically recognise or bind to a peptide having the N-terminus amino acid sequence selected from the group consisting of XMGNTGPTGAV (SEQ ID No. 2), wherein X represents any amino acid, GNTGPTGAV (SEQ ID NO. 3), MGQTGPTGAV (SEQ ID NO. 4), MGNSGPTGAV (SEQ ID NO. 5), and QGNTGPTGAV (SEQ ID NO. 6).
28-31. (canceled)
Description
FIGURES
[0080] The invention will now be described in the examples below which refer to the following figures:
[0081]
[0082]
[0083] Type IV collagen α2 chain was incubated with MMP-9 (A) or granzyme B (GzB) (B) for 72 hours and then C4-aa.sup.1355 ECLIA levels were measured.
[0084]
[0085] C4-aa.sup.1355 levels in serum at baseline and 3 weeks after Ipilimumab treatment (n=41). Serum levels were compared using Wilcoxon matched-pairs rank test.
[0086]
[0087] Overall survival for metastatic melanoma patients treated with Ipilimumab with pre-treatment levels in the upper quartiles (Q2+Q3+Q4) vs the lower quartile (Q1) for C4-aa.sup.1355 (cut off value: 14.5 ng/ml) (A) while for C4M it is the upper quartile (Q4) vs the lower quartiles (Q1+Q2+Q3) (cut off value: 35.0 ng/ml) (B). A log-rank test was used to determine differences between the survival curves where p-value of p<0.05 was considered statistically significant.
[0088]
[0089] Pearson's correlation analysis was performed to describe the relationship between C4-aa.sup.1355 and C4M levels in pre-treatment serum from metastatic melanoma treated with Ipilimumab (n=54).
[0090]
[0091]
[0092]
EXAMPLES
[0093] Various embodiments are described and disclosed in the following Examples, which are set forth to aid in the understanding of the present disclosure and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of the present disclosure nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
[0094] Materials and Methods
[0095] All reagents used for the experiments were standard chemicals from Merck (Whitehouse station, NJ, USA) and Sigma-Aldrich (St. Louis, Mo., USA) unless otherwise stated.
[0096] Peptide Identification by Mass Spectrometry
[0097] Type IV collagen from human placenta (Sigma Aldrich, cat. no. C5533) was proteolytically digested in the ratio 10:1 at 37° C. for 24 and 72 hours and then stored at −80° C. until mass spectrometry analysis.
[0098] One μg of sample (corresponding to digested or undigested collagen in 100 μl 50 mM Tris, 150 mM NaCl, pH 7.5 buffer) was reduced by 10 mM dithiothreitol for 30 minutes at 56° C. and alkylated by 40 mM iodoacetamide for 60 minutes in the dark at room temperature. Any remaining iodoacetamide was quenched by 10 mM dithiothreitol for 5 minutes at room temperature. The samples digested with Lys-C at 1:20 enzyme:substrate ratio (Wako chemicals, cat #125-05061) for 16 hours on a shaker at 37° C. After addition of 100 μl 1 M NaCl with 1% formic acid to the digests, these were run through 30 kDa filters (PALL Life Sciences, cat #OD030C34) to remove GAGs and desalted with reversed-phase Vydac UltraMicro Spin C18 columns (Harvard Apparatus, cat #74-7206) according to the manufacturer's instructions. Non-targeted mass spectrometry analysis was performed on a quadrupole Orbitrap benchtop mass spectrometer, QExactive, (Thermo Scientific) equipped with an Easy nano-LC 1000 system (ThermoFisher Scientific). Separation was performed on 75 μm×25 cm, Acclaim Pepmap™ RSLC C18 capillary columns packed with 2 μm particles (ThermoFisher Scientific). A spray voltage of +2000 V was used with a heated ion transfer setting of 275° C. for desolvation. The on-line reversed-phase separation was performed using a flow rate of 300 nl/min and a linear binary gradient 85 min was used. The gradient started with 3% solvent B for 4 minutes, then going to 35% solvent B in 64 minutes, after which it goes to 45% solvent B in 5 minutes. Finally, the organic solvent concentration was increased up to 90% in 5 minutes and kept at 90% for 7 minutes. An MS scan (400-1200 m/z) was recorded in the Orbitrap mass analyzer set at a resolution of 70,000 at 200 m/z, 1×10.sup.6 automatic gain control (AGC) target and 100 ms maximum ion injection time. The MS was followed by data-dependent collision-induced dissociation MS/MS scans at a resolution of 17,500 on the 15 most intense multiply charged ions at 2×10.sup.4 intensity threshold, 2 m/z isolation width and dynamic exclusion enabled for 30 s. Identification from discovery data was performed using the Homo sapiens proteome (UniProt proteome ID UP000005640) with Proteome Discoverer 2.1 software (ThermoFisher Scientific). The processing workflow consisted of the following nodes: Spectrum Selector for spectra pre-processing (precursor mass range: 300-30000 Da; S/N Threshold: 1.5), Sequest-HT search engine (Protein Database: see above; Enzyme: Lys-C (semi); Max. missed cleavage sites: 2; Peptide length range 6-144 amino acids; Precursor mass tolerance: 10 ppm; Fragment mass tolerance: 0.02 Da; Static modification: cysteine carbamidomethylation; and Percolator for peptide validation (FDR<1% based on peptide q-value). Results were filtered to keep only the Master protein with at least one unique peptide, and protein grouping was allowed according to the parsimony principle. For label-free quantification (LFQ), the sum of the top 3 peptides for each protein was taken to reflect the intensity of the protein. Peptide intensities were quantified using a proprietary algorithm developed in Proteome Discoverer 2.1 (ThermoFisher Scientific).
[0099] Selection of Peptides
[0100] The first six amino acids from the N-terminal and C-terminal of each peptide from type IV collagen identified by mass spectrometry were regarded as a protease generated neo-epitope. The protease generated sequences were analyzed for homology to other human proteins and species using the NPS@: Network Protein Sequence Analysis with the Uniprot/Swiss-Prot database (16). The amino acid sequence C-terminal from the cleavage site F.sup.1354↓M.sup.1355 (.sup.1355MGNTGPTGAV.sup.1364) was found unique for human type IV collagen α2 chain and selected as target for antibody production. Synthetic peptides used for monoclonal antibody production and technical evaluation of the ECLIA measuring protease mediated degradation of type IV collagen (C4-aa.sup.1355) were purchased from Genscript and shown in table 1.
TABLE-US-00008 TABLE 1 Synthetic peptides used for C4-aa.sup.1355 assay development and validation SEQ Peptide Amino acid sequence ID No. Selection peptide .sup.1355.MGNTGPTGAV.sup.1364 1 Immunogenic peptide MGNTGPTGAV-GGC-KLH N/A Biotinylated MGNTGPTAV-K-biotin N/A coating peptide Elongated peptide FMGNTGPTGAV 7 Truncated peptide GNTGPTGAV 3 Non-sense selection LLARDFEKNY 18 peptide Non-sense coating LLARDFEKNY-K- N/A peptide biotin Deselection 1 MGQTGPTGAV 4 Deselection 2 MGNSGPTGAV 5 Deselection 3 QGNTGPTGAV 6 KLH, Keyhole Limpet Hemocyanin
[0101] The target sequence was used as the selection peptide (MGNTGPTGAV (SEQ ID No. 1)). The immunogenic peptide (MGNTGPTGAV-GGC-KLH) was generated by covalently linking the selection peptide to Keyhole Limpet Hemocyanin (KLH) carrier protein with addition of glycine and cysteine residues in between to ensure right linking. A biotinylated peptide (MGNTGPTAV-K-biotin) was used as a coating peptide. The specificity of the antibody was tested by including an elongated peptide (FMGNTGPTGAV (SEQ ID No. 7)), a truncated peptide (GNTGPTGAV (SEQ ID No. 3)), a non-sense selection peptide (LLARDFEKNY (SEQ ID No. 18)) and a non-sense coating peptide (LLARDFEKNY-K-biotin). To test for potential cross-reactivity to other ECM proteins with similar sequences, three peptides with one amino acid mismatch at either position one (QGNTGPTGAV (SEQ ID No. 6)), three (MGQTGPTGAV (SEQ ID No. 4)) or four (MGNSGPTGAV (SEQ ID No. 5)) from the N-terminal were included in the specificity test. Antibody specificity was calculated as percentage of signal inhibition of two-fold diluted peptides.
[0102] Monoclonal Antibody Production and Clone Characterization
[0103] Female Balb/C mice of 6-7 weeks of age were immunized subcutaneously with 200 μl emulsified antigen containing 100 μg immunogenic peptide (MGNTGPTGAV-GGC-KLH) with Stimune Immunogenic adjuvant (Thermo fisher, cat. no. 7925000) repeatedly every second week until stable titer levels were obtained. The mouse with the highest antibody titer rested for four weeks and was then boosted intraperitoneally with immunogenic peptide. After three days, splenocytes were isolated and fused with murine SP2/0 myeloma cells to produce hybridoma cells as previously described (17). The hybridoma cells were cultured in 96-well microtiter plates and limited dilution was used to secure monoclonal growth. Supernatants from the monoclonal antibody producing hybridoma cells were screened for reactivity against the selection peptide and human serum samples in a preliminary competitive ELISA using biotinylated coating peptide on streptavidin-coated microtiter plates (Roche, cat. no. 11940279). The clone with the best reactivity towards the selection peptide was purified using protein-G-columns according to the manufacturer's instructions (GE Healthcare Life Sciences, cat. no. 17-0404-01).
[0104] C4-aa.sup.1355 ECLIA protocol
[0105] During assay development, an optimal incubation buffer, time, temperature and concentrations of antibody and coating peptide were determined and the finalized competitive C4-aa.sup.1355 ECLIA protocol was as follows: A MSD GOLD 96-well streptavidin pre-coated plate (Meso Scale Discovery, cat. no. L15SA-1) was incubated with 150 μl/well blocking buffer (10 mM phosphate-buffered saline (PBS) with bovine serum albumin (BSA) (5% w/v) and bronidox (0.36% v/v), 8 g/L NaCl, pH 7.4) for 60 minutes at 20° C. with shaking (300 rpm) in darkness. The plate was coated with 25 μl/well biotinylated coating peptide dissolved in assay buffer (50 mM PBS with BSA (1% w/v), Tween-20 (0.1% w/v) and bronidox (0.36% v/v), 8 g/L NaCl, pH 7.4) to a concentration at 2 ng/ml and incubated for 60 minutes at 20° C. with shaking (750 rpm) in darkness. Next, 25 μl/well selection peptide, assay controls or pre-diluted serum/plasma sample (1:2) were added followed by immediately addition of 25 μl/well of SULFO-TAG (MSD GOLD SULFO-TAG NHS-Ester Conjugation, Meso Scale Discovery, cat. no. R31AA-1) labelled monoclonal antibody diluted in assay buffer to a final concentration at 25 ng/ml and the plate incubated 20 hours at 4° C. with shaking (300 rpm) in darkness. All incubation steps were followed by washing the plate three times in washing buffer (20 mM Tris, 50 mM NaCl, pH 7.2). Finally, 150 μl/well of MSD GOLD Read Buffer (Meso Scale Discovery, cat. no. R92TG-2) was added and the plate was read immediately within 2 minutes in a Sector Imager 6000 (Meso Scale Discovery). SULFO-TAG enabled light emission when electricity was applied, and the light emission data was analyzed using the MSD Discovery Workbench 4.0 software. The analyte concentration was calculated using a 4-parametric curve fit model.
[0106] Technical Evaluation of the C4-aa.sup.1355 Assay
[0107] The lower limit of detection was determined from ten independent runs using the mean of the background plus 2.5 times the standard deviation. The upper limit of detection was determined from the same ten runs using the back-calibration concentration of the selection peptide at highest concentration minus 2.5 times the standard deviation. The intra- and inter-assay variation were determined by ten independent runs of seven samples in duplicates with concentrations covering the entire linear range of the standard curve. The samples consisted of four samples with different amount of selection peptide in assay buffer and three different healthy human serum samples. 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 the ten plates. To determine linearity of the assay, two-fold dilutions of human serum (n=3) or EDTA plasma samples (n=3) were performed and the linearity was calculated as a percentage recovery of undiluted sample. Analyte stability was tested by four repeated freeze/thaw cycles of human serum (n=3 at each cycle) and the analyte recovery was calculated with the first cycle as reference. Analyte stability was furthermore tested by incubated human serum samples (n=3 at each time point) at 4° C. or 20° C. for 24 or 48 hours and recovery was calculated with samples stored at −20° C. as reference. Interference was tested by adding a low/high content of biotin (3.0/9.0 ng/ml), lipemia (1.5/5.0 mg/ml) and hemoglobin (2.5/5.0 mg/ml) to a serum sample and the recovery was calculated with the serum sample as reference.
[0108] Cleavage of Type IV Collagen In Vitro
[0109] Recombinant type IV collagen α2 chain (MyBioSource) and MMP-9 (Giotto, cat. no. G04MP09C) or granzyme B (GzB) (Abcam, cat. no. ab168093) were incubated 10:1 (10 μg type IV collagen and 1 μg protease) in MMP-buffer (50 mM Tris-HCl, 150 nM NaCl, 10 mM CaCl.sub.2, 10 μM ZnCl, 0.05% Brij35, pH 7.5) or GzB buffer (50 mM Tris, 150 mM NaCl, pH 7.5), respectively, at 37° C. for 72 hours and then stored at −80° C. until analysis. Digestion of carboxymethylated transferrin with MMP-9 or GzB were included as positive controls, and MMP buffer added MMP-9 alone, and GzB buffer added GzB alone were included as negative controls. The activity of MMP-9 and GzB was confirmed by Coomassie blue staining (Data not shown).
[0110] Clinical Validation of the C4-aa.sup.1355 Assay
[0111] Serum samples were collected from stage IV melanoma patients (n=54) treated with Ipilimumab (3 mg/kg body weight) as standard of care at Herlev Hospital and Aarhus University Hospital, Denmark subsequent to informed consent. The study was approved by the Ethics Committee for The Capital Region of Denmark (H-2-2012-058) in compliance with the Helsinki Declaration of 1975. Serum samples were collected at baseline and 3 weeks after the first treatment (before the 2.sup.nd dose of treatment).
[0112] Serum samples from other cancer patients were obtained from the commercial vendor Asterand Bioscience (Detroit, Mich., USA) and included breast cancer (n=13), colorectal cancer (CRC) (n=7), gastric cancer (n=9), non-small cell lung cancer (NSCLC) (n=12), small cell lung cancer (SCLC) (n=7), melanoma (n=7), ovary cancer (n=10), pancreas cancer (n=2) and prostate cancer (n=13). Serum samples from healthy controls (n=40) were obtained from the commercial vender Valley BioMedical (Winchester, Va., USA). The samples were collected after informed content and approval by appropriate Institutional Review Board in compliance with the Helsinki Declaration.
[0113] C4M was assessed in serum samples from the cancer patients for comparison to the new developed biomarker C4-aa.sup.1355. The C4M competitive ELISA is a well-characterized assay based on a monoclonal antibody specific towards a neo-epitope of MMP-9 mediated degradation of type IV collagen manufactured by Nordic Bioscience (Herlev, Denmark) and measurements were performed according to the manufacturer's specifications (18).
[0114] Statistical Analyses
[0115] Wilcoxon matched pairs signed rank test was used to compare biomarker levels in melanoma patients at baseline with week 3. Kaplan-Meier survival curves were used to analyze overall survival (OS) in melanoma patients with pre-treatment levels in the upper quartiles (Q2+Q3+Q4) vs the lower quartile (Q1) for C4-aa.sup.1355 while it for C4M is the upper quartile (Q4) vs the lower quartiles (Q1+Q2+Q3).
[0116] The levels of C4-aa.sup.1355 in serum samples from the different cancer patients were compared to healthy controls using Kruskal-Wallis test adjusted for Dunn's multiple comparisons. The healthy controls were compared to the combined group of cancer patients using unpaired Mann-Whitney test. Pearson's correlation analysis was performed to describe the relationship between C4-aa.sup.1355 and C4M levels in serum from the metastatic melanoma and combined group of cancer patients, respectively. A p-value of p<0.05 was considered statistically significant. Graphs and statistical analyses were performed using GraphPad Prism version 7 (GraphPad Software, CA, USA).
[0117] Results
[0118] Specificity of the Novel C4-aa.sup.1355 Assay
[0119] The specificity of the monoclonal antibody in the new competitive C4-aa.sup.1355 ECLIA was tested. The selection peptide inhibited the signal in a dose-dependent manner whereas the elongated, truncated and non-sense selection peptide did not inhibit the signal (
[0120] Proteolytic Degradation of Type IV Collagen by MMP-9
[0121] To confirm that the C4-aa.sup.1355 antibody/assay recognized a protease generated type IV collagen neo-epitope, C4-aa.sup.1355 was measured in non-digested, MMP-9 digested and GzB digested type IV collagen. As shown in
[0122] Technical Evaluation of the C4-aa.sup.1355 Assay
[0123] Technical performance of the C4-aa.sup.1355 ECLIA assay was further evaluated through the different technical validation steps summarized in Table 2. The detection range of the assay was 0.6-832 ng/ml. The intra- and inter-assay variations were 6% and 8%, and below the acceptance criterion on 10% and 15%, respectively. Linearity was detected from undiluted to a 1:4 dilution with dilution recoveries at 94% and 106% for serum and EDTA plasma, respectively. After 4 freeze/thaw cycles, the analyte recovery in serum was 96%. After prolonged storage of human serum at 4° C. or 20° C. for 48 hours, the analyte recoveries were 122% and 109%, respectively. No interference was detected from low or high contents of lipemia or hemoglobin in serum with recoveries ranging from 92-111%. Low content of biotin neither interfered with the analyte whereas high content of biotin did with recoveries at 94% and 71%, respectively. The acceptance criterion of the recoveries was within 100±20%. Together, these results indicate that the C4-aa.sup.1355 ECLIA is a technical robust assay.
TABLE-US-00009 TABLE 2 Technical validation of the C4-aa.sup.1355 assay Technical validation step Results Detection range 0.6-832 ng/ml Intra-assay variation 6% Inter-assay variation 8% Dilution recovery of serum 94% Dilution recovery of EDTA plasma 106% Analyte recovery, 4 freeze/thaw cycles 96% Analyte recovery 24 h, 4° C./20° C. 109%/106% Analyte recovery 48 h, 4° C./20° C. 122%/109% Interference test Biotin recovery, low/high 94%/71% Lipemia recovery, low/high 111%/102% Hemoglobin recovery, low/high 99%/92% Percentages are reported as mean
[0124] Clinical Evaluation of the C4-Aa.sup.1355 Assay in Ipilimumab Treated Metastatic Melanoma Patients
[0125] To evaluate the biomarker potential of C4-aa.sup.1355, C4-aa.sup.1355 was measured in serum from metastatic melanoma patients at baseline and 3 weeks after Ipilimumab treatment. When the biomarkers levels were paired, C4-aa.sup.1355 levels were slightly elevated 3 weeks after treatment (p=0.090) (
[0126] Next, the association between the C4-aa.sup.1355 biomarker and survival outcomes were evaluated by Kaplan-Meier curves. High baseline levels (Q2+Q3+Q4) of C4-aa.sup.1355 were significantly associated with longer overall survival (OS) compared to low levels (Q1) (p=0.040) (
[0127] As the C4-aa.sup.1355 and C4M biomarker measure two different protease generated neo-epitopes on type IV collagen, the association between C4M and OS was also evaluated. These findings on C4M have previously published (.sup.8) and the results for C4M measured in the 54 patients are shown in
[0128] Furthermore, the correlation between the C4-aa.sup.1355 and C4M levels in the metastatic melanoma patients at baseline were investigated. C4-aa.sup.1355 and C4M did not correlate (r=0.021, p=0.883) (
[0129] Clinical Evaluation of the C4-aa.sup.1355 Assay in Other Cancer Patients
[0130] To further evaluate the biomarker potential of C4-aa.sup.1355, it was measured in serum from different cancer patients with either breast cancer, CRC, gastric cancer, NSCLC, SCLC, melanoma, ovary cancer, pancreas cancer or prostate cancer and in serum from healthy controls. Inter-patient variations in the C4-aa.sup.1355 biomarker levels in each group of patients (
[0131] As shown in the first cohort, again C4-aa.sup.1355 and C4M levels in these cancer patients, did not correlate (r=0.197, p=0.080) (
[0132] Serum C4-aa.sup.1355 (C4G) Measured at Baseline Predicts Outcome in Pancreas Ductal Adenocarcinoma (PDAC) Patients Treated with Chemotherapy
[0133] C4-aa.sup.1355 (C4G) was measured in pretreatment serum samples from 40 patients with pancreas ductal adenocarcinoma (PDAC). All patients were from the Danish BIOPAC study “BIOmarkers in patients with Pancreatic Cancer” (NCT03311776). Patients were recruited from six Danish hospitals from December 2008 until September 2017. PDAC patients had histologically confirmed tumors. The PDAC patients were treated with various types of chemotherapy according to national guidelines (www.gicancer.dk). The study was carried out in accordance with the recommendations of the Danish Regional Committee on Health Research Ethics. The BIOPAC protocol was approved by the Danish Regional Committee on Health Research Ethics (VEK ref. KA-20060113) and the Data Protection Agency (j.nr. 2006-41-6848). All subjects gave written informed consent in accordance with the Declaration of Helsinki, version 8. Blood samples were obtained at the time of diagnosis or before operation. Samples were processed according to nationally approved standard operating procedures for blood (www.herlevhospital.dk/biopac.dk). Serum samples and clinical data from patients were collected prospectively. Serum sample were measured blinded.
[0134] Results are shown according to stage of disease in
TABLE-US-00010 TABLE 3 Cox regression analysis for predicting overall survival outcome HR 95% CI p-value Univariate analysis C4-aa.sup.1355, Q2 + Q3 vs Q1 + Q4 0.37 0.17-0.81 0.0131 Multivariate analysis adjusted for stage of disease (early vs late) C4-aa.sup.1355, Q2 + Q3 vs Q1 + Q4 0.38 0.17-0.85 0.0179
[0135] Discussion and Conclusions
[0136] A robust and specific competitive ECLIA that enables non-invasive measurement of a neo-epitope generated by protease mediated degradation of type IV collagen (C4-aa.sup.1355) has been developed and validated. High baseline levels of C4-aa.sup.1355 in these melanoma patients were associated with clinical response (longer overall survival) to immune checkpoint inhibitor treatment. On the contrary, high baseline levels of C4M were associated with shorter overall survival. C4-aa.sup.1355 and C4M levels in these samples did not correlate. C4-aa.sup.1355 and C4M both measure a neo-epitope on type IV collagen but at two different sites, and interestingly these data suggest that these different cleavage products are released during two different pathologically events, one being associated with good outcome and one being associated with poor outcome when measured at baseline. Moreover, 3 weeks after immune checkpoint inhibitor treatment slightly elevated levels of C4-aa.sup.1355 were detected in serum from the metastatic melanoma patients compared to baseline.
[0137] The finding that C4-aa.sup.1355 associates with clinical response to immune checkpoint inhibitor treatment in these melanoma patients indicates that the C4-aa.sup.1355 assay has a biomarker potential in the immuno-oncology setting to identify cancer patients with a T-cell permissive tumor microenvironment responding to treatment. C4-aa.sup.1355 may reflect protease mediated T-cell transmigration from the circulation to the underlying stroma. Conversely, C4M associates with poor response supporting previous findings suggesting that C4M is linked to tumor activity and a reactive stroma (8-10).
[0138] Interestingly, this study indicates that one type of type IV collagen neo-epitope fragment is associated to tumorigenesis (C4M) and another type of type IV collagen neo-epitope fragment is associated to T-cell infiltration (C4-aa.sup.1355) supporting the value of measuring pathological specific neo-epitopes and not just the total protein.
[0139] C4-aa.sup.1355 was also elevated in other cancer types than melanoma suggesting that the biomarker has a potential in other indications. Furthermore, there was again no correlation between C4-aa.sup.1355 and C4M levels in these cancer patients verifying that these biomarkers reflect pathologically distinct aspect of the tumor microenvironment.
[0140] C4-aa.sup.1355 was found to be elevated in patient with early stage pancreas ductal adenocarcinoma (PDAC). In addition, those patients with an extreme level of the marker C4-aa.sup.1355 (i.e. in the 1st or 4th quartile) were found to have a reduced overall survival rate.
[0141] To our knowledge, this is the first study to show that this specific protease mediated degradation of type IV collagen (C4-aa.sup.1355) has biomarker potential in cancer and is associated with response to immune checkpoint inhibitor therapy.
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