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WO1996006856A1 - Anticorps a peptides et anti-peptides du recepteur dopaminergique - Google Patents

Anticorps a peptides et anti-peptides du recepteur dopaminergique Download PDF

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Publication number
WO1996006856A1
WO1996006856A1 PCT/US1995/011127 US9511127W WO9606856A1 WO 1996006856 A1 WO1996006856 A1 WO 1996006856A1 US 9511127 W US9511127 W US 9511127W WO 9606856 A1 WO9606856 A1 WO 9606856A1
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Prior art keywords
peptide
receptor
dopamine
antibodies
peptides
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PCT/US1995/011127
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Robert Webber
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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/286—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against neuromediator receptors, e.g. serotonin receptor, dopamine receptor

Definitions

  • the present invention relates to synthetic peptide analogues to regions of difference in dopamine receptors and to the anti-peptide antibodies which can be elicited with the peptide analogues.
  • Dopamine is a neurotransmitter in the central nervous system of the mammalian brain. Dopamine is believed to be involved in the regulation of a variety of functions including motor coordination, emotional stability, and reproduction regulation.
  • D 1 and D 5 receptors Five (5) different sub-types of dopamine receptors have been mapped and described. These receptors have been classified into two (2) major families: the D 1 family which includes the D 1 and D 5 receptors, and the D 2 family which includes the D 2 , D 3 , and D 4 receptors.
  • dopamine receptors are members of the G protein-coupled receptor gene superfamily, each having seven transmembrane spanning domains.
  • the D 2 receptor exists in two distinct forms (the long and short forms) which differ in the presence or absence of a 29 amino acid long segment. This difference is due to alternative splicing of the RNA.
  • Each of the distinct forms of the dopamine receptor possesses high sequence ho ology to all the other receptor forms.
  • the occurrence of high sequence homology has made the development of isoform specific antibodies difficult in the past, if not nearly impossible, by the use of the individual receptors themselves.
  • the present invention provides for synthetic peptide analogues to regions of difference of the dopamine receptors and anti-peptide antibodies elicited thereby. Peptide analogues to various regions of difference of dopamine receptors were constructed.
  • peptide analogues have been used to elicit antibodies after conjugation onto carrier proteins.
  • Each antiserum obtained from rabbits was tested by ELISA for the production of antibodies specific for the synthetic peptide analogue used as the im unogen.
  • the antisera resulting positively were tested for their ability to recognize mature whole protein in ELISA's and the denatured whole protein in SDS-PAGE/western immunoblots in whole rat brain homogenate, whole mouse brain homogenate, PC-12 cell lysate, and NRK cell lysate.
  • the antisera were also tested for their ability to immunostain fixed PC-12 cells and fixed NRK cells. Specificity was assessed by the ability of the immunogen peptide to block the anti-peptide antibody binding in these various assays.
  • Another object of the present invention is to provide dopamine receptor isoform specific anti-peptide antibodies specific for the regions of difference in the dopamine receptors which will be employed as diagnostic entities for disease states such as Parkinson's disease.
  • a further object of the present invention is to provide dopamine receptor peptides peculiar to the D.,-D 5 receptors of dopamine which may be employed in blocking experiments to show specificity.
  • FIG. 1 shows the amino acid sequences for peptides which are analogues for the D, dopamine receptor isoform.
  • FIG. 2 shows the amino acid sequences for peptides which are analogues for the D 2 dopamine receptor isoform.
  • FIG. 3 shows the amino acid sequences for peptides which are analogues for the D 3 dopamine receptor isoform.
  • FIG. 4 shows the amino acid sequences for peptides which are analogues for the D 4 dopamine receptor isoform.
  • FIG. 5 shows the amino acid sequences for peptides which are analogues for the D 5 dopamine receptor isoform.
  • FIG. 6 is a graphical representation of the ELISA titration of five dopamine " ⁇ receptor anti-peptide antisera for Rat Brain Homogenate e ">00 ngm/well.
  • FIG. 7 is a grap) al representation of the ELISA titration of six dopamine L 2 receptor anti-peptide antisera for Rat Brain Homogenate at 200 ngm/well.
  • FIG. 8 is a graphical representation of the ELISA titration of four dopamine D 2 receptor anti-peptide antisera for Rat Brain Homogenate at 200 ngm/well.
  • FIG. 9 is a graphical representation of the ELISA titration of four dopamine D 3 receptor anti-peptide antisera for Rat Brain Homogenate at 200 ngm/well.
  • FIG. 10 is a graphical representation of the ELISA titration of six dopamine D 4 receptor anti-peptide antisera for Rat Brain Homogenate at 200 ngm/well.
  • FIG. 11 is a graphical representation of the ELISA titration of five dopamine D 5 receptor anti-peptide antisera for Rat Brain Homogenate at 200 ngm/well.
  • FIG. 12 is a graphical representation of the ELISA titration of the peptide D 2L (243-254) cyclized antiserum for four different tissue and cell preparations at 200 ngm protein/well.
  • FIG. 13 is a graphical representation of the ELISA titration of the peptide D 2 (Acl75-182) antiserum for four different tissue and cell preparations at 200 ngm protein/well.
  • FIG. 14 is a graphical representation of the ELISA titration of the peptide D 5 (Ac23-35Cys 36 ) antiserum for four different tissue and cell preparations at 200 ngm protein/well.
  • FIG. 15 is a graphical representation of the ELISA titration of the peptide D 5 (Ac209-217) antiserum for four different tissue and cell preparations at 200 ngm protein/well.
  • FIGS. 16 A-O are photographs showing the indirect immunofluorescent staining of specific dopamine receptor isoforms by certain anti-peptide antibodies identified as numbered in Table II, in conjunction with FITC conjugated second antibody, described in Example 7.
  • FIG. 17 is a photograph of an SDS-PAGE/western immunoblot of PC-12 cell lysate with one of the D2, D3, and D5 isoform specific anti-peptide antibodies as described in Example 6.
  • Figs. 1-5 represent various synthetic peptide analogues constructed for each of the five isoforms of the dopamine receptor.
  • peptide depicted in Table I was synthesized by solid phase peptide synthesis utilizing the Fmoc protecting strategy.
  • Figs. 1-5 represent the amino acid sequences of Table I.
  • the Ac designation of Table I indicates acetylation of the amino terminus for the purpose of eliminating the charged end group in certain instances.
  • the synthetic peptides were cleaved from the solid support resin, isolated, and purified by standard procedures including preparative HPLC. The peptides were also analyzed for purity by analytical HPLC and for composition by amino acid analysis.
  • PS-3515 (Ac-165-173Cy; .174 AC-K-A-K-P-T-S-P-S-D-C
  • PS-3523 (Ac-25-34 ) Ac-S-D-G-K-A-D-R-P-H-Y 10, PS-3524 D 2L N-C-T-H-P-E-D-M-K-L-C-T
  • PS-3525 (Ac-25-34Cys 3 ;, 5 ⁇ 1) Ac-S-D-G-K-A-D-R-P-H-Y-C 12.
  • PS-3526 (Cys .2 ⁇ : 7 M 1-272-282 ) C-A-A-R-R-A-Q-E-L-E-M-E 13.
  • PS-3527 ( 2-12Cys 1 1 ⁇ 3) D-P-L-N-L-S- -Y-D-D-D-C 14.
  • PS-3528 (Ac-19-32Cys 3 J 3 ⁇ ) > Ac-S-R-P-F-N-G-S-D-G-K-A-D-R-P-C 15.
  • PS-3529 (AC-175-182 ) Ac-N-N-A-D-Q-N-E-C
  • PS-3531 (22-32) G-A-S-Q-A-R-P-H-A-Y-Y 17, PS-3532 (2-lOCys 1 ) A-S-L-S-Q-L-S-S-H-C 18, PS-3533 (Ac-17-29Cys 30 ) Ac-A-E-N-S-T-G-A-S-Q-A-R-P-H-C 19, PS-3534 (Ac-173-181) Ac-N-T-T-G-D-P-T-V-C
  • PS-3541 (Ac-22-35) AC-A-S-A-G-A-S-A-G-L-A-G-Q-G-A 21
  • PS-3542 (Ac-22-35Cys 36 ) Ac-A-S-A-G-A-S-A-G-L-A-G-Q-G-A-C 22
  • PS-3543 (2-10) G-N-R-S-T-A-D-A-D 23.
  • PS-3544 (Ac-l6-30Cys 31 ) Ac-R-G-P-A-A-G-A-S-A-G-A-S-A-G-L-C
  • PS-3545 (Ac-176-185) Ac-D-V-R-G-R-D-P-A-V-C
  • PS-3553 (Ac-23-35Cys 36 ) Ac-Q-G-N-A-V-G-G-S-A-G-A-P-P-C
  • Peptides numbered 1, 2, 4-6, 8-12, 14, 15, 18-21, 23-25, and 28-30 in Table I may be employed in the present application with the amino terminus acetylated or un- acetylated.
  • each synthetic peptide of Table I was conjugated onto a carrier protein, either bovine thyroglobulin (thyro) or keyhole limpet hemocyanin (KLH) .
  • the peptides were conjugated onto the carrier protein using either the EDAC or sulfo-MBS chemistries to construct the immunogen for the elicitation of antibodies.
  • the peptide/protein conjugates were used as immunogens in rabbits.
  • the different im unogens were used to immunize groups of 2-3 rabbits each.
  • the rabbits were immunized, boosted, and bled following a standard protocol.
  • the antiserum obtained from each bleed of each rabbit was tested by ELISA for the production of antibodies specific for the synthetic peptide analogue. Those antisera found positive for production of antibodies specific for the peptide portion of the immunogen were then assessed for their ability to recognize the whole protein. They were analyzed for their ability to recognize the native whole protein in ELISA's and the denatured whole protein in SDS-PAGE/ estern immunoblots in PC-12 cell lysate, in NRK cell lysate, in whole rat brain homogenates, and in whole mouse brain homogenate. They were also tested for their ability to stain fixed PC-12 cells and fixed NRK cells.
  • Table II represents the detection of dopamine receptor isoforms using these anti- peptide antibodies.
  • Antibody to Fragment refers to the anti-peptide antibody raised to the specific peptide immunogen of the peptides of Table I.
  • the peptides of Table I are identified according to dopamine receptor fragments.
  • FIG. 6-11 represents plots of the ELISA titration of D,, D 2 , D 3 , D 4 , and D 5 receptor anti-peptide antiserum at various dilutions for rat brain homogenate at 200 ngm/well. The optical density values were obtained from an ELISA plate reader, SLT Lab instruments, Easy Beam Reader Model, Salzburg, Austria. It may be observed that the positive ELISA response was indicated therein.
  • Figs. 12-15 show ELISA titration data for various antisera obtained from the particular peptide conjugate used as an immunogen as heretofore described.
  • the particular antisera is identified in Table II according to the corresponding receptor fragment named in Table I. These antisera were found to show positive results for rat brain homogenate and mouse brain homogenate.
  • PC-12 cell lysate was determined to contain D 2 , D 2L , D 3 , and D 5 isoforms, and NRK cell lysate was found to contain the D 2 , D 2L , D 4 , and D 5 isoforms (Table II).
  • Each of the peptides of Table I was conjugated onto either bovine thyroglobulin or keyhole limpet hemocyanin (KLH) using either EDAC or Sulfo-MBS as the cross linking reagent.
  • Peptides which contained a Cys residue were conjugated using sulfo-MBS and all the others were conjugated using EDAC.
  • the coupling ratios used were 200 molecules of antigenic peptide per molecule of thyroglobulin and 2000 molecules of antigenic peptide per molecule of KLH.
  • Each reaction was performed using standard procedures and routine reaction conditions.
  • the conjugates were isolated from the reaction mixture by gel filtration on Sephadex G-25 and lyophilized from a volatile buffer solution.
  • Row A Serial dilutions of pooled preimmune sera
  • Rows B-H Serial dilutions of the antiserum obtained from each animal of the group
  • Column 1 Blank (No serum bound to well)
  • Columns 2-12 2 fold serial dilutions of antiserum from 1:100 to 1:102,400
  • rat brain homogenate mouse brain homogenate
  • PC-12 cell lysate PC-12 cell lysate
  • NRK cell lysate The ELISA procedure was similar to that described in Example 4 above for the peptide screening ELISA, except the microtiter plates were sensitized at three different levels of total protein (200 ngm/well, 500 ngm/well and 1 ⁇ gm/well) with one of the four different cellular preparations.
  • At least one, and often numerous, antiserum from each group of rabbits was found to bind to the rat and mouse brain homogenates and to titer out like an antibody.
  • the binding and titration could be specifically blocked by pre-incubating the antiserum with the antigenic peptide used to elicit the anti-peptide antibodies.
  • Some of the antisera had very high titers (i.e., greater than 1:50,000).
  • Similar ELISA titration experiments were performed using PC-12 and NRK cell lysates a different pattern of response was found.
  • Some of the antisera which were found to react strongly with the rat and mouse brain homogenates bound very weakly or not at all, even in the 1 ⁇ gm/well experiments.
  • the rabbit anti-peptide antiserum specific for one of the isoforms of the dopamine receptor was diluted 1:500 with evaporated goat milk diluted 1:4 in TBS/Tween-20 buffer. This was applied to the membrane and allowed to bind for 2 hours before being washed 4 times with TBS/Tween-20.
  • Affinity purified HRP conjugated goat anti- rabbit IgG IgG was diluted 1:5,000 with evaporated goat milk diluted 1:4 in TBS/Tween 20 buffer. This solution was applied to the membrane and allowed to bind for 1 hour before being washed 3 times and then once overnight with TBS/Tween-20.
  • the membrane was developed using the enhanced DAB reaction in phosphate/citrate buffer, pH 5.0.
  • At least one and often numerous anti-peptide antiserum from each of the test groups was found to bind specifically to a protein with molecular weight of 52-58 kD in the rat and mouse brain homogenates: i.e., a protein with the correct size to be one of the dopamine receptors.
  • the binding could be specifically blocked by pre-incubating the anti-peptide antiserum with the antigenic peptide. Only the D 2 , D 2L , D 3 , and D 5 isoforms of the dopamine receptor were detected in PC-12 cell lysates by western immunoblot analysis, Fig. 17, and in the NRK cell lysates, only D 2 , D 2L , D 4 , and D 5 isoforms were detected by this procedure.
  • dopamine receptor isoform specific primary antibody (the anti-peptide antiserum) in PBS containing 0.1% Triton X- 100 and 2% NGS: incubate for 2 hours at room temperature.
  • mice are immunized with the same peptide/protein conjugates described in Example 2 by a modification of the immunization protocol described in Example 3.
  • the immunized mice are test bled from the eye orbital vein, and the anti-plasma obtained is screened by the ELISA procedures described in Examples 4 and 5 by changing only the HRP conjugated 2nd antibody, i.e., HRP- goat anti-mouse IgG 2nd antibody is used instead of the HRP-goat anti-rabbit IgG 2nd antibody.
  • mice found positive for the production of antibodies which recognize both the peptide immunogen and the whole rat brain homogenate are selected for use in the development of monoclonal antibodies.
  • the spleens of these mice are surgically removed using aseptic techniques and the splenocytes isolated.
  • the splenocytes are fused using polyethylene glycol with the mouse myeloma cell line SP2/0- Agl4.
  • the unfused SP2/0-Agl4 cells are killed during the sterile cell culture using the hypoxanthine, aminopterin, and thymidine (HAT) selection process.
  • the hybrids are screened using a modification of the ELISA screening protocols described in Examples 4 and 5 (see above) .
  • the cells in the positive wells are cloned by standard cell culture techniques to isolate a hydridoma cell line which is producing and secreting the monoclonal anti- peptide antibody specific for an isoform of the dopamine receptor.

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Abstract

Analogues peptidiques du récepteur dopaminergique correspondant à des régions présentant une différence dans les récepteurs D1-D5, et anticorps à anti-peptides mis à jour par lesdits analogues peptidiques, utiles dans les domaines de la recherche pharmaceutique et du diagnostic de maladies.
PCT/US1995/011127 1994-08-31 1995-08-30 Anticorps a peptides et anti-peptides du recepteur dopaminergique Ceased WO1996006856A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996037780A1 (fr) * 1995-05-24 1996-11-28 Deth Richard C Compositions et procedes pour deceler la schizophrenie
US5738998A (en) * 1995-05-24 1998-04-14 Deth; Richard C. Compositions and methods for diagnosing schizophrenia
US6080549A (en) * 1997-04-08 2000-06-27 Northeastern University Methods and materials for the diagnosis and treatment of schizophrenia and related disorders
WO2001042281A1 (fr) * 1999-12-06 2001-06-14 Hôpital Sainte-Justine Compositions destinees a traiter des anormalites en filtration glomerulaire, de persistance du canal arteriel et d'osteoporose

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL JOURNAL, Volume 289, issued 1993, P.L. CHAZOT et al., "Antisera Specific for D2 Dopamine Receptors", pages 789-794. *
BIOCHEMICAL SOCIETY TRANSACTIONS, Volume 19, issued 1991, P.L. CHAZOT et al., "Site-Specific Antibodies as Probes of the Structure and Function of the Brain D2 Dopamine Receptor", page 143S. *
COMPTES RENDUE ACADEMIE SCIENCE, PARIS, Volume 311, Series III, issued 1990, B. GIROS et al., "Clonage Du Gene Du Recepteur Dopaminergique D3 Humain et Identification de Son Chromosome", pages 501-508. *
EUROPEAN JOURNAL OF BIOCHEMISTRY, Volume 206, issued 1992, M.J. PLUG et al., "An Anti-Peptide Antibody that Recognizes the Dopamine D2 Receptor From Bovine Striatum", pages 123-130. *
MOLECULAR PHARMACOLOGY, Volume 37, Number 1, issued January 1990, T.M. STORMANN et al., "Molecular Cloning and Expression of a Dopamine D2 Receptor From Human Retina", pages 1-6. *
NATURE, Volume 347, issued 06 September 1990, A. DEARRY et al., "Molecular Cloning and Expression of the Gene for a Human D1 Dopamine Receptor", pages 72-76. *
NATURE, Volume 347, issued 06 September 1990, Q.-Y. ZHOU et al., "Cloning and Expression of Human and Rat D1 Dopamine Receptors", pages 76-80. *
NATURE, Volume 347, issued 06 September 1990, R.K. SUNAHARA et al., "Human Dopamine D1 Receptor Encoded by an Intronless Gene on Chromosome 5", pages 80-83. *
NATURE, Volume 350, issued 18 April 1991, H.H.M. VAN TOL et al., "Cloning of the Gene for a Human Dopamine D4 Receptor with High Affinity for the Antipsychotic Clozapine", pages 610-614. *
NATURE, Volume 350, issued 18 April 1991, R.K. SUNAHARA et al., "Cloning of the Gene for a Human Dopamine D5 Receptor With Higher Affinity for Dopamine Than D1", pages 614-619. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 86, issued December 1989, D.K. GRANDY et al., "Cloning of the cDNA and Gene for a Human D2 Dopamine Receptor", pages 9762-9766. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 88, issued October 1991, D.K. GRANDY et al., "Multiple Human D5 Dopamine Receptor Genes: a Functional Receptor and Two Pseudogenes", pages 9175-9179. *
THE EMBO JOURNAL, Volume 8, Number 13, issued 1989, R. DAL TOSO et al., "The Dopamine D2 Receptor: Two Molecular Forms Generated by Alternative Splicing", pages 4025-4034. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996037780A1 (fr) * 1995-05-24 1996-11-28 Deth Richard C Compositions et procedes pour deceler la schizophrenie
US5686255A (en) * 1995-05-24 1997-11-11 Deth; Richard C. Compositions and methods for diagnosing schizophrenia
US5738998A (en) * 1995-05-24 1998-04-14 Deth; Richard C. Compositions and methods for diagnosing schizophrenia
US6080549A (en) * 1997-04-08 2000-06-27 Northeastern University Methods and materials for the diagnosis and treatment of schizophrenia and related disorders
WO2001042281A1 (fr) * 1999-12-06 2001-06-14 Hôpital Sainte-Justine Compositions destinees a traiter des anormalites en filtration glomerulaire, de persistance du canal arteriel et d'osteoporose
US7442763B2 (en) 1999-12-06 2008-10-28 Hopital Sainte-Justine Compositions for treating abnormalities in glomerular filtration, patent ductus arteriosus and osteoporosis

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