CA2360511A1 - Methods for generating phosphorylation site-specific immunological reagents - Google Patents
Methods for generating phosphorylation site-specific immunological reagents Download PDFInfo
- Publication number
- CA2360511A1 CA2360511A1 CA002360511A CA2360511A CA2360511A1 CA 2360511 A1 CA2360511 A1 CA 2360511A1 CA 002360511 A CA002360511 A CA 002360511A CA 2360511 A CA2360511 A CA 2360511A CA 2360511 A1 CA2360511 A1 CA 2360511A1
- Authority
- CA
- Canada
- Prior art keywords
- polypeptide
- phosphorylated
- amino acid
- mimetic
- group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
Abstract
The present invention provides methods for generating phosphorylation site- specific immunological reagents. More specifically, a phosphopeptide mimetic is incorporated into a polypeptide in place of a phosphorylated amino acid. The polypeptide is used as antigen by standard methods to generate either monoclonal or polyclonal antibodies which cross-react with naturally phosphorylated polypeptide. The phosphopeptide mimetic preferably contains a non-hydrolyzable linkage from the appropriate carbon atom of the amino acid to a phosphate group. A preferred linkage is a CF2 group. Such a linkage is use d to generate the phosphoserine mimetic F2Pab, which is incorporated into a polypeptide sequence derived from p53 to produce antibodies which recognize a specific phosphorylation state of p53. A CF2 group linkage is also used to produce antibodies to produce the phosphothreonine mimetic F2Pmb, and to produce the phosphotyrosine mimetic, F2Pmp.
Claims (32)
1. A method for isolating polyclonal antibodies which bind specifically to a polypeptide phosphorylated at a particular amino acid, comprising:
a) providing a polypeptide containing a mimetic of the phosphorylated amino acid residue in a physiologically acceptable carrier;
b) immunizing an animal with the composition of step a);
c) collecting and preparing serum or ascitic fluid from the immunized animal of step b);
d) screening for the presence of antibodies specific for the phosphorylated polypeptide;
and e) when necessary, further enriching for antibodies specific for the phosphorylated polypeptide through conventional methods.
a) providing a polypeptide containing a mimetic of the phosphorylated amino acid residue in a physiologically acceptable carrier;
b) immunizing an animal with the composition of step a);
c) collecting and preparing serum or ascitic fluid from the immunized animal of step b);
d) screening for the presence of antibodies specific for the phosphorylated polypeptide;
and e) when necessary, further enriching for antibodies specific for the phosphorylated polypeptide through conventional methods.
2. The method of Claim 1 wherein binding of the polyclonal antibodies to the phosphorylated polypeptide is dependent on the amino acid sequences which flank the phosphorylated amino acid.
3. The method of Claim 1 wherein binding of the polyclonal antibodies to the phosphorylated polypeptide is independent of the sequences which flank the phosphorylated amino acid.
4. The method of Claim 1 wherein antibodies specific for the phosphorylated polypeptide are further enriched by affinity purification.
5. The method of Claim 1 wherein antibodies specific for the phosphorylated polypeptide are further enriched by depleting co-mingled antibodies which exhibit undesired binding activities.
6. The method of Claim 1 wherein the animal is further immunized with a natural phosphopeptide-conjugate comprised of an appropriate amino acid sequence as a boost.
7. The method of Claim 1 wherein the polypeptide of step a) is optionally modified prior to the immunization of step b).
8. The method of Claim 7 wherein the modification occurs through coupling of the polypeptide to a carrier protein selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin and purified protein derivative of tuberculin (PPD), using any bifunctional reagent.
9. The method of Claim 1 wherein the particular amino acid is selected from the group consisting of serine, threonine and tyrosine.
10. The method of Claim 1 wherein the mimetic contains a non-hydrolyzable linkage from a carbon atom to a phosphate group.
11. The method of Claim 10 wherein the non-hydrolyzable linkage comprises a CF2 group.
12. The method of Claim 11 wherein F2Pab is a mimetic for phosphoserine.
13. The method of Claim 11 wherein F2Pmp is a mimetic for phosphotyrosine.
14. The method of Claim 11 wherein F2Pmb is a mimetic for phosphothreonine.
15. The method of Claim 1 wherein the polypeptide of step a) is derived from p53.
16. The method of Claim 15 wherein the polypeptide is as specified in SEQ ID NO: 1.
17. The method of Claim 15 wherein the polypeptide is selected from the group consisting of SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 9, and 10.
18. A method for isolating monoclonal antibodies which bind specifically to a polypeptide which is phosphorylated at a particular amino acid residue, comprising:
a) providing a polypeptide containing a mimetic of the particular phosphorylated amino acid in a physiologically acceptable carrier;
b) immunizing an animal with the composition of step a);
c) collecting B-cells from the animal of step b) d) generating hybridomas by fusing B-cells from step c) with melanoma cells;
e) screening hybridomas of step d) for the production of antibodies specific for the phosphorylated polypeptide; and f) isolating and propagating hybridomas identified in step e).
a) providing a polypeptide containing a mimetic of the particular phosphorylated amino acid in a physiologically acceptable carrier;
b) immunizing an animal with the composition of step a);
c) collecting B-cells from the animal of step b) d) generating hybridomas by fusing B-cells from step c) with melanoma cells;
e) screening hybridomas of step d) for the production of antibodies specific for the phosphorylated polypeptide; and f) isolating and propagating hybridomas identified in step e).
19. The method of Claim 18 wherein binding of the monoclonal antibodies to the phosphorylated polypeptide is dependent on the amino acid sequences which flank the phosphorylated amino acid.
20. The method of Claim 18 wherein binding of the monoclonal antibodies to the phosphorylated polypeptide is independent of the sequences which flank the phosphorylated amino acid.
21. The method of Claim 18 wherein the animal is further immunized with a natural phosphopeptide-conjugate comprised of an appropriate amino acid sequence as a boost.
22. The method of Claim 18 wherein the polypeptide of step a) is modified prior to the immunization of step b).
23. The method of Claim 22 wherein the modification occurs through coupling of the polypeptide to a carrier protein selected from the group consisting of keyhole limpet hemacyanin (KLH), bovine serum albumin (BSA), ovalbumin and purified protein derivative of tuberculin (PPD), using any bifunctional reagent.
24. The method of Claim 18 wherein the particular amino acid residue is selected from the group consisting of serine, threonine and tyrosine.
25. The method of Claim 18 wherein the mimetic contains a non-hydrolyzable linkage from a carbon atom to a phosphate group.
26. The method of Claim 25 wherein the non-hydrolyzable linkage comprises a CF2 group.
27. The method of Claim 26 wherein F2Pab is a mimetic for phosphoserine.
28. The method of Claim 26 wherein F2Pmp is a mimetic for phosphotyrosine.
29. The method of Claim 26 wherein F2Pmb is a mimetic for phosphothreonine.
30. The method of Claim 18 wherein the polypeptide of step a) is derived from p53.
31. The method of Claim 30 wherein the polypeptide is as specified in SEQ ID NO: 1.
32. The method of Claim 30 wherein the polypeptide is selected from the group consisting of SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 9, and 10.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US09/236,415 US6309863B1 (en) | 1999-01-25 | 1999-01-25 | Methods for generating phosphorylation site-specific immunological reagents |
| US09/236,415 | 1999-01-25 | ||
| PCT/US2000/001796 WO2000043422A1 (en) | 1999-01-25 | 2000-01-25 | Methods for generating phosphorylation site-specific immunological reagents |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2360511A1 true CA2360511A1 (en) | 2000-07-27 |
| CA2360511C CA2360511C (en) | 2012-04-10 |
Family
ID=22889404
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2360511A Expired - Fee Related CA2360511C (en) | 1999-01-25 | 2000-01-25 | Methods for generating phosphorylation site-specific immunological reagents |
Country Status (16)
| Country | Link |
|---|---|
| US (1) | US6309863B1 (en) |
| EP (1) | EP1147137B9 (en) |
| JP (1) | JP3891542B2 (en) |
| AT (1) | ATE324382T1 (en) |
| AU (1) | AU767969B2 (en) |
| BR (1) | BR0008977A (en) |
| CA (1) | CA2360511C (en) |
| DE (1) | DE60027542T2 (en) |
| HU (1) | HUP0105309A3 (en) |
| IL (2) | IL144471A0 (en) |
| MX (1) | MXPA01007323A (en) |
| NO (1) | NO20013636L (en) |
| NZ (1) | NZ512868A (en) |
| UA (1) | UA73109C2 (en) |
| WO (1) | WO2000043422A1 (en) |
| ZA (1) | ZA200105520B (en) |
Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030162230A1 (en) * | 2000-09-27 | 2003-08-28 | Reagan Kevin J. | Method for quantifying phosphokinase activity on proteins |
| WO2003057238A1 (en) * | 2001-12-28 | 2003-07-17 | Oregon Health & Science University | Agents that recognize src when phosphorylated at serine 17 |
| US7452676B2 (en) * | 2003-01-16 | 2008-11-18 | Uti Limited Partnership | Monoclonal antibodies to RNA binding protein GW182 |
| US20090081659A1 (en) * | 2007-03-07 | 2009-03-26 | Cell Signaling Technology, Inc. | Reagents for the detection of protein phosphorylation in carcinoma signaling pathways |
| WO2009120801A2 (en) * | 2008-03-25 | 2009-10-01 | The Regents Of The University Of Michigan | Ikki inhibitor therapies and screening methods, and related ikki diagnostics |
| US8445706B2 (en) * | 2008-09-05 | 2013-05-21 | Board Of Trustees Of Northern Illinois University | Unnatural amino acids capable of covalently modifying protein phosphatases and their use as general and specific inhibitors and probes |
| WO2011149909A2 (en) * | 2010-05-24 | 2011-12-01 | Hunt Donald F | Class i mhc phosphopeptides for cancer immunotherapy and diagnosis |
| US10245255B2 (en) | 2011-02-14 | 2019-04-02 | The Regents Of The University Of Michigan | Compositions and methods for the treatment of obesity and related disorders |
| CA2886218A1 (en) | 2012-05-25 | 2013-11-28 | Phosimmune, Inc. | Identification of mhc class i phospho-peptide antigens from breast cancer utilizing shla technology and complementary enrichment strategies |
| EP3718556A3 (en) | 2012-08-31 | 2020-12-30 | University Of Virginia Patent Foundation | Target peptides for immunotherapy and diagnostics |
| AU2013312529A1 (en) | 2012-09-05 | 2015-04-16 | The University Of Birmingham | Target peptides for colorectal cancer therapy and diagnostics |
| ES2727898T3 (en) | 2013-05-02 | 2019-10-21 | Univ Michigan Regents | Deuterated Amlexanox with enhanced metabolic stability |
| EP3407974A4 (en) | 2016-01-29 | 2019-08-28 | The Regents Of The University Of Michigan | Amlexanox analogs |
| CN106046047B (en) * | 2016-05-26 | 2018-10-02 | 清华大学 | The method for preparing phosphorylation serine phosphonic acids analogies |
| WO2023049501A1 (en) * | 2021-09-27 | 2023-03-30 | Abbratech Inc. | Compositions and methods for linear and conformational site specific antibodies and methods of making the same |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4543439A (en) * | 1982-12-13 | 1985-09-24 | Massachusetts Institute Of Technology | Production and use of monoclonal antibodies to phosphotyrosine-containing proteins |
| DE4009848A1 (en) * | 1990-03-27 | 1991-10-02 | Boehringer Mannheim Gmbh | METHOD FOR DETECTING PROTEINS CONTAINING PHOSPHORYLATED TYROSINE |
| AU4281393A (en) * | 1992-04-10 | 1993-11-18 | Dana-Farber Cancer Institute, Inc. | Activation-state-specific phosphoprotein immunodetection |
| WO1994018324A2 (en) * | 1993-02-12 | 1994-08-18 | The Salk Institute For Biological Studies | Phospho-specific antibodies against transcription factors, more specifically against a creb derived peptide |
| US6495356B1 (en) * | 1999-11-30 | 2002-12-17 | The Regents Of The University Of California | Beryllofluoride analogues of acyl phosphate polypeptides |
-
1999
- 1999-01-25 US US09/236,415 patent/US6309863B1/en not_active Expired - Lifetime
-
2000
- 2000-01-25 UA UA2001075343A patent/UA73109C2/en unknown
- 2000-01-25 DE DE60027542T patent/DE60027542T2/en not_active Expired - Lifetime
- 2000-01-25 CA CA2360511A patent/CA2360511C/en not_active Expired - Fee Related
- 2000-01-25 EP EP00914437A patent/EP1147137B9/en not_active Expired - Lifetime
- 2000-01-25 AU AU35828/00A patent/AU767969B2/en not_active Ceased
- 2000-01-25 MX MXPA01007323A patent/MXPA01007323A/en not_active IP Right Cessation
- 2000-01-25 IL IL14447100A patent/IL144471A0/en active IP Right Grant
- 2000-01-25 JP JP2000594838A patent/JP3891542B2/en not_active Expired - Fee Related
- 2000-01-25 NZ NZ512868A patent/NZ512868A/en unknown
- 2000-01-25 BR BR0008977-0A patent/BR0008977A/en not_active IP Right Cessation
- 2000-01-25 WO PCT/US2000/001796 patent/WO2000043422A1/en active IP Right Grant
- 2000-01-25 HU HU0105309A patent/HUP0105309A3/en unknown
- 2000-01-25 AT AT00914437T patent/ATE324382T1/en not_active IP Right Cessation
-
2001
- 2001-07-04 ZA ZA200105520A patent/ZA200105520B/en unknown
- 2001-07-19 IL IL144471A patent/IL144471A/en not_active IP Right Cessation
- 2001-07-24 NO NO20013636A patent/NO20013636L/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| UA73109C2 (en) | 2005-06-15 |
| JP2003517451A (en) | 2003-05-27 |
| DE60027542T2 (en) | 2007-05-03 |
| BR0008977A (en) | 2002-02-13 |
| AU767969B2 (en) | 2003-11-27 |
| IL144471A0 (en) | 2002-05-23 |
| NZ512868A (en) | 2003-11-28 |
| DE60027542D1 (en) | 2006-06-01 |
| HUP0105309A2 (en) | 2002-04-29 |
| ATE324382T1 (en) | 2006-05-15 |
| HUP0105309A3 (en) | 2004-09-28 |
| EP1147137A4 (en) | 2004-12-29 |
| NO20013636L (en) | 2001-09-19 |
| EP1147137B1 (en) | 2006-04-26 |
| NO20013636D0 (en) | 2001-07-24 |
| AU3582800A (en) | 2000-08-07 |
| EP1147137B9 (en) | 2006-11-02 |
| WO2000043422A1 (en) | 2000-07-27 |
| JP3891542B2 (en) | 2007-03-14 |
| ZA200105520B (en) | 2002-01-17 |
| IL144471A (en) | 2006-06-11 |
| EP1147137A1 (en) | 2001-10-24 |
| US6309863B1 (en) | 2001-10-30 |
| CA2360511C (en) | 2012-04-10 |
| MXPA01007323A (en) | 2002-06-04 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed |
Effective date: 20150126 |