Compositions and methods for assessing the risk of cancer occurrence

Abstract

The present invention provides a method for evaluating the risk of occurrence of cancer in an individual.

Claims

1. A kit comprising at least one anti-progastrin antibody for evaluating risks of developing cancer in a subject who has not been previously diagnosed with cancer, wherein the anti-progastrin antibody is a monoclonal antibody produced by the hybridoma deposited at the CNCM, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris CEDEX 15, France, on 27 Dec. 2016, under reference I-5158.

2. A kit for evaluating risks of developing cancer in a subject who has not been previously diagnosed with cancer, comprising: (i) a first anti-progastrin antibody which binds to a first part of progastrin, wherein the anti-progastrin antibody is a monoclonal antibody produced by the hybridoma deposited at the CNCM, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris CEDEX 15, France, on 27 Dec. 2016, under reference I-5158, and (ii) a second anti-progastrin antibody which binds to a second part of progastrin.

3. The kit of claim 2, wherein the second anti-progastrin antibody is: a monoclonal antibody comprising: a heavy chain comprising the following three heavy chain complementarity determining regions (“CDR-H”), CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively, and a light chain comprising the following three light chain complementarity determining regions (“CDR-L”), CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively.

Description

FIGURE LEGENDS

(1) FIG. 1: Receiver operating characteristic (ROC) curve for colorectal cancer (upper panel) with the area under the ROC curve and the statistical analysis (lower panel).

(2) FIG. 2: median plasmatic concentration of progastrin in colorectal cancer patients (n=148), and in control patients (n=103) using a combination of an N-terminus polyclonal antibody and a C-terminus polyclonal antibody—Mann Whitney test two-tailed, ***p<0.0001.

(3) FIG. 3: Receiver operating characteristic (ROC) curve for hepatocellular carcinoma (upper panel) with the area under the ROC curve and the statistical analysis (lower panel).

(4) FIG. 4: median plasmatic concentration of progastrin in hepatocellular carcinoma patients (n=47), and in control patients (n=103) using a combination of an N-terminus polyclonal antibody and a C-terminus polyclonal antibody—Mann Whitney test two-tailed, ***p<0.0001

(5) FIG. 5: Receiver operating characteristic (ROC) curve for oesophagic cancer (upper panel) with the area under the ROC curve and the statistical analysis (lower panel).

(6) FIG. 6: median plasmatic concentration of progastrin in oesophagic cancer patients (n=12), and in control patients (n=103) using a combination of an N-terminus polyclonal antibody and a C-terminus polyclonal antibody—Mann Whitney test two-tailed, ***p<0.0001

(7) FIG. 7: Receiver operating characteristic (ROC) curve for gastric cancer (upper panel) with the area under the ROC curve and the statistical analysis (lower panel).

(8) FIG. 8: median plasmatic concentration of progastrin in gastric cancer patients (n=15), and in control patients (n=103) using a combination of an N-terminus polyclonal antibody and a C-terminus polyclonal antibody—Mann Whitney test two-tailed, **p<0.001

(9) FIG. 9: Receiver operating characteristic (ROC) curve for pancreatic cancer (upper panel) with the area under the ROC curve and the statistical analysis (lower panel).

(10) FIG. 10: median plasmatic concentration of progastrin in pancreatic cancer patients (n=44), and in control patients (n=103) using a combination of an N-terminus polyclonal antibody and a C-terminus polyclonal antibody—Mann Whitney test two-tailed, ***p<0.0001

(11) FIG. 11: Receiver operating characteristic (ROC) curve for ovarian cancer (upper panel) with the area under the ROC curve and the statistical analysis (lower panel).

(12) FIG. 12: median plasmatic concentration of progastrin in ovarian cancer patients (n=8), and in control patients (n=103) using a combination of an N-terminus polyclonal antibody and a C-terminus polyclonal antibody.

(13) FIG. 13: Schematic representation of a processing system according to a particular embodiment of the present invention

(14) FIG. 14: Functional graph representing a method according to a particular embodiment of the present invention.

(15) FIG. 15: Median plasmatic concentration of progastrin in various cancer type patients (n=231), and in control patients (n=322) using a combination of a polyclonal antibody and a monoclonal antibody.

(16) FIG. 16: Median plasmatic concentration of progastrin in various cancer type patients (n=10), using a combination of a polyclonal antibody and monoclonal antibody (mAb-pAb) or a combination of monoclonal antibodies (mAb-mAb)—Mann Whitney test two-tailed, NS p>0.05.

EXAMPLES

Example 1: Detection of Plasmatic Progastrin Concentration Using Polyclonal Antibodies

(17) Plasma progastrin levels were quantified by ELISA through the use of two specific anti-progastrin antibodies: capture antibodies are coated on the wells of the plate, whereas revelation antibodies are used to detect progastrin and mediates revelation of the signal.

(18) In the present example, quantification is based on the ELISA method which allows, through the use of a substrate whose reaction emits light, to assign a value proportional to the luminescence amount of antibodies bound to the antigen retained by capture antibodies.

(19) Material

(20) Reagents and apparatus are listed in Table 7:

(21) TABLE-US-00007 TABLE 7 Designation Provider Reférénce Plates MaxiSORP white Dutscher # 055221 Nunc, 96 wells Sodium Carbonate/ Sigma # 21851 Bicarbonate DPBS 1X Lonza # P04-36500 Tween-20 Biosolve # 20452335 BSA Euromedex # 04-100-810-C Streptavidin-HRP Pierce (Thermo) # 21130 SuperSignal ELISA Pierce (Thermo) # 37074 Femto Maximum Sensitivity Substrate Anti-ProGastrin Eurogentec / Polyclonal Antibody

(22) Polyclonal antibodies were obtained by immunizing a rabbit with N-terminal progastrin (SEQ ID N.sup.o 2) or with C-terminal progastrin corresponding to amino acids 71 to 80 of hPG and having the sequence FGRRSAEDEN (SEQ ID N.sup.o 40), according to standard protocols.

(23) The binding characteristics of polyclonal antibodies against progastrin used in this assay are the following: absence of binding to G34-Gly, G34, G17-Gly, G17, binding to full length progastrin.

(24) 96 wells plates are coated by preparing a solution of carbonate—sodium bicarbonate, 50 mM pH 9.6 by dissolving the contents of one capsule in 100 ml of MilliQ water. A solution of capture antibody (3 pg/ml), corresponding to polyclonal antibodies obtained by using the C-terminal of progastrin FGRRSAEDEN (SEQ ID N.sup.o 40) is prepared in carbonate buffer. 100 microliters of antibodies solution is added to each well and incubated at 4° C. for 16 hours (1 night). Plates are then blocked by eliminating the antibodies solution and wash 3 times with 300 μl 1×PBS/0.1% Tween-20, then adding 200 μl of blocking buffer (1×PBS/0.1% Tween-20/0.1% BSA) per well, and incubated 2 hours at 22° C. Blocking buffer is then eliminated, wells are washed 3 times with 300 μl 1×PBS/0.1% Tween-20.

(25) Plasma dilution is performed as follows: The plasma is used pure, diluted ½, ⅕ and 1/10. Dilutions are prepared from pure plasma in 1×PBS/0.1% Tween 20/0.1% BSA. For the control test, ELISA in the presence of a known concentration of progastrin, progastrin dilution is prepared as follows: stock recombinant PG (Full length human progastrin produced in E. coli and affinity purified with Glutathione agarose/Tag removal (Tev)/IMAC Counter purification/dialysis, from Institut Pasteur, Paris, France) is prepared at a concentration of 0.45 mg/ml (45 microM), in triplicate. Ranges of progastrin concentrations were prepared as follows: Solution A: Pre-dilution 1/10, 2 μl of stock+18 μl of the buffer Solution B: Pre-dilution 1/100, 10 μl of A+90 μl of the buffer Solution C: Pre-dilution 1/1000, 10 μl of B+90 μl of the buffer Solution D: 500 pM, 5.55 μl of C+494.5 μl of the diluent Solution E: 250 pM, 250 μl of D+250 μl of the diluent Solution F: 100 pM, 200 μl of E+300 μl of the diluent Solution G: 50 pM, 250 μl of F+250 μl of the diluent Solution H: 25 pM, 200 μl of G+200 μl of the diluent Solution I: 10 pM, 100 μl of H+150 μl of the diluent

(26) The range of recombinant PG is linear and can therefore be more or less extensive according to the antibody used.

(27) For the preparation of test samples, approximately 500 μl of each sample are set aside and stored until analysis (and confirmation if necessary) of the results. 100 μl of each point of the range and/or plasmas are assayed pure, diluted to ½, ⅕ and 1/10, and incubated for 2 hours at 22° C. on the plates.

(28) For the revelation of the test, the plates are washed 3 times with 300 μl 1×PBS/0.1% Tween-20. A solution of the polyclonal rabbit anti-progastrin antibody, wherein said antibodies have been obtained by using the N-terminal part of progastrin as an immunogen, coupled to biotin to 0.5 μg/ml, is prepared by dilution in 1×PBS/0.1% Tween-20/0.1% BSA. 100 μl of this solution is added to each well. Incubation takes place for 1 hour at 22° C. The revelation with streptavidin-HRP is performed by removing detection antibody and wash 3 times with 300 μl 1×PBS/0.1% Tween-20, then preparing a solution of Streptavidin-HRP at 20 ng/ml diluted in 1×PBS/0.1% Tween-20/0.1% BSA, wherein 100 Add 100 μl of this solution is added to each well, before incubation for 1 hour at 22° C.

(29) The detection consists of eliminating streptavidin-HRP and wash 3 times with 300 μl 1×PBS/0.1% Tween-20, then adding 100 μl of chemiluminescent substrate solution per well. The substrate solution is prepared by mixing equal volumes of the two solutions SuperSignal ELISA Femto kit, 20 ml+20 ml, 30 minutes before use and stored at room temperature in the dark. Luminescence is read after 5 minutes incubation at room temperature in the dark.

(30) For each condition, the test is performed in triplicate and the results of the ranges will be presented as a graph showing the change in luminescence depending on the progastrin concentration. For each plasma dilution, the concentration of progastrin is determined using the equation of the linear regression line of the corresponding range (range 1/10th for a sample diluted to 1/10th).

(31) Methods and Results

(32) Progastrin levels were determined in plasma samples from subjects who were known to have developed cancer later. Progastrin was captured with polyclonal antibodies specific for the C-terminus. Detection was performed with labelled polyclonal antibodies specific for the N-terminus.

(33) Importantly, at the time of sample collection, these subjects had never been diagnosed with cancer and did not show any symptom relating to cancer. The control was constituted by plasma samples from the general population.

(34) The results are shown in FIGS. 1-12. The median plasmatic concentration of progastrin was 17 pM in patients who developed colorectal cancer afterwards (n=148), 100 pM in patients who developed hepatocellular carcinoma (n=47), 42.3 pM in patients who developed oesophagic cancer (n=12), 17.90 pM in patients who developed gastric cancer (n=15), 16.6 pM in patients who developed pancreatic cancer (n=44), and 8.45 in patients who developed ovarian cancer (n=8). By comparison, the median plasmatic concentration of progastrin is 0 pM in control subjects (n=103).

(35) These data demonstrate that patients who will develop cancer have detectable levels of progastrin in their plasma whereas healthy control individuals have none. Progastrin can be detected even before any cancer can be diagnosed, making progastrin useful biomarker for the onset of cancer. ROC analysis confirmed the predictive nature of progastrin for each of the above-listed cancers.

(36) These data demonstrate that patients with a risk of developing cancer have higher concentration of progastrin in their plasma compared to healthy control individuals.

Example 2: Detection of Plasmatic Progastrin Concentration Using a Combination of Polyclonal Antibodies and Monoclonal Antibodies

(37) In the present example, plasma progastrin levels were quantified by ELISA through the use of antibody specific for human progastrin (hPG) pre-coated on a 96-well plate. Standards and samples are added to the wells, and any hPG present binds to the immobilized capture antibody. The wells are washed and an anti-hPG detection antibody horseradish peroxidase (HRP) conjugate is added, producing an antibody-antigen-antibody “sandwich.” After a second wash, TMB substrate solution is added, which produces a blue color in direct proportion to the amount of hPG present in the initial sample. The Stop Solution changes the color from blue to yellow, and the wells are read at 450 nm with a microplate reader.

(38) Polyclonal antibodies were obtained by immunizing a rabbit with N-terminal progastrin (SEQ ID N.sup.o 2) or with C-terminal progastrin corresponding to amino acids 71 to 80 of hPG and having the sequence FGRRSAEDEN (SEQ ID N.sup.o 40), according to standard protocols.

(39) Monoclonal antibodies were obtained by using hybridomas producing antibodies against N-terminal progastrin (SEQ ID N.sup.o 2) or against C-terminal progastrin corresponding to amino acids 71 to 80 of hPG and having the sequence FGRRSAEDEN (SEQ ID N.sup.o 40), according to standard protocols.

(40) The binding characteristics of polyclonal and monoclonal antibodies against progastrin used in this assay are the following: absence of binding to G34-Gly, G34, G17-Gly, G17, binding to full length progastrin.

(41) For the control test, ELISA in the presence of a known concentration of progastrin, progastrin dilution is prepared as follows: stock recombinant PG (Full length human progastrin produced in E. coli and affinity purified with Glutathione agarose/Tag removal (Tev)/IMAC Counter purification/dialysis, from Institut Pasteur, Paris, France) is prepared at a concentration of 0.45 mg/ml (45 microM), in triplicate. Ranges of progastrin concentrations were prepared as follows: Solution A: Pre-dilution 1/10, 2 μl of stock+18 μl of the buffer Solution B: Pre-dilution 1/100, 10 μl of A+90 μl of the buffer Solution C: Pre-dilution 1/1000, 10 μl of B+90 μl of the buffer Solution D: 500 pM, 5.55 μl of C+494.5 μl of the diluent Solution E: 250 pM, 250 μl of D+250 μl of the diluent Solution F: 100 pM, 200 μl of E+300 μl of the diluent Solution G: 50 pM, 250 μl of F+250 μl of the diluent Solution H: 25 pM, 200 μl of G+200 μl of the diluent Solution I: 10 pM, 100 μl of H+150 μl of the diluent

(42) The range of recombinant PG is linear and can therefore be more or less extensive according to the antibody used.

(43) Methods and Results

(44) Progastrin levels were determined in plasma samples from subjects who were known to have developed cancer later. Progastrin was captured with the C-terminus monoclonal antibody mAb 14 produced by hybridoma 2H9F4B7 described in WO 2011/083088 (Hybridoma 2H9F4B7 was deposited under the Budapest Treaty at the CNCM, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris CEDEX 15, France, on 27 Dec. 2016, under reference I-5158.). Detection was performed with labelled polyclonal antibodies specific for the N-terminus.

(45) Importantly, at the time of sample collection, these subjects had never been diagnosed with cancer and did not show any symptom relating to cancer. The control was constituted by plasma samples from the general population.

(46) The results are shown in FIG. 15. The median plasmatic concentration of progastrin was ranked between 2.750 and 21.5 pM in patients depending of the type of cancer (n=231). By comparison, the median plasmatic concentration of progastrin is 0 pM in control subjects (n=322).

(47) These data demonstrate that patients who will develop cancer have detectable levels of progastrin in their plasma whereas healthy control individuals have none. Progastrin can be detected even before any cancer can be diagnosed, making progastrin useful biomarker for the onset of cancer.

(48) These data demonstrate that patients with a risk of developing cancer have higher concentration of progastrin in their plasma compared to healthy control individuals.

Example 3: Detection of Plasmatic Progastrin Concentration Using a Combination of Monoclonal Antibodies

(49) In the present example, plasma progastrin levels were quantified by ELISA through the use of antibody specific for human progastrin (hPG) pre-coated on a 96-well plate. Standards and samples are added to the wells, and any hPG present binds to the immobilized capture antibody. The wells are washed and an anti-hPG detection antibody horseradish peroxidase (HRP) conjugate is added, producing an antibody-antigen-antibody “sandwich.” After a second wash, TMB substrate solution is added, which produces a blue color in direct proportion to the amount of hPG present in the initial sample. The Stop Solution changes the color from blue to yellow, and the wells are read at 450 nm with a microplate reader.

(50) Polyclonal antibodies were obtained by immunizing a rabbit with N-terminal progastrin (SEQ ID N.sup.o 2) or with C-terminal progastrin corresponding to amino acids 71 to 80 of hPG and having the sequence FGRRSAEDEN (SEQ ID N.sup.o 40), according to standard protocols.

(51) Monoclonal antibodies were obtained by using hybridomas producing antibodies against N-terminal progastrin (SEQ ID N.sup.o 2) or against C-terminal progastrin corresponding to amino acids 71 to 80 of hPG and having the sequence FGRRSAEDEN (SEQ ID N.sup.o 40), according to standard protocols.

(52) The binding characteristics of polyclonal and monoclonal antibodies against progastrin used in this assay are the following: absence of binding to G34-Gly, G34, G17-Gly, G17, binding to full length progastrin.

(53) For the control test, ELISA in the presence of a known concentration of progastrin, progastrin dilution is prepared as follows: stock recombinant PG (Full length human progastrin produced in E. coli and affinity purified with Glutathione agarose/Tag removal (Tev)/IMAC Counter purification/dialysis, from Institut Pasteur, Paris, France) is prepared at a concentration of 0.45 mg/ml (45 microM), in triplicate. Ranges of progastrin concentrations were prepared as follows: Solution A: Pre-dilution 1/10, 2 μl of stock+18 μl of the buffer Solution B: Pre-dilution 1/100, 10 μl of A+90 μl of the buffer Solution C: Pre-dilution 1/1000, 10 μl of B+90 μl of the buffer Solution D: 500 pM, 5.55 μl of C+494.5 μl of the diluent Solution E: 250 pM, 250 μl of D+250 μl of the diluent Solution F: 100 pM, 200 μl of E+300 μl of the diluent Solution G: 50 pM, 250 μl of F+250 μl of the diluent Solution H: 25 pM, 200 μl of G+200 μl of the diluent Solution I: 10 pM, 100 μl of H+150 μl of the diluent

(54) The range of recombinant PG is linear and can therefore be more or less extensive according to the antibody used.

(55) Methods and Results

(56) Progastrin levels were determined in plasma samples from subjects who were known to have developed cancer later. Progastrin was captured with the C-terminus monoclonal antibody mAb 14 produced by hybridoma 2H9F4B7 described in WO 2011/083088 (Hybridoma 2H9F4B7 was deposited under the Budapest Treaty at the CNCM, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris CEDEX 15, France, on 27 Dec. 2016, under reference I-5158.). Detection was performed with labelled monoclonal antibody mAb 16 described in WO 2011/083088 which is specific for the N-terminus.

(57) Importantly, at the time of sample collection, these subjects had never been diagnosed with cancer and did not show any symptom relating to cancer. The control was constituted by plasma samples from the general population.

(58) The results are shown in FIG. 16. The median plasmatic concentration of progastrin using mAb-pAb et mAb-mAb were similar in median (8.2 vs 6.6 pM respectively) and mean (18.99 vs 16.79 pM) respectively (n=10).

(59) These data demonstrate that mAb-pAb and mAb-mAb ELISA sandwich tests detect both progastrin in the plasma of patients. Importantly, no significant difference between both tests could be identified. In particular, the sensitivity of the mAb-pAb and mAb-mAb sandwiches were highly similar. Therefore mAb-mAb ELISA sandwich can be used reliably to detect progastrin in the plasma of patient even before any cancer can be diagnosed, making progastrin useful biomarker for the onset of cancer.

(60) These data demonstrate that patients with a risk of developing cancer have higher concentration of progastrin in their plasma compared to healthy control individuals.