EP0428518A1 - Für harvey-, kirsten- und n ras-proteine spezifische monoklonale antikörper und ihre verwendung als diagnostische reagentien - Google Patents

Für harvey-, kirsten- und n ras-proteine spezifische monoklonale antikörper und ihre verwendung als diagnostische reagentien

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Publication number
EP0428518A1
EP0428518A1 EP89905339A EP89905339A EP0428518A1 EP 0428518 A1 EP0428518 A1 EP 0428518A1 EP 89905339 A EP89905339 A EP 89905339A EP 89905339 A EP89905339 A EP 89905339A EP 0428518 A1 EP0428518 A1 EP 0428518A1
Authority
EP
European Patent Office
Prior art keywords
ras proteins
ras
epitope
ki2b
cells
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.)
Withdrawn
Application number
EP89905339A
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English (en)
French (fr)
Other versions
EP0428518A4 (en
Inventor
Walter Patrick Carney
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
OSI Pharmaceuticals LLC
Original Assignee
EI Du Pont de Nemours and Co
Oncogene Science Inc
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by EI Du Pont de Nemours and Co, Oncogene Science Inc filed Critical EI Du Pont de Nemours and Co
Publication of EP0428518A1 publication Critical patent/EP0428518A1/de
Publication of EP0428518A4 publication Critical patent/EP0428518A4/en
Withdrawn legal-status Critical Current

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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

Definitions

  • This invention relates to murine monoclonal antibodies and immunoreactive fragments specific for the Harvey, Kirsten and N ras proteins and, more particularly, to their use as diagnostic reagents.
  • Harvey, Kirsten and N ras proteins are immunologically related proteins and are collectively termed p21. They are products of the ras family of cellular genes which are found in a wide variety of nucleated mammalian cells. The ras genes appear to be frequent targets of genetic alterations that can lead normal cells along the pathway to malignancy. Ras oncogenes have been identified in a wide array of premalignant and malignant cells. The three members of the ras gene family, H, Ki, and N, are very highly conserved at the amino-terminal two-thirds of the molecule, but diverge somewhat over the remaining one-third of the molecule near the carboxyl-terminal end.
  • N ras The third member of the ras family, N ras, was so designated because it was identified from a human neuroblastoma (Shimizu et al., PNAS (USA) 80:2112 (1983)). It has not yet been found to have a retroviral counterpart.
  • the p21 proteins consist of about 188-189 amino acids having a molecular weight of about 21,000 daltons.
  • Viral and cellular ras genes encode membrane bound proteins ( illingham et al., Cell 19:1005 ⁇ l 9BO) ) which bind guanine nucleotides (Scolnick et al. r PNAS (USA) 76:5355 (1979);
  • Ki gene has the capacity to encode two distinct proteins referred to as Ki2A and Ki2B. Since this variable region exists among amino acids 160-180 at the C-terminal end of the ras p21, then it is theoretically possible to generate monoclonal antibodies that could specifically bind the individual H, Ki2A, Ki2B, and N ras p21s.
  • European Patent Application No. 86107244.5 discloses polypeptides having amino acid sequences derived from the variable regions of ras H , ras N , ras ⁇ KA and ras ⁇ KB protein families, immunogenic compositions wherein these polypeptides are covalently attached to immunogenic carriers, antibodies produced from such immunogens wherein these antibodies are specific for the ras oncogene from which the polypeptide sequence was derived and immunoassays employing these antibodies to distinguish among the individual p21 ras oncogene families.
  • the peptide structures disclosed correspond to amino acids 171-189 and 170-189 of p21 H ras, 170- 186 of p21 N ras, 171-186 of p21 Ki4A ras, and 170-185 of p21 Ki4B ras. Although hybridoma technology was disclosed, it appears that the peptide antibodies generated were polyclonal antibodies.
  • the anti-p21 sera was prepared by affinity purifying the rabbit sera and evaluating their specificity by biochemical assays. The specificity of these reagents, however, is questionable.
  • hybrido a cell lines which were found to secrete monoclonal antibodies reactive with the H ras p21s and the subject of this invention were deposited in the American Type Tissue Culture Collection (ATCC) under the Budapest Treaty on March 25, 1988.
  • Hybridoma H-770-1.1.4 was designated ATCC No. HB 9673.
  • Hybridoma H-784-4.7.7 was designated ATCC No. HB 9675.
  • 5 Hybridoma H-873-3.5.3 was designated ATCC No. HB 9674.
  • the hybridoma cell lines which were found to secrete monoclonal antibodies reactive with the N ras p21s and the subject of this invention were deposited in the American Type Tissue Culture Collection (ATCC) J O under the Budapest Treaty on March 25, 1988.
  • Hybridoma N-821-1.1.9 was designated ATCC No. HB 9671.
  • Hybridoma N-838-1.1.6 was designated ATCC No. HB 9672.
  • Hybridoma K2A-1 was designated ATCC No. HB10072, date of deposit was March 30, 1989.
  • Hybridoma K2A-2 was designated ATCC No. HB10065, date 0 of deposit was March 30, 1989.
  • Hybridoma K2B-1 was designated ATCC No. HB10064, date of deposit was March 30, 1989.
  • Hybridoma K2B-2 was designated ATCC No. HB10055, date of deposit was March 29, 1989.
  • Hybridoma K2B-3 was designated ATCC No. HB10066, date 0 of deposit was March 30, 1989.
  • Hybridoma K2B-4 was designated ATCC No. HB10085, date of deposit was March 31, 1989.
  • Hybridoma K2B-5 was designated ATCC No. HB10056, ' date of deposit was March 29, 1989
  • Hybridoma K2B-6 was designated ATCC No. HB10067, date of deposit was March 30, 1989.
  • Hybridoma K2B-7 was designated ATCC No. HB10091, date of deposit was March 31, 1989.
  • monoclonal antibodies and i munoreactive fragments thereof specific for Harvey, Kirsten and N ras proteins has widespread diagnostic and therapeutic rami ications.
  • Such monoclonal antibodies can be used to detect, quantify, and classify the three ras families of proteins in cells, tissues, and other bodily substances such as serum plasma, urine, effusions, and feces.
  • these monoclonal antibodies and fragments can be used to diagnose, stage, monitor, and classify ras proteins in normal cells, malignant cells, premalignant cells as well as in a variety of bodily substances.
  • these monoclonal antibodies and fragments can be useful as therapeutic tools to deliver drugs, radionuclides, and the like to malignant areas to arrest development.
  • the subject of this invention is the induction, production, and characterization of monoclonal antibodies (Mabs) that react with the individual H, Ki2A, Ki2B, and N ras p21s.
  • Mabs described in this invention are valuable tools for the detection, quantitation and classification of the individual ras p21s in normal, neoplastic, preneoplastic and dysplastic cells, as well as in bodily fluids, using a variety of techniques.
  • the anti-H specific Mabs Balb/c x C57B1/6 mice were immunized on several occasions with a synthetic peptide coupled to carrier protein.
  • Mabs specific for the H ras p21 were raised using a synthetic peptide corresponding to amino acids
  • Mabs H-770-1.1.4, H-784-4.7.7, and H-873-3.5.3 were selected because of their reactivity with the H- specific peptide and because of their lack of reactivity with the N-specific peptide. These Mabs were also selected because of their reactivity and specificity for the H ras cellular p21s and because of their lack of reactivity with the other cellular p21s as determined by biochemical assays, such as, im unoblots, immunoprecipitation, immunohistochemistry and immunoassays.
  • mice were immunized on several occasions with a synthetic peptide coupled to carrier protein.
  • Mabs specific for the N ras p21s were raised using a synthetic peptide corresponding to amino acids 163-180 of the N ras p21.
  • Spleen cells from mice immunized with an N-specific peptide were fused with Sp2/0 mouse myeloma cells. Two weeks later the culture supernatants from the hybridomas were screened by ELISA.
  • Mabs N-821-1.1.9 and N-838-1.1.6 were selected because of their reactivity with the N- specific peptide and because of their lack of reactivity with the H-specific peptide. These Mabs were also selected because of their specificity and reactivity for the N ras cellular p21s and because of their lack of reactivity with other cellular p21s as determined by biochemical assays such as, immunoblots, immunoprecipitation, immunohistochemistry and immunoassays . In the case of Mabs to the Ki2A and Ki2B Balb/c x C57B1/6 mice were immunized with synthetic peptides coupled to carrier proteins.
  • Mabs specific for the Ki2A ras p21s were raised using synthetic peptides corresponding to amino acids 163-180. Spleen cells from mice immunized with the Ki2A-specific peptides were fused with S ⁇ 2/0 mouse myeloma cells. After two weeks the culture supernatants from the hybridomas were screened by ELISA. Mabs were selected because of their reactivity with the Ki2A-specific peptides and because of their lack of reactivity with the H-specific, N-specific, and Ki2B-specific peptides. It is believed that these Mabs have specificity and reactivity for the Ki2A ras cellular p21s and lack reactivity with other cellular p21s as can be determined by immunoassays.
  • Ki2B ras p21s Mabs specific for the Ki2B ras p21s were raised using a synthetic peptide corresponding to amino acids 164-175 of the Ki ras p21.
  • Spleen cells from mice immunized with Ki2B-s ⁇ ecific peptides were fused with
  • Mabs were selected because of their reactivity with the Ki2B specific peptides and because of their lack of reactivity with the H-specific, N- specific, and Ki2A-specific peptides. It is believed these Mabs have specificity and reactivity for the K12B ras cellular p21s and lack reactivity with other cellular p21s as can be determined by immunoassays.
  • H or Ha can be used interchangeably to designate the Harvey family of ras proteins.
  • K or Ki can be used interchangeably to designate the Kirsten family of ras proteins and the designations 2A and 2B are used interchangeably with 4A and 4B respectively.
  • Immunizations In the case of Mab H-770-1.1.4, Balb/c x C57B1/6 mouse designated 4607 was immunized with the H- specific peptide coupled to carrier protein Keyhole Limpet Hemacyanin (KLH) via the glutaraldehyde method of A. Kagan et al..
  • the H-specific peptide was composed of 18 amino acids corresponding to positions 163-180 of the ras H p21.
  • the structure of the immunizing peptide was as follows: ⁇ Isoleucine-arginine-glutamine-histidine- lysine-leucine-arginine-lysine-leucine-asparagine- proline-proline-aspartic acid-glutamic acid-serine- glycine-proline-glycine ⁇ O.
  • Balb/c x C57B1/6 mouse designated 4615 was immunized with the H- specific peptide coupled to carrier protein KLH.
  • Balb/c x C57B1/6 mouse designated 4606 was immunized with the H- specific peptide coupled to carrier protein KLH.
  • N-specific peptide was composed of 18 amino acids corresponding to positions 163-180 of the N ras p21.
  • the structure of the N ras peptide was as follows: l ⁇ Isoleucine- arginine—glutamine-tyrosine-arginine-methionine-lysine- lysine-leucine-asparagine-serine-serine-aspartic acid- aspartic acid-glycine-threonine-glutamic acid- glycine 180 .
  • the immunizing N and H-specific peptides were coupled to carrier proteins KLH and BTG respectively to enhance i munogenicity of the peptide.
  • the first inoculation consisted of the peptide-carrier conjugate mixed with Complete Freunds Adjuvant. Total protein inoculated was 500 micrograms. Subsequent inoculations were given at two-week intervals. Three days before fusion mice were given an intraperitoneal inoculation of the appropriate immunogen. In the case of Mabs to the Ki2A p21,
  • the peptide corresponding to amino acids 163-180 was composed of 18 amino acids and had the following structure: ⁇ -" ⁇ Isoleucine-arginine- glutamine-tyrosine-arginine-leucine-lysine-lysine- isoleucine-serine-serine-glutamic acid-glutamic acid- lysine-threonine-proline-glycine-cysteine!80 ⁇ It
  • the peptide corresponding to amino acids 170-184 is composed of 15 amino acids and has the following structure: ⁇ 7 ⁇ Lysine-isoleucine-serine- lysine-glutamic acid-glutamic acid-lysine-threonine- proline-gl cine-cysteine-valine-lysine-isoleucine- lysine 184 .
  • mice In the case of Mabs to the Ki2B p2l, Balb/c x C57B1/6 mice designated 5606, 5602 and 6061 were immunized with peptides corresponding to amino acids 164-175. Peptides corresponding to amino acids 163-180 and 168-183 of the Ki2B p21 protein can also be used as an immunogen. Peptides were again coupled to carrier proteins prior to inoculation.
  • the peptide corresponding to amino acid positions 163-180 of the Ki2B ras p21 is composed of 18 amino acids and had the following structure: ⁇ Isoleucine- arginine-lysine-histidine-lysine- glutamic acid-lysine-methionine-serine-lysine-aspartic acid-glycine-lysine-lysine-lysine-lysine-lysine- lysine 180 .
  • the peptide corresponding to amino acid positions 164-175 of the Ki2B ras p21 is composed of 12 amino acids and had the following structure: ⁇ Arginine- lysine-histidine-lysine-glutamic acid- lysine-methionine-serine-lysine-aspartic acid-glycine- lysine 17 ⁇ .
  • the peptide corresponding to amino acid positions 168-183 of the Ki2B ras p21 is composed of 16 amino acids and has the following structure: ⁇ - ⁇ Glutamic acid-lysine-methionine-serine-lysine- aspart ic acid-glycine-lysine-lysine-lysine-lysine- lysine-lysine-serine-lysine-threonine 8 .
  • Ki2A and Ki2B-specific peptides were coupled to carrier proteins KLH or BTG to enhance immunogenicity of the peptide.
  • Spleen cell suspensions were prepared in serumless DMEM-high glucose medium and mixed with myeloma cells at a ratio of 4:1. This cell mixture was centrifuged at 1200 x g for 10 minutes at room temperature. After removal of the supernatant, the cells were resuspended by gently tapping the tube. The fi ⁇ sion procedure was initiated by adding 1.0 ml of 45% w/v polyethylene glycol 3350 ( ⁇ aker) at 37°C over a 30-second period.
  • the cells were occasionally mixed with a pipette tip for 90 seconds and 5 ml of serumless DMEM-high glucose medium was added over a 3-minute period. This was followed by the addition of 14 ml of DMEM-high glucose supplemented with 10% fetal calf serum, L- glutamine, hypoxanthine, aminopterin and thymidine (referred to as HAT medium) . The HAT medium was added over a 1-minute period.
  • HAT medium Appropriate volumes of HAT medium were added to cells and then the cells were centrifuged at 800 x g for 7 minutes at room temperature. Supernatants were aspirated and the.cell pellet disrupted with 10 ml of HAT medium. Peritoneal cells from Balb/c x C57B1/6 were added and the final volume adjusted so that two hundred thousand spleen cells were dispensed to each well. Approximately 14 days later, tissue culture supernatants from wells containing hybridoma colonies were tested by ELISA for the desired reactivity with peptides conjugated to carrier proteins. Screening Procedures and ELISA Protocol
  • the H-specific peptide described above was conjugated to the BTG carrier protein while the N-specific peptide was conjugated to the KLH carrier protein.
  • the rationale for coupling peptides to different carrier proteins for immunization and screening was to avoid selecting antibodies reactive with the carrier protein.
  • peptide-carrier conjugate Prior to screening hybridoma supernatants, 500 mg of the peptide-carrier conjugate was dispensed to 96 well microtiter plates for overnight incubation at 37°C. After incubation, plates were washed and unbound sites on the plates were blocked with bovine serum albumin (BSA) .
  • BSA bovine serum albumin
  • GAMHRP was determined by adding 50 microliters of the substrate o-phenylenediamine (OPD) in phosphate buffer containing 0.15% hydrogen peroxide. HRP, in combination with its substrate OPD, results in a yellow colored product. Development of the yellow product was allowed to occur at room temperature for
  • Mabs N-821-1.1.9 and N-838-1.1.6 were found to be reactive with the N ras specific peptide coupled to carrier protein and not reactive with the H, Ki2A and Ki2B peptides coupled to carrier protein.
  • Mabs Ki2A-l and Ki2A-2 were found to be reactive with the Ki2A specific peptide and not reactive with the H, N and Ki2B specific peptides also coupled to carrier protei .
  • Ki2B-l, Ki2B-2, Ki2B-3, Ki2B-4, Ki2-5, Ki2B- 6 and Ki2B-7 were found to be reactive with the Ki2B specific peptide (amino acids 164-175) and not reactive with the H, N, and Ki2A specific peptides also coupled to carrier proteins.
  • Ki2B specific peptide amino acids 164-175
  • the anti-N and anti-H Mabs were tested for specificity with free peptides (i.e., peptides not coupled to carrier proteins) to ensure that the Mabs were specific for the peptides of interest and not reactive with the bond attaching the carrier protein to the peptide.
  • Table 1 summarizes ELISA results of testing the anti-H and anti-N Mabs against the H, N, Ki2A and Ki2B specific peptides.
  • Ki2A and Ki2B are tested for specificity with peptides not coupled to carrier proteins. It is believed that Ki2A and Ki2B Mabs are specific for the peptides of interest and not reactive with the bond attaching the carrier protein to the peptide. Reactivity and Specificity of the Anti-Peptide Mabs for Cellular ras Proteins
  • Cell line designated 3T3-Nras overexpressed the N ras p21 protein.
  • Cell line (KNRK) designated 3T3-Ki2A expressed the viral form of the Ki p21 protein.
  • Cell line (SW480) designated 3T3-Ki2B expressed the cellular form of the Ki ras protein.
  • Nonradioactive extracts of the above-mentioned cells were incubated with an anti-H Mab, anti-N Mab, or an anti-p21 Mab designated Ras 10 for 1 hour.
  • the Ras 10 anti-p21 Mab reacted with normal and oncogenic forms of the ras p21 proteins, as discussed in co- pending patent application having Serial Number 071,175, filed on July 8, 1987.
  • the anti-p21 monoclonal antibodies described therein were generated by using a recombinant Ha-ras p-21 protein having an arginine amino acid as position 12 instead of glycine which is present in the normal Ha-ras p21 protein.
  • Hybridomas secreting these anti-p21 monoclonal antibodies were designated ras 8, ras 10, and ras 11, all of which were deposited in the American Type Tissue Culture Collection (ATCC) under the Budapest Treaty.
  • Hybridoma ras 8 was designated HB-9428
  • hybridoma ras 10 was designated HB-9426
  • hybridoma ras 11 was designated HB-9427.
  • membranes were incubated for 1 hour with either an anti-p21 Mab ras 10 or mouse serum which served as a negative control. After incubation with Ras 10 or a negative control antibody, membranes were washed three times with PBS-NP-40. Membranes were then incubated with an anti-mouse immunoglobulin coupled to HRP for 1 hour to detect the mouse Mabs. Membranes were then washed three times with PBS-NP-40 and incubated with 4-chloro-l-naphthol substrate to complete the reaction.
  • the ability to react in a particular analytical format is believed to be a function of how the epitope is presented.
  • two antibodies may recognize the same epitope and, yet, react differently in, for example, an immunoblot format.
  • Epitope specificity can be determined using conventional techniques such as a competition immunoassay.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
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  • Peptides Or Proteins (AREA)
EP19890905339 1988-04-22 1989-04-20 Monoclonal antibodies specific for the harvey, kirsten and n ras proteins and their use as diagnostic reagents Withdrawn EP0428518A4 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US334822 1981-12-28
US18519488A 1988-04-22 1988-04-22
US185194 1988-04-22
US33482289A 1989-04-11 1989-04-11

Publications (2)

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EP0428518A1 true EP0428518A1 (de) 1991-05-29
EP0428518A4 EP0428518A4 (en) 1992-02-19

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EP19890905339 Withdrawn EP0428518A4 (en) 1988-04-22 1989-04-20 Monoclonal antibodies specific for the harvey, kirsten and n ras proteins and their use as diagnostic reagents

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EP (1) EP0428518A4 (de)
AU (1) AU3533089A (de)
WO (1) WO1989010375A1 (de)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE68929511T2 (de) * 1988-04-22 2004-11-18 Bayer Corp. Nachweis, Quantifizierung und Klassifizierung von RAS-Proteinen in Körperflüssigkeiten und Geweben
JP2519801B2 (ja) * 1989-07-18 1996-07-31 住友電気工業株式会社 突切り用工具

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0108564A1 (de) * 1982-11-04 1984-05-16 The Regents Of The University Of California Methoden zur Feststellung von Oncogenese

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0203587A3 (de) * 1985-05-30 1989-08-09 F. Hoffmann-La Roche Ag Ras-Oncogene-Peptide und Antikörper

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0108564A1 (de) * 1982-11-04 1984-05-16 The Regents Of The University Of California Methoden zur Feststellung von Oncogenese

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ONCOGENE, vol. 1, no. 2, May 1987, pages 131-142, The Macmillan Press Ltd, Basingstoke, GB; D. BIZUB et al.: "Antisera to the variable region of ras oncogene proteins, and specific detection of H-ras expression in an experimental model of chemical carcinogenesis" *
See also references of WO8910375A1 *

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WO1989010375A1 (en) 1989-11-02
EP0428518A4 (en) 1992-02-19
AU3533089A (en) 1989-11-24

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