WO1991002536A1 - Compositions et procedes de detection et de traitement de l'infection due au virus d'epstein-barr et de troubles immuns - Google Patents

Compositions et procedes de detection et de traitement de l'infection due au virus d'epstein-barr et de troubles immuns Download PDF

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Publication number
WO1991002536A1
WO1991002536A1 PCT/US1990/004817 US9004817W WO9102536A1 WO 1991002536 A1 WO1991002536 A1 WO 1991002536A1 US 9004817 W US9004817 W US 9004817W WO 9102536 A1 WO9102536 A1 WO 9102536A1
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polypeptide
rcr2
residue
epstein
barr virus
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PCT/US1990/004817
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English (en)
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Margaret D. Moore
Neil D. Cooper
Glen R. Nemerow
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Scripps Clinic And Research Foundation
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to synthetic polypeptides, and polypeptide aggregates, that correspond to a B lymphocyte membrane receptor protein (CR2) for Epstein-Barr virus.
  • CR2 B lymphocyte membrane receptor protein
  • EBV Epstein-Barr virus
  • Epstein-Barr virus a human herpesvirus
  • EBV infection may facilitate the development of malignant diseases including nasopharyngeal carcinoma (Henle, W. , Science 157:1064-1065, 1967 and Raab-Traub, N. , et al, Int. J. Cancer 39:25-29, 1987), Burkitt's lymphoma (De-The, G. , et al, Advances in Comparative Leukemia Research.
  • EBV is also an opportunistic pathogen often associated with immunocompromised patients receiving allografts or with the acquired immune deficiency syndrome (AIDS) (Pelicci, P.G. , et al, J. EXP. Med. 164:2049-2060, 1986; Yarchoan, R. , et al, J. Clin. Invest. 78:439- 447, 1986; and Montagnier, L.
  • AIDS acquired immune deficiency syndrome
  • EBV is unique among the human herpes viruses in its selective tropism for B lymphocytes and epithelial cells. This is largely due to expression on the membrane of these cells of a specific EBV receptor which has shown to be antigenically, structurally and functionally identical to the CR2 glycoprotein which binds the C3d/C3dg complement fragments (Fingeroth, J.D., et al, Proc. Natl Acad. Sci.. USA 81:4510-4516, 1984; Nemerow, G.R. , et al, J. Virol. 55:347-351, 1985; Frade, R. , et al, Proc. Natl.
  • CR2 is involved in a pathway of B cell activation (Nemerow, G.R. , et al, supra, 1985; Chagelian & Fearnon, J. EXP. Med 163:101-115, 1986; Wilson, et al. Blood 66:824-829, 1985; Frade, R. , et al, Proc. Natl. Acad. Sci.. USA 82:1490-1493, 1985; and Melchers, et al, Nature 317:264-267, 1985). This property may play a crucial role in preparing the cell for transformation by EBV.
  • the major envelope protein of EBV is a primary target of neutralizing antibody in man and sub-human primates (Thorley-Lawson & Geilinger, Proc. Natl. Acad. Sci. U.S.A. 77:5307-5311, 1980).
  • Gp350/220 is also the ligand which mediates attachment of the virus to CR2 on B cells (Nemerow, G. , et al, J. Virol. 61:1416- 1420, 1987 and Tanner, J. , et al, Cell 50:203-213, 1987) and as such represents one of the few human herpesvirus glycoproteins for which a receptor binding function has been identified.
  • the binding of EBV to CR2 is specifically mediated by a 9 amino acid residue epitope located near the N-terminus of gp350/220 (Nemerow, G.R., et al., Cell 56:369-377, 1989) . Binding of EBV, via gp350/220, to CR2 is followed by endocytosis of the virus, de-envelopment and transformation (Nemerow, G.R. , et al, Virology 132:186-198, 1984; Tedder, T.F., et al, J. Immunol. 137:1387-1391, 1986; and Tanner, et al, supra, 1987). Purified CR2 (Nemerow, G.R.
  • the extracellular domain of the receptor contains 16 tandemly arranged short repeating regions of about 60 to 75 amino acids, followed by a hydrophobic transmembrane domain and a short, 34 amino acid residue cytoplasmic tail (Moore, M.D. et al., supra 1987, Weis, J.J. et al., J. Exp. Med. 167:1047-1066, 1988, and Fujisaku, A. et al., J. Biol. Chem. 264:2118-2125, 1989).
  • the present invention is directed to diagnostic and treatment methods that utilize synthetic polypeptides that have amino acid residue sequences which correspond to EBV-binding regions of the extracellular domain of CR2, polypeptide aggregates that contain amino acid residue sequences corresponding to the EBV-binding regions of CR2 and pharmacological compositions.
  • aqueous medium such as blood
  • a CR2 polypeptide of the present invention in contact with cells at risk for infection is admixed with a CR2 polypeptide of the present invention.
  • the CR2 polypeptide-containing admixture so formed is maintained for a time period sufficient to allow any Epstein-Barr virus present in the suspension to bind to the polypeptide.
  • a pharmacological composition containing CR2 polypeptides is preferably injected into the blood of a patient at risk for Epstein-Barr virus infection.
  • a method of treatment for immune disorders is also provided by the present invention. This method is directed to the administration to a patient of a therapeutically effective amount of a pharmacological composition of the present invention to bind to either a C3dg fragment of complement or antibodies directed against CR2.
  • a CR2 polypeptide is defined by the presence of an amino acid residue sequence corresponding to CR2 amino acid residues 1- 256 as shown in Figure 9.
  • a polypeptide aggregate is contemplated by the present invention that contains at least two CR2 polypeptides of the present invention operatively linked together.
  • a pharmacological composition is provided by the present invention that contains a physiologically tolerable carrier together with a substantially purified CR2 polypeptide and/or CR2 polypeptide aggregate as described herein as an active ingredient.
  • a method for the detection of Epstein-Barr virus in a fluid sample in which a fluid sample is contacted with a solid matrix that has a CR2 polypeptide attached thereto for a time period sufficient to allow any Epstein-Barr virus present in the sample to specifically bind to the polypeptide.
  • the solid matrix is then separated from the liquid sample, and washed (rinsed) to remove any unbound material.
  • the washed matrix is then contacted with a label that is capable of indicating the presence of any Epstein-Barr virus that is specifically bound to the matrix.
  • a method of detecting the presence of antibodies directed against CR2 in a fluid sample is contemplated.
  • a fluid sample is contacted with a solid matrix having a CR2 polypeptide of the present invention linked thereto for a time period sufficient to allow the formation of an immunoreaction product between the CR2 polypeptide and any anti-CR2 antibodies present in the sample.
  • the solid matrix is separated from the fluid sample and washed (rinsed) to remove any unbound material.
  • the washed matrix is then contacted with a label capable of indicating the presence of any antibodies bound to the matrix.
  • a diagnostic system in kit form contains a package containing a polypeptide of the present invention, preferably attached to a solid matrix and a label for indicating the presence of either an Epstein-Barr virus or an anti-CR2 antibody that binds to the polypeptide.
  • Figure 1 illustrates the circular dichroism spectral analysis of rCR2 performed as described in Example 3.
  • Figure 2 illustrates the binding of rCR2 polypeptide to gp350/220 (triangles) , C3dg (circles) and ovalbumin (squares) coated onto 96 well plates and determined as described in Example 4.
  • Figure 3 illustrates an ultracentrifugation analysis of rCR2 binding to gp350/220 (Panel A) and C3dg (Panel B) as described in Example 5.
  • Panel - ⁇ r [ 125 I]-gp350/220 was applied to linear sucrose gradients containing no rCR2 (open circles) , 3 nM rCR2 (closed circles) , 30 nM rCR2 (closed squares) , or 100 nM rCR2 (open squares) .
  • Figure 5 illustrates C3dg binding to rCR2 at low ionic strength as determined in Example 5.
  • Panel A shows the results of ultracentrifugation of C3dg and rCR2 admixtures in 5-15% sucrose density gradients with 150 mM NaCl (open circles) , 25 mM NaCl (closed circles) , and 10 mM NaCl (closed squares) , and the insert shows the relative amount (r) of C3dg bound per CR2 plotted against NaCl concentration.
  • Panel B shows a determination of the dissociation constant (K D ) of rCR2 for C3dg by extrapolation from lower salt concentrations.
  • the open circles represent the data from Panel A and the closed circles represent the data from a similar study performed using 1 uM C3dg.
  • Figure 6 illustrates rCR2 inhibition of binding of gp350/220 to B-lymphoblastoid cells as determined in Example 6.
  • Fluorescent microspheres coated with purified gp350/220 were reacted with CR2- negative SF9 cells (Panel A) or with CR2-positive Raji cells (Panels B, C and D) .
  • Gp350/220 coated microspheres were reacted with 2.0 ug (Panel C) or 10 ug (Panel D) of purified rCR2 prior to incubation with Raji cells.
  • Figure 7 illustrates the inhibition of EBV infectivity of human peripheral blood mononuclear cells (B cells) by rCR2 as determined in Example 7.
  • Figure 8 illustrates the inhibition of EBV infectivity by rCR2 for increasing amounts of EBV present as determined in Example 7. Varying amounts of EBV supernate were incubated with media alone (solid lines) or with 10 ug of purified rCR2 (broken lines) . Samples were then reacted with peripheral blood lymphocytes as described in FIGURE 7 and assessed for B cell transformation by colony outgrowth and stimulation of DNA synthesis.
  • Figure 9 illustrates the nucleotide sequence of a cDNA that codes for CR2, shown from left to right and in the direction of 5' terminus to 3' terminus using the single letter nucleotide base code.
  • the mature, leaderless, structural gene for CR2 begins at base 123 and ends at base 3320, with the numbers for base residue positions indicated in the right margin.
  • the amino acid residue sequence for CR2 is indicated by the single letter code above the nucleotide base sequence, with the numbers for each residue position indicated in the left margin.
  • the reading frame is indicated by placement of the deduced amino acid residue sequence above the nucleotide sequence such that the single letter that represents each amino acid is located above the middle base in the corresponding codon.
  • Figure 10 illustrates in schematic form the construction and resulting structure of the CR2 polypeptide expression vectors for producing rCR2-4, rCR2 and rCR2-10, where the vectors are labeled as pAC373 CR2(l-4), pAC373 CR2(1-16) and pAC373 CR2(1- 10) , respectively.
  • the expression vectors manipulated and produced by the construction process are indicated in the figure by the circles.
  • the construction proceeds by a series of steps as indicated by the arrows connecting the circles and the intermediate nucleic acid fragments in the figure, and the steps are described in detail in Example 1.
  • Landmark and utilized restriction enzyme recognition sites are indicated on the circles and fragments by words or by labeled lines intersecting the circles. Individual genes and their direction of transcription are indicated by the arrows in the circles.
  • CR2 Polypeptides Substantially purified polypeptides that correspond in amino acid residue sequence to regions of the extracellular domain of CR2 have been synthesized in the present invention, and are designated herein as CR2 polypeptides.
  • a CR2 polypeptide of the present invention includes an amino acid residue sequence that corresponds to the amino acid residue sequence shown in Figure 9 from residue 1 to residue 70.
  • a CR2 polypeptide includes an amino acid residue sequence that corresponds to the amino acid residue sequence shown in Figure 9 from residue 1 to residue 133, from residue 1 to residue 256, from residue 1 to residue 632, or from residue 1 to residue 1005.
  • Exemplary of two of the preferred embodiments are the CR2 polypeptides rCR2 and rCR2-4 that are described in Example 1.
  • the CR2 polypeptide can be produced as a fusion protein wherein one portion of the polypeptide has a sequence that corresponds to CR2 and another portion corresponds to the amino acid sequence of a protein other than CR2.
  • Representative of this embodiment is the CR2 polypeptide rCR2-10 which includes as its ⁇ arboxy terminus a portion of the polyhedrin gene as described in Example 1.
  • compositions and diagnostic kits containing the CR2 polypeptides of the present invention are provided, together with methods for the detection and treatment of EBV infections and immune disorders.
  • All amino acid residues identified herein are in the natural L-conformation.
  • abbreviations for amino acid residues are as shown in the following Table of Correspondence:
  • CR2 polypeptides are preferably produced by recombinant expression in the present invention.
  • a baculovirus expression system is used to generate milligram amounts of CR2 polypeptides.
  • a plasmid containing a cDNA that encodes a portion of the extracellular domain of CR2 was prepared and utilized to produce recombinant CR2 polypeptides as described in Example 1.
  • a recombinant DNA plasmid containing a cDNA that encodes CR2, is truncated to encode a CR2 polypeptide that comprises a region of the extracellular domain of CR2.
  • the CR2 polypeptide construct was then inserted downstream of an appropriate promoter in a transfer vector and integrated into a baculovirus.
  • the recombinant baculovirus is used to infect host insect cells which then secrete recombinantly produced CR2 polypeptides. Examples of the baculovirus-expression system can be found in Example 1.
  • CR2 polypeptide aggregate refers to a polymer containing at least two CR2 polypeptides corresponding to regions of the extracellular domain of CR2 operatively linked either in tandem or to a polypeptide carrier.
  • operatively linked refers to the covalent attachment of a CR2 polypeptide to another CR2 polypeptide or to a peptide, polypeptide or protein carrier.
  • the CR2 polypeptide aggregates of the present invention can contain a plurality of the same or different CR2 polypeptides, wherein the CR2 polypeptides are bound to each other or to a peptide, polypeptide or protein carrier.
  • Pharmacological compositions of the present invention contain a physiologically tolerable carrier together with a substantially purified CR2 polypeptide, or CR2 polypeptide aggregate, as described above, dissolved or dispersed therein as an active ingredient.
  • the pharmacological composition is not immunogenic when administered to a mammal or human patient.
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
  • compositions that contain active ingredients dissolved or dispersed therein are well understood in the art.
  • Such compositions are prepared as injectables either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared.
  • the active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.
  • the pharmacological composition of the present invention can include pharmaceutically acceptable salts of the components therein.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2- ethylamino ethanol, histidine, procaine and the like.
  • Physiologically tolerable carriers are well known in the art.
  • Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline.
  • aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose and other solutes.
  • Liquid compositions can also contain liquid phases in addition to and to the exclusion of water.
  • additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
  • the pharmacological compositions are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose and its grammatical equivalents, as used herein, refers to physically discrete units suitable as unitary dosages for human patients and other warm-blooded animals, each unit containing a predetermined effective and potentiating amount of active material calculated to produce the desired therapeutic effect in association with the required physiologically tolerable carrier.
  • the compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • the quantity to be administered depends on the subject to be treated, and the capacity of the subject to utilize the active ingredient. Precise amounts of active ingredients required to be administered depend on the judgment of the practitioner and are peculiar to each individual, and usually are in a range of about 100 ug to about 100 mg of CR2 polypeptide per ml of patient blood. Alternatively, continuous intravenous infusion sufficient to maintain therapeutically effective concentrations in the blood are contemplated.
  • Epstein-Barr virus infection of mammalian cells in an aqueous medium, such as blood, is inhibited by methods of the present invention.
  • aqueous medium containing mammalian cells, such as B lymphocytes is admixed with a therapeutically effective amount of a pharmacological composition of the present invention and maintained for a time period sufficient to allow the CR2 polypeptides of the composition to specifically bind any Epstein-Barr virus present in the aqueous medium.
  • the therapeutically effective amount of the CR2 polypeptide-containing composition utilized in this method is that which produces a concentration of CR2 polypeptide in the aqueous medium sufficient to bind essentially all of the EBV present, and usually is at a concentration of about 100 ug to about 1 mg per ml.
  • the therapeutically effective amount of the pharmacological composition administered is that which produces a blood concentration of CR2 polypeptides sufficient to specifically bind the circulating EBV present.
  • Such a blood concentration is usually about 100 ug to about 1 mg of CR2 polypeptide per ml. It is contemplated that multiple administrations of the pharmacological composition of this invention over an appropriate time period and at a dosage level determined by a medical practitioner for the patient will be undertaken for the inhibition of EBV infection in a human patient.
  • the terms “specifically bind”, and “specifically attach”, and grammatical forms thereof are used interchangeably and refer to non-random ligand binding, such as that which occurs between gp350/220 and CR2.
  • a pharmacological composition is administered to a patient in any manner that will efficaciously inhibit the infection of mammalian cells, such as B lymphocytes, by EBV.
  • the composition is administered by either intravenous injection of a unit dosage or continuous intravenous infusion of a predetermined concentration of CR2 polypeptides or CR2 polypeptide aggregates to a patient.
  • a method for treating an immune disorder characterized by the presence in a patient of circulating antibodies directed against CR2 present on B lymphocytes is provided.
  • CR2 polypeptide or CR2 polypeptide aggregate is administered to the patient in a therapeutically effective amount to effectively bind to the antibodies and thus inhibit or prevent the immunoreaction of the antibodies with B lymphocytes. It is preferred that a pharmacological composition administered in the present invention is not immunogenic.
  • a method for treating an immunological disorder or condition in a patient, in which an immunoreaction product containing a C3d or C3dg fragment of complement is present is provided.
  • a patient is administered a therapeutically effective amount of a CR2 polypeptide or polypeptide aggregate of the present invention to bind to the C3d or C3dg fragment of complement present in the immunoreaction product, and thus facilitate the alleviation of the symptoms of the disorder.
  • a diagnostic system in kit form of the present invention includes, in an amount sufficient for at least one assay, a CR2 polypeptide and/or CR2 polypeptide aggregate and/or composition of the present invention, as a separately packaged reagent. Instructions for use of the packaged reagent are also typically included.
  • a diagnostic system for assaying for the presence of or to quantitate anti- CR2 antibodies in a sample comprises a package containing at least one CR2 polypeptide of this invention.
  • a diagnostic system of the present invention for assaying for the presence or amount of EBV comprises a package containing a CR2 polypeptide, CR2 polypeptide aggregate or pharmacological composition of this invention.
  • a diagnostic system of the present invention further includes a label or indicating means capable of signaling the formation of a specifically bound complex containing a CR2 polypeptide or polypeptide aggregate of the present invention.
  • label and
  • indicating means in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex.
  • Illustrative labels include radionuclides, such as 125 I, and fluorescent radicals such as fluorescein isothiocyanate and the like.
  • the diagnostic systems can also include, preferably as a separate package, a specific binding agent.
  • a "specific binding agent” is a molecular entity capable of selectively binding a reagent species of the present invention but is not itself a protein expression product, polypeptide, or polypeptide aggregate of the present invention.
  • Exemplary specific binding agents are antibody molecules, complement proteins or fragments thereof, protein A and the like.
  • the specific binding agent can bind the CR2 polypeptide of this invention when it is present as part of a complex.
  • anti-human Fc antibodies are conveniently used.
  • the specific binding agent is labeled.
  • the agent when the diagnostic system includes a specific binding agent that is not labeled, the agent is typically used as an amplifying means or reagent.
  • the labeled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a reagent species-containing complex.
  • the diagnostic kits of the present invention can be used in an "ELISA" format to detect the presence or quantity of EBV or anti-CR2 antibodies in a body fluid sample such as blood serum, plasma or urine.
  • ELISA refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen or antibody present in a sample.
  • a description of the ELISA technique is found in Chapter 22 of the 4th Edition of Basic and Clinical Immunology by D.P. Sites, et al, published by Lange Medical Publications of Los Altos, CA in 1982, and in U.S. Patents No. 3,654,090; No. 3,850,752; and No.
  • the CR2 polypeptide, or polypeptide aggregate, of the present invention can be affixed to a solid matrix to form a solid support that is separately packaged in the subject diagnostic systems.
  • the reagent is typically affixed to the solid matrix by adsorption from an aqueous medium although other modes of affixation, well known to those skilled in the art can be used.
  • Useful solid matrices are well known in the art. Such materials include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, NJ) ; agarose; beads of polystyrene beads about 1 micron to about 5 millimeters in diameter available from Abbott Laboratories of North Chicago, IL; polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-based webs such as sheets, strips or paddles; or tubes, plates or the wells of a microliter plate such as those made from polystyrene or polyvinylchloride.
  • the reagent species, labeled specific binding agent or amplifying reagent of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form.
  • the indicating means is an enzyme
  • the enzyme's substrate can also be provided in a separate package of a system.
  • a solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.
  • the packages discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems. Such packages include glass and plastic (e.g., polyethylene, polypropylene and polycarbonate) bottles, vials, plastic and plastic- foil laminated envelopes and the like.
  • the present invention contemplates any method that results in detecting EBV or anti-CR2 antibodies in a body fluid sample using CR2 polypeptides or polypeptide aggregates of this invention.
  • exemplary methods are described herein, the invention is not so limited.
  • the presence of anti-CR2 antibodies in an antibody-containing bodily fluid is indicative of an autoimmune disorder in the host animal.
  • a bodily fluid such as blood, plasma or serum from the patient is contacted under biological assay conditions with a CR2 polypeptide or aggregate thereof for a period of time sufficient to form a polypeptide containing complex.
  • the presence of the complex is indicative of infection by EBV or of a CR2 autoimmune disorder, respectively.
  • the complex can be detected as described herein.
  • Biological assay conditions are those that maintain the biological activity of the CR2 polypeptide molecules of this invention and the EBV or anti-CR2 antibodies sought to be assayed.
  • Those conditions include a temperature range of about 4 C to about 45 C, preferably about 37 C, a pH value range of about 5 to about 9, preferably about 7, and an ionic strength varying from that of distilled water to that of about one molar sodium chloride, preferably about that of physiological saline. Methods for optimizing such conditions are well known in the art.
  • nucleotide sequence can be defined in terms of the amino acid residue sequence, i.e., protein or polypeptide, for which it codes.
  • An important and well known feature of the genetic code is its redundancy. That is, for most of the amino acids used to make proteins, more than one coding nucleotide triplet (codon) can code for or designate a particular amino acid residue. Therefore, a number of different nucleotide sequences can code for a particular amino acid residue sequence.
  • nucleotide sequences are considered functionally equivalent since they can result in the production of the same amino acid residue sequence in all organisms. Occasionally, a methylated variant of a purine or pyrimidine may be incorporated into a given nucleotide sequence. However, such methylations do not affect the coding relationship in any way.
  • a nucleotide sequence of the present invention encodes a CR2 polypeptide sequence of this invention.
  • Representative nucleotide sequences that encode a CR2 polypeptide of the present invention can include nucleotide sequences that correspond to the nucleotide base sequence shown in Figure 9.
  • a preferred CR2 polypeptide-encoding nucleotide sequence includes a sequence that corresponds to the sequence shown in Figure 9 from nucleotide base 123 to base 332, from base 123 to base 521, from base 123 to base 2018, or from base 123 to base 3137.
  • a DNA segment of the present invention that encodes an extracellular CR2 amino acid residue sequence and preferably an amino terminal sequence can easily be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci, et al, J. Am. Chem. Soc.. 103:3185 (1981). Of course. by chemically synthesizing the coding sequence, any desired modifications can be made simply by substituting the appropriate bases for those encoding the native amino acid residue sequence.
  • DNA segments consisting essentially of structural genes encoding the CR2 extracellular domain can be obtained by digesting B lymphocytes.
  • a nucleic acid construction of the present invention can be produced by operatively linking an expression vector to the nucleic acid sequence of the present invention, preferably in a baculovirus such as the Autographa californica nuclear polyhedrosis virus.
  • the term "operatively linked”, in reference to cDNA integration, describes that the nucleotide sequence is joined to the vector so that the sequence is under the transcriptional and translation control of the expression vector and can be expressed in a suitable host cell.
  • vector refers to a
  • a recombinant DNA molecule is a hybrid DNA molecule comprising at least two nucleotide sequences not normally found together in nature.
  • DNA segment of the present invention is operatively linked depends directly, as is well known in the art, on the functional properties desired, e.g., protein expression, and the host cell to be transformed. these being limitations inherent in the art of constructing recombinant DNA molecules.
  • Expression vectors which are relatively harmless in a host animal, including primates, are well known and can be used to induce an immune response to an expressed foreign polypeptide included in the vector.
  • vectors are provided containing convenient restriction sites for insertion of the desired DNA segment.
  • vaccinia virus vectors and baculovirus vectors.
  • a variety of methods have been developed to operatively link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted and to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
  • Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors.
  • the DNA segment, generated by endonuclease restriction digestion is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3', single-stranded termini with their
  • blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
  • Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies, Inc. , New Haven, CT. Also contemplated by the present invention are RNA equivalents of the above described recombinant DNA molecules.
  • the CR2 polypeptides are thus readily synthesized and expressed by an appropriate host cell such as the Spodoptera frugiperda (SF9) insect cells. Examples
  • CR2 was isolated from detergent lysates of Raji B lymphoblastoid cells by immunoaffinity chromatography.
  • Raji cells (2 x 10 10 ) were washed twice in cold (about 4 C) phosphate buffered saline (PBS, pH 7.4) and suspended in 400 ml of 10 mM phosphate buffer (pH 8.0) containing 3.3% Brij 96 (Sigma Chemical Co. , St. Louis, MO) , 3 mM sodium azide, 50 mM iodoacetamide, 5 mM phenylmethylsulfonyl fluoride, 5 mM EDTA and 0.2 trypsin inhibitory units of Aprotinin/ml (Sigma Chemical Co.).
  • the suspension was thoroughly mixed at 4 C for 30 min followed by high speed centrifugation to remove the nuclei and cell debris.
  • Sodium deoxycholate (DOC) in 10 mM Tris hydrochloride (pH 8.0) was added to the supernatant to obtain a final concentration of 1%.
  • This mixture was stirred at 4 C for 60 minutes, and then maintained, with constant mixing, for 18 hours with 2 ml of packed fixed Staphylococcus aureus (Pansorbin, Calbiochem-Behring, La Jolla, CA) .
  • the mixture was centrifuged and the supernatant was saved to be applied to an immunomatrix column having anti-CR2 monoclonal antibody HB-5 attached (HB-5 immunomatrix column) .
  • the immunomatrix was batch-washed four times with 200 ml of 10 mM Tris hydrochloride (pH 8.0) containing 0.5% DOC and four times with 200 ml of 10 mM Tris hydrochloride (pH 8.0) containing 150 mM NaCl and 0.5% DOC.
  • the immunomatrix was poured into a column.
  • the CR2-containing supernatant was applied to the HB-5 immunomatrix column and eluted with 7 to 10 ml of 50 mM diethylamine (pH 11.5) (Kodak, Rochester, NY) containing 0.5% DOC.
  • the pH of the CR2-containing eluate was adjusted to pH 8.0 by the addition of solid glycine.
  • the protein was rechromatographed on the immunomatrix column to remove minor contaminants.
  • the protein concentration of CR2 was estimated by comparing densitometric scans of CR2 with known amounts of standard markers (Bio-Rad
  • An ELISA was used to monitor purified CR2.
  • Various amounts of the purified CR2 (0.25 to 4 ng) in 100 uL aliquots in 0.1 M bicarbonate buffer (pH 9.5) were coated onto 96 well microtiter plates (Immulon II, Dynatech Laboratories Inc., Alexandria, VA) for 18 hours at 4 C.
  • the wells were then blocked with 0.5% nonfat dry milk in PBS (pH 7.4) containing 0.05% antifoam A (Sigma Chemical Co.) as blotto buffer for 1 hour at 37 C.
  • Anti-CR2 antibodies (1 ug) in 100 ul of blotto buffer were added to the wells and maintained for one hour at 22 C.
  • Kb representing the entire CR2 coding sequence was prepared as described by Moore et al. , Proc. Natl. Acad. Sci. USA. 84:9194-98, (1987).
  • a cDNA library was first constructed in lambda gtll (Stratagene, San Diego, CA) using mRNA isolated from Raji B lymphoblastoid cells. Complementary DNA was synthesized with a commercially available synthesis kit (cDNA synthesis system, Amersham, Arlington Heights, IL) . Packaging of recombinant cDNA clones with phage coat proteins was done as recommended by the manufacturer (Gigapack, Stratagene) . The library contains 2.5 x 10 6 recombinants.
  • Candidate CR2 cDNA clones were isolated from 85 mm plate lysates using affinity chromatography with anti-lambda phage antibody (LambdaSorb, Promega Biotec, Madison, WI) . Large scale preparation of lambda phage was carried out, digested with EcoR I and the cDNA inserts were isolated by preparative gel electrophoresis. Approximately 5 x 10 5 recombinant lambda phage were screened with a CR2 oligonucleotide probe in order to isolate a clone containing a 1.2 kilobase insert encoding CR2. The CR2 cDNA insert isolated from the clone was nick-translated and used to reprobe the Raji library to obtain additional cDNA inserts encoding CR2.
  • CR2 inserts were ligated into the EcoRI site of plasmid Bluescript (Stratagene) , designated plasmid Bluescript/CR2. c. Construction of CR2/Baculovirus
  • CR2 polypeptide rCR2 has an amino acid residue sequence that corresponds to the sequence shown in Figure 9 from residue 1 to residue 1005. rCR2 is expressed by the baculovirus expression vector pAC373 CR2(1-16) shown in the middle portion of Figure 10.
  • pAC373 CR2(1- 16) plasmid Bluescript/CR2 (pBS/CR2) was digested with Bgl 1 and Pst 1 to release the cDNA insert encoding the entire extracellular domain of CR2.
  • a synthetic oligonucleotide BamH I linker (5 1 - dCGGATCCG-3• ; Pharmacia/LKB Biotechnology, La Jolla, California) was ligated to the 5' end of the CR2 insert that had been previously digested with Bgll and blunt-ended with T4 DNA polymerase.
  • the construct was further modified by two successive oligonucleotide-based site-directed mutagenesis reactions according to the method of Kunkel, Proc. Natl. Acad. Sci.. USA. 82:488-492 (1985) .
  • the first mutagenesis step involved ablation of the BamH I site at nucleotide position No.
  • the second mutagenesis step was then carried out by incorporating a termination codon and BamH I site (TGAGGATCC) following nucleotide position No 3137, immediately preceding the transmembrane domain of CR2.
  • truncated CR2 fragment encoding the extracellular domain of CR2 was inserted into the BamH I cloning site of pAC373.
  • Recombinant plasmids containing a single copy of truncated CR2 in the correct orientation was identified by restriction mapping.
  • Transfer of the rCR2 sequence from the plasmid to the Auto rapha californica nuclear polyhedrosis virus (AcNPV) genome was carried out by cotransfection of Spodoptera fruigiperda (SF9) cells with 10 ug of rCR2 plasmid and 1 ug of AcNPV DNA using calcium phosphate. Recombinant viruses were selected and characterized.
  • rCR2-4 CR2 polypeptide rCR2-4 has an amino acid residue sequence that corresponds to the sequence shown in Figure 9 from residue 1 to residue 756. rCR2-4 is expressed by the baculovirus expression vector pAC373 CR2(l-4) shown in the left portion of Figure 10.
  • the preparation of pAC373 CR2(l-4) differed from the preparation of pAC373 CR2(1-16) in that the first mutagenesis was omitted, and the second mutagenesis step was conducted so as to incorporate a termination codon and BamH I site (TGAGGATCC) following nucleotide position No. 887. Subsequently the mutagenized and truncated CR2 polypeptide encoding fragment was inserted into the BamH I cloning site of pAC373 as shown for pAC373 CR2(1-16), the vector was manipulated as before to produce a recombinant virus.
  • rCR2-10 CR2 polypeptide rCR2-10 is a fusion protein that includes at its carboxy terminus a portion of the polyhedrin protein, and has at its amino terminus an amino acid residue sequence that corresponds to the sequence shown in Figure 9 from residue 1 to residue 632.
  • rCR2-10 is expressed by the baculovirus expression vector pAC373 CR2(1-10) shown in the right portion of Figure 10. The preparation of pAC373 CR2(1-16) in that no mutagenesis was conducted.
  • the BamH I linker modified plasmid pBS/CR2 was digested with BamH I and inserted into the BamH I cloning site of pAC373 that contained the polyhedrin structural gene and promoter.
  • the polyhedrin gene was fused to the carboxyl terminus of the CR2 polypeptide coding gene.
  • the resulting vector pAC373 CR2(1-10) was manipulated as before to produce recombinant virus. The virus was transfected into SF9 cells and rCR2-10 protein was expressed therefrom, harvested and purified as before.
  • Epstein-Barr virus membrane glycoprotein gp350/220 was isolated from GH3 19 rat pituitary tumor cells transfected with gp350/220 cDNA as described by Whang et al. , J. Virol. 61:1795-1807
  • the C3dg fragment of the third component of complement was purified from Mg +2 EGTA-activated aged human serum as described by Vik et al., J. Immunol. , 138:254-258 (1987).
  • rCR2 Soluble recombinant CR2 polypeptide (rCR2) corresponding to the extracellular region of CR2 was produced and purified as described in Example 1.
  • the molecular weight of rCR2 was determined by SDS gel electrophoresis to be 125 kDa. This value is higher than the molecular weight of 110 kDa predicted from the cDNA sequence for rCR2. The difference is presumed to be due to post-translational modification, such as glycosylation, of the soluble rCR2.
  • Soluble rCR2 (2 milliliters (ml) ) was applied to a 2.6 x 95 cm column of Sephacryl S-300 (Pharmacia, Piscataway, NJ) that had been equilibrated with 300 mM NaCl, 20 mM imidazole-HCl pH 7.3 at 4 C. Fractions (2.5 ml each) were collected at a flow rate of 17 ml/hour. [ 125 I]-labelled proteins were detected by gamma counting in a Micromedic Systems 4/600 gamma counter, and unlabelled protein in the fractions were detected by absorbance at 280 nm.
  • the Sephacryl S-300 column was calibrated with the following standards, where the diffusion coefficient (D 20>w ) is listed in parenthesis: thyroglobulin (2.54 x 10 "7 cm 2 /s) , Factor H (2.54 x 10 "7 cm 2 /s) , C3 (4.53 x 10 "7 cm 2 /s) , bovine serum albumin (5.94 x 10 "7 cm 2 /s) , and myoglobulin (1.13 xl0 "6 cm 2 /s) .
  • the diffusion coefficient (D 20 . folk) of rCR2 was determined to be 3.1 x 10 "7 cm 2 /s.
  • the Stokes radius for rCR2 was calculated from the D 20 . folk to be 69.3 Angstrom, and the frictional coefficient (f/fo) was determined as 2.1.
  • Soluble rCR2 (100 ul) was centrifuged through a 5-16% linear sucrose gradient (4 ml) prepared in phosphate-buffered saline (PBS) at 45,000 rpm in an SW-60 rotor (Beckman Instruments, Palo Alto, CA) for 18-24 hours at 4 C. Gradient fractions (160 ul) were collected and the sedimentation constant (s 20w ) for rCR2 was determined to be 4.5S.
  • Circular dichroism (CD) spectral analysis was carried out in an Aviv CD spectropolarimeter model 61 DS, standardized with d-10-camphosulfonic acid.
  • a sample of rCR2 (0.12 mg/ml) in 20 mM Tris HCl, pH 8.2 was scanned for CD between 187 and 260 nm in a 0.1 cm path length cuvette.
  • the CD spectrum for rCR2 ( Figure 1) is unlike that of most globular proteins because it is dominated by a maximum positive ellipticity at 229 nm and a maximum negative ellipticity between 200 and 205 nm.
  • the CD spectrum for rCR2 also indicates the absence of alpha-helix secondary structure. This spectrum is similar to that for the complement regulatory protein, factor H, that also contains repeating regions.
  • gp350/220 and C3dg obtained as described in Example 1, and ovalbumin were independently coated and immobilized onto separate wells of 96 well plates. Aliquots of rCR2 prepared in Example 1 were then added to each well at varied concentrations, maintained for 2 hrs to allow binding of rCR2 to the immobilized ligand, followed by rinsing to remove any unbound rCR2. The presence of bound rCR2 was determined by the CR2-specific ELISA described in Example la. The results are illustrated in Figure 2. The rCR2 bound to both gp350/220 and C3dg in a dose-dependent manner, while only background binding was observed with ovalbumin. Similarly, CR2 polypeptides rCR2-4 and rCR2-
  • rCR2-4 and rCR2-10 both exhibited binding to immobilized ligands comprised of EBV virus particles purified gp350/220 or complement C3dg. Therefore the exemplary CR2 polypeptides rCR2, rCR2-4 and rCR2-10 were shown to have the capacity to specifically bind to the target ligands for CR2.
  • S c S 0 (M C /M 0 )° *68
  • S c S 20jW of the complex
  • M c molecular weight of the complex
  • S 0 is the S 20jW of the rCR2
  • M 0 is the molecular weight of rCR2.
  • C3dg prepared as described in Example 1 was used in a sucrose gradient, as described above. Under conditions of physiologic pH and ionic strength, no binding of monomeric C3dg to rCR2 was detected ( Figure 3B) .
  • Micropheres preincubated with 2 ug rCR2 (Panel C) or 10 ug rCR2 (Panel D) were reacted with Raji cells, and show that rCR2 produced a dose-dependent inhibition of gp350/220 fluorescent microsphere binding to Raji cells, while no inhibition was observed with microspheres incubated with buffer alone.
  • EBV was preincubated with varying amounts of purified rCR2 prior to addition of unseparated peripheral blood mononuclear cells (6 x 10 5 cells) .
  • the mixture of cells with EBV and rCR2 were maintained for 1 hour at 4 C, washed and the cells (2 x 10 5 /plate, in triplicate) were then cultured for 14 to 21 days in complete RPMI medium with 0.1 ug/ml cyclosporine A and then assessed for transformation.
  • a preincubation of EBV with rCR2 resulted in dose-dependent inhibition of EBV infection as measured by both outgrowth of transformed colonies and by 3 H-thymidine incorporation.
  • rCR2 The ability of rCR2 to block infection was not due to toxic effects of rCR2 upon the cells because the highest dose of rCR2 used (7.5 ug) did not abolish infection of B cells if EBV was allowed to bind to B cells prior to exposure to rCR2.
  • EBV induction of B cells transformation was reduced by nearly 90% as illustrated in Figure 8.

Abstract

Sont des décrits des polypeptides synthétiques correspondants au domaine extracellulaire de la réceptrice CR2 de la membrane des lymphocytes B ainsi que des aggrégats de polypeptides, des compositions et des procédés de traitement de l'infection due au virus d'Epstein-Barr et de troubles immunologiques, ainsi qu'un test visant à déterminer la présence dudit virus.
PCT/US1990/004817 1989-08-23 1990-08-22 Compositions et procedes de detection et de traitement de l'infection due au virus d'epstein-barr et de troubles immuns WO1991002536A1 (fr)

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