WO1993005814A1 - Inhibition competitive des interactions entre lymphocytes t et b - Google Patents

Inhibition competitive des interactions entre lymphocytes t et b Download PDF

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Publication number
WO1993005814A1
WO1993005814A1 PCT/US1992/007994 US9207994W WO9305814A1 WO 1993005814 A1 WO1993005814 A1 WO 1993005814A1 US 9207994 W US9207994 W US 9207994W WO 9305814 A1 WO9305814 A1 WO 9305814A1
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cell
cells
fragment
soluble
binding
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PCT/US1992/007994
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Ivan Stamenkovic
Dennis C. Sgroi
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The General Hospital Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • B lymphocytes To differentiate into antibody producing cells, B lymphocytes must interact with helper T cells. Helper T cells are thought to stimulate B cells indirectly, by secreting B lymphotropic cytokines (see, e.g., Dutton et al., Prog, in Immunol.. 1:355-68 (1971); and Kishimoto and Hirano, Annu. Rev. Immunol.. 6_485-512 (1988)), and directly through physical cell-cell contact (see, e.g., Kupfer et al., Proc. Natl. Acad. Sci. , USA. 63;6060-83 (1986); and Noelle et al., J. Immunol. f 143;1807-14 (1989)).
  • B lymphotropic cytokines see, e.g., Dutton et al., Prog, in Immunol.. 1:355-68 (1971); and Kishimoto and Hirano, Annu. Rev. Immunol.. 6_
  • Mature B lymphocytes express a lineage-specific cell surface receptor, CD22, a 130/140kD heterodimer (Dorken et al., J. Immunol.. 136:4470-79 (1986)), composed of two independently expressed polypeptide chains (Boue and Lebien, J. Immunol.. 140:192-99 (1988)).
  • CD22 polypeptides are members of the immunoglobulin superfamily of integral membrane proteins (Stamenkovic and Seed, Nature. 344:74-77 (1990); Wilson et al., J. EXP. Med.. 173:137-46 (1991)).
  • the smaller form, CD22 ⁇ has an extracellular region composed of 5 Ig-like domains (Stamenkovic and Seed, supra) while the larger form, CD22 / 3, has two additional Ig-like domains (Wilson et al., supra) .
  • Both polypeptide chains are highly related to yelin associated glycoprotein (MAG) , neural cell adhesion molecule (N-CAM) , and the vascular adhesion molecule V-CAM/InCAm-110 (Stamenkovic and Seed, supra. Wilson et al., supra), consistent with a role in cell- cell adhesion.
  • MAG yelin associated glycoprotein
  • N-CAM neural cell adhesion molecule
  • V-CAM/InCAm-110 vascular adhesion molecule
  • CD22 ⁇ mediates adhesion to erythrocytes and monocytes (Stamenkovic and Seed, supra) while CD22/3 participates in B cell-B cell interactions (Wilson et al., supra) .
  • Cell surface expression of CD22 on B lymphocytes coincides with the capacity to respond to antigen (Pezzutto et al., J. Immunol.. 140:1791-95 (1988)), and the expression pattern of CD22 is reminiscent of that of IgM (Dorken et al. , supra) .
  • CD45 molecules have been shown to display phosphotyrosine phosphatase activity (Tonks et al., Biochemistry. 27:8695-701 (1988); Hunter, Cell,
  • CD45 may regulate early activation events in T cells.
  • CD45 isoforms participate in cell-cell interactions (Hunter, supra)
  • the corresponding ligands 0 on adjacent cells have remained elusive.
  • Humoral immunity is mediated by B cells
  • cellular immunity is mediated by T cells.
  • the interaction of antigen on an antigen-presenting cell with a helper T cell is a critical first step leading to activation of effector cells in both branches of the immune system.
  • T cells and B cells communicate with each other through various interactions involving receptors, e.g., the interaction between the T cell receptor and antigen, or the interaction between various cell adhesion molecules and their ligands, and through the secretion of various soluble factors.
  • Cell surface adhesion molecules play a role in the function and regulation of the immune response by enhancing the efficiency of interactions between lymphocytes and accessory cells or target cells, promoting interactions between leukocytes and endothelial cells, and by facilitating the recirculation of lymphocytes. Monoclonal antibodies to these adhesion molecules can inhibit the interaction between cells.
  • Various adhesion molecules have been identified, including a lymphocyte function-associated antigen-1 (LFA-1) and the T cell surface markers CD2, CD4, and CD8. Each adhesion molecule is thought to interact with a specific ligand on the surface of another cell.
  • helper-inducer T cell and lymphokine interleukin-1 IL-1
  • the activated helper- inducer T cells can produce soluble helper factors such as IL-4, IL-5, and IL-6, which induce the B cells to proliferate and differentiate into antibody producing cells (plasma cells) or memory cells.
  • B cells In a normal immune response, activation and proliferation of B cells occurs when antigen interacts with the B cell antigen receptor, or membrane bound immunoglobulin, followed by aggregation, or patching, of these receptors on the cell surface. In addition, other factors are required, such as the presence of T cells and macrophages as well as lymphokines that promote B cell growth and differentiation.
  • Tolerance in the normal immune system means that it does not respond destructively to self-antigens. An extreme situation occurs when this tolerance expands to non-self, and potentially all, antigens. Another mechanism that can cause such tolerance is ligand-induced inactivation, or antigen blockade. Immune tolerance may also be induced by other mechanisms, including the inhibition of B cell activation either by CD 8 + suppressor T cells or by inhibition of CD + helper-inducer T cell activation.
  • autoimmune diseases which are defined as any disease caused by immunologic reaction to self-antigens, i.e., the normal tolerance is lost or diminished.
  • Organ- specific autoimmune diseases include myasthenia gravis, thyroiditis, primary biliary cirrhosis, arteriosclerosis, and autoimmune hemolytic anemia.
  • Systemic autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus and rheumatic fever.
  • autoimmune disease Three mechanisms are principally responsible for inflammation and tissue injury in autoimmune disease: cell lysis and release of inflammatory mediators triggered by autoantibodies, immune complex disease, and cell-mediated immunity.
  • circulating autoantibodies react with modified or unmodified antigens on cell surfaces. .
  • the bound antibodies then stimulate the release of mediators of inflammation, trigger the complement pathway, or activate cytotoxic cells of the immune system.
  • complexes between autoantibodies and antigens form in a circulation or in intercellular fluids. These complexes then deposit in various tissues and cause inflammation and tissue injury.
  • sensitized T cells either injure cells directly or release ly phokines that amplify the inflammatory response.
  • CD22/3 mediates B cell interaction with CD4+, CD8+, and CD16+ (NK cells) T lymphocytes, as well as tonsilar B cells and B cell lines.
  • T and B cell adhesion to CD22 / 3 occurs via at least two known different sialylated ligands.
  • the T cell ligand recognized by CD22 / 3 is believed to be CD45RO, a cell surface phosphotyrosine phosphatase, associated with the helper T cell phenotype (Smith et al., Immunol.. 58:63-70 (1986); Streuli et al., J. EXP. Med.. 188:1548- 66 (1987); Tonks et al., Biochemistry. 27:8695-701 (1988)); and the B cell ligand is CD75, a cell surface ⁇ 2-6 sialyltransferase which is highly expressed on activated B cells.
  • CD223 constitutes the first CD45 ligand to be identified; its interaction with CQ22 ⁇ may regulate T cell activation.
  • the invention features a method of inhibiting, in a biological sample or system the binding of a CD22)8- bearing B cell to a second cell bearing a CD22J-specific ligand, by contacting the sample with a substance which binds to the CD22 / 3-speciifc ligand to competitively inhibit the binding of the B cell to the second cell.
  • This second cell may be a T cell or a B cell.
  • the preferred form of the inhibiting substance is a soluble protein including a portion of CD22 / 3 capable of binding to a CD22/3-specific ligand binding site on a T cell.
  • the inhibitory substance may also be an antibody to naturally occurring B cell CD22 / 3.
  • the invention also features a soluble protein fragment capable of binding to a CD22 / 3-specific ligand on a T cell.
  • this fragment excludes the transmembrane region of CD22 / 3 or includes only a portion of the transmembrane region small enough not to prevent solubilization of the fragment.
  • this fragment is at least 70% homologous with a region of CD22/3 and contains at least 322 amino acids.
  • the biological system noted above may be a human patient, in which case the method results in inhibition of the immune reponse of that patient.
  • the invention also features a therapeutic composition including one or more different soluble fragments as defined above in a pharmaceutically acceptable carrier.
  • the invention further features a method of inhibiting a human patient's immune response to alleviate an autoimmune disease, by administering to the patient an effective amount of this therapeutic composition.
  • This invention also features an expression vector including a DNA sequence encoding the soluble fragment and a cell comprising that expression vector.
  • A. soluble CD22 / 3 fragment according to the invention may be made by culturing this cell and isolating the soluble fragment therefrom.
  • the invention features methods of competitively inhibiting the binding of B cells to T cells, on other B cells, in a human patient, thereby preventing activation of both T and B cells, by administering an inhibiting amount of a composition including a soluble protein comprising a portion of CD223 capable of binding to a CD22 / 3-specific ligand on a T cell, and a pharmaceutically acceptable carrier.
  • Fig. la is an autoradiograph showing immunoprecipitation of CD22/3 v. CD22 ⁇ .
  • Fig. lb is a schematic representing the structures of CD22 and CD223.
  • Figs. 2a to 2h are a series of photomicrographs showing CD22 ⁇ -mediated adhesion of peripheral blood and tonsillar lymphocytes.
  • Fig. 3 is a set of four schematics representing the structures of truncated forms of CD223.
  • Fig. 4 is a graph showing the T and B lymphocyte- binding epitopes of CD22/3.
  • Fig. 5 is a graph showing the blocking of Molt-4 and Daudi cell adhesion to CD22 / 3 transfectants by monoclonal antibodies and neuraminidase treatment.
  • Fig. 6a is a schematic of a soluble D22 ⁇ protein.
  • Fig. 6b is an autoradiograph of two purified CD22/3 fusion proteins.
  • Fig. 7a is a graph showing the reactivity of UCLH- 1 monoclonal antibody with resting and activated T cells.
  • Fig. 7b is a graph showing the reactivity of resting T cells with CD22Rg, a CD22-immunoglobulin chimera.
  • Fig. 7c is a graph showing the reactivity of Molt- 4 cells with various CD22-immunoglobulin chimeras.
  • Fig. 8 is a series of graphs showing the comparison of CD45RO+/CD45RO-peripheral blood T cells and other T cells for reactivity with UCHL-1 and CD22Rg.
  • Figs. 9a to 9d are a series of graphs showing the reactivity of anti-CD75 monoclonal antibody and CD22Rg with CD75-transfected COS cells and B cell lines and antibody blocking of Daudi cell binding to CD22- / 3- expressing COS cells.
  • Fig. 10 is a schematic of the cDNA nucleotide and amino acid sequnces of the entire coding and 3' untranslated region of CD22 / 3.
  • CD22 / 3 was isolated from Nalm-6 and Raji cDNA libraries, constructed as described by Stamenkovic and Seed, J. Exp.
  • CD22F CGC GGG CTC GAG ACG CGG AAA CAG GCT TGC ACC CAG ACA CGA
  • CD22R CGC GGG CTG CAG GTC TGG GGA AAA CTC GGG GAC TTC CCT GGC
  • Reactions were done using amplitaq polymerase (Perkin Elmer) and buffers recommended by the vendor. Thirty cycles of amplification were carried out using the following scheme: 94 oC/lmin, 60 oC/2min, 72 oC/3min. Following the amplification, a fraction of the product was examined on a 1% agarose gel, and the remaining product was subjected to restriction nuclease digestion, after phenol-extraction and ethanol-precipitation. Two PCR products of 2.6 kb and 2.1 kb were obtained from the Raji cDNA library, while PCR products derived from Nalm-6 and Daudi cDNA libraries consisted of a single 2.6 kb and 2.1 kb fragment respectively.
  • Both 2.6 and 2.1 kb amplification products were subcloned into a CDM8 expression vector (Seed, Nature. 329:840-42 (1987)) and introduced into COS cells by the DEAE-Dextran method. Forty-eight hours following transfection, COS cells were tested for monoclonal antibody (mAb) reactivity.
  • mAb monoclonal antibody
  • Cells transfected with the 2.6 kb PCR amplification product derived from Nalm-6 and Raji cDNA libraries reacted with mAb to all of the reported CD22 epitopes (Schwartz-Albiez et al.. Leukocyte Typing IV. Oxford University Press, 65-67 (1989)), while cells expressing the 2.1 kb fragment reacted with only two of the mAb, HD39 and BL3C4, similar to the first reported CD22 CDNA isolate.
  • the lysates were centrifuged, precleared with 10 ⁇ g of isotype matched mouse IgG and protein A beads (Pierce) at 4°C overnight and incubated with 10 ⁇ g of Leu- 14 monoclonal antibody (mAb) and fresh protein A beads for 4 hours at 4°C. Beads were washed in lysis buffer, resuspended in loading buffer containing mercaptoethanol, and eluted by boiling. Eluates were electrophoresed on an 8% polyacrylamide gel and the dried gel was autoradiographed for 24h.
  • DNA sequence analysis, by the dideoxy method, of the larger CD22 insert showed an open reading frame identical to the reported CD22 / 3 sequence (Wilson et al., supra) , predicting an extracellular region composed of 7 Ig-like domains.
  • the sequence of the smaller insert was identical to that of CD22 ⁇ with the exception that extracellular Ig domains 3 and 4 were deleted (Fig. lb) .
  • the smaller isolate therefore corresponds to CD22 ⁇ .
  • CD22 / 3 Mediates the Adhesion of CD4+ T Cells and Tonsillar B Cells
  • COS cells transfected with CD223 were incubated with freshly isolated peripheral blood or tonsillar mononuclear cells under conditions previously described in Stamenkovic and Seed, Nature, 344:74-77 (1990) .
  • COS cells were transfected with CD22c., CD22 ⁇ or
  • CD20 by the DEAE-Dextran method, trypsinized 12 hours after transfection and replated in 6 cm plates at 25% confluence to facilitate rosette scoring, and cultured for 1-2 additional days before performing the adhesion assays.
  • Peripheral blood and tonsil mononuclear cells were separated on Ficoll Hypaque gradients, washed several times in PBS, and resuspended in Dulbecco's modified Eagle's medium (DMEM) in the presence of heparin (500 U/ml) .
  • DMEM Dulbecco's modified Eagle's medium
  • PHA blasts were obtained by incubating Ficoll-Hypaque separated peripheral blood mononuclear cells with 1 ⁇ g/ml PHA for 72 hours at 37°C in RPMI supplemented with 10% fetal bovine serum. 48 hours following transfection, COS cells were overlayed with PBL, tonsillar cells, or PHA blasts in 2 ml DMEM and 500 U/ml heparin, and incubated at 4°C for 30 min.
  • Non- adhering cells were removed by gentle washing with PBS and the remaining cells stained with fluorescein- or phycoerythrin labelled anti-CD3 (leu4) , anti-CD4 (leu- 3a), anti-CD8 (leu2) , anti-CD14 (leu-M3) , and anti-CD20 (leu-16) (Becton-Dickinson) mAb for 30 minutes at 22°C, washed in PBS, fixed in 4% formaldehyde and examined by fluorescence microscopy.
  • tonsillar lymphocyte rosettes which were also largely composed of CD3+ cells, contained a significant number of CD20+ cells (Figs. 2e and f) . All of the rosetting T cells, whether derived from PBLs or tonsils, belong to the CD4+ subset (Figs. 2g and h) .
  • truncated forms of CD22/3 comprising sequences encoding single or multiple Ig- domains of CD22 / 3 were ligated to sequences encoding the transmembrane and cytoplasmic sequences of CD32 as described in Stengelin et al., EMBO, 7:1053-59 (1988), and inserted into CDM8 expression vectors.
  • Fig. 3 shows the structure of four such truncated forms of CD22., along with the predicted amino acid sequences at the sites of fusion.
  • CD22 cDNA sequences were amplified by PCR using synthetic oligonucleotides complementary to . sequences flanking the cDNA regions to be amplified. Oligonucleotides were designed to allow the creation of restriction endonuclease cleavage sites at the 5' and 3' extremities of each amplified cDNA segment to facilitate subsequent insertion into CD32 expression vectors. 30 cycles were conducted consisting of 1 min. at 94°C, 2 min. at 60°C, and 3 min. at 72°C, using the reaction buffer recommended by the vendor (US Biochemical) . A CD22 primer encoding sequences at the 5' extremity of. the signal peptide and including an Xho I site was synthesized as follows: 5'-CGC GGG CTC GAG ATG CAT CTC CTC GGC CCC TGG CTC-3'
  • Reverse primers containing a Bgl II restriction site were synthesized with the following sequences:
  • CD22D2 5'-CTC GAG ATC TTT CAC GTT CAG CTG CAC CGT GTC ATT-3'
  • CD22D3 5'-CTC GAG ATC TTC CGG GGC ATA CTG CAC TTG CAG GAA-3'
  • CD22 PCR products were digested with Xho I and Bgl II and ligated to Xho I-BamHI-cut CD4-CD32 vector. Constructs containing CD22 Ig-domains 1; 1 and 2; 1, 2, and 3; and 1, 2, 3, and 4, were expressed in COS cells and tested for mAb binding. The reactivity results are shown in Fig. 3, on the right side. Domain 1 failed to show reactivity with anti-CD22 mAb, whereas domains 1 and 2 reacted with mAb Leu-14 and B1-3C4, which are thought to recognize two different CD22 epitopes (Schwarz-Albeiz et al. , supra) , and supported erythrocyte adhesion (data not shown) .
  • CD22 domains 1-3 and 1-4 were expressed in COS cells, reacted with all anti-CD22 mAb, and mediated adhesion of both Molt-4 and Daudi cells. Sequences required for reactivity with known antibodies, as well as T and B cell adhesion, are therefore encoded in the first three CD22 Ig-like domains (residues 1-302) .
  • T and B Cell Lines Bind to Different Epitopes of CD22_.
  • blocking assays were performed by treating CD22/3- transfected COS cells with a panel of anti-CD22 mAb, prior to incubation with Molt-4 or Daudi cells. Two of the mAbs, Leu-14 and B1-3C4 mAb, hich recognize both CD22 polypeptides, failed to inhibit adhesion of either cell line.
  • CD22 / 3-Mediated T Cell Adhesion is Blocked bv the CD45RO mAb UCHL-1
  • T cells adhering to CD22/3-transfected COS cells are CD4+, but not all CD4+ lymphocytes bind to CD22 / 3- transfectants (data not shown) .
  • T cell ligand of CD22 a panel of mAb to cell surface antigens expressed on subpopulations of CD4+ T cells were tested for blocking adhesion of Molt 4 cells to CD22?- transfected COS cells.
  • the panel included mAb to CD2, CD4, CD5, CD6, CD7 , CD8, CD18, CD44 and CD45 antigens. Only one monoclonal antibody, UCHL-1, which recognizes the restricted leukocyte common antigen isoform CD45RO (Smith et al., Immunol..
  • FIG. 5 peripheral blood, or tonsillar T cell binding.
  • the different lanes in Fig. 5 show the percentage of adhesion (compared to medium only) of Daudi and Molt-4 cells to CD22 / 3 transfectants in the presence of: lane 1, no treatment; lane 2, anti-CD44 mAb; lane 3, anti-CD5 mAb; lane 4, anti-CD8 mAb; lane 5, anti-CD45 mAb 2H4; lane 6, anti-CD45 mAb 4KB5; lane 7, anti-CD45 mAb UCHL-1; and lane 8, neuraminidase.
  • Transfected COS cells were pre-incubated with 50 ⁇ g of each anti-CD22 mAb for 45 min. at 22°C, washed in PBS, and overlayed with 5xl0 6 Molt-4 or Daudi cells in DMEM with 500U/ml heparin. Incubation proceeded for 30 min. as above. Rosettes were scored by recording the number of mononuclear cells in each individual rosette. Because of variation in COS cell size, 100-200 COS cells were scored per assay. Average scores obtained for adhesion in the presence of each mAb are expressed as a percentage of average scores obtained for adhesion in the presence of media alone.
  • CD2, CD4, CD4, CD6, CD7, CD8, CD18, CD44, CD45 and CD75 were obtained from the Fourth International Typing Leukocyte Workshop.
  • Antibodies to CD45RO (UCHL-1) and CD45RA (2H4) were a kind gift of Dr. Stuart Schlossman.
  • Antibody blocking was performed by pre-incubating transfected COS cells or PBL, TL, PHA blasts and cell lines with monoclonal antibodies at a concentration of 50 ⁇ g/ml for 45 min. at 22°C. Cells were washed in PBS and adhesion assays conducted as described above. Neuraminidase blocking assays
  • Neuraminidase treatment was done by incubating 5xl0 6 cells with 50 mU/ml neuraminidase from Vibrio cholerae at 37°C for 30 min.
  • transfected COS cells were treated with a non- blocking anti-CD22 mAb (leu-14) at a dilution of 1:500 in PBS, for 30 min. at 22°C immediately following the adhesion assay.
  • the cells were rinsed with PBS, incubated with a fluorescein-conjugated goat anti-mouse affinity purified antibody (Cappel) , rinsed with PBS, fixed in 4% formaldehyde and examined under a fluorescence microscope.
  • Cappel fluorescein-conjugated goat anti-mouse affinity purified antibody
  • CD22Rg soluble CD22 Ig chimeras
  • CD22Rg fusion proteins were efficiently secreted by COS cells and reacted with anti- CD22 monoclonal antibodies HD39 and HD6 (data not shown) .
  • Fig. 6b shows the molecular weight of these two proteins on an autoradiograph.
  • a CD8 fusion protein described in a previous study (Aruffo et al. , supra) was used as a control for non-specific, Fc-mediated interactions. All fusion proteins formed disulfide-linked di ers similar to immunoglobulins, and accumulated to concentration of 0.5- 2 ⁇ g/ml in COS cell supernatants at 7-10 days post transfection. To test for ligand reactivity, fusion proteins were used as supernatants or after purification on a protein A-sepharose column.
  • UCHL-1 and CD22Rg reactivity of resting T cells (a) , day 3 PHA-blasts (b) and T cells cultured for 16 days following PHA stimulation (c) were compared (Figs. 7a and b) .
  • the percentage of UCHL-1-reactive, resting T cells, day 3 PHA blasts and 16-day post-PHA stimulation T cells (71%, 84% and 93%, respectively) comparable to CD22Rg reactive fractions (69%, 81% and 88% respectively)
  • the relative intensity of reaction with CD45Rg of all three T cell populations (Figs. 7a and b) , was a reflection of the level of CD45R0 expression.
  • CD45R0 interacts with CD223
  • purified peripheral blood T cells were sorted into CD45RO+ and CD45RO- subpopulations and tested for CD22Rg reactivity and CD22/3-mediated adhesion. Only CD45R0+ cells were observed to react with CD22Rg and to form rosettes with CD223-expressing COS cells (Fig. 8) .
  • CD22.3Rg and CD22.4Rg constructs were introduced into COS cells by the DEAE-Dextran method, and supernatants collected 5-7 days post transfection. Supernatants were tested for CD22Rg production by labeling COS cells with 35S-cysteine-methionine (ICN) and precipitating the labeled soluble CD22 with protein A beads (Pierce) .
  • ICN 35S-cysteine-methionine
  • Purified peripheral T cells were obtained by depleting Ficoll-Paque-separated mononuclear cells of B cells using anti-human IgM antibody-coated magnetic beads
  • CD8Rg at 25-50 ⁇ g/ml, as above.
  • CD22Rg reactivity were measured by indirect fluorescence and flow cytometry. Purified peripheral T cells were sorted into CD45R0+ and CD45RO- populations on an EPICS cell sorter.
  • CD22Rg Blocks Anti-CD3-Mediated T Cell Activation Because CD45 is thought to be involved in regulating protein phosphorylation, interaction of CD45R0 with CD22 may play a role in T cell activation. Preliminary studies designed to determine a possible functional role of CD22 in T cell triggering, revealed that anti-CD3 mediated T cell activation is blocked by CD22Rg in a dose-dependent fashion (Table 1) .
  • Anti- CD45RO mAb UCHL-1 produced a less pronounced effect in soluble form at comparable doses but had a strong inhibitory effect when crosslinked to plates.
  • Human immunoglobulins and CD8Rg at comparable doses produced no •effect on T cell activation (Table 1) .
  • CD22Rg did not require crosslinking to block CD3-mediated activation.
  • Neither soluble CD22Rg nor soluble or plated UCHL-1 mAb had any effect on PHA-mediated T cell activation (data not shown) , consistent with the notion that triggering of CD45R0 by antibody or ligand modulates some but not all T cell activation pathways.
  • the amounts of antibody and CD22Rg used are indicated on Table 1.
  • the B Cell Ligand for CD22 is the ⁇ 2-6 Sialyltransferase CD75
  • CD22-transfected COS cells was abrogated by neuraminidase treatment. However, B cells do not express CD45R0
  • Fig. 9a shows the reactivity of CD75-transfected COS cells with anti-CD75 mAb HH-2 (continuous line) and with an unrelated, isotype-matched mouse antibody (dotted line) .
  • Fig. 9b shows the reactivity of these same transfected COS cells with CD22Rg (continuous line) ,
  • CD22Rg following treatment with neuraminidase (dotted line)
  • CD8Rg separcely dotted line
  • CD75 is identical to ⁇ .2-6 sialyltransferase (Stamenkovic et al., 1990, supra) . raises the possibility that the observed CD22Rg reactivity may be due to c.2-6 sialylation of an intrinsic COS or Hela cell surface molecule as a result of CD75 expression.
  • COS cells transfected with unrelated cDNA clones were incubated with varying concentrations of soluble sialyltransferase, which, in the presence of appropriate substrate, has been shown to retain its enzymatic activity (Weinstein et al., J. Biol. Chem.. 257:13835-44 (1982)). If CD22Rg recognizes a resident c.2-6 sialylated COS cell receptor, CD22Rg reactivity with COS cells subjected to soluble sialyltransferase would be expected. However, soluble sialyltransferase failed to induce COS cell reactivity with CD22Rg.
  • COS cells treated with soluble sialyltransferase or transfected with CD75 were treated for agglutination with sambucus nigra bark lectin (SNA) .
  • SNA specifically agglutinates c.2-6 sialylated glycoproteins (Shibuya, et al.. Arch. Bioche . Biophys.. 254:1-8 (1987)), but has virtually no effect on untreated COS cells, suggesting that COS cells do not constitutively express significant levels of c.2-6 sialylated molecules.
  • CD75-transfected and soluble sialyltransferase-treated COS cells both displayed agglutination in the presence of SNA (data not shown) , indicating that both the cell surface form and the soluble form of sialyltransferase mediate sialylation of COS cell glycoproteins.
  • 9c also shows the use of murine antibodies (dotted lines) as controls for UCHL-1 and HH-2 mAbs reactivity, and CD8Rg as a negative control (dotted line) for CD22Rg binding.
  • the mAbs were used at 5 ⁇ g/ml and CD22Rg and CD8Rg were used at 50 ⁇ g/ml.
  • COS cells transfected with an unrelated cDNA, encoding CD20 were treated with soluble sialyl transferase (Sigma, St. Louis, MO) at concentrations from 0.01 mM to l mM in DMEM/10%FBS for 30 min. to 2 hr. at 37°C, in the presence of CMP-sialic acid (Sigma) according to procedures of Weinstein et al., J ⁇ _ Bio. Chem.. 257:13835-44 (1982) .
  • COS cells treated with soluble sialyltransferase were compared for agglutination with CD75-transfected cells and untreated cells or tested for reactivity with CD22Rg by indirect immunofluorescence.
  • peripheral blood mononuclear cells isolated on Ficoll-Paque were used.
  • Cells were cultured in quadruplicate samples in 96 well microtiter plates at 2xl0 5 /ml, in RPMI medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum and gentamycin.
  • plates Prior to T cell stimulation, plates were coated with anti-CD3 mAb at a concentration of 10 ⁇ g/ml in PBS overnight at 4°C. After 3 days in culture, cells were pulsed for 6 hours with 0.5 ⁇ Ci/[ 3 H] thymidine/well. Cells were harvested with an automatic cell harvester and radioactivity was measured in a liquid scintillation counter.
  • UCHL-1 mAb, human IgG, CD22Rg and CD ⁇ Rg were used at 40 ⁇ g/ml or as indicated.
  • COS cells transfected with CD20, CD223, CD23, CD43, CD44, and CD75 were incubated with purified CD22Rg (25-50 ⁇ g/ml) for 1 hour at room temperature, rinsed with DMEM without serum, incubated with fluorescein-labeled affinity-purified goat-anti-human antibody for 30 min. at room temperature, rinsed, fixed in 4% formaldehyde and examined under a fluorescence microscope or by FACS scan.
  • Fig. 10 shows the nucleic acid sequence and corresponding amino acid sequence for the. CD22/3 cDNA and protein, as described in Wilson et al., supra.
  • the CD223 gene may be digested with restriction enzymes to generate a desired DNA fragment; the fragment may then be cloned, expressed, and the resulting protein fragment purified, all according to conventional techniques; e.g., see Maniatis et al. , Eds., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, NY (1982), and Pouwels et al., Eds., Cloning Vectors, Elsevier, Amsterdam (1987) .
  • the deduced a ino acid sequence of CD223 as shown in Fig. 10, may be used to generate a synthetic peptid .
  • CD22 / 3 interacts with different ligands on T, B, and myeloid cells.
  • CD45R0/CD4 T cells are known to provide help for B cell antibody production (Smith et al. , Immunol. , 58:63-70 (1986) .
  • CD45 molecules display phosphotyrosine phosphatase activity and are thought to regulate signal transduction in lymphocytes by enhancing or blocking cell activation induced through T or B cell surface antigens. The regulatory function is believed to result from interaction between intracellular portions of CD45 and various lymphocyte cell surface molecules.
  • cross-linking of CD45 with CD3 or CD2 inhibits the ability of anti-CD3 and anti-CD2 mAb to increase intracellular calcium fluxing and stimulate T cell activation.
  • cross-linking of CD45 with CD4 greatly augments the calcium fluxing produced upon cross- linking CD4 alone.
  • CD22/3 is the first CD45 ligand to be identified and apparently triggers CD45 to regulate T cell activation, and possibly, cytokine production.
  • soluble fragments or analogs of CD22 / 3 may be used in the therapeutic regulation of T cell activation.
  • T cell activation can be down- regulated, thus down-regulating the patient's system.
  • the invention provides a means for treatment of autoimmune diseases, e.g., rheumatoid arthritis, SLE, and Type I diabetes as well as alloqraft rejection, graft versus host disease, and other disease states in which it is advantageous to inhibit T-cell activation and/or T- cell activation of a B cell humoral response.
  • therapy according to the invention can also employ anti-CD22 / 3 antibodies, or antibodies to the T-cell or B-cell ligand for CD220.
  • Soluble proteins or protein fragments, as well as antibodies may be administered to a human patient in one of the conventional modes, e.g., orally, intravenously, parenterally, or transdermally in a sustained release formulation using a biodegradable biocompatible polymer, admixed with an appropriate pharmaceutically acceptable carrier or diluent, or by using micelles, gels, or liposomes.
  • the soluble protein or antibody can be administered to a human patient in a dosage of about 0.5 mg/kg/day to about 3.0 mg/kg/day.
  • the use of soluble CD223 may provide additional benefits in treating, e.g., alloqraft rejection, because its use does not cause the body to generate additional antibodies, because the soluble CD223 is recognized as a sel -antigen.

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Abstract

Procédé d'inhibition, dans un échantillon biologique, de la liaison d'un lymphocyte B porteur de CD22β à un deuxième lymphocyte porteur d'un ligand spécifique de CD22β, par la mise en contact de l'échantillon avec une substance qui se lie audit ligand spécifique de CD22β, afin d'inhiber de manière compétitive la liaison du lymphocyte B au deuxième lymphocyte. Celui-ci peut être par exemple, un lymphocyte T ou B. L'invention présente en outre un procédé d'inhibition compétitive de la liaison de lymphocytes B à des lymphocytes T chez un patient humain, empêchant l'activation de lymphocytes aussi bien T que B, par l'administration d'une quantité inhibitrice d'une composition comprenant une protéine soluble contenant un fragment de CD22β pouvant se lier à un ligand spécifique de CD22β sur un lymphocyte T, ainsi qu'un support acceptable en pharmacologie.
PCT/US1992/007994 1991-09-20 1992-09-21 Inhibition competitive des interactions entre lymphocytes t et b WO1993005814A1 (fr)

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US76344391A 1991-09-20 1991-09-20
US763,443 1991-09-20

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WO1993005814A1 true WO1993005814A1 (fr) 1993-04-01

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0660721A1 (fr) * 1993-05-21 1995-07-05 Dana Farber Cancer Institute Anticorps monoclonaux bloquant l'adhesion du ligand sur le recepteur cd22 dans les lymphocytes b matures
WO2004014422A1 (fr) * 2002-08-08 2004-02-19 The Corporation Of The Trustees Of The Order Of The Sisters Of Mercy In Queensland Methode d'immunomodulation
AU2003249776B2 (en) * 2002-08-08 2009-04-23 The Corporation Of The Trustees Of The Order Of The Sisters Of Mercy In Queensland A method of immunomodulation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FASEB JOURNAL, Volume 4, issued 26 April 1990, G.L. WILSON et al., "Identification of a Probable B Lymphocyte Homotypic Cell Adhesion Molecule, BL-CAM", page A1698, No. 23. *
JOURNAL OF EXPERIMENTAL MEDICINE, Volume 173, issued January 1991, G.L. WILSON et al., "cDNA Cloning CD22: A Mediator of B-B Cell Interactions", pages 137-146. *
JOURNAL OF IMMUNOLOGY, Volume 140, issued 01 January 1988, D.R. BOUE et al., "Structural Characterization of the Human B Lymphocyte Restricted Differentiation Antigen CD22", pages 192-199. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0660721A1 (fr) * 1993-05-21 1995-07-05 Dana Farber Cancer Institute Anticorps monoclonaux bloquant l'adhesion du ligand sur le recepteur cd22 dans les lymphocytes b matures
EP0660721A4 (fr) * 1993-05-21 2001-12-12 Dana Farber Cancer Inst Inc Anticorps monoclonaux bloquant l'adhesion du ligand sur le recepteur cd22 dans les lymphocytes b matures
WO2004014422A1 (fr) * 2002-08-08 2004-02-19 The Corporation Of The Trustees Of The Order Of The Sisters Of Mercy In Queensland Methode d'immunomodulation
AU2003249776B2 (en) * 2002-08-08 2009-04-23 The Corporation Of The Trustees Of The Order Of The Sisters Of Mercy In Queensland A method of immunomodulation

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