COMPOSITIONS AND METHODS FOR DETECTING LUNG CANCER

Abstract

The present invention relates to compositions and methods for the in vitro diagnosis of lung cancer, wherein said compositions comprise an antibody binding to progastrin and said methods comprise the use of an antibody binding to progastrin.

Claims

1. A method for the in vitro diagnosis of lung cancer in a subject, comprising the steps of: a) contacting said biological sample from said subject with at least one progastrin-binding molecule, b) detecting the binding of said progastrin-binding molecule to progastrin in said sample, wherein said binding indicates the presence of lung cancer in said subject.

2. The method of claim 1, wherein step b) further comprises determining the concentration of progastrin and wherein a concentration of progastrin at least 10 pM in said biological sample is indicative of the presence of lung cancer in said subject.

3. The method of claim 2, comprising the further steps of: c) determining a reference concentration of progastrin in a reference sample, d) comparing the concentration of progastrin in said biological sample with said reference concentration of progastrin, e) determining, from the comparison of step d), the presence of lung cancer.

4. The method of any one of claims 1 to 3, wherein said progastrin-binding molecule is an antibody, or an antigen-binding fragment thereof.

5. The method of any of claims 1 to 4, wherein said antibody, or antigen-binding fragment thereof, is selected among N-terminal anti-progastrin monoclonal antibodies and C-terminal anti-progastrin monoclonal antibodies.

6. The method of any of claims 1 to 5, wherein said antibody binding to progastrin is a monoclonal antibody chosen in the group consisting of: A monoclonal antibody comprising a heavy chain comprising at least one, preferentially at least two, preferentially three, of CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID No 4, 5 and 6, respectively, and a light chain comprising at least one, preferentially at least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No 7, 8 and 9, respectively, A monoclonal antibody comprising a heavy chain comprising at least one, preferentially at least two, preferentially three, of CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID No 10, 11 and 12, respectively, and a light chain comprising at least one, preferentially at least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No 13, 14 and 15, respectively, A monoclonal antibody comprising a heavy chain comprising at least one, preferentially at least two, preferentially three, of CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID No 16, 17 and 18, respectively, and a light chain comprising at least one, preferentially at least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No 19, 20 and 21, respectively, A monoclonal antibody comprising a heavy chain comprising at least one, preferentially at least two, preferentially three, of CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID No 22, 23 and 24, respectively, and a light chain comprising at least one, preferentially at least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No 25, 26 and 27, respectively, A monoclonal antibody comprising a heavy chain comprising at least one, preferentially at least two, preferentially three, of CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID No 28, 29 and 30, respectively, and a light chain comprising at least one, preferentially at least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No 31, 32 and 33, respectively, A monoclonal antibody comprising a heavy chain comprising at least one, preferentially at least two, preferentially three, of CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID No 34, 35 and 36, respectively, and a light chain comprising at least one, preferentially at least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No 37, 38 and 39, respectively, and 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 1-5158.

7. The method of any one of claims 1 to 6, wherein the determination of step a) includes: (i) contacting said sample with a first progastrin-binding molecule which binds to a first part of progastrin, and (ii) contacting said sample with a second progastrin-binding molecule which binds to a second part of progastrin.

8. The method of claim 7, wherein the first progastrin-binding molecule binds an epitope within the C-terminus of progastrin.

9. The method of any one of claim 7 or 8, wherein said progastrin-binding molecule 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 1-5158.

10. The method of any one of claims 7 to 9, wherein the second progastrin-binding molecule binds an epitope within the N-terminus of progastrin.

11. The method of any one of claims 7 to 10, wherein said second progastrin-binding molecule is a polyclonal antibody binding an epitope within the N-terminus of progastrin or a monoclonal antibody comprising a heavy chain comprising the following three CDRs, CDR-H1, CDR-H2 and CDR-H3 of amino acid sequences SEQ ID No 16, 17 and 18, respectively, and a light chain comprising the following three CDRs, CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No 19, 20 and 21, respectively.

12. The method of any one of claims 1 to 11, wherein the level of progastrin is determined in step a) with an ELISA.

13. The method of any one of claims 1 to 6, wherein said biological sample is contacted with a first molecule, which binds to a first part of progastrin, and with a second molecule, which binds to a second part of progastrin.

14. The method of any one of claims 1 to 7, wherein said biological sample is chosen among: blood, serum and plasma.

15. The method of any one of claims 1 to 8, wherein said biological sample is plasma, and wherein a concentration of progastrin of at least 10 pM is indicative of the presence of lung cancer in said subject.

16. Use of a progastrin-binding antibody, or an antigen-binding fragment thereof, as in any one of claims 10 to 14 for the in vitro diagnosis of lung cancer.

Description

FIGURE LEGENDS

[0157] FIG. 1

[0158] Progastrin concentration was measured in 40 plasma samples from lung cancer patients and 119 plasma samples from healthy donor using the ELISA Kit DECODE Lab (capture antibody: Mab14, detection antibody: anti-hPG polyclonal).

EXAMPLES

Example 1: Detection of Plasmatic Progastrin Concentration Using Polyclonal Antibodies

[0159] 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.

[0160] 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.

[0161] Material

[0162] Reagents and apparatus are listed in Table 7:

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

[0163] Polyclonal antibodies were obtained by immunizing a rabbit with N-terminal progastrin (SEQ ID No 2) or with C-terminal progastrin corresponding to amino acids 71 to 80 of hPG and having the sequence FGRRSAEDEN (SEQ ID No 40), according to standard protocols.

[0164] 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.

[0165] 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 g/ml), corresponding to polyclonal antibodies obtained by using the C-terminal of progastrin FGRRSAEDEN (SEQ ID No 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 l 1PBS/0.1% Tween-20, then adding 200 l of blocking buffer (1PBS/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 l 1PBS/0.1% Tween-20.

[0166] Plasma dilution is performed as follows: The plasma is used pure, diluted , and 1/10. Dilutions are prepared from pure plasma in 1PBS/0.1% Tween 20/0.1% BSA.

[0167] 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: [0168] Solution A: Pre-dilution 1/10, 2 l of stock+18 l of the buffer [0169] Solution B: Pre-dilution 1/100, 10 l of A+90 l of the buffer [0170] Solution C: Pre-dilution 1/1000, 10 l of B+90 l of the buffer [0171] Solution D: 500 pM, 5.55 l of C+494.5 l of the diluent [0172] Solution E: 250 pM, 250 l of D+250 l of the diluent [0173] Solution F: 100 pM, 200 l of E+300 l of the diluent [0174] Solution G: 50 pM, 250 l of F+250 l of the diluent [0175] Solution H: 25 pM, 200 l of G+200 l of the diluent [0176] Solution I: 10 pM, 100 l of H+150 l of the diluent

[0177] The range of recombinant PG is linear and can therefore be more or less extensive according to the antibody used.

[0178] For the preparation of test samples, approximately 500 l of each sample are set aside and stored until analysis (and confirmation if necessary) of the results. 100 l of each point of the range and/or plasmas are assayed pure, diluted to , and 1/10, and incubated for 2 hours at 22 C. on the plates.

[0179] For the revelation of the test, the plates are washed 3 times with 300 l 1PBS/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 g/ml, is prepared by dilution in 1PBS/0.1% Tween-20/0.1% BSA. 100 l 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 l 1PBS/0.1% Tween-20, then preparing a solution of Streptavidin-HRP at 20 ng/ml diluted in 1PBS/0.1% Tween-20/0.1% BSA, wherein 100 Add 100 l of this solution is added to each well, before incubation for 1 hour at 22 C.

[0180] The detection consists of eliminating streptavidin-HRP and wash 3 times with 300 l 1PBS/0.1% Tween-20, then adding 100 l 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.

[0181] 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).

[0182] Methods and Results

[0183] The median plasmatic concentration of progastrin is 0 pM in control patients (n=103), whereas a significant plasmatic concentration of progastrin can be detected in patients having lung cancer. Thus, patients with lung cancer have higher levels of progastrin in their plasma compared to healthy control individuals.

Example 2: Detection of Progastrin Concentration Using Monoclonal Anti-Progastrin Antibodies

[0184] The wells of Nunc MaxiSORP 96-well plates are coated with a first progastrin-specific antibody as follows. Anti-progastrin monoclonal antibodies specific for the carboxy-terminal region of progastrin are diluted to a concentration of 3 g/ml in a solution of 50 mM, pH 9.6 sodium carbonate/bicarbonate buffer in MilliQ water.

[0185] A total of 100 l of the antibody solution is then added to each well of the 96-well plates, and incubated overnight at 4 C. After binding, the antibody solution is removed from the wells, which are then washed three times with 100 l wash buffer (1PBS/0.1% Tween-20). A total of 100 l blocking buffer (1PBS/0.1% Tween-20/0.1% BSA) is then added to each well and incubated for 2 hours at 22 C. Blocking buffer is then removed and the wells washed three times with wash buffer. Plasma or serum samples isolated from patients is then added to the wells in a volume of 100 l in a dilution series, typically 1:1, 1:2, 1:5 and 1:10 dilutions, and is then incubated for 2 hours at 22 C. Plasma or serum samples are analyzed in duplicate.

[0186] Assays also include two standard curves. The first standard curve is prepared using dilutions of recombinant progastrin to a final amount of 1 ng, 0.5 ng, 0.25 ng, 0.1 ng, 0.05 ng, 0.01 ng, and 0 ng per well. The second standard curve, which serves as a negative control, is prepared from progastrin-negative human serum diluted in blocking buffer at the same dilutions as the test samples, i.e., 1:1, 1:2, 1:5 and 1:10. Alternatively, when plasma samples are being assayed, the second standard curve, which serves as a negative control, is prepared from progastrin-negative human plasma diluted in blocking buffer at the same dilutions as the test samples, i.e., 1:1, 1:2, 1:5 and 1:10.

[0187] After incubation with the plasma or serum samples is complete, the well contents are removed and the wells are washed three times with wash buffer, 100 l/well, after which progastrin bound to the first antibody is detected using a second antibody specific for progastrin, as follows.

[0188] Biotin-coupled anti-progastrin monoclonal antibodies specific for the amino-terminal region of progastrin are diluted in blocking buffer to a concentration of 0.1 to 10 l g/ml, depending on the antibody. A total of 100 l of the antibody solution is then added to each well, and incubated for 1 hour at 22 C.

[0189] After secondary antibody binding is complete, the plates are washed three times with wash buffer, 100 l/well, after which 100 l of a solution of streptavidin-HRP (25 ng/ml in blocking buffer) is added to each well and incubated for 1 hour at 22 C. After incubation with the streptavidin-HRP solution is complete, the plates are washed three times with wash buffer, 100 l/well. Thereafter, 100 l of chemiluminescent substrate prepared using a Pierce SuperSignal ELISA Femto Maximum Sensitivity Chemiluminescent Substrate kit, is added per well, incubated for 5 min at room temperature in the dark, and then read on a luminometer.

[0190] Based on the luminometer readings, linear regression analysis is used to derive the equation of the lines corresponding to the standard curve data. Using this equation, the concentration of progastrin in the various patient samples is then calculated.

[0191] The median plasmatic concentration of progastrin is calculated in patients having lung cancer and compared to the median plasmatic concentration of progastrin in plasma of control patients. Patients with lung cancer had elevated levels of progastrin in their plasma compared to healthy control individuals.

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

[0192] In the present example, plasma progastrin levels are 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 color from blue to yellow, and the wells are read at 450 nm with a microplate reader.

[0193] Polyclonal antibodies are obtained by immunizing a rabbit with N-terminal progastrin (SEQ ID No 2) or with C-terminal progastrin corresponding to amino acids 71 to 80 of hPG and having the sequence FGRRSAEDEN (SEQ ID No 40), according to standard protocols.

[0194] Monoclonal antibodies are obtained by using hybridomas producing antibodies against N-terminal progastrin (SEQ ID No 2) or against C-terminal progastrin corresponding to amino acids 71 to 80 of hPG and having the sequence FGRRSAEDEN (SEQ ID No 40), according to standard protocols.

[0195] 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.

[0196] 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 are prepared as follows:

[0197] Solution A: Pre-dilution 1/10, 2 l of stock+18 l of the buffer

[0198] Solution B: Pre-dilution 1/100, 10 l of A+90 l of the buffer

[0199] Solution C: Pre-dilution 1/1000, 10 l of B+90 l of the buffer

[0200] Solution D: 500 pM, 5,55 l of C+494.5 l of the diluent

[0201] Solution E: 250 pM, 250 l of D+250 l of the diluent

[0202] Solution F: 100 pM, 200 l of E+300 l of the diluent

[0203] Solution G: 50 pM, 250 l of F+250 l of the diluent

[0204] Solution H: 25 pM, 200 l of G+200 l of the diluent

[0205] Solution I: 10 pM, 100 l of H+150 l of the diluent

[0206] The range of recombinant PG is linear and can therefore be more or less extensive according to the antibody used.

[0207] Methods and Results

[0208] Progastrin levels are determined in plasma samples from subjects who were known to have developed lung cancer later. Progastrin is captured with the C-terminus monoclonal antibody mAb 14 produced by hybridoma 2H9F4B7 described in WO 2011/083088 (Hybridoma 2H9F4B7 is 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 1-5158.). Detection is performed with labelled polyclonal antibodies specific for the N-terminus.

[0209] The control is constituted by plasma samples from the general population.

[0210] The data demonstrate that patients with lung cancer have detectable levels of progastrin in their plasma whereas healthy control individuals have none.

Example 4: Detection of Plasmatic Progastrin Concentration Using DECODE Lab Kit

[0211] The test allows a measurement of hPG in plasma EDTA by ELISA.

[0212] The kit utilizes a capture antibody specific for hPG pre-coated on a 96-well plate. hPG present in standards and samples added to the wells bind to the immobilized capture antibody. The wells are washed and an anti-hPG detection antibody horseradish peroxidase (HRP) conjugate is added, resulting in an antibody-antigen-antibody complex. After a second wash, a 3,3,5,5-Tetramethylbenzidine (TMB) substrate solution is added to the well, producing a blue color in direct proportion to the amount of hPG present in the initial sample. The Stop Solution changes the colour from blue to yellow, and the wells are read at 450 nm with a microplate reader.

[0213] Methods and Results

[0214] 40 plasma samples from lung cancer patients and 119 plasma samples from healthy donor were used to measure the concentration of progastrin using the ELISA Kit DECODE Lab (capture antibody: Mab14, detection antibody: anti-hPG polyclonal) following manufacturer's recommendation.

[0215] Briefly: [0216] 1. Prepare all reagents, controls, and samples as directed in the previous section except the 1 Conjugate. [0217] 2. Remove excess strip from the microtiter plate frame, return them to the plate packet and store at 2-8 C. [0218] 3. Samples and controls must be tested in duplicate. Prepare the pre-loading of controls and samples by adding 65 l/replicate in wells of the 96-Well DeepWell Polypropylene Microplates. [0219] 4. Add 50 l of Sample dilution buffer to all the wells that will be used from the 96 pre-coated well plate strips included in the kit. [0220] 5. Transfer 50 l of the controls and samples with a multi-channel pipette (8 channels) from the pre-loading 96-Well DeepWell Polypropylene Microplates to the 96 pre-coated well plate strips included in the kit. The loading time should not exceed 10 minutes. [0221] 6. Cover the plate with plastic paraffin and incubate for 3 h5 min at 37 C. (2 C.). [0222] 7. Prepare the 1 Conjugate as described in section 10.2 [0223] 8. At the end of the incubation step, discard all the liquid from the wells by inverting the plate. Proceed to a thorough washing step by adding 300 l per well of 1 Wash solution. Discard the 1 wash solution by inverting the plate and thoroughly pat dry the microtiter plate frame upside down on absorbent paper. Repeat the washing step 6 times. At the end of the washing steps, ensure the complete removal of the liquid from the wells: all liquid has been successfully removed when no sign of liquid remains on the paper towel. The wash procedure is critical. Insufficient washing may result in poor precision and falsely elevated absorbance readings. [0224] 9. Add 100 l of the 1 Conjugate to each well. [0225] 10. Cover the plate with plastic paraffin and incubate 30 min3 min at 21 C. (5 C.). [0226] 11. At the end of the incubation step, discard all the liquid from the wells by inverting the plate. Proceed to a thorough washing step by adding 300 l per well of 1 Wash solution. Discard the 1 wash solution by inverting the plate and thoroughly pat dry the microtiter plate frame upside down on absorbent paper. Repeat the washing step 6 times. At the end of the washing steps, ensure the complete removal of the liquid from the wells: all liquid has been successfully removed when no sign of liquid remains on the paper towel. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. [0227] 12. Add 100 l of the Substrate solution to each well. Upon the addition of the Substrate solution, the content of the Positive Control 1 and Positive Control 2 wells should become blue. [0228] 13. Incubate for 15 min2 min at 21 C. (5 C.) in the dark. [0229] 14. Without removing the content, of the wells, add 100 l of the Stop solution to each well in order to stop the reaction. Upon the addition of the Stop solution, the content of the Positive Control 1 and Positive Control 2 wells should become yellow. [0230] 15. Read and record the O.D. at 450 nm.

[0231] As shown in FIG. 1, the median plasmatic concentration of progastrin was 0 pM in control patients (n=119), whereas a significant plasmatic concentration of progastrin could be detected in patients having lung cancer (n=40). Thus, patients with lung cancer have higher levels of progastrin in their plasma compared to healthy control individuals.

BIBLIOGRAPHIC REFERENCES

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