EP0417196A1 - Peptide immunogene de l'interleukine-3 humaine et anticorps monoclonaux relatifs - Google Patents

Peptide immunogene de l'interleukine-3 humaine et anticorps monoclonaux relatifs

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Publication number
EP0417196A1
EP0417196A1 EP19890906953 EP89906953A EP0417196A1 EP 0417196 A1 EP0417196 A1 EP 0417196A1 EP 19890906953 EP19890906953 EP 19890906953 EP 89906953 A EP89906953 A EP 89906953A EP 0417196 A1 EP0417196 A1 EP 0417196A1
Authority
EP
European Patent Office
Prior art keywords
peptide
human interleukin
antibody
vol
pgs
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.)
Pending
Application number
EP19890906953
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German (de)
English (en)
Inventor
John S. Abrams
Michael K. Pearce
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.)
Schering Biotech Corp
Original Assignee
Schering Biotech Corp
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 Schering Biotech Corp filed Critical Schering Biotech Corp
Publication of EP0417196A1 publication Critical patent/EP0417196A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5403—IL-3
    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]

Definitions

  • the invention relates generally to peptides useful in the generation of antibodies, and more particularly to an immunogenic peptide of human interleukin-3 and monoclonal antibodies specific therefor.
  • Circulating blood cells are constantly replaced by newly developed cells.
  • Replacement blood cells are formed in a process termed hematopoiesis which involves the production of at least eight mature blood cell lineages: red blood cells (erythrocytes) , macrophages (monocytes) , eosinophilic granulocytes, megakaryocytes (platelets) , neutrophilic granulocytes, basophilic granulocytes (mast cells) , T lymphocytes, and B lymphocytes: Burgess and Nicola, Growth Factors and Stem Cells (Academic Press, New York, 1983) .
  • CSFs colony stimulating factors
  • glycoproteins are so named because of the in vivo and in vitro assays used to detect their presence.
  • Techniques for the clonal culture of hematopoietic cells in semisolid culture medium have been especially important in the development of in vitro assays.
  • individual progenitor cells i.e., cells developmentally committed to a particular lineage, but still capable of proliferation
  • proliferate to form a colony of maturing progeny in a manner which is believed to be essentially identical to the comparable process in vivo.
  • CSFs The role of CSFs in hematopoiesis is the subject of many recent reviews, e.g.: Metcalf, The Hemopoietic Colony Stimulating Factors (Elsevier, New York, 1984); Metcalf, Proc. R. Soc. Lond. B, Vol. 230, pgs. 389-423 (1987); Clark et al., Science, Vol. 236, pgs. 1229-1237 (1987); and Sachs, Science, Vol. 238, pgs. 1374-1379 (1987).
  • CSFs As more CSFs become available, primarily through molecular cloning, interest has heightened in finding clinical applications for them. Because of physiological similarities to hormones (e.g., soluble factors, growth mediators, action via cell receptors) , potential uses of CSFs have been analogized to the current uses of hormones: e.g. Dexter, Nature, Vol. 321, pg. 198 (1986) .
  • hormones e.g., soluble factors, growth mediators, action via cell receptors
  • IL-3 interleukin-3
  • Yokota et al. Proc. Natl. Acad. Sci., Vol. 81, pgs. 1070-1074 (1984); Fung et al., Nature, Vol. 307, pgs. 233-237 (1984); Hapel et al., Blood, Vol. 65, pgs. 1453-1459 (1985); Ihle et al., J. Immunol., Vol. 129, pgs. 2431-2436 (1982); Ihle et al., J. Immunol. , Vol.
  • Murine IL-3 exhibits a broad spectrum of CSF activities, for it is capable of stimulating proliferation and development of the progenitor cells of all the recognized myeloid lineages, e.g.: Dexter, Nature, Vol. 309, pgs. 746-747 (1984) * ; Ihle et al., Lymphokines, Vol. 9, pgs. 153-200 (1984).
  • peptide antibodies are the most expedient manner of obtaining monoclonal antibodies specific for a protein of interest, particularly when the protein's gene has been cloned and sequenced, but sufficient quantities of the purified protein are not yet available for immunizations, e.g.: Walter et al., Genetic Engineering, Vol. 5, pgs. 61-91 (1983); Lerner et al. , Proc. Natl. Acad. Sci., Vol. 78, pgs. 3403-3407 (1981); and Bulinski, Internatl. Rev. Cytol., Vol. 103, pgs. 281-302 (1986).
  • a major problem of this approach is how to select from the deduced amino acid sequence a peptide which corresponds to an antigenic determinant of the protein of interest.
  • a large number of factors apparently determine which sequences of amino acids within a protein make up its antigenic determinants, and at present only a handful of imperfect criteria are available for correlating particular amino acid sequences with antigenic determinants, e.g.: Hopp et al. , Proc. Natl. Acad. Sci., Vol. 76, pgs. 3824-3828 (1981); Westof et al. , Nature, Vol. 311, pgs. 123-126 (1984); Fraga, Can. J. Che . , Vol. 60, pgs.
  • immunogens comprising conjugates between a carrier and the above peptide, and monoclonal antibodies specific for the above peptide.
  • imunogen refers to a substance which is capable of causing an immune response.
  • carrier refers to any substance which when chemically conjugated to a peptide of the invention permits a host organism immunized with the resulting conjugate to generate antibodies specific for the conjugated peptide.
  • Carriers include red blood cells, bacteriophages, proteins, or synthetic particles such as agarose beads.
  • carriers are proteins, such as serum albumin, gamma- globulin, keyhole limpet hemocyanin, throglobulin, ovalbumin, fibrinogen, yoglobin, or the like.
  • the peptide of the invention can be synthesized by standard techniques, e.g. Stewart and Young, Solid Phase Peptide Synthesis, 2nd Ed, (Pierce Chemical Company, Rockford, IL, 1984).
  • a commercial automated synthesizer is used, e.g. Vega Biochemicals (Tucson, AZ) models 296A or B, or Applied Biosystems, Inc. (Foster City, CA) model 43OA.
  • the peptide of the invention can be assembled by solid phase synthesis on a cross-linked polystyrene support starting from the carboxyl terminal residue and adding amino acids in a stepwise fashion until the entire 19-residue chain has been formed.
  • the following references are guides to the chemistry employed during synthesis: Merrifield, J. Amer. Chem. Soc. , Vol. 85, pg. 2149 (1963); Kent et al., pg. 185, in Peptides 1984, Ragnarsson, Ed. (Almquist and Weksell, Sweden, 1984) ; Kent et al., pg.
  • N -t-butyloxycarbonyl-amino acids were used with appropriate side-chain protecting groups stable to the conditions of chain assembly but labile to strong acids. After assembly of the protected peptide chain, the protecting groups were removed and the peptide-anchoring bond was cleaved by the use of low and then high concentrations of anhydrous hydrogen fluoride in the presence of a thioester scavenger: Tam et al., J. Amer. Che . Soc. , Vol. 105, pg. 6442 (1983).
  • Side-chain protecting groups used were Asp(OBzl), Glu(OBzl), Ser(Bzl), Thr(Bzl), Lys(Cl-Z), Tyr (Br-Z) , Arg(N G Tos), Cys(4-MeBzl) , and His(ImDNP).
  • the remaining amino acids had no protecting groups on their side-chains.
  • the peptide- resin was washed with DCM and then neutralized twice with 10 percent dusopropylethylamine (DIEA) (Aldrich) in dimethylformamide (DMF) (Applied Biosystems) , for 1 minute each. Neutralization was followed by washing with DMF. Coupling was performed with the preformed symmetric anhydride of the amino acid in DMF for 16 minutes.
  • the preformed symmetric anhydride was prepared on the synthesizer by dissolving 2 mmol of amino acid in 6 ml of DCM and adding 1 mmol of dicyclohexycarbodiimide (Aldrich) in 2 ml of DCM.
  • the activated amino acid was transferred to a separate vessel and the DCM was evaporated by purging with a continuous stream of nitrogen gas.
  • the DCM was replaced by DMF (6 ml total) at various stages during the purging.
  • the peptide-resin was washed with DCM, 10 percent DIEA in DCM, and then with DCM.
  • the same amino acid and the activating agent, dicyclohexylcarbodii ide were transferred sequentially to the reaction vessel. After activation in situ and coupling for 10 minutes, sufficient DMF was added to make a 50 percent DMF-DCM mixture, and the coupling was continued for 15 minutes.
  • Arginine was coupled a ⁇ a preformed ester with hydroxybenzotriazole (Aldrich) in DMF for 60 minutes and then recoupled in the same manner as the other amino acids. Asparagine and glutamine were coupled twice as preformed hydroxybenzotriazole esters in DMF: 40 minutes for each coupling. For all residues, the resin was washed after the second coupling and a sample was automatically taken for monitoring residual uncoupled ⁇ -amine by quantitative ninhydrin reaction (Sarin et al., cited above).
  • the four most commonly used methods for attaching a peptide to a carrier use (1) glutaraldehyde for amino coupling, e.g. as disclosed by Kagan and Glick, in Jaffe and Behrman, eds. Methods of Hormone Radioimmunoassay, pgs. 328-329 (Academic Press, N.Y. 1979), and Walter et al., Proc. Natl. Acad. Sci., Vol. 77, pgs. 5197-5200 (1980); (2) water-soluble carbodiimides for carboxyl-to-amino coupling, e.g. as disclosed by Hoare et al., J. Biol. Che . , Vol. 242, pgs.
  • BDB bis-diazobenzidine
  • MBS maleimidobenzoyl-N-hydroxysuccinimide ester
  • a general rule for selecting an appropriate method for coupling a given peptide to a protein carrier can be stated as follows: the amino acid chosen for coupling should occur only once in the sequence, preferably at the appropriate end of the segment. For example, BDB should not be used if a tyrosine residue occurs in the mam part of a sequence chosen for its potentially antigenic character. Similarly, centrally located ly ⁇ ines rule out the glutaraldehyde method, and the occurrence of aspartic or glutamic acid will frequently exclude the carbodiimide method. On the other hand, suitable amino acid residues can be positioned at either end of a chosen sequence segment as attachment sites, whether or not they naturally occur there in the chosen protein sequence.
  • the selected immunogenic peptide fragment does not extend to the carboxyl or amino terminus of IL- 3, then its unattached end may present a terminus that does not occur in IL-3; consequently the immunogenic peptide fragment will differ significantly at that terminus from the native IL-3. Any problems caused by this difference can be obviated to some extent if the carboxy terminus is chosen for attachment to the protein carrier and the free amino end is acetylated.
  • the coupling efficiency of the peptide of formula I to the carrier protein is conveniently measured by using a radioactively labeled peptide, prepared either by using a radioactive amino acid for one step of the synthesis or by labeling the completed peptide by the iodination of a tyrosine residue.
  • a radioactively labeled peptide prepared either by using a radioactive amino acid for one step of the synthesis or by labeling the completed peptide by the iodination of a tyrosine residue.
  • the presence of tyrosine in the peptide also allows one to set up a sensitive radioimmunoassay, if desirable. Therefore, it may be desirable to introduce tyrosine as a terminal residue if it is not part of the peptide sequence of the invention.
  • Preferred carriers are proteins, and preferred protein carriers include bovine serum albumin, myoglobulin, ovalbumin (OVA) , keyhole limpet hemocyanin (KLH) , or the like.
  • OVA ovalbumin
  • KLH keyhole limpet hemocyanin
  • Peptides can be linked to KLH through cysteines by MBS as disclosed by Liu et al.. Biochemistry, Vol. 18, pgs. 690-697 (1979) .
  • the peptides are dissolved in phosphate-buffered saline (pH 7.5), 0.1M sodium borate buffer (pH 9.0) or 1.0M sodium acetate buffer (pH 4.0).
  • the pH for the dissolution of the peptide is chosen to optimize peptide solubility.
  • the content of free cysteine for soluble peptides is determined by the method of Ellman, Arch. Biochem. Biophys., Vol. 82, pg. 7077 (1959) .
  • KLH-MB is then reacted with 5 mg peptide dissolved in 1 ml of the chosen buffer.
  • the pH is adjusted to 7-7.5 and the reaction is stirred for 3 hr at room temperature.
  • Coupling efficiency is monitored with radioactive peptide by dialysis of a ⁇ a ple of the conjugate against phosphate-buffered saline; it typically ranges from 8% to 60%.
  • polyclonal or monoclonal antibodies are produced by standard techniques, e.g.: as disclo ⁇ ed by Campbell in Monoclonal Antibody Technology (Elsevier, New York, 1984); Hurrell, ed., Monoclonal Hybridoma Antibodies: Techniques and Application ⁇ (CRC Pre ⁇ s, Boca Raton, FL, 1982) ; Schreier et al. Hybridoma Techniques (Cold Spring Harbor Laboratory, New York, 1980); U.S. Patent 4,562,003; or the like. In particular, U.S. Patent 4,562,003 i ⁇ incorporated by reference.
  • the fir ⁇ t step is to immunize a host animal to obtain a source of B lymphocytes.
  • the B lymphocytes are fused with an appropriate immortalizing cell line to form hybridoma ⁇ that secrete monoclonal antibodies.
  • Immortalizing cell line ⁇ are usually tumor cell lines, such as myelomas.
  • the host animals are rodents, and the immortalizing cell line al ⁇ o i ⁇ preferably derived from rodent cell ⁇ , e ⁇ pecially from the same rodent species.
  • hybridomas are ⁇ creened for tho ⁇ e producing antibodies against the peptide of the invention. Immunization, harvesting of lymphocytes, and cell fusion are all techniques well known in the art.
  • Immunization is carried out by a regimen of repeated injections into the host animal of the purified peptide- carrier conjugate, usually mixed with a suitable adjuvant. Immunization can be optimized by varying several factors, including the amount of antigen used for the primary injection and subsequent boosts, the route of injection, the time schedule for injecting and bleeding, and the use of adjuvant, e.g. Freund's complete or incomplete adjuvant. Techniques for fusion are also well known in the art, and in general involve mixing the cells with a fusing agent such as, most commonly, polyethylene glycol.
  • Successful hybridoma formation is assessed and selected by standard procedures such as, for example, HAT selection. From among proliferating hybridomas, those successfully secreting the desired antibody are selected by assaying the culture medium for their presence. Ordinarily this is done using immunoreaction-based as ⁇ ays, for example Western blot, ELISA, or RIA assay ⁇ .
  • the antibodie ⁇ can be recovered from the medium using standard protein purification techniques.
  • Both polyclonal and monoclonal antibodie ⁇ can be screened by ELISA.
  • the test is based on the tendency of acromolecules to adsorb nonspecifically to plastic. The irreversibility of this reaction, without loss of immunological activity, allows the formation of antigen- antibody complexes with a simple separation of such complexes from unbound material.
  • peptide conjugated to a carrier different from that used in immunization, or free peptide alone is adsorbed to the wells of a 96-well microtiter plate.
  • the adsorbed antigen is then allowed to react in the wells with dilutions of anti-peptide serum. Unbound antibody is washed away, and the remaining antigen-antibody complexes are allowed to react with antibody specific for the IgG of the immunized animal; this second antibody is conjugated to an enzyme such as alkaline phosphatase.
  • a visible colored reaction product produced when the enzyme substrate is added indicates which wells have bound antipeptide antibodies.
  • the use of a spectrophotometer allows better quantification of the amount of peptide- specific antibody bound. High-titer antisera yield a linear titration curve between 10 ⁇ 3 and 10 ⁇ 5 dilution ⁇ .
  • (C) indicates a cysteine bridge to the carrier protein.
  • OVA ovalbumin
  • MYO myoglobulin
  • 50 mg of ovalbumin (OVA) and 50 mg of myoglobulin (MYO) were each dissolved in 10 ml of 0.1M sodium bicarbonate, and reacted with 1 ml of 0.12 iodoacetamide solution (88 mg of iodoacetamide dissolved in 4 ml 0.1M sodium bicarbonate) for 1 hour at room temperature in a 15 ml Falcon tube (Falcon Plastics, Oxnard, CA) , or the like.
  • Each reaction mixture was dialyzed overnight again ⁇ t 4 liters of O.IM sodium bicarbonate at 4 ⁇ C.
  • the dialyzed iodoacetylated OVA and MYO were recovered, separately mixed with equal volumes (preferably 2 ml) of borate reduction buffer containing the peptide, and incubated overnight at room temperature.
  • the resulting conjugates were analyzed by SDS-PAGE (12.5% gel).
  • the conjugate-containing solution was diluted with PBS to 1 mg/ml, filtered sterile, and aliquots of convenient volumes (e.g. 500 microliters) were taken for immunizations, and/or stored at 4°C. Polyclonal antisera against the MYO conjugate were produced in rats.
  • the immunization schedule was as follows: Initially, 100 ⁇ g of the MYO conjugate in 0.5 ml PBS was mixed with 0.5 ml of Freund's Complete Adjuvant (FCA) and injected i.p. into the rat. Additionally, i.p. injections of identical composition were given on days 17 and 86, after which a positive reaction against PNLEAFNRAVKSLQNASAI was detected by ELISA.
  • FCA Freund's Complete Adjuvant
  • FCA Freund's Complete Adjuvant
  • FCA Freund's Complete Adjuvant
  • the rat polyclonal anti-serum was also used in a Western blot analy ⁇ is of electrophoretically separated recombinant human IL-3 (Otsuka et al., cited above). A commercially-obtained sheep anti-rat immunoglobulin reagent was used as the second-stage label.
  • the protein was transferred electrophoretically to a nitrocellulose membrane overnight at 0.2 A in 20 mM Tris base, 150 mM glycine and 20% methanol, at 4°C.
  • the membrane was blocked in 100 ml of 0.5% BSA in PBS.
  • the first stage antibodies were dilutions of the rat antisera against human IL-3 in PBS containing 0.1% BSA and 0.05% Tween 20.
  • the membrane was incubated in 50 ml of this solution for 2 hours and then washed in three change ⁇ of PBS-BSA-Tween buffer for 20 min. each.
  • a 50- ⁇ l volume of 1 5 -I-labeled sheep anti-rat Ig in 50 ml of PBS-BSA-Tween was used as the second-stage labeled antibody.
  • the blots were incubated for 2 hours and then washed again as described above. They were then dried briefly and exposed to X-ray film. The human recombinant IL-3 band was successfully identified by the antiserum.
  • the rat of Example I was given a final i.p. injection and an i.v. injection of identical composition to the prior injections (200 ⁇ g conjugate in all) on day 135.
  • the cell suspension (3.5 x 10 ⁇ 5 cells/ml) in HAT medium was distributed into 40 96-well plates.
  • Ten days later hybridoma supernatants were tested for their ability to bind to human IL-3 immobilized directly on microtiter plates (indirect ELISA) .
  • Bound antibody was detected by peroxidase conjugated goat anti-rat immunoglobulin with a standard protocol. Hybridomas secreting antibodies reacting with IL-3 were cloned by limiting dilution. MP3.8A5.12 was one such hybridoma selected by these procedure ⁇ . Antibodies from MP3.8A5.12 were determined to be of the IgG 2a isotype.
  • the hybridoma can be stored (e.g. -70°C in culture medium with 10% DMSO) and cultured using standard mammalian cell culture techniques (e.g., RPMI 1640 with 10% fetal bovine serum, supplemented with 1 mM glutamine and 50 mM 2- mercaptoethanol) .
  • the invention further provides a kit for detecting the presence of human interleukin-3 in a ⁇ ample suspected of containing human interleukin-3, the kit comprising: a first monoclonal antibody specific for a first antigenic determinant on human interleukin-3; a second antibody selected from the group consisting of a polyclonal antibody composition specific for human interleukin-3 and a second monoclonal antibody specific for a second antigenic determinant on human interleukin-3, the second antigenic determinant being different from the first antigenic determinant; a support means; and a signal-generating means.
  • the first monoclonal antibody can be the monoclonal antibody produced by hybridoma MP3.8A5.12 and the second antibody can be a polyclonal antibody composition specific for human interleukin-3.
  • the signal-generating means can comprise an enzyme operationally associated with said first monoclonal antibody, the enzyme being selected from the group consi ⁇ ting of peroxida ⁇ e, beta-galactosidase, and alkaline phosphatase. The general principle ⁇ for the pre ⁇ entation of such kits are well known.
  • the deposit has been modified to conform to the requirements of the Budapest Treaty on the Deposit of Microorganisms.

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Abstract

Peptide immunogène de l'interleukine-3 humaine (IL-3) à partir duquel on peut produire des anticorps peptidiques monoclonaux. Le peptide ci-décrit correspond à une sous-séquence interne de la protéine de l'IL-3. L'invention comprend également des conjugués du peptide immunogène et des supports, ainsi que des anticorps monoclonaux spécifiques pour le peptide immunogène.
EP19890906953 1988-05-26 1989-05-23 Peptide immunogene de l'interleukine-3 humaine et anticorps monoclonaux relatifs Pending EP0417196A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US19911088A 1988-05-26 1988-05-26
US199110 1994-02-22

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EP0417196A1 true EP0417196A1 (fr) 1991-03-20

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Family Applications (2)

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EP89305174A Ceased EP0351944A1 (fr) 1988-05-26 1989-05-23 Anticoprs monoclonaux contre peptides immunogènes humains d'interleukine-3
EP19890906953 Pending EP0417196A1 (fr) 1988-05-26 1989-05-23 Peptide immunogene de l'interleukine-3 humaine et anticorps monoclonaux relatifs

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EP89305174A Ceased EP0351944A1 (fr) 1988-05-26 1989-05-23 Anticoprs monoclonaux contre peptides immunogènes humains d'interleukine-3

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EP (2) EP0351944A1 (fr)
JP (1) JPH03504131A (fr)
AU (1) AU3752489A (fr)
WO (1) WO1989011489A1 (fr)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8911489A1 *

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WO1989011489A1 (fr) 1989-11-30
JPH03504131A (ja) 1991-09-12
AU3752489A (en) 1989-12-12
EP0351944A1 (fr) 1990-01-24

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