WO1991003251A1 - Compositions du ligand cr2 et procede de modulation des fonctions de cellules immunitaires - Google Patents

Compositions du ligand cr2 et procede de modulation des fonctions de cellules immunitaires Download PDF

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WO1991003251A1
WO1991003251A1 PCT/US1990/005027 US9005027W WO9103251A1 WO 1991003251 A1 WO1991003251 A1 WO 1991003251A1 US 9005027 W US9005027 W US 9005027W WO 9103251 A1 WO9103251 A1 WO 9103251A1
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ligand
amino acid
acid residue
formula
polypeptide
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PCT/US1990/005027
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Waldemar Lernhardt
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California Institute Of Biological Research
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Priority claimed from US07/404,679 external-priority patent/US5331090A/en
Priority claimed from US07/512,118 external-priority patent/US5310729A/en
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Publication of WO1991003251A1 publication Critical patent/WO1991003251A1/fr

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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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • 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
    • C07K16/249—Interferons
    • 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 the CR2 binding region of a CR2 ligand and to polypeptides and polypeptide aggregates that contain the binding region. More particularly, the present invention contemplates CR2 ligand-containing compositions, antibodies to CR2 ligands, rDNA's that express CR2 ligands and methods of using the ligands and the anti- ligand antibodies.
  • CR2 (CD21) is a cellular receptor on B lymphocytes that is implicated in their growth regulation and is linked to intercellular pathways involved in signaling B cell proliferation.
  • CR2 occurs on normal B lymphocytes and on B cell neoplasms. Cooper et al., Ann. Rev. Immunol., 6:85 (1988); Hatzfeld et al., J. Immunol., 140:170 (1988). CR2 functions as a receptor for several complement C3 activation products. The proteolytic C3 activation products iC3b, C3dg and C3d all bind to CR2 and have been shown to mediate both stimulating and inhibiting effects on lymphocytes. Weigle et al., in Complement r Muller-Eberhard, H.J. and Miescher, P.A. (eds.). p. 323, Springer-Verlag, Berlin (1985).
  • CR2 function and ligands that modulate that function are of great interest.
  • the CR2 receptor is also of clinical interest because it is the receptor for the human herpes virus, Epstein-Barr virus. Fingeroth et al. , Proc. Natl. Acad. Sci. USA, 86:242 (1989).
  • EBV is the causative agent of infectious mononucleosis, [Henle et al., Proc. Natl. Acad. Sci. USA, 59:94 (1968)] and is possibly a human cancer virus, because it has been linked to nasopharyngeal carcinoma and Burkitt's lymphoma. Henle et al..
  • EBV is thought to be associated with x- linked lymphoproliferative disease (Duncan's disease) [Purtillo et al.. Lancet i, 935 (1975)] and several human autoimmune disorders. Tosato et al.. Adv. Immunol., 37:99 (1985).
  • EBV may play a role in the onset of B cell neoplasia observed in a substantial number of patients with AIDS (Yarchoan et al., J. Clin. Invest., 78:439 (1986).
  • EBV In vitro infectious EBV is a T cell-independent B cell stimulator and transforms human B lymphocytes to immortal polyclonal lymphoblastoid cell lines. Cooper et al., Ann. Rev. Immunol., 6:85 (1988). Non- transforming virus is a T cell-dependent B cell activator. Cooper et al., Ann. Rev. Immunol., 6:85 (1988) . Drug inhibitors of EBV propagation that operate by interfering with virus binding to CR2 will therefore be of clinical relevance.
  • ligands that bind CR2 have been extensively characterized.
  • the exact sequence motif mediating binding of C3 fragments to CR2 has been elucidated by Lambris et al., Proc. Natl. Acad. Sci. USA, 82:4235 (1985) and has the amino acid residue sequence LYNVEA.
  • Peptides containing this motif have the ability to inhibit aggregated C3d-induced S phase entry of B cells [Lernhardt, et al., Immunol. Rev., 99:239 (1987)] and to inhibit alpha B cell growth factor activity. Melchers, et al., Proc. Natl. Acad. Sci. USA, 82:7681 (1985).
  • sequence motif mediating binding of EBV virus to CR2 is present on the gp350/220 protein and has the amino acid residue sequence EDPGFFNVE. Nemerow, et al.. Cell, 56:369 (1989).
  • the present invention describes DNA segments that encode a CR2 ligand and CR2 ligand polypeptides that contain the binding site.
  • Recombinant DNA (rDNA) molecules are also described that contain DNA segments that encode CR2 ligands, as well as rDNA expression vectors capable of expressing CR2 ligands in compatible hosts.
  • a CR2 ligand comprising a polypeptide having as a part of its amino acid residue sequence one or more CR2 binding sites represented by the formula: PAIVEAG or NSGVEA.
  • a CR2 ligand comprising a polypeptide is also described having as a part of its amino acid residue sequence one or more CR2 binding sites represented by the formula -QLNDLEA- or -QLNNLEA-.
  • a polypeptide aggregate is described having a plurality of polypeptides, each containing one CR2 binding site.
  • hybrid IFN ⁇ having a sequence of amino acid residues that form a heterologous CR2 binding site, which sequence is located within the hybrid at a position corresponding to residues 76 to 84 of IFNctA.
  • compositions containing CR2 ligands and methods of using those compositions to stimulate or inhibit B lymphocyte proliferation.
  • inhibitory CR2 ligands containing only one CR2 binding site are described that are useful to inhibit B lymphocyte proliferation, such as in patients with B cell lymphoma.
  • stimulatory CR2 ligands are described that are useful to stimulate B lymphocytes and myelomas, such as in patients with immunodeficiencies or to boost immunoglobulin secretion by hybridoma cultures.
  • a method of inhibiting infection in vitro or jLn vivo by Epstein-Barr virus (EBV) is also described in which CR2 ligands are administered to bind CR2 receptor and thereby competitively block EBV infection of host cells by blocking virus binding to the cell receptor, CR2.
  • Antibody and monoclonal antibody compositions are contemplated that contain antibody molecules that immunoreact with a CR2 ligand of this invention, and more particularly immunoreact a CR2 binding site for CR2.
  • Diagnostic systems and methods are also described for detecting the presence of CR2 ligand or anti-CR2 ligand antibodies in bodily fluid samples. The described systems and methods utilize the anti-CR2 ligand antibody compositions and CR2 ligands of the present invention.
  • a CR2 ligand of the present invention can be used therapeutically as an analog to an IFN ⁇ molecule that binds CR2 as described herein and in doing so avoids the side effects normally associated with therapies involving native IFN ⁇ , such as nausea, vomiting and diarrhea.
  • production costs for synthetic polypeptides can be considerably lower than for native IFN ⁇ .
  • Figure 1 illustrates in ten sheets.
  • Figure 1A through Figure 1J the nucleotide sequence of a cDNA that codes for a CR2 ligand of this invention, shown from left to right and in the direction of 5' terminus to 3' terminus using the single letter nucleotide base code.
  • the structural gene for the mature CR2 ligand begins at base 42 and ends at base 2141, with the position number of the first base residue in each row indicated to the left of the row showing the sequence.
  • the amino acid residue sequence for a CR2 ligand is indicated by the single letter code below the nucleotide base sequence, with the position number for the last residue in each row indicated to the right of the row showing the amino acid residue sequence.
  • 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 below the middle base in the corresponding codon.
  • Figure 2 illustrates in two sheets, Figure 2A and Figure 2B, the nucleotide sequence of a cDNA that codes for interferon alpha strain A (IFN ⁇ A) , shown from left to right and in the direction of 5' terminus to 3' terminus using the single letter nucleotide base code.
  • IFN ⁇ A interferon alpha strain A
  • the structural gene for the mature IFN ⁇ A begins at base 118 and ends at base 567, with the position number of the first base residue in each row indicated to left of the row showing the sequence.
  • the amino acid residue sequence for IFN ⁇ A is indicated by the single letter code below the nucleotide base sequence, with the position number for the last residue in each row indicated to the right of the row showing the amino acid residue sequence.
  • the reading frame is indicated by placement of the deduced amino acid residue sequence below the nucleotide sequence such that the single letter that represents each amino acid is located below the middle base in the corresponding codon.
  • the mature IFN ⁇ A protein amino acid residue sequence begins at residue 1 and ends at residue 150.
  • Figure 2 also shows the amino acid residue sequence for IFN ⁇ 88 as compared to the sequence for IFN ⁇ A, as indicated by the few single letters below the amino acid residue sequence for IFN ⁇ A. Only those residues that differ between IFN ⁇ 88 and IFN ⁇ A are shown, and blank spaces indicate that IFN ⁇ 88 has the same residue as IFN ⁇ A at that residue position.
  • Figure 3 illustrates in three sheets, Figure 3A through Figure 3C, the nucleotide sequence of a DNA segment that codes for a CR2 ligand comprising an alkaline phosphatase-IFN ⁇ fusion protein as described in Example 8.
  • the representation of the nucleotide bases, and the corresponding amino acid residues, is shown as described in the legend to Figure 2 except that the structural gene for the fusion protein begins at base 47 and ends at base 703, and the encoded amino acid residue sequence begins at residue 1 and ends at residue 219.
  • Figure 4 illustrates the two-step process used in the cassette mutagenesis procedure of Example 9 to produce a hybrid IFN ⁇ molecule having a modified CR2 binding site.
  • Step I involves creation of Sacl and BamHl restriction sites adjacent to the CR2 binding site coding region of rIFN ⁇ by site- directed mutagenesis using mismatched primers in a primer extension reaction.
  • Step II illustrates the annealing of an oligonucleotide coding for a synthetic polypeptide which includes the amino acid residue sequence -QLYNVEA- to the Sacl/BamHl digested rIFN ⁇ cDNA.
  • the base numbers correspond to the base squence numbers shown in Figure 3.
  • amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
  • amino acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or to a amino-ter inal NH 2 group or to a carboxy-terminal COOH group.
  • Polypeptide refers to a linear series of amino acid residues connected to one another by peptide bonds between the alpha-amino group and carboxy group of contiguous amino acid residues.
  • Protein refers to a linear series of greater than 50 amino acid residues connected one to the other as in a polypeptide.
  • Synthetic polypeptide refers to a chemically produced chain of amino acid residues linked together by peptide bonds that is free of naturally occurring proteins and fragments thereof.
  • Antibody The term antibody in its various grammatical forms refers to a composition containing immunoglobulin molecules and/or immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope.
  • Antibody Combining Site An "antibody combining site" is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.
  • Antibody Molecule The phase "antibody molecule" in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, referred to also as a fragment of an intact immunoglobulin molecule.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab', F(ab') 2 and F(v) .
  • Probe Binding conditions are those that maintain the binding activity of the probes of this invention and C3 gene expression product sought to be assayed. Probe binding conditions vary, as is well known in the art, - li ⁇
  • Immunoreaction conditions are those that maintain the immunological activity of the anti-CR2 ligand antibody molecules used in this invention and the CR2 ligand polypeptide sought to be assayed. Those conditions include a temperature range of about 4 degrees C (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.
  • Monoclonal Antibody in its various grammatical forms refers to an antibody containing having only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen, e.g., a bispecific monoclonal antibody.
  • Nucleotide A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose) , a phosphate, and a nitrogenous heterocyclic base.
  • the base is linked to the sugar moiety via the glycosidic carbon (1* carbon of the pentose) and that combination of base and sugar is a nucleoside.
  • nucleoside contains a phosphate group bonded to the 3' or 5' position of the pentose it is referred to as a nucleotide.
  • a sequence of operatively linked nucleotides is typically referred to herein as a "base sequence” or “nucleotide sequence”, and is represented herein by a formula whose left to right orientation is in the conventional direction of 5•-terminus to 3'- terminus.
  • Polynucleotide A nucleic acid molecule comprising a polymeric unit of DNA or RNA having a sequence of two or more operatively linked nucleotides that form a single linear strand of nucleotides, also referred to as an oligonucleotide.
  • Duplex A double-stranded nucleic acid molecule consisting of two strands of complementary polynucleotide hybridized together by the formation of a hydrogen bond between each of the complementary nucleotides present in a base pair of the duplex. Because the nucleotides that form a base pair can be either a ribonucleotide base or a deoxyribonucleotide base, the term "duplex" referring to either a DNA-DNA duplex comprising two DNA strands, or a RNA-DNA duplex comprising one DNA and one RNA strand.
  • Base Pair A partnership of adenine (A) with thymine (T) , or of cytosine (C) with guanine (G) in a double stranded DNA duplex.
  • Nucleic Acid A term to refer to any of a class of molecules that includes ribonucleic acid (RNA) , deoxynucleic acid (DNA) in its single or double stranded forms, and polynucleotides.
  • Isolated polynucleotide A term used to refer to a polynucleotide that is substantially free of contaminating proteins.
  • DNA segment A DNA-DNA duplex having a preselected conserved nucleotide sequence and a sequence coding for a CR2 ligand of the present invention.
  • nucleotide sequence In living organisms, the amino acid residue sequence of a protein or polypeptide is directly related via the genetic code to the deoxyribonucleic acid (DNA) sequence of the structural gene that codes for the protein and the mRNA from which it is translated.
  • a 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.
  • An isolated DNA segment of the present invention contains a nucleotide sequence that encodes a CR2 ligand polypeptide sequence of this invention.
  • the CR2 ligand-encoding DNA segment is no more than about 5,000, and preferably no more than about 2,500, nucleotides in length.
  • nucleotide sequences that encode a CR2 ligand polypeptide of the present invention can include nucleotide sequences that correspond to the nucleotide base sequence shown in Figure 1, Figure 2 or Figure 3.
  • a preferred CR2 ligand polypeptide-encoding nucleotide sequence includes a nucleotide sequence that encodes the amino acid residue sequence shown in Figure 1 from residue 389 to residue 394, or from residue 303 to residue 309.
  • a preferred DNA segment includes a nucleotide sequence that encodes the amino acid residue sequence from residue 1 to residue 700 shown in Figure 1.
  • CR2 ligand polypeptide-encoding nucleotide sequence includes a nucleotide sequence that encodes the amino acid residue sequence shown in Figure 2 from base 343 to base 363 corresponding in the amino acid residue sequences for either IFN ⁇ A or IFN ⁇ 88.
  • a preferred DNA segment includes a nucleotide sequence that encodes the amino acid residue sequence from residue 1 to residue 219 shown in Figure 3.
  • a DNA segment of this invention encodes a hybrid IFN ⁇ molecule having a CR2 ligand as described herein. An exemplary DNA segment is described in Example 9.
  • a DNA segment of the present invention that encodes a CR2 ligand polypeptide 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.
  • Reco binant DNA Molecules The present invention further contemplates a recombinant DNA (rDNA) that includes a DNA segment of the present invention operatively linked to a vector for replication and/or expression.
  • a preferred rDNA is characterized as being capable of directly expressing, in a compatible host, a CR2 ligand of the present invention.
  • directly expressing is meant that the mature polypeptide chain of the expressed CR2 ligand is formed by translation alone as opposed to proteolytic cleavage of two or more terminal amino acid residues from a larger translated precursor protein.
  • Preferred rDNA's of the present invention are the rDNA plasmids pBS 9-4, pCMV 9-4 and pMSG 9-4 described in Example 5.
  • an exemplary and preferred rDNA of the present invention is the rDNA molecule pCMV-IFN described in Example 8, and the rDNA molecule pCMV-mlFN described in Example 9.
  • a rDNA molecule of the present invention can be produced by operatively linking a vector to a DNA segment of the present invention.
  • vector refers to a nucleic acid molecule capable of transporting between different genetic environments another nucleic acid to which it has been operatively linked.
  • Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors".
  • expression vectors the term "operatively linked”, in reference to DNA segments, 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.
  • CR2 ligand-coding DNA segment is operatively linked depends directly, as is well known in the art, on the functional properties desired, e.g., replication or protein expression, and the host cell to be transformed, these being limitations inherent in the art of constructing recombinant DNA molecules.
  • the vector utilized includes a procaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra chromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a procaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra chromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a procaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra chromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith.
  • procaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA
  • Those vectors that include a procaryotic replicon can also include a procaryotic promoter capable of directing the expression (transcription and translation) of the CR2 ligand-coding segments in a bacterial host cell, such as E. coli transformed therewith.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenience restriction sites for insertion of a DNA segment of the present invention.
  • vector plasmids Typical of such vector plasmids are pUC8, pUC9, pBR322, and pBR329 available from BioRad Laboratories (Richmond, CA) and pPL and pKK223 available from Pharmacia (Piscataway, NJ) .
  • Expression vectors compatible with eucaryotic cells preferably those compatible with vertebrate cells, can also be used.
  • Eucaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment.
  • the eucaryotic cell expression vectors used include a selection marker that is effective in an eucaryotic cell, preferably a drug resistant selection marker.
  • a preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene. Southern et al., J. Mol. Appl. Genet., 1:327-341 (1982).
  • retroviral expression vector refers to a DNA molecule that includes a promoter sequences derived from the long terminal repeat (LTR) region of a retrovirus genome.
  • the expression vector is typically a retroviral expression vector that is preferably replication-incompetent in eucaryotic cells.
  • LTR long terminal repeat
  • complementary cohesive termini can be engineered into the CR2 ligand-coding DNA segments during a primer extension reaction by use of an appropriately designed polynucleotide synthesis primer, or by operatively linking a synthetic linker containing one or more restriction sites.
  • Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies, Ine. , New Haven, CT.
  • the vector, and DNA segment if necessary, is cleaved with a restriction endonuclease to produce termini complementary to those of the DNA segment.
  • the complementary cohesive termini of the vector and the DNA segment are then operatively linked (ligated) to produce a unitary double stranded DNA molecule.
  • the resulting construct is then introduced into an appropriate host to provide amplification and/or expression of the CR2 ligand-coding DNA segments, either separately or in combination.
  • Cellular hosts into which a CR2 ligand-coding DNA segment-containing construct has been introduced are referred to herein as having been "transformed” or as “transformants”, and such transformed cells, and cultures of said cells, are also contemplated by the present invention.
  • the host cell can be either procaryotic or eucaryotic.
  • Bacterial cells are preferred procaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strain DH5 available from Bethesda Research Laboratories, Inc. , Bethesda, MD.
  • Preferred eukaryotic host cells include yeast and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human cell line.
  • Particularly preferred vertebrate host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61 and NIH Swiss mouse embryo cells (NIH/3T3) available from the ATCC as CRL1658.
  • Transformation of appropriate cell hosts with a recombinant DNA molecule of the present invention is accomplished by methods that typically depend on the type of vector used.
  • transformation of procaryotic host cells see, for example, Cohen et al., Proc. Natl. Acad. Sci., USA, 69:2110 (1972); and Maniatis et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982) .
  • the transformation of vertebrate cells with retroviral vectors containing rDNAs see for example, Sorge et al., Mol. Cell. Biol., 4:1730-1737 (1984); Graham et al., Virol., 52:456 (1973); and Wigler et al., Proc. Natl. Acad. Sci. USA, 76:1373-1376 (1979).
  • Successfully transformed cells i.e., cells containing a CR2 ligand-coding DNA segment operatively linked to a vector
  • cells from a population subjected to transformation with a subject rDNA can be cloned to produce monoclonal colonies. Cells form those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using a method such as that described by Southern, J. Mol. Biol., 98:503 (1975) or Berent et al., Biotech., 3:208 (1985) .
  • CR2 ligand-coding DNA segment In addition to directly assaying for the presence of a CR2 ligand-coding DNA segment, successful transformation can be confirmed by methods that detect the expressed CR2 ligand polypeptide. For example, samples of cells suspected of being transformed are assayed for the presence of the CR2 ligand by testing for CR2 ligand binding activity, or by using an antibody that immunoreacts with the CR2 ligand.
  • CR2 Ligands D. CR2 Ligands
  • the present invention contemplates a polypeptide, referred to as a CR2 ligand, capable of specifically binding to the CR2 receptor, as a functional ligand, and thereby effect (modulate) changes in CR2 receptor- containing cell status, such as to induce or inhibit proliferation, to compete with other ligands of CR2 for binding, to inhibit EBV infection, and the like.
  • a subject CR2 ligand is further characterized by the presence of at least one of the following amino acid residue sequences: -PAIVEAG-, or
  • a CR2 ligand includes the amino acid residue sequence -NSGVEA- or -PAIVEAG- and corresponds in sequence to a portion of the amino acid residue shown in Figure 1.
  • a CR2 ligand has an amino acid residue sequence that corresponds, and preferably is identical, to the sequence shown in Figure 1 from residue 1 to residue 700.
  • Preferred CR2 ligands have an amino acid residue sequence that corresponds, and preferably is identical, to a sequence shown in one of the formulae:
  • PAIVEAGGM PAIVEAGGM
  • PAIVEAGGMQ PAIVEAGGMQ
  • a CR2 ligand is further characterized by the presence of a CR2 binding site that includes an amino acid residue sequence that corresponds, and is preferably identical, to a sequence represented by the formula —QLNDLEA- or -QLNNLEA-.
  • the overall amino acid residue sequence of a subject CR2 ligand is different from that of native IFN ⁇ .
  • a preferred CR2 ligand contains only a single CR2 binding site and has an amino acid residue sequence that is less than about 100 amino acid residues, preferably less than 50, and more preferably less than 20 residues in length.
  • Additional amino acid residues present on a CR2 ligand in addition to the above indicated sequences can be any residues sequence of residues, but preferably a sequence corresponding to a sequence of an IFN ⁇ having a CR2 binding site.
  • IFN ⁇ molecules that contain a CR2 binding site are any IFN ⁇ molecules having the sequence -QLNDLEA- or -QLNNLEA-, and include the known human IFN ⁇ molecules IFN ⁇ A (IFN ⁇ l) , IFN ⁇ 2, IFN ⁇ 4a, IFN ⁇ 4b, IFN ⁇ 6, IFN ⁇ 7, IFN ⁇ l ⁇ , IFN ⁇ 88, IFN (Ovch) , IFLrk, LeIF A, LeIF C, LeIF D, LeIF I and the like as described by Weissman et al.. Prog. Nucl. Acid Res. Mol. Biol.. 33:251-300 (1986).
  • a CR2 ligand includes the amino acid residue sequence -QLNDLEA- and corresponds in sequence, and preferably is identical to, a portion of the amino acid residue sequence of IFN ⁇ A shown in Figure 2.
  • a CR2 ligand in another embodiment includes the amino acid residue sequence -QLNNLEA- and corresponds in sequence, and preferably is identical to, a portion of the amino acid residue sequence of IFN ⁇ shown in Figure 2.
  • Preferred CR2 ligands have an amino acid residue sequence that corresponds, and preferably is identical, to a sequence shown in one of the formulae: QLNDLEA, QLNDLEAC, QLNDLEACV, QLNDLEACVI, QLNDLEACVIQ,
  • CR2 ligands are the polypeptides shown in Table 1. TABLE 1 IFN-Related CR2 Ligand Polypeptides
  • a subject polypeptide comprising a CR2 ligand includes any polypeptide, analog, fragment or chemical derivative of a polypeptide whose amino acid residue sequence is shown herein so long as the polypeptide is capable of binding CR2 and modulating the function of a CR2-containing cell in a manner disclosed herein. Therefore, a present polypeptide can be subject to various changes, substitutions, insertions, and deletions where such changes provide for certain advantages in its use.
  • analog includes any polypeptide having an amino acid residue sequence substantially identical to a sequence specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the ability of a CR2 ligand as described herein.
  • conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
  • CR2 ligands contemplated by the present invention have an amino acid residue sequence that corresponds to one of the sequences represented by the formulae: QSNGVEALT QNSGLEALT QNSGLEALI QSNGVEALI QNSVGEALI QPAIVEAG QNAIVEAG QPAIVEAL QPAIVEALI QPAIVEALT QPAILEAG QPAILEAGT QPAIVEAGI QNAIVEALI , and QNAIVEALT
  • Additional CR2 ligands contemplated by the present invention have an amino acid residue sequence that corresponds to one of the sequences represented by the formulae:
  • fragment refers to any subject polypeptide having an amino acid residue sequence shorter than that of a polypeptide whose amino acid residue sequence is shown herein.
  • “Chemical derivative” refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group.
  • Such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t- butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
  • Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives.
  • the imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine.
  • polypeptides of the present invention also include any polypeptide having one or more additions and/or deletions or residues relative to the sequence of a polypeptide whose sequence is shown herein, so long as the requisite activity is maintained.
  • modifications of a subject polypeptide are contemplated that are directed to affecting the polypeptide's stability in aqueous solutions, so long as the modifications do not substantially affect the capacity of the polypeptide to otherwise function as intended.
  • modifications to the amino terminal amino acid residue are contemplated that will inhibit susceptibility to degradation by amino peptidases, such as leucine amino peptidase, that are present in solutions in contact with the polypeptide when in use.
  • Such modifications include acetylation of the amino terminal residue, or the preparation of the amino terminal residue as a des-amino residue, lacking a free amino group. '
  • the present invention further includes a composition that includes a subject polypeptide in combination with one or more of a pH buffering agent, wetting agent, anti-oxidant, reducing agent, aqueous medium, and the like, such composition being formulated as an aqueous solution for a use as described herein or as a dry composition, such as a powder, that can be reconstituted to form an aqueous solution.
  • a subject polypeptide can be prepared using recombinant nucleic acid methodologies well known, some of which are disclosed herein above. For instance, DNA segments that encode a CR2 ligand are prepared and then ligated into an expression vector, and a host transformed therewith can be used to produce the polypeptide.
  • the recombinant expression vectors so formed that are capable of expressing a subject polypeptide and methods of their use for producing a subject polypeptide are contemplated as part of the present invention.
  • a subject polypeptide can also be prepared using the solid-phase synthetic technique initially described by Merrifield, in J. Am. Chem. Soc. ⁇ 5:2149- 2154 (1963) .
  • Other polypeptide synthesis techniques may be found, for example, in M. Bodanszky et al., Peptide Synthesis. John Wiley & Sons, 2d Ed. (1976) , as well as in other reference works known to those skilled in the art.
  • a summary of polypeptide synthesis techniques may be found in J. Stuart and J.D. Young, Solid Phase Peptide Synthesis f Pierce Chemical Company, Rockford, IL, 3d Ed., Neurath, H. et al., Eds., pp. 104-237, Academic Press, New York, NY
  • those synthetic methods comprise the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing polypeptide chain. Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group. A different, selectively removable protecting group is utilized for amino acids containing a reactive side group such as lysine.
  • the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group.
  • the protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably - 2 ⁇ -
  • a CR2 ligand is characterized by the presence of a plurality of polypeptide segments, each polypeptide segment being defined by the presence of one of the following amino acid residue sequences: -PAIVEAG-, or -NSGVEA-.
  • a CR2 ligand is characterized by the presence of a plurality of polypeptide segments, each polypeptide segment being defined by the presence of one of the following amino acid residue sequences: -QLNDLEA-, or -QLNNLEA-.
  • the included polypeptide segments can be adjacent and/or contiguous within the polypeptide chain, with adjacent segments being separated in the amino acid residue sequence of the polypeptide by one or more spacing residue.
  • the spacing residues make up a spacing segment in the range of about 1 to about 20, preferably about 5 to about 15, and more usually about 10, amino acid residues in length.
  • a subject polypeptide can contain a leader segment of 1 conveniently up to about 20, such as about 5, about 10 or about 15, amino acid residues located amino-terminal to the amino-terminal CR2 ligand-derived or spacing segment.
  • a subject polypeptide need not end with the carboxy-terminal residue of a CR2 ligand-derived segment or spacer segment.
  • a carboxy terminal tail segment can be present containing 1 conveniently up to about 20, such about 5, about 10 or about 15, amino acid residues.
  • Preferred polypeptides of the present invention having a plurality of segments are defined by the formula:
  • the sequence in parentheses corresponds in its entirety, and preferably is identical to, a portion of the amino acid residue sequence of CR2 ligand shown in Figure 1.
  • Preferred polypeptides are those whose formulas are shown in Table 3.
  • B is an amino-terminal NH 2 group or a previously discussed leader segment
  • J is a carboxy- terminal COOH group or a previously discussed tail segment
  • X and Z are first and second, respectively, spacing segments whose amino acid residue sequences can be the same or different
  • n is either 1 or 0 such that when n is 1, X is present, and when n is 0, X is not present
  • m is either 1 or 0 such that when m is 1, Z is present, and when m is 0, Z is not present
  • a is an integer from 2 to about 10, more preferably 2 to about 5 and usually 2 to 3, indicating the number of times the amino acid residue sequence in parenthesis is present (repeated) in the polypeptide primary structure.
  • a CR2 ligand in which the included CR2 ligand polypeptides described immediately above are present as a conjugate comprised of a plurality of said polypeptides operatively linked, by other than a peptide bond between the alpha-amino group and carboxy group of contiguous amino acid residues, where at least two of the linked polypeptides have an amino acid residue sequence corresponding to that represented by the formula: B-(X n -NSGVEA-Z a -J, wherein B, X, Z, J, n, m and a are defined as previously discussed except that a can also be the integer 1.
  • Preferred polypeptides in another embodiment of the present invention having a plurality of segments are defined by the formula: B-(X n -QLNULEA-Z m ) a -J.
  • U can be either aspartic acid (D) or asparagine (N) in each amino acid residue sequence in parenthesis;
  • n is either 1 or 0 such that when n is 1, X is present, and when n is 0, X is not present;
  • m is either 1 or 0 such that when m is 1, Z is present, and when m is 0, Z is not present; and
  • a is an integer from 2 to about 10, more preferably 2 to about 5 and usually 2 to 3, indicating the number of times the amino acid residue sequence in parenthesis is present (repeated) in the polypeptide primary structure.
  • the sequence in parenthesis corresponds in its entirety, and preferably is identical to, a portion of the amino acid residue sequence of CR
  • a CR2 ligand in which the included CR2 ligand polypeptides described above are present as a conjugate comprised of a plurality of said polypeptides operatively linked, by other than a peptide bond between the alpha-amino group and carboxy group of contiguous amino acid residues, where at least two of the linked polypeptides have an amino acid residue sequence corresponding to that represented by the formula:
  • B-(X n -QLNULEA-Z m ) a -J wherein B, X, Z, J, n, m and a are defined as previously discussed except that a can also be the integer 1.
  • a conjugate of this invention has a molecular weight of less than about 40,000 daltons, preferably less than about 20,000 daltons, and more preferably less than about 10,000 daltons.
  • a subject conjugate has a molecular weight of no more than about 15,000 daltons, preferably no more than about ⁇ ,000 daltons, and usually no more than about 4,000 daltons.
  • the conjugate is dimeric or trimeric, i.e., consists essentially of two or three polypeptide chains, respectively.
  • a polypeptide conjugate of this invention is further characterized by its ability to bind CR2 receptor and thereby modulate CR2 containing cells as disclosed herein.
  • the subject conjugates are also substantially free of toxicity toward lymphocytes at concentrations of about 20 micrograms per milliliter (ug/ml) .
  • the techniques of polypeptide conjugation or coupling through activated functional groups presently known in the art are particularly applicable. See, for example, Aurameas, et al., Scand. J. Immunol., 1:7-23 (1978), and U.S. Patent No. 4,493,795, No. 3,791,932 and No. 3,839,153.
  • a site directed coupling reaction can be carried out so that any loss of activity due to polypeptide orientation after coupling can be minimized. See, for example, Rodwell, et al., Biotech., 3:8 ⁇ 9- ⁇ 94 (1985), and U.S. Patent No. 4,671,958.
  • One or more additional amino acid residues may be added to the amino- or carboxy- termini of the polypeptide to assist in linking two or more polypeptides to form a conjugate.
  • a preferred method of linking is to form a disulfide bridge between two polypeptides that each have at least one cysteine residue, said bridge formed by exposure of the polypeptides to oxidizing conditions.
  • One preferred linking method involves air-oxidation of cysteine residues present on CR2 ligands in solution. Exemplary is the air-oxidation of rIFN ⁇ protein described in Example 8d, in which each rIFN ⁇ protein has a single CR2 binding site that, upon oxidation, forms a CR2 ligand having more than two CR2 binding sites.
  • a cysteine residue can be incorporated into the amino acid residue sequence providing the capacity for disulfide bridges therein.
  • the cysteine residue can be introduced at one or both termini of the polypeptide such that the aggregate formed upon oxidation is a dimer or multimer, respectively, of the cysteine-containing polypeptide, having end-to-end linkages at each termini containing a cysteine residue.
  • hybrid IFN ⁇ Molecules and Compositions The present invention also contemplates a hybrid interferon alpha (hybrid IFN ⁇ ) molecule that comprises an IFN ⁇ molecule having a heterologous CR2 binding site.
  • Human IFN ⁇ molecules are known to represent a family of proteins that are related by having homology in their amino acid residue sequences, and yet are distinct species based on their specific amino acid residue sequences and their biological activity.
  • CR2 binding sites are present within species of IFN ⁇ , one can alter the CR2 binding specificity and binding affinity of an IFN ⁇ molecule by substituting a heterologous CR2 binding site for the region of an IFN ⁇ molecule corresponding to amino acid residues 76 to 84 of IFN ⁇ A shown in Figure 2.
  • the hybrid IFN ⁇ molecule contains a CR2 binding site that increases the binding affinity with CR2, the hybrid IFN ⁇ molecule can be used effectively at lower concentrations as compared to the native IFN ⁇ counterpart. Reduced dosages provide the advantage that the administered patient is subjected to less side effects associated with IFN ⁇ therapies.
  • a heterologous CR2 binding site refers to a sequence of amino acid residues present in a CR2 ligand that (1) have the capacity to bind to CR2 and function as a CR2 ligand, and (2) are not found naturally in the CR2 ligand.
  • a hybrid IFN ⁇ is comprised of two parts, a heterologous CR2 binding site and the non-CR2 binding site portion of the IFN ⁇ molecule.
  • a hybrid IFN ⁇ is an IFN ⁇ molecule having a modified CR2 binding site, such that the modification produces an altered amino acid residue sequence within the CR2 binding site, which altered sequence is not normally found in the IFN ⁇ molecule.
  • Any CR2 binding site can be a heterologous CR2 binding site, so long as it is substituted into an IFN ⁇ molecule other than the IFN ⁇ species in which the CR2 binding site is found in nature.
  • CR2 binding sites suitable for use as heterologous CR2 binding sites include the binding sites defined by the IFN ⁇ -related CR2 ligands of the present invention, shown in Table 1, by the CR2 ligands p7, p8 and p9 in Table 2 derived from complement C3 or from CR2 binding protein (CBP) , and by the CR2 binding sequence of EBV's gp350/220 protein, including conservative substitutions thereof.
  • CBP CR2 binding protein
  • Preferred heterologous CR2 binding site- containing amino acid residue sequences suitable for use in a hybrid IFN ⁇ molecule are represented by a formula selected from the group consisting of: QLNDLEACV, QNNDLEACV, QLYNVEATS, QNNDVEATS,
  • An IFN ⁇ molecule suitable for use in producing a hybrid IFN ⁇ molecule can be any IFN ⁇ .
  • Numerous IFN ⁇ species have been identified whose nucleotide sequences are known, including the IFN ⁇ species identified by the designation IFN ⁇ Z, where Z is A, 1, 2, 4a, 4b, 5, 6, 7, 8, 16, 88, LeIF X, where X is A, B, C, D, F, G, H, I, J and the like. See Weismann et al., Prog. Nucl. Acid Res. Mol. Biol.. 33:251-300, 1986, for descriptions of the above IFN ⁇ species, including their nucleotide sequences.
  • the methods for producing a hybrid IFN ⁇ molecule include the recombinant nucleic acid methodologies described herein, and particularly by modification of existing rDNA molecules in the nucleotide base portion that encode a CR2 binding site as to encode a modified (heterologous) CR2 binding site.
  • Methods for modifying a rDNA to encode a modified CR2 binding site can include (1) substitutions of small DNA segments that encode a modified CR2 binding site into a larger DNA segment encoding an IFN ⁇ molecule, referred to as cassette mutagenesis, and (2) site-directed mutagenesis using mismatched primer extension reactions. Both of these methods are described in Example 9 to produce the exemplary hybrid IFN ⁇ , desiganted pCMV-mlFN ⁇ , having a modified (heterologous) CR2 binding site.
  • compositions of the present invention contain a physiologically tolerable carrier together with a CR2 ligand, a CR2 polypeptide conjugate, or a polyclonal or monoclonal anti-CR2 ligand antibody, as described herein, dissolved or dispersed therein as an active ingredient.
  • the therapeutic composition is not im unogenic when administered to a mammal or human patient for therapeutic purposes.
  • 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.
  • physiological effects such as nausea, dizziness, gastric upset and the like.
  • 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 preparation can also be emulsified.
  • 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 therapeutic 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, polyethylene glycol 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.
  • IFN ⁇ interferon alpha
  • the CR2 ligands of the present invention also contain a CR2 binding site, some of which are related to IFN ⁇ A or IFN ⁇ , and therefore are useful as analogs of interferon to the extent that these - 3 ⁇ -
  • interferons exert their action by complexing with (binding) CR2.
  • CR2 ligands have the capacity to induce (modulate) changes in the present status of a CR2-containing cell, such as normal or neoplastic B lymphocytes.
  • the CR2 ligands exhibit the modulating capacity on B lymphocytes from mammals such as mice, sheep, cattle, horses, and man. These changes include stimulation or inhibition of proliferation of the lymphocyte.
  • the capacity of a CR2 ligand to inhibit, as compared to the capacity to stimulate, B lymphocytes depends on the structure of the ligand, and particularly on the number of CR2 binding sites, and their relative spacing in the CR2 ligand.
  • the inhibitory or stimulatory capacity of a CR2 ligand of the present invention can be readily determined by a variety of methods, such as by monitoring changes in the amount of thymidine uptake by cultured B lymphocytes in the presence of various amounts of CR2 ligand, as disclosed in Examples 3 and 13.
  • the present invention provides for a method of inhibiting CR2 function on CR2-containing cells and comprises administering to a mammal a therapeutically effective amount of a physiologically tolerable composition containing an inhibitory CR2 ligand, thereby forming a competition reaction admixture in the mammal wherein the inhibitory CR2 ligand competes with native CR2 ligands for binding with the CR2.
  • the inhibitory CR2 ligand is administered to the mammal in a predetermined amount calculated to achieve the desired effect, i.e., in a therapeutically effective amount.
  • a CR2 ligand is inhibitory if it contains only one CR2 ligand binding site.
  • a CR2 ligand binding site present in one embodiment of a CR2 ligand has an amino acid residue sequence that corresponds, and is preferably identical, to a sequence represented by the formula -PAIVEAG- or -NSGVEA-.
  • inhibitory CR2 ligands are the polypeptides: NSGVEA, QNSGVEALT,
  • the above inhibitory CR2 ligand when used as an agent for inhibiting B lymphocyte proliferation, such as in a human patient displaying the symptoms of an autoimmune disease or in a patient with B cell lymphoma, is administered in an amount sufficient to achieve a plasma concentration of at least about O. ⁇ ug/ml, preferably at least about 0.10 ug/ml, more preferably at least about 2 ug/ml, and usually 3 or 4 ug/ml.
  • a CR2 binding site present in another embodiment of a CR2 ligand of the present invention has an amino acid residue sequence that corresponds, and is preferably identical, to a sequence represented by the formula -QLNDLEA- or -QLNNLEA-.
  • inhibitory CR2 ligands are the polypeptides: QLNDLEA,
  • the above inhibitory CR2 ligand is administered in an amount sufficient to achieve a plasma concentration of from about 0.1 ug/ml to about 100 ug/ml, preferably from about 1.0 ug/ml to about 50 ug/ml, more preferably at least about 2 ug/ml and usually 5 to 10 ug/ml.
  • the present invention provides for a method of stimulating proliferation of a CR2-containing cell, particularly a B lymphocyte in, for example, a human exhibiting B cell immunodeficiencies, a B cell hybridoma cell line in culture to boost production of antibody molecules, and the like.
  • the present invention contemplates a method of in vivo stimulating CR2-containing cells in a mammal that comprises administering a therapeutically effective amount of a physiologically tolerable composition containing a stimulatory CR2 ligand to a mammal in a predetermined amount calculated to achieve the desired effect.
  • a representative stimulatory CR2 ligand is a polypeptide having an amino acid residue sequence that corresponds to the sequence shown in Figure 1 from residue 1 to residue 700.
  • the stimulatory CR2 ligand is administered in an amount sufficient to achieve a plasma concentration of at least about O. ⁇ ug/ml, preferably at least about 0.1 ug/ml, more preferably at least about 2 ug/ml and usually 3 or 4 ug/ml.
  • a stimulating CR2 ligand is one having at least two CR2 binding sites, wherein each binding site has an amino acid residue sequence that corresponds, and is preferably identical, to a sequence represented by the formula -QLNDLEA- or -QLNNLEA-.
  • Preferred are the CR2 ligands described herein having a plurality of CR2 binding sites, and the rIFN ⁇ protein compositions described herein having at least two CR2 binding sites.
  • a stimulating CR2 ligand related to an INF ⁇ protein has the CR2 binding sites positioned in the ligand such that the binding sites as defined by the formulas immediately above are separated by about 10 to about 150 amino acid residues, preferably about 60 to 100 residues, and more preferably about 60 amino acid residues.
  • the IFN ⁇ -related stimulatory CR2 ligand When used as an agent to in vivo stimulate B lymphocyte proliferation, such as in a human displaying B cell immunodeficiencies, the IFN ⁇ -related stimulatory CR2 ligand is administered in an amount sufficient to achieve a plasma concentration of from about 0.01 ug/ml to about 100 ug/ml, preferably from about 1.0 ug/ml to about 50 ug/ml, more preferably at least about 2 ug/ml and usually 5 to 10 ug/ml.
  • An additional representative stimulatory CR2 ligand is the rIFN ⁇ protein composition having two CR2 binding sites as described in Example ⁇ d.
  • the present invention also contemplates a method of in vitro stimulating B cell hybridoma cells in culture.
  • a culture of B cell hybridoma cells is admixed with an effective amount of a stimulatory CR2 ligand and maintained under culture conditions for a time period sufficient to allow the admixed ligand to specifically bind any CR2 receptor present in the culture.
  • An effective amount is that amount which produces a concentration of ligand in the culture sufficient to bind essentially all of the CR2 receptor present.
  • the CR2 ligand is usually at a concentration of about 100 ug to about 1 mg per ml.
  • the concentration is typically about 0.01 ug to about 1 mg per ml preferably 0.1 ug to 50 ug, more preferably 1 to 10 ug/ml.
  • anti-CR2 ligand antibody molecules that immunoreact with either of the CR2 ligand polypeptides PAIVEAG, NSGVEA, QLNDLEACV or QLNNLEACV, and compositions containing those molecules, can be used in a method for inhibiting CR2 function, including the inhibition of B lymphocyte proliferation and other cellular responses including those that occur as a result of IFN ⁇ binding to CR2.
  • the physiologically tolerable composition administered contains a therapeutically effective amount of an anti-CR2 ligand antibody molecule of this invention in an amount sufficient to immunoreact with the CR2 ligand present in the patient, thereby competing with native CR2 ligand for binding to CR2 and inhibiting normal CR2 ligand- induced CR2 functions.
  • the anti-CR2 ligand antibody molecules immunoreactive with IFN ⁇ -derived polypeptides can be administered to block IFN ⁇ function by immunoreacting with the CR2 binding site present on IFN ⁇ , thereby preventing normal IFN ⁇ binding to its target, the CR2 receptor.
  • An effective amount of therapeutic antibody is that amount which produces a concentration of antibody sufficient to immunoreact with essentially all of the CR2 receptor present on available B lymphcytes of the administered patient, and usually is in the order of about 0.01 to 10, preferably one to several milligrams of active ingredient per kilogram bodyweight of individual per day, depending on the route of administration.
  • the present invention also contemplates the in vivo use of anti-CR2 antibody molecules to inhibit CR2 ligand induced proliferation of CR2 containing cells.
  • the anti-CR2 ligand antibody molecules can be used as an antidote to therapies in which a CR2 ligand, such as IFN ⁇ , is administered first to modulate CR2 function, and the antidote is thereafter administered to neutralize the modulating effect of therapeutically administered CR2 ligands.
  • anti-CR2 ligand antibody to be administered as an antidote depends on the CR2 ligand to be neutralized, and requires that the administered antibody (antidote) have the capacity to immunoreact with the CR2 ligand.
  • the therapeutic compositions containing CR2 ligand or anti-CR2 ligand antibody molecules are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier, or vehicle.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual.
  • suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.
  • a diagnostic method of this invention for detecting CR2 ligand in the subject's blood is useful to characterize the fate of the administered ligand.
  • Epstein-Barr virus (EBV) infection of mammalian cells in an aqueous suspension, such as blood, is inhibited by methods of the present invention.
  • the therapeutically effective amount of the CR2 ligand- containing composition utilized in this method is that which produces a concentration of CR2 ligand in the aqueous suspension sufficient to bind essentially all of the CR2 receptor present.
  • the CR2 ligand includes an amino acid residue sequence corresponding to the sequence -PAIVEAG- or -NSGVEA-
  • the CR2 ligand is usually present at a concentration of about 100 ug to about 1 mg per ml.
  • the CR2 ligand includes an IFN ⁇ -related sequence as described herein
  • the CR2 ligand is usually present at a concentration of about 1 ug to about 1 mg per ml preferably about 10 ug/ml.
  • the therapeutically effective amount of the therapeutic composition administered is that which produces a blood concentration of CR2 ligand sufficient to specifically bind the CR2 receptor present and available for EBV infection.
  • a blood concentration is usually about 100 ug to about 1 mg per ml where the CR2 ligand includes an amino acid residue sequence corresponding to the sequence -PAIVEAG- or -NSGVEA-.
  • the CR2 ligand blood concentration is usually about 1 ug to about 1 mg of CR2 ligand per ml, and preferably about 10 ug/ml.
  • a CR2 ligand is administered substantially concurrently with either recurrence of infection in a chronically infected patient or upon initial exposure to EBV, such as on receipt of tissue from an EBV-seropositive donor.
  • a CR2 ligand can be administered therapeutically to cure or ameliorate diseases in which EBV, or spread of infection, plays a role, such as mononucleosis, Burkitt's lymphoma and nasopharyngeal carcinoma.
  • a CR2 ligand suitable for use in the method is any of the several forms of
  • CR2 ligand described herein so long as it contains at least one CR2 binding site and therefore has the capacity to specifically bind CR2 and thereby compete with EBV for binding.
  • 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 CR2 ligand and CR2.
  • a method of inhibiting EBV infection comprises admixing CR2 receptor-containing cells with anti-CR2 ligand antibody molecules.
  • the method is practiced in the manner described above where CR2 ligand is used to inhibit EBV infection, except that the anti-CR2 ligand antibody molecules are substituted for CR2 ligand.
  • the mechanism of inhibiting action depends, however, on the ability of anti-CR2 ligand antibodies to immunoreact (bind) with a CR2 ligand such as the gp350/220 envelope protein on EBV and thereby block the ability to the CR2 ligand to bind the CR2 receptor on the target cell's surface.
  • the terms “specifically bind” and “specifically attach” refer to non-random immunoreaction that occurs between CR2 ligands, such as the EBV gp350/220 protein, and anti- CR2 ligand antibodies of this invention.
  • 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 a CR2 ligand or an anti-CR2 ligand antibody of the present invention to a patient in amounts described above for methods to therapeutically bind and effect CR2 function.
  • An antibody of the present invention is a composition containing antibody molecules that immunoreact with a CR2 ligand of the present invention (anti-CR2 ligand antibody molecules) .
  • a preferred antibody contains antibody molecules that immunoreact with a polypeptide having an amino acid residue sequence that corresponds to the sequence shown in Figure 1 from residue 1 to residue 700, from residue 303 to residue 309, from residue 389 to residue 394, or from residue 388 to residue 396.
  • a preferred antibody contains antibody molecules that immunoreact with a polypeptide having an amino acid residue sequence represented by the formula -QLNDLEA- or -QLNNLEA-, and more preferably immunoreact with a polypeptide having the sequence shown by the formula QLNDLEACV or QLNNLEACV.
  • anti-CR2 ligand antibody molecules do not immunoreact with a polypeptide having an amino acid residue sequence represented by the formula KFSTELYQ.
  • An antibody of the present invention is typically produced by immunizing a mammal with an inoculum containing a CR2 ligand and thereby induce in the mammal antibody molecules having immunospecificity for CR2 ligand.
  • the antibody molecules are then collected from the mammal and isolated to the extent desired by well known techniques such as, for example, by using DEAE Sephadex to obtain the IgG fraction.
  • the antibodies may be purified by immunoaffinity chromatography using solid phase-affixed immunizing CR2 ligand.
  • the antibody is contacted with the solid phase-affixed CR2 ligand for a period of time sufficient for the CR2 ligand to immunoreact with the antibody molecules to form a solid phase-affixed immunocomplex.
  • the bound antibodies are separated from the complex by standard techniques.
  • the antibody so produced can be used, inter alia, in the diagnostic methods and systems of the present invention to detect CR2 ligand present in a body sample, and in the therapeutic methods and systems to inhibit CR2 ligand function.
  • inoculum in its various grammatical forms is used herein to describe a composition containing a CR2 ligand of this invention as an active ingredient used for the preparation of antibodies against CR2 ligands.
  • a polypeptide can be used in various embodiments, e.g., alone or linked to a carrier as a conjugate, or as a polypeptide polymer.
  • the various embodiments of the polypeptides of this invention are collectively referred to herein by the term "polypeptide", and its various grammatical forms.
  • polypeptide that contains fewer than about 35 amino acid residues
  • One or more additional amino acid residues can be added to the amino- or carboxy-termini of the polypeptide to assist in binding the polypeptide to a carrier.
  • Cysteine residues added at the amino- or carboxy-termini of the polypeptide have been found to be particularly useful for forming conjugates via disulfide bonds.
  • Exemplary additional linking procedures include the use of Michael addition reaction products, di- aldehydes such as glutaraldehyde, Klipstein, et al., J. Infect.
  • KLH keyhole limpet hemocyanin
  • edestin thyroglobulin
  • albumins such as bovine serum albumin (BSA) or human serum albumin (HSA)
  • red blood cells such as sheep erythrocytes (SRBC)
  • SRBC sheep erythrocytes
  • tetanus toxoid cholera toxoid
  • polyamino acids such as poly (D-lysine: D-glutamic acid) , and the like.
  • carrier is more dependent upon the ultimate use of the inoculum and is based upon criteria not particularly involved in the present invention. For example, a carrier that does not generate an untoward reaction in the particular animal to be inoculated should be selected.
  • the present inoculum contains an effective, immunogenic amount of a polypeptide of this invention, typically as a conjugate linked to a carrier.
  • the effective amount of polypeptide per unit dose sufficient to induce an immune response to the immunizing polypeptide depends, among other things, on the species of animal inoculated, the body weight of the animal and the chosen inoculation regimen as is well known in the art.
  • Inocula typically contain polypeptide concentrations of about 10 micrograms to about 500 milligrams per inoculation (dose) , preferably about 50 micrograms to about 50 milligrams per dose.
  • unit dose refers to physically discrete units suitable as unitary dosages for animals, each unit containing a predetermined quantity of active material calculated to produce the desired immunogenic effect in association with the required diluent, i.e., carrier, or vehicle.
  • the specifications for the novel unit dose of an inoculum of this invention are dictated by and are directly dependent on (a) the unique characteristics of the active material and the particular immunologic effect to be achieved, and (b) the limitations inherent in the art of compounding such active material for immunologic use in animals, as disclosed in detail herein, these being features of the present invention.
  • Inocula are typically prepared from the dried solid polypeptide-conjugate by dispersing the polypeptide-conjugate in a physiologically tolerable (acceptable) diluent such as water, saline or phosphate-buffered saline to form an aqueous composition.
  • a physiologically tolerable (acceptable) diluent such as water, saline or phosphate-buffered saline to form an aqueous composition.
  • Inocula can also include an adjuvant as part of the diluent.
  • Adjuvants such as complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum are materials well known in the art, and are available commercially from several sources.
  • a monoclonal antibody is also contemplated by the present invention and is composed of monoclonal antibody molecules that immunoreact with a CR2 ligand of the present invention.
  • the monoclonal antibody molecules immunoreact with a polypeptide having an amino acid residue sequence that corresponds to the sequence shown in Figure 1 from residue 303 to residue 309, from residue 389 to residue 394, from residue 38 ⁇ to residue 396, or from residue 1 to residue 700.
  • monoclonal antibody molecules are contemplated that immunoreact with a polypeptide having an amino acid residue sequence represented by the formula -QLNDLEA- or -QLNNLEA-, and more preferably represented by the formula -QLNDLEACV- or -QLNNLEACV-, but do not immunoreact with a polypeptide having an amino acid residue sequence represented by the formula KFSTELYQ.
  • the monoclonal antibodies of this invention can be used in the same manner as disclosed herein for antibodies of the present invention.
  • a monoclonal antibody is typically composed of antibodies produced by clones of a single cell called a hybridoma that secretes (produces) but one kind of antibody molecule.
  • the hybridoma cell is formed by fusing an antibody-producing cell and a myeloma or other self-perpetuating cell line.
  • the preparation of such antibodies were first described by Kohler and Milstein, Nature 256:495-497 (1975), which description is incorporated by reference.
  • the hybridoma supernates so prepared can be screened for immunoreactivity with a CR2 ligand or for inhibition of binding of CR2 ligand to CR2.
  • Other methods of producing monoclonal antibodies, the hybridoma cell, and hybridoma cell cultures are also well known.
  • hybridoma cell and cultures containing a hybridoma cell that produce a monoclonal antibody of this invention.
  • a diagnostic system in kit form of the present invention includes, in an amount sufficient for at least one assay, a CR2 ligand and/or an anti- CR2 ligand antibody or monoclonal antibody of the present invention, as a separately packaged reagent. Instructions for use of the packaged reagent are also typically included.
  • Instructions for use typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like.
  • a diagnostic system for assaying for the presence of or to quantitate anti-CR2 ligand antibodies in a sample comprises a package containing at least one CR2 ligand of this invention.
  • a diagnostic system of the present invention for assaying for the presence or amount of CR2 ligand in a sample comprises a package containing an anti-CR2 ligand antibody composition of this invention.
  • a diagnostic system of the present invention further includes a label or indicating means capable of signaling the formation of an immuno ⁇ omplex comprised of either a CR2 ligand of this invention specifically bound to anti-CR2 ligand antibody molecules or an anti-CR2 ligand antibody molecule of this invention specifically bound to a CR2 ligand.
  • 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.
  • Any label or indicating means can be linked to or incorporated in an antibody molecule that is part of an antibody or monoclonal antibody used in the present invention, or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents.
  • Such labels are themselves well- known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins methods and/or systems.
  • the label can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer.
  • Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC) , fluorescein isothiocyanite (FITC) , 5-dimethylamine-l- naphthalenesulfonyl chloride (DANSC) , tetramethylrhoda ine isothiocyanate (TRITC) , lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like.
  • fluorochromes such as fluorescein isocyanate (FIC) , fluorescein isothiocyanite (FITC) , 5-dimethylamine-l- naphthalenesulfonyl chloride (DANSC) , t
  • the label is an enzyme, such as horseradish peroxidase (HRP) , glucose oxidase, alkaline phosphatase or the like.
  • HRP horseradish peroxidase
  • additional reagents are required to visualize the fact that a antibody-antigen complex (immunoreactant) has formed.
  • additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine.
  • An additional reagent useful with HRP is 2,2•-azino-di-(3-ethyl- benzthiazoline-6-sulfonic acid) (ABTS) .
  • Radioactive elements are also useful labeling agents and are used illustratively herein.
  • An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as 12 I, 125 I, 128 I, 132 I and 51 Cr represent one class of gamma ray emission- producing radioactive element indicating groups. Parti •cularly preferred i.s 125I.
  • Another group of useful labeling means are those elements such as 11 C, 18 F, 15 0 and 13 N which themselves emit positrons. The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body.
  • a beta emitter such as 111 indium or 3 H.
  • labeling of, polypeptides and proteins is well known in the art.
  • antibody molecules produced by a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium.
  • the techniques of protein conjugation or coupling through activated functional groups are particularly applicable. See, for example, Aurameas, et al., Scand. J. Immunol., 1:7-23 (1978), Rodwell et al., Biotech., 3:8 ⁇ 9- ⁇ 94 (1964), and U.S. Patent. No. 4,493,795.
  • 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 conjugate or 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 anti- CR2 ligand antibody molecules of this invention when it is present as part of an immunocomplex. When detecting patient anti-CR2 antibodies, anti-human Fc antibodies are conveniently used. In preferred embodiments 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 CR2 ligand or anti-CR2 ligand 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 19 ⁇ 2, and in U.S. Patents No. 3,654,090; No.
  • the CR2 ligand or an anti-CR2 ligand antibody molecule 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, - 5 ⁇ -
  • 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.
  • a package refers to a solid matrix or material such as gloss, plastic, paper, foil and the like capable of holding within fixed limits a diagnostic reagent such as a polypeptide, antibody or monoclonal antibody of the present invention.
  • a package can be a glass vial used to contain a contemplated diagnostic reagent or it can be a microtiter plate well to which microgram quantities of a contemplated diagnostic reagent have been operatively affixed, i.e., linked so as to be capable of being immunologically bound by an antibody or CR2 ligand to be detected.
  • the present invention also contemplates any diagnostic method that results in detecting CR2 ligand or anti-CR2 ligand antibodies in a body fluid sample using CR2 ligand or antibody molecule-containing compositions of this invention.
  • a diagnostic method that results in detecting CR2 ligand or anti-CR2 ligand antibodies in a body fluid sample using CR2 ligand or antibody molecule-containing compositions of this invention.
  • a bodily fluid sample such as blood, plasma or serum from the patient is contacted by admixture under biological assay conditions with an anti-CR2 ligand antibody molecule of the present invention to form an immunoreaction admixture.
  • the admixture is then maintained for a period of time sufficient to allow the formation of a CR2 ligand-antibody molecule immunoreaction product (immunocomplex) .
  • the complex can then be detected as described herein. The presence of the complex is indicative of CR2 ligand in the sample.
  • the detection of CR2 ligands in a body sample is utilized as a means to monitor the fate of therapeuticallly administered CR2 ligands according to the therapeutic methods disclosed herein.
  • a CR2 ligand of the present invention is admixed with a patient's bodily fluid sample to form the immunoreaction admixture.
  • the presence of an immunoreaction product is indicative of anti-CR2 ligand antibodies in the sample.
  • the diagnostic methods of the present invention are practiced in a manner whereby the immunocomplex is formed and detected in a solid phase, as disclosed for the diagnostic systems herein.
  • Biological assay conditions are those that maintain the biological activity of the CR2 ligand molecules and the anti-CR2 antibodies in the immunoreaction admixture. 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. Also contemplated are immunological assays capable of detecting the presence of immunoreaction product formation without the use of a label.
  • detection means include methods known as biosensors and include biosensing methods based on detecting changes in the reflectivity of a surface, changes in the absorption of an evanescent wave by optical fibers or changes in the propagation of surface acoustical waves.
  • a cDNA That Encodes A CR2 Ligand A lambda gtlO cDNA library obtained from a human B lymphoblastoid cell line Raji (ATCC #CCL 86) known to produce alpha-BCGF (B cell growth factor) activity [Corbel et al., Immunol. Rev., 78:51 (1983)] was obtained from Clontech (Palo Alto, CA) . Oligonucleotides encoding the LYNVEA motif on C3 fragments were used to screen the cDNA library and a clone, lambda 9-4, was ultimately isolated that contained an EcoR I insert of 3.1 kilobase pairs (kb) .
  • the insert was sequenced and found to contain an open reading frame that encodes a 700 amino acid residue protein.
  • the nucleotide sequence and encoded amino acid sequence for the lambda 9-4 insert are shown in Figure 1. A comparison of the lambda 9-4 insert sequence with sequence data banks showed no significant homology to any known protein or gene.
  • the CR2 ligand-encoding insert in the lambda 9-4 cDNA is designated a DNA segment that encodes a new CR2 ligand protein.
  • a B cell proliferation assay system was used to examine the ability of various CR2 ligand polypeptides to inhibit B lymphocyte proliferation.
  • Example 3 Each cell line was cultured as described in Example 3 with the following exceptions: human peripheral B cells were cultured with EBV (strain B95/ ⁇ ) at a multiplicity of infection in excess of 1.0 EBV units per cell and was admixed simultaneously with admixture of polypeptide; mouse splenic B cells were cultured with the admixed polypeptides in combination with lipopolysaccharide (LPS) at 25 ug/ml.
  • LPS lipopolysaccharide
  • b Inhibition is expressed as the amount of added polypeptide required to exhibit half-maximal inhibition: +: half-maximal inhibition at 200- 500 ug/ml; ++: at 50-200 ug/ml; +++: at 5-50 ug/ml.
  • Polypeptides were prepared as described in Example 1. N.T. indicates not tested.
  • the CR2 ligand polypeptides NSGVEA and QNSGVEALT also strongly inhibit B cell proliferation, as measured by the Raji cell assay. Thus, by this proliferation inhibition assay, inhibitory CR2 ligand polypeptides are identified. None of the polypeptides tested inhibited proliferation of CR2 receptor- negative human (HSB2) or murine (BW5147) T cell lines, indicating that the CR2 ligant polypeptides are specific for CR2-containing cells and do not inhibit proliferation due to toxicity of the polypeptide. 4. Inhibition of Direct Binding of CR2
  • Cultures containing 10 5 Raji or HSB2 cells were admixed with varying amounts (zero to 200 ug) of the polypeptides shown in Table 4 in a volume of 100 microliters (ul) of culture medium and the admixture was maintained at 37 C for 30 minutes to allow CR2 ligand polypeptides to bind any CR2 receptor present on the cells. Thereafter, 50 ul of C 3 -coated Zymosan composition was added to each culture, the cultures were subjected to centrifugation for one minute at 600 x g and then maintained at 37 C for 30 minutes. The maintained cultures were then washed with prewarmed culture medium to rinse off non-bound particles.
  • Washed cultures were observed using a microscope to detect and count the number of particles (rosettes) attached per cell. More than three rosettes per cell was counted as C 3 specific binding. Inhibition is expressed as the percent decrease in bound rosettes when comparing binding in the presence or absence of admixed polypeptide. The results are shown in Table 4.
  • the religated plasmid was digested with Xho I to form a linearized plasmid, and was then treated with mung bean nuclease to blunt end the previously cohesive Xho I termini.
  • the blunt-ended plasmid was then religated to form a circular plasmid, designated pBS 9-4.
  • the plasmid pBS 9-4 as presently constructed can express a fusion protein that consists of about 30 amino acid residues at the amino terminus of the fusion protein, that are derived from the beta- galactosidase gene present in the original plasmid Bluescript 2 SK+, and about 700 amino acids at the carboxy terminus of the fusion protein, that are derived from the lambda insert encoding the CR2 ligand protein.
  • CR2 Ligand Plasmid pBS 9-4 prepared in Example 5a, is introduced into the E. coli strain XLI- Blue (Stratagene) by transformation, and a culture of transformed bacteria is then cultured in the presence of IPTG to induce expression of fusion protein off of the beta-galactosidase promoter present on the expression vector.
  • Whole cell extracts from both induced and control (uninduced) cell cultures of the bacteria containing pBS 9-4 are then prepared and the extracts analyzed by polyacrylamide gel electrophoresis in the presence of SDS (PAGE-SDS) according to the procedure of Laemmli (Nature,
  • the resulting PAGE-SDS gels when strained with Coo issie blue, exhibit a band having a molecular weight of about 80,000 daltons that is present in higher amounts in the induced extracts relative to the uninduced extracts.
  • the 60,000 dalton band is the expected size for a fusion protein comprised of 30 amino acid residues from beta- galactosidase fused to 700 residues from the CR2 ligand-encoding gene.
  • the 80,000 dalton band is not present in induced extracts prepared using cells transformed with the control, plasmid Bluescript.
  • Eukaryotic Vector Preparation PCMV 9-4 The regliated plasmid produced after isoschitzomer ligation in Example 5a is digested with Xbal to form a linearized plasmid with cohesive Xbal termini. The cohesive termini are filled in by treating the linearized plasmid with the klenow fragment of E. coli DNA polymerase and then Xhol linkers are blunt end ligated onto the filled in termini of the linearized plasmid. The linker- modified plasmid is then digested with Xhol to produce cohesive termini and to excise an Xhol DNA segment of about 2.1 kb that includes the CR2 ligand-encoding gene.
  • the 2.1 kb DNA segment is isolated from the other nucleic acid products of the Xho I digestion by agarose gel electrophoresis, and the isolated segment is ligated into the Xho I site of the eukaryotic expression vector pCMVneo (Karasuyama et al., Eur. J. Immunol., 18:97, 198 ⁇ ) to form a mixture of plasmids in which the insert is present within pCMVneo in several possible orientations.
  • the formed mixture of plasmids are collectively transformed into E. coli. individually isolated, and individually characterized by restriction enzyme digestion with BamH I and Hinc II and electrophoresis on 1% agarose.
  • the isolated plasmid that exhibits a restriction pattern that includes a DNA segment of 1.6 kb and not 1.36 kb is selected as having the proper orientation for expression of the CR2 ligand encoding gene, and is designated pCMV 9-4.
  • pMSG 9-4 Plasmid pBS 9-4, prepared in Example 5a, is digested with Not I to form a linearized plasmid with cohesive Not I termini. The cohesive termini are filled in by treating the linearized plasmid with the klenow fragment of E. coli DNA polymerase and then Xhol linkers are blunt end ligated onto the filled in termini of the linearized plasmid.
  • the linker-modified plasmid is then digested with Xhol to produce cohesive Xhol termini and further digested with Sail to excise a DNA segment of about 2.1 kb having one Xhol and one Sail termini that includes the CR2 ligand-encoding gene.
  • the 2.1 kb DNA segment is isolated from the other nucleic acid products of the Xhol and Sail digestion by agarose gel electrophoresis, and the isolated segment is ligated into the eukaryotic expression vector pMSG (Pharmacia LKB Biotechnology, Piscataway, NJ) at the Xhol and Sail sites contained therein to form plasmid pMSG 9-4.
  • Example 5c is introduced into murine L cells (ATCC # CCL 1) by transfection using the calcium phosphate technique and selection in the presence of the drug G41 ⁇ to produce a transformed cell culture comprising L cells that contain the expression vector pCMV 9-4, designated as L/pCMV 9-4.
  • Culture supernatant is collected from a culture of L/pCMV 9-4 cells, and is analysed on PAGE-SDS as described in Example 5b. Electrophoresed proteins contained in the resulting PAGE-SDS gel are electrophoretically transferred from the gel to a nitrocellulose sheet according to the Western blot technique of Towbin et al. (Proc. Natl. Acad. Sci. USA, 75:4350-54, 1979), and the resulting blot (solid- phase affixed antigen) is then immunoreacted with an antibody composition containing anti-CR2 ligand antibody molecules prepared as in Example 6, to form an immunoblot containing an immunoreaction product affixed thereto.
  • the immunoreaction product is detected using a picoBlue Immunoscreening Kit (Stratagene) with anti-rabbit second antibody according to the manufacturer's instructions.
  • the results of detecting immunoreaction product in the immunoblot show a band having a molecular weight of about 77,000 daltons present in supernatant samples obtained from L/pCMV 9-4 cultures, but not present in control samples of supernatant from cultures of L/pCMVneo.
  • the 77,000 dalton band is the approximate size expected of an expressed CR2 ligand protein having 700 amino acid residues.
  • Plasmid pMSG 9-4 prepared in Example 5c, is introduced into L cells as described above for plasmid pCMV 9-4, except that selection is carried out as described by Mulligan et al. (Proc. Natl. Acid. Sci. USA, 7 ⁇ :2072, 19 ⁇ l) for transformants that contain the E. coli xanthine-guanine phosphoribosyltransferase gene (gpt) . Transformed cultures are cultured as for L/pCMV 9-4 and culture supernatant is harvested to produce expressed CR2 ligand from the L/pMSG 9-4 transformed cell cultures. Upon analysis by Western blotting, an expressed CR2 ligand is detected as a band having a molecular weight of about 77,000 daltons. 6. Production of Anti-CR2 Ligand Antibodies
  • Polypeptide NSGVEALI prepared as described in Example 2, was conjugated to bovine serum albumin (BSA) at an equal weight ratio using the glutaraldehyde coupling procedure of Aurameas et al. (Scand. I. Immunol., 1:7-23, 1978).
  • BSA bovine serum albumin
  • New Zealand white rabbits were each injected with an immunogen containing 2.5 mis of a PBS suspension containing 1.25 mis complete Freund's adjuvant and 0.5 mg of polypeptide in a polypeptide-BSA conjugate. Thereafter, each rabbit was boosted with the same immunogen, 7, 14, and 21 days after the first immunization, except that incomplete Freund's adjuvant was used.
  • CR2 was purified from the NP40-diluted supernatant by affinity chromatography in a column format using a mixture of anti-CR2 monoclonal antibodies [OKB-7 (Ortho-Immune, Rariton, NJ) and HB5 (Becton Dickinson, Cockeysville, MD) ] covalently bound to Affi-Gel 10 beads (BioRad, Richmond, CA) . Bound proteins were eluted in 50 mM diethylamine, pH 11.2, 0.1% Triton X-100 and the eluant was collected.
  • a second chromatography was performed by re-applying the eluted proteins to a second affinity column containing the OKB-7 and HB5 monoclonal antibodies. Proteins eluted from the second chromatography were dialyzed against PBS containing 0.1% Triton X-100 and stored at -80 C. The resulting twice affinity purified protein composition contained homogeneous CR2 as assessed by polyacrylamide gel electrophoresis in sodium dodecyl- sulfate (SDS-PAGE) and subsequent silver staining.
  • SDS-PAGE sodium dodecyl- sulfate
  • pCMV-IFN recombinant DNA molecule
  • rIFN ⁇ recombinant interferon alpha
  • This protein is a fusion protein having an amino acid residue sequence shown in Figure 3, including an amino terminal portion corresponding to a portion of placental alkaline phosphatase from residue 1 to residue 69, and having a carboxy terminal portion corresponding to IFN ⁇ A from residue 70 to residue 219.
  • pCMV-IFN includes a DNA segment having the nucleic acid sequence shown in Figure 3.
  • pCMV-IFN For the preparation of pCMV-IFN, a DNA segment that encodes the IFN ⁇ portion and including the nucleotide base residue sequence from base 254 to base 703 shown in Figure 3 was isolated from a cDNA clone provided by P. Gray. The isolated IFN ⁇ encoding DNA segment was then combined into the eukaryotic expression vector pCMVhyg (obtained from H. Karasuyama) so as to operatively link the IFN ⁇ coding DNA segment with a DNA segment that encodes an amino- terminal portion of alkaline phosphatase having the nucleotide base residue sequence from base 47 to base 253 shown in Figure 3, thereby forming pCMV-IFN. The resulting rDNA molecule, pCMV-IFN, encodes and expresses an alkaline phosphatase-IFN ⁇ fusion protein having the amino acid residue sequence shown in
  • FIG. 3 from amino acid residue 1 to residue 219, and is referred to as IFN ⁇ protein.
  • Example 8a Purification of IFN ⁇ Protein pCMV-IFN, prepared in Example 8a, was introduced into murine L cells by the calcium phosphate method, and successfully transfeeted L cells were isolated by selection for hygromycin resistance conferred by genetic markers present in the vector pCMV-IFN. The resulting transfected L cells were grown to confluence in roller bottles. Culture medium in the roller bottles was then replaced with Iscove's serum substituted medium containing huTransferrin (18 ug/ml) , BSA (100 ug/ml) and hygromycin B (500 ug/ml) , and the confluent L cells were maintained under normal culture conditions.
  • huTransferrin 18 ug/ml
  • BSA 100 ug/ml
  • hygromycin B 500 ug/ml
  • Culture medium supernatants were harvested every 24 hours and used as a source of IFN ⁇ protein. Under this regimen, steady IFN ⁇ protein production was observed for five days.
  • the collected supernatants from five days culturing were pooled and the IFN ⁇ protein in pooled supernatants was precipitated with ammonium sulf te.
  • the precipitated IFN ⁇ protein was collected, suspended in 0.1M potassium phosphate buffer, pH 6.0, containing 10% glycerol and dialyzed with 1000 volumes of the same buffer for 12 hours at 4 C. The dialyzed precipitate was chromatographed on a P60 column to form separate dimeric and monomeric IFN ⁇ fractions.
  • Example 8c The affinity purified IFN ⁇ protein so produced from clone LI-3 is referred to as affinity- purified recombinant IFN ⁇ (rIFN ⁇ ) .
  • the admixture was maintained for two hours at 37 C to allow the added CR2 in the liquid phase to specifically bind with the rIFN ⁇ present immobilized in the solid phase on the walls of the wells.
  • the wells were then washed with PBST to remove unbound CR2.
  • washed wells were incubated (admixed and maintained) successively for one hour at room temperature first with monoclonal anti-CR2 antibody HB5 (50 ng in 50 ul PBST/well) and then with rabbit anti-mouse antibody conjugated to alkaline phosphatase (50 ul of a 1:1000 solution in PBST/well) , the wells being washed with PBST after each incubation.
  • monoclonal anti-CR2 antibody HB5 50 ng in 50 ul PBST/well
  • rabbit anti-mouse antibody conjugated to alkaline phosphatase 50 ul of a 1:1000 solution in PBST/well
  • a chromogenic alkaline phosphatase substrate (100 ug p-nitrophenyl phosphate dissolved in 100 ul, 150 mM NaCl, 50 mM Tris-HCl, pH 8.5) was added to each well and the wells maintained for 60 minutes at 37 C to allow for the formation of p-nitrophenol as a means to detect the amount of immunoreaction product formed, and thereby the amount of specific CR2 binding to IFN ⁇ in the solid phase.
  • the formation of p-nitrophenol was determined by measuring optical absorbance at 405 nm in a Titertek ELISA reader (Titertek Multiskan MCC/340, Metea, VA) . Table 5 shows the results of the above detection of CR2 binding to rIFN ⁇ in the solid phase as determined by ELISA. TABLE 5 Binding of CR2 to IFN ⁇
  • rIFN ⁇ Compositions Containing At Least Two CR2 Binding Sites
  • rIFN ⁇ prepared in Example 8b is provided in a solution of PBS at a concentration of about 0.1 mg/ml. The solution is maintained at 23 C for 120 minutes to allow air oxidation to drive an oxidizing reaction between cysteine residues present in rIFNA ⁇ and form conjugates of rIFN ⁇ protein having disulfide bridges formed between rIFN ⁇ protein molecules. The resulting solution contains an rIFNA ⁇ composition having at least two CR2 binding sites per conjugate.
  • the DNA segment that encodes the rIFN ⁇ protein according to Example 8a is subcloned into the plasmid pBluescript (Stratagene Cloning Systems, San Diego, CA) . Sacl and BamHl restriction endonuclease sites are introduced adjacent to the CR2 binding site coding DNA sequence shown in Step I of Figure 4 by site-directed mutagenesis using two mismatch primers according to the scheme shown in Figure 4. By mutagenesis of the nucleotide at position 469, the base A is changed to a base G to create a Sacl endonuclease site.
  • the base T is converted to base a G and at the nucleotide position 508, the base A is converted to a base C.
  • These latter two changes create a BamHl endonuclease site as shown in Figure 4.
  • the resulting rDNA molecule containing the substituted bases and encoding rIFN ⁇ is then cleaved with Sacl and BamHl restriction endonucleases to allow for removal of the DNA segment encoding the CR2 binding site portion of rIFN ⁇ .
  • a 41 base long single-stranded oligonucleotide having the oligonucleotide sequence shown in Step II of Figure 4 and coding for the synthetic polypeptide including the amino acid residue sequence QLYNVEA, is annealed to the overhangs present on the rDNA molecule generated by the Sacl and BamHl restriction cuts to form a DNA segment encoding a modified rIFN ⁇ having a CR2 binding site amino acid residue sequence as shown in Figure 4.
  • the modified rIFN ⁇ -encoding DNA segment is removed from the plasmid pBluescript by digestion with the restriction enzymes Notl and Xhol and ligated into the Notl/Xhol restriction sites of the predigested expression plasmid pCMVhyg to form pCMV- lFN ⁇ .
  • the expression plasmid pCMV-mlFN ⁇ expresses an IFN ⁇ having a modified CR2 binding site, which site includes the amino acid residue sequence -QLYNVEA-.
  • pCMV-mlFN ⁇ is then tranfected into mouse L cells as described in Example 8b to produce an IFN ⁇ having a modified CR2 binding site.
  • Anti-CR2 Ligand Antibodies a. Preparation of Antibodies Synthetic polypeptides related to the
  • CR2 ligands C3 or CBP-A having the sequence of polypeptides p7 and p9 from Table 2 were independently conjugated to bovine serum albumin (BSA) at an equal weight ratio using the glutaraldehyde coupling procedure of Aura eas et al. (Scand. J. Immunol.. 1:7- 23, 1978).
  • BSA bovine serum albumin
  • New Zealand white rabbits were injected with an immunogen comprising 2.5 mis of a PBS suspension containing 1.25 mis complete Freund's adjuvant and 0.5 mg of polypeptide in the form of polypeptide-BSA conjugate. Thereafter, each rabbit was boosted with the same immunogen 7, 14, and 21 days after the first immunization except that incomplete Freund's adjuvant was used. About 28 days after the first immunization, antisera was collected from the immunized rabbits and analyzed in the ELISA assay described in Example 10b to detect anti-CR2 ligand antibody immunoreactivity.
  • the immunoreaction admixture was then maintained for two hours at 37 C to allow a first immunoreaction product to form.
  • the wells were then washed with PBST to remove unbound antibodies.
  • Fifty ul of a solution containing goat anti-rabbit alkaline phosphatase conjugate (Jackson Labs, 1:1000 solution in PBST) was added to each well to form a second immunoreaction admixture, and the admixture was maintained at room temperature to allow a second immunoreaction product to form.
  • the wells were washed with PBST and the second immunoreaction product was detected using a chromogenic substitute as described in Example 8c.
  • the immunoreactivity of the anti-CR2 ligand antibody preparations are shown below in Table 6.
  • the anti-polypeptide antisera prepared against the CR2 ligands p7 or p8 each immunoreacted with their homologous immunizing peptide and also immunoreacted to varying degrees with several other polypeptides bearing a CR2 binding site.
  • These same antisera did not contain polyclonal antibody molecules that significantly immunoreacted with a fibronectin fragment-derived polypeptide (plO) indicating the specificity of the immunoreaction.
  • the anti-CR2 ligand antibodies also immunoreacted strongly with the rIFN ⁇ prepared in Example ⁇ b. This latter immunoreaction demonstrates that native IFN ⁇ A contains a structural domain (i.e., a CR2 binding site) that crossreacts with antibodies immunoreactive with the CR2 ligands of this invention.
  • a PBST solution 50 ul containing about 100 ng of affinity-purified CR2, prepared as described in Example 7, was added to each well to form a first admixture and maintained for two hours at 37 C to allow the CR2 in solution to specifically bind to the solid phase CR2 ligand.
  • wells were incubated successively for one hour at room - ⁇ l -
  • CR2 binds with synthetic polypeptides related to the CR2 binding site of C3, IFN ⁇ A, CBP-A and CBP-B. These data demonstrate that the CR2 ligands of this invention, and other CR2 ligands, when used in a solid-phase binding assay, specifically interact (bind) with CR2 receptor. CR2 did not substantially bind with synthetic polypeptides derived from IFN ⁇ F (p6) or fibronectin fragments (plO) . These latter data are consistent with the observations on the effects of CR2 ligands on B cell proliferation as discussed below in Example 13. - 62 -
  • a binding reaction admixture containing rIFN ⁇ in the solid phase and CR2 in the liquid phase was prepared as described in Example ⁇ c except that a competing CR2 ligand shown in Table 8 was also included in the admixture at a concentration of 100 ug/ml. Thereafter, the binding assay was conducted as described in Example 8c to measure the amount of CR2 specifically bound to rIFN ⁇ . The results of inhibition of CR2 binding to rIFN ⁇ by CR2 ligand polypeptides is shown in Table 8.
  • CR2 ligands such as the polypeptides used above can inhibit the specific binding of CR2 to a native CR2 ligand such as the indicated interferon alpha species that bind CR2. Similar results were obtained when purified EBV gp350/220 protein was used in the liquid phase in place of CR2, indicating that CR2 ligands inhibit EBV binding to CR2.
  • a B cell proliferation assay system was used to examine the ability of various CR2 ligand polypeptides to inhibit B lymphocyte proliferation.
  • Cultures of the cell lines shown in Table 9 were established at a density of 2 x 10 4 cells per well of a 96 well microtiter plate in serum-free ISCOVE'S medium. Varying amounts (zero to 500 ug per ml of culture) of the polypeptides of Table 8 were added to the cultures, and the cell-polypeptide admixtures maintained for 24 to 48 hours at 37 C. Thereafter, 1 uCi of 3 H-thymidine label was admixed with each culture and maintained for four hours at 37 C to allow for label incorporation into proliferating cells. After four hours, the cultures were harvested and the incorporated label measured by standard techniques.
  • Example 13 with the following exceptions: human peripheral B cells were cultured with EBV (strain B95/8) at a multiplicity of infection in excess of 1.0 EBV units per cell and was admixed simultaneously with admixture of polypeptide; mouse splenic B cells were cultured with the admixed polypeptides in combination with lipopolysaccharide (LPS) at 25 ug/ml.
  • EBV strain B95/8
  • LPS lipopolysaccharide
  • b Inhibition is expressed as the amount of added polypeptide required to exhibit half-maximal inhibition: -: no inhibition at 500 ug polypeptide per ml; +: half-maximal inhibition at 200-500 ug/ml; ++: at 50-200 ug/ml; +++: at 5-50 ug/ml.
  • Polypeptides were prepared as described in Example 1. d N.T. indicates not tested.

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Abstract

On décrit des polypeptides synthétiques correspondant au site de liaison du récepteur CR2 à lymphocyte B, présent sur un ligand CR2, ainsi que des agrégats polypeptidiques, des compositions, des anticorps anti-polypeptidiques et des procédés de préparation et d'utilisation des polypeptides et des anticorps.
PCT/US1990/005027 1989-09-08 1990-09-04 Compositions du ligand cr2 et procede de modulation des fonctions de cellules immunitaires WO1991003251A1 (fr)

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US07/404,679 US5331090A (en) 1989-09-08 1989-09-08 CR2 ligand compositions and methods for modulating immune cell functions
US07/512,118 US5310729A (en) 1990-04-20 1990-04-20 Interferon-related polypeptides as CR2 ligands and their use for modulating immune cell functions
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5310729A (en) * 1990-04-20 1994-05-10 California Institute Of Biological Research Interferon-related polypeptides as CR2 ligands and their use for modulating immune cell functions
WO2000064936A2 (fr) * 1999-04-27 2000-11-02 Wieser Raimund J HOMODIMERES PEPTIDIQUES ET HETERODIMERES PEPTIDIQUES DERIVES DE L'INTERFERON α2
US6225292B1 (en) * 1997-06-06 2001-05-01 The Regents Of The University Of California Inhibitors of DNA immunostimulatory sequence activity
CN111848779A (zh) * 2019-08-07 2020-10-30 北京市农林科学院 一种鸡补体受体2(ChCR2)单克隆抗体及其应用

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5310729A (en) * 1990-04-20 1994-05-10 California Institute Of Biological Research Interferon-related polypeptides as CR2 ligands and their use for modulating immune cell functions
US6225292B1 (en) * 1997-06-06 2001-05-01 The Regents Of The University Of California Inhibitors of DNA immunostimulatory sequence activity
US8729039B2 (en) 1997-06-06 2014-05-20 The Regents Of The University Of California Use of inhibitory oligonucleotides to treat autoimmune disease
WO2000064936A2 (fr) * 1999-04-27 2000-11-02 Wieser Raimund J HOMODIMERES PEPTIDIQUES ET HETERODIMERES PEPTIDIQUES DERIVES DE L'INTERFERON α2
WO2000064936A3 (fr) * 1999-04-27 2001-02-22 Raimund J Wieser HOMODIMERES PEPTIDIQUES ET HETERODIMERES PEPTIDIQUES DERIVES DE L'INTERFERON α2
CN111848779A (zh) * 2019-08-07 2020-10-30 北京市农林科学院 一种鸡补体受体2(ChCR2)单克隆抗体及其应用

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