WO1991019006A1 - ANTICORPS MONOCLONAUX SERVANT A L'IDENTIFICATION ET A LA PREPARATION DE L'ONCOPROTEINE raf-1 - Google Patents

ANTICORPS MONOCLONAUX SERVANT A L'IDENTIFICATION ET A LA PREPARATION DE L'ONCOPROTEINE raf-1 Download PDF

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Publication number
WO1991019006A1
WO1991019006A1 PCT/US1991/003656 US9103656W WO9119006A1 WO 1991019006 A1 WO1991019006 A1 WO 1991019006A1 US 9103656 W US9103656 W US 9103656W WO 9119006 A1 WO9119006 A1 WO 9119006A1
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Prior art keywords
raf
hybridoma
monoclonal antibody
protein
monoclonal antibodies
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Application number
PCT/US1991/003656
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English (en)
Inventor
Ulf R. Rapp
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The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce
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Application filed by The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce filed Critical The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce
Publication of WO1991019006A1 publication Critical patent/WO1991019006A1/fr

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    • 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

Definitions

  • the present invention is related generally to the preparation of monoclonal antibodies. More
  • the present invention is related to providing monoclonal antibodies (Mab) which enable the identification, analysis and preparation of vertebrate raf-1 oncoprotein.
  • Mob monoclonal antibodies
  • the raf gene family mediates the integration of signals generated by various membrane bound receptor or second messenger systems and the transmission of growth signals from the cell membranes to the nucleus.
  • the raf proteins seem to alter the gene expression related to signal transduction pathways involved in the
  • a still further object of the present invention is to provide a method for detecting the presence of raf-1 oncoprotein in clinical specimens or samples.
  • Figure 1 shows the detection of raf proteins with monoclonal antibodies. Crude lysates of raf
  • overexpressor cells were run on 10% SDS-polyacrylamide gels, blotted onto nitrocellulose and probed with monoclonal antibodies as well as with polyclonal sera raised against C-terminal peptides specific for A-raf (antiserum #190) or c-raf (antiserum #187).
  • Lane 1 p48 kD rat c-raf protein of 208F-LTR cells: lane 2, p74 kD human c-raf protein of NIH/3T3-EC-4 cells; lane 3, 36 kD v-raf protein of CV2.1 cells; lane 4, p69 human A-raf protein expressed in E. coli; lane 5, p74 human c-raf protein expressed in E. coli; lane 6, 30kD v-raf protein expressed from plasmid pl86 in E. coli.
  • Figure 2 demonstrates epitope mapping of the monoclonal antibodies.
  • Panel 2A Western blots of E. coli expressed v-raf proteins of deletion clones derived from plasmid p186 by digestion with Exonuclease III and Mung Bean Nuclease. Numbering of deletion clones refers to the time points, when Exonuclease III digestion was stopped. Proteins were resolved on 20% acrylamide gels. A polyclonal rabbit serum raised against the p186 expressed raf protein (Kolch et al, 1988, Biochem. Biophys. Acta, 949:233-239) served as control.
  • Panel 2B Reactivity of serum antibodies from an immunized BALB/c mouse with deletion clone proteins. Proteins were run on a 15% acrylamide gel. The
  • Panel 2C Extension of the epitopes as defined by nucleotide sequencing of deletion clones. A comparison of the amino acid sequences of A-raf, B-raf, c-raf and v-raf is included. Amino acids identical to the v-raf sequence are indicated by dots.
  • Panel 3A, EC-4 cell lysates were immunoprecipitated with the
  • Figure 4 shows the comparison of the amino acid sequences of serine/threonine and tyrosine kinases between the conserved DFG and APE sequences.
  • the DFG and APE consensus as well as amino acids corresponding to threonine 241 of cAMP-dependent kinase are bolded.
  • the predicted common secondary structure motif is shown schematically; ---, beta sheet; @, random coil; T, turn; alpha helix.
  • Epitopes of the raf monoclonal antibodies are indicated below.
  • hybridomas PBA1, PBB1, 26/G2, URP26K and URP30K These monoclonal antibodies have specific binding affinity for different epitopes of the raf-1 oncoproteins.
  • a deposit of the hybridoma designated herein URP26K which secretes a unique Mab that immunoprecipitates, blots and recognizes a specific epitope of vertebrate raf-1 protein has been made at the ATCC, Rockville, MD, on April 18, 1990 under accession number HB 10451.
  • the deposit shall be viably maintained, replacing if it becomes non-viable during the life of the patent, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer and, upon issuance of the patent, made available to the public without restriction in accordance with the provisions of the law.
  • the Commissioner of Patents and Trademarks, upon request, shall have access to the deposit.
  • a truncated raf protein encoded by the expression vector pl86 was expressed in E. coli and purified as described by Kolch et al, supra. except that the final chromatographic step was substituted by preparative polyacrylamide electrophoresis and electroelution of the 30 kD raf protein.
  • raf protein Full length raf protein was derived from NIH 3T3 cells overexpressing wild-type c-raf-1 or the linker insertion mutant EC4 (Heidecker et al, in preparation; Rapp et al, supra). In addition, raf protein was obtained from three bacterial expression constructs. The first expresses a slightly truncated c-raf missing the first 25 condons, obtained by ligating the PvuII fragment of c-raf-1 clone p628 (Bonner et al, 1986,
  • substantially pure as used herein means the protein is as pure as can be obtained by standard isolation and purification techniques conventional in the art.
  • the myeloma cell line Ag8.653 and hybridoma cells were grown in RPMI (Gibco) plus 10% fetal calf serum (Gibco) and antibiotics.
  • the following raf expressing cell lines were used: NIH/3T3-3611 cells express p79 kD and p90 kD gag-v-raf fusion proteins (Rapp et al, 1983, Proc. Natl. Acad. Sci.
  • 208F- LTR cells express a truncation activated rodent c-raf p48 kD protein (Moelders et al, 1985, EMBO J., 4:693- 698; Schultz et al, 1988, Oncogene, 2:187-193);
  • NIH/3T3-EC-4 cells express very high levels of a p74 kD full-length human c-raf protein, which carries a linker insertion in CR3 (Rapp et al, 1988b, Cold Spring Harbor Symposia on Quantitative Biology. Vol. LIII, Cold Spring Harbor Laboratory: New York pp 173-184); CV2.1 cells express a 36 kD v-raf protein from a recombinant vaccina virus construct (Stephen M. Storm, Jakob gene 10 sequences. The same expression system was used to over express A-raf protein.
  • A-raf cDNA sequences (Beck et al, 1987, Nucleic Acids Res., 15:595-609) from the Apal site to the BamHI site were inserted into the BamHI site of pAR3039 after adding a BamHI linker to the blunted Apal site.
  • This construct expresses a fusion protein containing the first 10 amino acids of T7 gene 10 protein and all but the first 5 amino acids of A-raf. All three constructs over express raf
  • mice were immunized with 100 ⁇ g purified raf protein per injection.
  • the first injection was with complete Freund's Adjuvans, the second with incomplete and the last five days before the fusion in phosphate- buffered saline solution (PBS).
  • PBS phosphate- buffered saline solution
  • the spleen cells were fused with the myeloma cell line Ag8.653 by standard techniques and cultivated in 96-2311 plates in the presence of thymocyte feeder layers and HAT-selection medium (Boehringer). Hybridomas producing raf-specific antibodies were detected by ELISA and single cell cloned by limited dilution for further analysis.
  • 96-2311 polystyrene plates were coated with 10 ng p30-raf in 100 mM Na 2 CO 3 buffer (pH 9.5) overnight (12- 16 hrs) at 4°C. Plates were washed with TBS (50mM TrisHCl, pH7.5; 150 mM NaCl) containing 0.02% Tween-20 (Sigma). 100 ⁇ l hybridoma supernatant was added per well and incubated 1 hour at room temperature (22°- 24°C). After extensive washing the plates were
  • Nitrocellulose filters were coated with 1 ug goat anti-mouse Ig per well in a 96-well dot blot apparatus. 100 ⁇ l hybridoma supernatant was added per well and incubated for 1 hour at room temperature. The filter was washed extensively with TBS + 2% Tween-20 before a 1 hour incubation with [ 32 P]-labelled cell extracts containing high levels of raf phosphoproteins. Per filter 2x10 7 raf over expressor cells were phosphate- starved for 3 hours and labelled in vivo for 2 hours with 0.5 mCi/ml medium [ 32 P] orthophophoric acid.
  • Proteins were resolved on SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes . After transfer the filters were blocked with TBS+2%Tween for 10 minutes and incubated with antibody for 1 hours. Hybridoma supernatants were applied undiluted, ascites fluid or immune sera were diluted 1:1000 with TBS+2%Tween + 5% calf serum. After washing with TBS+2%Tween blots were incubated for 30 minutes with anti-mouse or anti-rabbit IgG coupled to alkaline phosphatase (Accurate Chemicals or BRL) and washed with TBS+2%Tween.
  • the blots were stained with BCIP (5-bromo-4-chloro-3-indolylphosphate p-toluidine salt) and NBT (nitroblue tetrazolium chloride) in alkaline phosphatase buffer (100 mM TrisHCl, pH9.5; 100 mM NaCl; 5mM MgCl 2 ). The reaction was stopped by rinsing with water and the blots were dried on Whatman 3MM paper. All incubations were at room temperature. Immunoprecipitations and In Vitro Kinase Assays
  • TBST TBS + 1% Triton-X100
  • immunoprecipitations typically 20 ⁇ l protein-G or protein-A goat anti-mouse IgG complex was used for 1 ml supernatant). Ascites or immune sera were used in a 1:300 dilution. Immunoprecipitates were washed three times in RIPA or TBST, respectively. For kinase assays the final wash was done in kinas buffer (TBST plus 5 mM MnCl 2 , 5 mM MgCl 2 , 2 mM DDT). The immunoprecipitate was resuspended in 10 ⁇ l kinase buffer containing 5 ⁇ Ci
  • Three derivatives of the expression vector p186 were constructed by excising appropriate restriction fragments such that the raf specific coding sequence was divided into three parts of about equal length.
  • Plasmid p186A lacks the amino-terminal Xbal-StuI fragment
  • p186D lacks the carboxyl-terminal SacII- BstEII fragment
  • p186C is missing two thirds of the raf sequence comprised in the StuI-BstEii fragment.
  • the deleted plasmids were size selected on a preparative agarose gel, ligated and transformed into E. coli. Individual colonies were screened by plasmid DNA minipreps and Western blotting of expressed deletion proteins. The exact extent of the deletion in clones of interest were determined by nucleotide sequencing with T7 DNA Polymerase.
  • pl86 created by excision of appropriate restriction fragments showed that all the antibodies were specific for a sequence encoded by a StuI-SacII restriction fragment.
  • pl86 was linearized at the SacII site and bidirectionally digested with Exonuclease III / Mung Bean Nuclease.
  • the resulting deletion clones were characterized by immunoblotting experiments and subsequent nucleotide sequence analysis of selected clones. The results are presented in Figure 2. It was found that the eptiopes of all five monoclonal antibodies map into a 16 amino acid long domain surrounding the sequence APE.
  • Panel 2A shows a selection of Western blots of pl86 deletion clones probed with monoclonal antibodies. All five monoclonal antibodies react with clones 20-5, 40-5 and 40-6, but none detects clones 80-10 and 80-13 (the numbering refers to the time points, when the Exonuclease III digestion was stopped). This epitope preference is reflected in a similar differential reactivity of the serum of
  • downstream region (recognized by PBB1 and 26/G2).
  • the upstream region of c-raf differs by one amino acid residue from A-raf and by three changes from B-raf.
  • PBA1 exhibits a higher affinity for c-raf than A-raf
  • its epitope likely comprises the divergent amino acid.
  • URP26K and URP30K probably map in the conserved part of the upstream sequence, since they detect c-raf and A-raf with equal avidity.
  • the sequence APE which is highly conserved amongst protein kinases, does not seem to be part of any epitope for the following reasons.
  • Peptide SP46 containing the sequence MAPEVIRMQ does not compete with PBA1, URP26K and URP30K. Both peptide SP26 and peptide SP67, whose raf specific sequence starts with
  • PEVIRMQ inhibit binding of 26/G2 and PBB1 to A-raf equally well.
  • A-raf has the sequence AAE instead of APE, alanine and proline can be excluded from the epitopes of 26/G2 and PBB1. Moreover, no
  • Panel 3A shows the in vitro kinase activity and pattern of protein phosphorylation in immunoprecipitates of EC4 cells. These cells contain high levels of a full-length non-transforming human c-raf protein expressed from a retroviral vector. Although the EC-4 protein carries a linker insertion, it biologically behaves like normal c-raf and is indistinguishable from endogenous c-raf in its response to mitogen mediated activation (unpublished results). An oncogenic version of c-raf is shown in panel 3B.
  • raf-1 specific MAbs now makes it possible to purify raf-1 protein from a variety of sources including prokaryotic (e.g., E. coli) and
  • Eukaryotic e.g., murine and insect cell lines
  • raf-1 protein can be detected in histological tissue preparations by immunohistochemical techniques and Western blotting. Using such
  • Purified raf-1 protein kinases prepared with the help of raf-1 specific Mabs of the present invention can also be processed for crystallographic studies and screening of activating and inhibitory chemicals which may be useful in chemotherapy of cancer.
  • the MAbs of the present invention react with all known raf family members (A-raf, B-raf, c-raf-1) in immunoprecipitation and immunoblot tests. Furthermore, they detect raf proteins in various species ranging from frog to man.
  • the MAbs of the present invention can be used for the screening of expression libraries and subsequent isolation and characterization of raf and new raf- related genes from different species.
  • a reagent kit for detecting the presence of raf-1 oncoproteins in a biological sample comprises
  • monoclonal antibodies of the present invention either alone or as a combination or mixture of several MAbs and instructional material to perform the test.
  • tests include any standard immunological assays, e.g., radioimmunological, immunofluorescence,
  • composition comprises immunoreactive amount of the monoclonal antibodies of the present invention, either alone or as a combination of the MAbs, in a

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Abstract

Cinq anticorps monoclonaux distincts qui immuno-précipitent l'oncoprotéine raf-1 ont été obtenus. Ces anticorps monoclonaux sont utiles à la détection de l'oncoprotéine raf-1 dans des échantillons cliniques.
PCT/US1991/003656 1990-06-01 1991-05-30 ANTICORPS MONOCLONAUX SERVANT A L'IDENTIFICATION ET A LA PREPARATION DE L'ONCOPROTEINE raf-1 WO1991019006A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999042837A1 (fr) * 1998-02-18 1999-08-26 Theryte Limited Traitement du cancer
US7112570B2 (en) * 2000-08-24 2006-09-26 Neuren Pharmaceuticals, Ltd. GPE analogs

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0612353B1 (fr) * 1991-09-16 1999-03-31 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES Procede de detection de genes c-raf-1

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4855406A (en) * 1986-07-11 1989-08-08 Noboru Yanaihara Et Al. Oncogene-related peptides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4855406A (en) * 1986-07-11 1989-08-08 Noboru Yanaihara Et Al. Oncogene-related peptides

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999042837A1 (fr) * 1998-02-18 1999-08-26 Theryte Limited Traitement du cancer
WO1999042834A2 (fr) * 1998-02-18 1999-08-26 Theryte Limited Traitement du cancer
WO1999042839A2 (fr) * 1998-02-18 1999-08-26 Theryte Limited Traitement du cancer
WO1999042839A3 (fr) * 1998-02-18 1999-10-28 Theryte Ltd Traitement du cancer
WO1999042834A3 (fr) * 1998-02-18 1999-11-25 Theryte Ltd Traitement du cancer
AU749180B2 (en) * 1998-02-18 2002-06-20 Theryte Limited Treating cancer
US7112570B2 (en) * 2000-08-24 2006-09-26 Neuren Pharmaceuticals, Ltd. GPE analogs

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