ANTIBODIES SPECIFICALLY BINDING THE CARBOXYMETHYLATED CATALYTIC SUBUNIT OF PROTEIN PHOSPHATASE 2A
20230121197 · 2023-04-20
Assignee
Inventors
Cpc classification
G01N33/57484
PHYSICS
G01N2800/042
PHYSICS
C07K2317/33
CHEMISTRY; METALLURGY
G01N2800/52
PHYSICS
G01N2800/325
PHYSICS
C07K2317/92
CHEMISTRY; METALLURGY
G01N2333/916
PHYSICS
G01N2500/02
PHYSICS
International classification
Abstract
The present invention relates to an antibody specifically binding the carboxymethylated catalytic subunit of protein phosphatase 2A (PP2Ac). Also provided are diagnostic uses of said antibody and screening methods employing the inventive antibody.
Claims
1. An antibody specifically binding the carboxymethylated catalytic subunit of protein phosphatase 2A (PP2Ac).
2. The antibody of claim 1 specifically binding the methylated carboxyl group of the C-terminal leucine, i.e. Leu309, of PP2Ac.
3. The antibody of claim 1 or 2 specifically binding an epitope comprised in the carboxymethylated C-terminal region of PP2Ac, wherein said C-terminal region has the sequence TPDYFL (SEQ ID NO:1).
4. The antibody of claim 3, wherein the epitope comprises (i) the methylated carboxyl group of the C-terminal leucine and (ii) the threonine and/or the proline within SEQ ID NO:1.
5. The antibody of any one of the preceding claims, wherein said antibody binds said carboxymethylated PP2Ac or, preferably, a peptide consisting of the last 11 amino acids of the carboxymethylated C-terminal region of PP2Ac (HVTRRTPDYFL; SEQ ID NO:18), with a dissociation constant (K.sub.D) of 200 nM, 100 nM, 80 nM, 60 nM, 40 nM, 20 nM, 15 nM or less, preferably 40 nM or less, more preferably 20 nM or less, e.g. about 11 nM.
6. The antibody of claim 5, wherein said dissociation constant (K.sub.D) is determined by surface plasmon resonance, preferably Biacore, more preferably a BiacoreT200 instrument, a Series S Sensor Chip CMS and a BiacoreT200 Evaluation Software, i.e. version 3.1.
7. The antibody of claim 5 or 6, wherein the conditions for determining said dissociation constant comprise: (i) a temperature of 25° C., (ii) said antibody at a concentration of 50 μg/mL, (iii) a peptide consisting of the last 11 amino acids of the carboxymethylated C-terminal region of PP2Ac (HVTRRTPDYFL-CH.sub.3; SEQ ID NO:18), preferably at least 4, preferably at least 6 different concentrations, wherein the lowest concentration is about 5 nM and the highest concentration is about 160 nM, and the other concentrations are preferably between said lowest and highest concentrations; and/or (iv) PBS with pH 7.4+0.005% Tween+0.1% BSA as a buffer; in particular such that all binding curves reach equilibrium and all requirements for a steady state analysis are fulfilled.
8. The antibody of any one of the preceding claims comprising a heavy chain variable region and a light chain variable region, wherein (a) the heavy chain variable region comprises at least one complementary determining region selected from a CDR-113, a CDR-112 and a CDR-H1, wherein (i) the CDR-H3 sequence is RFAY (SEQ ID NO:2), (ii) the CDR-H2 sequence is YISYDGSNNYNPSLKN (SEQ ID NO:3), (iii) the CDR-H1 sequence is SGYYWN (SEQ ID NO:4), and/or (b) the light chain variable region comprises at least one complementary determining region selected from a CDR-L3, a CDR-L2 and a CDR-L1, wherein the (iv) the CDR-L3 sequence is FQGSHVPWT (SEQ ID NO:5), (v) the CDR-L2 sequence is KVSNRFS (SEQ ID NO:6), and (vi) the CDR-L1 sequence is RSSQSIVHSNGNTYLE (SEQ ID NO:7).
9. The antibody of claim 8, wherein (a) the heavy chain variable region further comprises at least one framework region selected from a H-FR1, a H-FR2, a H-FR3 and a H-FR4, wherein the framework regions are directly adjacent to the CDRs according to the formula (H-FR1)-(CDR-H1)-(H-FR2)-(CDR-H2)-(H-FR3)-(CDR-H3)-(H-FR4), and/or (b) the light chain variable region further comprises at least one framework region selected from a L-FR1, a L-FR2, a L-FR3 and a L-FR4, wherein the framework regions are directly adjacent to the CDRs according to the formula (L-FR1)-(CDR-L1)-(L-FR2)-(CDR-L2)-(L-FR3)-(CDR-L3)-(L-FR4).
10. The antibody of claim 9, wherein the H-FR1 sequence is DVQLQESGPGLVKPSQSLSLTCSVTGYSIT (SEQ ID NO:8), the H-FR2 sequence is WIRQFPGNKLEWMG (SEQ ID NO:9), the H-FR3 sequence is RISITRDTSKNQFFLKLNSVTTEDTATYYCAG (SEQ ID NO:10), the H-FR4 sequence is WGQGTLVTVSA (SEQ ID NO:11), the L-FR1 sequence is DVLMTQTPLSLPVSLGDQASISC (SEQ ID NO:12), the L-FR2 sequence is WYLQKPGQSPKLLIY (SEQ ID NO:13), the L-FR3 sequence is GVPDRFSGSGSGTDFTLKINRVEAEDLGVYYC (SEQ ID NO:14), and the L-FR4 sequence is FGGGTKLEIK (SEQ ID NO:15)
11. The antibody of any one of the preceding claims, wherein the sequence of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4, heavy chain variable region and/or light chain variable region of said antibody is identical to the respective sequence of the monoclonal antibody produced by the single clone hybridoma cell line “7C10-C5” deposited under the accession number “DSM ACC3350” at “The Leipniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures”.
12. The antibody of any one of the preceding claims, wherein said antibody is a monoclonal antibody.
13. The antibody of any one of claims 1 to 12, wherein said antibody is an IgG1 or IgG2 such as IgG2a, preferably IgG1.
14. The antibody of any one of claims 1 to 12, wherein said antibody is a single-chain variable fragment (scFv) comprising at least one heavy chain variable region and at least one light chain variable region.
15. The antibody of claim 14, wherein said antibody is a bivalent scFv.
16. The antibody of any one of the preceding claims, wherein the heavy chain variable region has the amino acid sequence DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYIS YDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAGRFAYWGQ GTLVTVSA (SEQ ID NO:16) and/or the light chain variable region has the amino acid sequence TABLE-US-00021 (SEQ ID NO: 17) DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNT YLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS GTDFTLKINRVEAEDLGVYYCFQGSHVPWTFGGG TKLEIK.
17. The antibody of any one of the preceding claims, wherein said antibody does not specifically bind the non-methylated catalytic subunit of PP2A (PP2Ac), for example, as determined by Biacore according to claim 6 and under the conditions according to claim 7, except that said peptide is not methylated (HVTRRTPDYFL-OH; SEQ ID NO:18).
18. The antibody of any one of the preceding claims, wherein said antibody binds carboxymethylated PP2Ac preferably at least 4, 8, 16, 24, 26, 32, 40 or 48-fold, preferably at least 26-fold stronger than non-carboxymethylated PP2Ac, for example, as determined by an ELISA.
19. The antibody of any one of the preceding claims, wherein said antibody binds a peptide comprising the carboxymethylated C-terminal region of PP2Ac preferably at least 4, 8, 16, 24, 26, 32, 40 or 48-fold, preferably at least 26-fold, stronger than a corresponding peptide comprising the non-methylated C-terminal region of PP2Ac, wherein said C-terminal region of PP2Ac has the sequence TPDYFL (SEQ ID NO:1), for example, as determined by an ELISA, and preferably wherein both peptides have the same length.
20. The antibody of claim 19, wherein the C-terminal region of PP2Ac comprised in the peptide has the sequence HVTRRTPDYFL (SEQ ID NO:18).
21. The antibody of any one of the preceding claims, wherein said antibody binds a peptide consisting of the last 11 amino acids of the carboxymethylated C-terminal region of PP2Ac (HVTRRTPDYFL-CH.sub.3; SEQ ID NO:18) at least 4-, 6-, 8-, 10- or 12-fold, preferably at least 10-fold, e.g. about 12-fold, stronger than a peptide consisting of the last 11 amino acids of the carboxymethylated C-terminal region of PP4c TABLE-US-00022 (PSKKPVADYFL-CH.sub.3; SEQ ID NO: 20).
22. The antibody of claim 21, wherein the binding is determined by Biacore according to claim 6 under the conditions according to claim 7; preferably wherein each peptide is employed at different concentrations, e.g at least 4 different concentrations, wherein for the carboxymethylated peptide having the SEQ ID NO:18 the lowest concentration is about 5 nM and the highest concentration is about 160 nM, and wherein for the carboxymethylated peptide having the SEQ ID NO:20 the lowest concentration is about 50 nM and the highest concentration is about 800 nM.
23. The antibody of any one of the preceding claims, wherein said antibody binds a peptide comprising the C-terminal region of the carboxymethylated PP2Ac (SEQ ID NO:1) preferably at least 4, 8, 10, 16, 24, 32, 40 or 48-fold, preferably at least 10-fold, stronger than a peptide comprising the C-terminal region of the catalytic subunit of protein phosphatase 4 (PP4c), wherein the C-terminal region of PP4c has the sequence VADYFL (SEQ ID NO:19), and preferably wherein both peptides have the same length, for example, as determined by an ELISA.
24. The antibody of claim 23, wherein the C-terminal region of PP2Ac comprised in the peptide has the sequence HVTRRTPDYFL (SEQ ID NO:18) and the C-terminal region of PP4c comprised in the peptide has the sequence PSKKPVADYFL (SEQ ID NO:20).
25. The antibody of claim 23 or 24, wherein the carboxyl group of the C-terminal leucine of both peptides comprising either the C-terminal region of PP2Ac or PP4c is methylated.
26. The antibody of any one of the preceding claims, wherein said antibody binds a peptide comprising the C-terminal region of the carboxymethylated PP2Ac (SEQ ID NO:1) preferably at least 4, 8, 10, 16, 24, 32, 40 or 48-fold, preferably at least 10-fold, stronger than a peptide comprising the C-terminal region of the catalytic subunit of protein phosphatase 6 (PP6c), wherein the C-terminal region of PP6c has the sequence TTPYFL (SEQ ID NO:21), for example, as determined by an ELISA, and preferably wherein both peptides have the same length.
27. The antibody of claim 26, wherein the C-terminal region of PP2Ac comprised in the peptide has the sequence HVTRRTPDYFL (SEQ ID NO:18) and the C-terminal region of PP6c comprised in the peptide has the sequence IPPRTTTPYFL (SEQ ID NO:22).
28. The antibody of claim 26 or 27, wherein the carboxyl group of the C-terminal leucine of both peptides comprising either the C-terminal region of PP2Ac or PP6c is methylated.
29. The antibody of any one of the preceding claims, wherein said antibody binds a peptide comprising the carboxymethylated C-terminal region of PP2Ac preferably at least 4, 6, 8, 16, 24, 32, 40 or 48-fold, preferably at least 6-fold, stronger than a corresponding peptide comprising the amidated C-terminal region of PP2Ac, wherein said C-terminal region of PP2Ac has the sequence TPDYFL (SEQ ID NO:1) for example, as determined by an ELISA, and preferably wherein both peptides have the same length.
30. The antibody of any one of the preceding claims, wherein the binding strength of said antibody to a peptide comprising the carboxymethylated C-terminal region of PP2Ac (SEQ ID NO:1 or SEQ: ID NO:18) is not affected (+/−30%) when the tyrosine in said C-terminal region of PP2Ac is phosphorylated, for example, as determined by an ELISA.
31. The antibody of any one of the preceding claims, wherein the binding strength of said antibody to a peptide comprising the carboxymethylated C-terminal region of PP2Ac (SEQ ID NO:1 or SEQ: ID NO:18) is not affected (+/−30%), or is at most 4-fold, preferably at most 3-fold or 2-fold higher, when the most C-terminal threonine in said C-terminal region of PP2Ac is phosphorylated, for example, as determined by ELISA.
32. The antibody of any one of claims 7 to 31, wherein the peptide(s) is/are acetylated at the N-terminus.
33. The antibody of any one of claims 19, 23, 25, 26, 28, or 29 to 32, wherein the peptide comprising either the C-terminal region of PP2Ac, PP4c or PP6c consists of 6 to 16, preferably 9 to 13, preferably 11 amino acids.
34. The antibody of any one of claims 18 to 21 or 23 to 33, wherein the binding strength of said antibody to a certain peptide is determined by an ELISA, and preferably wherein said binding strength corresponds to the signal intensity determined by said ELISA.
35. The antibody of any one of the preceding claims, wherein said antibody is suitable for quantifying the amount of heterotrimeric PP2A holoenzyme and/or active PP2A in a biological sample.
36. The antibody of any one of the preceding claims, wherein said antibody has been raised against (i) a peptide comprising the sequence of the 5, preferably 6, C-terminal amino acids of the carboxymethylated PP2Ac or (ii) a peptide of the sequence TPDYFL (SEQ ID NO:1), PDYFL (SEQ ID NO:23) or RTPDYFL (SEQ ID NO:24), preferably SEQ ID NO:1, wherein the carboxyl group of the C-terminal leucine of said peptide is methylated.
37. The antibody of claim 36, wherein said peptide further includes at the N-terminus a cysteine followed by a β-alanine, and preferably wherein said peptide is coupled via the cysteine to a carrier protein, preferably to keyhole limpet hemocyanin (KLH).
38. A method for producing (i) an antibody specifically binding the carboxymethylated catalytic subunit of protein phosphatase 2A (PP2Ac), and/or (ii) the antibody of any one of claims 1 to 37, wherein said method comprises the steps of (a) immunizing a non-human mammal, preferably a mouse, with a peptide comprising the sequence of the 5, preferably 6, C-terminal amino acids of the carboxymethylated PP2Ac, (b) generating hybridoma clones from immune cells of said non-human animal, (c) selecting a hybridoma clone whereof the supernatant binds preferably at least 4, 8, 16, 20, 24, 26, 32, 40, 50, 100, 200 or 500-fold, preferably at least 100-fold, stronger to the PP2Ac of a cell which contains a PP2A specific methyltransferase (PPM1/LCMT-1) than to the PP2Ac of a cell which lacks said PP2A specific methyltransferase, and (d) obtaining said antibody from said selected hybridoma clone.
39. The method of claim 38, wherein the immunization peptide in (a) has the sequence TPDYFL (SEQ ID NO:1), PDYFL (SEQ ID NO:23) or RTPDYFL (SEQ ID NO:24), preferably SEQ ID NO:1, wherein the carboxyl group of the C-terminal leucine of said peptide is methylated.
40. The method of claim 38 or 39, wherein the immunization peptide in (a) further includes at the N-terminus a cysteine followed by a β-alanine, and preferably is coupled via the cysteine to a carrier protein, preferably to keyhole limpet hemocyanin (KLH).
41. The method of any one of claims 38 to 40, wherein the cell containing said PP2A specific methyltransferase lacks the PP2A specific demethylase (PPE-1/PME1).
42. The method of any one of claims 38 to 41, wherein the cells for assaying the binding of the hybridoma supernatant are yeast cells.
43. The method of any one of claims 38 to 42, wherein the PP2Ac of a cell is contained in the lysate of said cell, and preferably wherein the binding of the hybridoma supernatant to said PP2Ac is determined by western blotting.
44. An antibody obtainable by the method of any one of claims 38 to 42.
45. Use of an anti-carboxymethylated PP2Ac-specific antibody of any one of claims 1 to 37 for specifically detecting the carboxymethylated catalytic subunit of protein phosphatase 2A (PP2Ac) in a biological sample.
46. A method for specifically detecting the carboxymethylated catalytic subunit of protein phosphatase 2A (PP2Ac) in a biological sample, wherein said method comprises a step of contacting said biological sample with an anti-carboxymethylated PP2Ac-specific antibody of any one of claims 1 to 37.
47. The method of claim 47, wherein the biological sample does not have a pH greater than 8.0, preferably 8.5, and/or said sample is not treated with a liquid having a pH greater than 8.0, preferably 8.5, preferably wherein said sample is not treated with said liquid before and/or during contacting with said antibody.
48. An in vitro method for prognosing the outcome of a cancer in a patient, wherein said method comprises the steps of (a) determining the level of carboxymethylated PP2Ac in a sample from said patient by contacting said sample with an anti-carboxymethylated PP2Ac-specific antibody according to any one of claims 1 to 37; (b) comparing the level of carboxymethylated PP2Ac to a threshold value and/or reference level; and (c) prognosing the outcome of said cancer, wherein (i) a positive outcome is prognosed when the level of carboxymethylated PP2Ac is equal to or greater than said threshold value and/or reference level, and/or (ii) a negative outcome is prognosed when the level of carboxymethylated PP2Ac is lower than said threshold value and/or reference level.
49. The method of claim 48, wherein the positive outcome is survival of the patient, preferably recurrence free survival, for at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20 years, preferably at least 5 or 6 years, more preferably at least 10 years; and/or wherein the negative outcome is death and/or recurrence of the cancer in less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20 years, e.g. less than about 10 years, or less than about 5 or about 6 years.
50. An in vitro method for diagnosing whether a cancer is metastatic or prone to metastasize, wherein said method comprises the steps of (a) determining the level of carboxymethylated PP2Ac in a sample from said patient by contacting said sample with an anti-carboxymethylated PP2Ac-specific antibody according to any one of claims 1 to 37; (b) comparing the level of carboxymethylated PP2Ac to a threshold value and/or reference level; and (c) diagnosing whether the cancer is metastatic or prone to metastasize, wherein (i) it is diagnosed that said cancer is not metastatic or prone to metastasize when the level of carboxymethylated PP2Ac is equal to or greater than said threshold value and/or reference level, and/or (ii) it is diagnosed that said cancer is metastatic or prone to metastasize when the level of carboxymethylated PP2Ac is lower than said threshold value and/or reference level.
51. The method of any one of claims 48 to 50, wherein said threshold value refers to an Allred Score of 2 determined by immunohistochemistry staining with said anti-carboxymethylated PP2Ac-specific antibody, in particular wherein (i) an Allred Score of equal to or higher than 2 means that some cells in the sample, e.g. at least 0.1%, 0.5% or 1%, preferably at least 0.5%, of the cells show some staining, e.g. a weak, intermediate or high staining, and/or (ii) an Allied Score of lower than 2 means that essentially no cells in the sample, e.g. less than 0.1%, preferably less than 0.01%, more preferably no cells (0%), show at most a weak staining, preferably no staining.
52. The method of any one of claims 48 to 51, wherein said reference level is determined by analyzing the level of carboxymethylated PP2Ac in samples from a plurality of reference patients diagnosed with a cancer by contacting said sample with an anti-carboxymethylated PP2Ac-specific antibody according to any one of claims 1 to 37, wherein it is known whether the outcome of the cancer of said reference patients has been positive or negative, in particular wherein the reference level allows to separate the reference patients with a positive outcome from the reference patients with a negative outcome in an optimal way, and/or in particular wherein the level of carboxymethylated PP2Ac in the samples from said reference patients is determined by the same measurement method that is employed in said step (a).
53. A method of detecting an abnormal level of carboxymethylated PP2Ac in a sample from a patient, wherein said method comprises (a) measuring the level of carboxymethylated PP2Ac in a sample from said patient by contacting said sample with an anti-carboxymethylated PP2Ac-specific antibody according to any one of claims 1 to 37; and (b) determining whether the sample is abnormal, wherein the sample is determined to be abnormal if the level of carboxymethylated PP2Ac is at least about 20% lower than the amount determined for a reference sample.
54. The method of claim 53, wherein the sample is determined to be abnormal if the level of carboxymethylated PP2Ac is at least about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%, preferably at least about 60%, more preferably at least about 80%, e.g. about 80%, lower than the amount determined for the reference sample.
55. The method of claim 53 or 54, wherein the method further comprises: (c) reporting to said patient whether said sample is determined to be abnormal or normal.
56. The method of any one of claims 53 to 55, wherein said patient is suffering from a cancer, preferably a prostate cancer.
57. The method of any one of claims 53 to 56, wherein the reference sample is derived from at least one patient not suffering from a disease selected from the group consisting of the diseases of the following (i) and/or (ii): (i) cancer, a neurodegenerative disorder, diabetes and/or a heart disease; (ii) a disease that is associated with and/or is caused by a deregulated signaling pathway, wherein said deregulation comprises the hyperphosphorylation or hypophosphorylation of at least one component of said signaling pathway, and wherein said component can be dephosphorylated by protein phosphatase 2A (PP2A).
58. The method of any one of claims 53 to 57, wherein the reference sample is derived from at least one patient not suffering from a cancer.
59. The method of any one of claims 53 to 56, wherein the reference sample is derived from at least one patient not suffering from a metastatic cancer, preferably a metastatic prostate cancer.
60. The method of any one of claims 55 to 59, wherein said patient having reported an abnormal level of carboxymethylated PP2Ac is also reported to expect death and/or recurrence of the cancer in less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20 years, e.g. in less than about 10 years, or less than about 5 or about 6 years.
61. The method of any one of claims 55 to 59, wherein said patient having reported a normal level of carboxymethylated PP2Ac is also reported to expect survival, preferably recurrence free survival, for at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20 years, preferably at least 5 or 6 years, more preferably at least 10 years.
62. The method of any of claims 48 to 61, wherein said patient is suspected of having a metastatic cancer or developing a metastatic cancer.
63. The method of any one of claims 48 to 62, wherein said sample is a cancer sample.
64. The method of any one of claims 48 to 52 or 56 to 63, wherein said cancer is associated with and/or caused by hyperphosphorylation of androgen receptor, c-MYC, ERK, AKT, S6K, β-catenin, and/or Bcl2, preferably androgen receptor.
65. An in vitro method for prognosing the responsiveness of a cancer in a patient to treatment with an antiandrogen, wherein said method comprises the steps of (a) determining the level of carboxymethylated PP2Ac in a sample from said patient by contacting said sample with an anti-carboxymethylated PP2Ac-specific antibody according to any one of claims 1 to 37; (b) comparing the level of carboxymethylated PP2Ac to a reference level; and (c) prognosing whether the cancer is responsive, wherein (i) it is prognosed that said cancer is responsive when the level of carboxymethylated PP2Ac is equal to or greater than said reference level, and/or (ii) it is prognosed that said cancer is not responsive when the level of carboxymethylated PP2Ac is lower than said reference level; in particular wherein a response corresponds to (i) elimination of the cancer, (ii) prevention and/or elimination of metastases, (iii) a reduction of the cancer volume by at least 30% (iv) survival of the cancer patient for at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20 years, preferably at least 5 years; and/or (v) a decline of at least one cancer tumor marker, e.g. prostate-specific antigen (PSA), by at least 50%.
66. The method of claim 65, wherein said reference level is determined by analyzing the level of carboxymethylated PP2Ac in samples from a plurality of reference patients diagnosed with a cancer by contacting said sample with an anti-carboxymethylated PP2Ac-specific antibody according to any one of claims 1 to 37, wherein it is known whether the cancer of said reference patients has been responsive to an antiandrogen treatment or not, in particular wherein the reference level allows to separate the reference patients with a responsive cancer from the reference patients with an unresponsive cancer in an optimal way, and/or in particular wherein the level of carboxymethylated PP2Ac in the samples from said reference patients is determined by the same measurement method that is employed in said step (a).
67. The method of claim 65 or 66, wherein the antiandrogen is an antagonist of androgen receptor signaling, preferably an androgen receptor antagonist, more preferably enzalutamide.
68. A method for prognosing the progression of a disease in a subject, wherein said disease is selected from the group consisting of the diseases of the following (i) and/or (ii): (i) cancer, a neurodegenerative disorder, diabetes and/or a heart disease; (ii) a disease that is associated with and/or is caused by a deregulated signaling pathway, wherein said deregulation comprises the hyperphosphorylation or hypophosphorylation of at least one component of said signaling pathway, and wherein said component can be dephosphorylated by protein phosphatase 2A (PP2A), wherein said method comprises the steps of (a) determining in vitro the level of carboxymethylated PP2Ac in a sample by contacting said sample with an anti-carboxymethylated PP2Ac-specific antibody of any one of claims 1 to 37, and (b) evaluating whether the disease progression is favorable or unfavorable, and indicating a favorable progression if said disease is associated with hyperphosphorylation of said signaling pathway component and the level of said carboxymethylated PP2Ac is elevated, or said disease is associated with hypophosphorylation of said signaling pathway component and the level of said carboxymethylated PP2Ac is reduced, and/or indicating an unfavorable progression if said disease is associated with hyperphosphorylation of said signaling pathway component and the level of said carboxymethylated PP2Ac is reduced, or said disease is associated with hypophosphorylation of said signaling pathway component and the level of said carboxymethylated PP2Ac is elevated.
69. The method of claim 68, wherein an elevated level of said carboxymethylated PP2Ac corresponds to an Allied Score of 2 or greater, and/or wherein a reduced level of said carboxymethylated PP2Ac corresponds to an Allred Score lower than 2, preferably as defined in claim 51.
70. A method for predicting the responsiveness of a subject suffering from a disease to the treatment with a PP2A modulator, wherein said disease is selected from the group consisting of the diseases of the following (i) and/or (ii): (i) cancer, a neurodegenerative disorder, diabetes and/or a heart disease; (ii) a disease that is associated with and/or is caused by a deregulated signaling pathway, wherein said deregulation comprises the hyperphosphorylation or hypophosphorylation of at least one component of said signaling pathway, and wherein said component can be dephosphorylated by protein phosphatase 2A (PP2A), wherein said method comprises the steps of (a) determining in vitro the level of carboxymethylated PP2Ac in a sample by contacting said sample with an anti-carboxymethylated PP2Ac-specific antibody of any one of claims 1 to 37, and (b) evaluating whether said subject will be responsive to the treatment with said PP2A modulator, and indicating that said subject will be responsive if said disease is associated with hyperphosphorylation of said signaling pathway component, said PP2A modulator activates PP2A, and the level of said carboxymethylated PP2Ac is reduced, or said disease is associated with hypophosphorylation of said signaling pathway component, said PP2A modulator inhibits PP2A, and the level of said carboxymethylated PP2Ac is elevated, and/or indicating that said subject will not be responsive if said disease is associated with hyperphosphorylation of said signaling pathway component, said PP2A modulator activates PP2A, and the level of said carboxymethylated PP2Ac is elevated, or said disease is associated with hypophosphorylation of said signaling pathway component, said PP2A modulator inhibits PP2A, and the level of said carboxymethylated PP2Ac is reduced.
71. A method for determining the responsiveness of a subject suffering from a disease to the treatment with a PP2A modulator, wherein said disease is selected from the group consisting of the diseases of the following (i) and/or (ii): (i) cancer, a neurodegenerative disorder, diabetes and/or a heart disease; (ii) a disease that is associated with and/or is caused by a deregulated signaling pathway, wherein said deregulation comprises the hyperphosphorylation or hypophosphorylation of at least one component of said signaling pathway, and wherein said component can be dephosphorylated by protein phosphatase 2A (PP2A), wherein said method comprises the steps of (a) determining in vitro the level of carboxymethylated PP2Ac in sample by contacting said sample with an anti-carboxymethylated PP2Ac-specific antibody of any one of claims 1 to 37, wherein said biological sample is derived from a tissue that has been contacted in said subject with said PP2A modulator, (b) determining that said subject responds to the treatment with said PP2A modulator, if the level of carboxymethylated PP2Ac in said sample is altered.
72. The method of any one of claims 68 to 71, wherein the sample is a sample from the subject to be assessed.
73. The method of any one of claims 48 to 72, wherein the patient or subject is a human.
74. The method of any one of claims 68 to 73, wherein the carboxymethylated PP2Ac level in said sample is compared to the carboxymethylated PP2Ac level in a reference/control sample, thereby determining if the carboxymethylated PP2Ac level is reduced or increased.
75. The method of any one of claims 70 to 74, wherein the PP2A modulator is a PP2A activator.
76. The method of claim 75, wherein the PP2A activator is a small molecule, preferably a modified phenothiazine and/or a small molecule derived from phenothiazine.
77. The method of claim 75 or 76, wherein the PP2A activator is DT-061 (CAS No.: 1809427-19-7 or CAS No.: 1809427-18-6) or forskolin (Colforsin; (3R,4aR,5S,6S,6aS,10S,10aR,10bS)-dodecahydro-5,6,10,10b-tetrahydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-3-vinyl-1H-naphtho[2,1-b]pyran-5-yl acetat), preferably DT-061.
78. The method of any one claims 68 to 77, wherein said deregulation comprises the hyperphosphorylation of said component of said signaling pathway.
79. The method of any one claims 68 to 78, wherein said signaling pathway comprises a tyrosine kinase receptor, androgen receptor, Bcl2, PI3K, AKT, S6K, MAP, ERK, β-catenin and/or c-MYC, preferably wherein the tyrosine kinase receptor is EGFR.
80. The method of any one of claims 68 to 79, wherein the component that can be dephosphorylated by PP2A is selected from, c-MYC, ERK, AKT, S6K, β-catenin, androgen receptor and Bcl2.
81. The method of any one of claims 68 to 80, wherein the disease is a cancer, a neurodegenerative disorder, diabetes and/or a heart disease, preferably cancer.
82. The method of any one of claims 68 to 81, wherein the disease is a cancer that is associated with and/or caused by a hyperactive Akt, S6K and/or ERK/MAP signaling pathway(s) and/or the hyperphosphorylation of at least one component of said signaling pathways(s), wherein said component can be dephosphorylated by PP2A.
83. The method of any one of claims 68 to 82, wherein the disease is a cancer that is associated with and/or caused by abnormal androgen receptor signaling and/or hyperphosphorylation of androgen receptor.
84. The method of any one of claims 48 to 83, wherein the biological sample is derived from a diseased and/or pathogenic tissue from said subject.
85. The method of any one of claims 48 to 84, wherein the cancer is prostate cancer, lung adenocarcinoma, or breast cancer, preferably prostate cancer.
86. The method of any one of claims 68 to 84, wherein disease, in particular the neurodegenerative disorder, is Alzheimer's disease.
87. A method for evaluating whether a test agent modulates PP2Ac carboxymethylation, wherein said method comprises the steps of (a) assessing effects of said test agent on PP2Ac carboxymethylation status in a PP2Ac methylation assay, wherein said assay contains PP2Ac and (i) a PP2Ac methylase enzyme and/or a PP2Ac demethylase enzyme, and/or (ii) an A and B subunit of PP2A, and wherein the PP2Ac carboxymethylation status is determined by an anti-carboxymethylated PP2Ac-specific antibody of any one of claims 1 to 37; and (b) determining based on the assessed effects that said test agent modulates PP2Ac carboxymethylation or that said test agent does not modulate PP2Ac carboxymethylation.
88. A method for identifying agents that modulate PP2Ac carboxymethylation, wherein said method comprises the steps of (a) providing a plurality of candidate test agents; (b) assessing effects of an individual candidate test agent on PP2Ac carboxymethylation status in a PP2Ac methylation assay, wherein said assay contains PP2Ac and (i) a PP2Ac methylase enzyme and/or a PP2Ac demethylase enzyme; and/or (ii) an A and B subunit of PP2A, and wherein the PP2Ac carboxymethylation status is determined by an anti-carboxymethylated PP2Ac-specific antibody of any one of claims 1 to 37; and (c) identifying, based on the assessed effects, at least one agent that modulates PP2Ac methylation.
89. The method of claim 87 or 88, wherein said effects on PP2Ac carboxymethylation status comprise an increase or reduction of PP2Ac carboxymethylation compared to a reference and/or control.
90. The method of any one of claims 87 to 89, wherein the PP2Ac methylation assay comprises a cell and/or cell lysate and/or is performed in a cell and/or cell lysate.
91. The method of any one of claims 87 to 90, wherein assessing the effects of a test agent on the PP2Ac carboxymethylation status comprises the steps of (b′) contacting the PP2Ac methylation assay with said test agent, and subsequently (b″) contacting said PP2Ac methylation assay with an anti-carboxymethylated PP2Ac-specific antibody of any one of claims 1 to 37, thereby determining the PP2Ac carboxymethylation status.
92. A method for evaluating whether a test agent modulates the activity of PP2A, wherein said method comprises the steps of (a) evaluating whether said test agent modulates PP2Ac carboxymethylation according to the evaluation method of any one claims 87 or 89 to 91, and (b) determining that said test agent modulates the activity of PP2A, if said test agent modulates PP2Ac carboxymethylation.
93. A method for identifying an agent that modulates the activity of PP2A, wherein said method comprises the steps of (a) identifying an agent that modulates PP2Ac carboxymethylation according to the screening method of any one claims 88 to 92, and (b) selecting said identified agent.
94. The method of claim 92 or 93, wherein the agent or test agent activates PP2A and/or enhances the activity of PP2A, if said agent or test agent increases PP2Ac carboxymethylation.
95. The method of claim 92 or 93, wherein the agent or test agent inhibits the activity of PP2A and/or inhibits the activation of PP2A, if said agent or test agent reduces PP2Ac carboxymethylation.
96. The method of claim 93 for identifying an agent that activates PP2A and/or enhances the activity of PP2A, wherein said method comprises the steps of (a) identifying an agent that increases PP2Ac carboxymethylation according to the screening method of any one claims 88 to 92, and (b) selecting said identified agent.
97. The method of claim 93 for identifying an agent that inhibits the activity of PP2A and/or inhibits the activation of PP2A, wherein said method comprises the steps of (a) identifying an agent that reduces PP2Ac carboxymethylation according to the screening method of any one claims 88 to 92, and (b) selecting said identified agent.
98. The method of any one of claims 87 to 97, wherein the agent or test agent is a PP2A activator, if said agent/or est agent (i) activates the PP2Ac methylase enzyme and/or enhances the activity of the PP2Ac methylase enzyme; and/or (ii) inhibits the activity of the PP2Ac demethylase enzyme and/or inhibits the activation of the PP2Ac demethylase enzyme.
99. The method of any one of claims 87 to 98, wherein the agent or test agent is a PP2A activator, if said agent or test agent increases the amount of the trimeric PP2A holoenzyme.
100. The method of any one of claims 87 to 99, wherein the agent or test agent is a PP2A inhibitor, if said agent or test agent (i) activates the PP2Ac demethylase enzyme and/or enhances the activity of the PP2Ac demethylase enzyme; and/or (ii) inhibits the activity of the PP2Ac methylase enzyme and/or inhibits the activation of the PP2Ac methylase enzyme.
101. The method of any one of claims 87 to 100, wherein the agent or test agent is a PP2A inhibitor, if said agent or test agent reduces the amount of the trimeric PP2A holoenzyme.
102. The method of any one of claims 87 to 101, wherein, if said agent or test agent is a PP2A activator, said agent or test agent is DT-061 (CAS No.: 1809427-19-7 or CAS No.: 1809427-18-6) or forskolin (Colforsin; (3R,4aR,5S,6S,6aS,10S,10aR,10bS)-dodecahydro-5,6,10,10b-tetrahydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-3-vinyl-1H-naphtho[2,1-b]pyran-5-yl acetat), preferably DT-061.
103. The screening method of any one claims 88 to 102 further comprising a step of obtaining the identified agent.
104. The method of any one of claims 87 to 102 or the screening method of claim 103, wherein said agent or test agent is a medicament and/or a pharmaceutical.
105. An PP2A activator obtained according to claim 103 for use in treating a disease selected from the group consisting of the diseases of the following (i) and/or (ii): (i) cancer, a neurodegenerative disorder, diabetes and/or a heart disease; (ii) a disease that is associated with and/or is caused by a deregulated signaling pathway, wherein said deregulation comprises the hyperphosphorylation of at least one component of said signaling pathway, and wherein said component can be dephosphorylated by protein phosphatase 2A (PP2A).
106. An PP2A inhibitor obtained according to claim 103 for use in chemotherapy and/or radiotherapy.
107. A method for identifying a PP2A modulator, wherein said method comprises the steps of (a) contacting a cell or cell lysate containing an A, B and C subunit of PP2A with a candidate PP2A modulator, (b) contacting said cell or cell lysate with an anti-carboxymethylated PP2Ac-specific antibody of any one of claims 1 to 37, thereby determining the level of carboxymethylated PP2Ac in said cell or cell lysate, and (c) selecting said candidate PP2A modulator if the level of carboxymethylated PP2Ac in said cell or cell lysate is altered.
108. A screening method for evaluating whether a molecule modulates the activity of PP2A, wherein said method comprises the steps of (a) contacting a cell or cell lysate containing an A, B and C subunit of PP2A with said molecule, (b) contacting said cell or cell lysate with an anti-carboxymethylated PP2Ac-specific antibody of any one of claims 1 to 37, thereby determining the level of carboxymethylated PP2Ac in said cell or cell lysate, and (c) indicating that said molecule modulates the activity of PP2A if the level of carboxymethylated PP2Ac in said cell or cell lysate is altered.
109. The method of claim 107 or 108, wherein said PP2A modulator activates PP2A and/or enhances the activity of PP2A, said modulation is the activation of PP2A and/or the enhancement of the PP2A activity, and said alteration is an elevated carboxymethylated PP2Ac level.
110. The method of any one of claims 107 to 109, wherein said PP2A modulator or activator is DT-061 (CAS No.: 1809427-19-7 or CAS No.: 1809427-18-6) or forskolin (Colforsin; (3R,4aR,5S,6S,6aS,10S,10aR,10bS)-dodecahydro-5,6,10,10b-tetrahydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-3-vinyl-1H-naphtho[2,1-b]pyran-5-yl acetat), preferably DT-061.
111. A method for screening for (a) medicament(s) and/or drug(s), said method comprising the step of evaluating the capacity of said medicament or drug to stabilize and/or increase the methylation status of PP2Ac and/or increase methylated PP2Ac levels, wherein said stabilization, increase and/or methylation level is to be detected with an anti-carboxymethylated PP2Ac-specific antibody of any one of claims 1 to 37.
112. The method of claim 111, wherein said stabilization, said methylation status and/or said methylation level of PP2Ac is to be determined in a cell or tissue or in a cell lysate.
113. The method of claim 111 or 112, wherein said stabilization, said methylation status and/or said methylation level of PP2Ac is to be determined in or on an in vitro cell, in or on an in vitro tissue or in a cell or tissue of a test animal or in or on an isolated human cell and/or tissue sample.
114. The method of any one of claims 111 to 113, wherein said medicament and/or drug to be screened is an activator of protein phosphatase 2A.
Description
BRIEF DESCRIPTION OF THE FIGURES
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[0617] Other aspects and advantages of the invention will be described in the following examples, which are given for purposes of illustration and not by way of limitation. Each publication, patent, patent application or other document cited in this application is hereby incorporated by reference in its entirety.
EXAMPLES
[0618] Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting.
Example 1. Generation of the α-Carboxymethylation Specific Monoclonal Antibody 7C10-C5
[0619] The single clone hybridoma cell line “7C10-C5” (mouse Balb/c origin) as described herein in the Examples producing the α-carboxymethylation specific monoclonal antibody 7C10-C5 as characterized herein in the Examples, i.e. in Examples 2 and 3, has been deposited at the International Depository Authority “Leipniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig” under the accession number “DSM ACC3350”. The Leipniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig, has received said “7C10-C5” cell line with the accession number “DSM ACC3350” on 2019-01-16.
[0620] Immunization of Mice
[0621] To generate an antibody that specifically recognizes the α-carboxymethylated C-terminal leucine at position 309 (Leu.sup.309) of the a and f3 isoforms of the PP2A catalytic subunit (PP2Ac) mice were immunized with a synthetic peptide spanning amino acid 304 to 309 of the carboxyl terminus of the human PP2Ac subunit (sequence identical between α and β isoforms), in which the α-carboxyl group of the C-terminal leucine was methylated.
[0622] To elicit an immune response towards the α-carboxymethylated peptide, the peptide was crosslinked to the carrier protein keyhole limpet hemocyanin (KLH) via cysteine which was linked to the PP2Ac peptide by the non-proteinogenic amino acid β-alanine.
[0623] The Immunogen
[0624] Specifically, a peptide of the sequence C(βA)TPDYFL (SEQ ID NO:48), of which the last six amino acids correspond to the C-terminal 6 amino acids of mammalian or yeast PP2A catalytic subunit (PP2Ac) and which contains a carboxy-terminal methyl-esterified leucine (L-O-CH3) was synthesized by standard peptide synthesis by PiChem GmbH (Graz, Austria) and coupled via the amino-terminal cysteine to maleimide activated keyhole limpet hemocyanine (KLH). As a result, an immunogen of the sequence KLH-Cys-βAla-Thr-Pro-Asp-Tyr-Phe-Leu-CH3 (SEQ ID NO: 48) was obtained (the C-terminal 6 amino acid sequence of the mammalian or yeast PP2Ac including the carboxy-terminal methyl-esterified leucine is shown in bold).
[0625] The short PP2Ac specific sequence of only 6 amino acids was used to minimize the chance of obtaining antibodies that recognize the PP2Ac C-terminus independently of the methylated C-terminal leucine. Of note, for the generation of an earlier antibody (clone 2A10) binding to α-carboxymethyl-PP2Ac but cross-reacting with α-carboxymethyl-PP4 and weakly with α-carboxymethyl-PP6 (Tables 4 and 5 and
[0626] Immunization
[0627] 50 μg of said KLH-coupled peptide at a concentration of 0.5 μg/μl in PBS was mixed with adjuvant. In particular, the aqueous antigen solution and the adjuvant oil were emulsified by repeated cycles of sucking-up and pushing-out the oil-water mixture through a 23G (0.6 mm diameter) needle until a stable emulsion was formed. Blood samples were collected from the tail veins of four female cByJ.RBF-Rb(8.12)5Bnr/J mice at the age of 10 weeks (“pre-immune sera”), incubated for 1 h at 37° C. and centrifuged for 5 min at 22° C. at 14,000 rpm in a Beckman & Coulter Microfuge 18. The cleared blood sera were collected, sodium azide was added to a final concentration of 0.02% w/v, and the sera were stored at 4° C. Immediately after the collection of blood, the mice were immunized with 200 μl of antigen-adjuvant emulsion per mouse which was injected subcutaneously at the abdomen. 14 days after the first immunization (day 15), the mice were boosted with 100 μl of said KLH-coupled peptide at a concentration of 0.5 μg/μl in PBS emulsified with 100 μl adjuvant per mouse which was injected subcutaneously at the abdomen. 35 days after the first immunization (day 36), the mice were boosted a second time with 100 μl of said KLH-coupled peptide at a concentration of 0.5 μg/μl in PBS emulsified with 100 μl adjuvant per mouse which injected subcutaneously at the abdomen. 13 days after the second boost (day 49), blood samples of all mice were taken (“immune sera”, 2.sup.nd bleed) from the tail veins, incubated for 1 h at 37° C. and centrifuged for 5 min at 22° C. at 14,000 rpm in a Beckman & Coulter Microfuge 18. 95 days after the first immunization (day 96), the mice were boosted a third time with 90 μl of said KLH-coupled peptide at a concentration of 0.5 μg/μl in PBS mixed with 90 μl adjuvant per mouse which was injected subcutaneously at the abdomen. 10 days after the third boost (day 106), blood samples of all mice were taken (“immune sera”, 3.sup.rd bleed) from the tail veins, incubated for 1 h at 37° C. and centrifuged for 5 min at 22° C. at 14,000 rpm in a Beckman & Coulter Microfuge 18.
[0628] 162 days after the first immunization (day 163), mouse 4 (used for the generation of hybridoma cells) received a final boost with 40 μg of KLH-coupled peptide in 100 μl of PBS injected intravenously into the tail vein. 88 hours post injection (day 167) the mouse was sacrificed by cervical dislocation and the spleen removed surgically.
[0629] Screening of Polyclonal Antisera and Hybridoma Clone Supernatants
[0630] To identify antibodies specifically binding the carboxymethylated catalytic subunit of protein phosphatase 2A (PP2Ac) but not non-carboxymethylated PP2Ac, lysates of different yeast strains having either high levels of carboxymethylated PP2Ac or lacking carboxymethylated PP2Ac were employed for screening.
[0631] Yeast Strains
[0632] Yeast strain BY4741 expressing HA-tagged yeast PP2Ac (PPH21) was used as a background. Of note, the 6 C-terminal amino acids of mammalian/human PP2Ac are identical to the yeast PP2A catalytic subunit PPH21. The BY4741 wildtype (WT) strain contained carboxymethylated PP2Ac and non-carboxymethylated PP2Ac. The BY4741 ppe1Δ strain contained high levels of carboxymethylated PP2Ac because the ppe1 gene was deleted. The BY4741 ppm1Δ strain lacked carboxymethylated PP2Ac because the ppm1 gene was deleted. Said yeast strains were further used for the characterization of monoclonal antibodies, i.e. 7C10-C5, as described further below. PPE1/ppe1: Phosphoprotein Phosphatase Methylesterase; PPM1/ppm1: Protein Phosphatase Methyltransferase; HA: hemagglutinin.
[0633] Preparation of Yeast Cell Lysates for Immunoblot Analysis
[0634] Yeast cells were grown at 30° C. to exponential growth phase in drop-out complete medium (2.3 g/l Bacto yeast nitrogen base [Difco, 233520], 20 mg/l adenine, 20 mg/l L-arginine, 15 mg/l L-tyrosine, 15 mg/l L-isoleucine, 25 mg/l L-phenylalanine, 50 mg/l L-glutamic acid, 50 mg/l L-aspartic acid, 100 mg/l L-threonine, 200 mg/l L-serine, 75 mg/l L-valine, 75 mg/l L-methionine, 90 mg/l L-lysine, 20 mg/l uracil, 30 mg/l L-histidine, 0.05 M ammoniumsulfate) lacking L-leucine and containing 2% w/v glucose. Yeast cells were collected by centrifugation at 3,500 rpm at 4° C. in a Beckmann GS-6R centrifuge, washed with ice-cold water and the optical density at 600 nm (0D.sup.600) was measured in a Hitachi U-2000 spectrophotometer. Yeast cells equivalent to 50 OD.sup.600 were resuspended in 600 μl of yeast IP buffer (50 mM Tris pH 7.6, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 0.015-0.105 Trypsin Inhibitor Units per ml [TIU/ml] aprotinin [Sigma, A-6279], Complete protease inhibitor cocktail [Roche, 11836145001]), 400 μl of glass beads (0.5 mm diameter, Roth, N030.1) were added, and the cells were lysed in a MP Fastprep-24 at 6 m/s for 40 sec. Lysates were cleared by two times centrifugation for 10 min at 14,000 rpm at 4° C. in a Beckman & Coulter Microfuge 18 centrifuge. The protein concentration was determined with the Protein Assay Dye Reagent (Biorad, 500-0006) and measurement of absorbance at 595 nm in a Hitachi U-2000 spectrophotometer. For SDS-PAGE and Western blotting protein loading buffer was added to a final concentration of 0.112 M dithiothreitol, 2.22% w/v SDS and 11.1% glycerol, and protein samples were denatured by incubation for 5 min at 95° C.
[0635] Characterization of Polyclonal Antisera
[0636] To discriminate between carboxymethyl-specific versus carboxymethyl-unspecific polyclonal PP2Ac antibodies, the polyclonal antisera of the immunized mice were screened by Western blot analysis (
[0637] Specifically, the cleared blood sera were collected, sodium azide was added to a final concentration of 0.02% w/v, and the sera were tested for the presence of mouse PP2Ac specific IgG antibodies by immunoblotting (Western blot analysis) against 10% SDS-PAGE separated NIH3T3 mouse fibroblast whole cell lysate (prepared as in Example 2), as well as yeast lysates from the BY4741 ppe1Δ strain or the BY4741 ppm1Δ strain. 85×73 mm SDS polyacrylamide gels with 1 mm thick preparative combs (Bio-Rad, 165-2928) were casted with Bio-Rad Mini PROTEAN II electrophoresis cell systems. Whole cell lysate equivalent to 400 μg of total protein was mixed with denaturing buffer to a final concentration of 0.11 mol/l Dithiothreitol (DTT), 2.2% sodium dodecyl sulfate (SDS) and 11% (v/v) glycerol. Proteins were denatured by incubation at 95° C. for 5 min and separated in 0.025 M Tris/0.2 M glycine/0.01% w/v SDS pH 8.5 running buffer at a constant voltage of 100 V at 22° C. Proteins were transferred to Protran BA 83 nitrocellulose membrane (Whatman, 10401396) in 0.025 M Tris/0.19 M glycine/20% methanol pH 8.5 transfer buffer for 3h at a constant current of 0.5 A at 4° C. in Hoefer TE-Series Transphor Electrophoresis Units (Pharmacia Biotech, TE42). Membranes were washed with deionized water and stained with Ponceau S (2.97 mmol/l Ponceau S [Serva, 33429], 0.184 mol/l trichloroacetic acid [AppliChem, A1431], 0.137 mol/l sulfosalicylic acid [Merck, 1.00691]) for 2 min. Excess dye was washed off with deionized water and membranes were stored dry between two 3MM paper sheets at 22° C. Prior to usage, membranes were rehydrated by incubation for 2 min at 22° C. in PBS+0.1% Tween-20 (PBS-T). Membranes were blocked by incubation for 1 h at 22° C. in PBS-T+3% w/v skim milk powder (Merck, 1.15363). Blots were incubated with pre-immune or immune sera diluted 1:500 in PBS-T+0.5% skim milk powder in a Miniblotter system 28 channels dual blot MN28 unit (Immunetics, 168830) over night at 4° C. Membranes were washed 3×5 min with PBS-T at 22° C. For detection of primary mouse antibodies, membranes were incubated with peroxidase-conjugated AffiniPure goat anti-mouse IgG Fcγ fragment specific (Jackson ImmunoResearch, 115 008) secondary antibody diluted 1:10,000 in PBS-T+0.5% skim milk powder for 1 h at 22° C. Membranes were washed three times 10 min with PBS-T at 22° C. and bound antibodies were visualized by enhanced chemoluminescence with ECL Western Blotting Detection Reagents (GE Healthcare, RPN2106) and exposure of Fuji Medical X-ray films (FUJIFILM Corporation, Super HR-E, 47410 08471).
[0638] All four mice showed a robust immune response against mammalian (NIH3T3 mouse fibroblast) as well as yeast PP2A catalytic subunit (yeast strains). However, the polyclonal antisera of the immunized mice detected the PP2Ac subunit independent of its carboxymethylation state (
[0639] Generation of Hybridoma Mix Clones
[0640] First, splenocytes were fused with mouse myeloma cells to generate hybridoma cells. Specifically, the spleen of mouse 4 was placed in 10 ml of 37° C. warm Dulbecco's Modified Eagle's medium (DMEM; Sigma, D5671), cut in small pieces with a sterile pair of scissors and grinded between two sterile frosted microscope slides (Menzel Glaser Superfrost Plus, Thermo Scientific, J1800AMNZ) until no macroscopic pieces of splenic tissue were visible. The cell suspension was filtered through a 100 μm nylon cell strainer (BD Falcon, Ref. 352360) and the filter was washed two times with 10 ml of 37° C. warm DMEM. Cells were centrifuged for 5 min at 1200 rpm in a Heraeus Megafuge 1.0 at 22° C., resuspended in 3 ml of ice-cold red blood cell lysis buffer (Sigma, R7757) and incubated for 90 sec. The cell suspension was filled up to 30 ml with 37° C. warm DMEM and centrifuged for 5 min at 1200 rpm in Heraeus Megafuge 1.0 at 22° C. The splenocytes were counted with a 0.0025 mm.sup.2 glass counting chamber (0,100 mm depth; Bikker, Labor Optik). X63-Ag8.653 mouse myeloma cells were grown at 37° C. in a 5% CO2 atmosphere on Vents Nunclon TC 140/20 petri dishes (Nunc, 168381) for a minimum of 3 passages after thawing in DMEM+10% fetal bovine serum (Sigma, F7524)+2 mM Glutamax (Gibco, 35050-038)+100 units/ml Penicillin/0.1 mg/ml Streptomycin (Sigma, P4333)+1 mM sodium pyruvate (Sigma, S8636). X63-Ag8.653 cells were harvested by rinsing off the petri dish, centrifuged for 5 min at 1200 rpm in a Heraeus Megafuge 1.0 at 22° C., resuspended in 30 ml of 37° C. warm DMEM, counted with a 0.0025 mm.sup.2 glass counting chamber (0,100 mm depth; Burker, Labor Optik) and centrifuged again for 5 min at 1200 rpm in a Heraeus Megafuge 1.0 at 22° C. Splenocytes and myeloma cells were mixed at a ratio of 2.5:1, centrifuged for 5 min at 1200 rpm and fused by resuspending and incubating for 90 sec at 37° C. the cells in 1 ml of polyethylenglycol (PEG) 1450 (50% w/v solution in PBS; Sigma, P7181). After 90 sec, the cell suspension was diluted stepwise with 1 ml of 37° C. warm DMEM, followed by 5 ml of 37° C. warm DMEM and followed again by 10 ml of 37° C. warm DMEM and was then incubated at 37° C. for 5 min. Cells were centrifuged for 5 min at 1200 rpm in a Heraeus Megafuge 1.0 at 22° C. and were resuspended in DMEM+10% HyClone Fetal Clone I (Thermo Scientific, SH30080.03)+2 mM Glutamax+100 units/ml Penicillin/0.1 mg/ml Streptomycin+1 mM sodium pyruvate+5% BM Condimed H1 Hybridoma Cloning Supplement (Roche, 11088947001)+0.1 mM hypoxanthine/0.4 μM aminopterin/16 μM thymidine (provided as HAT 50× stock; Life Technologies, 21060-017). 10.sup.5 cells per well were seeded onto 96-well petri-dishes (TC Microwell 96F, Nunc, 167000). Cells were grown for 7 days at 37° C. in a 5% CO.sub.2 atmosphere and the supernatants were tested for the presence of carboxymethyl PP2Ac specific IgG antibodies by immunoblotting and ELISA of above-described yeast cell lysates (
[0641] Specifically, the supernatants were first tested by immunoblotting against SDS-PAGE separated yeast BY4741 wildtype strain lysate to determine the presence of PP2Ac specific IgG antibodies. Secondly, positive hybridoma supernatants were tested similarly against SDS-PAGE separated lysates from the BY4741 ppe1Δ strain or the BY4741 ppm1Δ strain to determine the presence of antibodies which are specific for carboxymethylated PP2Ac (
[0642] In parallel to the immunoblotting screening, hybridoma supernatants were tested for the presence of TPDYFL-CH3 (SEQ ID NO:1) specific IgG antibodies by ELISA. Specifically, flat-bottom Nunc-Immuno Medisorp 96-well ELISA plates (Thermo; 467320) were coated over night at 4° C. with 50 μl of either a peptide of the sequence C(βA)TPDYFL (SEQ ID NO:48) at a concentration of 3 μg/ml in 10 mM sodium phosphate buffer pH 7.0 or with a peptide of the sequence C(βA)TPDYFL-CH.sub.3 (SEQ ID NO:48) at a concentration of 3 μg/ml in 10 mM sodium phosphate buffer pH 7.0. The wells were washed once with PBS, blocked with 180 μl of 2% bovine serum albumin (Sigma, A9647) in PBS for 1 hour at RT and washed again once with PBS. The wells were incubated with 50 μl of undiluted splenic fusion hybridoma supernatants for 2 hours at RT, washed 3 times with PBS, incubated for 1 hour at RT with 50 μl peroxidase-conjugated AffiniPure goat anti-mouse IgG Fcγ fragment specific (Jackson ImmunoResearch, 115-035-008) secondary antibody diluted 1:10,000 in PBS and washed 3 times with PBS. Bound antibodies were detected colorimetrically by addition of 50 μl of 33 μg/ml 3′,5′,5′,5′-tetramethylbenzidine (TMB) (Sigma, T2885) in 0.1 M sodium acetate pH 6.0 and 0.01% H.sub.2O.sub.2 (Sigma, H1009). The colorimetric reaction was stopped after 28 min by addition of 50 μl 0.5 M H.sub.2SO.sub.4, and absorbance was measured at 450 nm with a Perkin Elmer Wallac Victor 2 1420 Multilabel Counter.
[0643] It was found by Western blotting that the supernatant of hybridoma mix clones 7G8, 7C10, 8F1 and 3A4 detected PP2Ac only in the BY4741 ppe1Δ strain that contains high levels of carboxymethylated PP2Ac subunit but not in the BY4741 ppm1Δ strain that lacks carboxymethylated PP2Ac (
[0644] Furthermore, it was found by ELISA that supernatants of hybridoma mix clones 5A4, 5A5, 5H8, 6F12, 6H11, 7C10, 7G1, 7G8 and 8F1 detected the carboxymethylated peptide (C(βA)TPDYFL-CH.sub.3) (SEQ ID NO:48) but did not detect the non-carboxymethylated peptide (C(βA)TPDYFL) (SEQ ID NO:48).
[0645] Those results indicated that mix clones 7G8, 7C10, 8F1, 3A4, 5A4, 5A5, 5H8, 6F12, 6H11 and 7G1 were specific for the carboxymethylated PP2Ac. Since the specificity of mix clone 7C10 was determined by Western blotting and ELISA, 7C10 was selected for further monoclonalization.
[0646] Generation of the Single Clone 7C10-C5
[0647] Mix clone 7C10 was monoclonalized and single clone 7C10-C5 was isolated. Specifically, hybridoma cells growing in tissue culture 96-well containing supernatant that was tested positive for the presence of antibodies specific for carboxymethylated PP2Ac were resuspended in DMEM+10% HyClone Fetal Clone I (Thermo Scientific, SH30080.03)+2 mM Glutamax +100 units/ml Penicillin/0.1 mg/ml Streptomycin+1 mM sodium pyruvate+5% BM Condimed H1 Hybridoma Cloning Supplement (Roche, 11088947001)+0.1 mM hypoxanthine/0.4 μM aminopterin/16 μM thymidine (referred to as “Hybridoma growth medium”) and counted with a 0.0025 mm.sup.2 glass counting chamber (0,100 mm depth; Bürker, Labor Optik). The appropriate volume of cell suspension was diluted in 30 ml of Hybridoma growth medium to yield a concentration of 1 cell in 200 μl of Hybridoma growth medium, and 300 μl of cell suspension per well were seeded onto 96-well petri-dishes (TC Microwell 96F, Nunc, 167000). Cells were grown for 7 days at 37° C. in a 5% CO.sub.2 atmosphere and the supernatants were tested for the presence of anti-carboxymethylated PP2Ac subunit IgG antibodies by immunoblotting against lysates from the BY4741 ppe1Δ strain as described above for the screening of the hybridoma supernatants. Wells containing supernatant that was tested positive for the presence of anti-carboxymethylated PP2Ac antibodies were further examined under the microscope for the number of hybridoma clones growing. One well with a single clone growing was selected for expansion and further propagation to obtain the 7C10-C5 monoclonal antibody.
Example 2. Characterization of the Binding Specificity of the 7C10-C5 Monoclonal Antibody
[0648] Confirmation of the Carboxymethylation Specificity of 7C10-C5
[0649] 7C10-C5 Western Blotting with Yeast Strain Lysates
[0650] The carboxymethyl-PP2Ac specificity of the 7C10-C5 monoclonal antibody was confirmed by Western blot analysis of lysates from the wildtype BY4741 strain, the BY4741 ppe1Δ strain and the BY4741 ppm1Δ strain. Furthermore, lysates from the BY4741 ppe1Δ strain were analyzed in which the methylation of the C-terminal carboxyl group of PP2Ac was chemically removed (hydrolyzed) by NaOH treatment (
[0651] Specifically, in one assay 50 μl of protein lysate corresponding to 180 μg of protein from the BY4741 ppe1Δ strain was incubated with 2 μl of 2 M NaOH, resulting in a pH ˜9-10 as determined with pH-indicator paper pH 1-14 (Merck, 1.10232.0001) for either 5 min or 15 min on ice. Lysates were then neutralized by addition of 4 μl 1 M HCl plus 14 μl 1 M Tris pH 6.8 to pH ˜7 as determined with pH-indicator paper pH 1-14 and boiled with Laemmli buffer. In a further assay, said protein lysate was incubated with 6 μl of 2M NaOH, resulting in a pH >11 as determined with pH-indicator paper pH 1-14 for either 5 min or 15 min on ice. Lysates were then neutralized by addition of 12 μl 1 M HCl plus 36 μl 1 M Tris pH 6.8 to pH ˜7 as determined with pH-indicator paper pH 1-14 and boiled with Laemmli buffer.
[0652] In particular, it was confirmed that the 7C10-C5 antibody only detected carboxymethylated PP2Ac (ppe1Δ strain) but not non-carboxymethylated PP2Ac (ppm1Δ strain; ppe1Δ strain +NaOH, 6 μl;
[0653] 7C10-C5 ELISA with Peptides
[0654] 7C10-C5 was tested for its specificity to the unmodified, methylated or amidated carboxyl group of Leu.sup.309 at the carboxy-terminus of PP2Ac by ELISA with 3 different undecapeptides (11-mers) (
[0655] Specifically, flat-bottom Nunc-Immuno Medisorp 96-well ELISA plates (Thermo; 467320) were coated o/n at 4° C. with 50 μl of either a peptide of the sequence ac-HVTRRTPDYFL-OH (L309; unmodified) (SEQ ID NO:18) at a concentration of 2 μg/ml, a peptide of the sequence ac-HVTRRTPDYFL-CH.sub.3 (meL309; methylated) (SEQ ID NO:18) at a concentration of 2 μg/ml in TBS (137 mM NaCl (AppliChem, #131659, 2.7 mM KCl (AppliChem, #131494), 24.8 mM Tris (AppliChem, #A1086), pH 7.4 with HCl) or a peptide of the sequence ac-HVTRRTPDYFL-NH.sub.2 (amL309; amidated) (SEQ ID NO:18) at a concentration of 2 μg/ml in TBS. Of note, the “ac-” in the peptides described here and in other Examples refers to an acetylated N-terminus. Said acetylation neutralizes the positive charge at the N-terminus, and the free charged N-terminus is thereby removed.
[0656] Then, the wells were washed once with TBS, blocked with 200 μl of 2% bovine serum albumin (BSA) (Sigma, A9647) in TBS for 1 hour at RT and washed again once with TBS. The wells were incubated with 50 μl of hybridoma supernatants 7C10-C5, 1:50 dilution in of 1% BSA in TBS for 1 hour at RT, washed 3 times with TBS, incubated for 1 hour at RT with 50 μl peroxidase-conjugated AffiniPure goat anti-mouse IgG Fcγ fragment specific (Jackson ImmunoResearch, 115-035-008) secondary antibody diluted 1:10,000 in TBS and washed 3 times with TBS. Bound antibodies were detected colorimetrically by addition of 50 μl of 33 μg/ml 3′,5′,5′,5′-tetramethylbenzidine (TMB) (Sigma, T2885) in 0.1 M sodium acetate pH 6.0 and 0.01% H.sub.2O.sub.2 (Sigma, H1009). The colorimetric reaction was stopped after 10 min by addition of 50 μl 0.5 M H.sub.2SO.sub.4, and measured at 450 nm and 560 nm with a Perkin Elmer VICTOR® Nivo™ microplate reader. The results from 560 nm read were subtracted from the 450 nm read.
[0657] It was found that the 7C10-C5 antibody detects the corresponding α-carboxymethylated PP2Ac peptide with 26-fold higher signal intensity than the non-methylated peptide. Furthermore, the peptide with the methylated α-carboxyl-group was recognized with 6-fold higher signal intensity than the amidated peptide (
[0658] Preparation of Mammalian Cell Lysates for Immunoblot Analysis
[0659] NIH3T3 mouse fibroblasts and human embryonic kidney cells (HEK293Trex) were grown in DMEM+10% FCS+2 mM L-glutamine (Sigma, G2150)+100 units/ml Penicillin/0.1 mg/ml Streptomycin at 37° C. in a 7.5% CO.sub.2 atmosphere. HAP1 wild type and HAP1 Lcmt-1-KO cells (Leucine carboxyl methyltransferase 1 deleted by CRISPR/Cas9; Horizon Discovery, #C631 and #HZGHC004373c001) were grown in Iscove's Modified Dulbecco's Medium (IMDM, Thermo Fischer Scientific, Life Technologies #12440053) supplemented with 10% (v/v) Fetal Calf Serum (FCS) (Sigma #F7524, lot 104M3333), GlutaMAX™ (Thermo Fischer Scientific, Life Technologies #35050-38, lot 1895829), and Penicillin-Streptomycin solution (Sigma, #P4333, lot 125M4781V) in a 5% CO.sub.2 atmosphere. Of note, HAP1 is a near-haploid human cell line that was derived from the male chronic myelogenous leukemia (CML) cell line KBM-7 (Carette (2011), Nature 477(7364):340-3). For cell lysis, exponentially growing cells were washed once with ice-cold PBS, once with ice-cold IP-Wash buffer (20 mmol/l Tris pH 8.0, 135 mmol/l NaCl, 10% glycerol) and lysed on the petri-dish in ice-cold IP-Lyse buffer (18 mmol/l Tris pH 8.0, 122 mmol/l NaCl, 9% glycerol, 1% w/v NP-40) for 20 min at 4° C. rocking. Lysed cells were transferred to Eppendorf 1.5 ml tubes and centrifuged for 15 min at 14,000 rpm at 4° C. in a Beckman & Coulter Microfuge 18 centrifuge. The protein concentration was determined with the Protein Assay Dye Reagent (Biorad, 500-0006) and measurement of absorbance at 595 nm in a Hitachi U-2000 spectrophotometer. For SDS-PAGE and Western blotting, protein loading buffer was added to a final concentration of 0.112 M dithiothreitol, 2.22% w/v SDS and 11.1% glycerol, and protein samples were denatured by incubation for 5 min at 95° C.
[0660] 7C10-C5 Western Blotting with Mammalian Cells
[0661] To confirm the ELISA results with methylated and non-methylated full-length PP2Ac, the carboxy-terminal methyl group was chemically removed from cellular PP2Ac by treating lysates of two different human cell lines, HAP1, and HEK293Trex (HEK) with NaOH as described in Favre (1994), J Biol Chem 269, 16311-16317. In brief, 100 μl of HAP1 wild type or HEK293T cell lysates corresponding to 200 μg of protein was mixed with 1M NaOH to a final concentration of 0.2M and incubated for 10 min at RT. The reaction was neutralized by adding HCL to a final concentration of 0.2M and diluted to 200 μl with IP Lyse. The control reaction was treated with preneutralization solution (0.2M NaOH and 0.2M HCL) for 10 min at room temperature and diluted to 200 μl with IP Lyse. For immunoblot analysis, protein loading buffer was added to the protein samples and proteins were denatured by incubation at 95° C. for 5 min.
[0662] The methyl-PP2Ac specific antibody 7C10-C5 only detected methylated PP2Ac in the untreated cell lysates but not in the NaOH treated lysates, whereas actin and total PP2Ac levels (detected by the anti-total PP2Ac antibody H8) remained unchanged by the NaOH treatment (
[0663] It has been reported previously that the deletion of the PP2A methyltransferase LCMT-1 in mouse embryonic fibroblasts decreases the carboxymethylation of PP2Ac by >95% (Hwang (2016), J Biol Chem 291, 21008-21019). In agreement with the results of the NaOH experiments, no carboxymethylated PP2Ac specific signals were detected with the 7C10-C5 antibody in HAP1 cells that lack the PP2A methyltransferase LCMT-1 (Lcmt-1-KO), further confirming the specificity of the 7C10-C5 antibody for carboxymethylated PP2Ac (
[0664] No Impairment of 7C10-C5 Binding Specificity by Co-Cocurrent Phosphorylation of Tyrosine 307 or Threonine 304 of PP2Ac
[0665] Additional post-translational modifications to the C-terminus of PP2Ac include two reported phosphorylation sites at threonine 304 (Thr.sup.304) and tyrosine 307 (Tyr.sup.307). To test whether the carboxymethyl-specific binding of 7C10-C5 is affected by these modifications, ELISAs were performed with 6 different undecapeptides (11-mers). (
[0666] To further determine if the binding of the 7C10-C5 antibody depends on the peptide concentration, ELISAs were performed with 4 different undecapeptides. Specifically, flat-bottom Nunc-Immuno Medisorp 96-well ELISA plates (Thermo; 467320) were coated o/n at 4° C. with 50 μl of either a peptide of the sequence ac-HVTRRTPDYFL-OH (L309) (SEQ ID NO:18) at a concentration of 8, 2, 0.5, 0.125, 0.03125 or 0.0078125 μg/ml in TBS (137 mM NaCl (AppliChem, #131659, 2.7 mM KCl (AppliChem, #131494), 24.8 mM Tris (AppliChem, #A1086), pH 7.4 with HCl), a peptide of the sequence ac-HVTRRTPDYFL-CH.sub.3 (meL309) (SEQ ID NO:18) at a concentration of 8, 2, 0.5, 0.125, 0.03125 or 0.0078125 μg/ml in TBS, with a peptide of the sequence ac-HVTRRpTPDYFL-CH.sub.3 (pT304-meL309) (SEQ ID NO:116) at a concentration of 8, 2, 0.5, 0.125, 0.03125 or 0.0078125 μg/ml in TBS or with a peptide of the sequence ac-HVTRRTPDpYFL-CH.sub.3 (pY307-meL309) (SEQ ID NO:117) at a concentration of 8, 2, 0.5, 0.125, 0.03125 or 0.0078125 μg/ml in TBS (
[0667] The wells were washed once with TBS, blocked with 200 μl of 2% bovine serum albumin (BSA) (Sigma, A9647) in TBS for 1 hour at RT and washed again once with TBS. The wells were incubated with 50 μl of 7C10-C5 hybridoma supernatant 1:50 diluted in 1% BSA in TBS for 1 hour at RT, washed 3 times with TBS, incubated for 1 hour at RT with 50 μl peroxidase-conjugated AffiniPure goat anti-mouse IgG Fcγ fragment specific (Jackson ImmunoResearch, 115-035-008) secondary antibody diluted 1:10,000 in TBS and washed 3 times with TBS. Bound antibodies were detected colorimetrically by addition of 50 μl of 33 μg/ml 3′,5′,5′,5′-tetramethylbenzidine (TMB) (Sigma, T2885) in 0.1 M sodium acetate pH 6.0 and 0.01% H.sub.2O.sub.2 (Sigma, H1009). The colorimetric reaction was stopped after 10 min by addition of 50 μl 0.5 M H.sub.2SO.sub.4, and absorbance was measured at 450 nm and 560 nm with a Perkin Elmer VICTOR® Nivo™ microplate reader. The results from 560 nm read were subtracted from the 450 nm read.
[0668] It was found that the 7C10-C5 antibody detects the α-carboxymethylated PP2Ac peptide with 1.6-fold higher signal intensity, when Thr.sup.304 is phosphorylated. (
[0669] No Cross-Reactivity of 7C10-C5 with PP4c or PP6c
[0670] It was found that the monoclonal antibody 7C10-C5 detects α-carboxymethylated PP2Ac with at least 10-fold higher signal intensity than α-carboxymethylated PP4c or α-carboxymethylated PP6c. In particular, the monoclonal antibody 7C10-C5 detected α-carboxymethylated PP2Ac with at least 16-fold higher signal intensity than α-carboxymethylated PP6c.
[0671] 7C10-C5 Western Blot with Mammalian Cells
[0672] The catalytic subunits of protein phosphatase 4 (PP4c) and 6 (PP6c) are closely related to PP2Ac with approximately 50% sequence identity. PP2Ac, PP4c and PP6c share the C-terminal DYFL or YFL motif, respectively (
[0673] Specifically, 500 μl corresponding to 1 mg of whole cell protein lysates of NIH3T3 mouse fibroblasts were incubated with anti-hemagglutinin (HA) antibody (clone 12CA5) crosslinked to BSA-coated protein A-Sepharose beads (GE-Healthcare) to immunoprecipitate HA-epitope tagged PP2Ac, PP4c or PP6c for 1 h. The Immune complexes were washed once with 1 ml with IP-Lyse, and 3 times with 1 ml with Tris-buffered saline (25 mM Tris, 135 mM NaCl, 2.6 mM KCL pH=7.4 with HCl). For immunoblot analysis protein loading buffer was added to the immunoprecipitate and proteins were denatured by incubation at 95° C. for 5 min. IP-Lyse and protein loading buffers were as described above for the preparation of mammalian cell lysates for immunoblot analysis.
[0674] It was found that 7C10-C5 did not detect PP4c or PP6c, and thus is specific for carboxymethylated PP2Ac, whereas clone 2A10 cross-reacted with PP4c or PP2Ac. Quantification of the signal strength revealed that 2A10 bound PP4ac with about 25% of the strength compared to PP2Ac, and PP6c with about 10% of the strength compared to PP2Ac (
[0675] Since no discernable PP4c or PP6c signal was obtained with the 7C10-C5 antibody, the data suggest that 7C10-C5 might bind carboxymethylated PP2Ac 100-fold or even 500-fold stronger than carboxymethylated PP4c or PP6c. Consistent with what we observed for the PP2Ac, both antibodies recognized their targets in a carboxymethylation dependent manner because neither 7C10-C5 nor 2A10 detected PP2Ac or PP4Ac when the carboxymethylation was chemically removed by NaOH treatment (
[0676] 7C10-C5 ELISA with Peptides
[0677] To further characterize the carboxymethyl-PP2Ac specificity of monoclonal antibody 7C10-C5 ELISAs were performed on carboxymethylated versus non-methylated PP2Ac, PP4c and PP6c C-terminal undecapeptides.
[0678] Specifically, flat-bottom Nunc-Immuno Medisorp 96-well ELISA plates (Thermo; 467320) were coated o/n at 4° C. with 50 μl of either a peptide of the sequence ac-HVTRRTPDYFL-OH (L309) (SEQ ID NO:18) at a concentration of 2 μg/ml in TBS (137 mM NaCl (AppliChem, #131659, 2.7 mM KCl (AppliChem, #131494), 24.8 mM Tris (AppliChem, #A1086), pH 7.4 with HCl), a peptide of the sequence ac-HVTRRTPDYFL-CH.sub.3 (meL309) (SEQ ID NO:18) 2 μg/ml in TBS, with a peptide of the sequence (ac-PSKKPVADYFL (L307) (SEQ ID NO:20) at a concentration of 2 μg/ml in TBS or with a peptide of the sequence ac-PSKKPVADYFL-CH3 (meL307) (SEQ ID NO:20) at a concentration of 2 μg/ml in TBS, with a peptide of the sequence ac-IPPRTTTPYFL (L305) (SEQ ID NO:22) at a concentration of 2 μg/ml in TBS or with a peptide of the sequence ac-IPPRTTTPYFL-CH.sub.3 (meL305) (SEQ ID NO:22) at a concentration of 2 μg/ml in TBS. The wells were washed once with TBS, blocked with 200 μl of 2% bovine serum albumin (BSA) (Sigma, A9647) in TBS for 1 hour at RT and washed again once with TBS. The wells were incubated with 50 μl of hybridoma supernatant 7C10-C5, 1:50 diluted in TBS with 1% BSA for 1 hour at RT, washed 3 times with TBS, incubated for 1 hour at RT with 50 μl peroxidase-conjugated AffiniPure goat anti-mouse IgG Fcγ fragment specific (Jackson ImmunoResearch, 115-035-008) secondary antibody diluted 1:10,000 in TBS and washed 3 times with TBS. Bound antibodies were detected colorimetrically by addition of 50 μl of 33 μg/ml 3′,5′,5′,5′-tetramethylbenzidine (TMB) (Sigma, T2885) in 0.1 M sodium acetate pH 6.0 and 0.01% H.sub.2O.sub.2 (Sigma, H1009). The colorimetric reaction was stopped after 10 min by addition of 50 μl 0.5 M H.sub.2SO.sub.4, and absorbance was measured at 450 nm and 560 nm with a Perkin Elmer VICTOR® Nivo™ microplate reader. The results from 560 nm read were subtracted from the 450 nm read.
[0679] These experiments further confirmed the high specificity of 7C10-C5 for carboxymethylated PP2Ac (
Example 3. Structural Characterization of the 7C10-C5 Monoclonal Antibody
[0680] Isotyping of the 7C10-C5 Monoclonal Antibody
[0681] The heavy chain and light chain isotypes of clone 7C10-C5 were determined with the ImmunoPure Monoclonal Antibody Isotyping Kit II (Pierce, 37502) by following the protocol for antigen-independent procedure for isotype determination as instructed by the manufacturer's manual on flat-bottom Nunc-Immuno Medisorp 96-well ELISA plates (Thermo; 467320). Instead of the alkaline phosphatase-conjugated goat-anti-rabbit IgG secondary antibody provided in the Isotyping kit, peroxidase-conjugated AffiniPure goat anti-rabbit IgG Fc fragment specific (Jackson ImmunoResearch, 111-035-008) secondary antibody diluted 1:10,000 was used. Instead of the PNPP substrate solution provided in the Isotyping kit, 50 μl of 33 μg/ml TMB (Sigma, T2885) in 0.1 M sodium acetate pH 6.0 and 0.01% 14202 (Sigma, H1009) was used for the colorimetric detection. The colorimetric reaction was stopped by addition of 50 μl 0.5 M H.sub.2SO.sub.4, and absorbance was measured at 450 nm with a Perkin Elmer Wallac Victor 3 1420 Multilabel Counter.
[0682] It was determined that the isotype of the 7C10-C5 monoclonal antibody is IgG1.
[0683] Determination of the 7C10-C5 Heavy and Light Chain Variable Regions and Complementary Determining Regions (CDRs)
[0684] RNA was isolated from 5×10.sup.7 cells of the carboxymethyl-PP2Ac specific hybridoma cell line clone 7C10-C5 using the RNeasy Mini Kit (Qiagen) following the protocol as instructed by the manufacturer's manual. The purified RNA was resuspended in 50 μl RNase free water and 800 ng RNA was taken for cDNA synthesis using the Accu Script 1.sup.st Strand cDNA Synthesis Kit (Agilent) in a 20 μl reaction mix using random hexamers following the protocol as instructed by the manufacturer's manual. For cloning of the variable region of the antibody, the heavy and light chain variable regions were amplified by Polymerase chain reaction (PCR). Specifically, a forward primer mix binding to the V(D)J leader sequences of the heavy chain and a reverse primer binding to the constant region of the heavy chain were used to amplify the heavy chain variable region, and a forward primer mix binding to the V(D)J leader sequences of the light chain and a reverse primer binding to the constant region of the light chain were used to amplify the light chain variable region as demonstrated in von Boehmer (2016), Nat Protoc 11, 1908-1923. The used primers (ordered from SigmaAldrich-Merck) are shown in Table 1:
TABLE-US-00005 TABLE 1 Primer sets for the PCR of the heavy and light chain regions FORWARD HEAVY 5′.fwdarw.3′ FORWARD LIGHT 5′.fwdarw.3′ 1MFH_I AGGAACTGCAGGTGTCC (SEQ ID 1mFK_I RGTGCAGATTTTCAGCTTCCTGCT NO: 53) (SEQ ID NO: 65) 1MFH_II CAGCTACAGGTGTCCACTCC 1mFK_II TGGACATGAGGGCYCCTGCTCAGT (SEQ ID NO: 54) (SEQ ID NO: 66) 1MFH_III TGGCAGCARCAGCTACAGG (SEQ 1mFK_III CTSTGGTTGTCTGGTGTTGAYGGA ID NO: 55) (SEQ ID NO: 67) 1MFH_IV CTGCCTGGTGACATTCCCA (SEQ 1mFK_IV GTTGCTGCTGCTGTGGCTTACA (SEQ ID NO: 56) ID NO: 68) 1MFH_V CCAAGCTGTGTCCTGTC (SEQ ID 1mFK_V GTATCTGGTACCTGTGG (SEQ ID NO: 57) NO: 69) 1MFH_VI TTTTAAAAGGTGTCCAGKGT 1mFK_VI TGCCTGTTAGGCTGTTGGTGCT (SEQ (SEQ ID NO: 58) ID NO: 70) 1MFH_VII CCTGTCAGTAACTRCAGGTGTCC 1mFK_VII GCTCAGTTCCTTGGTCTCCTGTTGC (SEQ ID NO: 59) (SEQ ID NO: 71) IMFH_VIII TTTTAAAAGGGGTCCAGTGT 1mFK_VIII TGGGTGCTGCTGCTCTGGGT (SEQ ID (SEQ ID NO: 60) NO: 72) 1MFH_IX CGTTCCTGGTATCCTGTCT (SEQ 1mFK_IX CAGTTCCTGTTTCTGTTARTGCTCTGG IDNO: 61) (SEQ ID NO: 73) 1MFH_X ATGAAGTTGTGGYTRAACTGG 1mFK_X TGCTCTGGTTATATGGTGCTGATGGG (SEQ ID NO: 62) (SEQ ID NO: 74) 1MFH_XI TGTTGGGGCTKAAGTGGG (SEQ ID NO: 63) REVERSE HEAVY 5′.fwdarw.3′ REVERSE LIGHT 5′.fwdarw.3′ 1MRG AGAAGGTGTGCACACCGCTGGAC 1mRK ACTGAGGCACCTCCAGATGTT (SEQ (SEQ ID NO: 64) ID NO: 75)
[0685] The PCR consisted of 2 μl cDNA from the 200 Accu Script reaction volume, 0.60 forward primer mix (10 μM stock) and 0.40 reverse primer (10 μM stock), 100 2× OneTaq® Quick-Load 2× Master Mix (New England BioLabs NEB, #M0482) and 7 μl nuclease free water (Sigma-Aldrich #W4502). Reactions were done in a Biometra TRIO thermocycler starting with a DNA denaturation step at 95° C. for 5 minutes followed by 35 cycles of denaturation of the DNA at 94° C. for 30 sec., annealing at 52° C. for 30 sec., and elongation at 72° C. for 1 minutes. A 10 minutes 72° C. step completed the reaction. The PCR products were separated on a 1.5% TAE (25 mM Tris (AppliChem #A1086), 0.114% Glacial Acetic Acid (AppliChem #A3701), 0.1 mM EDTA pH 8.0 (AppliChem #2937) agarose gel (Sigma, A9539) and stained with ethidium bromide (Sigma #E1510). Agarose gel slices containing the PCR products corresponding to the light chain variable fragments (about 350 bp) and the heavy chain variable fragments (about 400 bp) were cut out with a sterile scalpel blade and purified using a Wizard® DNA Clean Up Kit (Promega, #A9282), following the protocol as instructed by the manufacturer's manual, and eluted with 50 μl nuclease free water (Sigma-Aldrich, #W4502). The purified DNA fragments were cloned into the pJet 1.2 cloning vector following the protocol of the CloneJET PCR Cloning Kit (Thermo, #K1231). 2.5 μl of the ligation mixture was used for bacterial transformation using heat shock competent bacteria. E. coli HB101 cells were grown in Leuria Broth (LB) medium (1% (wt/vol) Tryptone (AppliChem #A1553), 0.5% (wt/vol) Yeast extract (AppliChem #A1552), 0.5% (wt/vol) NaCl (AppliChem #131659) to the early exponential phase (0D600 0.3-0.4) followed by centrifugation at 1000 g for 10 minutes at 4° C. The pellet was then resuspended at one-tenth of its volume in ice cold transformation and storage buffer (1% (wt/vol) Trypton, 0.5% (wt/vol) Yeast extract, 0.5% (wt/vol) NaCl, 10% (wt/vol) PEG (Mw 3350), 5% (vol/vol) 1M MgCl (Merk #105833), 5% (vol/vol) DMSO (added after autoclavation), and pH 6.5 HCl), aliquoted, and frozen at −20° C. 100 μl of this heat shock competent E. coli were thawed on ice, the DNA was added and the cells were kept on ice for another 30 minutes. Bacteria were then heat shocked at 42° C. for 1 minute, put on ice again for 10 minutes, resuspended in 500 μl LB medium without antibiotics for recovery, incubated at 37° C. for 30 minutes, plated out on LB agar plates (LB medium inclusive 1.5% Agar (AppliChem #A0949) with 0.1 mg/ml Ampicillin (GERBU, #1046) and stored over night at 37° C. Individual bacterial colonies were screened for inserted light or heavy chain DNA fragments by colony PCR as described in the CloneJET PCR Cloning Kit (Thermo, #K1231) with the enclosed pJet primers. Positive clones were subsequently inoculated in 5 ml LB with Ampicillin and grown over night at 37° C. Plasmid DNA was prepared with the Qiagen Miniprep Kit (Qiagen, #27106) as instructed by the manufacturer's manual, and after purification eluted with 50 μl nuclease free water (Sigma-Aldrich, #W4502). The DNA was prepared for sequencing as demanded by the sequencing company (LGC genomics). 10 μl with 100 ng/μ1 DNA was mixed with 4 μl primer (10 μM) using either the pJET1.2 forward sequencing primer (5′-CGACTCACTATAGGGAGAGCGGC-3′) (SEQ ID NO:76) or the pJET1.2 reverse sequencing primer (5′-AAGAACATCGATTTTCCATGGCAG-3′) (SEQ ID NO:77) to get sequences from both sides. The obtained sequences for the heavy and light variable fragments were checked with the V-Quest tool from the IMGT homepage (www.imgt.org; Brochet (2008), Nucleic Acids Res 36, W503-508; Giudicelli (2011), Cold Spring Harb Protoc 2011, 695-715).
[0686] Heavy and light chain variable regions were again cloned as a single cell variable fragment (scFv) as described below in Example 4. The sequences of the heavy and light chain variable regions of the 7C10-C5 were determined as described in Example 3 and Example 4. Although the sequence determined according to Example 3 could have also been used, the sequence of the scFv plasmid described in Example 4 below was actually used for annotating the heavy and light chain variable regions, i.e. the CDRs and FRs. Specifically, the obtained sequences for the heavy and light chain variable regions were checked with the V-Quest tool from the IMGT homepage (www.imgt.org) and position and length of the CDRs were additionally analyzed with the sequence annotation tool on the abysis hompage (http://www.abysis.org/) which uses inter alia the Chothia, Kabat, and IMGT numbering (Tables 2 and 3).
[0687] The nucleotide sequence of the 7C10-C5 heavy chain variable region was determined to be:
TABLE-US-00006 (SEQ ID NO: 39) GATGTACAGCTTCAGGAGTCAGGACCTGGCCTCGTG AAACCTTCTCAGTCTCTGTCTCTCACCTGCTCTGT CACTGGCTACTCCATCACCAGTGGTTATTACTGGA ACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAA TGGATGGGCTACATAAGCTACGACGGTAGCAATAA CTACAACCCATCTCTCAAAAATCGAATCTCCATCA CTCGTGACACATCTAAGAACCAGTTTTTCCTGAAG TTGAATTCTGTGACTACTGAGGACACAGCTACATA TTACTGTGCTGGACGGTTTGCTTACTGGGGCCAAG GGACTCTGGTCACTGTCTCTGCA;
[0688] the amino acid sequence of the 7C10-C5 heavy chain variable region was determined to be:
TABLE-US-00007 (SEQ ID NO: 16) DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWTRQ FPGNKLEWMGYISYDGSNNYNPSLKNRISITRDTSKNQFF LKLNSVTTEDTATYYCAGREAYWGQGTLVTVSA;
[0689] the nucleotide sequence of the 7C10-C5 light chain variable region was determined to be:
TABLE-US-00008 (SEQ ID NO: 40) GATGTTTTGATGACCCAAACTCCACTCTCCCTGCC TGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCA GATCTAGTCAGAGCATTGTACATAGTAATGGAAAC ACCTATTTAGAATGGTACCTGCAGAAACCAGGCCA GTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACC GATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGT GGATCAGGGACAGATTTCACACTCAAGATCAACAG AGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCT TTCAAGGTTCACATGTTCCGTGGACGTTCGGTGG AGGCACCAAGCTGGAAATCAAA;
[0690] and
[0691] the amino acid sequence of the 7C10-C5 light chain variable region was determined to be:
TABLE-US-00009 (SEQ ID NO: 17) DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLE WYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTL KINRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK;
[0692] wherein in the amino acid sequences, underlined letters indicate the CDRs according to Kabat and bold letters indicate the CDRs according to Chotia; and in the nucleotide sequences italic letters indicate the joining (J) region. The J region is annotated according to the IMGT nomenclature.
[0693] Alternatively, the amino acid sequence of the 7C10-C5 heavy chain variable region may comprise a serine instead of a glycine at position 10.
[0694] The complementary determining regions (CDRs) and the framework regions (FRs) of the heavy and light chain variable regions according to Kabat, Chotia, AbM, Contact and IMGT numbering systems/definitions are depicted in Tables 2 and 3. Of note, the Kabat definition is based on sequence variability and is the most commonly used, the Chothia definition is based on the location of the structural loop regions, the AbM definition is a compromise between the two used by Oxford Molecular's AbM antibody modelling software, and the contact definition is based on an analysis of the available complex crystal structures (also see http://www.bioinf.org.uk/abs/; and Swindells (2017), J Mol Biol. 2017; 429(3):356-364). The IMGT definition is according to Brochet (2008), Nucleic Acids Res 36, W503-508; and Giudicelli (2011), Cold Spring Harb Protoc 2011, 695-715.
TABLE-US-00010 TABLE 2 Complementary determining regions (CDRs) and the framework regions (FRs) of the heavy chain variable region of 7C10-C5. Defini- Region tion Sequence Fragment Residues Length HFR1 Chothia DVQLQESGPGLVKPSQSLS 1-25 25 LTCSVT----- (SEQ ID NO: 31) AbM DVQLQESGPGLVKPSQSLS 1-25 25 LTCSVT----- (SEQ ID NO: 31) Kabat DVQLQESGPGLVKPSQSLS 1-30 30 LTCSVTGYSIT (SEQ ID NO: 8) Contact DVQLQESGPGLVKPSQSLS 1-29 29 LTCSVT GYSI- (SEQ ID NO: 78) IMGT DVQLQESGPGLVKPSQSLS 1-25 25 LTCSVT (SEQ ID NO: 31) CDR- Chothia GYSITSGY--- 26-33 8 H1 (SEQ ID NO: 27) AbM GYSITSGYYWN 26-36 11 (SEQ ID NO: 79) Kabat -----SGYYWN 31-36 6 (SEQ ID NO: 4) Contact ----TSGYYWN 30-36 7 (SEQ ID NO: 80) IMGT GYSITSGYY-- 26-34 9 (SEQ ID NO: 81) HFR2 Chothia YWNWIRQFPGNKLEWMGYI 34-52 19 (SEQ ID NO: 32) AbM ---WIRQFPGNKLEWMG-- 37-50 14 (SEQ ID NO: 9) Kabat ---WIRQFPGNKLEWMG-- 37-50 14 (SEQ ID NO: 9) Contact ---WIRQFPGNKLE----- 37-47 11 (SEQ ID NO: 82) IMGT -WNWIRQFPGNKLEWMGY- 35-51 17 (SEQ ID NO: 83) CDR- Chothia -----SYDGS--------- 53-57 5 H2 (SEQ ID NO: 26) AbM ---YISYDGSNN------- 51-59 9 (SEQ ID NO: 84) Kabat ---YISYDGSNNYNPSLKN 51-66 16 (SEQ ID NO: 3) Contact WMGYISYDGSNN------- 48-59 12 (SEQ ID NO: 85) IMGT ----ISYDGSN-------- 52-58 7 (SEQ ID NO: 86) HFR3 Chothia NNYNPSLKNRISITRDTS 58-98 41 KNQFFLKLNSVTTEDTAT YYCAG (SEQ ID NO: 33) AbM --YNPSLKNRISITRDTSK 60-98 39 NQFFLKLNSVTTEDTATYY CAG (SEQ ID NO: 87) Kabat ---------RISITRDTSK 67-98 32 NQFFLKLNSVTTEDTATYY CAG (SEQ ID NO: 10) Contact --YNPSLKNRISITRDTSK 60-96 37 NQFFLKLNSVTTEDTATYY C-- (SEQ ID NO: 88) IMGT -NYNPSLKNRISITRDTSK 59-96 38 NQFFLKLNSVTTEDTATYY C-- (SEQ ID NO: 89) CDR- Chothia --RFAY 99-102 4 H3 (SEQ ID NO: 25) AbM --RFAY 99-102 4 (SEQ ID NO: 2 or 25) Kabat --RFAY 99-102 4 (SEQ ID NO: 2) Contact AGRFA- 97-101 5 (SEQ ID NO: 90) IMGT AGRFAY 97-102 6 (SEQ ID NO: 91) HFR4 Chothia -WGQGTLVTVSA 103-113 11 (SEQ ID NO: 34) AbM -WGQGTLVTVSA 103-113 11 (SEQ ID NO: 11/34) Kabat -WGQGTLVTVSA 103-113 11 (SEQ ID NO: 11) Contact YWGQGTLVTVSA 102-113 12 (SEQ ID NO: 92) IMGT -WGQGTLVTVSA 103-113 11 (SEQ ID NO: 11/34)
[0695] Alternatively, the HFR1 of 7C10-C5 may comprise a serine instead of a glycine at position 10 and thus read:
TABLE-US-00011 Defini- Region tion Sequence Fragment Residues Length HFR1 Chothia DVQLQESGPSLVKPSQ SLSLTCSVT----- (SEQ ID NO: 46) 1-25 25 AbM DVQLQESGPSLVKPS QSLSLTCSVT----- (SEQ ID NO: 46) 1-25 25 Kabat DVQLQESGPSLVKPS QSLSLTCSVTGYSIT (SEQ ID NO: 45) 1-30 30 Contact DVQLQESGPSLVKPS QSLSLTCSVTGYSI- (SEQ ID NO: 93) 1-29 29 IMGT DVQLQESGPSLVKPS QSLSLTCSVT----- (SEQ ID NO: 46) 1-25 25
TABLE-US-00012 TABLE 3 Complementary determining regions (CDRs) and the framework regions (FRs) of the light chain variable region of 7C10-C5. Defini- Region tion Sequence Fragment Residues Length LFR1 Chothia DVLMTQTPLSLPVSLGD 1-23 23 QASISC------ (SEQ ID NO: 35) AbM DVLMTQTPLSLPVSLGD 1-23 23 QASISC------ (SEQ ID NO: 12/35) Kabat DVLMTQTPLSLPVSLGD 1-23 23 QASISC------ (SEQ ID NO: 12) Contact DVLMTQTPLSLPVSLGD 1-29 29 QASISCRSSQSI (SEQ ID NO: 94) IMGT DVLMTQTPLSLPVSLGD 1-26 26 QASISCRSS--- (SEQ ID NO: 95) CDR-L1 Chothia RSSQSIVHSNGNTYLE-- 24-39 16 (SEQ ID NO: 30) AbM RSSQSIVHSNGNTYLE-- 24-39 16 (SEQ ID NO: 7/30) Kabat RSSQSIVHSNGNTYLE-- 24-39 16 (SEQ ID NO: 7) Contact ------VHSNGNTYLEWY 30-41 12 (SEQ ID NO: 96) IMGT ---QSIVHSNGNTY---- 27-37 11 (SEQ ID NO: 97) LFR2 Chothia --WYLQKPGQSPKLLIY 40-54 15 (SEQ ID NO: 36) AbM --WYLQKPGQSPKLLIY 40-54 15 (SEQ ID NO: 13/36) Kabat --WYLQKPGQSPKLLIY 40-54 15 (SEQ ID NO: 13) Contact ----LQKPGQSPK---- 42-50 9 (SEQ ID NO: 98) IMGT LEWYLQKPGQSPKLLIY 38-54 17 (SEQ ID NO: 99) CDR-L2 Chothia ----KVSNRFS 55-61 7 (SEQ ID NO: 29) AbM ----KVSNRFS 55-61 7 (SEQ ID NO: 6/29) Kabat ----KVSNRFS 55-61 7 (SEQ ID NO: 6) Contact LLIYKVSNRF- 51-60 10 (SEQ ID NO: 100) IMGT ----KVS---- 55-57 3 LFR3 Chothia -----GVPDRFSGSGSGTDF 62-93 32 TLKINRVEAEDLGVYYC (SEQ ID NO: 37) AbM -----GVPDRFSGSGSGTDF 62-93 32 TLKINRVEAEDLGVYYC (SEQ ID NO: 14/37) Kabat -----GVPDRFSGSGSGTDF 62-93 32 TLKINRVEAEDLGVYYC (SEQ ID NO: 14) Contact ----SGVPDRFSGSGSGTDF 61-93 33 TLKINRVEAEDLGVYYC (SEQ ID NO: 101) IMGT NRFSGVPDRFSGSGSGTDFT 58-93 36 LKINRVEAEDLGVYYC (SEQ ID NO: 102) CDR-L3 Chothia FQGSHVPWT 94-102 9 (SEQ ID NO: 28) AbM FQGSHVPWT 94-102 9 (SEQ ID NO: 5/28) Kabat FQGSHVPWT 94-102 9 (SEQ ID NO: 5) Contact FQGSHVPW- 94-101 8 (SEQ ID NO: 103) IMGT FQGSHVPWT 94-102 9 (SEQ ID NO: 5/28) LFR4 Chothia -FGGGTKLEIK 103-112 10 (SEQ ID NO: 38) AbM -FGGGTKLEIK 103-112 10 (SEQ ID NO: 15/38) Kabat -FGGGTKLEIK 103-112 10 (SEQ ID NO: 15) Contact TFGGGTKLEIK 102-112 11 (SEQ ID NO: 104) IMGT -FGGGTKLEIK 103-112 10 (SEQ ID NO: 15/38)
[0696] The CDRs and FRs in Tables 2 and 3 are shown for illustrative purposes with IMGT according to Brochet (2008), loc. cit.; and Giudicelli (2011) loc cit. In the context of the invention, the CDRs and FRs of the inventive antibody/antibodies are defined according to the Kabat numbering system.
[0697] Therefore, the CDRs of 7C10-C5 according to Kabat numbering were determined as following:
[0698] the CDR-H3 sequence is RFAY (SEQ ID NO:2),
[0699] the CDR-H2 sequence is YISYDGSNNYNPSLKN (SEQ ID NO:3),
[0700] the CDR-H1 sequence is SGYYWN (SEQ ID NO:4),
[0701] the CDR-L3 sequence is FQGSHVPWT (SEQ ID NO:5),
[0702] the CDR-L2 sequence is KVSNRFS (SEQ ID NO:6), and
[0703] the CDR-L1 sequence is RSSQSIVHSNGNTYLE (SEQ ID NO:7).
[0704] Accordingly, the FRs of 7C10-C5 according to Kabat numbering were determined as following:
[0705] the H-FR1 sequence is preferably DVQLQESGPGLVKPSQSLSLTCSVTGYSIT (SEQ ID NO:8) or alternatively DVQLQESGPSLVKPSQSLSLTCSVTGYSIT (SEQ ID NO:45),
[0706] the H-FR2 sequence is WIRQFPGNKLEWMG (SEQ ID NO:9),
[0707] the H-FR3 sequence is RISITRDTSKNQFFLKLNSVTTEDTATYYCAG (SEQ ID NO:10),
[0708] the H-FR4 sequence is WGQGTLVTVSA (SEQ ID NO:11),
[0709] the L-FR1 sequence is DVLMTQTPLSLPVSLGDQASISC (SEQ ID NO:12),
[0710] the L-FR2 sequence is WYLQKPGQSPKLLIY (SEQ ID NO:13),
[0711] the L-FR3 sequence is GVPDRFSGSGSGTDFTLKINRVEAEDLGVYYC (SEQ ID NO:14), and
[0712] the L-FR4 sequence is FGGGTKLEIK (SEQ ID NO:15).
[0713] Accordingly, the CDRs of 7C10-C5 according to Chothia numbering were determined as following:
[0714] the CDR-H3 sequence is RFAY (SEQ ID NO:25),
[0715] the CDR-H2 sequence is SYDGS (SEQ ID NO:26),
[0716] the CDR-H1 sequence is GYSITSGY (SEQ ID NO:27),
[0717] the CDR-L3 sequence is FQGSHVPWT (SEQ ID NO:28),
[0718] the CDR-L2 sequence is KVSNRFS (SEQ ID NO:29), and
[0719] the CDR-L1 sequence is RSSQSIVHSNGNTYLE (SEQ ID NO:30).
[0720] Accordingly, the FRs of 7C10-C5 according to Chothia numbering were determined as following:
[0721] the H-FR1 sequence is preferably DVQLQESGPGLVKPSQSLSLTCSVT (SEQ ID NO:31) or alternatively DVQLQESGPSLVKPSQSLSLTCSVT (SEQ ID NO:46),
[0722] the H-FR2 sequence is YWNWIRQFPGNKLEWMGYI (SEQ ID NO:32),
[0723] the H-FR3 sequence is NNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAG (SEQ ID NO:33),
[0724] the H-FR4 sequence is WGQGTLVTVSA (SEQ ID NO:34),
[0725] the L-FR1 sequence is DVLMTQTPLSLPVSLGDQASISC (SEQ ID NO:35),
[0726] the L-FR2 sequence is WYLQKPGQSPKLLIY (SEQ ID NO:36),
[0727] the L-FR3 sequence is GVPDRFSGSGSGTDFTLKINRVEAEDLGVYYC (SEQ ID NO:37), and
[0728] the L-FR4 sequence is FGGGTKLEIK (SEQ ID NO:38).
Example 4. Generation and Characterization of Single-Chain Variable Fragments (scFvs) of 7C10-C5
[0729] Cloning of the Single-Chain Variable Fragments (scFv)
[0730] For cloning of the single-chain variable fragments (scFv), the sequences of the first 20 base pairs (bp) of the heavy or light chain variable regions (SEQ ID NO:39 and SEQ ID NO:40, respectively) were taken as forward primers. As reverse primer for the light chain variable fragment a sequence in the joining region and for the heavy chain a sequence at the beginning of the constant region was used.
[0731] Additionally, the light chain forward primer contained a part of a signal peptide. Of note, the signal peptide is thought to improve expression of the antibody (Haryadi (2015); PLoS One 10, e0116878). The sequence of the light chain forward primer was: 5′-CCTGGGGCTCCTGCTGCTCTGGCTCTCAGGTGCCAGATGT-gatgttttgatgacccaaac-3′ (SEQ ID NO:105), with the signal peptide (overhang) in capital letters.
[0732] The light chain reverse primer additionally contained the linker sequence between the heavy and light chain variable fragments. The sequence of the light chain reverse primer was: 5′-GGAAGATCTAGAGGAACCACCCCCACCACCGCCCGAGCCACCGCCACCAGAGG-atttgatttccagcttggtgcc-3′) (SEQ ID NO:106), with the linker sequence (overhang) in capital letters.
[0733] The heavy chain forward primer also contained the linker sequence between the heavy and light chain variable fragments and had the sequence 5′-GGTGGTTCCTCTAGATCTTCCCTCgatgtacagcttcaggagtc-3′ (SEQ ID NO:107), with the linker sequence (overhang) in capital letters.
[0734] The heavy chain reverse primer additionally contained the sequence for a FseI restriction enzyme cutting site and had the sequence
TABLE-US-00013 (SEQ ID NO: 108) 5’-CTGGCCGGCCTGGCCACTAGT gacagatgggggtgtcgttttggc-3’,
with the overhang in capital letters and the FseI restriction site underlined.
[0735] For the PCR, 2 μl cDNA (derived from the single hydridoma clone 7C10-C5) from the 20 μl Accu Script reaction volume (prepared as described above in Example 3) were mixed with 2 μl forward primer (10 μM) and 2 μl reverse primer (10 μM), 10 μl 5×Q5 Reaction Buffer (NEB #B9027), 0.41 dNTPs (10 μM), 33 μl nuclease free water (Sigma-Aldrich, #W4502) and 0.5 μl Q5® High-Fidelity DNA Polymerase (Neb, #M0491L). Reactions were performed in a Biometra TRIO thermocycler starting with a DNA denaturation step at 98° C. for 1 min followed by 30 cycles of denaturation of the DNA at 98° C. for 15 sec. annealing at 56° C. for 15 sec., and elongation at 72° C. for 30 sec. A 3 minutes 72° C. step completed the reaction. The PCR products were separated on a 1.5% TAE (for 1 Liter: 4.84 g Tris (AppliChem #A1086), 1.14 ml Glacial Acetic Acid, 2 ml 0.5M EDTA pH 8.0) agarose gel (Sigma, A9539) and stained with ethidium bromide. The bands corresponding to the light chain variable fragment and the heavy chain variable fragment were cut out and purified using a Wizard® DNA Clean Up Kit (Promega, #A9282), following the protocol as instructed, and eluted in 50 μl nuclease free water (Sigma, #W4502).
[0736] Next, the light and heavy chain variable fragments were joined by fusion PCR. 100 ng of the respective light chain variable fragment with a part of the signal peptide on the 5′ end and the overlapping linker sequence at the 3 ‘-end and 100 ng of the respective heavy variable fragment with the overlapping linker sequence at the 5’-end were mixed with 2 μl signal peptide forward primer (10 μM) containing a BlpI restriction enzyme cutting site (5′-ATGGACATGAGGGTCCCTGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTC-3′) (SEQ ID NO:109) and 2 μl heavy chain reverse primer containing a FseI restriction enzyme cutting site (5′-GAGGAGGAGGAGGAGGAGCCTGGCCGGCCTGGCCACTAGTG-3′) (SEQ ID NO:110) (10 μM), 10 μl 5× Q5 Reaction Buffer (NEB #B9027), 0.2 μl dNTPs (10 μM), 33 μl nuclease free water (Sigma-Aldrich, #W4502) and 0.5 μl Q5® High-Fidelity DNA Polymerase (Neb, #M0491L). Reactions were done in a Biometra TRIO thermocycler starting with a DNA denaturation step at 98° C. for 1 min followed by 30 cycles of denaturation of the DNA at 98° C. for 15 sec. annealing at 56° C. for 15 sec., and elongation at 72° C. for 30 sec. A 3 minutes 72° C. step completed the reaction. The PCR products were separated on a 1.5% TAE agarose gel (Sigma, A9539) stained with ethidium bromide. The corresponding band (about 862 bp) was cut out and purified using a Wizard® DNA Clean Up Kit (Promega, #A9282), following the protocol as instructed, and eluted in 50 μl nuclease free water (Sigma-Aldrich, #W4502). The purified fragments were cloned into the pJet 1.2 cloning vector following the protocol of the CloneJET PCR Cloning Kit (Thermo, #K1231). 2.5 μl of the ligation mixture was used for bacterial transformation. 100 μl of heat shock competent E. coli HB101 were thawed on ice, the DNA was added and the cells were kept for another 30 minutes on ice. A heat shock for 1 minute at 42° C. was performed and after 10 minutes on ice the bacteria were resuspended in 500 μl LB medium without antibiotics for recovery. After 30 minutes at 37° C. bacteria were plated out on LB plates with Ampicillin and incubated over night at 37° C. Colonies were screened for insertions by colony PCR as described in the CloneJET PCR Cloning Kit with the enclosed pJet primers. Positive clones were subsequently inoculated in 5 ml LB with Ampicillin, grown over night and DNA was eluted in 50 μl nuclease free water (Sigma-Aldrich, #W4502) after purification with a Qiagen Miniprep Kit by following the manufacture's manual (Qiagen, #27106). The gained DNA was prepared for sequencing as demanded by the sequencing company (LGC genomics). 10 μl with 100 ng/μl DNA was mixed with 4 μl primer (10 μM) using either the pJET1.2 forward sequencing primer (5′-CGACTCACTATAGGGAGAGCGGC-3′) (SEQ ID NO:76) or the pJET1.2 reverse sequencing primer (5′-AAGAACATCGATTTTCCATGGCAG-3′) (SEQ ID NO:77) to get sequences from both sides.
[0737] Cloning of Single-Chain Variable Fragments (scFvs) of 7C10-C5 into a Mammalian Expression Vector
[0738] Monovalent 7C10-C5 scFv
[0739] For mammalian expression, the monovalent 7C10-C5 scFv was cloned into the pcDNA3.1(+)/Hygro vector (Thermo Fisher) already containing the Signal Peptide with the BlpI restriction site and a FseI restriction site before the 6×Histidine (His) Tag and hemagglutinin (HA) tag. The pcDNA3.1(+) vector was cut with BlpI and FseI. 3 μg plasmid was mixed with 50 NEB CutSmart® buffer (10×), 0.50 BlpI (NEB R0585S) and FseI (NEB R0588L), filled up with nuclease free water (Sigma-Aldrich, #W4502) to 50 μl, and incubated at 37° C. overnight. The monovalent 7C10-C5 scFv obtained with the fusion PCR described above was cut by mixing 1 μg of the PCR with 50 10×CutSmart® buffer (NEB B7204S), 0.50 BlpI (NEB R0585S) and FseI (NEB R0588L), filled up with nuclease free water to 500, and incubated at 37° C. over night. The restriction digest was separated on a 1% TAE (25 mM Tris (AppliChem #A1086), 0.114% Glacial Acetic Acid (AppliChem #A3701), 0.1 mM EDTA pH 8.0 (AppliChem #2937) agarose gel (Sigma, A9539) and stained with ethidium bromide (Sigma #E1510). The correct bands (5761 bp for the pcDNA 3 vector backbone and 819 bp for the 7C10-C5 scFv) were cut out and purified using a Wizard® DNA Clean Up Kit (Promega #A9282), following the protocol as instructed, and eluted in 30 μl nuclease free water (Sigma #W4502). Ligation was performed mixing 50 ng of the vector, 21 ng of the 7C10-C5 scFv, 20 of T4 DNA Ligase Buffer (NEB) and 1 μl of T4 DNA Ligase (NEB M0202L), filled up with nuclease free water to 20 μl and incubated at 22° C. for 1 hour. 10 μl of the Ligation mixture was used for bacterial transformation. 100 μl of heat shock competent E. coli HB101 were thawed on ice, the DNA was added and the cells were kept for another 30 minutes on ice. A heat shock for 1 minute at 42° C. was performed and after 10 minutes on ice the bacteria were resuspended in 500 μl LB medium without antibiotics for recovery. After 30 minutes at 37° C. bacteria were plated out on LB plates with Ampicillin and incubated over night at 37° C. 3 Clones were subsequently inoculated in 5 ml LB with Ampicillin, grown over night and DNA was eluted in 50 μl nuclease free water after purification with a Qiagen Miniprep Kit by following the manufacture's manual (Qiagen, #27106). The gained DNA was prepared for sequencing as demanded by the sequencing company (LGC genomics). 10 μl with 100 ng/μ1 DNA was mixed with 4 μl primer (10 μM) using either the pcDNA3 forward sequencing primer (5′-TAATACGACTCACTATAGGG-3′) (SEQ ID NO:111) or the pcDNA3 reverse sequencing primer (5′-TAGAAGGCACAGTCGAGG-3′) (SEQ ID NO:112) to get sequences from both sides. The obtained sequences for the heavy and light variable fragments were checked with previously obtained sequences.
[0740] Bivalent 7C10-C5 scFv
[0741] To obtain a bivalent 7C10-C5 scFV, two scFvs were linked together by amplifying the scFvs from the pcDNA3.1(+)/Hygro 7C10-C5 by PCR. The 5′-terminal primer of the first scFv was binding the signal peptide encoding sequence (3′-ATGGACATGAGGGTCCCTGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTC-5′) (SEQ ID NO:109) and the 3′-terminal primer at the heavy chain constant region comprised an additional sequence for a (Gly-Ser)×15 linker between the two scFvs (5′-CCTGAACCGGACCCAGATCCGCTGCCACTACCAGACCCTGATCCGGAGCCAGAAC CGACAGATGGGGGTGTCG-3′) (SEQ ID NO:113). The 5′-terminal primer of the second scFv started with the overlapping linker sequence, comprised the rest of the linker sequence, and was binding the scFv encoding sequence at the 5′-end of the variable fragment (5′-GGATCTGGGTCCGGTTCAGGCTCAGGAAGTGGGAGCGGATCAGGGTCCGGGTCA GATGTTTTGATGACCCAAAC-3′) (SEQ ID NO:114). The 3′-terminal primer reverse primer contained a FseI restriction enzyme cutting site (5′-GAGGAGGAGGAGGAGGAGCCTGGCCGGCCTGGCCACTAGTG-3′) (SEQ ID NO:110). 50 ng pcDNA3.1(+)/Hygro 7C10-C5 vector, 10 μl 5×Q5 Reaction Buffer (NEB #B9027), 0.41 dNTPs (10 μM) nuclease free water filled up to 50 μl (Sigma-Aldrich, #W4502) and 0.5 μl Q5® High-Fidelity DNA Polymerase (Neb, #M0491L) were mixed. Reactions were done in a Biometra TRIO thermocycler starting with a DNA denaturation step at 98° C. for 1 min followed by 30 cycles of denaturation of the DNA at 98° C. for 15 sec. annealing at 56° C. for 15 sec., and elongation at 72° C. for 30 sec. A 3 minutes 72° C. step completed the reaction. The PCR products were separated on a 1% TAE agarose gel stained with ethidium bromide. The correct bands (about 878 for the first and 851 bp for the second scFv) were cut out and purified using a Wizard® DNA Clean Up Kit (Promega #A9282), following the protocol as instructed, and eluted in 50 μl nuclease free water (Sigma-Aldrich, #W4502). For linking of the two scFv, 100 ng of the first and 100 ng of the second scFv encoding DNA fragments were mixed with 2 μl of the 5′-terminal primer of the first scFv (10 μM), 2 μl of the 3′ terminal primer of the second scFv (10 μM), 10 μl Q5 buffer (5×), 0.2 μl of dNTPs (10 mM) and filled up with nuclease free water (Sigma-Aldrich, #W4502) to 50 μl. Reactions were done in a X thermocylcer starting with 1 minutes 98° C. followed by 20 cycles of denaturation of the DNA at 98° C. for 15 sec., annealing at 56° C. for 15 sec., and elongation at 72° C. for 45 seconds. A 3 minutes 72° C. step completed the reaction. The PCR products were separated on a 1% TAE agarose gel (Sigma, A9539) stained with ethidium bromid (X). The band corresponding to the combined bivalent scFv DNA fragment (about 1709 bp) was cut out and purified using a Wizard® DNA Clean Up Kit (Promega #A9282), following the protocol as instructed, and eluted in 50 μl nuclease free water (Sigma-Aldrich, #W4502).
[0742] The bivalent scFv 7C10-C5 was cloned into the pcDNA3.1(+)/Hygro vector (Thermo Fisher) containing the signal peptide containing the BlpI restriction site and a FseI restriction site before the 6× Histidine (His) Tag and hemagglutinin (HA) tag. The pcDNA3.1(+) vector was cut with BlpI and FseI. 3 μg plasmid was mixed with 5 μl NEB CutSmart® buffer (10×), 0.5 μl BlpI (NEB R0585S) and FseI (NEB R0588L), filled up with nuclease free water (Sigma-Aldrich, #W4502) to 50 μl, and incubated at 37° C. overnight. The bivalent 7C10-C5 scFv obtained with the fusion PCR described above was cut by mixing 1 μg of the PCR with 5 μl 10× CutSmart® buffer (NEB B7204S), 0.5 μl BlpI (NEB R0585S) and FseI (NEB R0588L), filled up with nuclease free water to 50 μl, and incubated at 37° C. overnight. The restriction digests were separated on a 1% TAE (25 mM Tris (AppliChem #A1086), 0.114% Glacial Acetic Acid (AppliChem #A3701), 0.1 mM EDTA pH 8.0 (AppliChem #2937) agarose gel (Sigma, A9539) and stained with ethidium bromide (Sigma #E1510). The correct bands (5761 bp for the pcDNA 3 vector backbone and 1665 bp for the 7C10-C5 bivalent scFv) were cut out and purified using a Wizard® DNA Clean Up Kit (Promega #A9282), following the protocol as instructed, and eluted in 30 μl nuclease free water (Sigma #W4502). Ligation was performed mixing 50 ng of the vector, 43 ng of the 7C10-C5 bivalent scFv, 2 μl of T4 DNA Ligase Buffer (NEB) and 1 μl of T4 DNA Ligase (NEB M0202L), filled up with nuclease free water to 20 μl and incubated at 22° C. for 1 hour. 10 μl of the Ligation mixture was used for bacterial transformation. 100 μl of heat shock competent E. coli HB101 were thawed on ice, the DNA was added and the cells were kept for another 30 minutes on ice. A heat shock for 1 minute at 42° C. was performed and after 10 minutes on ice the bacteria were resuspended in 500 μl LB medium without antibiotics for recovery. After 30 minutes at 37° C. bacteria were plated out on LB plates with Ampicillin and incubated overnight at 37° C. 3 Clones were subsequently inoculated in 5 ml LB with Ampicillin, grown over night and DNA was eluted in 50 μl nuclease free water after purification with a Qiagen Miniprep Kit by following the manufacture's manual (Qiagen, #27106). The gained DNA was prepared for sequencing as demanded by the sequencing company (LGC genomics). 10 μl with 100 ng/μ1 DNA was mixed with 4 μl primer (10 μM) using either the pcDNA3 forward sequencing primer (5′-TAATACGACTCACTATAGGG-3′) (SEQ ID NO:111) or the pcDNA3 reverse sequencing primer (5′-TAGAAGGCACAGTCGAGG-3′) (SEQ ID NO:112) to get sequences from both sides. The obtained sequences for the heavy and light variable fragments were checked with previously obtained sequences.
[0743] Expression of Recombinant Mono- and Bivalent scFv 7C10-C5 Constructs.
[0744] For mammalian transfection, the clones with the correct expression constructs for the monovalent or bivalent 7C10-C5 recombinant antibodies were subsequently inoculated in 100 ml LB with Ampicillin, grown overnight and DNA was eluted in 200 μl nuclease free water after purification with a Qiagen Midiprep Kit by following the manufacture's manual (Qiagen, #12943).
[0745] HEK293T cells were grown in DMEM+10% FCS+2 mM L-glutamine (Sigma, G2150)+100 units/ml Penicillin/0.1 mg/ml Streptomycin in a humidified incubator controlled at 37° C. with 7.5% CO2. For subculturing, cells were passaged every 2 to 3 days at a ratio of 1:3 to 1:8. 1.3×106 HEK 293T were seeded in 6 cm dishes in 5 ml growth media the day before transfection. Cells were transfected by mixing 600 μl of OptiMEM (Gibco™ 31985047, #11524456) with 5 μg of DNA (monovalent or bivalent 7C10-C5 recombinant antibody constructs) 2 seconds of vortex-mixing, addition of 7.5 μl of TurboFectin 8.0 (Origin #TF81001) mixing by 5×inverting the tube, incubating the transfection mix for 15 min at room temperature and adding it to the cells. 72 hours later the cell supernatant was centrifuged at 1200 rpm in a Beckman SC6R table top centrifuge, and the supernatant was used for the ELISA and Western blot experiments.
[0746] Structural Characterization of Recombinant Mono- and Bivalent scFv 7C10-C5 Antibodies
[0747] Monovalent scFv Sequence
[0748] The recombinant monovalent scFv antibody was determined to have the following nucleotide and amino acid sequences:
TABLE-US-00014 (SEQ ID NO: 41) ATGGACATGAGGGTCCCTGCTCAGCTCCTGGGGCT CCTGCTGCTCTGGCTCTCAGGTGCCAGATGTGAT GTTTTGATGACCCAAACTCCACTCTCCCTGCCTGT CAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGAT CTAGTCAGAGCATTGTACATAGTAATGGAAACACC TATTTAGAATGGTACCTGCAGAAACCAGGCCAGTC TCCAAAGCTCCTGATCTACAAAGTTTCCAACCGAT TTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGA TCAGGGACAGATTTCACACTCAAGATCAACAGAGT GGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTC AAGGTTCACATGTTCCGTGGACGTTCGGTGGAGGC ACCAAGCTGGAAATCAAATCCTCTGGTGGCGGTG GCTCGGGCGGTGGTGGGGGTGGTTCCTCTAGATCT TCCCTCGATGTACAGCTTCAGGAGTCAGGACCTGG CCTCGTGAAACCTTCTCAGTCTCTGTCTCTCACCT GCTCTGTCACTGGCTACTCCATCACCAGTGGTTAT TACTGGAACTGGATCCGGCAGTTTCCAGGAAACAA ACTGGAATGGATGGGCTACATAAGCTACGACGGTA GCAATAACTACAACCCATCTCTCAAAAATCGAATC TCCATCACTCGTGACACATCTAAGAACCAGTTTTT CCTGAAGTTGAATTCTGTGACTACTGAGGACACAG CTACATATTACTGTGCTGGACGGTTTGCTTACTGG GGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAA AACGACACCCCCATCTGTCACTAGTGGCCAGGCCG GCCAGCACCATCACCATCACCATGGCGCATACCCG TACGACGTTCCGGACTACGCTTCTTAG; (SEQ ID NO: 42) MDMRVPAQLLGLLLLWLSGARCDVLMTQTPLSLPV SLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQS PKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKINRV EAEDLGVYYCFQGSHVPWTFGGGTKLEIKSSGGG GSGGGGGGSSRSSLDVQLQESGPGLVKPSQSLSLT CSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYDG SNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDT ATYYCAGRFAYWGQGTLVTVSAAKTTPPSVTSGQA GQHHHHHHGAYPYDVPDYAS*;
[0749] wherein the signal peptide is in italics, the light chain variable region in bold, the linker double-underlined, the heavy chain variable region single-underlined, and the 3′ or C-terminal part including an 6× His-tag and a HA-tag in standard font. The * denotes the stop codon.
[0750] Bivalent scFv Sequence
[0751] The recombinant bivalent scFv antibody was determined to have the following nucleotide and amino acid sequences:
TABLE-US-00015 (SEQ ID NO: 43) ATGGACATGAGGGTCCCTGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCTCAGGTG CCAGATGTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTT GGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTA ATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAA GCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTC AGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAACAGAGTGGAG GCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTGGA CGTTCGGTGGAGGCACCAAGCTGGAAATCAAATCCTCTGGTGGCGGTGGCTCG GGCGGTGGTGGGGGTGGTTCCTCTAGATCTTCCCTCGATGTACAGCTTCAGGAGT CAGGACCTGGCCTCGTGAAACCTTCTCAGTCTCTGTCTCTCACCTGCTCTGTCACT GGCTACTCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAA ACAAACTGGAATGGATGGGCTACATAAGCTACGACGGTAGCAATAACTACAACC CATCTCTCAAAAATCGAATCTCCATCACTCGTGACACATCTAAGAACCAGTTTTTC CTGAAGTTGAATTCTGTGACTACTGAGGACACAGCTACATATTACTGTGCTGGAC GGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGAC ACCCCCATCTGTC
GATGTTTTGA TGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCAT CTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTA GAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAG TTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGG GACAGATTTCACACTCAAGATCAACAGAGTGGAGGCTGAGGATCTGGGAGT TTATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACC AAGCTGGAAATCAAATCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGGGTGGT TCCTCTAGATCTTCCCTCGATGTACAGCTTCAGGAGTCAGGACCTGGCCTCGTGA AACCTTCTCAGTCTCTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCACCAGT GGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGG GCTACATAAGCTACGACGGTAGCAATAACTACAACCCATCTCTCAAAAATCGAAT CTCCATCACTCGTGACACATCTAAGAACCAGTTTTTCCTGAAGTTGAATTCTGTGA CTACTGAGGACACAGCTACATATTACTGTGCTGGACGGTTTGCTTACTGGGGCCA AGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCACTAGT GGCCAGGCCGGCCAGCACCATCACCATCACCATGGCGCATACCCGTACGACGTTC CGGACTACGCTTCTTAG; (SEQ ID NO: 44) MDMRVPAQLLGLLLLWLSGARCDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGN TYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGV YYCFQGSHVPWTFGGGTKLEIKSSGGGGSGGGGGGSSRSSLDVQLQESGPGLVKPS QSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYDGSNNYNPSLKNRISITRD TSKNQFFLKLNSVTTEDTATYYCAGRFAYWGQGTLVTVSAAKTT
DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTY LEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYY CFQGSHVPWTFGGGTKLEIKSSGGGGSGGGGGGSSRSSLDVQLQESGPGLVKPSQS LSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYDGSNNYNPSLKNRISITRDTS KNQFFLKLNSVTTEDTATYYCAGRFAYWGQGTLVTVSAAKTTPPSVTSGQAGQHHH HHHGAYPYDVPDYAS*;
[0752] wherein the signal peptide is in italics, the light chain variable region in bold, linker A double-underlined, the heavy chain variable region single-underlined, linker B double-underlined and in italics, and the remainder (the 3′ or C-terminal part including an 6×His-tag and a HA-tag; and a small part before linker B) in standard font. The * denotes the stop codon.
[0753] Carboxymethylation Specificity of the Recombinant scFv 7C10-C5 Antibodies
[0754] The recombinant anti-carboxymethyl PP2Ac antibodies (mono- or bivalent scFv 7C10-C5) were tested for carboxymethyl-PP2Ac specificity by Western blot analysis and ELISA (
[0755] ScFv 7C10-C5 Western Blotting
[0756] 100 ng of BSA crosslinked with peptides (L309: CGEPHVTRRTPDYFL (SEQ ID NO:49), Y307F: CGEPHVTRRTPDFFL (SEQ ID NO.50), pY307: CGEPHVTRRTPDpYFL (SEQ ID NO:51) or meL309: CGEPHVTRRTPDYFL-CH.sub.3) (SEQ ID NO:49) was separated by 10% SDS-PAGE and transferred to nitrocellulose membrane (0.2 μm, GE Healthcare). Of note, the Y307F peptide cannot be phosphorylated at position 307 due to the presence of a phenylalanine. The membranes were blocked in 3% non-fatty dry milk (NFDM) in TBS-Tween for 1 h at RT and incubated with single clone hybridoma cell culture supernatant, clone 7C10-C5 diluted 1:100 in 0.5% NFDM in TBS-T (7C10 X63), recombinant single chain variable fragment of antibody 7C10-C5, undiluted (7C10 scFvs), recombinant bivalent single chain variable fragment 7C10-C5, undiluted (7C10 bi-scFvs) or single clone hybridoma cell culture supernatant, clone 1D7 (binding to non-carboxymethylated PP2Ac) 1:100 in 0.5% NFDM in TBS-T o/n at 4° C. After washing 3×5 min in TBS-T, blots incubated with recombinant antibodies were incubated with mouse anti-HA monoclonal antibody 16B12 (recognizing the hemagglutinin (HA) tag on the recombinant antibody), 1:10000 in 0.5% NFDM in TBS-T 2 h at room temperature. After washing 3×5 min in TBS-T, incubation with anti-mouse-HRP coupled secondary antibody for 1 h at RT and 3×10 min washes in TBS-T was performed. ECL was performed with GE Healthcare ECL reagents (RPN2106) mixed 1:3 with Clarity™ Western ECL Substrate, 500 ml #1705061 (Biorad). Signals were detected by exposure of X-ray films. Molecular weights in
[0757] It was found that, similarly to the monoclonal antibody 7C10-C5 from the hybridoma supernatant, both recombinant monovalent or bivalent scFv 7C10-C5 antibodies specifically recognized the carboxymethylated meL309 peptide, but none of the non-carboxymethylated peptides, regardless of phosphorylation at position 307. In contrast, the anti-non-carboxymethylated PP2Ac antibody 1D7 did not recognize the meL309 peptide.
[0758] Scfv 7C10-C5 ELISA
[0759] Nunc Medisorp 96-well ELISA flat-bottom plates were coated with C-terminal PP2Ac peptides either free or linked to BSA. Specifically, the peptides were: unmodified L309 (CGEPHVTRRTPDYFL) (SEQ ID NO:49), Y307F (CGEPHVTRRTPDFFL) (SEQ ID NO:50), pY307 (CGEPHVTRRTPDpYFL) (SEQ ID NO:51) or carboxymethylated meL309 (CGEPHVTRRTPDYFL-CH.sub.3) (SEQ ID NO:49), on the ELISA plate at 8 μg/ml in TBS (free) or 1 μg/ml in TBS (linked to BSA) at 4° C. overnight. After washing twice with TBS, the wells were incubated with 2% BSA in TBS for 1 h at RT. After washing once with TBS, the wells were incubated with single clone hybridoma cell culture supernatant, clone 7C10-C5 1:50 (7C10 X63), recombinant single chain variable fragment of antibody 7C10-C5, undiluted (7C10 scFvs), or recombinant bivalent single chain variable fragment of antibody 7C10-C5, undiluted (7C10 bi-scFvs). After washing 3× with TBS, wells incubated with recombinant antibodies were incubated with monoclonal antibody 16B12, 1:10000 (recognizing the hemagglutinin tag on the recombinant antibody) in 1% BSA TBS 1 h at room temperature. After washing 3× in TBS, incubation with anti-mouse-HRP coupled secondary antibody for 1 h at RT was performed. Bound antibodies were detected by colorimetric reaction with 3,3′,5,5′-Tetramethylbenzidine as substrate, and absorbance was measured at 450 nm. Values were normalized to meL309 which was arbitrarily set to 1. Average and standard deviation of N=3 experiments are shown.
[0760] It was found that, similarly to the monoclonal antibody 7C10-C5 from the hybridoma supernatant, both recombinant monovalent or bivalent scFv 7C10-C5 antibodies specifically recognized the carboxymethylated meL309 peptide, but none of the non-carboxymethylated peptides, regardless of phosphorylation at position 307. Of note, the bivalent scFv has bound stronger to the meL309 peptide than the monovalent scFv.
[0761] In conclusion, both recombinant monovalent or bivalent scFvs of 7C10-C5 are highly specific for the α-carboxymethylated PP2Ac. This confirms that the constant region is not required for the anti-carboxymethylated PP2Ac binding specificity of 7C10-C5.
Example 5. Carboxymethylated PP2Ac Levels Detected by a Truly Specific Anti-Methyl-PP2Ac Antibody (7C10-C5) Indicate PP2A Activity
[0762] Small molecule activators of PP2A (SMAPs) have been previously developed, with the lead drug DT-061 that shows anti-tumor efficacy in cancer mouse models (Allen-Petersen (2019), Cancer Res 79, 209-219; Kastrinsky (2015), Bioorganic & medicinal chemistry 23, 6528-6534; McClinch (2018), Cancer Res 78, 2065-2080; Sangodkar (2017), The Journal of clinical investigation 127, 2081-2090; Tohme (2019), JCI insight 4). DT-061 binds to and activates PP2A holoenzymes such as the AB56αC holoenzyme (Allen-Petersen (2019), Cancer Res 79, 209-219), whose assembly and activation in vivo is regulated by the LCMT-1 catalysed α-carboxymethylation of PP2Ac. The inventors have therefore assessed if the DT-061-mediated stabilization/activation of PP2A holoenzymes can be determined by monitoring the methylation state of the PP2A catalytic subunit in cells/tissues from an in vivo DT-061 treated xenograft tumor model, using the 7C10-C5 antibody.
[0763] Xenograft Tumor Model
[0764] Specifically, for xenograft tumor formation and in vivo DT-061 treatment studies, 5×10.sup.6 H358 (NCI-H358; ATCC® CRL-5807™) cells were resuspended in a 1:1 mix of RPMI:Matrigel and subcutaneously injected into the right flank of 6-8 week old Balb/c nu/nu mice. Tumors were monitored and measured twice a week until study enrollment. For the DT-061 time-course analysis, tumors were grown to 300 mm.sup.3, then randomized into a control N,N-Dimethylacetamide (DMA; solvent for DT-061) 24 hour treatment group or a DT-061 (5 mg/kg BID) treatment group which included a 1 hour, 2 hour, 3 hour, 6 hour, 12 hour, and 24 hour group (7 total groups for randomization). After the respective treatment incubation, mice were sacrificed, and serum and tumor tissue were harvested. Tumor tissue was both formalin fixed for IHC and snap frozen, along with collected serum, in liquid nitrogen for immunoblotting or molecule exposure analysis, respectively. Serum was analyzed by Agilux Laboratories Inc. to determine serum concentrations of DT-061. For in vivo treatment studies, DT-061 was dissolved in 10% DMA, 10% solutol, and 80% water.
[0765] Immunostaining of Xenograft Tumors
[0766] Next, the immunohistochemical analysis of xenograft tumors was performed. In brief, five micron sections from formalin-fixed paraffin-embedded (FFPE) specimens were deparaffinized in Histo-Clear and ethanol, exposed to 0.3% hydrogen peroxide to block endogenous peroxidase activity, and incubated for antigen retrieval with a citrate-based antigen unmasking solution (H-3300, Vector Laboratories) in a pressure cooker. Tumor sections were blocked with Mouse Ig Blocking Reagent (BMK-2202, Vector Laboratories), and incubated with anti-carboxymethyl PP2Ac specific antibody (7C10-C5) or an antibody recognizing total PP2Ac (ab106262) at 4° C. for two nights. Washes were performed in PBS. Slides were subsequently developed using secondary anti-mouse immunoglobulins/HRP (K1497, Agilent), DAB peroxidase substrate as the chromagen, and hematoxylin for counterstaining nuclei. Slides were sealed, and randomized for subsequent blinded review. Two independent researchers scored the level of carboxymethylated PP2Ac staining (with 7C10-C5) in one section per tumor (min. 6 tumors per treatment/control group). Specifically, each IHC sample was scored in a blinded fashion where each slide was categorized the following way: little to no staining (0), less than 50% of the slide had a moderate to low intensity stain (1), or more than 50% of the slide had a moderate to high intensity stain (2). The scores per slide were then combined so that each slide had a grade from 0-4 (2×0-2). Scores from 0-2 are considered low staining, 2-4 as moderate, and 4-6 as high intensity staining. Based on these scores the data was plotted as the percentage of slides from each time point that fell into each group (
[0767] Immunohistochemical Analysis of Rabbit Monoclonal E155 and Mouse Monocolonal 7C10-C5
[0768] Immunohistochemical staining was performed on the DAKO Autostainer (DAKO, Carpinteria, Calif.) using Envision+ or liquid streptavidin-biotin and diaminobenzadine (DAB) as the chromogen. De-paraffinized sections were labeled with mouse monoclonal antibody 7C10-C5 (1:200) overnight at 4 C. Microwave 10 mM Tris HCl, pH9 containing 1 mM EDTA epitope retrieval was used prior to staining. Appropriate negative (no primary antibody) and positive controls (pancreas) were stained in parallel with each set of slides studied. Peptide competition studies were performed using 2 peptides (DPAPRRGEPHVTRRTPDYFL*) (SEQ ID NO:115) differing in the methylation status at position L (marked with a *);
[0769] Stained slides were scanned using an APERIO AT2 (Leica) and the digital images imported into QuPath (Bankhead et al., 2017). TMA cores were analyzed for percentage of cells staining and intensity. In
[0770] Analysis of DT-061 Treated Xenograft Tumors
[0771] Immunohistochemical (IHC) analysis of xenograft DT-061 treated tumors with an anti-methyl-PP2Ac antibody of the invention (i.e here 7C10-C5) revealed an increase in α-carboxymethylated PP2Ac at early time points (one, two, and three hours) after single dose DT-061 treatment followed by a return to basal levels by 12 hours post treatment (
[0772] The potent oncoprotein c-MYC is one of the most well characterized substrates of AB56αC holoenzymes. Dephosphorylation of c-MYC at serine 62 by AB56αC leads to the polyubiquitination and degradation of c-MYC (Arnold and Sears, 2006). It was therefore checked if monitoring carboxymethylated PP2Ac levels with the 7C10-C5 antibody allows to assess the (de)phosphorylation of the PP2A substrate c-MYC. Immunohistochemical analysis from the single dose treated xenograft tumors showed a significant decrease of c-MYC at early time points (one, two, and three hours) following DT-061 exposure which then return to basal levels by 6 hours (
[0773] In other words, a truly anti-methyl-PP2Ac-specific antibody (here: monoclonal antibody 7C10-C5) allows to detect the increase of the α-carboxymethylation of PP2Ac caused by PP2A activators, i.e. a SMAP, which correlates with the stabilization of specific trimeric PP2A holoenzymes (including AB56αC holoenzymes) and the dephosphorylation of specific substrates of the drug-stabilized holoenzyme (for example, c-MYC). The α-carboxymethylation of PP2Ac detected by the specific anti-methyl-PP2Ac antibody of the invention (i.e. 7C10-C5) may thus be used for screening drugs which potentially target PP2A, and i.e. modulate PP2A activity. In particular, potential PP2A activators may be tested if they enhance PP2A activity and hence may improve therapeutic efficacy and target engagement of PP2A for the treatment of certain diseases, such as inter alia cancer, wherein increased α-carboxymethylated PP2Ac levels correspond to an increased PP2A activity. In other words, the α-carboxymethylation level of PP2Ac as detected the truly anti-methyl-PP2Ac specific antibody of this invention (i.e. monoclonal antibody 7C10-C5) is a marker (in particular a pharmacodynamic) marker for PP2A activation including, but not limited to, SMAP enhanced stabilization of active AB56αC holoenzymes.
Example 6. The α-Carboxymethylation Level of PP2Ac as Detected by an Antibody Specific for Methyl-PP2Ac (Anti-Methyl-PP2Ac-Specific Antibody of this Invention, in Particular Monoclonal Antibody 7C10-C5) is a Prognostic Biomarker in Cancer and Other Diseases
[0774] The assembly of trimeric tumor-suppressive PP2A holoenzymes is promoted/facilitated by PP2Ac α-carboxymethylation. The PP2Ac α-carboxymethylation levels correlate positively with the levels of methylation-sensitive trimeric PP2A holoenzymes. A truly specific anti-methyl-PP2Ac antibody (7C10-C5) can be used to determine the α-carboxymethylated PP2Ac levels in normal and disease tissue by IHC analyses of tissue microarrays of cancer, normal tissues and tissues obtained from other disease states including but not limited to heart disease, Alzheimers disease, and diabetes. The PP2A methylation status in disease tissue can be graded using the Allred scoring system that scores the proportion of cells that stain by IHC as well as the intensity of the IHC staining. Specifically, the Allied scoring system is based on the percentage of cells that stain by immunohistochemistry for carboxymethylated PP2Ac (on a scale of 0 to 5) and the intensity of that staining (on a scale of 0 to 3), for a possible total score of 8. Allied scoring is calculated the following way:
[0775] Proportion Score:
[0776] 0—No cells, or essentially no cells (e.g. ≤0.01%) stain positively
[0777] 1—≤1% (e.g. 0.5-1%) of cells stain positively
[0778] 2—1-10% (i.e. >1% and ≤10%; e.g. 2-10%) of cells stain positively
[0779] 3—11-33% of cells stain positively
[0780] 4—34-66% of cells stain positively
[0781] 5—67-100% of cells stain positively;
[0782] Intensity Score:
[0783] 0—Negative.
[0784] 1—Weak.
[0785] 2—Intermediate.
[0786] 3—Strong.
[0787] For example, if 67-100% of cells stain positively and the staining intensity in the sample is at maximum, the Allred score is 8 (5+3).
[0788] For example, Allied scores of 0-3 may indicate low PP2Ac α-carboxymethylation levels and low/very low amounts of tumor-suppressive PP2A holoenzymes and Allred scores of at least 6-8 may indicate intermediate to high α-carboxymethylation levels and high/very high amounts of tumor-suppressive PP2A holoenzymes. However, the thresholding of Allred scores may vary and/or be adjusted according to the sample or disease. For example, it is also possible that an Allred score below 2 (i.e. 0 or 1) indicates very low PP2Ac α-carboxymethylation levels, essentially no carboxymethylated PP2Ac, or no carboxymethylated PP2Ac, and an Allied score of at least 2 (i.e. 2, 3, 4, 5, 6, 7, or 8) indicates low/intermediate to high α-carboxymethylation levels.
[0789] A truly specific anti-methyl-PP2Ac antibody (7C10-C5) is particularly useful for investigating cancers (lung adenocarcinoma, breast and prostate cancer) with oncogenic hyper-activated Akt, S6K and ERK/MAP kinase signaling pathways, because methylation-sensitive PP2A holoenzymes are known to negatively regulate these signaling pathways by dephosphorylating substrates downstream of the hyperactivated receptor tyrosine kinases (Jackson (2012), Neoplasia 14, 585-599). A critical step in tumor initiation and metastasis is the capacity of transformed cells to grow anchorage-independently, which is facilitated by resisting anoikis, a programmed cell death induced by the detachment from the extracellular matrix (ECM). Knockdown of the PP2A methyltransferase LCMT-1 or overexpression of the PP2A methylesterase PME1, which both lead to the reduction of the α-carboxymethylation levels of PP2Ac and as a consequence to the reduction of methylation-sensitive holoenzymes, enhance transformation/promote anchorage-independent growth by activating the AKT and p70/p85 S6K pathways (by preventing the inhibition of AKT and S6K) (Jackson (2012), Neoplasia 14, 585-599). Along these lines, inhibition/low activity of PP2A increases cancer cell survival under suspension conditions and also the tumor initiation capacities of cancer cells (Liu (2016), Nat Commun 7, 11798). Of note, although these authors claimed (by using the rabbit monoclonal antibody E155) that Tyr.sup.307 phosphorylation of PP2Ac is causing the inhibition of PP2Ac, re-evaluating the data based on the actual specificity of E155 (see
[0790] The experiments revealed that 7 out of 12 non-metastatic (localized) prostate cancer tissue samples had Allred scores between 2 and 7, whereas 11 out of 12 metastatic prostate cancer tissues (progressed tumor) had an Allied score of 0 suggesting a correlation between disease stage and the levels of α-carboxymethylated PP2Ac in prostate cancer tissue (
Example 7. The α-Carboxymethylation Level of PP2Ac Detected by a Truly Anti-Methyl-PP2Ac Specific Antibody (Like 7C10-C5) Allows the Prediction of Responsiveness to Cancer and Other Disease Therapy
[0791] The α-carboxymethylated PP2Ac levels may be used for predicting, e.g. based on the Allied scores of the IHC analyses, the likelihood of a therapeutic response to drug-induced inhibition of signaling pathways that are known to be suppressed/negatively regulated by carboxymethylation-regulated, tumor-suppressive PP2A holoenzymes, for example the epidermal growth factor receptor (EGFR), the AKT, c-MYC, ERK, and β-catenin pathways because α-carboxymethylated PP2Ac levels are indicative of the levels of trimeric tumor-suppressive PP2A holoenzymes present in cancer/disease cells. The α-carboxymethylated PP2Ac levels determined by an anti-methyl-PP2Ac specific antibodies of this invention (like, e.g. 7C10-C5) may allow to predict the efficacy of kinase inhibitors in turning off the hyperactivated proliferation pathways in cells. In order to reset an hyperactivated kinase pathway to ground-state, it may not be sufficient to only inhibit a certain kinase because an active phosphatase, i.e. PP2A, may be needed to further dephosphorylate the still (hyper)phosphorylated substrate(s). Disease tissue with a high Allred score of 6-8 (with high α-carboxymethylated PP2Ac levels) may predict a high likelihood of response to cancer therapy, because high levels of α-carboxymethylated PP2Ac are indicative of high levels of active, tumor-suppressive PP2A holoenzymes that efficiently dephosphorylate the hyperphosphorylated substrates, thereby shutting down the hyperactivated pathways. In turn, low Allred scores between 0-3, i.e. 0 or 1, indicate low/very low levels of α-carboxymethylated PP2Ac or no carboxymethylated PP2Ac correlating with low PP2A activity and thus may predict a low likelihood of response to cancer therapy.
[0792] When SMAPs (e.g. DT-061) or other PP2A activating/modulating drugs such as forskolin are used in therapies, the α-carboxymethylation level of PP2Ac of tumor tissue as determined by the 7C10-C5 antibody might serve as a decision criterion for identifying treatment responsive patient populations (predictive biomarker). For example, when the carboxymethylation level of PP2Ac is low, the patient may be responsive to activation of PP2A. In other words, determining the α-carboxymethylation level of PP2Ac of tumor tissues of patients with the 7C10-C5 antibody may allow stratifying the patients into “likely drug (e.g. DT-061) responders” having low carboxymethylated PP2Ac levels and “unlikely drug (e.g. DT-061) responders” already having high carboxymethylated PP2Ac levels. Moreover, a drug induced increase of α-carboxymethylation PP2Ac levels might indicate (re-) activation of PP2A tumor suppressive functions.
Example 8. Prior Art Anti-PP2Ac Antibodies do not Specifically Bind Carboxymethylated PP2Ac
[0793] Phosphorylation of PP2Ac subunit either on Tyr.sup.307 or an undefined threonine residue has been primarily associated with the inhibition of its catalytic activity (Chen et al., 1992; Chen et al., 1994; Damuni et al., 1994; Guo and Damuni, 1993). The most frequently cited antibody (112 publications) for the detection of pTyr.sup.307 is a rabbit monoclonal antibody clone E155 generated by Epitomics (catalog #1155-1, Abeam). High levels of Tyr.sup.307 phosphorylation detected by E155 were interpreted as evidence for PP2A inhibition and were claimed to correlate with poor outcome/overall survival in different human cancer types (Chen et al., 2017; Cristobal et al., 2014; Rincon et al., 2015). Later-on, the antibody has been re-examined, and it was clarified that E155 is not specific for pTyr.sup.307. Furthermore, it was found by the inventors that commercial pTyr.sup.307 antibodies including E155 and mouse monoclonal F-8 from Santa Cruz Biotechnology (SCBT) are not specific for pTyr.sup.307 but recognize the non-methylated C-terminus of PP2Ac and are impaired in recognition of PP2Ac by α-carboxymethylation of PP2Ac at Leu.sup.309 and to a lesser extent also by phosphorylation of Thr.sup.304.
[0794] Prior Art Antibodies Described as “Anti-Carboxymethylated PP2Ac Antibodies”
[0795] The only commercially available antibody which binds α-carboxymethylated PP2Ac specificity but not non-carboxymethylated PP2Ac is 2A10 that is sold by several companies including Upstate Biotechnology (now Merck-Millipore), Abcam, Biolegend/Covance, ImmuQuest and Santa Cruz Biotechnology. This clone, however, possesses cross-reactivity with the methylated PP4c and weakly also with methylated PP6c (
[0796] Later, a monoclonal antibody, termed 4D9 was reported to have specificity for the methylated PP2Ac (Tolstykh (2000), Embo J 19, 5682-5691). In contrast to 7C10-C5 (and also 2A10) this clone was raised against an amidated and not α-carboxymethylated C-terminal PP2Ac peptide (299-309). Amidation neutralizes the charge of the α-carboxyl-group and is thought to mimic the functional consequences of the in vivo occurring methylation. Thus, the 4D9 antibody may be specific for the uncharged, amidated C-terminus but not the α-carboxymethylated C-terminus of PP2Ac subunit per se. Its cross-reactivity with the PP2A-like phosphatases PP4c and PP6c that share the terminal 3 amino acids (
[0797] Anti-Non-Carboxymethylated PP2Ac Antibodies
[0798] Examples of antibodies with a preference for non-methylated PP2Ac are mouse monoclonal antibody 4B7 (Yu (2001), Mol Biol Cell 12, 185-199), mouse monoclonal antibody 1D7 generated by the Ogris lab but also the rabbit monoclonal E155 antibody from Abeam and the mouse monoclonal F-8 from SCBT that were thought to be specific for pTyr.sup.307. The inventors have analyzed the detection properties of 5 commercial antibodies that were raised against the unmodified C-terminus of PP2Ac and found that all of them displayed various degrees of being impaired by α-carboxymethylation of PP2Ac and cross-reactivities with PP4, and in addition their PP2Ac recognition was hampered by the phosphorylation of Tyr.sup.307 and Thr.sup.304 as summarized in Table 4.
[0799] Characterization of Prior Art Antibodies
[0800] E155 ELISAs
[0801] E155 was tested for its specificity to phosphorylation and methylation on the carboxy-terminus of PP2A on 6 different undecapeptides by ELISA (
[0802] In a further experiment, flat-bottom Nunc-Immuno Medisorp 96-well ELISA plates (Thermo; 467320) were coated o/n at 4° C. with 50 μl of either a peptide of the sequence ac-HVTRRTPDYFL-OH (L309) (SEQ ID NO:18) at a concentration of 2 μg/ml in TBS (137 mM NaCl (AppliChem, #131659, 2.7 mM KCl (AppliChem, #131494.), 24.8 mM Tris (AppliChem, #A1086), pH 7.4 with HCl), a peptide of the sequence ac-HVTRRTPDYFL-CH.sub.3 (meL309) (SEQ ID NO:18) at a concentration of 2 μg/ml in TBS (137 mM NaCl (AppliChem, #131659.1214, 2.7 mM KCl (AppliChem, #131494.1211) pH 7.4 with HCl), with a peptide of the sequence ac-HVTRRTPDpYFL-OH (pY307-L309) (SEQ ID NO:117) at a concentration of 2 μg/ml in TBS or with a peptide of the sequence ac-HVTRRTPDpYFL-CH.sub.3 (pY307-meL309) (SEQ ID NO:117) at a concentration of 2 μg/ml in TBS (
[0803] Furthermore, E155 was tested in a peptide dilution series for its affinity to 4 different undecapeptides (
[0804] E155 was tested in a further experiment (
[0805] 4B7 and 1D7 ELISAs
[0806] ELISAs have been performed as described in Example 2, “No impairment of 7C10-C5 binding specificity by co-cocurrent phosphorylation of tyrosine 307 or threonine 304 of PP2Ac”,but instead of the 7C10-C5 antibody with either 50 μl of monoclonal antibody 4B7 (SCBT, sc-13601, lot 0716) 1 μg/ml in 1% BSA in TBS for 1 hour at RT (
[0807] Results of ELISAs
[0808] The characterization of the antibodies with a preference for non-methylated PP2Ac, such as 4B7 and 1D7, but also the former “pTyr.sup.307” antibody E155 by ELISA revealed that none of them possesses an exclusive specificity for the non-methylated PP2Ac (
[0809] Western Blotting
[0810] To test these antibodies in the western blot analysis with methylated and non-methylated full-length PP2Ac the methyl group was chemically removed from cellular PP2Ac by treating lysates of the human cell line, HAP1, with NaOH, as described in Example 2. The antibodies 4B7, 1D7, E155 and F-8 detected low levels of PP2Ac in the untreated cell lysates and after removal of PP2Ac methylation with NaOH high levels of PP2Ac, whereas actin and total PP2Ac levels remained unchanged by the NaOH treatment (
TABLE-US-00016 TABLE 4 Summary of detection properties and cross-reactivities of C-terminal PP2Ac antibodies. This Table shows an overview of the properties of the tested c-terminal PP2A C antibodies. Antibodies screened for binding methylated PP2Ac pThr.sup.304 pTyr.sup.307 PP4 PP6 unmodified meLeu.sup.309 meLeu.sup.309 meLeu.sup.309 meLeu.sup.307 meLeu.sup.305 7C10-C5 — *** **** *** — — 2A10 — *** nd nd *** ** clone unmodified meLeu.sup.309 pThr.sup.304 pTyr.sup.307 PP4 PP6 Antibodies screened for binding non-methylated PP2Ac 4B7 *** — ** *** — — 1D7 *** — ** *** — — Antibodies raised against pTyr.sup.307 PP2Ac E155 *** * ** *** — — F8 *** — — *** — — PP2A Antibodies raised against the unmodified C-terminus of PP2Ac 1D6 *** * ** — *** — 7A6 *** * ** — * — G-4 *** * ** — * — 52F8 *** ** nd nd — — 2038 *** * nd nd * * Summary of results, **** >100%, *** 80-100%, ** 40-80%, * 15-40%, — <15%, and “nd” not determined
Example 9. Illustrative Prognostic Assays Employing a Truly Anti-Methyl-PP2Ac Specific Antibody (Like 7C10-C5 of the Present Invention)
[0811] Reduction of the α-carboxymethylation levels of PP2Ac leads to the reduction of methylation-sensitive holoenzymes and enhances transformation and promotes anchorage-independent growth by activating pro-survival and pro-growth signaling pathways. In addition, inhibition of PP2A increases anchorage independent growth and augments the tumor initiation capacity of cancer cells (Liu (2016), Nat Commun 7, 11798). These findings indicate that a decrease of PP2Ac methylation in cancer cells promotes both their ability to form tumors and to metastasize.
[0812] PP2A has been implicated in the pathogenesis of prostate cancer. PP2A directly binds to and dephosphorylates the androgen receptor (Yang (2007), Mol Cell Biol 27, 3390-3404) and its downstream targets including Akt, Erk, c-MYC and Bcl2 (Kiely (2015), Cancers (Basel) 7, 648-669). Small-molecule activators of PP2A (SMAPs) show profound anti-cancer efficacy in preclinical models of castration-resistant prostate cancer (CRPC) (McClinch (2018), Cancer Res 78, 2065-2080).
[0813] Immunohistochemical (IHC) tissue microarray analyses of prostate cancer tissues with a truly anti-methyl-PP2Ac specific antibody (i.e., 7C10-C5) revealed that 7 out of 12 non-metastatic prostate cancer tissue samples had Allied scores between 2 and 7, whereas 11 out of 12 metastatic prostate cancer tissues had an Allred score of 0 suggesting a correlation between disease stage and the levels of α-carboxymethylated PP2Ac in prostate cancer tissue and that the complete loss of carboxymethylation is a common event in metastatic prostate cancer (
[0814] Prognostic IHC Analysis:
[0815] Step A: Prostate cancer patients, whose cancer has not yet spread outside the prostate (Stage Ti and T2a-c), has not spread to lymph nodes (N0) or elsewhere in the body [M0] (American Joint Committee on Cancer/AJCC TNM Staging) will be studied.
[0816] Step B: Immunohistochemical (IHC) analyses of formalin-fixed paraffin-embedded (FFPE) samples from tumor biopsies (10-12 needle core biopsies for systematic mapping of the prostate) or resected tumor material from at least 4 different tumor sites
[0817] Step C: 7C10-C5 IHC staining to determine the methyl-PP2Ac levels; General PP2Ac antibody IHC staining to determine the total-PP2Ac levels; Counterstain with hematoxylin;
[0818] Step D: IHC staining will be graded according to the Allred scoring system that scores the proportion of cells that are methyl-PP2Ac positive as well as the intensity of the methy-PP2Ac staining.
[0819] All red scoring stratifies the methyl-PP2Ac status of a prostate cancer into cancers with a high risk to metastasize to those with a low risk.
[0820] Proportion Score
[0821] 0—No cells or essentially no cells (e.g. ≤0.01%) are methyl-PP2Ac+
[0822] 1—≤1% (e.g. 0.5-1%) of cells are methyl-PP2Ac+
[0823] 2—1-10% (i.e. >1% and ≤10%; e.g. 2-10%) of cells are methyl-PP2Ac+
[0824] 3—11-33% of cells are methyl-PP2Ac+.
[0825] 4—34-66% of cells are methyl-PP2Ac+.
[0826] 5—67-100% of cells are methyl-PP2Ac+.
[0827] Intensity Score
[0828] 0—Negative methyl-PP2Ac staining.
[0829] 1—Weak methyl-PP2Ac staining.
[0830] 2—Intermediate methyl-PP2Ac staining.
[0831] 3—Strong methyl-PP2Ac staining.
[0832] Allred Score
[0833] 0-1—High probability to spread/metastasize.
[0834] 2-6—Intermediate risk to spread/metastasize.
[0835] 7-8—Low probability to spread/metastasize.
[0836] In addition, a low Allred score may be predictive of PP2A reactivating therapy response for these specific prostate cancer patients. In other words, if the sample from these specific prostate cancer patients has a low Allred score (i.e. 0 or 1), said cancer patients are predicted to be responsive for a PP2A reactivating therapy, e.g. using SMAPs.
[0837] This example of the use of the methyl-PP2Ac levels determined by a truly specific anti-methyl-PP2Ac antibody (7C10-C5) to assess the metastatic risk (and thus the prognosis) may be extended to all other cancers in whose pathogenesis the tumor suppressor PP2A has been shown to play role.
[0838] Additionally, the predictive nature of IHC score to PP2A reactivator therapy response may be extended to all cancers and other diseases treated with these therapies.
Example 10. Illustrative Screen for Compounds with PP2A Activating Potential Using a Truly Antimethyl-PP2Ac-Specific Antibody (Like 7C10-C5)
[0839] In context of the present invention, also screening methods for potentially useful pharmaceuticals are provided. Such pharmaceuticals and/or medicaments may, inter alia, comprise activators of protein phosphatase 2A (PP2A). Without being bound by theory, such medicaments (i.e. activators of PP2A) are in particular useful in context of the present invention, since the protein phosphatase 2A complex is capable of stabilizing and/or increasing the methylation status of PP2Ac, in particular the stabilization of alpha-carboxymethylation of PP2Ac as described herein. Such a “stabilization” of said alpha-carboxymethylation of PP2Ac may lead to advantageous effects in disorders wherein it is desired to de-phosphorylate a hyperactive/hyperphosphorylated component of a disorder-related signaling pathway, like a disordered and/or modified signaling pathway in cancer. Accordingly, binding molecules as described in context of this invention, in particular the antimethyl-PP2Ac-specific antibody are particularly useful in drug screenings. One illustrative example of such a drug screening is provided herein below. In this context, a known activator of protein phosphatase 2A (PP2A) is employed as standard control, i.e. as a drug that fulfils this desired function. Such an activator may be DT-061, an activator of protein phosphatase 2A (PP2A) and proposed in the therapy of KRAS-mutant and MYC-driven tumorigenesis; see inter alia, Kauko (2018) “PP2A inhibition is a druggable MEK inhibitor resistance mechanism in KRAS-mutant lung cancer cells”. Sci Transl Med. 18; 10(450); McClinch (2018) Cancer Res.;78(8):2065-2080. The “read-out” of such drug screening methods may be the increase of methyl PP2Ac levels in response to the test compound, inter alia, versus (a) control compound(s). Control compounds may comprise negative controls, like compounds that do not lead to an increase of methyl PP2Ac levels and positive controls, like compounds that are known to lead to an increase of methyl PP2Ac levels (like activator of protein phosphatase 2A (PP2A), like DT-061 (CAS NO 1809427-19-7)) Other “positive controls may comprise known drugs that lead to an increase and/or stabilization of carboymehtylated PP2Ac, like PP2A activators such as phenothiazine derived small molecule PP2A activators (SMAPs) (Allen-Petersen (2019), Cancer Res 79, 209-219; Sangodkar (2017), J Clin Invest. 127, 2081-2090; Gutierrez (2014), J Clin Invest. 124(2), 644-55; Kastrinsky (2015), Bioorg Med Chem. October 1; 23(19):6528-34), drugs that counteract the endogenous PP2A inhibitors SET (I2PP2A, UniProt: Q01105) and CIP2A (UniProt: Q8TCG1-1). In the description herein above, further potential drugs are described. In such drug screenings, the inventive antibodies being specific for methyl-PP2Ac, in particular specific alpha-carboxymethylated PP2Ac, are particularly useful.
[0840] Also an illustrative in vitro drug screening assay is provided herein below wherein the antibodies of the present invention are useful.
[0841] Screening In Vivo Using Tumor Xenografts in Mice:
[0842] Step A: 5×10.sup.6 H358 cells (Sangodkar (2017), The Journal of clinical investigation 127, 2081-2090) or LNCap cells (McClinch (2018), Cancer Res 78, 2065-2080) will be subcutaneously injected into 6-8 week old athymic nude mice.
[0843] Step B: For an DT-061 time-course analysis, tumors may be grown to 300 mm3, then randomized into a control (DMA (Dimethylacetamide) 24 hour treatment group, a novel compound (5 mg/kg BID), and a DT-061 (5 mg/kg BID) treatment group which includes a 1 hour, 2 hour, 3 hour, 6 hour, 12 hour, and 24 hour group (13 total groups, 10 for randomization). After the respective treatment incubation, mice will be sacrificed, serum and tumor tissue were will be harvested.
[0844] Step C: Tumor tissue may be both formalin-fixed for IHC and snap frozen, along with collected serum, in liquid nitrogen for immunoblotting or molecule exposure analysis. For in vivo treatment studies, compounds as well as DT-061 may be dissolved in 10% DMA, 10% solutol, and 80% water.
[0845] PP2A activators equivalent and/or even superior to the existing DT-061 may be selected by the following 2 selection criteria:
[0846] 1. Faster (than with DT-061) kinetics of methyl PP2Ac increase in response to novel compound compared to DT-061 and vehicle control determined by IHC of formalin-fixed tumor tissue and by western blot analysis with 7C10-C5 and an antibody specific for total PP2Ac such as mouse monoclonal H-8, with non-methyl specific mouse monoclonal antibody 1D7 and a loading control antibody specific for actin or tubulin.
[0847] 2. Greater (than with DT-061) increase of methyl PP2Ac levels in response to novel compound compared to DT-061 and vehicle control and determined by IHC of formalin-fixed tumor tissue and by western blot analysis with 7C10-C5 and an antibody specific for total PP2Ac such as mouse monoclonal H-8, with non-methyl specific mouse monoclonal antibody 1D7 and a loading control antibody specific for actin or tubulin.
[0848] In Vitro Drug Screening
[0849] The effect of novel compounds on PP2Ac carboxymethylation may be screened in a 96 well plate assay using LNCaP cells. Cells may be plated in 96-well plates at a density of 5000 cells per well. After 24 hours of plating, cells may be treated with increasing concentrations of novel compounds starting at doses with 1 μM up to 100 μM for 1 h-3 h (always compared to identical concentrations of DT-061 as well as to vehicle control),
[0850] Cells may be washed 2× with PBS and fixed in 3.7% formaldehyde/PBS for 15 min, quenched with 50 mM NH.sub.4Cl/PBS for 10 min, permeabilized with 0.2% Triton X-100/PBS for 10 min, blocked with 0.2% gelatine/PBS for 1 hour and then incubated with the primary antibodies 7C10-C5, 118 (for total PP2Ac), Abcam antibody (Cat #ab74272) for androgen receptor and Abcam (Cat #ab32072) for c-MYC expression diluted in 0.2% gelatine/PBS for 2 hours. Incubation with secondary antibodies, Alexa Fluor 594 goat anti-mouse IgG (H+L) (1:500, Molecular Probes), Alexa Fluor 488 donkey anti-mouse (H+L) (1:500, Molecular Probes), or donkey anti-goat IgG (H+L) Texas Red (1:200, Jackson ImmunoResearch) may be done for 1 hour. The DNA may be counterstained with Hoechst 33342 and the cover-slips mounted with Vectashield (Vector Laboratories). All steps should be performed at RT. Cells may be analyzed using an imaging instrument. 7C10-C5 fluorescence intensity may be normalized to the 118 fluorescence intensity.
[0851] The PP2A activating potential of novel compounds may be determined based on the respective normalized 7C10-C5 fluorescence values that may be compared to the methyl-PP2Ac increase achieved with the lead compound DT-061 and only those compounds with similar or higher induced increase of methyl-PP2Ac may be further considered.
[0852] Furthermore, it has been shown that DT-061 induced PP2A activation leads to lower levels of the androgen receptor, which is a known substrate of PP2A (McClinch (2018) Cancer Res.; 78(8):2065-2080) and also the known PP2A substrate c-MYC (Leonard (2020) Cell 30; 181(3):688-701).
[0853] Thus, the levels of androgen receptor (as determined by androgen receptor antibody (Abcam Cat #ab74272)) and the levels of c-MYC (as determined by c-MYC antibody (Abcam Cat #ab32072)) in the compound treated cells may be compared to the DT-061 induced levels and only those compounds with similar or higher induced downregulation of androgen receptor may be further considered.
Example 11. About PP2A Inhibitors
[0854] Current PP2A inhibitors are not holoenzyme specific but act on the catalytic subunit. Most of them are highly toxic to cells because PP2A is an essential phosphatase. A less toxic inhibitor has been developed termed LB-100, which has been used in combination with chemo- and radiotherapy enhancing their activity. LB-100 prevents DNA repair by PP2A allowing for malignant cells to progress through the cell cycle with damaged DNA, which leads to tumor cell apoptosis. More recently it has been shown that LB-100 is not specific for PP2Ac but also inhibits the catalytic subunit of PP5, a related phosphatase (D'Arcy (2019), Mol Cancer Ther 18, 556-566).
Example 12. Carboxymethyl PP2Ac IHC Staining Using the 7C10-C5 Antibody in a Panel of Human Dysplastic and Cancer Tissues
[0855] Immunohistochemical staining was performed on the DAKO Autostainer (DAKO, Carpinteria, Calif.) using Envision+ and diaminobenzadine (DAB) as the chromogen. De-paraffinized sections were labeled with a mouse monoclonal antibody to methyl-PP2Ac (clone 7C10-C5, 1:200) overnight at 4 C. Microwave 10 mM Tris HCl, pH9 containing 1 mM EDTA epitope retrieval was used prior to staining. Appropriate negative (no primary antibody) and positive controls (pancreas) were stained in parallel with each set of slides studied. Stained slides were scanned using an APERIO AT2 (Leica) and the digital images imported into QuPath (Bankhead (2017), Scientific Reports 7: 16878). TMA cores were analyzed for percentage of cells staining and intensity.
Example 13. Use of an Anti-Methyl-PP2Ac Specific Antibody of the Invention for Prognosing Survival of Prostate Cancer Patients
[0856] IHC analysis of 50 prostate cancer biopsies/resected tumor material with a Gleason score of 7 (Gleason (1974) J Urol. 111(1):58-64 and Gordetsky (2016) Diagn Pathol. 11:25) using a truly specific anti-methyl-PP2Ac antibody (7C10-C5) revealed a higher survival rate, i.e. recurrence free survival rate, for those patients whose tumor material stained with an Allred score of at least 2 (60% survival at 4000 days post biopsy), than for those patients whose tumor material stained with an Allied score of <2 (30% survival at 4000 days post biopsy) (
[0857] Moreover, since the prostate cancer samples were considered to be at the same stage by using the Gleason score, it is evident that assessing carboxymethylated PP2Ac levels in cancer samples, e.g. prostate cancer samples, using a truly specific anti-methyl-PP2Ac antibody (7C10-C5) allows to obtain a much more precise prognosis for the progression of the cancer (than by conventional staging alone or, by using other anti-PP2Ac antibodies that are not truly specific for carboxymethylated PP2Ac as suggested by the data shown, e.g., in Example 6 and
[0858] Thus, a further illustrative prognostic assay is contemplated:
[0859] Step A: Prostate cancer patients, whose cancer has not yet spread outside the prostate (Stage Ti and T2a-c) with a Gleason score 8 or less (Gleason (1974) J Urol. 111(1):58-64 and Gordetsky (2016) Diagn Pathol. 11:25) and which has not spread to lymph nodes (N0) or elsewhere in the body [M0] (American Joint Committee on Cancer/AJCC TNM Staging) may be studied.
[0860] Step B: Obtaining or providing formalin-fixed paraffin-embedded (FFPE) samples from tumor biopsies (10-12 needle core biopsies for systematic mapping of the prostate) or resected tumor material from at least 4 different tumor sites for immunohistochemical (IHC) analyses.
[0861] Step C: IHC staining with the 7C10-C5 antibody to determine the methyl-PP2Ac levels; General PP2Ac antibody IHC staining to determine the total-PP2Ac levels; Counterstain with hematoxylin;
[0862] Step D: IHC staining may be graded according to the Allied scoring system that scores the proportion of cells that are methyl-PP2Ac positive as well as the intensity of the methy-PP2Ac staining.
[0863] Allred scoring allows to categorize a cancer, i.e. a prostate cancer, based on its methyl-PP2Ac status into (1) the group of cancers with a high risk to metastasize and/or cancers associated with lower patient survival rate (prognosis of a negative outcome and/or unfavorable progression) and (2) the group of cancers with a low risk to metastasize and/or cancers associated with a higher patient survival rate (prognosis of a positive outcome and/or favorable progression).
[0864] Allred Scoring:
[0865] Proportion Score
[0866] 0—No cells or essentially no cells (e.g. ≤0.01%) are methyl-PP2Ac+
[0867] 1—≤1% (e.g. 0.5-1%) of cells are methyl-PP2Ac+
[0868] 2—1-10% (i.e. >1% and ≤10%; e.g. 2-10%) of cells are methyl-PP2Ac+
[0869] 3—11-33% of cells are methyl-PP2Ac+.
[0870] 4—34-66% of cells are methyl-PP2Ac+.
[0871] 5—67-100% of cells are methyl-PP2Ac+.
[0872] Intensity Score
[0873] 0—Negative methyl-PP2Ac staining.
[0874] 1—Weak methyl-PP2Ac staining.
[0875] 2—Intermediate methyl-PP2Ac staining.
[0876] 3—Strong methyl-PP2Ac staining.
[0877] Allred Score:
[0878] <2: High probability to spread/metastasize, lower patient survival rate (prognosis of a negative outcome and/or unfavorable progression);
[0879] ≥2: Lower probability to spread/metastasize, higher survival rate (prognosis of a positive outcome and/or favorable progression).
[0880] In particular, if the Allred score is below 2, a 50% reduced survival rate 10 years post biopsy may be expected compared to samples with an Allied score of at least 2.
[0881] In addition, a low Allred score may be predictive of PP2A reactivating therapy response for these specific prostate cancer patients. In other words, if the sample from these specific prostate cancer patients has a low Allred score (i.e. 0 or 1), said cancer patients are predicted to be responsive for a PP2A reactivating therapy, e.g. using SMAPs.
[0882] The use of the methyl-PP2Ac levels determined by 7C10-C5 to assess the metastatic risk (and thus the prognosis) may be extended to all other cancers in whose pathogenesis the tumor suppressor PP2A has been shown to play role.
[0883] Additionally, the predictive nature of IHC score to PP2A reactivator therapy response may be extended to all cancers and other diseases treated with these therapies.
Example 14. Use of an anti-methyl-PP2Ac specific antibody of the invention for Prognosing Responsiveness to Antiandrongen Therapy of Prostate Cancer
[0884] Profiling of Enzalutamide Resistant Prostate Cancer
[0885] The data shown in Examples 6 and 13 suggest that loss/reduction of PP2Ac carboxymethylation may be causally involved in the aggravation of the disease. The sole enzyme responsible for the carboxymethylation of PP2Ac (as well as for PP4c and PP6c) is the essential Leucine Carboxyl Methyltransferase 1 (LCMT-1) and for the demethylation the Phosphatase Methylesterase (PME-1). Loss/reduction of PP2Ac carboxymethylation could be due to inhibition and/or reduction of LCMT-1 or overexpression and/or hyperactivation of PME-1, which would lead to the loss/reduction of tumor-suppressive PP2A holoenzymes and therefore promote tumorigenesis (Pusey (2016), Tumour Biol 37, 11835-11842).
[0886] Iglesias-Gato et al. carried out a system wide quantitative proteomic analysis of metastatic prostate tumors, localized prostate tumors, and control adjacent prostate tissue (Iglesias-Gato (2018), Clin Cancer Res. 1; 24(21).) The inventors of the present invention assessed these quantitative proteomic data to evaluate changes in PP2A subunits, PP2A regulators, and PP2A substrates. The inventor's analysis surprisingly uncovered that there is a negative correlation between the expression of leucine carboxyl methyltransferase 1 (LCMT1) and the expression of androgen receptor indicating that reduction/loss of PP2A α-carboxymethylation may lead to increased AR expression levels (
[0887] To evaluate the role of LCMT1 in enzalutamide resistance, the inventors examined enzalutamide resistant cell lines derived by long-term exposure to enzalutamide. Previous studies have shown that these cell lines display increased PSA and androgen receptor (AR) levels as well as decreased PP2Ac levels (Rasool (2019), Cancer Discov. 9(11)).
[0888] In context of the present invention, examination of enzalutamide resistant derivatives of LNCaP, LNCaP-AR, LAPC4 prostate cancer cell lines by quantitative real-time PCR and western blotting surprisingly showed decreased LCMT1 mRNA expression and protein levels compared to non-enzalutamide resistant control cell lines (
[0889] However, no anti-human LCMT antibody is currently available for immunohistochemistry (IHC). Thus, the inventors hypothesized whether carboxymethylation of PP2Ac determined by the inventive antibody provided herein (e.g. 7C10-C5) is associated with reduced LCMT expression in the context of prostate cancer. The inventors found that this was surprisingly the case, as detailed out in the following. Thus, the inventors surprisingly found that carboxymethylation of PP2Ac measured with the inventive antibody provided herein (e.g. 7C10-C5) may be used as a predictive marker for prognosing whether a cancer such as a prostate cancer is responsive to antiandrogen treatment, e.g. treatment with an androgen receptor antagonist such as enzalutamide.
[0890] LCMT1 Alters Sensitivity to Enzalutamide in Prostate Cancer Cells
[0891] Given the decrease in carboxymethylated PP2Ac in metastatic prostate cancer (see, e.g. Example 6) and the decrease in LCMT1 in enzalutamide resistant prostate cancer cells, the inventors sought to understand whether decreased LCMT1 drives enzalutamide resistance in prostate cancer cells. Knockdown of LCMT1 resulted in decreased methylation of PP2Ac and increase in phosphorylation of androgen receptor (AR) and c-MYC as assessed by western blotting (
[0892] Furthermore, without being bound by theory, reduced LCMT1 levels and correspondingly reduced carboxymethylated PP2Ac levels could lead to a PP2A bias towards methylation independent B subunits which could result in increased MYC stabilization and increased AR phosphorylation with or without changes in total AR levels, and/or loss of methylation dependent PP2A holoenzymes that have been shown to dephosphorylate c-MYC at serine 62 (Nat Cell Biol. 2004 April; 6(4):308-18. doi: 10.1038/ncb1110. Epub 2004 Mar. 14. and Mol Cell Biol. 2006 April; 26(7):2832-44. doi: 10.1128/MCB.26.7.2832-2844.2006.) and prime it for polyubiquitination and proteasomal degradation.
[0893] Altogether, this suggests that decreased levels of carboxymethylated PP2Ac measured using the inventive antibody (e.g. 7C10-C5) may be used as a predictive marker in human prostate cancer specimens to determine the responsiveness and/or response to antiandrogens, i.e. androgen receptor targeted drugs.
[0894] Materials and Methods
[0895] Cell Culture
[0896] Human cancer cell lines LNCaP, LNCAP-AR, and LAPC4 were purchased from ATCC cultured in RPMI, supplemented with 10% FBS (ThermoFisher, SH3007003) and 1% penicillin/streptomycin (GE Healthcare, SV30010). Lentiviral transduction with shLCMT1 or non-targeting control (shNTC) was done in 6 well plate for 24 hours. Cells were sorted for GFP expression. Knockdown was confirmed by western blotting. For western blotting, cells were washed 2× in PBS upon collection and then lysed in RIPA buffer (ThermoFisher Scientific, Waltham, Mass.) containing phosphatase and protease inhibitors (Roche, Basel, Switzerland). Proteins lysates were separated by SDS-PAGE 12% polyacrylamide gels (Bio-Rad, Hercules, Calif.) and transferred to nitrocellulose membranes (Bio-Rad, Hercules, Calif.). Membranes were probed with the inventive specific anti-carboxymethylated 7C10-C5 antibody, anti-total PP2Ac (Cell Signaling) antibody, anti-phospho AR s81 antibody (Abcam), anti-total androgen receptor (AR) (EMD Millipore) antibody, anti-phospho c-MYC s62 (Abcam) antibody, anti total c-MYC (Cell Signaling) antibody, or anti-vinculin antibody (Santa Cruz). Primary antibodies were probed with either goat anti-mouse (Abcam) or donkey anti-rabbit (GE Healthcare) conjugated to horseradish peroxidase and imaged and quantified using the Bio-Rad ChemiDoc XRS chemiluminescence imager and software. All values were normalized to vinculin, and expressed as fold change relative to control.
[0897] Cell Viability and Colony Formation
[0898] Cells were treated with increasing dose of Enzalutamide for 96 hours and cell viability was assessed through cell titer glo (Promega). For colony formation assays, cells were plated at a low density in 6-well plates. After 48 hours, cells were treated with DMSO (Sigma-Aldrich) or 20 uM Enzalutamide for 10-12 days. Drug medium was refreshed every 48 hours. Cells were fixed and stained with 1% crystal violet solution (Sigma-Aldrich). Quantification was performed through the cell counter function on ImageJ (imagej.nih.gov/ij/).
Example 15. Binding Affinities of the 7C10-C5 Antibody to Carboxymethylated PP2Ac or Carboxymethylated PP4c as Determined by Surface Plasmon Resonance
[0899] The differential affinity of anti-carboxymethylated PP2Ac antibodies, 7C10-C5 and 2A10, to carboxymethylated PP2A catalytic subunit versus noncarboxymethylated PP2A catalytic subunit was determined by surface plasmon resonance (SPR) on a Biacore T200 (GE Healthcare) at the Core Facility Biomolecular & Cellular Analysis (University of Natural Resources and Life Sciences Vienna BOKU). The affinity of 7C10-C5 for carboxymethylated and noncarboxymethylated catalytic subunit of PP4 (PP4c), a PP2A-related phosphatase, was also determined. This analysis revealed a binding strength as follows: 7C10-C5 with carboxymethylated PP2Ac (K.sub.D in low nM range, 11 nM)>7C10-C5 with carboxymethylated PP4c (K.sub.D in higher nM range, 132 nM)>2A10 with carboxymethylated PP2Ac (K.sub.D 448 nM)>2A10 with carboxymethylated PP4c (K.sub.D 1130 nM) and a negligible/no binding of 7C10-C5 as well as of 2A10 to the nonmethylated PP2Ac and PP4c (Table 5). The K.sub.D of the binding of 7C10-C5 (or 2A10) to non-carboxymethylated PP2Ac or PP4c could not be determined because at the highest analyte sample concentration (5000 nM) only very weak binding signals were detected indicating K.sub.D values of at least >5000 nM or higher.
[0900] Thus, carboxymethylated PP2Ac was bound by 7C10-C5 41-fold, i.e. 40.7-fold, stronger than by 2A10. Furthermore, 7C10-C5 was binding to carboxymethylated PP2Ac 12-fold stronger than to carboxymethylated PP4c. In contrast, 2A10 was binding to carboxymethylated PP2Ac only 2.5-fold stronger than to carboxymethylated PP4c.
[0901] Furthermore, the data indicate that 7C10-C5 may bind to carboxymethylated PP2Ac at least about 400-fold stronger than to non-carboxymethylated PP2Ac.
[0902] Hence, these data further demonstrate that 7C10-C5 binds to carboxymethylated PP2Ac with much higher affinity than 2A10, that 7C10-C5 has a strongly reduced cross-reactivity with carboxymethylated PP4c compared to 2A10, and that 7C10-C5 does not cross-react with non-carboxymethylated PP2Ac or PP4c. In brief, the SPR analysis confirms that 7C10-C5 is highly and truly specific for carboxymethylated PP2Ac, whereas 2A10 is much less specific (i.e. due to considerable cross-reactivity with PP4c) and further has a lower affinity to carboxymethylated PP2Ac.
TABLE-US-00017 TABLE 5 7C10-C5 and 2A10 surface plasmon resonance (SPR) analysis Steady state analysis KD Rmax (M) (RU) 50 μg/ml mAB 7C10-C5 PP2A Me 5-160 nM 1.100E−08 25.14 capturing PP4 Me 50-800 nM 1.320E−07 20.17 150 sec 2A10 PP2A Me 30-480 nM 4.480E−07 15.95 PP4 Me 400-6400 nM 1.130E−06 10.97 PP2A Me: 360 sec contact time, 40 μL/min PP4 Me: 360 sec contact time, 40 μL/min
[0903] Material and Methods
[0904] Series S Sensor Chip CM5 (GE Healthcare, 10296958) Flow cells (e.g. Flow cells 3 and 4 for the data shown in Table 5 and
[0905] The following 11-mer peptides corresponding to the carboxymethylated or non-methylated carboxy-terminus of PP2A and PP4 catalytic subunits were used as antigens:
TABLE-US-00018 PP2Ac: (SEQ ID NO: 18) ac-HVTRRTPDYFL-OH mePP2Ac: (SEQ ID NO: 18) ac-HVTRRTPDYFL-CH3 PP4c: (SEQ ID NO: 20) ac-PSKKPVADYFL-OH mePP4c: (SEQ ID NO: 20) ac-PSKKPVADYFL-CH3
[0906] Analyte sample concentration (antigen) should be within a range of 0.1 to 10 times the K.sub.D and equilibrium should be reached at all concentrations that are used for K.sub.D calculation. Furthermore, to obtain a robust fit of a plot of Req against concentration for affinity determination, it is important that the range of analyte concentration is wide enough to reveal the full curvature of the plot (i.e. to reach saturation). Thus, the analytes (11-mer peptides) were used in different concentrations which is important to obtain accurate K.sub.D values, wherein the optimal concentrations depend on the affinity of the binding partners.
[0907] The concentration ranges were: [0908] mePP2Ac 5 nM, 10 nM, 20 nM, 40 nM, 80 nM and 160 nM for 7C10-C5 [0909] mePP2Ac 30 nM, 60 nM, 120 nM, 240 nM and 480 nM for 2A10 [0910] mePP4c 50 nM, 100 nM, 200 nM, 400 nM, 800 nM for 7C10-C5 [0911] mePP4c 400 nM, 800 nM, 1600 nM, 3200 nM and 6400 nM for 2A10
[0912] Antibody capturing time was adjusted to reach approx. 1200 RU, antigen association time was 360 sec, and dissociation time was 450 sec. The flow rate was 40 μL/min. Regeneration of the chip was performed by injecting 10 mM Glycin-HCl pH 1.7 for 180 sec with a flow rate of 20 μL/min. Flow cell 1 and 3, respectively, were used as reference cells. All experiments were performed in multi cycle kinetics mode.
[0913] Running buffer: PBS pH 7.4 (PAN-Biotech cat: P04-36500, lot: 5270820)+0.005% Tween (Roth cat: 9127)+0.1% BSA (Albumin, fraction V, protease free >98%, Roth, cat: T844.2, charge 299286373)
[0914] SPR experiments were performed with the BiacoreT200 instrument (Cytiva). All measurements were performed at 25° C. The sensorgrams obtained were analyzed with BiacoreT200 Evaluation Software (version 3.1). The value of the equilibrium dissociation constant (K.sub.D) was obtained by fitting a plot of response at equilibrium (Req) against the respective concentration of the analyte. All binding curves reached equilibrium and all requirements for a steady state analysis were fulfilled (
Example 16. Recombinant 7C10-C5 Antibody Containing an IgG2 Portion
[0915] Cloning of Recombinant 7C10-C5 into pTrioz mIgG2a
[0916] pTrioz mIgG2a: see Invivogen, https://www.invivogen.com/ptrioz-migg2a
[0917] For cloning of the pTrioz 7C10 mIgG2a, the sequences of the first 20 base pairs (bp) of the heavy or light chain variable regions (SEQ ID NO:39 and SEQ ID NO:40, respectively) were taken as forward primers. As reverse primer for the light or heavy chain variable fragment a sequence in the joining region was used and cloned using the NEBuilder® HiFi DNA Assembly (NEB #E2621) into the pTrioz SP mIgG2a which already contained the sequence of signal peptide for the light chain (L1) and heavy chain (IL2) in the 5′ region of the IgCK and IgG2a sequences, respectively.
[0918] Additionally, the light chain forward primer contained a part of a signal peptide, which was cloned into the pTrioz. Of note, the signal peptide (L1) is thought to improve expression of the antibody (Haryadi (2015); PLoS One 10, e0116878). The sequence of the light chain forward primer was: 5′-GGCTCTCAGGTGCCAGATGTgatgttttgatgacccaaac-3′ (SEQ ID NO:127), with the signal peptide (overhang) in capital letters.
[0919] The light chain reverse primer additionally contained the overlapping region of the light chain constant domain IgCK present in the pTrioz. The sequence of the light chain reverse primer was: 5′-ACAGTTGGTGCAGCATCTGCtttgatttccagcttggtg-3′) (SEQ ID NO:128), with the IgCK sequence (overhang) in capital letters.
[0920] The heavy chain forward primer contained a part of a signal peptide (IL2), which was cloned into the pTrioz. Of note, the signal peptide sequence, IL2 was derived from the pFUSE-mIgG2a-Fc2 (InvivoGene) which is a cloning plasmid for the generation and secretion of a Fc-fusion protein expressing the Fc region (CH2 and CH3 domains) of the murine IgG2a heavy chain and the hinge region. The sequence of the heavy chain forward primer was: 5′-CTTGCACTTGTCACGAATTCGgatgtacagcttcaggagtc-3′ (SEQ ID NO:129), with the signal peptide sequence (overhang) in capital letters.
[0921] The heavy chain reverse primer additionally contained the overlapping region of the heavy chain constant domain IgG2a present in the pTrioz. The sequence of the heavy chain reverse primer was: 5′-AGACCGATGGGGCTGTTGTTTTAGCtgcagagacagtgaccag-3′ (SEQ ID NO:130), with the mIgG2a (overhang) in capital letters.
[0922] For the PCR, 100 ng of plasmid DNA from the Monovalent 7C10-C5 scFv (prepared as described above in Example 4) were mixed with 41 forward primer (10 μM) and 2 μl reverse primer (10 μM), 10 μl 5× Q5 Reaction Buffer (NEB #B9027), 0.41 dNTPs (10 μM), 33 μl nuclease free water (Sigma-Aldrich, #W4502) and 0.5 μl Q5® High-Fidelity DNA Polymerase (NEB, #M0491L). Reactions were performed in a Biometra TRIO thermocycler starting with a DNA denaturation step at 98° C. for 1 min followed by 31 cycles of denaturation of the DNA at 98° C. for 15 sec. annealing at 52° C. for 15 sec., and elongation at 72° C. for 30 sec. A 5 minutes 72° C. step completed the reaction. The PCR products were separated on a 1.5% TAE (for 1 Liter: 4.84 g Tris (AppliChem #A1086), 1.14 ml Glacial Acetic Acid, 2 ml 0.5M EDTA pH 8.0) agarose gel (Sigma, A9539) and stained with ethidium bromide. The bands corresponding to the light chain variable fragment and the heavy chain variable fragment were cut out and purified using a Wizard® DNA Clean Up Kit (Promega, #A9282), following the protocol as instructed, and eluted in 50 μl nuclease free water (Sigma, #W4502).
[0923] Next, the 7C10-C5 light and heavy chain variable fragments were cloned with the NEBuilder® HiFi DNA Assembly (NEB #E2621) into the pTrioz SP mIgG2a, which was cut with AscI, cutting between the light chain signal peptide (L1) and IgCK, and AgeI cutting between heavy chain signal peptide (IL2) and IgG2a sequence. 50 fmol of the respective light chain variable fragment with the overlapping L1 signal peptide on the 5′-end and the overlapping IgCK sequence at the 3 ‘-end and 50 fmol of the respective heavy variable fragment with the overlapping IL2 signal peptide sequence at the 5’-end and the overlapping IgG2a sequence at the 3′-end were mixed with 50 fmol of the purified fragments of the AscI and AgeI cut and gel purified fragments of the pTrioz SP mIgG2a vector and was incubated with the 2× HiFi DNA Assembly master mix in a final volume of 20 μl at 50° C. for 1 hour.
[0924] The NEBuilder mixture was used for bacterial transformation. 100 μl of heat shock competent E. coli HB101 were thawed on ice, the DNA was added and the cells were kept for another 20 minutes on ice. A heat shock for 1 minute at 42° C. was performed and after 10 minutes on ice the bacteria were resuspended in 500 μl LB medium without antibiotics for recovery. After 30 minutes at 30° C. bacteria were plated out on LB plates with Zeocin (50 μg/ml) and incubated over night at 30° C. Clones were subsequently inoculated in 5 ml LB with Zeocin, grown over night and DNA was eluted in 50 μl nuclease free water after purification with a Qiagen Miniprep Kit by following the manufacture's manual (Qiagen, #27106). The gained DNA was prepared for sequencing as demanded by the sequencing company (Microsynth). 10 μl with 100 ng/μl DNA was mixed with 4 μl primer (10 μM) using the EF1 alpha forward sequencing primer (5′-TAATACGACTCACTATAGGG-3′) (SEQ ID NO:111) to get sequences of the L1 signal peptide, light chain variable fragment and IgCK and the pTrioz H forward sequencing primer (5′-TTTGAGCGGAGCTAATTC-3′) (SEQ ID NO:126) to get sequences of the signal peptide, heavy variable fragments and beginning of the IgG2a region. The obtained sequences for the heavy and light variable fragments were checked with and matched the previously obtained sequences.
[0925] Expression of Recombinant 7C10-C5 Constructs.
[0926] For mammalian transfection, the clone with the correct expression constructs for the 7C10-C5 recombinant antibodies were subsequently inoculated in 100 ml LB with Ampicillin, grown overnight and DNA was eluted in 200 μl nuclease free water after purification with a Qiagen Midiprep Kit by following the manufacture's manual (Qiagen, #12943).
[0927] HEK293T cells were grown in DMEM+10% FCS+2 mM L-glutamine (Sigma, G2150)+100 units/ml Penicillin/0.1 mg/ml Streptomycin in a humidified incubator controlled at 37° C. with 7.5% CO2. For subculturing, cells were passaged every 2 to 3 days at a ratio of 1:3 to 1:8. 1.3×10.sup.6 HEK 293T cells were seeded in 6 cm dishes in 5 ml growth media the day before transfection. Cells were transfected by mixing 600 μl of OptiMEM (Gibco™ 31985047, #11524456) with 5 μg of DNA (7C10-C5 recombinant antibody constructs) 2 seconds of vortex-mixing, addition of 7.5 μl of TurboFectin 8.0 (Origin #TF81001) mixing by 5× inverting the tube, incubating the transfection mix for 15 min at room temperature and adding it to the cells. 72 hours later the cell supernatant was centrifuged at 1200 rpm in a Beckman SC6R table top centrifuge, and the supernatant was used for the ELISA (and Western blot) experiments.
[0928] Sequences of the Recombinant Light Chain Variable Fragment and mIgCK of Antibody 7C10-C5 (See
TABLE-US-00019 DNA sequence of the recombinant 7C10-C5 light chain (SEQ ID NO: 122) ATGGACATGAGGGTCCCTGCTCAGCTCCTGGGGCT CCTGCTGCTCTGGCTCTCAGGTGCCAGATGTGATG TTTTGATGACCCAAACTCCACTCTCCCTGCCTGTC AGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATC TAGTCAGAGCATTGTACATAGTAATGGAAACACCT ATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCT CCAAAGCTCCTGATCTACAAAGTTTCCAACCGATT TTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGAT CAGGGACAGATTTCACACTCAAGATCAACAGAGTG GAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCA AGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCA CCAAGCTGGAAATCAAAGCAGATGCTGCACCAACT GTATCCATCTTCCCACCATCCAGTGAGCAGTTAAC ATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACA ACTTCTACCCCAAAGACATCAATGTCAAGTGGAAG ATTGATGGCAGTGAACGACAAAATGGCGTCCTGAA CAGTTGGACTGATCAGGACAGCAAAGACAGCACCT ACAGCATGAGCAGCACCCTCACGTTGACCAAGGAC GAGTATGAACGACATAACAGCTATACCTGTGAGGC CACTCACAAGACATCAACTTCACCCATTGTCAAGA GCTTCAACAGGAATGAGTGTTAG Signal Peptide L1 is in italics; the variable domain of the 7C10-C5 light chain is in bold Amino acid sequence of the recombinant 7C10-C5 light chain (SEQ ID NO: 123) MDMRVPAQLLGLLLLWLSGARCDVLMTQTPLSLPV SLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQS PKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKINRV EAEDLGVYYCFQGSHVPWTFGGGTKLEIKADAAPT VSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWK IDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKD EYERHNSYTCEATHKTSTSPIVKSFNRNEC Signal Peptide L1 is in italics; the variable domain of the 7C10-C5 light chain is in bold
[0929] Sequence of the Recombinant Heavy Chain Variable Fragment and mIgG2a of Antibody 7C10-C5 (see
TABLE-US-00020 DNA sequence of the recombinant 7C10-C5 heavy chain coding sequence (SEQ ID NO: 124) ATGTACAGGATGCAACTCCTGTCTTGCATTGCACT AAGTCTTGCACTTGTCACGAATTCGGATGTACAGC TTCAGGAGTCAGGACCTGGCCTCGTGAAACCTTCT CAGTCTCTGTCTCTCACCTGCTCTGTCACTGGCTA CTCCATCACCAGTGGTTATTACTGGAACTGGATCC GGCAGTTTCCAGGAAACAAACTGGAATGGATGGGC TACATAAGCTACGACGGTAGCAATAACTACAACCC ATCTCTCAAAAATCGAATCTCCATCACTCGTGACA CATCTAAGAACCAGTTTTTCCTGAAGTTGAATTCT GTGACTACTGAGGACACAGCTACATATTACTGTGC TGGACGGTTTGCTTACTGGGGCCAAGGGACTCTGG TCACTGTCTCTGCAGCTAAAACAACAGCCCCATCG GTCTATCCACTGGCCCCTGTGTGTGGAGATACAAC TGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGG GTTATTTCCCTGAGCCAGTGACCTTGACCTGGAAC TCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCC AGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCA GCTCAGTGACTGTAACATCGAGCACCTGGCCCAGC CAGTCCATCACCTGCAATGTGGCCCACCCGGCAAG CAGCACCAAGGTGGACAAGAAAATTGAGCCCAGAG GGCCCACAATCAAGCCCTGTCCTCCATGCAAATGC CCAGCACCTAACCTCTTGGGTGGACCATCCGTCTT CATCTTCCCTCCAAAGATCAAGGATGTACTCATGA TCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTG GATGTGAGCGAGGATGACCCAGATGTCCAGATCAG CTGGTTTGTGAACAACGTGGAAGTACACACAGCTC AGACACAAACCCATAGAGAGGATTACAACAGTACT CTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCA GGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGG TCAACAACAAAGACCTCCCAGCGCCCATCGAGAGA ACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCC ACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGA TGACTAAGAAACAGGTCACTCTGACCTGCATGGTC ACAGACTTCATGCCTGAAGACATTTACGTGGAGTG GACCAACAACGGGAAAACAGAGCTAAACTACAAGA ACACTGAACCAGTCCTGGACTCTGATGGTTCTTAC TTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAA CTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGG TCCACGAGGGTCTGCACAATCACCACACGACTAAG AGCTTCTCCCGGACTCCGGGTAAATAA Signal Peptide IL2 is in italics; the variable domain of the 7C10-C5 heavy chain coding sequence is in bold Amino acid sequence of the recombinant 7C10-C5 heavy chain (SEQ ID NO: 125) MYRMQLLSCIALSLALVTNSDVQLQESGPGLVKPS QSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMG YISYDGSNNYNPSLKNRISITRDTSKNQFFLKLNS VTTEDTATYYCAGRFAYWGQGTLVTVSAAKTTAPS VYPLAPVCGDTTGSSVTLGCLVKGYFPE PVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTV TSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIK PCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSP IVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTH REDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKD LPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQ VTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPV LDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGL HNHHTTKSFSRTPGK Signal Peptide IL2 is in italics; the variable domain of the 7C10-C5 heavy chain coding sequence is in bold
[0930] Recombinant 7C10-C5 Antibody is Specific for the α-Carboxymethylated PP2Ac as Determined by ELISA
[0931] Nunc Medisorp 96-well ELISA flat-bottom plates were coated with peptides either free or linked to BSA, L309 (CGEPHVTRRTPDYFL) (SEQ ID NO:49) or meL309 (CGEPHVTRRTPDYFL-CH.sub.3) (SEQ ID NO:49), on the ELISA plate at 0.5 μg/ml in TBS (free) or 1 μs/ml in TBS (linked to BSA) at 4° C. over night. After washing twice with TBS, the wells were incubated with 2% BSA in TBS for 1 h at RT. After washing once with TBS, the wells were incubated with single clone hybridoma cell culture supernatant, clone 7C10-C5 1:50 (7C10 X63), or recombinant antibody 7C10-C5 undiluted (rec 7C10). After washing 3× with TBS, incubation with anti-mouse-HRP coupled secondary antibody for 1 h at RT was performed. Bound antibodies were detected by colorimetric reaction with 3,3′,5,5′-Tetramethylbenzidine as substrate, and absorbance was measured at 450 nm. Values were normalized to meL309 incubated with 7C10 X63, which was arbitrarily set to 1. Average and standard deviation of N=3 experiments are shown (
[0932] Recombinant 7C10-C5 Antibody is Specific for the α-Carboxymethylated PP2Ac as Determined by Western Blotting with Mammalian Cell Lysates
[0933] To confirm the methyl specificity of the recombinant 7C10-C5, the carboxy-terminal methyl group was chemically removed from cellular PP2Ac by treating lysates of HEK293Trex with NaOH as described in Favre (1994), J Biol Chem 269, 16311-16317. In brief, 100 μl of HEK293T cell lysate corresponding to 200 μg of protein was mixed with 1M NaOH to a final concentration of 0.2M and incubated for 10 min at RT. The reaction was neutralized by adding HCL to a final concentration of 0.2M and diluted to 200 μl with IP Lyse. The control reaction was treated with preneutralization solution (0.2M NaOH and 0.2M HCL) for 10 min at room temperature and diluted to 200 μl with IP Lyse. For immunoblot analysis, protein loading buffer was added to the protein samples and proteins were denatured by incubation at 95° C. for 5 min. 15 μg of untreated and NaOH treated protein samples were loaded on a 10% SDS-PAG.
[0934] Like the X63 supernatant, the recombinant methyl-PP2Ac specific antibody 7C10-C5 only detected methylated PP2Ac in the untreated cell lysates but not in the NaOH treated lysates, the non-methyl specific antibody 1D7 detected the NaOH treated samples, whereas an anti-total PP2Ac antibody (H8) detected PP2A in treated and untreated samples (