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WO1993016102A1 - Cd26 humain et procedes d'utilisation - Google Patents

Cd26 humain et procedes d'utilisation Download PDF

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Publication number
WO1993016102A1
WO1993016102A1 PCT/US1992/002892 US9202892W WO9316102A1 WO 1993016102 A1 WO1993016102 A1 WO 1993016102A1 US 9202892 W US9202892 W US 9202892W WO 9316102 A1 WO9316102 A1 WO 9316102A1
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Prior art keywords
cell
polypeptide
immunoprecipitate
sample
fragment
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PCT/US1992/002892
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English (en)
Inventor
Chikao Morimoto
Stuart F. Schlossman
Toshiaki Tanaka
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Dana-Farber Cancer Institute, Inc.
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Publication of WO1993016102A1 publication Critical patent/WO1993016102A1/fr

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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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/289—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD45
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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)

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 .
  • DPPIV Dipeptidyl peptidase IV
  • CD26 is recognized by a second monoclonal antibody, anti-lF7 (Morimoto et al., J. Immunol .
  • the invention features a polypeptide fragment of CD26 having an amino acid sequence substantially identical to the amino acid sequence of CD26, except that amino acid residues 3-9 of the latter sequence have been deleted ( ⁇ 3-9, SEQ ID NO: 2) .
  • the polypeptide has an amino acid sequence substantially identical to the amino acid sequence of CD26, except that amino acid residues 3-9 of the latter sequence have been deleted ( ⁇ 3-9, SEQ ID NO: 2) .
  • the polypeptide has an amino acid sequence substantially identical to the amino acid sequence of CD26, except that amino acid residues 3-9 of the latter sequence have been deleted ( ⁇ 3-9, SEQ ID NO: 2) .
  • the polypeptide has an amino acid sequence substantially identical to the amino acid sequence of CD26, except that amino acid residues 3-9 of the latter sequence have been deleted ( ⁇ 3-9, SEQ ID NO: 2) .
  • the polypeptide has an amino acid sequence substantially identical to the amino acid sequence of CD26, except that amino acid residues 3-9 of the latter sequence have been deleted ( ⁇ 3-9, SEQ ID NO: 2)
  • the invention features a nucleic acid encoding a polypeptide fragment of CD26 having an amino acid sequence substantially identical to the amino acid sequence of ⁇ 3-9 (SEQ ID NO: 2) .
  • the invention features a plasmid which includes this nucleic acid, and preferably also an expression control sequence.
  • the invention features a polypeptide fragment of CD26 having an amino acid sequence substantially identical to the amino acid sequence of CD26 except that residues 24-34 of the latter sequence are deleted ( ⁇ 24-34, SEQ ID NO: 3).
  • the polypeptide has an amino acid Osequence 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, l- 36, or 1-37.
  • the invention features a nucleic acid encoding a polypeptide fragment of CD26 having an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 3 ( ⁇ 24-34) .
  • the invention features a plasmid which includes the nucleic acid, and preferably also an expression control sequence.
  • SUBSTITUTE SHEET 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.
  • substantially pure is meant a polypeptide or protein which has been separated from biological acromolecules, (e.g., other proteins, carbohydrates, etc.) with which it naturally occurs.
  • 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 owned pharmaceutically acceptable pharmaceutically acceptable pharmaceutically acceptable pharmaceutically acceptable pharmaceutically acceptable pharmaceutically acceptable pharmaceutically acceptable pharmaceutically acceptable pharmaceutically acceptable pharmaceutically acceptable pharmaceutically acceptable pharmaceutically acceptable pharmaceutically acceptable pharmaceutically acceptable pharmaceutically acceptable pharmaceutically acceptable pharmaceutically acceptable pharmaceutically acceptable pharmaceuticallycerin, 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
  • SUBSTITUTESHEET 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 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 (or analogs thereof) capable of disrupting the naturally-occurring binding interaction between CD45 and 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.
  • SUBSTITUTESHEET are identical to the corresponding ones in CD26. It is important that the substitutions do not eliminate the ability of the polypeptide fragment to interfere with the naturally occurring association between CD26 and CD45. In some instances, 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".
  • Polypeptides and analogs which disrupt the interaction between CD26 and CD45 can be identified using the immunoprecipitation assay described herein 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:
  • the invention features a method which includes:
  • 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:
  • SUBSTITUTE SHEET (b) transfecting the cell with a nucleic acid encoding CD26 and a nucleic acid encoding CD45.
  • the invention includes a method of generating a hybridoma cell, which method includes:
  • 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 hybrido as.
  • the resulting hybridomas 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.
  • SUBSTITUTE SHEET Also within the invention are cell-free preparations of CD26, or a fragment thereof, complexed with CD45, or a fragment thereof.
  • 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.
  • 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.
  • SUBSTITUTESHEET 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).
  • 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 lc 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 lc 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 /xg/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- L ,* 1F7, IgG- L ,- 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;
  • 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 by 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., Ceil 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. l, short underlines) suggests that CD26 is a type II membrane protein.
  • N-terminal 6 amino acid residues are predicted to be cytoplas ic, 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: ah 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. 1) .
  • the N- terminal region contains 8 of the 10 potential attachment sites for N-linked glycans (Fig. 1, 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) . Ho ology 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
  • SUBSTITUTESHEET aminopeptidase SUBSTITUTESHEET 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.
  • Recently the structures for 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 pSR 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 -5. ⁇ oRI 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 digested with Pvul
  • 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
  • SUBSTITUTESHEET 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.
  • SUBSTITUTESHEET proteins were separated by 8% SDS-PAGE under reducing conditions.
  • DPP-IV 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.
  • DPPIV enzymatic activity was associated with a 160 kDa protein in both transfectants (lanes 2 and 3) and PHA blasts (lane 4) , but not in parental Jurkat cells (lane 1) , or vector-only transfected cells. It should be noted that the DPPIV enzyme activity was stable in both non-reducing and reducing conditions but disappeared after boiling of the samples. While the apparent molecular weight of CD26 was 160,000 for preparations that were not boiled prior to electrophoresis, the molecular weight of CD26 antigen was 110,000 if the
  • indo-1 pentaacetoxymethyl ester Calbiochem, San Diego, CA
  • flow cyto etry 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, Burlinga e, CA) .
  • the RW24B6 anti-CD3 antibody was titrated in this system to determine the submitogenic dose for triggering each cell type.
  • Antibody concentrations were l ⁇ g/ml for anti-lF7 and 20 ng/ml for anti-CD3.
  • 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 . 145:3963, 1990), 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. 5, incubation of the clone 26.C28 transfectants with solid-phase-immobilized anti-lF7 and anti-CD3, which mimicked the crosslinking by anti-mouse antibody, induced the production of a significant amount of IL-2 (striped
  • sample buffer 2% SDS, 10% glycerol, 0.1M Tris [pH 6.8] 0.02% bromophenol blue
  • 2- mercaptoethanol 5% 2- mercaptoethanol
  • cell lysates were transferred to nitrocellulose, and developed using 125 ⁇ - 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
  • SUBS ⁇ TUT ⁇ SHEET 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,
  • 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 HC1 [pH 7.4]) or digitonin lysis buffer (1% digitonin, 0.12% Triton X-100, 150mM NaCl, lmM PMSF, 20mM Triethanolamine [pH 7.8]) using anti-CD26 (Tal, Coulter Immunology, Hialeah, FL; or 1F7, Dr.
  • peripheral blood T cells were labeled and lysed in digitonin lysis buffer as described above.
  • 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.
  • 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% bro ophenol 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, an * ti-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) .
  • One of the potential substrates for the CD45 PTPase is the tyrosine kinase p56 lck (Osergaard et al., Proc .
  • 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 p 56 ic 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. The immunoprecipitates were then washed extensively with lysis buffer prior to incubation with 30 ⁇ l of 25 mM Hepes containing 0.1% NP-40, and 10 ⁇ Ci of [ ⁇ - 32 P]ATP (ICN,
  • 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 lck 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 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.
  • increased numbers of 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.
  • CD26 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
  • SUBSTITUTE SHEET 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 -1 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
  • SUBSTITUTESHEET generated by the Xbal treatment of the original plasmid CDM7-CD26 is inserted into the Xbal 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 Xbal 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 Xial site just in front of the CD26 cDNA.
  • the Mlul-Xbal CMV promoter DNA fragment of this plasmid DNA is exchanged with the Hindlll-Xbal 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 Xbal-Dralll DNA fragment derived from the mutant cDNAs which encoded the mutant part and the wild type 2.0 kb i.ralII-HindIII 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 RcSRc.-26. ⁇ 3-9 and RcSR ⁇ -26. ⁇ 24-34 are used to transfect Jurkat cells or CHO cells.
  • 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.
  • CD26 and CD45 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.
  • SUBSTITUTE SHEET 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 l: anti-CDl (T6) as negative control; lane 2: anti-lF7; lane 3: anti-Tal; lane 4: anti-5F8 (another anti-CD26 monoclonal antibody);
  • SUBSTITUTE SHEET 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) .
  • Similar results were seen when the cells were human thymocytes or from the human T cell lines H9 or Peer IV (data not shown) .
  • the 43kDa band was sometimes more distinct than those shown in lanes 3 and 4 of Fig. 12.
  • a third band at approximately 70 kDa is sometimes observed in these CD26 immunoprecipitation experiments. Because they are found in association with the 110 kDa CD26 molecule, both the 43 kDa molecule and the 70 kDa molecule may play important roles in T cell activation. Compounds (such as fragments or analogs of CD26) which interfere with the association of CD26 with either p43 or the 70 kDa molecule may be detected by means of a screening assay patterned on those described above with respect to CD26 and CD45.
  • SUBSTITUTE SHEET 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 would be useful for use 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
  • SUBSTITUTESHEET characterized by the over- or under-expression of CD26 in a given cell type, such as T cells, would include the following steps:
  • Trp lie Ser Asp His Glu Tyr Leu Tyr Lys Gin Glu Asn Asn lie 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

Fragment polypeptidique de CD26 (ou de ses analogues), pouvant détruire l'interaction de liaison naturelle entre CD45 et CD26, et procédé de triage de tels composés pour identifier des composés pouvant inhiber la liaison de CD26 à CD45, le procédé consistant à: a) prendre un premier et un second échantillon de cellules exprimant à la fois CD26 et CD45; b) incuber le premier échantillon en présence du composé potentiel; c) incuber le second échantillonen l'absence du composé potentiel; d) générer un premier immunoprécipité par l'addition d'une première aliquote d'un anticorps anti-CD26 au premier échantillon; e) générer un second immunoprécipité par l'addition d'une seconde aliquote de l'anticorps au second échantillon; et f) déterminer si la quantité de CD45 présent dans le premier immunoprécipité est inférieure à la quantité de CD45 dans le second immunoprécipité, la présence d'une quantité inférieure de CD45 dans le premier immunoprécipité, par rapport au second, indiquant que le composé inhibe la liaison.
PCT/US1992/002892 1992-02-06 1992-04-09 Cd26 humain et procedes d'utilisation WO1993016102A1 (fr)

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WO2023143245A1 (fr) * 2022-01-30 2023-08-03 江苏众红生物工程创药研究院有限公司 Application de cd45 en tant que biomarqueur dans le criblage de l'efficacité et de la précision d'un anticorps cd26 ou d'un dérivé de celui-ci dans le traitement de tumeurs

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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7230074B2 (en) 1991-10-22 2007-06-12 Trustees Of Tufts College Inhibitors of dipeptidyl-aminopeptidase type IV
US6825169B1 (en) 1991-10-22 2004-11-30 Trustees Of Tufts College Inhibitors of dipeptidyl-aminopeptidase type IV
US5965532A (en) * 1996-06-28 1999-10-12 Trustees Of Tufts College Multivalent compounds for crosslinking receptors and uses thereof
US6875737B1 (en) 1996-06-28 2005-04-05 Trustees Of Tufts College Multivalent compounds for crosslinking receptors and uses thereof
US6503882B2 (en) 1997-05-07 2003-01-07 Trustees Of Tufts College Treatment of HIV
US6692753B2 (en) 1997-05-07 2004-02-17 Trustees Of Tufts College Potentiation of the immune response
US6100234A (en) * 1997-05-07 2000-08-08 Tufts University Treatment of HIV
US6258597B1 (en) 1997-09-29 2001-07-10 Point Therapeutics, Inc. Stimulation of hematopoietic cells in vitro
US6703238B2 (en) 1997-09-29 2004-03-09 Point Therapeutics, Inc. Methods for expanding antigen-specific T cells
US7276371B2 (en) 1997-09-29 2007-10-02 Point Therapeutics, Inc. Stimulation of hematopoietic cells in vitro
US7067489B2 (en) 1998-05-04 2006-06-27 Point Therapeutics, Inc. Hematopoietic stimulation
US6300314B1 (en) 1998-05-04 2001-10-09 Point Therapeutics, Inc. Hematopoietic stimulation
US6770628B2 (en) 1998-05-04 2004-08-03 Point Therapeutics, Inc. Hematopoietic stimulation
US6355614B1 (en) 1998-06-05 2002-03-12 Point Therapeutics Cyclic boroproline compounds
US6890904B1 (en) 1999-05-25 2005-05-10 Point Therapeutics, Inc. Anti-tumor agents
US6949514B2 (en) 1999-05-25 2005-09-27 Point Therapeutics, Inc. Anti-tumor agents
US7259138B2 (en) 1999-05-25 2007-08-21 Point Therapeutics, Inc. Anti-tumor agents
US7282484B2 (en) 1999-05-25 2007-10-16 Point Therapeutics, Inc. Anti-tumor agents
US7462698B2 (en) 2005-07-22 2008-12-09 Y's Therapeutics Co., Ltd. Anti-CD26 antibodies and methods of use thereof
US8030469B2 (en) 2005-07-22 2011-10-04 Sbi Incubation Co., Ltd. Anti-CD26 antibodies and methods of use thereof
WO2015089881A1 (fr) * 2013-12-19 2015-06-25 江苏众红生物工程创药研究院有限公司 Anticorps humain anti-cd26 et son application
WO2023143245A1 (fr) * 2022-01-30 2023-08-03 江苏众红生物工程创药研究院有限公司 Application de cd45 en tant que biomarqueur dans le criblage de l'efficacité et de la précision d'un anticorps cd26 ou d'un dérivé de celui-ci dans le traitement de tumeurs

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