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WO1994009132A1 - Antigenes cd26 humain et procedes d'utilisation - Google Patents

Antigenes cd26 humain et procedes d'utilisation Download PDF

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WO1994009132A1
WO1994009132A1 PCT/US1993/007923 US9307923W WO9409132A1 WO 1994009132 A1 WO1994009132 A1 WO 1994009132A1 US 9307923 W US9307923 W US 9307923W WO 9409132 A1 WO9409132 A1 WO 9409132A1
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leu
ser
immunoprecipitate
tyr
thr
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Chikao Morimoto
Stuart Schlossman
Toshiaki Tanaka
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Dana Farber Cancer Institute Inc
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Dana Farber Cancer Institute Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70589—CD45
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the field of the invention is human T cell activation antigens.
  • CD26 is a human T cell activation antigen originally identified by its reactivity with the monoclonal antibody Tal (Fox et al., J. Immunol . 133:1250, 1984). CD26 has recently been shown to be identical to human dipeptidyl peptidase IV (EC 3.4.14.5) (Ulmer et al., Scand . J. Immunol . 31:429, 1990; Barton et al., J . Leukocyte Biol . 48:291, 1990).
  • Dipeptidyl peptidase IV is a serine exopeptidase which is capable of cleaving x-proline or x-alanine (where x is any amino acid) from the amino terminus of certain peptides.
  • CD26 is recognized by a second monoclonal antibody, anti-lF7 (Morimoto et al., J. Immunol . 143:3430, 1989). Dang et al. (J. Immunol . 144:4092, 1990) report that solid phase-immobilized anti-lF7 mAb is capable of inducing proliferation of human CD4 + T lymphocytes in conjunction with submitogenic doses of anti-CD3 or anti-CD2 antibodies. They suggest that the CD26 antigen is involved in CD3- and CD2-induced human CD4 + T cell activation. Summary of the Invention In one aspect, the invention features a polypeptide fragment of CD26 lacking amino acid residues 3-9 of the latter sequence.
  • fragment is meant a portion of CD26 that represents at least 50 consecutive residues of CD26. Such a fragment will preferably represent at least 100 residues of CD26, more preferably at least 200, and most preferably at least 500; it preferably includes the DPPIV active site residues at residues 627-631.) Such a fragment, in which the amino acid residues to the carboxy terminal side of residue 37 are preferably intact, is encoded by the nucleic acid sequence shown as CD26 ⁇ 3-9 (SEQ ID NO: 2) .
  • the polypeptide has an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 2; the polypeptide is soluble under physiological conditions; and the polypeptide is substantially pure. Also within the invention is the product of signal peptidase proteolytic cleavage of this polypeptide, which would be a form of CD26 lacking residues 1-34, 1-35, 1- 36, or 1-37.
  • the invention features a polypeptide fragment of CD26 lacking residues 24-34 of the latter sequence.
  • a fragment in which the amino acid residues to the carboxy terminal side of residue 37 are preferably intact, is encoded by the nucleic acid sequence shown as CD26 ⁇ 24-34 (SEQ ID NO: 3) .
  • the polypeptide has an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 3; the polypeptide is soluble under physiological conditions; and the polypeptide is substantially pure.
  • the product of signal peptidase proteolytic cleavage of this polypeptide which would be a form of CD26 lacking residues 1-34, 1-35, 1- 36, or 1-37.
  • the invention features a plasmid encoding a polypeptide fragment of CD26 having an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 2 (CD ⁇ 3-9) or 3 (CD ⁇ 24-34) ; this plasmid preferably includes an expression control sequence.
  • Polypeptide fragments of CD26 which are soluble under physiological conditions generally lack most or all of the hydrophobic amino acid residues found near the amino terminus of the polypeptide depicted in SEQ ID NO: 1. This can be accomplished by genetically manipulating a nucleic acid encoding CD26 to delete the hydrophobic residues, or to delete enough of the N-terminal amino acids (e.g., residues 3-9 or 24-34) to leave the resulting polypeptide susceptible to cleavage by signal peptidase.
  • Other fragments of CD26 which are within the invention include those in which all or part of the putative dipeptidyl aminopeptidase catalytic site (Gly 627 to Gly 631 ) is deleted.
  • Such fragments which include inter alia the deletion mutant shown in Fig. 15 (SEQ ID NO: 11) ; fragments having additional deletions such as those in ⁇ 3-9 (SEQ ID NO: 2) and ⁇ 24-34 (SEQ ID NO: 3) ; and those missing the entire signal peptide region up to Ala 35 , Thr 36 , Ala 37 or Asp 38 , would constitute enzymatically inactive fragments of CD26 useful in the screening assays of the invention, as well as for inhibiting complex formation between CD26 and/or CD45 and p43.
  • a mutant form of CD26 (or a fragment thereof) which lacks DPPIV activity can be generated by replacing one of the residues in the active site with a different amino acid (e.g., by replacing Ser 62g with Ala) .
  • substantially pure is meant a polypeptide or protein which has been separated from biological macromolecules, (e.g., other proteins, carbohydrates, etc.) with which it naturally occurs.
  • biological macromolecules e.g., other proteins, carbohydrates, etc.
  • a protein or polypeptide of interest is substantially pure when less than 25% (preferably less than 15%) of the dry weight of the sample consists of such other macromolecules.
  • physiological conditions an aqueous solution, whether in vivo or in vitro, having a pH and salt concentration similar to that found in serum.
  • Phosphate buffered saline is an example of a commonly used buffer in which a polypeptide that is soluble under physiological conditions would be soluble.
  • substantially identical to CD26 is meant that at least 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 99%, of the amino acid sequence is identical to that of the corresponding portion of CD26, and any non-identical amino acids in the sequence are amino acid substitutions, preferably conservative, which do not eliminate the biological activity of the molecule.
  • plasmid is meant an extrachromosomal DNA molecule which includes sequences that permit replication within a particular host cell.
  • an expression control sequence is meant a nucleotide sequence which includes recognition sequences for factors that control expression of a protein coding sequence to which it is operably linked. Accordingly, an expression control sequence generally includes sequences for controlling both transcription and translation: for example, promoters, ribosome binding sites, repressor binding sites, and activator binding sites.
  • the invention features a polypeptide fragment of CD26 capable of disrupting the naturally-occurring binding interaction between CD45 and CD26.
  • Polypeptides which disrupt the interaction between CD26 and CD45 can be identified, for example, using the immunoprecipitation assay described below.
  • the invention features a method for screening candidate compounds to identify compounds capable of inhibiting the binding of CD26 to CD45, which method includes the steps of:
  • CD45 present in the second immunoprecipitate the presence of a lesser amount of CD45 in the first immunoprecipitate than in the second immunoprecipitate indicating that the candidate compound inhibits the binding.
  • an anti-CD26 antibody is one capable of forming a specific immune complex with CD26, i.e., the antibody binds directly to CD26 but does not substantially bind directly to other molecules in the assay of the invention.
  • the invention features a method for screening candidate compounds to identify compounds capable of inhibiting the binding of CD26 to CD45, which method includes the steps of: (a) providing a first and a second sample of cells expressing both CD26 and CD45;
  • the invention features a monoclonal antibody which, when contacted under physiological conditions with a cell (preferably a eukaryotic cell such as a mammalian cell) expressing CD26 and CD45, interferes with the association of CD26 and CD45; and a method for assaying for such an antibody.
  • a cell preferably a eukaryotic cell such as a mammalian cell
  • the invention features a method which includes:
  • Eennccooddiinngg CD26 such that the cell expresses CD26 on its surface.
  • the invention features a method which includes: (a) providing a cell which expresses CD26 on its surface; and
  • the invention includes a cell transfected with a nucleic acid encoding CD26, the cell expressing both CD26 and CD45 on its surface; and a cell transfected with a nucleic acid encoding CD45, the cell expressing both CD26 and CD45 on its surface.
  • the cells are T-cells such as Jurkat cells.
  • the invention features a method which includes: (a) providing a cell which expresses neither CD26 nor CD45 on its surface; and
  • the invention includes a method of generating a hybridoma cell, which method includes:
  • (c) fusing a B lymphocyte from the subject animal with a cell from an immortal cell line (i.e., a line of cells which can be maintained indefinitely in culture) to produce a hybridoma cell.
  • an immortal cell line i.e., a line of cells which can be maintained indefinitely in culture
  • the invention features a hybridoma cell generated by:
  • (c) fusing a B lymphocyte from the subject animal with a cell from an immortal cell line to produce a hybridoma cell, wherein the hybridoma cell produces a monoclonal antibody specific for CD26.
  • Applicable methods of inducing an immune response in an animal by using cells as the antigen, and fusing B lymphocytes with immortal cells to produce hybridoma cells are well known to those of ordinary skill in the art of making hybridomas.
  • the resulting hybrido as are then cloned and screened for production of monoclonal antibodies which bind to cells expressing the CD26 antigen, but not to identical cells which do not express the CD26 antigen.
  • cell-free preparations of CD26, or a fragment thereof, complexed with CD45, or a fragment thereof are also within the invention.
  • Such complexes may be conveniently prepared by recombinant expression of each of the relevant polypeptides in a manner that prevents their being anchored to the cellular membrane (e.g., by use of a soluble fragment of each) , or by isolation of the full-length proteins from a cell membrane preparation, and by combining the two polypeptides to form the desired complex either before or after removal of contaminating cellular constituents.
  • Such complexes would be useful, e.g., for generating monoclonal antibodies specific for the complex, and for screening for compounds capable of interfering with the association of CD26 and CD45.
  • the screening assay described above for compounds capable of inhibiting the interaction of CD26 and CD45 can be readily adapted to detect compounds (including fragments of CD26 or p43) capable of inhibiting the interaction of CD26 and p43.
  • a therapeutic composition containing a fragment of CD26 (e.g., water- soluble CD26) , in a pharmaceutically acceptable carrier (e.g.
  • Such a therapeutic composition can be used in a method for modulating the immune response of a patient (e.g., enhancing the immune response of an immunosuppressed patient) by administering the composition by any appropriate means to the patient.
  • the compounds of the invention are, when combined with a pharmaceutically acceptable carrier, also useful as vaccine adjuvants, to be administered to an individual vaccinee in conjunction with (i.e., immediately before, after, or along with) a vaccine antigen in order to enhance the immune response produced by such antigen.
  • vaccine antigens which may be used with the adjuvant of the invention include those containing chemically inactivated or genetically engineered viral or bacterial products, such as diphtheria or pertussin toxoid or recombinant viral proteins, and those containing live but attenuated virus or bacteria.
  • the assays described herein may be used to screen candidate immunosuppressive compounds by a method including the steps of (a) contacting a lymphocyte with CD26 or a fragment of CD26 in the presence of a candidate compound, and (b) determining whether the candidate compound inhibits the CD26-induced proliferation of the lymphocyte, such inhibition being an indication that the candidate compound has immunosuppressive activity.
  • the assays may instead be used to screen CD26 fragments for immunostimulatory activity.
  • One such assay would include the following steps: (a) contacting a lymphocyte with a candidate CD26 fragment, and (b) determining whether the fragment increases the rate of proliferation of the lymphocyte, such increase being an indication that the fragment has immunostimulatory activity.
  • a solid matrix material e.g. Affi-Gel m (Bio-rad) to which CD26 or a fragment thereof is attached.
  • CD26 is known to play a role in T cell activation. By interfering with the normal functioning of CD26, one can control the process of T cell activation, and thus prevent such unwanted immune responses as transplant rejection and certain autoimmune diseases.
  • the information disclosed herein concerning proteins with which CD26 associates on the T cell provides the means for designing and screening compounds that interfere with CD26 function in the cell.
  • Fig. 1 depicts the nucletide sequence and deduced amino acid sequence (SEQ ID NO:l) of the cDNA clone for human CD26.
  • Fig. 2 depicts the results of an indirect fluoresence staining assay.
  • Fig. 3 is a pair of photographs of gels illustrating the results of immunoprecipitation analysis (panel A) and enzymatic activity analysis (panel B) .
  • Fig. 4 is a set of graphs depicting the results of a [Ca 2+ ] i mobilization assay.
  • Fig. 5 is a graph illustrating the effect of various treatments on interleukin-2 production.
  • Fig. 6 is a photograph of a gel illustrating the results of immunoblotting analysis.
  • Fig. 7 depicts the results of FACS analysis.
  • Figs. 8-12 are photographs of gels illustrating the results of immunoprecipitation assays.
  • Fig. 13 is a representation of the amino acid sequence of CD26 in which the deleted amino acids of ⁇ 3-9 (SEQ ID NO: 2) are indicated by a box, and the probable proteolytic cleavage sites of the signal peptidase are indicated by arrows.
  • Fig. 14 is a representation of the amino acid sequence of CD26 in which the deleted amino acids of ⁇ 24- 34 (SEQ ID NO: 3) are indicated by a box, and the probable proteolytic cleavage sites of the signal peptidase are indicated by arrows.
  • Fig. 15 depicts the amino acid sequence of a CD26 fragment lacking a portion of the carboxy terminal region of CD26 (SEQ ID NO: 11) .
  • Fig. 16 is a graph illustrating the effect of soluble CD26, soluble CD45, and soluble CD4 on PBL proliferation.
  • CD26 Described below is the cloning and sequencing of a full-length CD26 cDNA. Also described are a series of experiments which demonstrate that: (1) modulation of CD26 from the surface of T lymphocytes leads to enhanced CD3 ⁇ phosphorylation and increased CD4-associated p56 lck tyrosine kinase activity; (2) CD26 is comodulated with CD45; and (3) CD26 and CD45 are closely associated.
  • modulation of CD26 from the surface of T lymphocytes leads to enhanced CD3 ⁇ phosphorylation and increased CD4-associated p56 lck tyrosine kinase activity
  • CD26 is comodulated with CD45
  • CD26 and CD45 are closely associated.
  • Human peripheral blood mononuclear cells PBMC
  • E rosette-positive cells PBMC
  • PHA-activated T cells for use in the experiments described below were prepared as follows.
  • Human PBMC were isolated from healthy volunteer donors by Ficoll-Hypaque density gradient centrifugation (LKB Biotechnology, Inc. , Piscataway, NJ) . Unfractionated mononuclear cells were separated into E rosette-positive (E+) and E rosette-negative (E-) populations, and the E+ cells were depleted of contaminating monocytes as described (Morimoto et al. , J " . Immunol . 134:3762, 1985; Morimoto et al. , J. Immunol .
  • T cells were used for experiments involving T cells in this report.
  • E+ cells were stimulated with PHA (0.25 g/ml) and rIL-2 (40 U/ml) for 7 days in RPMI 1640 medium supplemented with 10% human AB serum, 4mM L- glutamine, 25 mM HEPES buffer, 0.5% sodium bicarbonate, and 1% penicillin/streptomycin (culture medium) and used as PHA blasts.
  • the monoclonal antibodies used were anti- CD26 (Tal/4EL-lC7, IgG 1 ; 1F7, IgG ⁇ j 5F8, IgG- ⁇ , and anti- CD3 (T3/R 24B6; IgG 2b ) (Fox et al., J. Immunol . 133:1250, 1984; Morimoto et al., J. Immunol . 143:3430, 1989; Morimoto et al., J . Immunol . 134:3762, 1985) .
  • Anti-CD29 (4B4; IgG- j (Morimoto et al., J . Immunol. 134:3762, 1985) was used as an isotype-matched control throughout. Isolation of a CD26 cDNA
  • a cDNA library was constructed from mRNA isolated from human PHA-activated T cells using the CDM7 vector. Briefly, poly(A)+ RNA was prepared from 4-day-old PHA-activated T cells by the guanidinium isothiocyanate method (Chirgwin et al., Biochem . 18:5294, 1979), and an expression library was prepared as previously described, except that the vector CDM7, a precursor to CDM8 lacking polyo a sequences, was employed (Aruffo et al., Proc . Natl . Acad . Sci . USA 84:8573, 1987; Serai et al. , Proc . Natl . Acad .
  • Recombinant hybrid plasmids were transfected into COS cells, and CD26 expressing cells were immunoselected with the monoclonal antibody, anti- Tal (Aruffo et al., supra ; Seed et al., supra) . Reactive cells were retained on antibody coated dishes, and plasmids were recovered from transfected cells. Plasmid DNAs, were further selected by three additional rounds of transfection and immunoselection. Two of eight clones thus isolated were found to encode anti-Tal reactive determinants. The two clones were identical byi restriction enzyme fragment mapping.
  • the predicted CD26 polypeptide has a single stretch of hydrophobic amino acids in the N-terminal region between residues 7 and 28 (Fig. 1, boxed), which is sufficiently long and hydrophobic to span a lipid bilayer (Davis et al., Cell 41:607, 1985).
  • the sequence is preceded by six N-terminal residues which contain polar and charged residues, and is followed by charged residues that would not allow cleavage by signal peptidase (von Heijne, Nucl . Acids Res . 14:4683, 1986).
  • This sequence thus has the characteristics of a signal sequence of a type II membrane protein, which serves both to direct the translocation of the nascent protein across the membrane of the rough endoplasmic reticulum, and to anchor the mature protein in the membrane (Hong et al., supra , 1990; Shipp et al., Proc . Natl . Acad. Sci . USA 85:4819, 1988; Thomas et al., J “ . Clin . Invest . 83:1299, 1989) . Furthermore, the fact that potential N- glycosylation sites are located in the carboxy side of the hydrophobic core (Fig. 1, short underlines) suggests that CD26 is a type II membrane protein.
  • N-terminal 6 amino acid residues are predicted to be cytoplasmic, and the next 22 amino acids, which are primarily hydrophobic, are predicted to transverse the cytoplasmic membrane.
  • the 738 C-terminal amino acids constitute the predicted extracellular domain of CD26.
  • the predicted extracellular domain of CD26 may be conveniently divided into three regions: an N-terminal glycosylated region (residues 29 to 323) , a relatively cysteine-rich middle section (residues 324 to 551) , and a C-terminal region (residues 552 to 766) (Fig. l) .
  • the N- terminal region contains 8 of the 10 potential attachment sites for N-linked glycans (Fig. l, short underlines) (Marshall, Ann . Rev. Biochem . 41:673, 1972), and one of the 12 cysteine residues (Fig. 1, asterisks).
  • the subsequent cysteine-rich section contains 9 cysteines but only one N-linked glycosylation site.
  • the C-terminal region contains two cysteines, one N-linked glycosylation site and a potential catalytic site (Fig. 1, double underline) , the sequence G-W-S-Y-G at position 627 to 631.
  • This sequence fits the consensus G-X-S-X-G found in the active sites of serine proteases and esterases, although tryptophan and tyrosine flanking the catalytic serine are unusual residues at these positions (Brenner, Nature 334:528, 1988). Homology with the Other Proteins.
  • the predicted amino acid sequence of the human CD26 antigen (SEQ ID NO: 1) is 85% homologous to the deduced rat DPPIV enzyme sequence predicted from cDNAs isolated from rat liver and kidney libraries.
  • sequences are identical from residues 624 to 724, and 94% homologous from residues 552 to 766.
  • This C-terminal region is 46% homologous to a region of the predicted yeast aminopeptidase B (DPAPB) sequence (Roberts et al., J . Cell . Biol . 108:1363, 1989).
  • CD26 amino acid residues 107 to 233 are 36% homologous to DPAPB.
  • the yeast DPAPB enzyme is also a type II membrane dipeptidyl aminopeptidase, and is involved in the maturation of the yeast pheromone alpha factor.
  • the putative catalytic sequence G-W-S-Y-G is conserved between human and rat CD26/DPPIV and yeast DPAPB.
  • CD10 and CD13 were determined by cDNA cloning (Shipp et al., supra , Thomas et al., supra) .
  • These antigens are ectoenzymes which have neutral endopeptidase [EC. 3.4.24.11] and aminopeptidase N [EC. 3.4.11.2] activities, respectively.
  • CD10 and CD13 are also type II membrane proteins, there is no significant sequence homology between these enzymes and CD26.
  • CD26 antigen is known to be a functional collagen receptor (Dang et al., J " . Exp. Med . 172:649, 1990), a homology search did not find significant homology with any other known collagen- binding proteins such as fibronectin, CDllb and the integrins. Characterization of CD26 Antigen expressed on Transfected Jurkat Cells
  • the human T cell leukemia line, Jurkat was transfected with the expression plasmid pSRQ:26, in which the CD26 cDNA was placed under the control of the SR ⁇ promoter. Briefly, the CD26 cDNA insert was cloned into the PstI and EcoRI sites of the plasmid pCDLSR 296 (Takebe et al., Mol . Cell . Biol . 8:466, 1988) by blunt-end ligation to create the CD26 expression plasmid, pSR -26.
  • pSR ⁇ -26 digested with Sail
  • pSV2neo-SP confers neomycin resistance to host cells; Streuli et al., EMBO J. 8:787, 1989
  • Pvul digested with Pvul
  • Transfectants were initially selected in RPMI1640 supplemented with 10% fetal calf serum. 4mM glutamine and 1.0 mg/ml Geneticin (Gibco/BRL, Bethesda, MD) . Subsequently, the concentration of Geneticin was gradually decreased to 0.25 mg/ml during the selection period. Geneticin- resistant clones were further screened for CD3 and CD26 antigen expression by cell-surface staining as described below. Transfectants were maintained in the above medium containing 0.25 mg/ml Geneticin.
  • Fig. 2 Parental Jurkat cells do not express detectable amounts of the CD26 antigen as determined by cell surface staining (Fig. 2) , or by a binding assay with radiolabeled Tal monoclonal antibody. Northern blotting analysis revealed that this cell line also does not express CD26 mRNA even after phorbol 12-myristate 13- acetate (PMA) treatment, which is known to induce CD26 expression (Dang et al., J. Immunol . 145:3963, 1990). Referring to Fig. 2, the Jurkat-CD26 transfectant 26.C28 had high expression of the CD26 antigen. On the other hand, another Jurkat-CD26 clone, 26.24, expressed only moderate levels of the antigen. Both transfectants were reactive with three anti-CD26 monoclonal antibodies (Tal, 1F7, and 5F8) which define three distinct CD26 antigen epitopes.
  • DPPIV enzymatic activity was measured using an Enzyme Overlay Membrane system (EOM, Enzyme System Products, Dublin, CA) . Briefly, lysates were incubated with SDS sample buffer for 1 hr at room temperature and separated by SDS-PAGE under non-reducing conditions. Following electrophoresis, the EOM moistened with 0.5M Tris-HCl, pH 7.8, was placed on the surface of the gel and this sandwich was incubated for 20 min in a humidified box at 37°C. The reaction was monitored by long wavelength ultraviolet light. Referring to Fig.
  • indo-1 pentaacetoxymethyl ester Calbiochem, San Diego, CA
  • indo-1 pentaacetoxymethyl ester Calbiochem, San Diego, CA
  • flow cytometry were performed as described by (Blue et al., J. Immunol . 140:376, 1988).
  • Indo-1-loaded cells were preincubated for 1-2 minutes with antibodies and the basal intracellular calcium levels were determined for 33 seconds before the addition of polyclonal goat anti-mouse antibody (10 ⁇ g/ml) (Tago, Burlingame, CA) .
  • the RW24B6 anti-CD3 antibody was titrated in this system to determine the submitogenic dose for triggering each cell type.
  • Antibody concentrations were 1 ⁇ .g/ml for anti-lF7 and 20 ng/ml for anti-CD3.
  • the anti- CD26 and anti-CD3 crosslinking induced a strong initial [Ca 2+ ] i increase (Fig. 4) .
  • crosslinking induced a sustained increase of the [Ca 2+ ] i level as well (Fig. 4).
  • the differential pattern of [Ca 2+ ] i mobilization of the two transfectants may be attributed to the difference in the amount of CD26 antigen expressed by these two transfectants.
  • the enhanced [Ca 2+ ] i mobilization was specific because, as was reported for peripheral blood T cells (Dang et al., J. Immunol .
  • crosslinking of the CD26 antigen alone did not induce [Ca 2+ ] i mobilization.
  • crosslinking of anti-CD26 and anti-CD3 did not enhance the [Ca 2+ ] i mobilization of nontransfected or vector-only transfected Jurkat cells, and crosslinking of the isotype-matched control antibody, anti-4B4, did not result in enhanced [Ca 2+ ] i mobilization of the transfectants. Similar to the data observed with transfectants, a small but significant transient rise in [Ca 2+ ] i mobilization was observed in normal resting T cells following CD26 and CD3 crosslinking.
  • IL-2 production by transfected cells cultured in antibody-coated plates was measured as described by Dang et al., J. Immunol . 144:4092, 1990), except that the cell concentration was adjusted to 2xl0 6 cell/ml. After 24 hr of culture, supernatants were assayed for IL-2 production using ELISA (R&D system, Minneapolis, MN) . Referring to Fig.
  • sample buffer 2% SDS, 10% glycerol, 0.1M Tris [pH 6.8] 0.02% bro ophenol blue
  • 2- mercaptoethanol 5% 2- mercaptoethanol
  • cell lysates were transferred to nitrocellulose, and developed using 125 i- labelled anti-phosphotyrosine (UBI, NY; 100,000 cpm/ml in PBS containing 1% BSA) .
  • Affinity-purified anti- phosphotyrosine was iodinated to a specific radioactivity of 10-20 ⁇ Ci/ ⁇ g protein using iodobeads (Pierce Chemical Co. , Rockford, IL) .
  • a 21 kD tyrosine phosphoprotein (p21) , which has been previously identified in T cells stimulated with various stimuli as phosphorylated CD3 ⁇ (Vivier et al., supra , 1990; Vivier et al., J. Immunol . 146:1142, 1991; Ashwell et al., Annu . Rev . Immunol . 8:139, 1990), was detected at a constitutive level in samples not treated with anti-CD26 (lane 1) .
  • Anti-CD26 treatment significantly increased the phosphorylation of CD3 ⁇ over the constitutive level after 1 hour of anti-CD26 incubation (lane 2) .
  • the level of phosphorylated CD3 ⁇ gradually increased with time, reaching a maximum level after 4 hours of anti-CD26 incubation (lanes 3 and 4; 2 and 4 hours of anti-CD26 treatment respectively) , and gradually decreased upon longer incubation (lanes 5 and 6; 6 and 8 hours of anti- CD26 treatment respectively) .
  • CD26 cytoplasmic domain of CD26 (DPPIV) in the rat includes only six amino acid residues
  • DPPIV cytoplasmic domain of CD26
  • CD45 another cell surface molecule
  • Anti-CD26 (1F7) induced modulation was performed as previously described (by Dang et al. J . Immunol . 145:3963, 1990). Briefly, peripheral blood T cells were incubated overnight at 37°C in medium containing anti-CD26 (1F7) at 1:100 ascites dilution. Cells were then collected, washed and stained with anti- CD26 (1F7) and FITC-conjugated goat anti-mouse IgG; or they were stained with anti-CD45RA (2H4)-PE, anti-CD2-PE, anti-CD3-PE (Coulter) or biotinylated anti-CD45RO (UCHL- 1) and PE-conjugated avidin.
  • Fig. 7 The negative control of each fluorescence was less than 5%.
  • the FACS analysis presented in Fig. 7 are representative of three separate experiments. As shown in Fig. 7, overnight incubation with anti-CD26 led to a significant reduction in CD26 expression on T cells. Interestingly, while CD26 modulation did not have any detectable effect on CD2, CD3 or CD45RA expression, the expression of CD45RO, particularly the high fluorescence peak of CD45RO, was markedly reduced. In addition, modulation of CD2, CD3, or CD4 with respective antibodies had no effect on CD45RO expression. Thus, the co ⁇ modulation of CD45RO induced by anti-CD26 treatment appears to be specific for this structure.
  • Peripheral blood T cells (50xl0 6 ) were labeled at the surface by lactoperoxidase-catalyzed iodination and immunoprecipitated from NP-40 lysis buffer (0.5% NP-40, 140mM NaCl, ImM PMSF, 5mM EDTA, 50mM Tris HCl [pH 7.4]) or digitonin lysis buffer (1% digitonin, 0.12% Triton X-100, 150mM NaCl, ImM PMSF, 20mM Triethanolamine [pH 7.8]) using anti-CD26 (Tal, Coulter Immunology, Hialeah, FL; or 1F7, Dr.
  • the lysates were precleared by four successive immunoprecipitations with anti-CD45 (GAP 8.3, American Type Culture Collection, Bethesda, MD) or anti- CD1 (T6) and then precipitated by anti-CD26 and anti- CD45.
  • anti-CD45 GAP 8.3, American Type Culture Collection, Bethesda, MD
  • anti- CD1 T6
  • V8 protease from S . aureus was carried out during gel electrophoresis as described by Cleveland et al. (J " . Biol . Chem . 252:1102, 1977). After the first gel electrophoresis, gel slices containing the high molecular weight proteins co-precipitated with CD26 and CD45 proteins were excised and polymerized into the stacking gel of a 15% SDS-polyacrylamide gel. 2.5 ⁇ g of V8 protease in 10 ⁇ l of sample buffer (0.1% SDS, 0.125M Tris-HCl [pH 6.8], 10% glycerol, 0.1% bromophenol blue) were added to wells above the polymerized gel slices.
  • FIG. 8 presents the results of immunoprecipitation analysis without prior depletion.
  • Surface labeled T- lymphocytes were solubilized in NP-40 (lanes 1-4) or digitonin (lanes 5-8) and immunoprecipitated with anti- CD1 (T6) as a negative control (lanes 1 and 5); anti-CD26 (1F7, lanes 2 and 6); anti-CD26 (Tal, lanes 3 and 7); or anti-CD45 (GAP 8.3, lanes 4 and 8).
  • Fig. 9 presents the results of immunoprecipitation analysis of samples previously depleted for CD45 using anti-CD45 antibody (GAP 8.3, lanes 4-6) or, as a control, CD-I using anti-CDl antibody (T6, lanes 1-3). After depletion, anti-CD26 (1F7, lanes 1 and 4), anti-CD26 (Tal, lanes 2 and 5), or anti-CD45 (GAP 8.3, lanes 3 and 6) was used for immunoprecipitation. As can be seen in Fig. 9, depletion of CD45 resulted in a complete loss of the high molecular weight structures in the CD26 immunoprecipitate (lanes 4, 5).
  • CD45 has PTPase activity which regulates T cell activation pathways through dephosphorylation of phosphotyrosine (Charboneau et al., Proc . Natl . Acad . Sci . USA 85:7182, 1988; Ledbetter et al., Proc. Natl . Acad . Sci . , USA 85:8628; Pingel et al.. Cell 58:1055, 1989; Koretzky et al., Nature 346:66, 1990) .
  • CD45 PTPase One of the potential substrates for the CD45 PTPase is the tyrosine kinase p56 lck (Osergaard et al., Proc . Natl . Acad . Sci . USA 86:8959, 1989; Mustelin et al., Proc. Natl . Acad . Sci . USA 86:6302, 1989), which itself may be involved in the CD3 chain phosphorylation (Veillette et al. , Nature 338:257, 1989).
  • CD26 may function in this system by enhancing CD3 phosphorylation through its association with CD45. If this model is correct, incubation with anti-CD26 (1F7) should alter p56 lck kinase activity as measured by in vitro autophosphorylation.
  • CD4 was immunoprecipitated from lysates containing equivalent amounts of total protein (500 ⁇ g) by a combination of anti-CD4 (19thy5D7; IgG2) and protein A-Sepharose.
  • CD26 is broadly distributed on non-hematopoietic cells. However, since the expression of CD45 is largely restricted to leukocytes, the association between CD26 and CD45 is probably found only on leukocytes. On the other hand, membrane-linked PTPases such as CD45 have been found on non-hematopoietic cells (Streuli et al., J. Exp. Med . 168:1553, 1988; Streuli et al., Proc . Natl . Acad . Sci . USA 86:8698, 1989; Lau et al. Biochem J.
  • CD26 is associated with the membrane-linked PTPase on nonhematopoietic cells.
  • anti-CD26- induced modulation resulted in enhanced CD3 phosphorylation and increased p56 lc PTK activity. Both observations are consistent with the enhanced proliferative response of T cells following CD26 modulation. These observations further suggest that the physical association of CD26 with CD45 may be key for CD26-mediated T cell signaling pathways.
  • CD26 is known to be the membrane-associated ectoenzyme DPPIV which can cleave N-terminal dipeptides from polypeptides with either L-proline or L-alanine at the penultimate position.
  • DPPIV membrane-associated ectoenzyme
  • CD26/DPPIV binding of the natural substrate to the DPPIV enzyme may lead to cleavage and alteration in the biologic activity of the ligand.
  • CD26 modulates the enzymatic activity of the CD45 PTPase or perhaps affects the accessibility of critical substrates. This process would then enhance T cell activation via the CD3 or CD2 pathway and could amplify the immune response in vivo.
  • CD26+ T lymphocytes have been found in both inflamed tissues and peripheral blood of patients with multiple sclerosis, Graves' Disease and rheumatoid arthritis (Hafler et al., N . Engl . J. Med . 312:1405, 1985; Nakao et al., J. Rheumatol . 16:904, 1989; Eguchi et al., J. Immunol . 142:4233, 1989), suggesting that these CD26+ T cells may play an important role in chronic inflammation and in subsequent tissue damage. Soluble CD26 Fragments
  • Soluble fragments of CD26 are useful for interfering with CD26 activity.
  • CD26 is a type II membrane protein
  • the signal sequence used to transfer the protein across a membrane also serves as an anchor to the membrane. The cleavage of the signal sequence after protein transfer which usually occurs for other secreted proteins does not occur in type II transmembrane proteins.
  • soluble forms of CD26 can be prepared by making its signal/anchor sequence accessible to a cellular proteolytic cleavage system.
  • the putative signal sequence of CD26 was shortened, as described below, since the 23 amino acid CD26 signal sequence is longer than most natural occuring cleavable signal sequences (von Heijne et al., J. Mol . Biol . 184:99, 1985). This is expected to result in proteolytic cleavage of the expressed polypeptide at or near one of the residues Ala Thr Ala corresponding to positions 35-37 of wild type CD26, yielding a soluble fragment of CD26 having at its amino terminus Ala 35 , Thr 36 , Ala 37 or Asp 38 of wild type CD26.
  • a first soluble CD26 construct is created by deleting the codons corresponding to amino acids 3-9 of intact CD26 (shown as the boxed amino acids in Fig. 13) .
  • the amino terminal sequence of the expressed polypeptide is MKGLLG— (SEQ ID NO: 4) rather than the original MKTPWKVLLGLLG— (SEQ ID NO: 5) , and the potential proteolytic cleavage sites are shown as arrows in Fig. 13.
  • This deletion mutant is prepared by oligonucleotide directed mutagenesis (see below) using the following oligonucleotide:
  • a second construct is generated by taking advantage of the following rules proposed for signal peptide cleavage: (1) the residue in position -l must be small, i.e., either Ala, Ser, Gly, Thr, Cys, Gin; (2) the residue in position -3 must not be aromatic (Phe, His, Tyr, Trp) , charged (Asp, Glu, Lys, Arg) , or large and polar (Asn, Gin) ; and (3) Pro must not be present at positions -3 through -1 (von Heijne, Nuc . Acids Res . 14:4683, 1986). Following these rules, we have designed a CD26 cDNA construct lacking codons corresponding to amino acids 24 to 34 of wild type CD26 (illustrated as the boxed amino acids in Fig. 14) . This deletion mutant encodes the amino acid sequence
  • This mutant is prepared by oligonucleotide- directed mutagenesis (see below) using the following oligonucleotide: 5'-ACCATCATCACCGTGGCTACAGCTGACAGT- 3' (SEQ ID NO: 9) . Site-directed mutagenesis is performed as follows.
  • the 3.0 kb CD26 cDNA fragment generated by the XJbal treatment of the original plasmid CDM7-CD26 is inserted into the XJbal site of pTZ19u (Bio-rad) .
  • a recombinant plasmid which inserts the cDNA inverse to the lacZ gene on the plasmid is identified by restriction enzyme mapping and used for subsequent mutagenesis.
  • oligonucleotide-directed mutagenesis is performed by the method of Kunkel (Proc . Natl . Acad . Sci . USA 82:488, 1985), using a commercially available kit (BioRad, Richmond, CA) .
  • a new expression vector is constructed. First the XJbal CD26 cDNA fragment of pTZ19u-CD26 and the Hindlll-Xbal vector fragment of Rc/CMV (Invitrogene, San Diego, CA) are treated with Klenow enzyme and ligated. The resulting plasmid is screened by restriction enzyme mapping for the insertion of the CD26 cDNA fragment under the control of the CMV promoter. This construct leaves one Xjal site just in front of the CD26 cDNA.
  • the Miul-X al CMV promoter DNA fragment of this plasmid DNA is exchanged with the JEfindlll-XJbal SR ⁇ promoter DNA fragment of pSR -26 to give a final expression vector RcSR -26.
  • the above mutant CD26 cDNAs are transferred to this expression vector.
  • the Xjal-Dralll DNA fragment derived from the mutant cDNAs which encoded the mutant part and the wild type-2.0 kb DraIII-Hindl ⁇ lI DNA fragment are ligated with the Xbal-Hindlll vector fragment of RcSR ⁇ -26.
  • the expression plasmid which has the ⁇ 3-9 or ⁇ 24-34 mutant CD26 cDNA is identified by restriction enzyme mapping and DNA sequencing.
  • the resultant plasmids RcSRo;-26. ⁇ 3-9 and RcSR ⁇ -26. ⁇ 24-34 are used to transfect Jurkat cells or CHO cells.
  • Jurkat cells are transfected with these plasmids as described above except pSVneo-sp is omitted from the donor DNA mixture since the RcSR ⁇ plasmid already carries the neo resistance marker.
  • Neo-resistant clones are screened by metabolic labelling and immunoprecipitation (Harlow et al., eds. Antibodies : a laboratory manual , Cold Spring Harbor Laboratory, 1988) for the expression of soluble CD26.
  • the transfectants which produce a large amount of soluble CD26 are used for protein production.
  • CHO cells transfected with the DNA mixture of pMT2 and RcSR ⁇ -26. ⁇ 3-9 or RcSR ⁇ -26. ⁇ 24-34 are selected for their growing ability in ⁇ -medium and the production of soluble CD26.
  • the expression of the soluble protein is amplified by culturing the transfected CHO cells in medium containing an increasing amount of MTX.
  • both Jurkat cells and CHO cells can provide the soluble form of CD26, the protein produced by Jurkat cells is preferred because of its human T cell origin.
  • polypeptide fragments of CD26 can be produced by standard methods of protein synthetic chemistry, using the information disclosed herein to design appropriate polypeptides and assay them for biological activity.
  • a preferred method of producing such fragments is by the use of recombinant DNA techniques.
  • the sequence of CD26 given in Fig. 1 can be used to design oligonucleotides encoding fragments of CD26 containing deletions of nonessential CD26 amino acid residues from the beginning, the end, and/or any central portion of the protein; such oligonucleotides are chemically synthesized by known methods and inserted into expression vectors for expression of a polypeptide fragment of CD26.
  • CD26 coding regions of CD26 expression plasmids may be altered by site-directed mutagenesis, as disclosed above for two such fragments of CD26, or by insertion of a stop codon at an appropriate place in the coding sequence.
  • the CD26 fragment can then be produced in transfected cultured cells in large quantities, purified by standard methods, and tested in an assay such as the immunoprecipitation assay described above, which is useful for identifying fragments capable of disrupting the interaction of CD26 and CD45.
  • peripheral blood T cells which express both CD26 and CD45 (or any mammalian cells transfected with cDNAs encoding CD26 and CD45 so that both proteins are functionally expressed on the cells' surfaces) are incubated in the presence and absence of a CD26 polypeptide fragment.
  • the cells are lysed in digitonin lysis buffer, and anti-CD45 monoclonal antibody is used to immunoprecipitate CD45 and any proteins associated with CD45.
  • the amount of CD26 that co-precipitates with CD45 in the presence of a given polypeptide fragment can be determined by known methods (e.g., by densitometer readings of the labelled bands on an SDS-PAGE gel analyzing the constituents of an immunoprecipitate) and compared to the amount that co-precipitates with CD45 in the absence of the polypeptide fragment.
  • an anti-CD26 antibody can instead be used and measure the relative amounts of CD45 that co-precipitate with CD26 in the presence and absence of the given polypeptide fragment. If an anti-CD26 antibody is used, it is preferred that the antibody does not substantially bind to the competitor CD26 polypeptide; such binding interferes with the assay. In either case, CD26 polypeptide fragments which interfere with the interaction between CD26 and CD45 will decrease co- precipitation.
  • Fig. 12 illustrates one such experiment, in which E+ cells were labeled by lactoperoxidase-catalyzed iodination and lysed in NP-40 lysis buffer for immunoprecipitation as described above. Precipitates were analyzed by 9% SDS-PAGE.
  • Lane 1 anti-CDl (T6) as negative control; lane 2: anti-lF7; lane 3: anti-Tal; lane 4: anti-5F8 (another anti-CD26 monoclonal antibody); lane 5: anti-CD29 (4B4) as control.
  • anti-lF7 brought down an obvious 43kDa structure (lane 2) from surface-labeled T cells.
  • this structure was detected faintly following anti-Tal or anti-5F8 precipitation (lanes 3 and 4) .
  • This band was not detected following anti-CDl or anti-CD29 precipitation (lanes 1 and 5) .
  • P43 may be purified by affinity chromatography, using an anti-CD26 monoclonal antibody to purify the CD26-p43 complex from T cell membranes. P43 may then be separated from CD26 by SDS-PAGE, followed by HPLC if further purification is necessary. Affinity chromatography with monoclonal antibodies, SDS-PAGE, and HPLC are all standard methods well known to those of ordinary skill in the art.
  • Hybridization probes based upon a partial amino acid sequence of the purified protein may be used to select p43 cDNA from a T cell library.
  • the partial amino acid sequence can be used to design PCR primers for priming synthesis of a partial p43 cDNA on mRNA templates, using standard methods, and the resulting partial cDNA used as a probe to detect full-length p43 cDNA in a T cell library.
  • This cDNA can be inserted in an expression plasmid and used to transfect cells which do not naturally express the p43 gene.
  • Such cells may be used as an antigen to develop anti-p43 monoclonal antibodies, and also as a means to study the role of p43 in T cell activation. They can also be used in the screening assay referred to above.
  • Analysis of the degree of expression of CD26 in any given cell type or tissue type can be accomplished using the standard technique of Northern blotting, probing with a labelled, single stranded nucleic acid molecule derived from the coding region of CD26 cDNA.
  • the probe would have a sequence based upon the sense strand of SEQ ID NO: 1, which is complementary to CD26 mRNA, and preferably would be at least 8 nucleotides in length (more preferably at least 14 nucleotides, and most preferably at least 30) .
  • the probe may contain most or all of the entire coding sequence of CD26 cDNA.
  • Such an assay which would be useful for diagnosing conditions characterized by the over- or under-expression of CD26 in a given cell type, such as T cells, would include the following steps: (a) providing a biological sample containing mRNA of a cell;
  • CHO cells stably expressing CD26 ⁇ 3-9 were cultured in serum-free medium (CHO-S-SFM; GIBCO/BRL) containing 0.5 ⁇ M methotrexate.
  • the culture supernatant was collected and proteins were precipitated using 75% ammonium sulfate.
  • the resulting pellet was solubilized in PBS, dialyzed against PBS, and loaded on a ConA- Sepharose column (Pharmacia, Piscataway, NJ) equilibrated with 2xPBS/0.02% sodium azide.
  • the column was washed with the equilibration buffer, and protein was eluted with 2xPBS/0.2M methyl ⁇ -D-mannopyranoside/0.02% sodium azide.
  • a DPPIV assay (described below) was used to identify the CD26-containing fractions, which were pooled and loaded directly on a BSA-conjugated AffigelTM 10 column (Bio-rad) equilibrated with PBS/0.02% sodium azide.
  • the flowthrough fraction was collected and applied to a lF7-conjugated AffigelTM 10 column equilibrated with PBS.
  • the column was washed with PBS, and soluble CD26 was eluted with PBS/3M sodium thiocyanate.
  • the fractions containing DPPIV activity were pooled and dialyzed against PBS.
  • the resulting soluble CD26 was more than 95% pure as judged by gel electrophoresis.
  • Soluble CD26 was shown to stimulate antigen- dependent proliferation of peripheral blood lymphocytes in vitro . Assays were performed in triplicate wells in round-bottom plates using 0.2 ml/well standard culture medium consisting of RPMI1640 supplemented with 10% human AB serum, 4 mM L-glutamine, 25 mM HEPES buffer (Microbiological Associates), 0.5% sodium bicarbonate, and 50 ⁇ g/ml of Gentamicin (GIBCO) . The cell concentration was 1.2xl0 5 cells/well. Tetanus toxoid (Connaught Lab, Inc.) dialyzed against PBS was added to some of the wells to make a final concentration of 0.2 or 0.1 L.T.
  • the toxoid serves as soluble antigen in this assay.
  • Purified soluble CD26 antigen, soluble LCA (leucocyte common antigen; CD45) , or soluble CD4 was added at a final concentration of 1 ⁇ g/ml or 25 ⁇ g/ml * After 7 days culture in a C0 2 incubator, the cells were pulsed with 1 ⁇ Ci/well of 3 H-thymidine. After a 16 hr incubation, the cells were harvested and the 3 H- thymidine incorporation was measured using a scintillation counter. As shown in Fig.
  • soluble CD26 prepared as described above stimulated PBL proliferation in a dose-dependent manner. This stimulation was greater than that observed when soluble CD45 or soluble CD4 was used instead of soluble CD26.
  • This assay can be used to screen fragments of CD26 to identify molecules capable of stimulating antigen- dependent immune cell proliferation. In addition, it can be used to assay for compounds capable of inhibiting soluble CD26-stimulated proliferation of lymphocytes.
  • an anti-CD3 antibody such as OKT3 (Kung et al. U.S. Patent 4,658,019, 4,361,549, and 4,654,210) for the tetanus toxoid in this assay, the ability of fragments of CD26 to stimulate antigen-independent immune cell proliferation can be determined.
  • CD26-629A Although CD26-629A apparently lacks DPPIV activity, cells expressing CD26-629A were recognized by three anti-CD26 antibodies (1F7, Tal(4EL), and 5F8) which recognize wild-type CD26, indicating that the mutant protein is expressed in the transformed cells, and suggesting that the mutation does not have a substantial effect on protein conformation.
  • Jurkat cells expressing wild-type CD26 were stimulated with anti-CD3 antibody (OKT3) and either anti- CD26 antibody (1F7) or PMA produced substantially more IL-2 than cells expressing CD26-629A or cells transformed with vector only. This suggests that the DPPIV activity of CD26 is important for both CD26-dependent and CD26- independent activation.
  • the compounds of the invention can be used for treatment of disease conditions characterized by immunosuppression: e.g., AIDS or AIDS-related complex, other virally- or environmentally-induced conditions, and certain congenital immune deficiencies.
  • the compounds may also be employed to increase immune function that has been impaired by the use of immunosuppressive drugs such as certain chemotherapeutic agents, and therefore are particularly useful when given in conjunction with such drugs.
  • immunosuppressive drugs such as certain chemotherapeutic agents
  • the compounds of the invention will boost the immune response triggered by the vaccine and thus increase the vaccine's protective potency. This would be particularly beneficial where the vaccinee is incapable of generating an optimal immune response without the use of such an adjuvant, as is the case for newborns or for persons undergoing renal dialysis or transplantation, or where the vaccine antigen is one which is poorly immunogenic.
  • the compounds of the invention will be suspended in a pharmaceutically-acceptable carrier (e.g., physiological saline) and administered orally or by intravenous infusion, or injected subcutaneously, intramuscularly, or intraperitoneally.
  • a pharmaceutically-acceptable carrier e.g., physiological saline
  • Optimal formulation and dosage can be readily determined by one of ordinary skill in the art of pharmacology, taking into account such factors as the biological half-life of the compound and the degree of immunostimulation desired. It is expected that a typical dose for a severely immunocompromised patient will be approximately 0.01 to 100 ⁇ g/kg/day. When utilized as a vaccine adjuvant, a typical single dose of the compound of the invention would be 0.1 to 100 ⁇ g.
  • CD26 soluble forms of CD26
  • intact CD26 or a form of CD26 which retains the membrane-anchoring amino terminal portion of native CD26, as well as all critical portions of the remainder of the molecule can be incorporated into red cell "ghosts" or liposomes, so that the protein is expressed on the surface of the ghosts or liposomes.
  • This form of CD26 is then suspended in a pharmaceutically acceptable carrier and introduced into the patient as described above, so that it can interact with the patient's immunological cells in vivo.
  • peripheral blood lymphocytes can be withdrawn from the patient and treated with a CD26 compound of the invention (whether in soluble or membrane-bound form, or attached to a solid support by standard methodologies) ex vivo , prior to introducing the newly-stimulated lymphocytes into the same or a different patient.
  • a CD26 compound of the invention whether in soluble or membrane-bound form, or attached to a solid support by standard methodologies
  • the assay for enhancement of lymphocyte proliferation with soluble CD26 can be utilized to screen for compounds which inhibit such enhancement, and which therefore could be used to interfere with CD26-stimulated proliferation of lymphocytes in vivo .
  • the information provided above concerning the location of the DPPIV active site of CD26 provides a starting place for the design of compounds which will bind to the active site and thus potentially inhibit the stimulatory activity of CD26.
  • Such compounds can first be tested for their ability to bind to CD26 by passing each such compound over a CD26 affinity column; compounds which bind to the column can then be assayed for their ability to inhibit soluble CD26-enhanced proliferation of lymphocytes in vitro, as described above.
  • Such inhibitory compounds would be useful for the treatment of conditions characterized by an unwanted immune response: for example, autoimmune diseases such as systemic lupus erythematosis and rheumatoid arthritis.
  • the invention also includes analogs of CD26 and of fragments of CD26.
  • analogs refers to polypeptide fragments of CD26 having conservative and/or non-conservative substitutions for some of the amino acids of naturally-occurring CD26, having D-amino acids in place of some or all of the corresponding L-amino acids, or having non-peptide bonds in place of some of the peptide bonds of CD26.
  • Techniques for producing such analogs are well known in the art, and can be readily accomplished by those of ordinary skill.
  • Preferably at least 85%, more preferably at least 95%, and most preferably at least 99%, of the amino acids in the analog are identical to the corresponding ones in CD26.
  • the substitutions do not eliminate the ability of the polypeptide fragment to interfere with the naturally occurring association between CD26 and CD45, or the ability of the compound to stimulate proliferation of lymphocytes.
  • the removal of peptide bonds from a polypeptide compound is a desirable goal because the presence of such bonds may leave the compound susceptible to attack by proteolytic enzymes. Additionally, such peptide bonds may affect the biological availability of the resulting therapeutic molecules. The removal of peptide bonds is part of a process referred to as "depeptidization".
  • AAA GGC ACA GAT GAT GAT GCT ACA GCT GAC AGT CGC AAA ACT TAC ACT CTA 145 Lys Gly Thr Asp Asp Ala Thr Ala Asp Ser Arg Lys Thr Tyr Thr Leu 30 35 40 45
  • Trp lie Ser Asp His Glu Tyr Leu Tyr Lys Gin Glu Asn Asn He Leu 65 70 75
  • AGT GGA AGA TGG AAC TGC TTA GTG GCA CGG CAA CAC ATT GAA ATG AGT 1057 Ser Gly Arg Trp Asn Cys Leu Val Ala Arg Gin His He Glu Met Ser 335 340 345

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Abstract

L'invention se rapporte à un fragment polypeptidique ou analogue de l'antigène CD26 capable d'interrompre l'interaction de liaison naturelle entre CD45 et CD26, et à un procédé de dépistage de ces composés afin d'identifier les composés capables d'inhiber la liaison de CD26 à CD45. Ce procédé consiste à: a) produire un premier et un second échantillons de cellules exprimant à la fois CD26 et CD45; b) incuber le premier échantillon en présence d'un composé potentiel; c) incuber le second échantillon en l'absence du composé potentiel; d) générer un premier immunoprécipité en ajoutant au premier échantillon une première aliquote d'un anticorps anti-CD26; e) générer un second immunoprécipité en ajoutant au second échantillon une seconde aliquote de l'anticorps; et f) déterminer si la quantité de CD45 présent dans le premier immunoprécipité est inférieure à la quantité de CD45 présent dans le second immunoprécipité, la présence d'une quantité moindre de CD45 dans le premier immunoprécipité inférieure à celle du second immunoprécipité indiquant que le composé potentiel empêche la liaison.
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