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WO1992000092A1 - Ligand pour recepteur a cd28 sur des lymphocytes b et procedes - Google Patents

Ligand pour recepteur a cd28 sur des lymphocytes b et procedes Download PDF

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Publication number
WO1992000092A1
WO1992000092A1 PCT/US1991/004682 US9104682W WO9200092A1 WO 1992000092 A1 WO1992000092 A1 WO 1992000092A1 US 9104682 W US9104682 W US 9104682W WO 9200092 A1 WO9200092 A1 WO 9200092A1
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WIPO (PCT)
Prior art keywords
cells
antigen
amino acid
receptor
ligand
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PCT/US1991/004682
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English (en)
Inventor
Peter S. Linsley
Jeffrey A. Ledbetter
Nitin K. Damle
William Brady
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Bristol-Myers Squibb Company
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Priority to CA2086325A priority Critical patent/CA2086325C/fr
Priority to JP3513625A priority patent/JPH06508501A/ja
Publication of WO1992000092A1 publication Critical patent/WO1992000092A1/fr

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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • 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
    • C07K14/70521—CD28, CD152
    • 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
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the present invention relates to the identification of an interaction between the CD28 receptor and its ligand, the B7 antigen, and to a method for regulating cellular interactions using the antigen, fragments and derivatives thereof.
  • T cell T lymphocyte
  • T cell T lymphocyte
  • cyto ines or lymphokines soluble immune mediators
  • This response is regulated by several T-cell surface receptors, including the T-cell receptor complex (Weiss et al., Ann. Rev. Immuno1. 4:593- 619 (1986)) and other "accessory" surface molecules (Springer et al., (1987) supra) .
  • CD cell surface differentiation
  • CD28 antigen a homodimeric glycoprotein of the immunogloh lin superfamily (Aruffo and Seed, Proc. Natl. Acad. Sci. 84:8573-8577 (1987)) found on most mature human T cells (Damle et al., J. Immunol. 131:2296-2300 (1983)). Current evidence suggests that this molecule functions in an alternative T cell activation pathway distinct from that initiated by the T-cell receptor complex (June et al., Mol. Cell. Biol. 7:4472-4481 (1987)).
  • Monoclonal antibodies (mAbs) reactive with CD28 antigen can augment T cell responses initiated by various polyclonal stimuli (reviewed by June et al., supra) . These stimulatory effects may result from mAb-induced cytokine production (Thompson et al., Proc. Natl. Acad. Sci 86:1333-1337 (1989); Lindsten et al. , Science 244:339-343 (1989)) as a consequence of increased mRNA stabilization (Lindsten et al., (1989), supra).
  • Anti-CD28 mAbs can also have inhibitory effects, i.e., they can block autologous mixed lymphocyte reactions (Damle et al., Proc. Natl. Acad. Sci. 78:5096-6001 (1981)) and activation of antigen- specific T cell clones (Lesslauer et al. , Eur. J. Immunol. 16:1289-1296 (1986)).
  • CD28 antigen could conceivably function as a cytokine receptor, although this seems unlikely since it shares no homology with other lymphokine or cytokine receptors (Aruffo and Seed, (1987) supra) .
  • CD28 might be a receptor which mediates cell-cell contact ("intercellular adhesion") .
  • Intercellular adhesion Antigen-independent intercellular interactions involving lymphocyte accessory molecules are essential for an immune response (Springer et al., (1987), supra) .
  • binding of the T cell-associated protein, CD2, to its ligand LFA-3, a widely expressed glycoprotein is important for optimizing antigen-specific T cell activation (Moingeon et al. , Nature 339:314 (1988)).
  • Another important adhesion system involves binding of the LFA-1 glycoprotein found on lymphocytes, macrophages, and granulocytes (Springer et al., (1987), supra; Shaw and Shimuzu (1988), supra) to its ligands ICAM-1 (Makgoba et al.. Nature 331:86-88 (1988)) and ICAM-2 (Staunton et al.. Nature 339:61-64 (1989)).
  • the T cell accessory molecules CD8 and CD4 strengthen T cell adhesion by interaction with MHC class I (Norment et al.. Nature 336:79-81 (1988)) and class II (Doyle and Strominger, Nature 330:256-259 (1987)) molecules, respectively.
  • the VLA glycoproteins are integrins which appear to mediate lymphocyte functions requiring adhesion to extracellular matrix components (Hemler, Immunology Today 9:109-113 (1988)).
  • the CD2/LFA-3, LFA-l/ICAM-1 and ICAM- 2, and VLA adhesion systems are distributed on a wide variety of cell types (Springer et al., (1987), supra; Shaw and Shimuzu, (1988,) supra and Hemler, (1988), supra) .
  • Intercellular adhesion interactions mediated by integrins are strong interactions that may mask other intercellular adhesion interactions.
  • interactions mediated by integrins require divalent cations (Kishimoto et al.. Adv. Immunol. 46:149-182 (1989) . These interactions may mask other intercellular adhesion interactions that are divalent cation independent. Therefore, it would be useful to develop assays that permit identification of non-integrin mediated ligand/receptor interactions.
  • T cell interactions with other cells such as B cells are essential to the immune response.
  • Levels of many cohesive molecules found on T cells and B cells increase during an immune response (Springer et al., (1 37) , supra; Shaw and Shimuzu, (1988) , supra; Hemler (1988) , supra) . Increased levels of these molecules may help explain why activated B cells are more effective at stimulating antigen-specific T cell proliferation than are resting B cells (Kaiuchi et al., J. Immunol. 131:109- 114 (1983); Kreiger et al., J. Immunol 135:2937-2945 4
  • Optimal activation of B lymphocytes and their subsequent differentiation into immunoglobulin secreting cells is dependent on the helper effects of major histocompatibility complex (MHC) class II antigen (Ag)- reactive CD4 positive T helper (CD4 + T h ) cells and is mediated via both direct (cognate) T h -B cell intercellular contact-mediated interactions and the elaboration of antigen-nonspecific cytokines (non-cognate activation; see, e.g. Noel and Snow, Immunol. Today 11:361 (1990)).
  • MHC major histocompatibility complex
  • CD4 + T h CD4 positive T helper
  • T h cells have synthesis and directional exocytosis is initiated and sustained via cognate interactions between antigen-primed T h cells and antigen-presenting B cells (Moller, supra) .
  • the successful outcome of T h -B interactions requires participation of transmembrane receptor-ligand pairs of co-stimulatory accessory/adhesion molecules on the surface of T h and B cells which include CD2 (LFA-2) ; CD58 (LFA-3), CD4:MHC class II, CDlla/CD18 (LFA-1):CD54 (1CAM- 1).
  • T h During cognate T h :B interaction, although both T h and B cells cross-stimulate each other, their functional differentiation is critically dependent on the provision by T h cells of growth and differentiation- inducing cytokines such as IL-2, IL-4 and IL-6 (Noel, supra. Kupfer et al., supra. Brian, supra and Moller, supra) . Studies by Poo et al.
  • T h :B interaction indicates that interaction of the T cell receptor complex (TcR) with nominal Ag-MHC class II on B cells results in focused release of T h cell-derived cytokines in the area of T h and B cell contact (vectorially oriented exocytosis) . This may ensure the activation of only B cells presenting antigen to T h cells, and also avoids activation of bystander B cells.
  • TcR T cell receptor complex
  • T helper cell T h
  • APC antigen-presenting cells
  • Freeman et al. (J. Immunol. 143(8) :2714-2722 (1989)) isolated and sequenced a cDNA clone encoding a B cell activation antigen recognized by mAb B7 (Freeman et al., J. Immunol. 138:3260 (1987)). COS cells transfected with this cDNA have been shown to stain by both labeled mAb B7 and mAb BB-1 (Clark et al.. Human Immunol. 16:100- 113 (1986); Yokochi et al., J. Immunol. 128:823 (1981)); Freeman et al., (1989) supra; and Freedman et al.. (1987), supra) ) . Expression of the B cell activation antigen has been detected on cells of other lineages. For example, studies by Freeman et al. (1989) have shown that monocytes express low levels of mRNA for B7.
  • CD4 the receptor for HIV-1
  • hybrid fusion molecules consisting of DNA sequences encoding portions of the extracellular domain of CD4 receptor fused to antibody domains (human immunoglobulin C gamma 1), as described by Capon et al.. Nature 337:525-531 (1989).
  • CD28 antigen has functional and structural characteristics of a receptor
  • a natural ligand for this molecule has not been identified. It would be useful to identify ligands that bind with the CD28 antigen and other receptors and to use such ligand(s) to regulate cellular responses, such as T cell and B cell interactions, for use in treating pathological conditions.
  • the present invention identifies the B7 antigen as a ligand recognized by the CD28 receptor.
  • the B7 antigen, or its fragments or derivatives are reacted with CD28 positive T cells to regulate T cell interactions with other cells.
  • the CD28 receptor, its fragments or derivatives are reacted with B7 antigen to regulate interactions of B7 positive cells with T cells.
  • antibodies or other molecules reactive with the B7 antigen or CD28 receptor may be used to inhibit interaction of cells associated with these molecules, thereby regulating T cell responses.
  • a preferred embodiment of the invention provides a method for regulating CD28 specific T cell interactions by reacting CD28 positive T cells with B7 antigen, or its fragments or derivatives, so as to block the functional interaction of T cells with other cells.
  • the method for reacting a ligand for CD28 with T cells may additionally include the use of anti-CD monoclonal antibodies such as anti-CD2 and/or anti-CD3 monoclonal antibody.
  • the invention provides a method for regulating immune responses by contacting CD28 positive T cells with fragments containing at least a portion of the DNA sequence encoding the amino acid sequence corresponding to the extracellular domain of B7 antigen.
  • derivatives of B7 antigen may be used to regulate immune responses, wherein the derivatives are fusion protein constructs including at least a portion of the extracellular domain of B7 antigen and another protein, such as human immunoglobulin C gamma 1, that alters the solubility, binding affinity and/or valency of B7 antigen.
  • DNA encoding amino acid residues from about position 1 to about position 215 of the sequence corresponding to the extracellular domain of B7 antigen is joined to DNA encoding amino acid residues of the sequences corresponding to the hinge, CH2 and CH3 regions of human Ig C ⁇ l to form a DNA fusion product which encodes B7Ig fusion protein.
  • DNA encoding amino acid residues from about position 1 to about position 134 of the sequence corresponding to the extracellular domain of the CD28 receptor is joined to DNA encoding amino acid residues of the sequences corresponding to the hinge, CH2 and CH3 regions of human Ig C ⁇ l to form a CD28Ig fusion protein.
  • fragments or derivatives of the CD28 receptor may be reacted with B cells to bind the B7 antigen and regulate T cell/B cell interactions.
  • the methods for regulating T cell interactions may be further supplemented with the addition of a cytokine.
  • the invention provides a method for treating immune system diseases mediated by T cell by administering B7 antigen, including B7Ig fusion protein, to react with T cells by binding the CD28 receptor.
  • a method for inhibiting T cell proliferation in graft versus host disease wherein CD28 positive T cells are reacted with B7 antigen, for example in the form of the B7Ig fusion protein, to bind to the CD28 receptor, and an immunosuppressant is administered.
  • B7 antigen for example in the form of the B7Ig fusion protein
  • the invention also provides a cell adhesion assay to identify ligands that interact with target receptors that mediate intercellular adhesion, particularly adhesion that is divalent cation independent.
  • Figure 1 are bar graphs showing the results of cellular adhesion experiments using CD28 positive (CD28 +) and CD28 negative (CD28') CHO cells as described in Example 1, infra.
  • Figure 2 are micrographs of the cellular adhesion studies of Figure 1, as described in Example 1, infra.
  • Figure 3 are bar graphs of experiments testing the ability of different human cell lines and normal and activated murine spleen B cells to adhere to CD28 + CHO cells, as described in Example 1, infra.
  • Figure 4 is a graph of the effects of blocking by mAbs on CD28-mediated adhesion to human B cells, as described in Example 1, infra.
  • Figure 5 is a bar graph of the results of adhesion between COS cells transfected with B7 antigen and CD28 + or CD28 " CHO cells, as described in Example 1, infra.
  • Figure 6 is a bar graph demonstrating the effect of anti- CD28 and anti-B7 mAbs on T cell proliferation as described in Example 2, infra.
  • Figure 7 is graphs showing the effects of DR7-primed CD4 + CD45RO + T h cells on differentiation of B cells into immunoglobulin secreting cells, as described in Example 2, infra (7a: IgM production by SKW B cells; 7b: IgG production by CESS B cells) .
  • Figure 8 is graphs showing the effect of anti-CD28 and anti-B7 mAbs on the T h -induced production of immunoglobulin by B cells as described in Example 2, infra (8a: IgM production, 8b: IgG production) .
  • Figure 10 is a photograph of a gel obtained from purification of B7Ig and CD28 protein fusion constructs as described in Example 3, infra.
  • Figure 11 depicts the results of FACS R analysis of binding of the B7Ig and CD28Ig fusion proteins to transfected CHO cells as described in Example 3, infra.
  • Figure 12 is a graph illustrating competition binding analysis of 125 I-labeled B7Ig fusion protein to immobilized CD28Ig fusion protein as described in Example 3, infra.
  • Figure 13 is a graph showing the results of Scatchard analysis of B7Ig fusion protein binding to immobilized CD28Ig fusion protein as described in Example 3, infra-
  • Figure 14 is a graph of FACS R profiles of B7Ig fusion protein binding to PHA blasts as described in Example 3, infra.
  • Figure 15 is an autoradiogram of 125 I-labeled proteins immunoprecipitated by B7Ig as described in Example 3, infra.
  • Figure 16 is a graph showing the effect of B7Ig binding to CD28 on CD28-mediated adhesion as described in Example 3, infra.
  • Figure 17 is a photograph of the results of RNA blot analysis of the effects of B7 on accumulation of IL-2 mRNA as described in Example 3, infra.
  • This invention is directed to the identification of a ligand reactive with CD28 antigen (hereafter referred to as "CD28 receptor”), and to methods of using the ligand and its fragments and derivatives, including fusion proteins. Also disclosed is a cell adhesion assay method to detect ligands for cell surface receptors.
  • CD28 receptor CD28 antigen
  • the ligand for CD28 was identified by the experiments described herein, as the B7/BB-1 antigen isolated by Freeman et al., (Freedman et al., and Freeman et al., supra. both of which are incorporated by reference herein) .
  • B7 antigen the ligand for CD28, identified as the B7/BB-1 antigen, is referred to herein as the "B7 antigen”.
  • fragment means a portion of the amino acid sequence corresponding to the B7 antigen or CD28 receptor.
  • a fragment of the B7 antigen useful in the method of the present invention is a polypeptide containing a portion of the amino acid sequence corresponding to the extracellular portion of the B7 antigen, i.e. the DNA encoding amino acid residues from position 1 to 215 of the sequence corresponding to the B7 antigen described by Freeman et al., supra.
  • a fragment of the CD28 antigen that may be used is a polypeptide containing amino acid residues from about position 1 to about position 134 of the sequence corresponding to the CD28 receptor as described by Aruffo and Seed, Proc. Natl. Acad. Sci. (USA) 84:8573-8577 (1987) .
  • a derivative of the B7 antigen useful in the method of the present invention is a B7Ig fusion protein that comprises a polypeptide corresponding to the extracellular domain of the B7 antigen and an immunoglobulin constant region that alters the solubility, affinity and/or valency (valency is defined herein as the number of binding sites available per molecule) of the B7 antigen.
  • derivative also includes monoclonal antibodies reactive with the B7 antigen or CD28 receptor, or fragments thereof, and antibodies reactive with the B7Ig and CD28Ig fusion proteins of the invention.
  • the B7 antigen and/or its fragments or derivatives for use in the present invention may be produced in recombinant form using known molecular biology techniques based on the cDNA sequence published by Freeman et al., supra. Specifically, cDNA sequences encoding the amino acid sequence corresponding to the B7 antigen or fragments or derivatives thereof can be synthesized by the polymerase chain reaction (see U.S. Patent No. 4,683,202) using primers derived from the published sequence of the antigen (Freeman et al..supra) .
  • cDNA sequences can then be assembled into a eukaryotic or prokaryotic expression vector and the resulting vector can be used to direct the synthesis of the ligand for CD28 by appropriate host cells, for example COS or CHO cells.
  • CD28 receptor and/or its fragments or derivatives may also be produced using recombinant methods.
  • DNA encoding the amino acid sequence corresponding to the extracellular domain of the B7 antigen, containing amino acids from about position 1 to about position 215, is joined to DNA encoding the amino acid sequences corresponding to the hinge, CH2 and CH3 regions of human Ig O ⁇ l, using PCR, to form a construct that is expressed as B7Ig fusion protein.
  • DNA encoding the amino acid sequence corresponding to the B7Ig fusion protein has been deposited with the American Type Culture Collection (ATCC) in Rockville, Maryland, under the Budapest Treaty on May 31, 1991 and accorded accession number 68627.
  • DNA encoding the amino acid sequence corresponding to the extracellular domain of the CD28 receptor containing amino acids from about position 1 to about position 134, is joined to DNA encoding the amino acid sequences corresponding to the hinge, CH2 and CH3 regions of human Ig C ⁇ l using PCR to form a construct expressed as CD28Ig fusion protein.
  • DNA encoding the amino acid sequence corresponding to the CD28Ig fusion protein has been deposited in the ATCC, in Rockville, Maryland under the Budapest Treaty on May 31, 1991 and accorded accession number 68628.
  • cDNA clones containing DNA encoding CD28 and B7 proteins are obtained to provide DNA for assembling CD28 and B7 fusion proteins as described by Aruffo and Seed, Proc. Natl. Acad. Sci. USA 84:8573-8579 (1987) (for CD28) ; and Freeman et al., J. Immunol. 143:2714-2722 (1989) (for B7) , incorporated by reference herein.
  • cDNA clones may be prepared from RNA obtained from cells expressing B7 antigen and CD28 receptor based on knowledge of the published sequences 14 for these proteins (Aruffo and Seed, and Freeman, supra) using standard procedures.
  • the cDNA is amplified using the polymerase chain reaction ("PCR") technique (see U.S. Patent Nos. 4,683,195 and 4,683,202 to Mullis et al. and Mullis & Faloona, Methods Enzv ol. 154:335-350 (1987)) using synthetic oligonucleotides encoding the sequences corresponding to the extracellular domain of the CD28 and B7 proteins as primers. PCR is then used to adapt the fragments for ligation to the DNA encoding amino acid fragments corresponding to the human immunoglobulin constant ⁇ 1 region, i.e.
  • PCR polymerase chain reaction
  • vectors containing DNA encoding the amino acid sequences corresponding to the fusion constructs of the invention are transformed into suitable host cells, such as the bacterial cell line MC1061/p3 using standard procedures, and colonies are screened for the appropriate plasmids.
  • transfection is performed using standard techniques appropriate to such cells.
  • transfection into mammalian cells is accomplished using DEAE-dextran mediated transfection, CaP0 4 co-precipitation, lipofection, electroporation, or protoplast fusion, and other methods known in the art including: lysozyme fusion or erythrocyte fusion, scraping, direct uptake, osmotic or sucrose shock, direct microinjection, indirect microinjection such as via erythrocyte-mediated techniques, and/or by subjecting host cells to electric currents.
  • the above list of transfection techniques is not considered to be exhaustive, as other procedures for introducing genetic information into cells will no doubt be developed.
  • Expression plasmids containing cDNAs encoding sequences corresponding to CD28 and B7 for cloning and expression of CD28Ig and B7Ig fusion proteins include the OMCD28 and OMB7 vectors modified from vectors described by Aruffo and Seed, Proc. Natl. Acad. Sci. USA (1987) , supra. (CD28) ; and Freeman et al., (1989), supra. (B7) , both of which are incorporated by reference herein.
  • Preferred host cells for expression of CD28Ig and B7Ig proteins include COS and CHO cells.
  • Useful host cell lines include Chinese hamster ovary (CHO) , monkey kidney (COS) , VERO and HeLa cells. In the present invention, cell lines stably expressing the fusion constructs are preferred.
  • Expression vectors for such cells ordinarily include promoters and control sequences compatible with mammalian cells such as, for example, CMV promoter (CDM8 vector) and avian sarcoma virus (ASV) ( ⁇ LN vector) .
  • CMV promoter CDM8 vector
  • ASV avian sarcoma virus
  • Other commonly used early and late promoters include those from Simian Virus 40 (SV 40) (Fiers, et al.. Nature 273:113 (1973)), or other viral promoters such as those derived from polyoma, Adenovirus 2, and bovine papilloma virus.
  • SV 40 Simian Virus 40
  • the controllable promoter, hMTII Kerin, et al.. Nature 299:797-802 (1982) may also be used.
  • eukaryotic cells such as COS or CHO cells
  • other eukaryotic microbes may be used as hosts.
  • Laboratory strains of Saccharomvces cerevisiae. Baker's yeast are most used although other strains such as Schizosaccharomyces pombe may be used.
  • Vectors employing, for example, the 2 ⁇ origin of replication of Broach, Meth. Enz. 101:307 (1983), or other yeast compatible origins of replications see, for example, Stinchcomb et al.. Nature 282:39 (1979)); Tschempe et al.. Gene 10:157 (1980); and Clarke et al., Meth. Enz ⁇ 101:300 (1983) may be used.
  • Control sequences for yeast vectors include promoters for the synthesis of glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149 (1968); Holland et al.. Biochemistry 17:4900 (1978)). Additional promoters known in the art include the CMV promoter provided in the CDM8 vector (Toyama and Okayama, FEBS 268:217-221 (1990); the promoter for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073 (1980)), and those for other glycolytic enzymes.
  • promoters which have the additional advantage of transcription controlled by growth conditions are the promoter regions for alcohol dehydrogenase 2, iso ⁇ ytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and enzymes responsible for maltose and galactose utilization. It is also believed terminator sequences are desirable at the 3' end of the coding sequences. Such terminators are found in the 3' untranslated region following the coding sequences in yeast-derived genes.
  • prokaryotic cells may be used as hosts for expression.
  • Prokaryotes most frequently are represented by various strains of E. coli; however, other microbial strains may also be used.
  • Commonly used prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems (Chang et al.. Nature 198: 1056 (1977)), the tryptophan (trp) promoter system (Goeddel et al.. Nucleic Acids Res. 8:4057 (1980)) and the lambda derived P L promoter and N-gene ribosome binding site (Shimatake et al.. Nature 292:128 (1981)).
  • the nucleotide sequences encoding the amino acid sequences corresponding to the CD28Ig and B7Ig fusion proteins may be expressed in a variety of systems as set forth below.
  • the cDNA may be excised by suitable restrictio n enzymes and ligated into suitable prokaryotic or eukaryotic expression vectors for such expression.
  • CD28 receptors occur in nature as dimers, it is believed that successful expression of these proteins requires an expression system which permits these proteins to form as dimers. Truncated versions of these proteins (i.e. formed by introduction of a stop codon into the sequence at a position upstream of the transmembrane region of the protein) appear not to be expressed.
  • the expression of CD28 antigen in the form of a fusion protein permits dimer formation of the protein.
  • expression of CD28 antigen as a fusion product is preferred in the present invention.
  • the CD28 receptor is not readily expressed as a mature protein using direct expression of DNA encoding the amino acid sequence corresponding to the truncated protein.
  • DNA encoding the amino acid sequence corresponding to the extracellular domain of CD28 and including the codons for a signal sequence such as oncostatin M in cells capable of appropriate processing is fused with DNA encoding amino acids corresponding to the Fc domain of a naturally dimeric protein. Purification of the fusion protein products after secretion from the cells is thus facilitated using antibodies reactive with the anti- immunoglobulin portion of the fusion proteins. When secreted into the medium, the fusion protein product is recovered using standard protein purification techniques, for example by application to protein A columns.
  • monoclonal antibodies reactive with the B7 antigen and CD28 receptor, and reactive with B7Ig and CD28Ig fusion proteins may be produced by hybridomas prepared using known procedures, such as those introduced by Kohler and Milstein (see Kohler and Milstein, Nature, 256:495-97 (1975), and modifications thereof, to regulate cellular interactions.
  • the animal which is primed to produce a particular antibody.
  • the animal can be primed by injection of an immunogen (e.g. the B7Ig fusion protein) to elicit the desired immune response, i.e. production of eintibodies reactive with the ligand for CD28, the B7 antigen, from the primed animal.
  • a primed animal is also one which is expressing a disease. Lymphocytes derived from the lymph nodes, spleens or peripheral blood of primed, diseased animals can be used to search for a particular antibody.
  • the lymphocyte chromosomes encoding desired immunoglobulins are immortalized by fusing the lymphocytes with myeloma cells, generally in the presence of a fusing agent such as polyethylene glycol (PEG) .
  • a fusing agent such as polyethylene glycol (PEG) .
  • PEG polyethylene glycol
  • Any of a number of myeloma cell lines may be used as a fusion partner according to standard techniques; for example, the P3-NSl/l-Ag4-l, P3- x63-Ag8.653, Sp2/0-Agl4, or HL1-653 myeloma lines. These myeloma lines are available from the ATCC, Rockville, Maryland.
  • the resulting cells which include the desired hybridomas, are then grown in a selective medium such as HAT medium, in which unfused parental myeloma or lymphocyte cells eventually die. Only the hybridoma cells survive and can be grown under limiting dilution conditions to obtain isolated clones.
  • the supernatants of the hybridomas are screened for the presence of the desired specificity, e.g. by immunoassay techniques using the B7Ig fusion protein that has been used for immunization. Positive clones can then be subcloned under limiting dilution conditions, and the monoclonal antibody produced can be isolated.
  • the individual cell line may be propagated in vitro .
  • the culture medium containing high concentrations of a single specific monoclonal antibody can be harvested by decantation, filtration, or centrifugation.
  • fragments of these antibodies containing the active binding region of the extracellular domain of B7 or CD28 antigen such as Fab, F(ab') 2 and Fv fragments, may be produced.
  • Such fragments can be produced using techniques well established in the art
  • CD28 receptor and its ligand may function in vivo by mediating T cell interactions with other cells such as B cells.
  • the functional consequences of these interactions may be induced or inhibited using ligands that bind to the native CD28 receptor or the B7 antigen.
  • anti-CD28 monoclonal antibodies reactive with the CD28 receptor in vivo.
  • anti-CD28 mAbs may exert either stimulatory or inhibitory effects on T cells, depending, in part, on the degree of crosslinking or "aggregation" of the CD28 receptor (Damle, J. Immunol. 140:1753-1761 (1988); Ledbetter et al.. Blood 75(7):1531- 1539 (1990)) it is expected that the B7 antigen, its fragments and derivatives, will act to stimulate or inhibit T cells in a manner similar to the effects observed for an anti-CD28 monoclonal antibody, under similar conditions in vivo.
  • B7 antigen e.g. as a soluble B7Ig fusion protein to react with CD28 positive T cells
  • B7 antigen e.g. as a soluble B7Ig fusion protein
  • bind the CD28 receptor on the T cells will result in inhibition of the functional responses of T cells.
  • binding of introduced B7 antigen in the form of a fusion protein that binds to CD28 receptor on CD28 positive T cells should interfere, i.e. inhibit, the T cell interactions with B cells.
  • CD28 antigen or its fragments and derivatives in vivo, for example in the form of a soluble CD28Ig fusion protein, will result in binding of the soluble CD28Ig to B7 antigen, preventing the endogenous stimulation of CD28 receptor by B7 positive cells such as activated B cells, and interfering with the interaction of B7 positive cells with T cells.
  • B7 positive cells such as activated B cells
  • the B7 antigen, and/or its fragments or derivatives may be used to stimulate T cells, for example by immobilizing B7 antigen or B7Ig fusion protein, for reacting with the T cells.
  • the activated T cells stimulated in this manner in vitro may be used in vivo in adoptive therapy.
  • the B7 antigen and/or fragments or derivatives of the antigen may be used to react with T cells to regulate immune responses mediated by functional T cell responses to stimulation of the CD28 receptor.
  • the B7 antigen may be presented for reaction with CD28 positive T cells in various forms.
  • the B7 antigen may be encapsulated, for example in liposomes, or using cells that have been genetically engineered, for example using gene transfer, to express the antigen for stimulation of the CD28 receptor on T cells.
  • the CD28 receptor, and/or its fragments or derivatives may also be used to react with cells expressing the B7 antigen, such as B cells. This reaction will result in inhibition of T cell activation, and inhibition of T cell dependent B cell responses, for example as a result of inhibition of T cell cytokine production.
  • other reagents such as molecules reactive with B7 antigen or the CD28 receptor are used to regulate T and/or B cell responses.
  • antibodies reactive with the CD28Ig fusion proteins, and Fab fragments of CD28Ig may be prepared using the CD28Ig fusion protein as immunogen, as described above. These anti-CD28 antibodies may be screened to identify those capable of inhibiting the binding of the B7 antigen to CD28 antigen. The antibodies or antibody fragments such as Fab fragments may then be used to react with the T cells, for example, to inhibit CD28 positive T cell proliferation.
  • Fab fragments of the 9.3 monoclonal antibody, or Fab fragments of the anti-CD28Ig monoclonal antibodies as described herein is expected to prevent binding of CD28 receptor on T cells to B7 antigen, for example on B cells. This will result in inhibition of the functional response of the T cells.
  • anti-B7 monoclonal antibodies such as BB-1 mAb, or anti-B7Ig monoclonal antibodies prepared as described above using B7Ig fusion protein as immunogen, may be used to react with B7 antigen positive cells such as B cells to inhibit B cell interaction via the B7 antigen with CD28 positive T cells.
  • the B7 antigen may be used to identify additional compounds capable of regulating the interaction between the B7 antigen and the CD28 antigen.
  • Such compounds may include soluble fragments of the B7 antigen or CD28 antigen or small naturally occurring molecules that can be used to react with B cells and/or T cells.
  • soluble fragments of the ligand for CD28 containing the extracellular domain (e.g. amino acids 1-215) of the B7 antigen may be tested for their effects on T cell proliferation.
  • the ligand for CD28, B7 antigen is used for regulation of CD28 positive (CD28 + ) T cells.
  • the B7 antigen is reacted with T cells in vitro to crosslink or aggregate the CD28 receptor, for example using CHO cells expressing B7 antigen, or immobilizing B7 on a solid substrate, to produce activated T cells for administration in vivo for use in adoptive therapy.
  • adoptive therapy T lymphocytes are taken from a patient and activated in vitro with an agent. The activated cells are then reinfused into the autologous donor to kill tumor cells (see Rosenberg et al.. Science 223:1318-1321 (1986)).
  • the method can also be used to produce cytotoxic T cells useful in adoptive therapy as described in copending U.S. Patent application serial no. 471,934, filed January 25, 1990, incorporated by reference herein.
  • the ligand for CD28, its fragments or derivatives may be introduced in a suitable pharmaceutical carrier in vivo f i.e. administered into a human subject for treatment of pathological conditions such as immune system diseases or cancer.
  • a suitable pharmaceutical carrier in vivo i.e. administered into a human subject for treatment of pathological conditions such as immune system diseases or cancer.
  • Introduction of the ligand in vivo is expected to result in interference with T cell/B cell interactions as a result of binding of the ligand to T cells.
  • the prevention of normal T cell/B cell contact may result in decreased T cell activity, for example, decreased T cell proliferation.
  • cytokines including, but not limited to, 24 interleukins, e.g. interleukin ("IL")-2, IL-3, IL-4, IL- 6, IL-8, growth factors including tumor growth factor (“TGF”) , colony stimulating factor (“CSF”) , interferons (“IFNs”), and tumor necrosis factor (“TNF”) to promote desired effects in a subject.
  • TGF tumor growth factor
  • CSF colony stimulating factor
  • IFNs interferons
  • TNF tumor necrosis factor
  • ligands for CD28 such as B7Ig fusion proteins and Fab fragments may thus be used in place of cytokines such as IL-2 for the treatment of cancers in vivo.
  • ligand for CD28 is introduced in vivo it is available to react with CD28 antigen positive T cells to mimic B cell contact resulting in increased production of cytokines which in turn will interact with B cells.
  • the effect of administration of the B7 antigen, its fragments or derivatives in vivo is stimulatory as a result of aggregation of the CD28 receptor.
  • the T cells are stimulated resulting in an increase in the level of T cell cytokines, mimicking the effects of T cell/B cell contact on triggering of the CD28 antigen on T cells.
  • inhibitory effects may result from blocking by the B7 antigen of the CD28 triggering resulting from T cell/B cell contact.
  • the B7 antigen may block T cell proliferation.
  • Introduction of the B7 antigen in vivo will thus produce effects on both T and B cell mediated immune responses.
  • the ligand may also be administered to a subject in combination with the introduction of cytokines or other therapeutic reagents.
  • cytokines such as B7 lymphomas, carcinomas, and T cell leukemias
  • ligands reactive with the B7 antigen such as anti-B7Ig monoclonal antibodies, may be used to inhibit the function of malignant B cells.
  • the ligand for CD28 of the invention may be useful for in vivo regulation of cytokine levels in response to the presence of infectious agents.
  • the ligand for CD28 may be used to increase antibacterial and antiviral resistance by stimulating tumor necrosis factor (TNF) and IFN production. TNF production seems to play a role in antibacterial resistance at early stages of infection
  • TNF may play a role in antiviral immunity.
  • Gamma interferon is also regulated by CD28 (Lindsten et al., supra) . Because mRNAs for alpha and beta IFNs share potential regulatory sequences in their 3' untranslated regions with cytokines regulated by CD28, levels of these cytokines may also be regulated by the ligand for CD28. Thus, the ligand for CD28 may be useful to treat viral diseases responsive to interferons (De Maeyer and De Maeyer-Guignard, in Interferons and Other Regulatory Cytokines, Wiley Publishers, New York (19i * 8)).
  • the ligand for CD28 may also be used to substitute for alpha-IFN for the treatment of cancers, such as hairy cell leukemia, melanoma and renal cell carcinoma (Goldstein and Laszio, CA: a Cancer Journal for Clinicians 38:258-277 (1988)), genital warts and Kaposi's sarcoma.
  • cancers such as hairy cell leukemia, melanoma and renal cell carcinoma (Goldstein and Laszio, CA: a Cancer Journal for Clinicians 38:258-277 (1988)), genital warts and Kaposi's sarcoma.
  • B7Ig fusion proteins as described above may be used to regulate T cell proliferation.
  • the soluble CD28Ig and B7Ig fusion proteins may be used to block T cell proliferation in graft versus host (GVH) disease which accompanies allogeneic bone marrow transplantation.
  • the CD28-mediated T cell proliferation pathway is cyclosporine-resistant, in contrast to proliferation driven by the CD3/Ti cell receptor complex (June et al., 1987, supra) . Cyclosporine is relatively ineffective as a treatment for GVH disease (Storb, Blood 68:119-125 (1986)).
  • GVH disease is thought to be mediated by T lymphocytes which express CD28 antigen (Storb and Thomas, Immunol.
  • B7 antigen in the form of B7Ig fusion protein, or in combination with immunosuppressants such as cyclosporine, for blocking T cell proliferation in GVH disease.
  • B7Ig fusion protein may be used to crosslink the CD28 receptor, for example by contacting T cells with immobilized B7Ig fusion protein, to assist in recovery of immune function after bone marrow transplantation by stimulating T cell proliferation.
  • the fusion proteins of the invention may be useful to regulate granulocyte macrophage colony stimulating factor (GM-CSF) levels for treatment of cancers (Brandt et al., N. Eng. J. Med. 318:869-876
  • GM-CSF granulocyte macrophage colony stimulating factor
  • Regulation of T cell interactions by the methods of the invention may thus be used to treat pathological conditions such as autoimmunity, transplantation, infectious diseases and neoplasia.
  • CD28-mediated adhesion in T cell and B cell function was investigated using procedures used to demonstrate intercellular adhesion mediated by MHC class I (Norment et al., (1988) supra) and class II (Doyle and Strominger, (1987) supra) molecules with the CD8 and CD4 accessory molecules, respectively.
  • the CD28 antigen was expressed to high levels in Chinese hamster ovary (CHO) cells and the transfected cells were used to develop a CD28- mediated cell adhesion assay, described infra. With this assay, an interaction between the CD28 antigen and its ligand expressed on activated B lymphocytes, the B7 antigen, was demonstrated.
  • the CD28 antigen expressed in CHO cells, was shown to mediate specific intracellular adhesion with human lymphoblastoid and leukemic B cell lines, and with activated murine B cells. CD28-mediated adhesion was not dependent upon divalent cations. A mAb, BB-1, reactive with B7 antigen was shown to inhibit CD28- mediated adhesion. Transfected COS cells expressing the B7 antigen were also shown to adhere to CD28 + CHO cells; this adhesion was blocked by mAbs to CD28 receptor and B7 antigen. The specific recognition by CD28 receptor of B7 antigen, indicated that B7 antigen is the ligand for the CD28 antigen.
  • results presented herein demonstrate that antibodies reactive with CD28 and B7 antigen specifically block helper T h -mediated immunoglobulin production by allogeneic B cells, providing evidence of the role of CD28/B7 interactions in the collaboration between T and B cells.
  • B7Ig and CD28Ig fusion proteins were constructed by fusing DNA encoding the extracellular domains of B7 antigen or the CD28 receptor to DNA encoding portions of human immunoglobulin C gamma 1. These fusion proteins were used to further demonstrate the interaction of the CD28 receptor and its ligand, the B7 antigen.
  • the cell adhesion assay method of the invention permits identification and isolation of ligands for target cell surface receptors mediating intercellular adhesion, particularly divalent cation independent adhesion.
  • the target receptor may be an antigen or other receptor on lymphocytes such as T or B cells, on monocytes, on microorganisms such as viruses, or on parasites.
  • the method is applicable for detection of ligand involved in ligand/receptor interactions where the affinity of the receptor for the ligand is low, such that interaction between soluble forms of the ligand and target receptor is difficult to detect.
  • adhesion interactions between other ligands and receptors that are divalent cation dependent may "mask" other interactions between ligands for target receptors, such that these interactions are only observed when divalent cations are removed from the system.
  • the cell adhesion assay utilizes cells expressing target cell surface receptor and cells to be tested for the presence of ligand mediating adhesion with the receptor.
  • the cells expressing target receptor may be cells that are transfected with the receptor of interest, such as Chinese hamster ovary (CHO) or COS cells.
  • the cells to be tested for the presence of ligand are labeled, for example with 51 Cr, using standard methods and are incubated in suitable medium containing a divalent cation chelating reagent such as ethylenediamine tetraacetic acid (EDTA) or ethyleneglycol tetraacetic acid (EGTA) .
  • EDTA ethylenediamine tetraacetic acid
  • EGTA ethyleneglycol tetraacetic acid
  • the assay may be performed in medium that is free of divalent cations, or is rendered free of divalent cations, using methods known in the art, for example using ion chromatography.
  • Use of a divalent cation chelating reagent or cation-free medium removes cation-dependent adhesion interactions permitting detection of divalent cation-independent adhesion interactions.
  • the labeled test cells are then contacted with the cells expressing target receptor and the number of labeled cells bound to the cells expressing receptor is determined by measuring the label, for example using a gamma counter.
  • a suitable control for specificity of adhesion can be used, such as a blocking antibody, which competes with the ligand for binding to the target receptor.
  • CD28 receptor antigen binds to a cell surface ligand
  • cells expressing the ligand should adhere more readily to cells expressing CD28 receptor than to cells which do not.
  • a cDNA clone encoding CD28 under control of a highly active promoter (Aruffo and Seed, (1987) supra) together with a selectable marker (pSV2dhfr) (Mulligan and Berg, Science 209:1414-1422 (1980)) was transfected into dihydrofolate reductase (dhfr)-deficient CHO cells.
  • CEM, Jurkat, HSB2, THP-1 and HL60 cells were cultured in complete RPMI medium (RPMI containing 10% fetal bovine serum (FBS) , 100 U/ml penicillin and 100 ⁇ g/ml streptomycin.
  • RPMI fetal bovine serum
  • penicillin 100 U/ml penicillin
  • streptomycin 100 ⁇ g/ml streptomycin.
  • Dhfr-deficient Chinese hamster ovary (CHO) cells Urlaub and Chasin, Proc. Natl. Acad Sci..
  • 77:4216-4220 (1980) were cultured in Maintenance Medium (Ham's F12 medium (GIBCO, Grand Island, NY) supplemented with 10% FBS, 0.15 mM L-proline, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin) .
  • Dhfr-positive transfectants were selected and cultured in Selective Medium (DMEM, supplemented with 10% FBS, 0.15 mM L-proline, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin) .
  • Spleen B cells were purified from Balb/c mice by treatment of total spleen cells with an anti-Thy 1.2 mAb (30H12) (Ledbetter and Herzenberg, Immunol. Rev. 47:361-389 (1979)) and baby rabbit complement.
  • the resulting preparations contained approximately 85% B cells, as judged by FACS R analysis following staining with fluorescein isothiothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin (TAGO) .
  • FITC fluorescein isothiothiocyanate
  • TAGO goat anti-mouse immunoglobulin
  • Monoclonal Antibodies Monoclonal antibody (mAb) 9.3 (anti-CD28) (ATCC No. HB 10271, Hansen et al.,
  • mAb 9.3 F(ab') 2 fragments were prepared as described by Parham, in J. Immunol. 131:2895- 2902 (1983). Briefly, purified mAb 9.3 was digested with pepsin at pH 4.1 for 75 min. followed by passage over protein A Sepharose to remove undigested mAb. A number of mAbs to B cell-associated antigens were screened for their abilities to inhibit CD28-mediated adhesion.
  • mAbs B43 (CD19) ; BL-40 (CD72); AD2, 1E9.28.1, and 7G2.2.11 (CD73) ; EBU-141, LN1 (CDW75) ; CRIS-1 (CD-76) ; 424/4A11, 424/3D9 (CD77) Leu 21, Ba, 1588, LO-panB-1, FN1, and FN4 (CDw78) ; and M9, G28-
  • cDNA clones encoding the amino acid sequences corresponding to T cell antigens CD4, CD5 and CD28 in the expression vector p ⁇ H3M (Aruffo and Seed (1987), supra) ) were provided by Drs. S. Aruffo and B. Seed, Massachusetts General Hospital, Boston, MA.
  • An expressible cDNA clone in the vector CDM8 encoding the amino acid sequence corresponding to B7 antigen was provided by Dr. Gordon Freeman (Dana Farber Cancer Institute, Boston, MA) .
  • Dhfr-deficient CHO cells were co-transfected with a mixture of 9 ⁇ g of plasmid ⁇ H3M-CD28 (Aruffo and Seed, (1987) supra) and 3 ⁇ g of plasmid pSV2dhfr (Mulligan and Berg, (1980) , supra) using the calcium phosphate technique (Graham and Van Der Eb, Virology 52:456-467 (1973)). Dhfr-positive colonies were isolated and grown in Selective Medium containing increasing amounts of methotrexate (Sigma Chemical Co., St. Louis, MO) .
  • COS cells were transfected with B7, CD4 or CD5 cDNAs as described by Malik et al.. Molecular and Cellular Biology 9:2847-2853 (1989). Forty-eight to seventy-two hours after transfection, cells were collected by incubation in PBS containing 10 mM EDTA, and used for flow cytometry analysis or in CD28-mediated adhesion assays as described, infra.
  • CD28 " levels of the CD28 receptor were isolated from amplified populations by FACS R sorting following indirect immunostaining with mAb 9.3. After two rounds of FACS R selection, the CD28 + population stained unifoirmly positive with FITC-conjugated mAb 9.3 (mean channel, 116 in linear fluorescence units) , while the CD28 " population stained no brighter (mean channel, 3.9) than unstained cells (mean channel, 3.7). Staining by CD28 + CHO cells was approximately ten-fold brighter than phytohemagglutin- stimulated T cells (mean channel, 11.3). The CD28 + and
  • CD28 populations stably maintained their phenotypes after more than 6 months of continuous culture in Selective Medium containing 1 ⁇ M of methotrexate.
  • CD28-Mediated Adhesion Assay Cells to be tested for adhesion were labeled with 51 Cr (0.2-1 mCi) to specific activities of 0.2-2 cpm/cell.
  • Labeled and washed cells were preincubated in complete RPMI containing 10 ⁇ g/ml of mAb Wl, and unless otherwise indicated, 10 mM EDTA.
  • mAb 9.3 or mAb 9.3 F(ab') 2 was added to some samples at 10 ⁇ g/ml, for approximately 1 hr at 23°C.
  • T51 lymphoblastoid cells were found to adhere more to CD28 + CHO cells, than to CD28 " CHO cells. Furthermore, adhesion of T51 cells to CD28 + CHO cells was partially blocked by mAb 9.3, while adhesion to CD28 " CHO cells was not consistently affected. Adhesion was not affected by control mAb L6 (ATCC No. HB 8677, Hellstrom et al., Cancer Res. 46:3917-3923 (1986)), which is of the same isotype as mAb 9.3 (IgG2a) . These experiments suggested that T51 cells adhered specifically to CD28 + CHO cells. Since blocking of adhesion by mAb 9.3 was incomplete, ways to increase the specificity of the CD28 adhesion assay were explored.
  • CD28-mediated adhesion was greatly increased in the presence of EDTA (Figure 1) .
  • Adhesion to CD28 + cells in the presence of EDTA was 17- fold greater than to CD28 " cells in the presence of EDTA, compared with 5.5-fold greater in its absence.
  • Adhesion to CD28 + cells in the presence of mAb 9.3 plus EDTA was reduced by 93%, compared with 62% in the presence of mAb alone.
  • CD28-mediated adhesion of T51 cells in the presence of EDTA could also be seen quite clearly by microscopic examination following immunohistological staining of T51 cells.
  • the Ligand For CD28 is a B Cell Activation Marker
  • CD28-mediated adhesion in EDTA made it possible to more readily detect adhesion by cells other than T51.
  • additional cell lines were tested, including three lymphoblastoid lines (T51, 1A2, and 5E1) ; four Burkett's Lymphoma lines (Daudi, Raji, Jijoye, and Namalwa) ; one acute lymphoblastic (B cell) leukemia (REH) ; three T cell leukemias (CEM, Jurkat and HSB2) ; and two monocytic leukemias (THP-1 and HL60) .
  • B cell acute lymphoblastic leukemia
  • CEM T cell leukemias
  • CEM T cell leukemias
  • HSB2 T cell leukemias
  • THP-1 and HL60 monocytic leukemias
  • CD28-specific adhesion i.e., adhesion being greater than 70% inhibitable by mAb 9.3
  • T51, 5E1, Raji, and Jijoye cells were observed with T51, 5E1, Raji, and Jijoye cells. Daudi cells also showed specific adhesion, although to a lesser extent. Other cell lines did not show specific CD28-mediated adhesion, although some (e.g., Namalwa) showed relatively high non-specific adhesion.
  • Primary mouse splenic B cells did not show CD28-mediated adhesion, but acquired the ability to adhere following activation with LPS.
  • lymphoblastoid lines showed CD28-mediated adhesion, while the U937 cell line, unstimulated human tonsil B cells, and phytohemagglutinin stimulated T cells did not show adhesion. These experiments indicate that a ligand for CD28 is found on the cell surface of activated B cells of human or mouse origin.
  • CD28-Mediated Adhesion is Specifically Blocked by a mAb fBB-1) to B7 Antigen
  • lymphoblastoid cell lines having mutations in other known cellular adhesion molecules were measured using the adhesion assay described above.
  • the 616 lymphoblastoid line (MHC class II-deficient) (Gladstone and Pious, Nature 271:459-461 (1978)) bound to CD28 + CHO cells equally well or better than parental T51 cells.
  • a CD18-deficient cell line derived from a patient with leukocyte adhesion deficiency (Gambaro cells) (Beatty et al.. Lancet 1:535-537 (1984)) also adhered specifically to CD28.
  • MHC class II and CD18 molecules do not mediate adhesion to CD28.
  • a panel of mAbs to B cell surface antigens were then tested for their ability to inhibit CD28-mediated adhesion of T51 cells.
  • a total of 57 mAbs reactive with T51 cells were tested, including mAbs to the B cell-associated antigens CD19, CD20, CD21, CD22, CD23, CD37, CD39, CD40, CD71, CD72, CD73, CDW75, CD76, CD77, CDw78, IgM, and IgD; other non-lineage- restricted antigens CD18, CD32, CD45, CD54, and CD71; CD44 and another integrin; MHC class I and class II antigens; and 30 unclustered B cell associated antigens.
  • mAb 9.3 was most effective at blocking, but mAb BB-1 was able to block approximately 60% of adhesion at concentrations less than 1 ⁇ g/ml. mAb HIDE also partially blocked adhesion at all concentrations tested. When EDTA was omitted from the adhesion assay, blocking by class I mAbs was consistently less, and required higher mAb concentrations, than mAbs 9.3 or BB-1.
  • AML Acute Monocytic leukemia
  • adhesion was not blocked by non-reactive, isotype matched controls, mAb W5 (IgM) (Linsley, (1986) supra) and mAb L6 (IgG2A) (Hellstrom et al. , (1986) supra) , or by mAb HIDE, which reacts with class I antigens on COS cells.
  • CD28-mediated adhesion by B7 transfected cells could also be clearly seen by microscopic examination of the CHO cell monolayers after the assay. When COS cells were transfected with expressible CD4 or CD5 cDNA clones, no CD28-mediated adhesion was detected. Expression of CD4 and CD5 was confirmed by FACS R analysis following immunofluorescent staining.
  • the above assay for intracellular adhesion mediated by the CD28 receptor demonstrated CD28-mediated adhesion by several lymphoblastoid and leukemic B cell lines, and by primary murine spleen cells following activation with LPS. These results indicate the presence of a natural ligand for the CD28 receptor on the cell surface of some activated B lymphocytes.
  • CD28 Aruffo and Seed, (1987) supra
  • B7 Freeman et al., (1989) supra
  • their interaction represents another example of heterophilic recognition between members of this gene family (Williams and Barclay (1988) , supra) .
  • CD28-mediated adhesion differs in several respects from other cell adhesion systems as shown in the above results.
  • CD28-mediated adhesion was not blocked by mAbs to other adhesion molecules, including mAbs to ICAM- 1 (LB-2), MHC Class II (HBlOa) CD18 (60.3), CD44 (HERMES- 1 homing receptor), and an integrin (P3E3, P4C2, P4G9) .
  • CD28-mediated adhesion was also resistant to EDTA and EGTA, indicating that this system does not require divalent cations, in contrast to integrins (Kishimoto et al.. Adv. Immunol.
  • Non-CD28 mediated adhesion systems may also be responsible for the incomplete blockage by mAb BB-1 of B cell adhesion ( Figure 4) . That this mAb is more effective at blocking adhesion by transfected COS cells ( Figure 5) may indicate that non-CD28 mediated systems are less effective in COS cells.
  • CD28-mediated adhesion appears more restricted in its cellular distribution to T and B cells as compared to other adhesion molecules.
  • CD28 receptor is primarily expressed by cells of the T lymphocyte lineage.
  • the B7 antigen is primarily expressed by cells of the B lymphocyte lineage. Consistent with this distribution, the ligand for CD28 was only detected on cells of B lymphocyte lineage.
  • CD28 expression has been detected on plasma cells (Kozbor et al. , J. Immunol 138:4128-4132 (1987)) and B7 on cells of other lineages, such as monocytes (Freeman et al., (1989) supra) .
  • CD28 and B7 antigen are known to mediate T cell-B cell interactions during an immune response and the levels of several of these, including CD28 and B7 antigen, have been reported to increase following activation. Increased levels of these molecules may help explain why activated B cells are more effective at stimulating antigen-specific T cell proliferation than are resting B cells. Because the B7 antigen is not expressed on resting B cells, CD28-mediated adhesion may play a role in maintaining or amplifying the immune response, rather than initiating it. Such a role is also consistent with the function of CD28 in regulating lymphokine and cytokine levels (Thompson et al. , (1989), supra; and Lindsten et al., (1989), supra) . EXAMPLE 2 Characterization of Interaction Between CD28 Receptor and B7 Antigen
  • This example used alloantigen-driven maturation of B cells as a model system to demonstrate the involvement of the CD28 receptor on the surface of major histocompatibility complex (MHC) class II antigen- reactive CD4 positive T helper (T h ) cells and antigen presenting B cells during the T h -B cell cognate interaction leading to B cell differentiation into immunoglobulin-secreting cells (IgSC) .
  • MHC major histocompatibility complex
  • T h antigen- reactive CD4 positive T helper
  • IgSC immunoglobulin-secreting cells
  • CM Complete culture medium
  • RPMI 1640 Irvine Scientific, Santa Ana, CA
  • penicillin G 100 ⁇ g/ml of penicillin G
  • streptomycin 100 ⁇ g/ml of streptomycin
  • 2 mM L-glutamine 5 X 10 *5 M 2-ME
  • 10% FBS 10% FBS
  • EBV-transformed B cell lines CESS HLA-AS1, A3; B5, B17; DR7) , JIJOYE, and SKW6.4 (HLA-Ala; B27, B51; DR7), were obtained from the ATCC.
  • EBV- transformed B cell lines ARENT HLA-A2; B38, B39, DRw6
  • MSAB HLA-A1, A2; B57; DR7 were provided by Dr. E. G. Engleman, Stanford University School of Medicine, Stanford, CA.
  • Hybridomas 0KT4 (IgG anti-CD4) , OKT8 (IgG anti-CD8) and HNK1 (IgM anti-CD57) were obtained from the ATCC and ascitic fluids from these hybridomas were generated in pristane-primed BALB/c mice.
  • Production and characterization of anti-CD28 mAb 9.3 (IgG2a) has been described by Ledbetter et al., J. Immunol. 135:2331 (1985); Hara et al., J. EXP. Med. 161:1513 (1985) and Martin et al. , J. Immunol. 136:3282 (1986), incorporated by reference herein.
  • mAb 4H9 (IgG2a anti-CD7) as described by Damle and Doyle, J. Immunol 143:1761 (1989), incorporated by reference herein, was provided by Dr.
  • PBMC Peripheral blood mononuclear cells
  • T cells Proliferative responses of T cells.
  • fifty-thousand CD4 + CD45R0 + T cells were stimulated by culturing with 1 x 10 4 irradiated (8000 rad from a 137 Cs source) EBV-transformed allogenic B cells (or 2.5 X 10 4 non-T cells) in 0.2 ml of CM in round-bottom microtiter wells in a humidified 5% C0 2 and 95% air atmosphere in the presence of 10 ⁇ g/ml of mAb reactive with CD7, CD28, CD57 or B7 antigen.
  • CD4 + CD45R0 + T cells also were also independently stimulated with 100 ⁇ g/ml of soluble purified protein derivative of tuberculin (PPD, Connough Laboratories, Willowdale, Ontario, Canada) in the presence of 1 X 10 4 irradiated (3000 rad) autologous non-T cells in the presence of the above mAbs.
  • EBV- transformed B cell lines were used as stimulator cells in these experiments because these B cells exhibit various features of activated B cells such as the expression of high levels of MHC class II and B7 molecules (Freeman et al., J. Immunol. 139:3260 (1987); and Yokochi et al., Ji. Immunol. 128:823 (1981)).
  • Figure 6 shows the results of these experiments.
  • the addition of anti-B7 mAb (BB1; IgM) but not that of isotype-matched anti-CD57 HNK1; IgM) resulted in the inhibition of T cell proliferation.
  • the inhibitory effects of anti-CD28 mAb 9.3 on the MLR responses of T cells are consistent with previous observations reported by Damle et al. , J. Immunol.
  • DR7-primed CD4* T h cells were derived from the allogeneic MLC consisting of responder CD4 + CD45RO + T cells (HLA-A26, A29; B7, B55; DR9, DR10) and irradiated MSAB (DR7 + ) B cells as stimulator cells as described by Damle et al. , J. Immunol. 133:1235 (1984), incorporated by reference herein.
  • the isolation of resting CD4 + CD45R0 + T cells and that of DR7-primed CD4 + CD45RO* T lymphoblasts using discontinuous Percoll density gradient centrifugation was also as described by Damle, supra (1984) .
  • DR7- primed CD4 + T h cells were continuously propagated in the presence of irradiated MSAB B cells and 50 U/ml of IL-2. Prior to their functional analysis, viable DR7-primed T h cells were isolated by Ficoll-Hypaque gradient centrifugation and maintained overnight in CM without DR7 + feeder cells or IL-2, after which immunoglobulin secreted in the cell-free supernatant (SN) was quantitated using a solid-phase ELISA.
  • SN cell-free supernatant
  • IgM-producing SKW or IgG-producing CESS were cultured with varying numbers of DR7-primed CD4 + CD45RO + T h cells for 96 h after which cell-free SN from these cultures were collected and assayed for the quantitation of IgM (SKW cultures) or IgG (CESS cultures) using solid-phase ELISA.
  • Exogenous IL-6 (1-100 U/ml) induced Ig production by these B cells was also used as a positive control to monitor the non-cognate Ig production by these B cell lines.
  • Ig production by freshly isolated resting CD4 + CD45RO + T h cells autologous to the DRt-primed CD4 + T h cells was also simultaneously examined as a control for DR7-primed CD4 + T h cells.
  • Ig guantitation IgG or IgM in culture SN were measured using solid-phase ELISA as described by Volkman et al., Proc. Natl. Acad. Sci. USA 78:2528 (1981), incorporated by reference herein. Briefly, 96-well flat- bottom miorotiter ELISA plates (Corning, Corning NY) were coated with 200 ⁇ l/well of sodium carbonate buffer (pH 9.6) containing 10 ⁇ g/ml of affinity-purified goat anti- human Ig or IgM Ab (Tago, Burlingame, CA) incubated overnight at 4° C, and then washed with PBS and wells were further blocked with 2% BSA in PBS (BSA-PBS) .
  • BSA BSA in PBS
  • HRP horseradish peroxidase
  • F(ab') 2 fraction of affinity-purified goat anti-human IgG or IgM Ab Tago
  • the plates were then washed, and 100 ⁇ l/well of o-phenylenediamine (Sigma, St. Louis, MO) solution (0.6 mg/ml in citrate-phosphate buffer with pH
  • Figure 7 shows the Ig production by either B cell line as a function of the concentration of DR7- primed T h with optimal Ig production induced at either 1:1 or 1:2 T h :B ratios. At T h :B ratios higher than 1:1 inhibition of Ig production was observed. Hence, all further experiments were carried out using a T h :B ratio of 1:2. As shown in Figure 7, these unprimed resting CD4 + T h cells slightly induced IgM production by SKW B cells but has no effect on the IgG production by CESS B cells in 4- day cultures. This slight helper effect observed with unprimed CD4 + CD45RO + population during the Ig induction cultures.
  • CD28 and B7 during cognate T h :B- induced Ig production were further examined using anti- CD28 and anti-B7 mAbs.
  • Both CESS and SKW B cells constitutively express B7 antigen on their surface and thus, represent a source of uniformly activated B cell populations for use in T h -B cognate interactions or in cytokine-driven non-cognate maturation.
  • DR7 + B cells CESS or SKW
  • Ig production (IgM, Figure 8a and IgG, Figure 8b) at the end of 3-day cultures was quantitated in cell-free SN.
  • Figure 8 shows that both anti-CD28 and anti-B7 mAbs but not their isotype-matched mAb controls (anti-CD7 and anti-CD57, respectively) inhibited T h induced Ig production by B cells in a does-dependent manner.
  • anti-CD28 mAb-mediated inhibition of Ig production was stronger than that by anti-B7 mAb.
  • Interaction between CD28 receptor and B7 antigen may influence the production of cytokines and thus B cell differentiation.
  • Ligation of CD28 by B7 during T h :B collaboration may facilitate sustained synthesis and delivery of cytokines for their utilization during the differentiation of B cells into immunoglobulin secreting cells.
  • the lack of inhibition by anti-CD28 and anti-B7 mAbs of cell dependent differentiation of CESS or SKW B cells induced with exogenous IL-4 or IL-6 suggests that CD28:B7 interaction controls either production of these cytokines, or their targeted delivery to B cells, or both of these events.
  • CD28 and B7 are most likely not restricted to T h :B cell interactions, and applies more generally to other antigen-presenting cells such as monocyte/M ⁇ , dendritic cells, and epidermal Langerhans cells.
  • Ligation of a nominal antigen presented in conjunction with MHC class II molecules on the surface of antigen-presenting cells by the TcR/CD3 complex on the surface of T h cells may lead to elevated expression of B7 antigen by these cells, which, via the interaction with CD28, then facilitates the production of various cytokines by T h . This in turn drives both growth and differentiation of both T h and B cells.
  • fusion proteins of B7 and CD28 with human immunoglobulin C gamma 1 (human Ig C ⁇ l) chains were constructed and expressed and used to measure the specificity and apparent affinity of interaction between these molecules.
  • Purified B7Ig fusion protein, and CHO cells transfected with B7 antigen were used to investigate the functional effects of this interaction on T cell activation and cytokine production.
  • B7Ig and CD28Ig fusion proteins were prepared as follows. DNA encoding the amino acid sequence corresponding to the extracellular domain of the respective protein (B7 and CD28) was joined to DNA encoding the amino acid sequences corresponding to the hinge, CH2 and CH3 regions of human immunoglobulin C ⁇ l. This was accomplished as follows.
  • Expression plasmids were used containing cDNA encoding the amino acid sequence corresponding to CD28 (pCD28) as described by Aruffo and Seed, Proc. Natl. Acad. Sci. USA 84:8573 (1987), incorporated by reference, and provided by Drs. Aruffo and Seed, Mass General Hospital, Boston, MA.
  • OMCD28 and 0MB7 constructs were made (OMCD28 and 0MB7) in which stop codons were introduced upstream of the transmembrane domains and the native signal peptides were replaced with the signal peptide from oncostatin M (Malik et al., Mol. Cell Biol. 9:2847 (1989)). These were made using synthetic oligonucleotides for reconstruction (OMCD28) or as primers (OMB7) for PCR.
  • OMCD28 synthetic oligonucleotides for reconstruction
  • OMB7Ig fusion constructs were made in two parts. The 5' portions were made using OMCD28 and OMB7 as templates and the oligonucleotide,
  • CTAGCCACTGAAGCTTCACCATGGGTGTACTGCTCACAC (SEQ ID N0:1) (corresponding to the oncostatin M signal peptide) as a forward primer, and either TGGCATGGGCTCCTGATCAGGCTTAGAAGGTCCGGGAAA (SEQ ID NO:2), or, TTTGGGCTCCTGATCAGGAAAATGCTCTTGCTTGGTTGT (SEQ ID NO:3) as reverse primers, respectively.
  • Products of the PCR reactions were cleaved with restriction endonucleases (Hind III and Bell) as sites introduced in the PCR primers and gel purified.
  • the 3' portion of the fusion constructs corresponding to human Ig C ⁇ l sequences was made by a coupled reverse transcriptase (from Avian myeloblastosis virus; Life Sciences Associates, Bayport, NY)-PCR reaction using RNA from a myeloma cell line producing human-mouse chimeric mAb L6 (provided by Dr. P. Fell and M. Gayle, Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA) as template.
  • the oligonucleotide, AAGCAAGAGCATTTTCCTGATCA GGAGCCCAAATCTTCTGACAAAACTCACACATCCCCACCGTCCCCAGCACCTGAACT CCTG (SEQ ID NO:4), was used as forward primer, and CTTCGACCAGTCTAGAAGCATCCTCGTGCGACCGCGAGAGC (SEQ ID NO:5) as reverse primer. Reaction products were cleaved with Bell and Xbal and gel purified. Final constructs were assembled by ligating Hindlll/Bcll cleaved fragments containing CD28 or B7 sequences together with Bcll/Xbal cleaved fragment containing Ig C ⁇ l sequences into Hindlll/Xbal cleaved CDM8.
  • Ligation products were transformed into MC1061/p3 E. coli cells and colonies were screened for the appropriate plasmids. Sequences of the resulting constructs were confirmed by DNA sequencing.
  • the DNA used in the B7 construct encodes amino acids from about position 1 to about position 215 of the sequence corresponding to the extracellular domain of the B7 antigen, and for CD28, the DNA encoding amino acids from about position 1 to about position 134 of the sequence corresponding to the extracellular domain of the CD28 receptor.
  • CD5Ig was constructed in identical fashion, USing ( ⁇ TTGCACAGTCAAGCTTCCATGCCCATGGGTTCTCTGGCCACCTTG (SEQ ID NO:6), as forward primer and
  • ATCCACAGTGCAGTGATCATTTGGATCCTGGCATGTGAC (SEQ ID NO:7) as reverse primer.
  • the PCR product was restriction endonuclease digested and ligated with the Ig C ⁇ l fragment as described above.
  • the resulting construct (CD5Ig) encodes an amino acid sequence containing residues from about position 1 to about position 347 of CD5, two amino acids introduced by the construction procedure (amino acids DQ) , followed by the Ig C ⁇ l hinge region.
  • cDNA constructs were made encoding molecules truncated at the NH 2 -terminal side of their transmembrane domains.
  • the native signal peptides were replaced with the signal peptide from oncostatin M (Malik, supra. 1989) , which mediates efficient release of secreted proteins in transient expression assays.
  • the cDNAs were cloned into an expression vector, transfected into COS cells, and spent culture medium was tested for secreted forms of B7 and CD28. In this fashion, several soluble forms of B7 were produced, but in repeated attempts, soluble CD28 molecules were not detected.
  • the next step was to construct receptor Ig C ⁇ l fusion proteins.
  • the Ig hinge disulfides were mutated to serine residues to abolish intrachain disulfide bonding.
  • the resulting fusion proteins were produced in COS cells and purified by affinity chromatography on immobilized protein A as described below. Yields of purified protein were typically 1.5-4.5 mg/liter of spent culture medium.
  • PCR Polymerase Chain Reaction
  • COS monkey kidney cells
  • COS monkey kidney cells
  • seed and Aruffo Proc. Natl. Acad. Sci. 84:3365 (1987)
  • Plasmid DNA was added (approximately 15 ⁇ g/dish) in a volume of 5 ml of serum-free DMEM containing 0.1 mM cloroquine and 600 ⁇ g/ml DEAE Dextran, and cells were incubated for 3-3.5 h at 37°C.
  • Transfected cells were then briefly treated (approximately 2 min) with 10% dimethyl sulfoxide in PBS and incubated at 37°C for 16-24 h in DMEM containing 10% FCS. At 24 h after transfection, culture medium was removed and replaced with serum-free DMEM (6 ml/dish) . Incubation was continued for 3 days at 37°C, at which time the spent medium was collected and fresh serum-free medium was added. After an additional 3 days at 37°C, the spent medium was again collected and cells were discarded.
  • CHO cells expressing CD28, CD5 or B7 were isolated as described by Linsley et al., (1991) supra. as follows: Briefly, stable transfectants expressing CD28, CD5, or B7, were isolated following cotransfection of dihydrofolate reductase-deficient Chinese hamster ovary (dhfr " CHO) cells with a mixture of the appropriate expression plasmid and the selectable marker, pSV2dhfr, as described above in Example 1. Transfectants were then grown in increasing concentrations of methotrexate to a final level of 1 ⁇ M and were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 0.2 mM proline and 1 ⁇ M methotrexate.
  • FBS fetal bovine serum
  • CHO lines expressing high levels of CD28 (CD28 + CHO) or B7 (B7 + CHO) were isolated by multiple rounds of fluorescence-activated cell sorting (FACS R ) following indirect im unostaining with mAbs 9.3 or BB-1.
  • FACS R fluorescence-activated cell sorting
  • Amplified CHO cells negative for surface expression of CD28 or B7 (dhfr* CHO) were also isolated by FACS R from CD28-transfected populations.
  • Transfected CHO cells or activated T cells were analyzed by indirect immunostaining. Before staining, CHO cells were removed from their culture vessels by incubation in PBS containing 10 mM EDTA.
  • FITC-conjugated second step reagent goat anti-mouse Ig serum for murine mAbs, or goat anti-human Ig C ⁇ serum for fusion proteins (Tago, Inc. , Burlingame, CA) . Fluorescence was analyzed on 10,000 stained cells using a FACS IV" cell sorter (Becton Dickinson and Co. , Mountain View, CA) equipped with a four decade logarithmic amplifier.
  • the first, second and third collections of spent serum-free culture media from transfected COS cells were used as sources for the purification of Ig fusion proteins.
  • medium was applied to a column (approximately 200-400 ml medium/ml packed bed volume) of immobilized protein A (Repligen Corp., Cambridge, MA) equilibrated with 0.05 M sodium citrate, pH 8.0.
  • immobilized protein A Repligen Corp., Cambridge, MA
  • the B7Ig fusion protein migrated during SDS-PAGE under nonreducing conditions predominantly as a single species of M p 70,000, with a small amount of material migrating as a M r approximately 150,000 species. After reduction, a single M r approximately 75,000 species was observed.
  • CD28Ig migrated as a Mr approximately 140,000 species under non- reducing conditions and a M r approximately 70,000 species after reduction, indicating that it was expressed as a homodimer. Since the Ig C ⁇ l hinge cysteines had been mutated, disulfide linkage probably involved cysteine residues which naturally form interchain bonds in the CD28 homodimer (Hansen et al., Immunogenetics 10:247 (1980)) .
  • Binding of B7Ig and CD28Ig to CHO cells Binding of CD28Ig and B7Ig fusion proteins was detected by addition of FITC-conjugated goat anti-human Ig second step reagent as described above. B7Ig was bound by CD28 + CHO, while CD28Ig was bound by B7 + CHO. B7Ig also bound weakly to B7 + CHO ( Figure 11) , suggesting that this molecule has a tendency to form homophilic interactions. No binding was detected of chimeric mAb L6 containing human Ig C ⁇ l, or another fusion protein, CD5Ig. Thus B7Ig and CD28Ig retain binding activity for their respective counter- receptors.
  • B7lg was either iodinated or metabolically labeled with [ 35 S]methionine, and radiolabeled derivatives were tested for binding to immobilized CD28Ig or to CD28 + CHO cells.
  • B7Ig Radiolabeling of B7Ig.
  • Purified B7Ig 25 ⁇ g in a volume of 0.25 ml of 0.12 M sodium phosphate, pH 6.8 was iodinated using 2 mCi 125 I and 10 ⁇ g of chloramin T. After 5 min at 23°C, the reaction was stopped by the addition of 20 ⁇ g sodium metabisulfite, followed by 3 mg of KI and 1 mg of BSA. Iodinated protein was separated from untreated 125 I by chromatography on a 5-ml column of Sephadex G-10 equilibrated with PBS containing 10% FCS. Peak fractions were collected and pooled. The specific activity of 125 I-B7Ig labeled in this fashion was 1.5 x 10 6 cpm/pmol.
  • B7Ig was also metabolically labeled with [ 35 S]methionine.
  • COS cells were transfected with a plasmid encoding B7Ig as described above.
  • [ 35 S]methionine ⁇ 800 Ci/mmol; Amersham 58
  • Binding Assays For assays using immobilized CD28Ig, 96-well plastic dishes were coated for 16-24 h with a solution containing CD28Ig (0.5 ⁇ g in a volume of 0.05 ml of 10 mM Tris, pH 8). Wells were then blocked with binding buffer (DMEM containing 50 mM BES, pH 6.8, 0.1% BSA, and 10% FCS) (Sigma Chemical Co., St.
  • Radioactivity was then solubilized by addition of 0.5 N NaOH, and quantified by liquid scintillation or gamma counting.
  • radioactivity is expressed as a percentage of radioactivity bound to wells treated without competitor (7,800 cpm). Each point represents the mean of duplicate determinations; replicates generally vaxied from the mean by ⁇ 20%. Concentrations were calculated based on a M r of 75,000 per binding site for mAbs and 51,000 per binding site for B7Ig. When binding of 125 I-B7 to CD28* CHO cells was measured, cells were seeded (2.5 x 10 4 /well) in 96-well plates 16-24 h before the start of the experiment. Binding was otherwise measured as described above.
  • B7Ig bound to immobilized CD28Ig, and to CD28 + CHO cells, it was not known whether B7Ig could bind to CD28 naturally expressed on T cells. This is an important consideration since the level of CD28 on transfected cells was approximately 10-fold higher than that found on PHA-activated T cells as shown above in Example 1. PHA-activated T cells were prepared as follows.
  • PBL were isolated by centrifugation through Lymphocyte Separation Medium (Litton Bionetics, Kensington, MD) and cultured in 96- well, flat-bottomed plates (4 x 10 4 cells/well, in a volume of 0.2 ml) in RPMI containing 10% FCS. Cellular proliferation of quadruplicate cultures was measured by upt ⁇ e of C 2 ⁇ ]thymidine during the last 5 h of a 3 day (d) culture.
  • PHA-activated T cells were prepared by culturing PBL with 1 ⁇ g/ml PHA (Wellcome) for 5 d, and 1 d in medium lacking PHA. Viable cells were collected by sedimentation through Lymphocyte Separation Medium before use.
  • PHA-activated T cells were then tested for binding of B7Ig (10 ⁇ g/ml) by FACS R analysis after indirect immunofluorescence as described above. Where indicated ( Figure 14), mAbs 9.3 or BB-1 were also added at 10 ⁇ g/ml to cells simultaneously with B7Ig. Bound mAb was detected with a FITC-conjugated goat anti-human Ig C ⁇ l reagent.
  • B7Ig-binding proteins was also determined by immunoprecipitation analysis of 125 I-surface labeled cells as follows.
  • PHA-activated T cells were cell-surface labeled with 125 I using lactoperoxidase and H 2 0 2 as described by Vitetta et al., J. Exp. Med. 134:242 (1971), incorporated by reference herein. Aliquots of a nonionic detergent extract of labeled cells (approximately 3 X 10 8 cpm in a volume of 0.12 ml) were prepared as described by Linsley et al., J. Biol. Chem.
  • CD28 is the major receptor for B7Ig on PHA-activated T cells.
  • mAbs to CD28 have potent biological activities on T cells, suggesting that interaction of CD28 with its natural ligand(s) may also have important functional consequences.
  • B7Ig could block the CD28-mediated adhesion assay described above.
  • the adhesion of 51 Cr- labeled PM lymphoblastoid cells to monolayers of CD28 + CHO cells was measured as described above, in the presence of the indicated amounts of mAb 9.3 or B7Ig.
  • Data are expressed in Figure 16 as a percentage of cells bound in the absence of competitor (40,000 cpm or approximately 1.1 X 10 5 cells) . Each point represents the mean of triplicate determinations; coefficients of variation were ⁇ 25%.
  • B7Ig blocked CD28- mediated adhesion somewhat less effectively than mAb 9.3 (half-maximal inhibition at 200 nM as compared with 10 nM for mAb 9.3).
  • the relative effectiveness of these molecules at inhibiting CD28-mediated adhesion was similar to their relative binding affinities in competition binding experiments ( Figure 12) .
  • CD28Ig failed to inhibit CD28-mediated adhesion at concentrations of up to 950 nM, suggesting that much higher levels of CD28Ig were required to compete with the high local concentrations of CD28 present on transfected cells.
  • B7 + CHO and control dhfr* CHO cells were irradiated with 1,000 rad before mixing with PBL at a 4:1 ratio of PBL/CHO cells. After culture for 3 d, proliferation was measured by uptake of l ⁇ ] thymidine for 5 h. Values shown are means of determinations from quadruplicate cultures (SEM ⁇ 15%) .
  • B7Ig in solution at concentrations of 1-10 ⁇ g/ml showed only a modest enhancement of proliferation even though the anti-CD28 mAb 9.3 was effective. Because CD28 crosslinking has been identified as an important determinant of CD28 signal transduction (Ledbetter et al.. Blood 75:1531 (1990)), B7Ig was also compared to 9.3 when immobilized on plastic wells (Table 2, Exp. 1).
  • B7Ig was able to enhance proliferation and compared favorably with mAb 9.3.
  • B7* CHO cells also were tested and compared with control dhfr * CHO cells for costimulatory activity on resting lymphocytes (Table 2, Exp. 2).
  • proliferation was seen with dhfr* CHO cells in the absence of anti-CD3 mAb because of residual incorporation of [ 3 H]thymidine after irradiation of these cells.
  • the stimulation by dhfr* cells was not enhanced by anti-CD3 mAb and was not observed in other experiments (Tables 3 and 4) where transfected CHO cells were added at lower ratios.
  • B7* CHO cells were very effective at costimulation with anti-CD3 mAb, indicating that cell surface B7 had similar activity in this assay as the anti-CD28 mAbs.
  • B7* CHO cells were also tested as to whether they could directly stimulate proliferation of resting PHA blasts which respond directly to CD28 crosslinking by mAb 9.3. Again, the B7* CHO cells were very potent in stimulating proliferation (Table 3) and were able to do so at very low cell numbers (PHA blast:B7* CHO ratios of >800:1). The control CD5* CHO cells did not possess a similar activity. (In a number of different experiments neither dhfr CHO, CD5* CHO, nor CD7* CHO cells stimulated T cell proliferation. These were therefore used interchangeably as negative controls for effects induced by B7* CHO cells.
  • B7* CHO The stimulatory activity of B7* CHO was further shown to result from CD28/B7 interaction, since mAb BBl inhibited stimulation by the B7* CHO cells without affecting background proliferation in the presence of CD7* CHO cells (Table 4).
  • mAb LB-1 (Yokochi et al., supra) . an IgM mAb to a different B cell antigen, did not inhibit proliferation.
  • mAb 9.3 (Fab fragments) inhibited proliferation induced by B7* CHO and as well as background proliferation seen with CD7* CHO cells.
  • PHA blasts (5 X 10 7 ) were mixed with transfected CHO cells at a ratio of 40:1 T cells/CHO cells, and/or mAbs as indicated in Figure 17.
  • mAb 9.3 was used at 10 ⁇ g/ml.
  • mAb BB-1 was added at 20 ⁇ g/ml 1 h before addition of B7* CHO cells.
  • mAb 9.3 was crosslinked, goat anti-mouse Ig (40 ⁇ g/ml) was added 10 min after addition of mAb 9.3.
  • Cells were incubated for 6 h at 37°C and RNA was isolated and subjected to RNA blot analysis using 32 P-labeled IL-2 or GAPDH probes as described below.
  • RNA was prepared from stimulated PHA blasts by the procedure described by Chomczynki and Sacchi, Anal.
  • RNA samples Equal loading of RNA samples was verified both by rRNA staining and by hybridization with a rat glyceraldehyde-6-phosphate dehydrogenase probe (GAPDH, an approximately 1.2-kb cDNA fragment provided by Dr. A Purchio, Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA) .
  • GPDH rat glyceraldehyde-6-phosphate dehydrogenase probe
  • B7* CHO cells but not CD7* CHO cells, induced accumulation of IL-2 mRNA transcripts. Induction by B7* CHO cells was partially blocked by mAb BB-1. Induction by B7* CHO cells was slightly better than achieved by mAb 9.3 in solution, but less effective than mAb 9.3 after crosslinking with goat anti-mouse Ig. Thus, triggering of CD28 by cell surface B7 on apposing cells stimulated IL-2 mRNA accumulation.
  • the apparent K d value for the interaction of soluble Ig C ⁇ fusions of CD28 and B7 approximately 200 nM) is within the range of affinities observed for mAbs (2-10,000 nM; Alzari et al., Annu. Ref. Immunol. 6:555 (1988)) and compares favorably with the affinities estimated for other lymphoid adhesion molecules.
  • CD28 and B7 are found at relatively low levels on resting lymphoid cells (Lesslauer et al., Eur. J. Immuno. 16:1289 (1986); Freeman et al., supra 1989), they may be less involved than other adhesion systems (Springer Nature (Lond) . 346:425 (1990)) in initiating intercellular interactions.
  • the primary role of CD28/B7 interactions may be to maintain or amplify a response subsequent to induction of these counter-receptors on their respective cell types.
  • Binding of B7 to CD28 on T cells was costimulatory for T cell proliferation (Tables 2-4) suggesting that some of the biological effects of anti- CD28 mAbs result from their ability to mimic T cell activation resulting from natural interaction between CD28 and its counter-receptor, B7.
  • mAb 9.3 has greater affinity for CD28 than does B7Ig ( Figures 15 and 16) , which may account for the extremely potent biological effects of this mAb (June et al. , supra 1989) in costimulating polyclonal T cell responses.
  • anti-CD28 mAbs are inhibitory for antigen- specific T cell responses (Damle et al., Proc. Natl.
  • CD28/B7 interactions may also be important for B cell activation and/or differentiation.
  • mAbs 9.3 and BB-1 block T h cell- induced Ig production by B cells. This blocking effect may be due in part to inhibition by these mAbs of production of T h -derived B cell-directed cytokines, but may also involve inhibition of B cell activation by interfering with direct signal transduction via B7.
  • the inhibition of anti-CD28 and anti-B7 mAbs on the cognate T h :B interaction also provide the basis for employing the CD28Ig and B7Ig fusion proteins, and monoclonal antibodies reactive with these proteins, to treat various autoimmune orders associated with exaggerated B cell activation such as insulin-dependent diabetes mellitus, myasthenia gravis, rheumatoid arthritis and systemic lupus erythematosus (SLE) .
  • various autoimmune orders associated with exaggerated B cell activation such as insulin-dependent diabetes mellitus, myasthenia gravis, rheumatoid arthritis and systemic lupus erythematosus (SLE) .
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Abstract

L'invention identifie l'antigène B7 comme étant un ligand réagissant avec le récepteur à CD28 sur des lymphocytes T. On décrit des fragments et des dérivés de l'antigène B7 et du récepteur à CD28, y compris des protéines de fusion ayant des séquences d'acide aminé correspondant aux domaines extracellulaires de B7 ou CD28 joints à des séquences d'acide aminé codant des parties de l'immunoglobuline humaine Cη1. L'invention concerne également des procédés d'utilisation de l'antigène B7, de ses fragments et dérivés, et du récepteur à CD28, de ses fragments et dérivés, ainsi que des anticorps et d'autres molécules réagissant avec l'antigène B7 et/ou le récepteur à CD28, afin de réguler les réponses de lymphocytes T positives à CD28, et les réponses immunitaires induites par des lymphocytes T. En outre, l'invention concerne une méthode d'analyse permettant de détecter des ligands réagissant avec des récepteurs cellulaires induisant une adhérence intercellulaire.
PCT/US1991/004682 1990-07-02 1991-07-01 Ligand pour recepteur a cd28 sur des lymphocytes b et procedes WO1992000092A1 (fr)

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Cited By (63)

* Cited by examiner, † Cited by third party
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WO1994001547A3 (fr) * 1992-07-09 1994-03-31 Cetus Oncology Corp Procede permettant de produire des anticorps contre des molecules de surface des cellules
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WO1994029436A1 (fr) * 1993-06-04 1994-12-22 The United States Of America Represented By The Secretary Of The Navy Procedes de stimulation selective de la proliferation des lymphocytes t
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CA2086325A1 (fr) 1992-01-03
EP0537293A1 (fr) 1993-04-21
CA2086325C (fr) 2010-10-05
JPH06508501A (ja) 1994-09-29
EP0537293A4 (en) 1993-09-08
JP2002186486A (ja) 2002-07-02

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