WO1995003408A1 - B7-2: ctl a4/cd 28 counter receptor - Google Patents

B7-2: ctl a4/cd 28 counter receptor Download PDF

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Publication number
WO1995003408A1
WO1995003408A1 PCT/US1994/008423 US9408423W WO9503408A1 WO 1995003408 A1 WO1995003408 A1 WO 1995003408A1 US 9408423 W US9408423 W US 9408423W WO 9503408 A1 WO9503408 A1 WO 9503408A1
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Prior art keywords
peptide
cell
cells
nucleic acid
tumor
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PCT/US1994/008423
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English (en)
French (fr)
Inventor
Gordon J. Freeman
Lee M. Nadler
Gary S. Gray
Edward Greenfield
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Dana-Farber Cancer Institute
Repligen Corporation
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Publication date
Priority claimed from US08/101,624 external-priority patent/US5942607A/en
Priority to AU74052/94A priority Critical patent/AU7405294A/en
Priority to EP94924017A priority patent/EP0711345A1/de
Priority to JP7505397A priority patent/JPH09500788A/ja
Application filed by Dana-Farber Cancer Institute, Repligen Corporation filed Critical Dana-Farber Cancer Institute
Publication of WO1995003408A1 publication Critical patent/WO1995003408A1/en
Priority to US08/479,744 priority patent/US6084067A/en
Priority to US09/206,132 priority patent/US6723705B1/en
Priority to US09/425,762 priority patent/US6605279B2/en
Priority to AU72086/00A priority patent/AU772061B2/en
Priority to US10/429,079 priority patent/US7459544B2/en
Priority to US10/767,561 priority patent/US20050129670A1/en
Priority to AU2004200586A priority patent/AU2004200586C1/en

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    • 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
    • C07K16/2827—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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2267/03—Animal model, e.g. for test or diseases
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
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    • A01K2267/0331—Animal model for proliferative diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0381—Animal model for diseases of the hematopoietic system
    • 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
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the first signal which confers specificity to the immune response, is mediated via the T cell receptor (TCR) following recognition of foreign antigenic peptide presented in the context of the major histocompatibility complex (MHC).
  • TCR T cell receptor
  • MHC major histocompatibility complex
  • Costimulation is neither antigen-specific, nor MHC restricted and is thought to be provided by one or more distinct cell surface molecules expressed by APCs (Jenkins, M.K., et al. (1988) J. Immunol. 14Q, 3324-3330; Linsley, P.S., et al. (1991) J. Exp. Med. 121, 721-730; Gimmi, CD., et al., (1991) Proc. Natl. Acad. Sci. USA. 88, 6575-6579; Young, J.W., et al. (1992) J. Clin. Invest. 9.0, 229-237; Koulova, L., et al.
  • B7 is the counter-receptor for two ligands expressed on T lymphocytes.
  • the first ligand, termed CD28, is constitutively expressed on resting T cells and increases after activation.
  • CTLA4 The second ligand, termed CTLA4 is homologous to CD28 but is not expressed on resting T cells and appears following T cell activation (Brunet, J.F., et al., (1987) Nature 328, 267- 270).
  • DNA sequences encoding the human and murine CTLA4 protein are described in Dariavich, et al. (1988) Eur. J. Immunol. 18(12), 1901-1905; Brunet, J.F., et al. (1987) supra; Brunet, J.F. et al. (1988) Immunol. Rev. 103:21-36; and Freeman, G.J., et al. (1992) J. Immunol. 142, 3795-3801.
  • B7 has a higher affinity for CTLA4 than for CD28 (Linsley, P.S., et al., (1991) J. Exp. Med. 174, 561-569), the function of CTLA4 is still unknown
  • This invention pertains to isolated nucleic acids encoding novel molecules which costimulate T cell activation.
  • Preferred costimulatory molecules include antigens on the surface of B lymphocytes, professional antigen presenting cells (e.g., monocytes, dendritic cells, Langerhan cells) and other cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes) which present antigen to immune cells, and which bind either CTLA4, CD28, both CTLA4 and CD28 or other known or as yet undefined receptors on immune cells.
  • professional antigen presenting cells e.g., monocytes, dendritic cells, Langerhan cells
  • other cells e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes
  • Such costimulatory molecules are referred to herein as CTLA4/CD28 binding counter-receptors or B lymphocyte antigens, and are capable of providing costimulation to activated T cells to thereby induce T cell proliferation and/or cytokine secretion.
  • Preferred B lymphocyte antigens include B7-2 and B7-3 and soluble fragments or derivatives thereof which bind CTLA4 and/or CD28 and have the ability to inhibit or induce costimulation of immune cells.
  • an isolated nucleic acid which encodes a peptide having the activity of the human B7-2 B lymphocyte antigen is provided.
  • the nucleic acid is a cDNA molecule having a nucleotide sequence encoding human B7-2, as shown in Figure 8 (SEQ ID NO:l).
  • the nucleic acid is a cDNA molecule having a nucleotide sequence encoding murine B7-2, as shown in Figure 14 (SEQ ID NO:22).
  • the invention also features nucleic acids which encode a peptide having B7-2 activity and at least about 50%, more preferably at least about 60% and most preferably at least about 70% homologous with an amino acid sequence shown in Figure 8 (SEQ ID NO:2) or an amino acid sequence shown in Figure 14 (SEQ ID NO:23).
  • Nucleic acids which encode peptides having B7-2 activity and at least about 80%, more preferably at least about 90%, more preferably at least about 95% and most preferably at least about 98% or at least about 99% homologous with an amino acid sequence shown in Figure 8 (SEQ ID NO:2) or an amino acid sequence shown in Figure 14 (SEQ ID NO:23) are also within the scope of the invention.
  • the peptide having B7-2 activity is encoded by a nucleic acid which hybridizes under high or low stringency conditions to a nucleic acid which encodes a peptide having an amino acid sequence of Figure 8 (SEQ ID NO:2) or a peptide having an amino acid sequence shown in Figure 14 (SEQ ID NO:23).
  • the invention further pertains to an isolated nucleic acid comprising a nucleotide sequence encoding a peptide having B7-2 activity and having a length of at least 20 amino acid residues.
  • Peptides having B7-2 activity and consisting of at least 40 amino acid residues in length, at least 60 amino acid residues in length, at least 80 amino acid residues in length, at least 100 amino acid residues in length or at least 200 or more amino acid residues in length are also within the scope of this invention.
  • Particularly preferred nucleic acids encode a peptide having B7-2 activity, a length of at least 20 amino acid residues or more and at least 50% or greater homology (preferably at least 70%) with a sequence shown in Figure 8 (SEQ ID NO:2).
  • the invention features an isolated DNA encoding a peptide having B7-2 activity and an amino acid sequence represented by a formula:
  • Y consists essentially of amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2).
  • X n and Z m are additional amino acid residue(s) linked to Y by an amide bond.
  • X n and Z m are amino acid residues selected from amino acid residues contiguous to Y in the amino acid sequence shown in Figure 8 (SEQ ID NO:2).
  • X n is amino acid residue(s) selected from amino acids contiguous to the amino terminus of Y in the sequence shown in Figure 8 (SEQ ID NO:2), i.e., selected from amino acid residue 23 to 1.
  • Z m is amino acid residue(s) selected from amino acids contiguous to the carboxy terminus of Y in the sequence shown in Figure 8 (SEQ ID NO:2), i.e., selected from amino acid residue 246 to 329.
  • the invention also features an isolated DNA encoding a B7-2 fusion protein which includes a nucleotide sequence encoding a first peptide having B7-2 activity and a nucleotide sequence encoding a second peptide corresponding to a moiety that alters the solubility, binding affinity, stability or valency of the first peptide.
  • the first peptide having B7-2 activity includes an extracellular domain portion of the B7-2 protein (e.g., about amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2)) and the second peptide is an immunoglobulin constant region, for example, a human C ⁇ l or C ⁇ 4 domain, including the hinge, CH2 and CH3 region, to produce a B7-2 immunoglobulin fusion protein (B7-2Ig)(see Capon et al. (1989) Nature 211, 525-531 and Capon U.S. 5,116,964).
  • B7-2Ig B7-2 immunoglobulin fusion protein
  • nucleic acids obtained in accordance with the present invention can be inserted into various expression vectors, which in turn direct the synthesis of the corresponding protein or peptides in a variety of hosts, particularly eucaryotic cells, such as mammalian and insect cell culture, and procaryotic cells such as E. coli.
  • Expression vectors within the scope of the invention comprise a nucleic acid encoding at least one peptide having the activity of a novel B lymphocyte antigen as described herein, and a promoter operably linked to the nucleic acid sequence.
  • the expression vector contains a DNA encoding a peptide having the activity of the B7-2 antigen and a DNA encoding a peptide having the activity of another B lymphocyte antigen, such as the previously characterized B7 activation antigen, referred to herein as B7-1.
  • B7-1 another B lymphocyte antigen
  • Such expression vectors can be used to transfect host cells to thereby produce proteins and peptides, including fusion proteins, encoded by nucleic acids as described herein.
  • Nucleic acid probes useful for assaying a biological sample for the presence of B cells expressing the B lymphocyte antigens B7-2 and B7-3 are also within the scope of the invention.
  • the invention further pertains to isolated peptides having the activity of a novel B lymphocyte antigen, including the B7-2 and B7-3 protein antigens.
  • a preferred peptide having B7-2 activity is produced by recombinant expression and comprises an amino acid sequence shown in Figure 8 (SEQ ID NO: 2).
  • Another preferred peptide having B7-2 activity comprises an amino acid sequence shown in Figure 14 (SEQ ID NO:23).
  • a particularly preferred peptide having the activity of the B7-2 antigen includes at least a portion of the mature form of the protein, such as an extracellular domain portion (e.g., about amino acid residues 24-245 of SEQ ID NO:2) which can be used to enhance or suppress T-cell mediated immune responses in a subject.
  • Other preferred peptides having B7-2 activity include peptides having an amino acid sequence represented by a formula: X n -Y-Z m
  • Y is amino acid residues selected from the group consisting of: amino acid residues 55-68 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 81-89 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 128-142 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 160-169 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 188-200 of the sequence shown in Figure 8 (SEQ ID NO:2); and amino acid residues 269-282 of the sequence shown in Fig ⁇ re 8 (SEQ ID NO:2).
  • X n and Z m are additional amino acid residue(s) linked to Y by an amide bond and are selected from amino acid residues contiguous to Y in the amino acid sequence shown in Figure 8 (SEQ ID NO:2).
  • X n is amino acid residue(s) selected from amino acids contiguous to the amino terminus of Y in the sequence shown in Figure 8 (SEQ ID NO:2).
  • Z m is amino acid residue(s) selected from amino acids contiguous to the carboxy terminus of Y in the sequence shown in Figure 8 (SEQ ID NO:2).
  • Fusion proteins or hybrid fusion proteins including a peptide having the activity of a novel B lymphocyte antigen are also featured.
  • a fusion protein comprising a first peptide which includes an extracellular domain portion of a novel B lymphocyte antigen fused to second peptide, such as an immunoglobulin constant region, that alters the solubility, binding affinity, stability and/or valency of the first peptide are provided.
  • a fusion protein is produced comprising a first peptide which includes amino acid residues of an extracellular domain portion of the B7-2 protein joined to a second pepide which includes amino acid residues of a sequence corresponding to the hinge, CH2 and CH3 regions of C ⁇ l or C ⁇ 4 to form a B7-2Ig fusion protein.
  • a hybrid fusion protein is produced comprising a first peptide which includes an extracellular domain portion of the B7-1 antigen and an extracellular domain portion of the B7-2 antigen and a second peptide which includes amino acid residues corresponding to the hinge, CH2 and CH3 of C ⁇ l (see e.g., Linsley et al. (1991) J. Exp. Med.
  • Isolated peptides and fusion proteins of the invention can be administered to a subject to either upregulate or inhibit the expression of one or more B lymphocyte antigens or block the ligation of one or more B lymphocyte antigens to their natural ligand on immune cells, such as T cells, to thereby provide enhancement or suppression of cell-mediated immune responses in vivo.
  • Another embodiment of the invention provides antibodies, preferably monoclonal antibodies, specifically reactive with a peptide of a novel B lymphocyte antigen or fusion protein as described herein.
  • Preferred antibodies are anti-human B7-2 monoclonal antibodies produced by hybridoma cells HF2.3D1, HA5.2B7 and HA3.1F9. These hybridoma cells have been deposited with the American Type Culture Collection at ATCC Accession
  • a still further aspect of the invention involves the use of the nucleic acids of the invention, especially the cDNAs, to enhance the immunogenicity of a mammalian cell.
  • the mammalian cell is a tumor cell, such as a sarcoma, a lymphoma, a melanoma, a neuroblastoma, a leukemia or a carcinoma, or an antigen presenting cell, such as a macrophage, which is transfected to allow expression of a peptide having the activity of a novel B lymphocyte antigen of the invention on the surface of the cell.
  • Macrophages that express a peptide having the activity of a B lymphocyte antigen can be used as antigen presenting cells, which, when pulsed with an appropriate pathogen- related antigen or tumor antigen, enhance T cell activation and immune stimulation.
  • Mammalian cells can be transfected with a suitable expression vector containing a nucleic acid encoding a peptide having the activity of a novel B lymphocyte antigen, such as the B7-2 antigen, ex vivo and then introduced into the host mammal, or alternatively, cells can be transfected with the gene in vivo via gene therapy techniques.
  • a novel B lymphocyte antigen such as the B7-2 antigen
  • cells can be transfected with the gene in vivo via gene therapy techniques.
  • the nucleic acid encoding a peptide having B7-2 activity can be transfected alone, or in combination with nucleic acids encoding other costimulatory molecules.
  • the invention also provides methods for inducing both general immunosuppression and antigen-specific tolerance in a subject by, for example, blocking the functional interaction of the novel B lymphocyte antigens of the invention, e.g., B7-2 and B7-3, to their natural ligand(s) on T cells or other immune system cells, to thereby block co-stimulation through the receptor-ligand pair.
  • novel B lymphocyte antigens of the invention e.g., B7-2 and B7-3
  • inhibitory molecules that can be used to block the interaction of the natural human B7-2 antigen to its natural ligands include a soluble peptide having B7-2 binding activity but lacking the ability to costimulate immune cells, antibodies that block the binding of B7-2 to its ligands and fail to deliver a co-stimulatory signal (so called "blocking antibodies", such as blocking anti-B7-2 antibodies), B7-2-Ig fusion proteins, which can be produced in accordance with the teachings of the present invention, as well as soluble forms of B7-2 receptors, such as CTLA4Ig or CD28Ig.
  • blocking antibodies such as blocking anti-B7-2 antibodies
  • B7-2-Ig fusion proteins which can be produced in accordance with the teachings of the present invention, as well as soluble forms of B7-2 receptors, such as CTLA4Ig or CD28Ig.
  • Such blocking agents can be used alone or in combination with agents which block interaction of other costimulatory molecules with their natural ligands (e.g., anti-B7 antibody).
  • Inhibition of T cell responses and induction of T cell tolerance according to the methods described herein may be useful prophylactically, in preventing transplantation rejection (solid organ, skin and bone marrow) and graft versus host disease, especially in allogeneic bone marrow transplantation.
  • the methods of the invention may also be useful therapeutically, in the treatment of autoimmune diseases, allergy and allergic reactions, transplantation rejection, and established graft versus host disease in a subject.
  • Another aspect of the invention features methods for upregulating immune responses by delivery of a costimulatory signal to T cells through use of a stimulatory form of B7-2 antigen, which include soluble, multivalent forms of B7-2 protein, such as a peptide having B7-2 activity and B7-2 fusion proteins.
  • Delivery of a stimulatory form of B7-2 in conjunction with antigen may be useful prophylactically to enhance the efficacy of vaccination against a variety of pathogens and may also be useful therapeutically to upregulate an immune response against a particular pathogen during an infection or against a tumor in a tumor-bearing host.
  • the invention also features methods of identifying molecules which can inhibit either the interaction of B lymphocyte antigens, e.g., B7-2, B7-3, with their receptors or interfere with intracellular signalling through their receptors.
  • B lymphocyte antigens e.g., B7-2, B7-3
  • Methods for identifying molecules which can modulate the expression of B lymphocyte antigens on cells are also provided.
  • methods for identifying cytokines produced in response to costimulation of T cells by novel B lymphocyte antigens are within the scope of the invention.
  • Figure 1A-B are graphic representations of the responses of CD28 + T cells, as assessed by 3H-thymidine incorporation or IL-2 secretion, to costimulation provided by either B7 (B7-1) transfected CHO cells (panel a) or syngeneic activated B lymphocytes (panel b) cultured in media, anti-CD3 alone, or anti-CD3 in the presence of the following monoclonal antibodies or recombinant proteins: ⁇ B7 (133, anti-B7-l); CTLA4Ig; Fab ⁇ CD28; control Ig fusion protein (isotype control for CTLA4Ig); or ⁇ B5 (anti-B5, the isotype control for anti-B7-l).
  • Figure 2A-C are graphs of log fluorescence intensity of cell surface expression of B7-1 on splenic B cells activated with surface immunoglobulin (sig) crosslinking.
  • the total (panel a), B7-1 positive (B7-l + , panel b) and B7-1 negative (B7-1", panel c) activated B cells were stained with anti-B7-l monoclonal antibody (133) and fluoroscein isothiocyanate (FITC) labeled goat anti-mouse immunoglobulin and analyzed by flow cytometry.
  • sig surface immunoglobulin
  • Figure 3A-B are graphic representations of the responses of CD28 + T cells, as assessed by - ⁇ H-thymidine incorporation and IL-2 secretion, to costimulation provided by B7-l + (panel a) or B 7-1" (panel b) activated syngeneic B lymphocytes cultured in media, anti-CD3 alone, or anti-CD3 in the presence of the following monoclonal antibodies or recombinant proteins: ⁇ BB-1 (133, anti-B7-l and anti-B7-3); ⁇ B7 (anti-B7-l); CTLA4Ig; Fab ⁇ CD28; control Ig fusion protein or ⁇ B5 (anti-B5).
  • Figure 4 is a graphic representation of the cell surface expression of B7-1, B7-3 and total CTLA4 counter-receptors on fractionated B7-l + and B7-1" activated B lymphocytes.
  • Figure 5 is a graphic representation of temporal surface expression of B7-1
  • CTLA4Ig and mAbs BB-1 and 133 B7-3 (CTLA4Ig and mAb BBl) and B7-2 (CTLA4Ig) counter-receptors on splenic B cells activated by sig crosslinking.
  • Figure 6 is a graphic representation of temporal surface expression of B7-1 (CTLA4Ig and mAbs BB-1 and 133), B7-3 (CTLA4Ig and mAb BBl) and B7-2 (CTLA4Ig) counter-receptors on splenic B cells activated by MHC class II crosslinking.
  • Figure 7A-B are graphic representations of the response of CD28 + T cells, as assessed by ⁇ H-thymidine incorporation and IL-2 secretion, to costimulation provided by syngeneic B lymphocytes activated by sig crosslinking for 24 hours (panel a) or 48 hours (panel b) and cultured in media, anti-CD3 alone, or anti-CD3 in the presence of the following monoclonal antibodies or recombinant protein: ⁇ B7(133, anti-B7-l); ⁇ BBl (anti-B7-l, anti-B7-3) CTLA4Ig; Fab ⁇ CD28; and ⁇ B5(anti-B5).
  • Figure 8 is the nucleotide and deduced amino acid sequence of the human B lymphocyte antigen B7-2 (hB7-2-clone29).
  • Figure 9 is a graphic representation of COS cells transfected with control plasmid (pCDNAI), plasmid expressing B7-1 (B7-1), or plasmid expressing B7-2 (B7-2) stained with either control mAb (IgM), anti-B7-l (mAbs 133 and BB-1), recombinant protein CTLA4Ig, or isotype matched control Ig protein followed by the appropriate second FITC labelled immunoglobulin and analyzed by flow cytometry.
  • control mAb IgM
  • anti-B7-l mAbs 133 and BB-1
  • CTLA4Ig recombinant protein
  • isotype matched control Ig protein followed by the appropriate second FITC labelled immunoglobulin and analyzed by flow cytometry.
  • Figure 10A-B show RNA blot analyses of B7-2 expression in unstimulated and anti- Ig activated human spenic B cells and cell lines (panel a) and human myelomas (panel b).
  • Figure 11 is a graphic representation of the proliferation of CD28+ T cells, as assessed by - ⁇ -thymidine incorporation or IL-2 secretion, to submitogenic stimulation with phorbol myristic acid (PMA) and COS cells transfected with vector alone or vectors directing the expression of either B7-1 or B7-2.
  • PMA phorbol myristic acid
  • Figure 12 is a graphic representation of the inhibition by mAbs and recombinant proteins of the proliferation of CD28+ T cells, as assessed by -1H-thymidine incorporation and IL-2 secretion, to stimulation by PMA and COS cells transfected with vector alone (vector), or with a vector expressing B7-1 (B7-1) or B7-2 (B7-2).
  • Inhibition studies were performed with the addition of either no antibody (no mAb), anti-B7 mAb 133 (133), anti-B7 mAb BB-1 (BBl), anti-B5 mAb (B5), Fab fragment of anti-CD28 (CD28 Fab), CTLA4Ig (CTLA4Ig), or Ig control protein (control Ig) to the PMA stimulated COS cell admixed CD28+ T cells.
  • Figure 13 shows the sequence homology between the human B7-2 protein (h B7-2) deduced amino acid sequence (SEQ ID NO: 2) and the amino acid sequence of both the human B7-1 protein (h B7-1) (SEQ ID NO: 28 and 29) and the murine B7-1 protein (m B7) (SEQ ID NO: 30 and 31).
  • Figure 14 is the nucleotide and deduced amino acid sequence of the murine B7-2 antigen (mB7-2) (SEQ ID NO: 22 and 23).
  • Figure 75 is a graphic representation of the competitive inhibition of binding of biotinylated-CTLA4Ig to immobilized B7-2 Ig by B7 family-Ig fusion proteins.
  • the Ig fusion proteins examined as competitors were: full-length B7-2 (hB7.2), full-length B7-1 (hB7.1 ), the variable region-like domain of B7-2 (hB7.2N) or the constant region-like domain of B7-2 (hB7.2C).
  • Figure 16A-B are graphic representations of the competitive inhibition of binding of biotinylated-B7-l-Ig (panel A) or B7-2-Ig (panel B) to immobilized CTLA4-Ig by increasing concentrations of unlabelled B7-1-Ig (panel A) or B7-2-Ig (panel B).
  • the experimentally determined IC50 values are indicated in the upper right corner of the panels.
  • Figure 77 depicts flow cytometric profiles of cells stained with an anti-hB7-2 monoclonal antibody, HA3.1F9.
  • Cells stained with the antibody were CHO cells transfected to express human B7-2 (CHO-hB7.2), ⁇ IH 3T3 cells transfected to express human B7-2 (3T3-hB7.2) and control transfected ⁇ IH 3T3 cells (3T3-neo).
  • the anti-hB7.2 antibody B70 was used as a positive control.
  • Figure 18 depicts flow cytometric profiles of cells stained with an anti-hB7-2 monoclonal antibody, HA5.2B7.
  • Cells stained with the antibody were CHO cells transfected to express human B7-2 (CHO-hB7.2), ⁇ IH 3T3 cells transfected to express human B7-2 (3T3-hB7.2) and control transfected ⁇ IH 3T3 cells (3T3-neo).
  • the anti-hB7.2 antibody B70 was used as a positive control.
  • Figure 19 depicts flow cytometric profiles of cells stained with an anti-hB7-2 monoclonal antibody, HF2.3D1.
  • Cells stained with the antibody were CHO cells transfected to express human B7-2 (CHO-hB7.2), ⁇ IH 3T3 cells transfected to express human B7-2 (3T3-hB7.2) and control transfected ⁇ IH 3T3 cells (3T3-neo).
  • the anti-hB7.2 antibody B70 was used as a positive control.
  • Figure 20 is a graphic representation of tumor cell growth (as measured by tumor size) in mice following transplantation of J558 plasmacytoma cells or J558 plasmacytoma cells transfected to express B7-1 (J558-B7.1) or B7-2 (J558-B7.2).
  • B lymphocyte activation antigen B7 (referred to herein as B7-1), human B lymphocytes express other novel molecules which costimulate T cell activation. These costimulatory molecules include antigens on the surface of B lymphocytes, professional antigen presenting cells (e.g., monocytes, dendritic cells, Langerhan cells) and other cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes) which present antigen to immune cells, and which bind either CTLA4, CD28, both CTLA4 and CD28 or other known or as yet undefined receptors on immune cells.
  • professional antigen presenting cells e.g., monocytes, dendritic cells, Langerhan cells
  • other cells e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes
  • Costimulatory molecules within the scope of the invention are referred to herein as CTLA4/CD28 ligands (counter-receptors) or B lymphocyte antigens.
  • Novel B lymphocyte antigens which provide cotimulation to activated T cells to thereby induce T cell proliferation and/or cytokine secretion include the B7-2 (human and murine) and the B7-3 antigens described and characterized herein.
  • the B lymphocyte antigen B7-2 is expressed by human B cells at about 24 hours following stimulation with either anti-immunoglobulin or anti-MHC class II monoclonal antibody.
  • the B7-2 antigen induces detectable IL-2 secretion and T cell proliferation.
  • human B cells express both B7-1 and a third CTLA4 counter-receptor, B7-3, identified by a monoclonal antibody BB-1 , which also binds B7-1
  • the B7-3 antigen is also expressed on B7-1 negative activated B cells and can costimulate T cell proliferation without detectable IL-2 production, indicating that the B7-1 and B7-3 molecules are distinct.
  • B7-3 is expressed on a wide variety of cells including activated B cells, activated monocytes, dendritic cells, Langerhan cells and keratinocytes. At 72 hours post B cell activation, the expression of B7-1 and B7-3 begins to decline. The presence of these costimulatory molecules on the surface of activated B lymphocytes indicates that T cell costimulation is regulated, in part, by the temporal expression of these molecules following B cell activation.
  • nucleic acids comprising a nucleotide sequence encoding a novel costimulatory molecule, such as the B lymphocyte antigen, B7-2, fragments of such nucleic acids, or equivalents thereof.
  • a novel costimulatory molecule such as the B lymphocyte antigen, B7-2
  • fragments of such nucleic acids or equivalents thereof.
  • nucleic acid as used herein is intended to include such fragments or equivalents.
  • the term "equivalent” is intended to include nucleotide sequences encoding functionally equivalent B lymphocyte antigens or functionally equivalent peptides having an activity of a novel B lymphocyte antigen, i.e., the ability to bind to the natural ligand(s) of the B lymphocyte antigen on immune cells, such as CTLA4 and/or CD28 on T cells, and inhibit (e.g., block) or stimulate (e.g., enhance) immune cell costimulation.
  • Such nucleic acids are considered equivalents of the human B7-2 nucleotide sequence provided in Figure 8 (SEQ ID NO:l) and the murine B7-2 nucleotide sequence provided in Figure 14 (SEQ ID NO:22) and are within the scope of this invention.
  • the nucleic acid is a cDNA encoding a peptide having an activity of the B7-2 B lymphocyte antigen.
  • the nucleic acid is a cDNA molecule consisting of at least a portion of a nucleotide sequence encoding human B7-2, as shown in Figure 8 (SEQ ID NO:l) or at least a portion of a nucleotide sequence encoding murine B7-2, as shown in Figure 14 (SEQ ID NO:22).
  • a preferred portion of the cDNA molecule of Figure 8 (SEQ ID NO: 1) or Figure 14 (SEQ ID NO:22) includes the coding region of the molecule.
  • nucleic acid of the invention encodes a peptide having an activity of B7-2 and comprising an amino acid sequence shown in Figure 8 (SEQ ID NO:2) or Figure 14 (SEQ ID NO:23).
  • Preferred nucleic acids encode a peptide having B7-2 activity and at least about 50% homology, more preferably at least about 60% homology and most preferably at least about 70% homology with an amino acid sequence shown in Figure 8
  • Nucleic acids which encode peptides having B7-2 activity and at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homologous with a sequence set forth in Figure 8 (SEQ ID NO:2) are also within the scope of the invention.
  • Homology refers to sequence similarity between two peptides having the activity of a novel B lymphocyte antigen, such as B7-2, or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same nucleotide base or amino acid, then the molecules are homologous at that position. A degree (or percentage) of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
  • nucleic acid which hybridizes under high or low stringency conditions to a nucleic acid which encodes a peptide having all or a portion of an amino acid sequence shown in Figure 8 (SEQ ID NO:2) or a peptide having all or a portion of an amino acid sequence shown in Figure 14 (SEQ ID NO:23).
  • Appropriate stringency conditions which promote DNA hybridization for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C to high stringency conditions, at about 65°C
  • nucleic acids encoding a peptide having an activity of a novel B lymphocyte antigen, as described herein, and having a sequence which differs from nucleotide sequence shown in Figure 8 (SEQ ID NO: 1 ) or Figure 14 (SEQ ID NO:22) due to degeneracy in the genetic code are also within the scope of the invention.
  • Such nucleic acids encode functionally equivalent peptides (e.g., a peptide having B7-2 activity) but differ in sequence from the sequence of Figure 8 or Figure 14 due to degeneracy in the genetic code. For example, a number of amino acids are designated by more than one triplet.
  • Codons that specify the same amino acid may occur due to degeneracy in the genetic code.
  • DNA sequence polymorphisms within the nucleotide sequence of a B7-2 may result in "silent" mutations in the DNA which do not affect the amino acid encoded.
  • DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the B7-2 antigen will exist within a population.
  • nucleotides up to about 3-4% of the nucleotides
  • nucleic acids encoding peptides having the activity of a novel B lymphocyte antigen may exist among individuals within a population due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of the invention.
  • isoforms or related, cross-reacting family members of the novel B lymphocyte antigens described herein are defined as proteins related in function and amino acid sequence to a B lymphocyte antigen (e.g., the B7-2 antigen), but encoded by genes at different loci.
  • a "fragment" of a nucleic acid encoding a novel B lymphocyte antigen is defined as a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the entire amino acid sequence of the B lymphocyte antigen and which encodes a peptide having an activity of the B lymphocyte antigen (i.e., the ability to bind to the natural ligand(s) of the B lymphocyte antigen on immune cells, such as CTLA4 and/or CD28 on T cells and either stimulate or inhibit immune cell costimulation).
  • a peptide having B7-2 activity binds CTLA4 and/or CD28 and stimulates or inhibits a T cell mediated immune response, as evidenced by, for example, cytokine production and/or T cell proliferation by T cells that have received a primary activation signal.
  • the nucleic acid fragment encodes a peptide of the B7-2 antigen which retains the ability of the antigen to bind CTLA4 and/or CD28 and deliver a costimulatory signal to T lymphocytes.
  • the nucleic acid fragment encodes a peptide including an extracellular portion of the human B7-2 antigen (e.g., approximately amino acid residues 24-245 of the sequence provided in Figure 8 (SEQ ID NO:2)) which can be used to bind CTLA4 and/or CD28 and, in monovalent form, inhibit costimulation, or in multivalent form, induce or enhance costimulation.
  • an extracellular portion of the human B7-2 antigen e.g., approximately amino acid residues 24-245 of the sequence provided in Figure 8 (SEQ ID NO:2)
  • nucleic acid fragments encode peptides of at least 20 amino acid residues in length, preferably at least 40 amino acid residues and length, and more preferably at least 60 amino acid residues in length. Nucleic acid fragments which encode peptides of at least 80 amino acid residues in length, at least 100 amino acid residues in length, and at least 200 or more amino acids in length are also within the scope of the invention. Particularly preferred nucleic acid fragments encode a peptide having the activity of human B7-2 and an amino acid sequence represented by a formula:
  • Y comprises amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2).
  • X n and Z m are additional amino acid residue(s) linked to Y by an amide bond.
  • X n and Z m are selected from amino acid residues contiguous to Y in the amino acid sequence shown in Figure 8 (SEQ ID NO:2).
  • X n is amino acid residue(s) selected from amino acids contiguous to the amino terminus of Y in the sequence shown in Figure 8 (SEQ ID NO:2), i.e., from amino acid residue 23 to 1.
  • Z m is amino acid residue(s) selected from amino acids contiguous to the carboxy terminus of Y in the sequence shown in Figure 8 (SEQ ID NO:2), i.e., from amino acid residue 246 to 329.
  • Nucleic acid fragments within the scope of the invention include those capable of hybridizing with nucleic acid from other animal species for use in screening protocols to detect novel proteins that are cross-reactive with the B lymphocyte antigens described herein. These and other fragments are described in detail herein. Generally, the nucleic acid encoding a fragment of a B lymphocyte antigen will be selected from the bases coding for the mature protein, however, in some instances it may be desirable to select all or part of a fragment or fragments from the leader sequence or non-coding portion of a nucleotide sequence.
  • Nucleic acids within the scope of the invention may also contain linker sequences, modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of recombinant protein or fragments thereof. These and other modifications of nucleic acid sequences are described in further detail herein.
  • a nucleic acid encoding a peptide having an activity of a novel B lymphocyte antigen may be obtained from mRNA present in activated B lymphocytes. It should also be possible to obtain nucleic acid sequences encoding B lymphocyte antigens from B cell genomic DNA.
  • the gene encoding the B7-2 antigen can be cloned from either a cDNA or a genomic library in accordance with protocols herein described.
  • a cDNA encoding the B7-2 antigen can be obtained by isolating total mRNA from an appropriate cell line. Double stranded cDNAs can then prepared from the total mRNA.
  • the cDNAs can be inserted into a suitable plasmid or viral (e.g., bacteriophage) vector using any one of a number of known techniques.
  • Genes encoding novel B lymphocyte antigens can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention.
  • the nucleic acids of the invention can be DNA or RNA.
  • a preferred nucleic acid is a cDNA encoding the human B7-2 antigen having the sequence depicted in Figure 8 (SEQ ID NO:l).
  • Another preferred nucleic acid is a cDNA encoding the murine B7-2 antigen having the sequence shown on Figure 14 (SEQ ID NO:22).
  • This invention further pertains to expression vectors containing a nucleic acid encoding at least one peptide having the activity of a novel B lymphocyte antigen, as described herein, operably linked to at least one regulatory sequence.
  • "Operably linked” is intended to mean that the nucleotide acid sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence (e.g., in cis or trans). Regulatory sequences are art-recognized and are selected to direct expression of the desired protein in an appropriate host cell. Accordingly, the term regulatory sequence includes promoters, enhancers and other expression control elements.
  • the expression vector includes a nucleic acid encoding at least a portion of the B7-2 protein, such as an extracellular domain portion.
  • the expression vector includes a DNA encoding a peptide having an activity of the B7-2 antigen and a DNA encoding a peptide having an activity of another B lymphocyte antigen, such as B7-1.
  • cDNAs encoding the human B7-1 and mouse B7-1 antigens are shown in SEQ ID NO:28 and SEQ ID NO:30, respectively.
  • the deduced amino acid sequences of these antigens are also shown in SEQ ID NO:29 and SEQ ID NO:31, respectively.
  • Such expression vectors can be used to transfect cells to thereby produce proteins or peptides, including fusion proteins or peptides encoded by nucleic acid sequences as described herein. These and other embodiments are described in further detail herein.
  • the invention also features methods of producing peptides having an activity of a novel B lymphocyte antigen.
  • a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding a peptide having an activity of the B7-2 protein can be cultured in a medium under appropriate conditions to allow expression of the peptide to occur.
  • one or more expression vectors containing DNA encoding a peptide having an activity of B7-2 and DNA encoding another peptide, such as a peptide having an activity of a second B lymphocyte antigen (e.g., B7-1, B7-3) can be used to transfect a host cell to coexpress these peptides or produce fusion proteins or peptides.
  • a recombinant expression vector containing DNA encoding a B7-2 fusion protein is produced.
  • a B7-2 fusion protein can be produced by recombinant expression of a nucleotide sequence encoding a first peptide having B7-2 activity and a nucleotide sequence encoding second peptide corresponding to a moiety that alters the solubility, affinity, stability or valency of the first peptide, for example, an immunoglobulin constant region.
  • the first peptide consists of a portion of the extracellular domain of the human B7-2 antigen (e.g., approximately amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2)).
  • the second peptide can include an immunoglobulin constant region, for example, a human C ⁇ l domain or C ⁇ 4 domain (e.g., the hinge, CH2 and CH3 regions of human IgC ⁇ l, or human IgC ⁇ 4, see e.g., Capon et al. US 5,116,964, incorporated herein by reference).
  • a resulting B7-2Ig fusion protein may have altered B7-2 solubility, binding affinity, stability and/or valency (i.e., the number of binding sites available per molecule) and may increase the efficiency of protein purification. Fusion proteins and peptides produced by recombinant technique may be secreted and isolated from a mixture of cells and medium containing the protein or peptide.
  • the protein or peptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated.
  • a cell culture typically includes host cells, media and other byproducts. Suitable mediums for cell culture are well known in the art. Protein and peptides can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins and peptides. Techniques for transfecting host cells and purifying proteins and peptides are described in further detail herein.
  • Particularly preferred human B7-2Ig fusion proteins include the extracellular domain portion or variable region-like domain of human B7-2 coupled to an immunoglobulin constant region.
  • the immunoglobulin constant region may contain genetic modifications which reduce or eliminate effector activity inherent in the immunoglobulin structure.
  • DNA encoding the extracellular portion of human B7-2 (hB7-2), as well as DNA encoding the variable region-like domain of human B7-2 (hB7.2N) or the constant region ⁇ like domain of human B7-2 (hB7.2C) can be joined to D ⁇ A encoding the hinge, CH2 and CH3 regions of human IgC ⁇ l and/or IgC ⁇ 4 modified by site directed mutagenesis.
  • the preparation and characterization of these fusion proteins is described in detail in Example 7.
  • Transfected cells which express peptides having an activity of one or more B lymphocyte antigens (e.g., B7-2, B7-3) on the surface of the cell are also within the scope of this invention.
  • a host cell such as a COS cell is transfected with an expression vector directing the expression of a peptide having B7-2 activity on the surface of the cell.
  • a transfected host cell can be used in methods of identifying molecules which inhibit binding of B7-2 to its counter-receptor on T cells or which interfere with intracellular signaling of costimulation to T cells in response to B7-2 interaction.
  • a tumor cell such as a sarcoma, a melanoma, a leukemia, a lymphoma, a carcinoma or a neuroblastoma is transfected with an expression vector directing the expression of at least one peptide having the activity of a novel B lymphocyte antigen on the surface of the tumor cell.
  • MHC major histocompatibility complex
  • MHC class II ⁇ and ⁇ chain proteins for example MHC class II ⁇ and ⁇ chain proteins or an MHC class I ⁇ chain protein, and, if necessary, a ⁇ 2 microglobulin protein.
  • MHC major histocompatibility complex
  • Such transfected tumor cells can be used to induce tumor immunity in a subject.
  • the nucleic acid sequences of the invention can also be chemically synthesized using standard techniques.
  • Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et aL U.S. Patent No. 4,598,049; Caruthers ej aL U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071, incorporated by reference herein).
  • a peptide having an activity of a B lymphocyte antigen may differ in amino acid sequence from the B lymphocyte antigen, such as the human B7-2 sequence depicted in Figure 8 (SEQ ID NO:2), or murine B7-2 sequence depicted in Figure 14 (SEQ ID NO:22), but such differences result in a peptide which functions in the same or similar manner as the B lymphocyte antigen or which has the same or similar characteristics of the B lymphocyte antigen.
  • a peptide having an activity of the B7-2 protein is defined herein as a peptide having the ability to bind to the natural ligand(s) of the B7-2 protein on immune cells, such as CLTA4 and/or CD28 on T cells and either stimulate or inhibit immune cell costimulation.
  • a peptide having B7-2 activity binds CTLA4 and/or CD28 and stimulates or inhibits a T cell mediated immune response (as evidenced by, for example, cytokine production and/or proliferation by T cells that have received a primary activation signal).
  • One embodiment provides a peptide having B7-2 binding activity, but lacking the ability to deliver a costimulatory signal to T cells.
  • Such a peptide can be used to inhibit or block T cell proliferation and/or cytokine secretion in a subject.
  • a peptide having both B7-2 binding activity and the ability to deliver a costimulatory signal to T cells is used to stimulate or enhance T cell proliferation and/or cytokine secretion in a subject.
  • B7-2 protein Various modifications of the B7-2 protein to produce these and other functionally equivalent peptides are described in detail herein.
  • the term "peptide” as used herein, refers to peptides, proteins and polypeptides.
  • a peptide can be produced by modification of the amino acid sequence of the human B7-2 protein shown in Figure 8 (SEQ ID NO:2) or the murine B7-2 protein shown in Figure 14 (SEQ ID NO:23), such as a substitution, addition or deletion of an amino acid residue which is not directly involved in the function of B7-2 (i.e., the ability of B7-2 to bind CTLA4 and/or CD28 and/or stimulate or inhibit T cell costimulation).
  • Peptides of the invention are typically at least 20 amino acid residues in length, preferably at least 40 amino acid residues in length, and most preferably 60 amino acid residues in length.
  • Peptides having B7-2 activity and including at least 80 amino acid residues in length, at least 100 amino acid residues in length, or at least 200 or more amino acid residues in length are also within the scope of the invention.
  • a preferred peptide includes an extracellular domain portion of the human B7-2 antigen (e.g., about amino acid residues 24-245 of the sequence shown in Figure 8 (SEQ ID NO:2).
  • Other preferred peptides have an amino acid sequence represented by a formula:
  • Y is amino acid residues selected from the group consisting of: amino acid residues 55-68 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 81-89 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 128-142 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 160-169 of the sequence shown in Figure 8 (SEQ ID NO:2); amino acid residues 188-200 of the sequence shown in Figure 8 (SEQ ID NO:2); and amino acid residues 269-282 of the sequence shown in Figure 8 (SEQ ID NO:2).
  • X n and Z m are additional amino acid residues linked to Y by an amide bond.
  • X n and Z m are amino acid residues selected from amino acids contiguous to Y in the amino acid sequence shown in Figure 8 (SEQ ID NO:2).
  • X n is amino acid residues selected from amino acids contiguous to the amino terminus of Y in the sequence shown in Figure 8 (SEQ ID NO:2).
  • Z is amino acid residues selected from amino acids contiguous to the carboxy terminus of Y in the sequence shown in Figure 8 (SEQ ID NO: 2).
  • Another embodiment of the invention provides a substantially pure preparation of a peptide having an activity of a novel B lymphocyte antigen such as B7-2 or B7-3.
  • a preparation is substantially free of proteins and peptides with which the peptide naturally occurs in a cell or with which it naturally occurs when secreted by a cell.
  • isolated refers to a nucleic acid, protein or peptide having an activity of a novel B lymphocyte antigen, such as B7-2, substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • An isolated nucleic acid is also free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the organism from which the nucleic acid is derived.
  • Suitable cells for use in isolating nucleic acids encoding peptides having an activity of a novel B lymphocyte antigen include cells capable of producing mRNA coding for B lymphocyte antigens (e.g., B7-1, B7-2, B7-3) and appropriately translating the mRNA into the corresponding protein.
  • B lymphocyte antigens e.g., B7-1, B7-2, B7-3
  • One source of mRNA is normal human splenic B cells, either resting or activated by treatment with an anti-immunoglobulin antibody or an anti-MHC class II antibody, or from subsets of neoplastic B cells.
  • RNA levels increasing 4-fold from resting levels following stimulation.
  • Total cellular RNA can be obtained using standard techniques from resting or activated B cells during these intervals and utilized in the construction of a cDNA library.
  • various subsets of neoplastic B cells may express B7-2 and B7-3 and can alternatively serve as a source of the mRNA for construction of a cDNA library.
  • tumor cells isolated from patients with non-Hodgkins lymphoma express B7-1 mRNA.
  • B cells from nodular, poorly differentiated lymphoma (NPDL), diffuse large cell lymphoma (LCL) and Burkitt's lymphoma cell lines are also suitable sources of human B7-1 mRNA and, potentially B7-2 and B7-3 mRNA.
  • Myelomas generally express B7-2, but not B7-1 mRNA, and, thus can provide a source of B7-2 mRNA.
  • the Burkitt's lymphoma cell line Raji is one source of B lymphocyte antigen mRNA.
  • B7-2 mRNA is obtained from a population of both resting and activated normal human B cells.
  • Activated B cells can be obtained by stimulation over a broad spectrum of time (e.g., from minutes to days) with, for example, an anti-immunoglobulin antibody or an anti-MCH class II antibody.
  • Total cellular mRNA can be isolated by a variety of techniques, e.g., by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al., Biochemistry 18, 5294- 5299 (1979).
  • Poly (A+) mRNA is prepared and purified for use in a cDNA library construction using oligo (dT) cellulose selection.
  • cDNA is then synthesized from the poly(A+) RNA using oligo(dT) priming and reverse transcriptase.
  • Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, MD
  • AMV reverse transcriptase available from Seikagaku America, Inc., St. Moscow, FL are preferably employed.
  • the mRNA/DNA hybrid molecule is converted to double stranded DNA using conventional techniques and incorporated into a suitable vector.
  • the experiments herein employed E. coli DNA polymerase I and ribonuclease H in the conversion to double stranded cDNA.
  • Cloning of the cDNAs can be accomplished using any of the conventional techniques for joining double stranded DNA with an appropriate vector.
  • the use of synthetic adaptors is particularly preferred, since it alleviates the possibility of cleavage of the cDNA with restriction enzyme prior to cloning.
  • non-self complementary, kinased adaptors are added to the DNA prior to ligation with the vector.
  • Virtually any adaptor can be employed.
  • non-self complementary BstXI adaptors are preferably added to the cDNA for cloning, for ligation into a pCDM8 vector prepared for cloning by digestion with BstXI.
  • ⁇ ucaryotic cDNA can be expressed when placed in the sense orientation in a vector that supplies an appropriate eucaryotic promoter and origin of replication and other elements including enhancers, splice acceptors and/or donor sequences and polyadenylation signals.
  • the cDNAs of the present invention are placed in suitable vectors containing a eucaryotic promoter, an origin of replication functional in E. coli, an SV40 origin of replication which allows growth in COS cells, and a cDNA insertion site.
  • suitable vectors include ⁇ H3 (Seed and Aruffo, Proc. Natl. Acad. Sci., 84:3365-3369 (1987)), ⁇ H3m (Aruffo and Seed, Proc. Natl.
  • pCDM7 and pCDM8 Seed, Nature, 329:840-841 (1987), with the pCDM8 vector being particularly preferred (available commercially from Invitrogen, San Diego, CA).
  • the thus prepared cDNA library is then used to clone the gene of interest by expression cloning techniques.
  • a basic expression cloning technique has been described by Seed and Aruffo, Proc. Natl. Acad. Sci. USA, 84:3365-3369 (1987) and Aruffo and Seed, Proc. Natl. Acad. Sci. USA, 84:8573-8577 (1987), although modifications to this technique may be necessary.
  • plasmid DNA is introduced into a simian COS cell line (Gluzman, Cell 23:175 (1981)) by known methods of transfection (e.g., D ⁇ A ⁇ -Dextran) and allowed to replicate and express the cDNA inserts.
  • the transfectants expressing B7-1 antigen are depleted with an anti-B7-l monoclonal antibody (e.g., 133 and Bl.l) and anti- murine IgG and IgM coated immunomagnetic beads.
  • Transfectants expressing human B7-2 antigen can be positively selected by reacting the transfectants with the fusion proteins CTLA4Ig and CD28Ig, followed by panning with anti-human Ig antibody coated plates.
  • CTLA4Ig and CD28Ig fusion proteins were used in the examples described herein, given the cross-species reactivity between B7-1 and, for example murine B7-1, it can be expected that other fusion proteins reactive with another cross-reactive species could be used.
  • episomal DNA is recovered from the panned cells and transformed into a competent bacterial host, preferably E. coli. Plasmid DNA is subsequently reintroduced into COS cells and the cycle of expression and panning repeated at least two times. After the final cycle, plasmid DNA is prepared from individual colonies, transfected into COS cells and analyzed for expression of novel B lymphocyte antigens by indirect immunofluorescence with, for example, CTLA4Ig and CD28Ig.
  • Plasmids are prepared from those clones which are strongly reactive with the CTL A4Ig and/or CD28Ig. These plasmids are then sequenced. Any of the conventional sequencing techniques suitable for sequencing tracts of DNA about 1.0 kb or larger can be employed.
  • a human B7-2 clone (clone29) was obtained containing an insert of 1,120 base pairs with a single long open reading frame of 987 nucleotides and approximately 27 nucleotides of 3' noncoding sequences ( Figure 8, SEQ ID NO:l).
  • the predicted amino acid sequence encoded by the open reading frame of the protein is shown below the nucleotide sequence in Figure 8.
  • the encoded human B7-2 protein is predicted to be 329 amino acid residues in length (SEQ ID NO:2). This protein sequence exhibits many features common to other type I Ig superfamily membrane proteins.
  • Protein translation is predicted to begin at the methionine codon (ATG, nucleotides 107 to 109) based on the DNA homology in this region with the consensus eucaryotic translation initiation site (see Kozak, M. (1987) Nucl. Acids Res. 15.: 8125-8148).
  • the amino terminus of the B7-2 protein (amino acids 1 to 23) has the characteristics of a secretory signal peptide with a predicted cleavage between the alanines at positions 23 and 24 (von Heijne (1987) Nucl. Acids Res. 14:4683). Processing at this site would result in a B7-2 membrane bound protein of 306 amino acids having an unmodified molecular weight of approximately 34 kDa.
  • This protein would consist of an approximate extracellular Ig superfamily V and C like domains of from about amino acid residue 24 to 245, a hydrophobic transmembrane domain of from about amino acid residue 246 to 268, and a long cytoplasmic domain of from about amino acid residue 269 to 329.
  • the homologies to the Ig superfamily are due to the two contiguous Ig-like domains in the extracellular region bound by the cysteines at positions 40 to 110 and 157 to 218.
  • the extracellular domain also contains eight potential N-linked glycosylation sites and, like B7-1, is probably glycosylated. Glycosylation of the human B7-2 protein may increase the molecular weight to about 50-70 kDa.
  • the cytoplasmic domain of human B7-2 while somewhat longer than B7-1, contains a common region of multiple cysteines followed by positively charged amino acids which presumably function as signaling or regulatory domains within an antigen-presenting cell (APC).
  • APC antigen-presenting cell
  • B lymphocyte antigens from Other Mammalian Species The present invention is not limited to human nucleic acid molecules and contemplates that novel B lymphocyte antigen homologues from other mammalian species that express B lymphocyte antigens can be cloned and sequenced using the techniques described herein.
  • B lymphocyte antigens isolated for one species e.g., humans
  • which exhibit cross-species reactivity may be used to modify T cell mediated immune responses in a different species (e.g., mice).
  • Isolation of cDNA clones from other species can also be accomplished using human cDNA inserts, such as human B7-2 cDNA, as hybridization probes.
  • a murine B7-2 clone (mB7-2, clone 4) was obtained containing an insert of 1,163 base pairs with a single long open reading frame of 927 nucleotides and approximately 126 nucleotides of 3' noncoding sequences ( Figure 14, SEQ ID NO:22).
  • the predicted amino acid sequence encoded by the open reading frame of the protein is shown below the nucleotide sequence in Figure 14.
  • the encoded murine B7-2 protein is predicted to be 309 amino acid residues in length (SEQ ID NO:23). This protein sequence exhibits many features common to other type I Ig superfamily membrane proteins.
  • Protein translation is predicted to begin at the methionine codon (ATG, nucleotides 1 11 to 113) based on the DNA homology in this region with the consensus eucaryotic translation initiation site (see Kozak, M. (1987) Nucl. Acids Res. 11:8125-8148).
  • the amino terminus of the murine B7-2 protein (amino acids 1 to 23) has the characteristics of a secretory signal peptide with a predicted cleavage between the alanine at position 23 and the valine at position 24 (von Heijne (1987) Nucl. Acids Res. 14:4683).
  • This protein would consist of an approximate extracellular Ig superfamily V and C like domains of from about amino acid residue 24 to 246, a hydrophobic transmembrane domain of from about amino acid residue 247 to 265, and a long cytoplasmic domain of from about amino acid residue 266 to 309.
  • the homologies to the Ig superfamily are due to the two contiguous Ig-like domains in the extracellular region bound by the cysteines at positions 40 to 110 and 157 to 216.
  • the extracellular domain also contains nine potential N-linked glycosylation sites and, like murine B7-1, is probably glycosylated.
  • Glycosylation of the murine B7-2 protein may increase the molecular weight to about 50-70 kDa.
  • the cytoplasmic domain of murine B7-2 contains a common region which has a cysteine followed by positively charged amino acids which presumably functions as signaling or regulatory domain within an APC
  • Comparison of both the nucleotide and amino acid sequences of murine B7-2 with the GenBank and EMBL databases yielded significant homology (about 26% amino acid sequence identity) with human and murine B7- 1.
  • Murine B7-2 exhibits about 50% identity and 67% similarity with its human homologue, hB7-2.
  • coli DH106/p3 transfected with a vector (plasmid pmBx4) containing a cDNA insert encoding murine B7-2 (clone 4) was deposited with the American Type Culture Collection (ATCC) on August 18, 1993 as Accession No. 69388.
  • ATCC American Type Culture Collection
  • Nucleic acids which encode novel B lymphocyte antigens from other species can be used to generate either transgenic animals or "knock out" animals which, in turn, are useful in the development and screening of therapeutically useful reagents.
  • a transgenic animal e.g., a mouse
  • a transgenic animal is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage.
  • a transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops.
  • murine B7-2 cDNA or an appropriate sequence thereof can be used to clone genomic B7-2 in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express B7-2 protein.
  • Methods for generating transgenic animals, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009.
  • particular cells would be targeted for B7-2 transgene incorporation with tissue specific enhancers, which could result in T cell costimulation and enhanced T cell proliferation and autoimmunity.
  • Transgenic animals that include a copy of a B7-2 transgene introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased B7 expression.
  • Such animals can be used as tester animals for reagents thought to confer protection from, for example, autoimmune disease.
  • reagents thought to confer protection from, for example, autoimmune disease.
  • an animal is treated with the reagent and a reduced incidence of the disease, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the disease.
  • the non-human homologues of B7-2 can be used to construct a B7-2 "knock out" animal which has a defective or altered B7-2 gene as a result of homologous recombination between the endogenous B7-2 gene and altered B7-2 genomic DNA introduced into an embryonic cell of the animal.
  • murine B7-2 cDNA can be used to clone genomic B7-2 in accordance with established techniques.
  • a portion of the genomic B7-2 DNA e.g., such as an exon which encodes an extracellular domain
  • flanking DNA typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector (see e.g., Thomas, K.R. and Capecchi, M. R. (1987) Cell 51:503 for a description of homologous recombination vectors).
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see e.g., Li, E. et al. (1992) Cell £2:915).
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152).
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal.
  • Progeny harbouring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA.
  • Knockout animals can be characterized for their ability to accept grafts, reject tumors and defend against infectious diseases and can be used in the study of basic immunobiology.
  • Host cells transfected to express peptides having the activity of a novel B lymphocyte antigen are also within the scope of the invention.
  • the host cell may be any procaryotic or eucaryotic cell.
  • a peptide having B7-2 activity may be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO) and NS0 cells.
  • Other suitable host cells may be found in Goeddel, (1990) supra or are known to those skilled in the art.
  • expression in eucaryotic cells such as mammalian, yeast, or insect cells can lead to partial or complete glycosylation and/or formation of relevant inter- or intra-chain disulfide bonds of recombinant protein.
  • yeast S. cerivisae examples include pYepSecl (Baldari. et aL, (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et aL, (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA).
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et aL, (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V.A., and Summers, M.D., (1989) Virology 170:31-39).
  • COS cells Gluzman, Y., (1981) Ce// 23:175- 182
  • pCDM8 Seed, B., (1987) Nature 322:840
  • CHO dhfr Chinese Hamster Qvary
  • pMT2PC Kaufman et aL (1987), EMBOJ. 6:187-195
  • a preferred cell line for production of recombinant protein is the NSO myeloma cell line available from the ECACC (catalog #85110503) and described in Galfre, G.
  • Vector DNA can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofectin, or electroporation. Suitable methods for transforming host cells can be found in Sambrook et aL (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.
  • the expression vector's control functions are often provided by viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and most frequently, Simian Virus 40.
  • a gene that contains a selectable marker i.e., resistance to antibiotics
  • Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Selectable markers may be introduced on the same plasmid as the gene of interest or may be introduced on a separate plasmid. Cells containing the gene of interest can be identified by drug selection; cells that have incorporated the selectable marker gene will survive, while the other cells die.
  • the surviving cells can then be screened for production of novel B lymphocyte antigens by cell surface staining with ligands to the B cell antigens (e.g., CTLA4Ig and CD28Ig).
  • the protein can be metabolically radiolabeled with a labeled amino acid and immunoprecipitated from cell supernatant with an anti-B lymphocyte antigen monoclonal antibody or a fusion protein such as CTLA4Ig or CD28Ig.
  • Fusion vectors add a number of amino acids usually to the amino terminus of the expressed target gene.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the target recombinant protein; and 3) to aid in the purification of the target recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the target recombinant protein to enable separation of the target recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-tranferase, maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • E. coli expression systems include the inducible expression vectors pTrc (Amann et aL, (1988) Gene (52:301-315) and pET 11 (Studier et al, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89; commercially available from Novagen).
  • pTrc vector system the inserted gene is expressed with a pelB signal sequence by host RNA polymerase transcription from a hybrid trp-lac fusion promoter. After induction, the recombinant protein can be purified from the periplasmic fraction.
  • the target gene is expressed as non-fusion protein by transcription from the T7 gnlO-lac 0 fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl).
  • This viral polymerase is supplied by host E. coli strains BL21(DE3) or HMS174(DE3) from a resident ⁇ prophage harboring a T7 gnl under the transcriptional control of the lacUV 5 promoter.
  • the recombinant protein can be purified from inclusion bodies in a denatured form and, if desired, renatured by step gradient dialysis to remove denaturants.
  • One strategy to maximize recombinant B7-2 expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185. Academic Press, San Diego, California (1990) 119-128).
  • Another strategy would be to alter the nucleic acid sequence of the B7-2 gene or other DNA to be inserted into an expression vector so that the individual codons for each amino acid would be those preferentially utilized in highly expressed E. coli proteins (Wada et aL, ( 1992) Nuc. Acids Res. 20:2111-2118).
  • Such alteration of nucleic acid sequences of the invention could be carried out by standard DNA synthesis techniques.
  • Novel B lymphocyte antigens and portions thereof, expressed in mammalian cells or otherwise can be purified according to standard procedures of the art, including ammonium sulfate precipitation, fractionation column chromatography (e.g. ion exchange, gel filtration, electrophoresis, affinity chromatography, etc.) and ultimately, crystallization (see generally, "Enzyme Purification and Related Techniques", Methods in Enzymology, 22:233-577 (1971)).
  • fractionation column chromatography e.g. ion exchange, gel filtration, electrophoresis, affinity chromatography, etc.
  • crystallization see generally, "Enzyme Purification and Related Techniques", Methods in Enzymology, 22:233-577 (1971).
  • the recombinantly produced B lymphocyte antigens or portions thereof can be utilized in compositions suitable for pharmaceutical administration as described in detail herein. VII. Modifications of Nucleic Acid and Amino Acid Sequences of the Invention and Assays for B7 Lymph
  • nucleic acids encoding peptides having the activity of a novel B lymphocyte antigen can be isolated by the above process. Different cell lines can be expected to yield DNA molecules having different sequences of bases. Additionally, variations may exist due to genetic polymorphisms or cell- mediated modifications of the genetic material. Furthermore, the DNA sequence of a B lymphocyte antigen can be modified by genetic techniques to produce proteins or peptides with altered amino acid sequences. Such sequences are considered within the scope of the present invention, where the expressed peptide is capable of either inducing or inhibiting activated T cell mediated immune responses and immune function.
  • a number of processes can be used to generate equivalents or fragments of an isolated DNA sequence.
  • Small subregions or fragments of the nucleic acid encoding the B7-2 protein for example 1-30 bases in length, can be prepared by standard, synthetic organic chemical means.
  • the technique is also useful for preparation of antisense oligonucleotides and primers for use in the generation of larger synthetic fragments of B7-2 DNA.
  • PCR polymerase chain reaction
  • a DNA can be synthesized which does not encode the transmembrane and cytoplasmic regions of the protein.
  • This DNA molecule can be ligated into an appropriate expression vector and introduced into a host cell such as CHO, where the B7-2 protein fragment is synthesized and secreted.
  • the B7-2 protein fragment can then readily be obtained from the culture media.
  • mutations can be introduced into a DNA by any one of a number of methods, including those for producing simple deletions or insertions, systematic deletions, insertions or substitutions of clusters of bases or substitutions of single bases, to generate variants or modified equivalents of B lymphocyte antigen DNA.
  • changes in the human B7-2 cDNA sequence shown in Figure 8 (SEQ ID NO:l) or murine B7-2 cDNA sequence shown in Figure 14 (SEQ ID NO:22) such as amino acid substitutions or deletions are preferably obtained by site-directed mutagenesis.
  • Site directed mutagenesis systems are well known in the art. Protocols and reagents can be obtained commercially from Amersham International PLC, Amersham, U.K.
  • Peptides having an activity of a novel B lymphocyte antigen i.e., the ability to bind to the natural ligand(s) of a B lymphocyte antigen on T cells and either stimulate (amplify) or inhibit (block) activated T cell mediated immune responses, as evidenced by, for example, cytokine production and/or T cell proliferation by T cells that have received a primary activation signal are considered within the scope of the invention.
  • peptides that bind to T lymphocytes may be capable of delivering a costimulatory signal to the T lymphocytes, which, when transmitted in the presence of antigen and class II MHC, or other material capable of transmitting a primary signal to the T cell, results in activation of cytokine genes within the T cell.
  • a costimulatory signal to the T lymphocytes, which, when transmitted in the presence of antigen and class II MHC, or other material capable of transmitting a primary signal to the T cell, results in activation of cytokine genes within the T cell.
  • such a peptide can be used in conjunction with class I MHC to thereby activate CD8 + cytolytic T cells.
  • soluble, monomeric forms of the B7-2 protein may retain the ability to bind to their natural ligand(s) on CD28 + T cells but, perhaps because of insufficient cross-linking with the ligand, fail to deliver the secondary signal essential for enhanced cytokine production and cell division.
  • Such peptides which provide a means to induce a state of anergy or tolerance in the cells, are also considered within the scope of the invention.
  • the peptides for those which retain a characteristic B lymphocyte antigen activity as described herein can be accomplished using one or more of several different assays.
  • the peptides can be screened for specific reactivity with an anti-B7-2 monoclonal antibody reactive with cell surface B7-2 or with a fusion protein, such as CTLA4Ig or CD28Ig.
  • appropriate cells such as COS cells, can be transfected with a B7-2 DNA encoding a peptide and then analyzed for cell surface phenotype by indirect immunofluorescence and flow cytometry to determine whether the peptide has B7-2 activity.
  • Cell surface expression of the transfected cells is evaluated using a monoclonal antibody specifically reactive with cell surface B7-2 or with a CTLA4Ig or CD28Ig fusion protein. Production of secreted forms of B7-2 is evaluated using anti-B7-2 monoclonal antibody or CTLA4Ig or CD28 fusion protein for immunoprecipitation.
  • assays take advantage of the functional characteristics of the B7-2 antigen.
  • the ability of T cells to synthesize cytokines depends not only on occupancy or cross-linking of the T cell receptor for antigen (the "primary activation signal” provided by, for example anti-CD3, or phorbol ester to produce an "activated T cell”), but also on the induction of a costimulatory signal, in this case, by interaction with a B lymphocyte antigen, such as B7-2, B7-1 or B7-3.
  • B7-2 binding of B7-2 to its natural ligand(s) on, for example, CD28 + T cells, has the effect of transmitting a signal to the T cell that induces the production of increased levels of cytokines, particularly of interleukin-2, which in turn stimulates the proliferation of the T lymphocytes.
  • Other assays for B7-2 function thus involve assaying for the synthesis of cytokines, such as interleukin-2, interleukin-4 or other known or unknown novel cytokines, and/or assaying for T cell proliferation by CD28 + T cells which have received a primary activation signal.
  • T cells can be provided with a first or primary activation signal by anti-T3 monoclonal antibody (e.g. anti-CD3) or phorbol ester or, more preferably, by antigen in association with class II MHC.
  • T cells which have received a primary activation signal are referred to herein as activated T cells.
  • B7-2 function is assayed by adding a source of B7-2 (e.g., cells expressing a peptide having B7-2 activity or a secreted form of B7-2) and a primary activation signal such as antigen in association with Class II MHC to a T cell culture and assaying the culture supernatant for interleukin-2, gamma interferon, or other known or unknown cytokine.
  • a source of B7-2 e.g., cells expressing a peptide having B7-2 activity or a secreted form of B7-2
  • a primary activation signal such as antigen in association with Class II MHC
  • any one of several conventional assays for interleukin-2 can be employed, such as the assay described in Proc. Natl. Acad. Sci. USA, 86:1333 (1989) the pertinent portions of which are incorporated herein by reference.
  • a kit for an assay for the production of interferon is also available from Genzyme Corporation (Cambridge, MA.).
  • T cell proliferation can also be measured as described in the Examples below. Peptides that retain the characteristics of the B7-2 antigen as described herein may result in increased per cell production of cytokines, such as IL-2, by T cells and may also result in enhanced T cell proliferation when compared to a negative control in which a costimulatory signal is lacking.
  • the same basic functional assays can also be used to screen for peptides having B7-2 activity, but which lack the ability to deliver a costimulatory signal, but in the case of such peptides, addition of the B7-2 protein will not result in a marked increase in proliferation or cytokine secretion by the T cells.
  • the ability of such proteins to inhibit or completely block the normal B7-2 costimulatory signal and induce a state of anergy can be determined using subsequent attempts at stimulation of the T cells with antigen presenting cells that express cell surface B7-2 and present antigen. If the T cells are unresponsive to the subsequent activation attempts, as determined by IL-2 synthesis and T cell proliferation, a state of anergy has been induced.
  • a peptide having the activity of a novel B lymphocyte antigen for such purposes as increasing solubility, enhancing therapeutic or prophylactic efficacy, or stability (e.g., shelf life ex vivo and resistance to proteolytic degradation in vivo).
  • modified peptides are considered functional equivalents of the B lymphocyte antigens as defined herein.
  • a peptide having B7-2 activity can be modified so that it maintains the ability to co-stimulate T cell proliferation and/or produce cytokines.
  • Those residues shown to be essential to interact with the CTLA4/CD28 receptors on T cells can be modified by replacing the essential amino acid with another, preferably similar amino acid residue (a conservative substitution) whose presence is shown to enhance, diminish, but not eliminate, or not effect receptor interaction.
  • those amino acid residues which are not essential for receptor interaction can be modified by being replaced by another amino acid whose incorporation may enhance, diminish, or not effect reactivity.
  • Another example of modification of a peptide having the activity of a novel B lymphocyte antigen is substitution of cysteine residues preferably with alanine, serine, threonine, leucine or glutamic acid residues to minimize dimerization via disulfide linkages.
  • amino acid side chains of a peptide having B7-2 activity can be chemically modified. Another modification is cyclization of the peptide.
  • peptides having B7-2 activity can be modified to incorporate one or more polymorphisms in the amino acid sequence of the antigen resulting from any natural allelic variation.
  • D-amino acids, non-natural amino acids, or non-amino acid analogs can be substituted or added to produce a modified protein within the scope of this invention.
  • the peptides can be modified using polyethylene glycol (PEG) according to the method of A. Sehon and co-workers (Wie et aL, supra) to produce a peptide conjugated with PEG.
  • PEG polyethylene glycol
  • hexa-histidine can be added to the peptide for purification by immobilized metal ion affinity chromatography (Hochuli, E. et aL, (1988) Bio/Technology 6:1321-1325).
  • specific endoprotease cleavage sites can be introduced between the sequences of a fusion moiety and the peptide. It may be necessary to increase the solubility of a peptide by adding functional groups to the peptide, or by omitting hydrophobic regions of the peptide.
  • the nucleic acids of this invention are useful diagnostically, for tracking the progress of disease, by measuring the activation status of B lymphocytes in biological samples or for assaying the effect of a molecule on the expresssion of a B lymphocyte antigen (e.g., detecting cellular mRNA levels).
  • the nucleic acid sequences are labeled with a detectable marker, e.g., a radioactive, fluorescent, or biotinylated marker and used in a conventional dot blot or Northern hybridization procedure to probe mRNA molecules of total or poly(A+) RNAs from a biological sample.
  • the peptides and fusion proteins produced from the nucleic acid molecules of the present invention can also be used to produce antibodies specifically reactive with B lymphocyte antigens.
  • B lymphocyte antigens For example, by using a full-length B7-2 protein, or a peptide fragment thereof, having an amino acid sequence based on the predicted amino acid sequence of B7-2, anti-protein/anti-peptide polyclonal antisera or monoclonal antibodies can be made using standard methods.
  • a mammal e.g., a mouse, hamster, or rabbit
  • an immunogenic form of the protein or peptide which elicits an antibody response in the mammal e.g., a mouse, hamster, or rabbit
  • the immunogen can be, for example, a recombinant B7-2 protein, or fragment thereof, a synthetic peptide fragment or a cell that expresses a B lymphocyte antigen on its surface.
  • the cell can be for example, a splenic B cell or a cell transfected with a nucleic acid encoding a B lymphocyte antigen of the invention (e.g., a B7-2 cDNA) such that the B lymphocyte antigen is expressed on the cell surface.
  • the immunogen can be modified to increase its immunogenicity.
  • techniques for conferring immunogenicity on a peptide include conjugation to carriers or other techniques well known in the art.
  • the peptide can be administered in the presence of adjuvant.
  • the progress of immunization can be monitored by detection of antibody titers in plasma or serum.
  • Standard ELISA or other immunoassay can be used with the immunogen as antigen to assess the levels of antibodies.
  • antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera.
  • antibody producing cells lymphocytes
  • myeloma cells can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells.
  • myeloma cells can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells.
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the peptide and monoclonal antibodies isolated.
  • antibody as used herein is intended to include fragments thereof which are also specifically reactive with a peptide having the activity of a novel B lymphocyte antigen or fusion protein as described herein.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments.
  • the antibody of the present invention is further intended to include bispecific and chimeric molecules having an anti-B lymphocyte antigen (i.e., B7-2, B7-3) portion.
  • Anti-human B7-2 monoclonal antibodies produced by hybridomas HA3.1F9, HA5.2B7 and HF2.3D1. The preparation and characterization of these antibodies is described in detail in Example 8. Monoclonal antibody HA3.1F9 was determined to be of the IgGl isotype; monoclonal antibody HA5.2B7 was determined to be of the IgG2b isotype; and monoclonal anibody HF2.3D1 was determined to be of the IgG2a isotype. Hybidoma cells were deposited with the American Type Culture Collection, which meets the requirements of the Budapest Treaty, on July 19, 1994 as ATCC
  • chimeric antibody derivatives i.e., antibody molecules that combine a non-human animal variable region and a human constant region.
  • Chimeric antibody molecules can include, for example, the antigen binding domain from an antibody of a mouse, rat, or other species, with human constant regions.
  • a variety of approaches for making chimeric antibodies have been described and can be used to make chimeric antibodies containing the immunoglobulin variable region which recognizes the gene product of the novel B lymphocyte antigens of the invention.
  • the monoclonal or chimeric antibodies specifically reactive with a peptide having the activity of a B lymphocyte antigen as described herein can be further humanized by producing human variable region chimeras, in which parts of the variable regions, especially the conserved framework regions of the antigen-binding domain, are of human origin and only the hypervariable regions are of non-human origin.
  • humanized chimeric antibodies are provided by Morrison, S. L. (1985) Science 222:1202-1207 and by Oi et al. (1986) BioTechniques 4:214.
  • Such altered immunoglobulin molecules may be made by any of several techniques known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci.
  • Humanized antibodies which have reduced immunogenicity are preferred for immunotherapy in human subjects. Immunotherapy with a humanized antibody will likely reduce the necessity for any concomitant immunosuppression and may result in increased long term effectiveness for the treatment of chronic disease situations or situations requiring repeated antibody treatments.
  • a human monoclonal antibody directed against a human protein can be generated.
  • Transgenic mice carrying human antibody repertoires have been created which can be immunized with a human B lymphocyte antigen, such as B7-2. Splenocytes from these immunized transgenic mice can then be used to create hybridomas that secrete human monoclonal antibodies specifically reactive with a human B lymphocyte antigen (see, e.g., Wood et al. PCT publication WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. PCT publication WO 92/03918; Kay et al. PCT publication 92/03917; Lonberg, N. et al.
  • combinatorial antibody display An alternative method, referred to as the "combinatorial antibody display” method, has been developed to identify and isolate antibody fragments having a particular antigen specificity, and can be utilized to produce monoclonal antibodies that bind a B lymphocyte antigen of the invention (for descriptions of combinatorial antibody display see e.g., Sastry et al. (1989) PNAS £6:5728; Huse et al. (1989) Science 246:1275; and Orlandi et al. (1989) PNAS £6:3833). After immunizing an animal with a B lymphocyte antigen, the antibody repertoire of the resulting B-cell pool is cloned.
  • Methods are generally known for directly obtaining the DNA sequence of the variable regions of a diverse population of immunoglobulin molecules by using a mixture of oligomer primers and PCR.
  • mixed oligonucleotide primers corresponding to the 5' leader (signal peptide) sequences and/or framework 1 (FR1) sequences, as well as primer to a conserved 3' constant region primer can be used for PCR amplification of the heavy and light chain variable regions from a number of murine antibodies (Larrick et al. (1991) Biotechniques 11:152-156).
  • a similar strategy can also been used to amplify human heavy and light chain variable regions from human antibodies (Larrick et al.
  • RNA is isolated from activated B cells of, for example, peripheral blood cells, bone marrow, or spleen preparations, using standard protocols (e.g., U.S. Patent No. 4,683,202; Orlandi, et al. PNAS (1989) £6:3833-3837; Sastry et al., PNAS (1989) £6:5728-5732; and Huse et al. (1989) Science 246:1275-1281.) First-strand cDNA is synthesized using primers specific for the constant region of the heavy chain(s) and each of the K and ⁇ light chains, as well as primers for the signal sequence. Using variable region
  • PCR primers the variable regions of both heavy and light chains are amplified, each alone or in combinantion, and ligated into appropriate vectors for further manipulation in generating the display packages.
  • Oligonucleotide primers useful in amplification protocols may be unique or degenerate or incorporate inosine at degenerate positions. Restriction endonuclease recognition sequences may also be incorporated into the primers to allow for the cloning of the amplified fragment into a vector in a predetermined reading frame for expression.
  • the V-gene library cloned from the immunization-derived antibody repertoire can be expressed by a population of display packages, preferably derived from filamentous phage, to form an antibody display library.
  • the display package comprises a system that allows the sampling of very large diverse antibody display libraries, rapid sorting after each affinity separation round, and easy isolation of the antibody gene from purified display packages.
  • kits for generating phage display libraries e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01 ; and the Stratagene - wr 24 TM phage display kit, catalog no.
  • examples of methods and reagents particularly amenable for use in generating a diverse antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271 ; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No.
  • V region domains of heavy and light chains can be expressed on the same polypeptide, joined by a flexible linker to form a single-chain Fv fragment, and the scFV gene subsequently cloned into the desired expression vector or phage genome.
  • a flexible linker to form a single-chain Fv fragment
  • the scFV gene subsequently cloned into the desired expression vector or phage genome.
  • VJJ and VL domains of an antibody, joined by a flexible (Gly4-Ser)3 linker can be used to produce a single chain antibody which can render the display package separable based on antigen affinity.
  • Isolated scFV antibodies immunoreactive with a peptide having activity of a B lymphocyte antigen can subsequently be formulated into a pharmaceutical preparation for use in the subject method.
  • the antibody library is screened with a B lymphocyte antigen protein, or peptide fragment thereof, to identify and isolate packages that express an antibody having specificity for the B lymphocyte antigen.
  • Nucleic acid encoding the selected antibody can be recovered from the display package (e.g., from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques.
  • the antibodies of the current invention can be used therapeutically to inhibit T cell activation through blocking recepto ⁇ ligand interactions necessary for costimulation of the T cell.
  • blocking antibodies can be identified by their ability to inhibit T cell proliferation and/or cytokine production when added to an in vitro costimulation assay as described herein.
  • the ability of blocking antibodies to inhibit T cell functions may result in immunosuppression and/or tolerance when these antibodies are administered in vivo.
  • polyclonal or monoclonal antibodies of the current invention such as an antibody specifically reactive with a recombinant or synthetic peptide having B7-2 activity or B7-3 activity can also be used to isolate the native B lymphocyte antigen from cells.
  • antibodies reactive with the peptide can be used to isolate the naturally-occurring or native form of B 7-2 from activated B lymphocytes by immunoaffinity chromatography.
  • the native form of B7-3 can be isolated from B cells by immunoaffinity chromatography with monoclonal antibody BB-1.
  • nucleic acid sequences and novel B lymphocyte antigens described herein can be used in the development of therapeutic reagents having the ability to either upregulate (e.g., amplify) or downregulate (e.g., suppress or tolerize) T cell mediated immune responses.
  • upregulate e.g., amplify
  • downregulate e.g., suppress or tolerize
  • peptides having B7-2 activity including soluble, monomeric forms of the B7-2 antigen or a B7-2 fusion protein, e.g., B7-2Ig, and anti-B7-2 antibodies that fail to deliver a costimulatory signal to T cells that have received a primary activation signal, can be used to block the B7-2 ligand(s) on T cells and thereby provide a specific means by which to cause immunosuppression and/or induce tolerance in a subject.
  • Such blocking or inhibitory forms of B lymphocyte antigens and fusion proteins and blocking antibodies can be identified by their ability to inhibit T cell proliferation and or cytokine production when added to an in vitro costimulation assay as previously described herein.
  • stimulatory forms of B7-2 such as an intact cell surface B7-2, retain the ability to transmit the costimulatory signal to the T cells, resulting in an increased secretion of cytokines when compared to activated T cells that have not received the secondary signal.
  • fusion proteins comprising a first peptide having an activity of B 7-2 fused to a second peptide having an activity of another B lymphocyte antigen (e.g., B7-1) can be used to modify T cell mediated immune responses.
  • B7-1 B lymphocyte antigens
  • B7-2 and B7-1 B7-2 and B7-1
  • blocking antibodies e.g., anti-B7-2 and anti-B7-l monoclonal antibodies
  • a therapeutically active amount of one or more peptides having B7-2 activity and or B7-1 activity can be used in conjunction with other immunomodulating reagents to influence immune responses.
  • other immunomodulating reagents include blocking antibodies, e.g., against CD28 or CTLA4, against other T cell markers or against cytokines, fusion proteins, e.g., CTLA4Ig, or immunosuppressive drugs, e.g., cyclosporine A or FK506.
  • the peptides produced from the nucleic acid molecules of the present invention may also be useful in the construction of therapeutic agents which block T cell function by destruction of the T cell.
  • secreted forms of a B lymphocyte antigen can be constructed by standard genetic engineering techniques.
  • a soluble form of B7-1, B7-2 or B7-3 can be constructed by standard genetic engineering techniques.
  • a toxin such as ricin
  • an agent capable of preventing T cell activation can be made.
  • Infusion of one or a combination of immunotoxins, e.g., B7-2-ricin, B7-1 -ricin, into a patient may result in the death of T cells, particularly of activated T cells that express higher amounts of CD28 and CTLA4.
  • Soluble forms of B7-2 in a monovalent form alone may be useful in blocking B7-2 function, as described above, in which case a carrier molecule may also be employed.
  • Another method of preventing the function of a B lymphocyte antigen is through the use of an antisense or triplex oligonucleotide.
  • an oligonucleotide complementary to the area around the B7-1, B7-2 or B7-3 translation initiation site e.g., for B7-1, TGGCCCATGGCTTCAGA, (SEQ ID NO:20) nucleotides 326-309 and for B7-2, GCCAAAATGGATCCCCA (SEQ ID NO:21)
  • One or more antisense oligonucleotides can be added to cell media, typically at 200 ⁇ g/ml, or administered to a, patient to prevent the synthesis of B7-1, B7-2 and/or B7-3.
  • the antisense oligonucleotide is taken up by cells and hybridizes to the appropriate B lymphocyte antigen mRNA to prevent translation.
  • an oligonucleotide which binds double-stranded DNA to form a triplex construct to prevent DNA unwinding and transcription can be used. As a result of either, synthesis of one or more B lymphocyte antigens is blocked.
  • B lymphocyte antigens Given the structure and function of the novel B lymphocyte antigens disclosed herein, it is possible to downregulate the function of a B lymphocyte antigen, and thereby downregulate immune responses, in a number of ways. Downregulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response.
  • the functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.
  • Tolerance which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
  • Downregulating or preventing one or more B lymphocyte antigen functions e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft- versus-host disease (GVHD).
  • GVHD graft- versus-host disease
  • blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
  • rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.
  • a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody
  • B7 lymphocyte antigen e.g., B7-1, B7-3 or blocking antibody
  • Blocking B lymphocyte antigen function in this manner prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.
  • the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.
  • Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.
  • To acheive sufficient immunosuppression or tolerance in a subject it may also be necessary to block the function of a combination of B lymphocyte antigens.
  • B7-2 and B7-1, B7-2 and B7-3, B7-1 and B7-3 or B7-2, B7-1 and B7-3 by administering a soluble form of a combination of peptides having an activity of each of these antigens or a blocking antibody (separately or together in a single composition) prior to transplantation.
  • inhibitory forms of B lymphocyte antigens can be used with other suppressive agents such as blocking antibodies against other T cell markers or against cytokines, other fusion proteins, e.g., CTLA4Ig, or immunosuppressive drugs.
  • the efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.
  • the functionally important aspects of B7-1 are conserved structurally between species and it is therefore likely that other B lymphocyte antigens can function across species, thereby allowing use of reagents composed of human proteins in animal systems.
  • appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science, 212: 789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci. USA, £2: 11102- 11105 (1992).
  • murine models of GVHD see Paul ed., Fundamental
  • Blocking B lymphocyte antigen function e.g., by use of a peptide having B7-2 activity alone or in combination with a peptide having B7-1 activity and/or a peptide having B7-3 activity, may also be therapeutically useful for treating autoimmune diseases.
  • Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
  • reagents which block costimulation of T cells by disrupting recepto ⁇ ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell- derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.
  • the efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases.
  • Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).
  • the IgE antibody response in atopic allergy is highly T cell dependent and, thus, inhibition of B lymphocyte antigen induced T cell activation may be useful therapeutically in the treatment of allergy and allergic reactions.
  • An inhibitory form of B7-2 protein such as a peptide having B7-2 activity alone or in combination with a peptide having the activity of another B lymphocyte antigen, such as B7-1, can be administered to an allergic subject to inhibit T cell mediated allergic responses in the subject.
  • Inhibition of B lymphocyte antigen costimulation of T cells may be funeralied by exposure to allergen in conjunction with appropriate MHC molecules.
  • Allergic reactions may be systemic or local in nature, depending on the route of entry of the allergen and the pattern of deposition of IgE on mast cells or basophils. Thus, it may be necessary to inhibit T cell mediated allergic responses locally or systemically by proper administration of an inhibitory form of B7-2 protein.
  • Inhibition of T cell activation through blockage of B lymphocyte antigen function may also be important therapeutically in viral infections of T cells.
  • AIDS acquired immune deficiency syndrome
  • Blocking B7-2 function could lead to a lower level of viral replication and thereby ameliorate the course of AIDS.
  • B7-1, B7-2 and B7-3 it may also be necessary to block the function of a combination of B lymphocyte antigens i.e., B7-1, B7-2 and B7-3.
  • HTLV-I infected T cells express B7-1 and B7-2.
  • This expression may be important in the growth of HTLV-I infected T cells and the blockage of B 7-1 function together with the function of B7-2 and or B7-3 may slow the growth of HTLV-I induced leukemias.
  • stimulation of viral replication by T cell activation may be induced by contact with a stimulatory form of B7-2 protein, for such purposes as generating retroviruses (e.g., various HIV isolates) in sufficient quantities for isolatation and use.
  • Upregulation of a B lymphocyte antigen function may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. Viral infections are cleared primarily by cytolytic T cells. In accordance with the present invention, it is believed that B7-2 and thus, B7-1 and B7-3 with their natural ligand(s) on T cells may result in an increase in the cytolytic activity of at least some T cells.
  • B7-2, B7-1, and B7-3 are involved in the initial activation and generation of CD8+ cytotoxic T cells.
  • a soluble peptide having B7-2 activity alone, or in combination with a peptide having the activity of another B lymphocyte antigen, in a multi-valent form, to stimulate T cell activity through the costimulation pathway would thus be therapeutically useful in situations where more rapid or thorough clearance of virus would be beneficial.
  • viral skin diseases such as Herpes or shingles, in which cases the multi-valent soluble peptide having B7-2 activity or combination of such peptide and/or a peptide having B7-1 activity and/or a peptide having B7-3 activity is delivered topically to the skin.
  • systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.
  • anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide having B7-2 activity (alone or in combination with a peptide having B7-1 activity and/or a peptide having B7-3 activity) or together with a stimulatory form of a soluble peptide having B7-2 activity (alone or in combination with a peptide having B7-1 activity and/or a peptide having B7-3 activity) and reintroducing the in vitro activated T cells into the patient.
  • viral antigen-pulsed APCs either expressing a peptide having B7-2 activity (alone or in combination with a peptide having B7-1 activity and/or a peptide having B7-3 activity) or together with a stimulatory form of a soluble peptide having B7-2 activity (alone or in combination with a peptide having B7-1 activity and/or a
  • Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a peptide having the activity of a B lymphocyte antigen as described herein such that the cells express all or a portion of a B lymphocyte antigen on their surface, e.g., B7-2 or B7-3, and reintroduce the transfected cells into the patient.
  • the infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
  • Stimulatory forms of B lymphocyte antigens may also be used prophylactically in vaccines against various pathogens.
  • Immunity against a pathogen e.g., a virus, could be induced by vaccinating with a viral protein along with a stimulatory form of a peptide having B7-2 activity or another peptide having the activity of B lymphocyte antigen in an appropriate adjuvant.
  • an expression vector which encodes genes for both a pathogenic antigen and a peptide having the activity of a B lymphocyte antigen e.g., a vaccinia virus expression vector engineered to express a nucleic acid encoding a viral protein and a nucleic acid encoding a peptide having B7-2 activity as described herein, can be used for vaccination.
  • Presentation of B7-2 with class I MHC proteins by, for example, a cell transfected to coexpress a peptide having B7-2 activity and MHC class I ⁇ chain protein and ⁇ 2 microglobulin may also result in activation of cytolytic CD8+ T cells and provide immunity from viral infection.
  • Pathogens for which vaccines may be useful include hepatitis B, hepatitis C, Epstein-Barr virus, cytomegalovirus, HIV-1, HIV-2, tuberculosis, malaria and schistosomiasis.
  • a stimulatory form of one or more soluble peptides having an activity of a B lymphocyte antigen can be administered to a tumor-bearing patient to provide a costimulatory signal to T cells in order to induce anti-tumor immunity.
  • the inability of a tumor cell to trigger a costimulatory signal in T cells may be due to a lack of expression of a costimulatory molecule, failure to express a costimulatory molecule even though the tumor cell is capable of expressing such a molecule, insufficient expression of a costimulatory molecule on the tumor cell surface or lack of expression of an appropriate costimulatory molecule (e.g. expression of B7 but not B7-2 and/or B7-3).
  • a tumor cell is modified to express B7-2 and/or B7-3 by transfection of the tumor cell with a nucleic acid encoding B7-2 and/or B7-3 in a form suitable for expression of B7-2 and/or B7-3 on the tumor cell surface.
  • the tumor cell is modified by contact with an agent which induces or increases expression of B7- 2 and/or B7-3 on the tumor cell surface.
  • B7-2 and/or B7-3 is coupled to the surface of the tumor cell to produce a modified tumor cell.
  • Tumor cells can be modified ex vivo to express B7-2 or B7-3, alone or in combination or in combination with B7-1 by transfection of isolated tumor cells with a nucleic acid encoding B7-2 and/or B7-3 and B7-1 in a form suitable for expression of the molecule on the surface of the tumor cell.
  • transfection refers to the introduction of exogenous nucleic acid into a mammalian cell and encompass a variety of techniques useful for introduction of nucleic acids into mammalian cells including electroporation, calcium-phosphate precipitation, DEAE-dextran treatment, lipofection, microinj ection and infection with viral vectors. Suitable methods for transfecting mammalian cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory press (1989)) and other laboratory textbooks.
  • the nucleic acid to be introduced may be, for example, DNA encompassing the gene(s) encoding B7-2 and/or B7-3, sense strand RNA encoding B7-2 and/or B7-3 or a recombinant expression vector containing a cDNA encoding B7-2 and/or B7-3.
  • the nucleotide sequence of a cDNA encoding human B7-2 is shown in the Sequence Listing.
  • a preferred approach for introducing nucleic acid encoding B7-2 and/or B7-3 into tumor cells is by use of a viral vector containing nucleic acid, e.g. a cDNA, encoding B7-2 and/or B7-3.
  • viral vectors which can be used include retroviral vectors (Eglitis, M.A., et al., Science 230, 1395-1398 (1985); Danos, O. and Mulligan, R., Proc. Natl. Acad. Sci. USA 85, 6460-6464 (1988); Markowitz, D., et al., J. Virol.
  • adenoviral vectors Rosenfeld, M.A., et al., Cell 68, 143-155 (1992)
  • adeno-associated viral vectors Tratschin, J.D., et al., Mol. Cell. Biol. 5, 3251-3260 (1985)
  • Infection of tumor cells with a viral vector has the advantage that a large proportion of cells will receive nucleic acid, thereby obviating a need for selection of cells which have received nucleic acid, and molecules encoded within the viral vector, e.g. by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.
  • B7-2 and/or B7-3 can be expressed on a tumor cell using a plasmid expression vector which contains nucleic acid, e.g. a cDNA, encoding B7-2 and/or B7-3.
  • a plasmid expression vector which contains nucleic acid, e.g. a cDNA, encoding B7-2 and/or B7-3.
  • Suitable plasmid expression vectors include CDM8 (Seed, B., Nature 329, 840 (1987)) and pMT2PC (Kaufman, et aL, EMBOJ. 6, 187-195 (1987)).
  • Suitable vectors and methods for expressing nucleic acids in host cells, such as tumor cells are described in further detail herein.
  • tumor cells When transfection of tumor cells leads to modification of a large proportion of the tumor cells and efficient expression of B7-2 and/or B7-3 on the surface of tumor cells, e.g. when using a viral expression vector, tumor cells may be used without further isolation or subcloning.
  • a homogenous population of transfected tumor cells can be prepared by isolating a single transfected tumor cell by limiting dilution cloning followed by expansion of the single tumor cell into a clonal population of cells by standard techniques.
  • a tumor cell can be modified to trigger a costimulatory signal in T cells by inducing or increasing the level of expression of B7-2 and/or B7-3 on a tumor cell which is capable of expressing B7-2 and/or B7-3 but fails to do so or which expresses insufficient amounts of B7-2 and/or B7-3 to activate T cells.
  • An agent which stimulates expression of B7-2 and/or B7-3 can be used in order to induce or increase expression of B7-2 and/or B7-3 on the tumor cell surface.
  • tumor cells can be contacted with the agent in vitro in a culture medium.
  • the agent which stimulates expression of B7-2 and/or B7-3 may act, for instance, by increasing transcription of B7-2 and/or B7-3 gene, by increasing translation of B7-2 and/or B7-3 mRNA or by increasing stability or transport of B7-2 and or B7-3 to the cell surface.
  • expression of B7 can be upregulated in a cell by a second messenger pathway involving cAMP. Nabavi, N., et al. Nature 360, 266-268 (1992).
  • B7-2 and B7-3 may likewise be inducible by cAMP.
  • a tumor cell can be contacted with an agent, which increases intracellular cAMP levels or which mimics cAMP, such as a cAMP analogue, e.g.
  • B7-2 and/or B7-3 can stimulate expression of B7-2 and/or B7-3 on the tumor cell surface.
  • expression of B7 can be induced on normal resting B cells by crosslinking cell-surface MHC class II molecules on the B cells with an antibody against the MHC class II molecules. Kuolova, L., et al., J. Exp. Med. 173, 759-762 (1991).
  • B7-2 and B7-3 can be induced on resting B cells by crosslinking cell-surface MHC class II molecules on the B cells.
  • a tumor cell which expresses MHC class II molecules on its surface can be treated with anti-MHC class II antibodies to induce or increase B7-2 and or B7-3 expression on the tumor cell surface.
  • interleukin-4 which has been found to induce expression of B7-2 on B cells, may be used to upregulate expression of B7-2 on tumor cells (Stack R.M., et al., J. Cell. Biochem. Suppl 1(18):434 (1994).
  • Another agent which can be used to induce or increase expression of B7-2 and/or B7- 3 on a tumor cell surface is a nucleic acid encoding a transcription factor which upregulates transcription of the gene encoding the costimulatory molecule.
  • This nucleic acid can be transfected into the tumor cell to cause increased transcription of the costimulatory molecule gene, resulting in increased cell-surface levels of the costimulatory molecule.
  • a tumor cell is modified to be capable of triggering a costimulatory signal in T cells by coupling B7-2 and/or B7-3 to the surface of the tumor cell.
  • B7-2 and/or B7-3 molecules can be obtained using standard recombinant DNA technology and expression systems which allow for production and isolation of the costimulatory molecule(s).
  • B7-2 and/or B7-3 can be isolated from cells which express the costimulatory molecule(s) using standard protein purification techniques.
  • B7-3 protein can be isolated from activated B cells by immunoprecipitation with an anti-B7-3 antibody such as the BBl monoclonal antibody. The isolated costimulatory molecule is then coupled to the tumor cell.
  • Coupled refers to a chemical, enzymatic or other means (e.g., antibody) by which B7-2 and or B7-3 is linked to a tumor cell such that the costimulatory molecule is present on the surface of the tumor cell and is capable of triggering a costimulatory signal in T cells.
  • B7-2 and/or B7-3 can be chemically crosslinked to the tumor cell surface using commercially available crosslinking reagents (Pierce, Rockford IL).
  • Another approach to coupling B7-2 and/or B7-3 to a tumor cell is to use a bispecific antibody which binds both the costimulatory molecule and a cell- surface molecule on the tumor cell. Fragments, mutants or variants of B7-2 and/or B7-3 which retain the ability to trigger a costimulatory signal in T cells when coupled to the surface of a tumor cell can also be used.
  • Another aspect of the invention is a tumor cell modified to express multiple costimulatory molecules.
  • the temporal expression of costimulatory molecules on activated B cells is different for B7, B7-2 and B7-3.
  • B7-2 is expressed early following B cell activation, whereas B7-3 is expressed later.
  • the different costimulatory molecules may thus serve distinct functions during the course of an immune response.
  • An effective T cell response may require that the T cell receive costimulatory signals from multiple costimulatory molecules.
  • the invention encompasses a tumor cell which is modified to express more than one costimulatory molecule.
  • a tumor cell can be modified to express both B7-2 and B7-3.
  • a tumor cell modified to express B7- 2 can be further modified to express B7-1.
  • a tumor cell modified to express B7-3 can be further modified to express B7-1.
  • a tumor cell can also be modified to express B7-1, B7-2 and B7-3.
  • a tumor cell can be modified to express multiple costimulatory molecules (e.g., B7-1 and B7-2) by any of the techniques described herein.
  • a tumor cell Before modification, a tumor cell may not express any costimulatory molecules, or may express certain costimulatory molecules but not others.
  • tumor cells can be modified by transfecting the tumor cell with nucleic acid encoding a costimulatory molecule(s), by inducing the expression of a costimulatory molecule(s) or by coupling a costimulatory molecule(s) to the tumor cell.
  • a tumor cell transfected with nucleic acid encoding B7-2 can be further transfected with nucleic acid encoding B7-1.
  • the cDNA sequence and deduced amino acid sequence of human B7-1 is shown in the Sequence Listing.
  • more than one type of modification can be used.
  • a tumor cell transfected with a nucleic acid encoding B7-2 can be stimulated with an agent which induces expression of B7-1.
  • tumor cells which express a costimulatory molecule and which express one or more MHC molecules on their surface to trigger both a costimulatory signal and a primary, antigen-specific, signal in T cells.
  • tumor cells may be unable to express MHC molecules, may fail to express MHC molecules although they are capable of expressing such molecules, or may express insufficient amounts of MHC molecules on the tumor cell surface to cause T cell activation.
  • Tumor cells can be modified to express either MHC class I or MHC class II molecules, or both.
  • One approach to modifying tumor cells to express MHC molecules is to transfect the tumor cell with one or more nucleic acids encoding one or more MHC molecules.
  • an agent which induces or increases expression of one or more MHC molecules on tumor cells can be used to modify tumor cells.
  • Inducing or increasing expression of MHC class II molecules on a tumor cell can be particularly beneficial for activating CD4 + T cells against the tumor since the ability of MHC class II + tumor cells to directly present tumor peptides to CD4 + T cells bypasses the need for professional MHC class II + APCs. This can improve tumor immunogenicity because soluble tumor antigen (in the form of tumor cell debris or secreted protein) may not be available for uptake by professional MHC class II + APCs.
  • One embodiment of the invention is a modified tumor cell which expresses B7-2 and/or B7-3 and one or more MHC class II molecules on their cell surface.
  • MHC class II molecules are cell-surface ⁇ / ⁇ heterodimers which structurally contain a cleft into which antigenic peptides bind and which function to present bound peptides to the antigen-specific TcR.
  • Multiple, different MHC class II proteins are expressed on professional APCs and different MHC class II proteins bind different antigenic peptides. Expression of multiple MHC class II molecules, therefore, increases the spectrum of antigenic peptides that can be presented by an APC or by a modified tumor cell.
  • the ⁇ and ⁇ chains of MHC class II molecules are encoded by different genes.
  • the human MHC class II protein HLA-DR is encoded by the HLA-DR ⁇ and HLA-DR ⁇ genes.
  • Another embodiment of the invention is a modified tumor cell which expresses B7-2 and/or B7-3 and one or more MHC class I molecules on the cell surface. Similar to MHC class II genes, there are multiple MHC class I genes and many polymorphic alleles of these genes are found in human and other species. Like MHC class II proteins, class I proteins bind peptide fragments of antigens for presentation to T cells. A functional cell-surface class I molecule is composed of an MHC class I ⁇ chain protein associated with a ⁇ 2- microglobulin protein.
  • Tumor cells can be modified ex vivo to express one or more MHC class II molecules by transfection of isolated tumor cells with one or more nucleic acids encoding one or more MHC class II ⁇ chains and one or more MHC class II ⁇ chains in a form suitable for expression of the MHC class II molecules(s) on the surface of the tumor cell.
  • Both an ⁇ and a ⁇ chain protein must be present in the tumor cell to form a surface heterodimer and neither chain will be expressed on the cell surface alone.
  • the nucleic acid sequences of many murine and human class II genes are known. For examples see Hood, L., et al. Ann. Rev. Immunol.
  • the introduced MHC class II molecule is a self MHC class II molecule.
  • the MHC class II molecule could be a foreign, allogeneic, MHC class II molecule.
  • a particular foreign MHC class II molecule to be introduced into tumor cells can be selected by its ability to induce T cells from a tumor-bearing subject to proliferate and/or secrete cytokines when stimulated by cells expressing the foreign MHC class II molecule (i.e. by its ability to induce an allogeneic response).
  • the tumor cells to be transfected may not express MHC class II molecules on their surface prior to transfection or may express amounts insufficient to stimulate a T cell response.
  • tumor cells which express MHC class II molecules prior to transfection can be further transfected with additional, different MHC class II genes or with other polymorphic alleles of MHC class II genes to increase the spectrum of antigenic fragments that the tumor cells can present to T cells.
  • a preferred variant is an MHC class II molecule in which the cytoplasmic domain of either one or both of the ⁇ and ⁇ chains is truncated. It is known that truncation of the cytoplasmic domains allows peptide binding by and cell surface expression of MHC class II molecules but prevents the induction of endogenous B7 expression, which is triggered by an intracellular signal generated by the cytoplasmic domains of the MHC class II protein chains upon crosslinking of cell surface MHC class II molecules. Kuolova.
  • B7-2 and B7-3 are also induced by crosslinking surface MHC class II molecules, and thus truncation of MHC class II molecules may also prevent induction of B7-2 and/or B7-3.
  • endogenous costimulatory molecules for instance to restrain potential downregulatory feedback mechanisms.
  • Transfection of a tumor cell with a nucleic acid(s) encoding a cytoplasmic domain-truncated form of MHC class II ⁇ and ⁇ chain proteins would inhibit endogenous B7-1 expression and possibly also endogenous B7-2 and B7-3 expression.
  • Such variants can be produced by, for example, introducing a stop codon in the MHC class II chain gene(s) after the nucleotides encoding the transmembrane spanning region.
  • the cytoplasmic domain of either the chain or the ⁇ chain protein can be truncated, or, for more complete inhibition of B7 (and possibly B7-2 and/or B7-3) induction, both the ⁇ and ⁇ chains can be truncated. See e.g.
  • Tumor cells can be modified to express an MHC class I molecule by transfection with a nucleic acid encoding an MHC class I ⁇ chain protein.
  • nucleic acids see Hood, L., et al. Ann. Rev. Immunol. 1, 529-568 (1983) and Auffray, C. and Strominger, J.L., Advances in Human Genetics 15, 197-247 (1987).
  • the tumor cell if it does not express ⁇ -2 microglobuhn, it can also be transfected with a nucleic acid encoding the ⁇ -2 microglobuhn protein.
  • nucleic acids see Gussow, D., et al., J. Immunol. 139, 3132-3138 (1987) and Parnes, J.R., et al., Proc. Natl. Acad. Sci. USA 78, 2253-2257 (1981).
  • MHC class II molecules increasing the number of different MHC class I genes or polymorphic alleles of MHC class I genes expressed in a tumor cell can increase the spectrum of antigenic fragments that the tumor cells can present to T cells.
  • the transfections can be performed simultaneously or sequentially. If the transfections are performed simultaneously, the molecules can be introduced on the same nucleic acid, so long as the encoded sequences do not exceed a carrying capacity for a particular vector used. Alternatively, the molecules can be encoded by separate nucleic acids. If the transfections are conducted sequentially and tumor cells are selected using a selectable marker, one selectable marker can be used in conjunction with the first introduced nucleic acid while a different selectable marker can be used in conjunction with the next introduced nucleic acid.
  • MHC molecules class I or class II
  • the expression of MHC molecules (class I or class II) on the cell surface of a tumor cell can be determined, for example, by immunoflourescence of tumor cells using fluorescently labeled monoclonal antibodies directed against different MHC molecules.
  • Monoclonal antibodies which recognize either non-polymorphic regions of a particular MHC molecule (non-allele specific) or polymorphic regions of a particular MHC molecule (allele- specific) can be used and are known to those skilled in the art.
  • MHC Molecules on a Tumor Cell
  • an agent which stimulates expression of MHC molecules in order to induce or increase expression of MHC molecules on the tumor cell surface.
  • tumor cells can be contacted with the agent in vitro in a culture medium.
  • An agent which stimulates expression of MHC molecules may act, for instance, by increasing transcription of MHC class I and/or class II genes, by increasing translation of MHC class I and/or class II mRNAs or by increasing stability or transport of MHC class I and/or class II proteins to the cell surface.
  • cytokine interferon- ⁇ One agent which upregulates MHC class I and class II molecules on a wide variety of cell types is the cytokine interferon- ⁇ .
  • tumor cells modified to express B7-2 and/or B7-3 and B7-1 can be further modified to increase expression of MHC molecules by contact with interferon- ⁇ .
  • Another agent which can be used to induce or increase expression of an MHC molecule on a tumor cell surface is a nucleic acid encoding a transcription factor which upregulates transcription of MHC class I or class II genes.
  • a nucleic acid can be transfected into the tumor cell to cause increased transcription of MHC genes, resulting in increased cell-surface levels of MHC proteins.
  • MHC class I and class II genes are regulated by different transcription factors. However, the multiple MHC class I genes are regulated coordinately, as are the multiple MHC class II genes. Therefore, transfection of a tumor cell with a nucleic acid encoding a transcription factor which regulates MHC gene expression may increase expression of several different MHC molecules on the tumor cell surface.
  • Another embodiment of the invention provides a tumor cell modified to express a T cell costimulatory molecule (e.g., B7-2 and/or B7-3 and B7-1) and in which expression of an MHC class II-associated protein, the invariant chain, is inhibited.
  • T cell costimulatory molecule e.g., B7-2 and/or B7-3 and B7-1
  • MHC class II-associated protein the invariant chain
  • an oligonucleotide complementary to nucleotides near the translation initiation site of the Ii mRNA can be synthesized.
  • One or more antisense oligonucleotides can be added to media containing tumor cells, typically at a concentration of oligonucleotides of 200 ⁇ g/ml. The antisense oligonucleotide is taken up by tumor cells and hybridizes to Ii mRNA to prevent translation.
  • a recombinant expression vector in which a nucleic acid encoding sequences of the Ii gene in an orientation such that mRNA which is antisense to a coding or regulatory region of the Ii gene is produced.
  • Tumor cells transfected with this recombinant expression vector thus contain a continuous source of Ii antisense nucleic acid to prevent production of Ii protein.
  • Ii expression in a tumor cell can be inhibited by treating the tumor cell with an agent which interferes with Ii expression.
  • an agent which interferes with Ii expression for example, a pharmaceutical agent which inhibits Ii gene expression, Ii mRNA translation or Ii protein stability or intracellular transport can be used.
  • the tumor cells to be modified as described herein include tumor cells which can be transfected or treated by one or more of the approaches encompassed by the present invention to express B7-2 and or B7-3, alone or in combination with B7-1. If necessary, the tumor cells can be further modified to express MHC molecules or an inhibitor of Ii expression.
  • a tumor from which tumor cells are obtained can be one that has arisen spontaneously, e.g in a human subject, or may be experimentally derived or induced, e.g. in an animal subject.
  • the tumor cells can be obtained, for example, from a solid tumor of an organ, such as a tumor of the lung, liver, breast, colon, bone etc.
  • Malignancies of solid organs include carcinomas, sarcomas, melanomas and neuroblastomas.
  • the tumor cells can also be obtained from a blood-borne (ie. dispersed) malignancy such as a lymphoma, a myeloma or a leukemia.
  • the tumor cells to be modified include those that express MHC molecules on their cell surface prior to transfection and those that express no or low levels of MHC class I and/or class II molecules.
  • a minority of normal cell types express MHC class II molecules. It is therefore expected that many tumor cells will not express MHC class II molecules naturally.
  • These tumors can be modified to express B7-2 and/or B7-3 and MHC class II molecules.
  • Several types of tumors have been found to naturally express surface MHC class II molecules, such as melanomas (van Duinen et al., Cancer Res.
  • Tumor cells which naturally express class II molecules can be modified to express B7-2 and/or B7-3, and, in addition, other class II molecules which can increase the spectrum of TAA peptides which can be presented by the tumor cell. Most non-malignant cell types express MHC class I molecules.
  • Types of tumors in which MHC class I expression has been shown to be inhibited include melanomas, colorectal carcinomas and squamous cell carcinomas, van Duinen et al., Cancer Res. 48, 1019-1025, (1988); Moller et al., Int. J. Cancer 6, 155-162, (1991); Csiba, A., et al., Brit. J. Cancer 50, 699-709 (1984); Holden, C.A., et al. J. Am. Acad. Dermatol. 9., 867-871 (1983).
  • a tumor cell which fails to express class I molecules or which expresses only low levels of MHC class I molecules can be modified by one or more of the techniques described herein to induce or increase expression of MHC class I molecules on the tumor cell surface to enhance tumor cell immunogenicity. (10). Modification of Tumor Cells In Vivo
  • Another aspect of the invention provides methods for increasing the immunogenicity of a tumor cell by modification of the tumor cell in vivo to express B7-2 and/or B7-3 and B7- 1 to trigger a costimulatory signal in T cells.
  • tumor cells can be further modified in vivo to express MHC molecules to trigger a primary, antigen-specific, signal in T cells.
  • Tumor cells can be modified in vivo by introducing a nucleic acid encoding B7-2 and/or B7-3 and B7-1 into the tumor cells in a form suitable for expression of the costimulatory molecule(s) on the surface of the tumor cells.
  • nucleic acids encoding MHC class I or class II molecules or an antisense sequence of the Ii gene can be introduced into tumor cells in vivo.
  • a recombinant expression vector is used to deliver nucleic acid encoding B7-2 and/or B7-3 and B7-1 to tumor cells in vivo as a form of gene therapy.
  • Vectors useful for in vivo gene therapy have been previously described and include retroviral vectors, adenoviral vectors and adeno-associated viral vectors. See e.g. Rosenfeld, M.A., Cell 68, 143-155 (1992); Anderson, W.F., Science 226, 401-409 (1984); Friedman, T., Science 244, 1275-1281 (1989).
  • nucleic acid can be delivered to tumor cells in vivo by direct injection of naked nucleic acid into tumor cells. See e.g. Acsadi, G., et al., Nature 332, 815-818 (1991). A delivery apparatus is commercially available (BioRad).
  • the nucleic acid can be complexed with a carrier such as a liposome. Nucleic acid encoding an MHC class I molecule complexed with a liposome has been directly injected into tumors of melanoma patients. Hoffman, M., Science 256, 305- 309 (1992).
  • Tumor cells can also be modified in vivo by use of an agent which induces or increases expression of B7-2 and/or B7-3 and B7-1 (and, if necessary, MHC molecules) as described herein.
  • the agent may be administered systemically, e.g. by intravenous injection, or, preferably, locally to the tumor cells.
  • the modified tumor cells of the invention are useful for stimulating an anti-tumor T cell-mediated immune response by triggering an antigen-specific signal and a costimulatory signal in tumor-specific T cells.
  • effector populations of T cells are generated.
  • These effector T cell populations can include both CD4+ T cells and CD8+ T cell.
  • the effector populations are responsible for elimination of tumors cell, by, for example, cytolysis of the tumor cells. Once T cells are activated, expression of a costimulatory molecule is not required on a target cell for recognition of the target cell by effector T cells or for the effector functions of the T cells.
  • the anti- tumor T cell-mediated immune response induced by the modified tumor cells of the invention is effective against both the modified tumor cells and unmodified tumor cells which do not express a costimulatory molecule.
  • the density and/or type of MHC molecules on the cell surface required for the afferent and efferent phases of a T cell-mediated immune response can differ. Fewer MHC molecules, or only certain types of MHC molecules (e.g. MHC class I but not MHC class II) may be needed on a tumor cell for recognition by effector T cells than is needed for the initial activation of T cells. Therefore, tumor cells which naturally express low amounts of MHC molecules but are modified to express increased amounts of MHC molecules can induce a T cell-mediated immune response which is effective against the unmodified tumor cells.
  • tumor cells which naturally express MHC class I molecules but not MHC class II molecules which are then modified to express MHC class II molecules can induce a T cell-mediated immune response which includes effector T cell populations which can eliminate the parental MHC class I+, class II- tumor cells.
  • compositions of modified tumor cells in a biologically compatible form suitable for pharmaceutical administration to a subject in vivo comprises an amount of modified tumor cells and a physiologically acceptable carrier.
  • the amount of modified tumor cells is selected to be therapeutically effective.
  • biologically compatible form suitable for pharmaceutical administration in vivo means that any toxic effects of the tumor cells are outweighed by the therapeutic effects of the tumor cells.
  • a "physiologically acceptable carrier” is one which is biologically compatible with the subject. Examples of acceptable carriers include saline and aqueous buffer solutions. In all cases, the compositions must be sterile and must be fluid to the extent that easy syringability exists.
  • subject is intended to include living organisms in which tumors can arise or be experimentally induced. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
  • a therapeutically effective dose of modified tumor cells may vary according to such factors as age, sex and weight of the individual, the type of tumor cell and degree of tumor burden, and the immunological competency of the subject. Dosage regimens may be adjusted to provide optimum therapeutic responses. For instance, a single dose of modified tumor cells may be administered or several doses may be administered over time. Administration may be by injection, including intravenous, intramuscular, intraperitoneal and subcutaneous injections. (13).
  • T lymphocytes can be obtained from a subject, for example, from peripheral blood. Peripheral blood can be further fractionated to remove red blood cells and enrich for or isolate T lymophocytes or T lymphocyte subpopulations. T cells can be activated in vitro by culturing the T cells with tumor cells obtained from the subject (e.g.
  • the term "stimulatory form” means that the costimulatory molecule is capable of crosslinking its receptor on a T cell and triggering a costimulatory signal in T cells.
  • the stimulatory form of the costimulatory molecule can be, for example, a soluble multivalent molecule or an immobilized form of the costimulatory molecule, for instance coupled to a solid support. Fragments, mutants or variants (e.g.
  • fusion proteins of B7-2 and/or B7-3 which retain the ability to trigger a costimulatory signal in T cells can also be used.
  • a soluble extracellular portion of B7-2 and/or B7-3 is used to provide costimulation to the T cells.
  • the modified tumor cells of the present invention can be used to increase tumor immunogenicity, and therefore can be used therapeutically for inducing or enhancing T lymphocyte-mediated anti-tumor immunity in a subject with a tumor or at risk of developing a tumor.
  • a method for treating a subject with a tumor involves obtaining tumor cells from the subject, modifying the tumor cells ex vivo to express a T cell costimulatory molecule, for example by transfecting them with an appropriate nucleic acid, and administering a therapeutically effective dose of the modified tumor cells to the subject.
  • nucleic acids to be introduced into a tumor cell include nucleic acids encoding B7-2 and/or B7-3, alone or together with nucleic acids encoding B7-1, MHC molecules (class I or class II) or Ii antisense sequences as described herein.
  • tumor cells after tumor cells are obtained from a subject, they can be modified ex vivo using an agent which induces or increases expression of B7-2 and/or B7-3 (and possibly also using agent(s) which induce or increase B7-1 or MHC molecules).
  • Tumor cells can be obtained from a subject by, for example, surgical removal of tumor cells, e.g. a biopsy of the tumor, or from a blood sample from the subject in cases of blood-borne malignancies.
  • the cells used to induce the tumor can be used, e.g. cells of a tumor cell line.
  • Samples of solid tumors may be treated prior to modification to produce a single-cell suspension of tumor cells for maximal efficiency of transfection.
  • Possible treatments include manual dispersion of cells or enzymatic digestion of connective tissue fibers, e.g. by collagenase.
  • Tumor cells can be transfected immediately after being obtained from the subject or can be cultured in vitro prior to transfection to allow for further characterization of the tumor cells (e.g. determination of the expression of cell surface molecules).
  • the nucleic acids chosen for transfection can be determined following characterization of the proteins expressed by the tumor cell. For instance, expression of MHC proteins on the cell surface of the tumor cells and/or expression of the Ii protein in the tumor cell can be assessed.
  • Tumors which express no, or limited amounts of or types of MHC molecules (class I or class II) can be transfected with nucleic acids encoding MHC proteins; tumors which express Ii protein can be transfected with Ii antisense sequences.
  • tumor cells can be screened for introduction of the nucleic acid by using a selectable marker (e.g. drug resistance) which is introduced into the tumor cells together with the nucleic acid of interest.
  • a selectable marker e.g. drug resistance
  • the modified tumor cells Prior to administration to the subject, can be treated to render them incapable of further proliferation in the subject, thereby preventing any possible outgrowth of the modified tumor cells.
  • Possible treatments include irradiation or mitomycin C treatment, which abrogate the proliferative capacity of the tumor cells while maintaining the ability of the tumor cells to trigger antigen-specific and costimulatory signals in T cells and thus to stimulate an immune response.
  • the modified tumor cells can be administered to the subject by injection of the tumor cells into the subject.
  • the route of injection can be, for example, intravenous, intramuscular, intraperitoneal or subcutaneous.
  • Administration of the modified tumor cells at the site of the original tumor may be beneficial for inducing local T cell-mediated immune responses against the original tumor.
  • Administration of the modified tumor cells in a disseminated manner, e.g. by intravenous injection may provide systemic anti-tumor immunity and, furthermore, may protect against metastatic spread of tumor cells from the original site.
  • the modified tumor cells can be administered to a subject prior to or in conjunction with other forms of therapy or can be administered after other treatments such as chemotherapy or surgical intervention.
  • an effective T cell response may require exposure of the T cell to more than one type of costimulatory molecule.
  • the temporal sequence of exposure of the T cell to different costimulatory mocules may be important for generating an effective response. For example, it is known that upon activation, a B cell expresses B7-2 early in its response (about 24 hours after stimulation). Subsequently, B7-1 and B7-3 are expressed by the B cell (about 48-72 hours after stimulation). Thus, a T cell may require exposure to B7-2 early in the induction of an immune response by exposure to B7-1 and/or B7-3 in the immune response.
  • modified tumor cells can be administered at different times to a subject to generate an effective immune response against the tumor cells.
  • tumor cells modified to express B7-2 can be administered to a subject.
  • a tumor cell from the same tumor but modified to express B7- 3 (alone or in conjunction with B7-1) can be administered to the subject.
  • Another method for treating a subject with a tumor is to modify tumor cells in vivo to express B7-2 and or B7-3, alone or in conjunction with B7-1, MHC molecules and/or an inhibitor of Ii expression.
  • This method can involve modifying tumor cells in vivo by providing nucleic acid encoding the protein(s) to be expressed using vectors and delivery methods effective for in vivo gene therapy as described in a previous section herein.
  • one or more agents which induce or increase expression of B7-2 and/or B7-3, and possibly B7-1 or MHC molecules can be administered to a subject with a tumor.
  • the modified tumor cells of the current invention may also be used in a method for preventing or treating metastatic spread of a tumor or preventing or treating recurrence of a tumor.
  • anti-tumor immunity induced by B7-1 -expressing tumor cells is effective against subsequent challenge by tumor cells, regardless of whether the tumor cells of the re-exposure express B7-1 or not.
  • administration of modified tumor cells or modification of tumor cells in vivo as described herein can provide tumor immunity against cells of the original, unmodified tumor as well as metastases of the original tumor or possible regrowth of the original tumor.
  • the current invention also provides a composition and a method for specifically inducing an anti-tumor response in CD4 + T cells.
  • CD4 + T cells are activated by antigen in conjunction with MHC class II molecules. Association of peptidic fragments of TAAs with MHC class II molecules results in recognition of these antigenic peptides by CD4 + T cells.
  • Providing a subject with tumor cells which have been modified to express MHC class II molecules along with B7-2 and/or B7-3, or modified in vivo to express MHC class II molecules along with B7-2 and/or B7-3, can be useful for directing tumor antigen presentation to the MHC class II pathway and thereby result in antigen recognition by and activation of CD4 + T cells specific for the tumor cells.
  • CD4+ or CD8 + T cells in vivo by administration of anti-CD4 or anti-CD8 antibodies, can be used to demonstrate that specific anti-tumor immunity is mediated by a particular (e.g. CD4 + ) T cell subpopulation.
  • a particular (e.g. CD4 + ) T cell subpopulation e.g. CD4 +
  • modified tumor cells of the invention from one human subject as an immunogen to induce anti -tumor immunity in another human subject is prohibited by histocompatibility differences between unrelated humans.
  • use of modified tumor cells from one individual to induce anti- tumor immunity in another individual to protect against possible future occurrence of a tumor may be useful in cases of familial malignancies.
  • the tumor-bearing donor of tumor cells to be modified is closely related to the (non-tumor bearing) recipient of the modified tumor cells and therefore the donor and recipient share MHC antigens.
  • a strong hereditary component has been identified for certain types of malignancies, for example certain breast and colon cancers.
  • tumor cells from that individual could be modified to express B7-2 and/or B7-3, alone or in combination with B7-1 and administered to susceptible, histocompatible family members to induce an anti-tumor response in the recipient against the type of tumor to which the family is susceptible. This anti-tumor response could provide protective immunity to subsequent development of a tumor in the immunized recipient.
  • a subject e.g. a mouse
  • the modified tumor cells of the invention before being challenged with unmodified tumor cells.
  • the subject is initially exposed to TAA peptides on tumor cells together with B7-2 and/or B7-3, and B7-1 which activates TAA-specific T cells.
  • the activated T cells are then effective against subsequent challenge with unmodified tumor cells.
  • the subject's immune system will be exposed to unmodified tumor cells before exposure to the modified tumor cells of the invention.
  • the subject is initially exposed to TAA peptides on tumor cells in the absence of a costimulatory signal.
  • TAA-specific T cell tolerance is likely to induce TAA-specific T cell tolerance in those T cells which are exposed to and are in contact with the unmodified tumor cells. Secondary exposure of the subject to modified tumor cells which can trigger a costimulatory signal may not be sufficient to overcome tolerance in TAA-specific T cells which were anergized by primary exposure to the tumor.
  • Use of modified tumor cells to induce anti-tumor immunity in a subject already exposed to unmodified tumor cells may therefore be most effective in early diagnosed patients with small tumor burdens, for instance a small localized tumor which has not metastasized. In this situation, the tumor cells are confined to a limited area of the body and thus only a portion of the T cell repertoire may be exposed to tumor antigens and become anergized.
  • modified tumor cells in a systemic manner may expose non-anergized T cells to tumor antigens together with B7-2 and or B7-3 alone, or in combination with B7-1 thereby inducing an anti- tumor response in the non-anergized T cells.
  • the anti-tumor response may be effective against possible regrowth of the tumor or against micrometastases of the original tumor which may not have been detected.
  • additional signals such as a cytokine, may need to be provided to the subject together with the modified tumor cells.
  • IL-2 has been shown to be capable of restoring the alloantigen-specific responses of previously anergized T cells in an in vitro system when exogenous IL-2 is added at the time of secondary alloantigenic stimulation. Tan, P., et al. J. Exp. Med. Ill, 165-173 (1993).
  • Another approach to generating an anti-tumor T cell response in a subject despite tolerance of the subject's T cells to the tumor is to stimulate an anti-tumor response in T cells from another subject who has not been exposed to the tumor (referred to as a naive donor) and transfer the stimulated T cells from the naive donor back into the tumor-bearing subject so that the transferred T cells can mount an immune response against the tumor cells.
  • An anti-tumor response is induced in the T cells from the naive donor by stimulating the T cells in vitro with the modified tumor cells of the invention.
  • Such an adoptive transfer approach is generally prohibited in outbred populations because of histocompatibity differences between the transferred T cells and the tumor-bearing recipient.
  • a tumor-bearing subject (referred to as the host) is prepared for and receives an allogeneic bone marrow transplant from a naive donor by a known procedure.
  • Preparation of the host involves whole body irradiation, which destroys the host's immune system, including T cells tolerized to the tumor, as well as the tumor cells themselves.
  • Bone marrow transplantation is accompanied by treatment(s) to prevent graft versus host disease such as depletion of mature T cells from the bone marrow graft, treatment of the host with immunosuppressive drugs or treatment of the host with an agent, such as CTLA4Ig, to induce donor T cell tolerance to host tissues.
  • T cells from the naive donor are stimulated in vitro with tumor cells from the host which have been modified, as described herein, to express B7-2 and/or B7-3.
  • the donor T cells are initially exposed to tumor cells together with a costimulatory signal and therefore are activated to respond to the tumor cells.
  • These activated anti-tumor specific T cells are then transferred to the host where they are reactive against unmodified tumor cells. Since the host has been reconstituted with the donor's immune system, the host will not reject the transferred T cells and, additionally, the treatment of the host to prevent graft versus host disease will prevent reactivity of the transferred T cells with normal host tissues.
  • the peptides of the invention are administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo to either enhance or suppress T cell mediated immune response.
  • biologically compatible form suitable for administration in vivo is meant a form of the protein to be administered in which any toxic effects are outweighed by the therapeutic effects of the protein.
  • subject is intended to include living organisms in which an immune response can be elicited, e.g., mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
  • Administration of a peptide having the activity of a novel B lymphocyte antigen as described herein can be in any pharmacological form including a therapeutically active amount of peptide alone or in combination with a peptide having the activity of another B lymphocyte antigen and a pharmaceutically acceptable carrier.
  • Administration of a therapeutically active amount of the therapeutic compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessay to achieve the desired result.
  • a therapeutically active amount of a peptide having B7-2 activity may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of peptide to elicit a desired response in the individual. Dosage periods may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • the active compound (e.g., peptide) may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration.
  • the active compound may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.
  • a peptide having B7-2 activity may be necessary to coat the peptide with, or co-administer the peptide with, a material to prevent its inactivation.
  • a peptide hving B7-2 activity may be administered to an individual in an appropriate carrier, diluent or adjuvant, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes.
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon.
  • Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n- hexadecyl polyethylene ether.
  • Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol.
  • Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Strejan et aL, (1984) J. Neuroimmunol 2:27).
  • the active compound may also be administered parenterally or intraperitoneally.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, asorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating active compound (e.g., peptide having B7-2 activity) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • active compound e.g., peptide having B7-2 activity
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze- drying which yields a powder of the active ingredient (e.g., peptide) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the protein may be orally administered, for example, with an inert diluent or an assimilable edible carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • nucleic acid sequences encoding peptides having the activity of novel B lymphocyte antigens as described herein can be used to identify cytokines which are produced by T cells in response to stimulation by a form of B lymphocyte antigen, e.g., B7-2.
  • T cells can be suboptimally stimulated in vitro with a primary activation signal, such as phorbol ester, anti-CD3 antibody or preferably antigen in association with an MHC class II molecule, and given a costimulatory signal by a stimulatory form of B7-2 antigen, for instance by a cell transfected with nucleic acid encoding a peptide having B7-2 activity and expressing the peptide on its surface or by a soluble, stimulatory form of the peptide.
  • a primary activation signal such as phorbol ester, anti-CD3 antibody or preferably antigen in association with an MHC class II molecule
  • a stimulatory form of B7-2 antigen for instance by a cell transfected with nucleic acid encoding a peptide having B7-2 activity and expressing the peptide on its surface or by a soluble, stimulatory form of the peptide.
  • Known cytokines released into the media can be identified by ELISA or by the ability of an antibody which
  • An in vitro T cell costimulation assay as described above can also be used in a method for identifying novel cytokines which may be induced by costimulation. If a particular activity induced upon costimulation, e.g., T cell proliferation, cannot be inhibited by addition of blocking antibodies to known cytokines, the activity may result from the action of an unkown cytokine. Following costimulation, this cytokine could be purified from the media by conventional methods and its activity measured by its ability to induce T cell proliferation.
  • T cells would be given the primary activation signal and contacted with a selected cytokine, but would not be given the costimulatory signal. After washing and resting the T cells, the cells would be rechallenged with both a primary activation signal and a costimulatory signal. If the T cells do not respond (e.g., proliferate or produce IL-2) they have become tolerized and the cytokine has not prevented the induction of tolerance. However, if the T cells respond, induction of tolerance has been prevented by the cytokine.
  • T cells do not respond (e.g., proliferate or produce IL-2) they have become tolerized and the cytokine has not prevented the induction of tolerance. However, if the T cells respond, induction of tolerance has been prevented by the cytokine.
  • cytokine blocking antibody a cytokine blocking antibody
  • Another application of the peptide having the activity of a novel B lymphocyte antigen of the invention is the use of one or more of these peptides in screening assays to discover as yet undefined molecules which are inhibitors of costimulatory ligand binding and/or of intracellular signaling through T cells following costimulation.
  • a solid-phase binding assay using a peptide having the activity of a B lymphocyte antigen, such as B7-2 could be used to identify molecules which inhibit binding of the antigen with the appropriate T cell ligand (e.g., CTLA4, CD28).
  • an in vitro T cell costimulation assay as described above could be used to identify molecules which interfere with intracellular signaling through the T cells following costimulation as determined by the ability of these molecules to inhibit T cell proliferation and/or cytokine production (yet which do not prevent binding of B lymphocyte antigens to their receptors).
  • the compound cyclosporine A inhibits T cell activation through stimulation via the T cell receptor pathway but not via the CD28/CTLA4 pathway. Therefore, a different intracellular signaling pathway is involved in costimulation.
  • Molecules which interfere with intracellular signaling via the CD28/CTLA4 pathway may be effective as immunosuppressive agents in vivo (similar to the effects of cyclosporine A).
  • the monoclonal antibodies produced using the proteins and peptides of the current invention can be used in a screening assay for molecules which modulate the expression of B lymphocyte antigens on cells.
  • molecules which effect intracellular signaling which leads to induction of B lymphocyte antigens e.g. B7-2 or B7-3
  • B7-2 or B7-3 can be identified by assaying expression of one or more B lymphocyte antigens on the cell surface. Reduced immunofluorescent staining by an anti-B7-2 antibody in the presence of the molecule would indicate that the molecule inhibits intracellular signals.
  • Molecules which upregulate B lymphocyte antigen expression result in an increased immunofluorescent staining.
  • the effect of a molecule on expression of a B lymphocyte antigen, such as B7- 2 can be determined by detecting cellular B7-2 mRNA levels using a B7-2 cDNA as a probe.
  • a cell which expresses a peptide having B7-2 activity can be contacted with, a molecule to be tested, and an increase or decrease in B7-2 mRNA levels in the cell detected by standard technique, such as Northern hybridization analysis or conventional dot blot of mRNA or total poly(A + )RNAs using a B7-2 cDNA probe labeled with a detectable marker.
  • Molecules which modulate B lymphocyte antigen expression may be useful therapeutically for either upregulating or downregulating immune responses alone or in conjunction with soluble blocking or stimulating reagents.
  • a molecule which inhibits expression of B7-2 could be administered together with a B7-2 blocking reagent for immunosuppressive purposes.
  • Molecules which can be tested in the above-described assays include cytokines such as IL-4, ⁇ INF, IL-10, IL-12, GM-CSF and prostagladins.
  • Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation from single cell suspensions of normal human spleens and were separated into E- and E+ fractions by rosetting with sheep red blood cells (Boyd, A.W., et al. (1985) J. Immunol. 134. 1516). B cells were purified from the E- fraction by adherence of monocytes on plastic and depletion of residual T, natural killer cells (NK) and residual monocytes by two treatments with anti-MsIgG and anti-MsIgM coated magnetic beads (Advanced Magnetics, Cambridge, MA), using monoclonal antibodies: anti-CD4, -CD8, -CDl lb, -CD 14 and -CD 16.
  • CD4+ T cells were isolated from the E+ fraction of the same spleens after adherence on plastic and depletion of NK, B cells and residual monocytes with magnetic beads and monoclonal antibodies: anti-CD20, -CDl lb, -CD8 and -CD16.
  • CD28+ T cells were identically isolated from the E+ fraction using anti-CD20, -CDl lb, -CD 14 and -CD 16 monoclonal antibodies. The efficiency of the purification was analyzed by indirect immunofluorescence and flow cytometry using an EPICS flow cytometer (Coulter). B cell preparations were >95% CD20+, ⁇ 2% CD3+, ⁇ 1% CD14+.
  • CD4+ T cell preparations were >98% CD3+, >98% CD4+, ⁇ 1% CD8+, ⁇ 1% CD20+, ⁇ 1% CD 14+.
  • CD28+ T cell preparations were >98% CD3+, >98% CD28+, ⁇ 1% CD20+, ⁇ 1% CD14+.
  • IgM Monoclonal antibodies were used as purified Ig unless indicated otherwise: anti- B7:133, IgM is a blocking antibody and has been previously described (Freedman, A.S. et al. (1987) Immunol. Ill, 3260-3267); anti-B7:Bl.l, IgGl (RepliGen Corp., Cambridge, MA) (Nickoloff, B., et al (1993) Am. J. Pathol. 142, 1029-1040) is a non-blocking monoclonal antibody; BB-1 : IgM is a blocking antibody (Dr. E. Clark, University of Washington, Seattle, WA) (Yokochi, T., et al. (1982) J. Immunol.
  • anti-CD20 BI, IgG2a (Stashenko, P., et al.(1980) J. Immunol. 121, 1678-1685); anti-B5: IgM (Freedman, A., et al. (1985) J. Immunol. 114, 2228-2235); anti-CD8: 7PT 3F9, IgG2a; anti-CD4: 19Thy5D7, IgG2a; anti-CDl lb: Mol, IgM and anti-CD14: Mo2, IgM (Todd, R, et al. (1981) J. Immunol. 126.
  • anti-MHC class II 9-49, IgG2a (Dr R. Todd, University of Michigan, Ann Arbor) (Todd, R.I., et al. (1984) Hum Immunol. 1__, 23-40; anti-CD28: 9.3, IgG2a (Dr. C. June, Naval Research Institute, Bethesda) (Hansen, J.A., et al. (1980) Immunogenetics. 1Q, 247-260); anti-CDl 6: 3G8, IgGl (used as ascites) (Dr. J.
  • CHO Cell Transfection B7-1 transfectants were prepared from the B7-1 negative Chinese hamster ovary (CHO) cell line, fixed with paraformaldehyde and used as previously described (Gimmi, CD., et al. Proc. Natl. Acad. Sci USA ££, 6575-6579).
  • B cells activated for 72 hours were used as total activated B cell populations or were indirectly stained with anti-B7 (B 1.1 ) monoclonal antibody and fluorscein isothiocyanate (FITC) labeled goat anti-mouse immunoglobulin (Fisher, Pittsburgh, PA), and fractionated into B7-1+ and B7-1- populations by flow cytometric cell sorting (EPICS Elite flow cytometer, Coulter).
  • B7 B 1.1
  • FITC fluorscein isothiocyanate
  • Cell suspensions were stained by two step indirect membrane staining with lO ⁇ g/ml of primary monoclonal antibody followed by the appropriate secondary reagents.
  • immunoreactivity with anti-B7 (133) and BB-1 monoclonal antibodies was studied by indirect staining using goat anti-mouse Ig or immunoglobulin FITC (Fisher) as secondary reagent and immunoreactivity with fusion proteins was studied using biotinylated CTLA4Ig or biotinylated control-Ig and streptavidin-phycoerythrin as secondary reagent.
  • P » BS containing 10% AB serum was used as diluent and wash media. Cells were fixed with 0.1% paraformaldehyde and analyzed on a flow cytometer (EPICS Elite Coulter).
  • T cells were cultured at a concentration of lxl ⁇ 5 cells per well in 96-well flat bottom microtiter plate at 37°C for 3 days in 5% CO2- Syngeneic activated B cells (total B cell population or B7+ and B7- fractions) were irradiated (2500 rad) and added into the cultures at a concentration of lxl ⁇ 5 cells per well. Factors under study were added to the required concentration for a total final volume of 200 ⁇ l per well. When indicated, T cells were incubated with anti-CD28 Fab (final concentration of lO ⁇ g/ml), for 30 minutes at 4°C, prior to addition in experimental plates.
  • anti-CD28 Fab final concentration of lO ⁇ g/ml
  • CHO-B7 or B cells were incubated with CTLA4Ig or control-Ig (lO ⁇ g/ml) for 30 minutes at 4°C Thymidine incorporation as an index of mitogenic activity, was assessed after incubation with l ⁇ Ci (37kBq) of ⁇ methyl- ⁇ K ⁇ thymidine (Du Pont, Boston, MA) for the last 15 hours of the culture.
  • the cells were harvested onto filters and the radioactivity on the dried filters was measured in a Pharmacia beta plate liquid scintilation counter.
  • IL-2 and IL-4 concentrations were assayed by ELISA (R&D Systems, Minneapolis, MN and BioSource, Camarillo, CA) in culture supernatants collected at 24 hours after initiation of the culture.
  • FIG. 1 depicts the costimulatory response of human splenic CD28+ T cells, submitogenically activated with anti-CD3 monoclonal antibody, to either B7 (B7-1) transfected CHO cells (CHO-B7) or syngeneic splenic B cells activated with anti-Ig for 72 hours. -1H-Thymidine incorporation was assessed for the last 15 hours of a 72 hours culture.
  • IL-2 was assessed by ELISA in supernatants after 24 hours of culture (Detection limits of the assay: 31-2000 pg/ml).
  • Figure 1 is representative of seventeen experiments. Submitogenically activated CD28+ T cells proliferated and secreted high levels of IL-
  • anti-B7-l monoclonal antibody could completely abrogate both proliferation and IL-2 secretion delivered by CHO-B7
  • anti-B7-l monoclonal antibody consistently inhibited proliferation induced by activated B cells by only 50% whereas IL-2 secretion was totally inhibited.
  • both CTLA4Ig and Fab anti-CD28 monoclonal antibody completely blocked proliferation and IL-2 secretion.
  • anti-B7-l monoclonal antibody inhibited proliferation only 50% but consistently abrogated IL-2 secretion.
  • CTLA4Ig binding or blockade of CD28 with Fab anti-CD28 monoclonal antibody completely inhibited both proliferation and IL-2 secretion.
  • Control monoclonal antibody and control-Ig were not inhibitory.
  • CTLA4/CD28 binding costimulatory ligand(s) which might account for the residual, non-B7 mediated proliferation delivered by B7+ B cells, the effect of BB-1 monoclonal antibody on proliferation and IL-2 secretion was examined.
  • Figure 3b displays the costimulatory potential of B7-1- activated human splenic B cells. Irradiated B7-1- activated (72 hr) B cells could also deliver a significant costimulatory signal to submitogenically activated CD4+ lymphocytes. This costimulation was not accompanied by detectable IL-2 ( Figure 3b) or IL-4 accumulation and anti-B7-l monoclonal antibody did not inhibit proliferation. However, CTLA4Ig, Fab anti-CD28 monoclonal antibody, and BB-1 monoclonal antibody all completely inhibited proliferation.
  • FIG. 4 shows the cell surface expression of B7-1, B7-2 and B7-3 on fractionated B7-1+ and B7-1" activated B cell.
  • B7-1+ activated splenic B cells stained with anti-B7-l (133) monoclonal antibody, BB-1 monoclonal antibody, and bound CTLA4-Ig.
  • B7- activated splenic B cells did not stain with anti-B7-l (133) monoclonal antibody but did stain with BB-1 monoclonal antibody and CTLA4Ig.
  • CTLA4 binding counter-receptor(s) which: 1) can induce submitogenically activated T cells to proliferate without detectable IL- 2 secretion; and 2) are identified by the BB-1 monoclonal antibody but not anti-B7-l monoclonal antibody.
  • CTLA4/CD28 ligands can be distinguished on the basis of their temporal expression after B cell activation and their reactivity with CTLA4Ig and anti- B7 monoclonal antibodies.
  • the results of Figure 4 are representative of five experiments.
  • anti-B7-l (133) monoclonal antibody inhibited proliferation approximately 50% but totally blocked IL-2 accumulation.
  • BB-1 monoclonal antibody totally inhibited both proliferation and IL-2 secretion.
  • CTLA4Ig and Fab anti-CD28 also totally blocked proliferation and IL-2 production.
  • 72 hour activated B cells induced T cell response identical to that induced by 48 hour activated B cells. Similar results are observed if the submitogenic signal is delivered by phorbol myristic acid (PMA) and if the human splenic B cells are activated by MHC class II rather than Ig crosslinking.
  • PMA phorbol myristic acid
  • MHC class II rather than Ig crosslinking.
  • B7 B7-1
  • BB-1 monoclonal antibody BB-1
  • the original anti-B 7 monoclonal antibody, 133 was generated by immunization with anti-immunoglobulin activated human B lymphocytes whereas the BB-1 monoclonal antibody was generated by immunization with a baboon cell line.
  • the BB-1 monoclonal antibody must identify an epitope on human cells that is conserved between baboons and humans.
  • B7 transfected COS cells were found to be identically stained with the anti-B7 (133) and BB-1 monoclonal antibodies and that they both precipitated the identical broad molecular band (44-54kD) strongly suggesting that they identified the same molecule (Freeman, G.J., et al. (1989) J. Immunol. 143. 2714-2722). This observation was unexpected since the gene encoding the molecule identified by the BB-1 monoclonal antibody had been previously mapped to chromosome 12 (Katz, F.E., et al. (1985) Eur. J. Immunol.
  • B7 gene was located by two groups on chromosome 3 (Freeman, G.J., et al. (1992) Blood. 22, 489-494; Selvakumar, A., et al. (1992) lmmunogenetics 2__, 175-181.). Subsequently, additional discrepancies between the phenotypic expression of B7 (B7-1) and the molecule identified by the BB-1 monclonal antibody were noted. BB-1 monoclonal antibody stained thymic epithelial cells (Turka, L.A., et al. (1991) J. Immunol. 146. 1428-36; Munro, J.M., et al.
  • BB-1 monoclonal antibody recognizes an epitope on the B7-1 protein and that this epitope is also found on a distinct B7-3 protein, which also has costimulatory function. Phenotypic and blocking studies demonstrate that the BB-1 monoclonal antibody could detect one (on B7 negative cells) or both (on B7 positive cells) of these proteins. In contrast, the anti-B7 monoclonal antibodies, 133 and Bl.l detect only the B7-1 protein.
  • B cells express at least two distinct CTLA4 binding counter-receptors, one identified by both anti-B7 and BB-1 monclonal antibodies and the other identified only by BB-1 monoclonal antibody.
  • the B7-2 antigen is not detectable on activated B cells after 12 hours, but by 24 hours it is strongly expressed and functional. This molecule appears to signal via CD28 since proliferation and IL-2 production are completely blocked by Fab anti-CD28 monoclonal antibody. At 48 hours post activation, IL-2 secretion seems to be accounted for by B7-1 costimulation, since anti-B7 monoclonal antibody completely inhibits IL-2 production.
  • B7 B7-1 is neither expressed (Freedman, A.S. et al. (1987) Immunol. 137. 3260-3267; Freedman, A.S., et al. (1991) Cell. Immunol. 137. 429-437) nor capable of costimulating T cell proliferation or IL-2 secretion until 48 hours post B-cell activation.
  • Previous studies have shown that activation of T cells via the TCR in the absence of costimulation (Gimmi, CD., et al. (1993) Proc. Natl. Acad. Sci USA 20:6586-6590; Schwartz, R.H., et al. (1989) Cold Spring Harb. Symp Quant.
  • B7 (B7-1) deficient mouse has been developed and its antigen presenting cells were found to still bind CTLA4Ig (Freeman and Sharpe manuscript in preparation). This mouse is viable and isolated mononuclear cells induce detectable levels of IL-2 when cultured with T cells in vitro. Therefore, an alternative CD28 costimulatory counter-receptor or an alternative IL-2 producing pathway must be functional.
  • CTLA4/CD28 ligands capable of inducing IL-2 exist, and taken together with the results presented herein, suggest that all three CTLA4 binding counter-receptors may be critical for the induction of T cell immunity. Furthermore, their blockade will likely be required for the induction of T cell anergy. Identical results have been observed in the murine system with the identification of two CTLA4 binding ligands, corresponding to the human B7-1 and B7-2 molecules. APCs in the B7 deficient mouse bind to the CTLA4 and can induce IL-2 secretion. Taken together, these observations indicate that multiple CTLA-4 binding counter-receptors exist and sequentially costimulate T cell activation in the murine system.
  • a cDNA library was constructed in the pCDM8 vector (Seed, Nature, 329:840 (1987)) using poly (A) + RNA from the human anti-IgM activated B cells as described (Aruffo et al, Proc. Natl. Acad. Sci. USA, 84:3365 (1987)).
  • Splenic B cells were cultured at 2x1 ⁇ 6 cells/ml in complete culture media, ⁇ RPMI 1640 with 10% heat inactivated fetal calf serum (FCS), 2mM glutamine, 1 mM sodium pyruvate, penicillin (100 units/ml), streptomycin sulfate (lOO ⁇ g/ml) and gentamycin sulfate (5 ⁇ g/ml) ⁇ , in tissue culture flasks and were activated by crosslinking of sig with affinity purified rabbit anti-human IgM coupled to Affi-Gel 702 beads (Bio-Rad), Richmond, CA) (Boyd, A.W., et al., (1985) J. Immunol. 114,1516). Activated B cells were harvested after 1/6, 1/2, 4, 8 12, 24, 48, 72 and 96 hours.
  • RNA was prepared by homogenizing activated B cells in a solution of 4M guanidine thiocyanate, 0.5% sarkosyl, 25mM EDTA, pH 7.5, 0.13% Sigma anti-foam A, and 0.7% mercaptoethanol.
  • RNA was purified from the homogenate by centrifugation for 24 hour at 32,000 rpm through a solution of 5.7M CsCl, 1 OmM EDTA, 25mM Na acetate, pH 7. The pellet of RNA was dissolved in 5% sarkosyl, ImM EDTA, lOmM Tris, pH 7.5 and extracted with two volumes of 50% phenol, 49% chloroform, 1% isoamyl alcohol. RNA was ethanol precipitated twice.
  • RNA used in cDNA library construction was purified by two cycles of oligo (dT)-cellulose selection.
  • Complementary DNA was synthesized from 5.5 ⁇ g of anti-IgM activated human B cell poly(A) + RNA in a reaction containing 50mM Tris, pH 8.3, 75mM KC1, 3mM MgCl2, lOmM dithiothreitol, 500 ⁇ M dATP, dCTP, dGTP, dTTP, 50 ⁇ g/ml oligo(dT) 12-18, 180 units/ml RNasin, and 10,000 units/ml Moloney-MLV reverse transcriptase in a total volume of 55 ⁇ l at 37 for 1 hr.
  • the cDNA was converted to double- stranded DNA by adjusting the solution to 25mM Tris, pH 8.3, 1 OOmM KC1, 5mM MgCl2, 250 ⁇ M each dATP, dCTP, dGTP, dTTP, 5mM dithiothreitol, 250 units/ml DNA polymerase I, 8.5 units/ml ribonuclease H and incubating at 16 for 2 hr.
  • EDTA was added to 18mM and the solution was extracted with an equal volume of 50% phenol, 49% chloroform, 1% isoamyl alcohol.
  • DNA was precipitated with two volumes of ethanol in the presence of 2.5M ammonium acetate and with 4 micrograms of linear polyacrylamide as carrier.
  • cDNA was synthesized from 4 ⁇ g of anti-IgM activated human B cell poly(A) + RNA in a reaction containing 50mM Tris, pH 8.8, 50 ⁇ g/ml oligo(dT) 12-18, 327 units/ml RNasin, and
  • the reverse transcriptase was inactivated by heating at 70 for 10 min.
  • the cDNA was converted to double-stranded DNA by adding 320 ⁇ l H2O and 80 ⁇ l of a solution of 0.1M Tris, pH 7.5, 25mM MgCl2, 0.5M KC1, 250 ⁇ g/ml bovine serum albumin, and 50mM dithiothreitol, and adjusting the solution to 200 ⁇ M each dATP, dCTP, dGTP, dTTP, 50 units/ml DNA polymerase I, 8 units/ml ribonuclease H and incubating at o
  • Non-selfcomplementary BstXI adaptors were added to the DNA as follows:
  • the double-stranded cDNA from 6 ⁇ g of poly(A) + RNA was incubated with 3.6 ⁇ g of a kinased oligonucleotide of the sequence CTTTAGAGCACA (SEQ ID NO: 15) and 2.4 ⁇ g of a kinased oligonucleotide of the sequence CTCTAAAG (SEQ ID NO: 16) in a solution containing 6mM Tris, pH 7.5, 6mM MgCl2, 5mM NaCl, 350 ⁇ g/ml bovine serum albumin, 7mM mercaptoethanol, O.lmM ATP, 2mM dithiothreitol, ImM spermidine, and 600 units T4 o
  • DNA ligase in a total volume of 0.45ml at 15 C for 16 hours. EDTA was added to 34mM and the solution was extracted with an equal volume of 50% phenol, 49% chloroform, 1% isoamyl alcohol. DNA was precipitated with two volumes of ethanol in the presence of 2.5M ammonium acetate.
  • DNA larger than 600bp was selected as follows: The adaptored DNA was redissolved in lOmM Tris, pH 8, ImM EDTA, 600mM NaCl, 0.1% sarkosyl and chromatographed on a Sepharose CL-4B column in the same buffer. DNA in the void volume of the column (containing DNA greater than 600bp) was pooled and ethanol precipitated.
  • the pCDM8 vector was prepared for cDNA cloning by digestion with BstXI and purification on an agarose gel. Adaptored DNA from 6 ⁇ g of poly(A) + RNA was ligated to 2.25 ⁇ g of BstXI cut pCDM8 in a solution containing 6mM Tris, pH 7.5, 6mM MgCl2, 5mM NaCl, 350 ⁇ g/ml bovine serum albumin, 7mM mercaptoethanol, O.lmM ATP, 2mM dithiothreitol, ImM spermidine, and 600 units T4 DNA ligase in a total volume of 1.5ml at 15° for 24 hr.
  • Plasmid DNA was prepared from a 500 ml culture of the original transformation of the cDNA library. Plasmid DNA was purified by the alkaline lysis procedure followed by twice banding in CsCl equilibrium gradients (Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1987)).
  • the detached cells were treated with 10 ⁇ g/ml/CTLA4Ig and CD28Ig for 45 minutes at 4°C Cells were washed and distributed into panning dishes coated with affinity-purified Goat anti-human IgG antibody and allowed to attach at room temperature. After 3 hours, the plates were gently washed twice with PBS/0.5mM EDTA, pH 7.4/0.02% Na azide, 5% FCS and once with 0.15M NaCl, 0.01 M Hepes, pH 7.4, 5% FCS. Episomal DNA was recovered from the panned cells and transformed into E. coli DH10B/P3.
  • the plasmid DNA was re-introduced into COS cells via spheroplast fusion as described (Seed et al, Proc. Natl. Acad. Sci. USA, 84:3365 (1987)) and the cycle of expression and panning was repeated twice.
  • the detached COS cells were first incubated with CC-B7-1 mAbs (133 and Bl.l, 10 ⁇ g/ml), and COS cells expressing B7-1 were removed by ⁇ - mouse IgG and IgM coated magnetic beads.
  • COS cells were then treated with 10 ⁇ g/ml of human CTLA4Ig (hCTLA4Ig) and human CD28Ig (hCD28Ig) and human B7-2 expressing COS cells were selected by panning on dishes with goat anti-human IgG antibody plates.
  • plasmid DNA was prepared from individual colonies and transfected into COS cells by the DEAE-Dextran method. Expression of B7-2 on transfected COS cells was analyzed by indirect immunofluorescence with CTLA4Ig.
  • Plasmid DNA was prepared from individual colonies. A total of 4 of 48 candidate clones contained a cDNA insert of approximately 1.2 kb. Plasmid DNA from these four clones was transfected into COS cells. All four clones were strongly positive for B7-2 expression by indirect immunofluorescence using CTLA4Ig and flow cytometric analysis.
  • CDM8 RGV(2) (SEQ ID NO:5) 5'd[ACTGGTAGGTATGGAAGATCC]3' HBX29-5P (2R) (SEQ ID NO:6) 5'd[ATGCGAATCATTCCTGTGGGC]3' HBX29-5P (2F) (SEQ ID NO:7) 5'd[AAAGCCCACAGGAATGATTCG]3' HBX29-5P (SEQ ID NO:8) 5'd[CTCTCAAAACCAAABCCTGAG]3' 5PA (SEQ ID NO:9) 5'd[TTAGGTCACAGCAGAAGCAGC]3'
  • 5PA (3FA) (SEQ ID NO: 10) 5'd[TCTGGAAACTGACAAGACGCG]3'
  • HBX29-5P(1R) (SEQ ID NO:l 1) 5'd[CTCAGGCTTTGGTTTTGAGAG]3 * HBX29-3P(1R) (SEQ ID NO: 12) 5'd[CACTCTCTTCCCTCTCCATTG]3' HBX29-5P(3R) (SEQ ID NO: 13) 5'd[GACAAGCTGATGGAAACGTCG]3' HBX29-3P(1P) (SEQ ID NO:14) 5*d[CAATGGAGAGGGAAGAGAGTG]3'
  • the human B7-2 clone 29 contained an insert of 1,120 base pairs with a single long open reading frame of 987 nucleotides and approximately 27 nucleotides of 3' noncoding sequences ( Figure 8 (SEQ ID NO: 1)).
  • the predicted amino acid sequence encoded by the open reading frame of the protein is shown below the nucleotide sequence in Figure 8.
  • the encoded protein, human B7-2 is predicted to be 329 amino acids in length (SEQ ID NO:2). This protein sequence exhibits many features common to other type 1 Ig superfamily embrane proteins. Protein translation is predicted to begin at the ATG codon (nucleotide 107- 109) based on DNA homology in this region with the consensus eukaryotic translation initiation site (Kozak, M.
  • the amino terminus of the human B7-2 protein (amino acids 1 to 23) has the characteristics of a secretory signal peptide with a predicted cleavage between the alanines at positions 23 and 24 (von Heijne (1986) Nucl. Acids Res. 14:4683). Processing at this site would result in a human B7-2 membrane bound protein of 306 amino acid with an unmodified molecular weight of approximately 34 kDa.
  • This protein would consist of an extracellular Ig superfamily V and C like domains, of from about amino acid residue 24-245, a hydrophobic transmembrane domain of from about amino acid residue 246-268 and a long cytoplasmic domain of from about amino acid residue 269-329.
  • the homologies to the Ig superfamily are due to the two contiguous Ig-like domains in the extracellular region bound by the cysteines at positions 40 to 110 and 157 to 218.
  • the extracellular domain also contains eight potential N-linked glycosylation sites.
  • FIG. 13 represents the comparison of the amino acid sequences for human B7-2 (hB7-2) (SEQ ID NO:2), human B7-1 (hB7-l) (SEQ ID NO: 28 and 29) and murine B7 (mB7) (SEQ ID NO: 30 and 31).
  • the amino acid sequences for the human B7-1 and murine B7 can be found in Genbank at Accession #M27533 and X60958 respectively.
  • Vertical lines in Figure 13 show identical amino acids between the hB7-2 and hB7-l or mB7. Identical amino acids between hB7-l and mB7 are not shown.
  • the hB7-2 protein exhibits the same general structure as hB7-l as defined by the common cysteines (positions 40 and 110, IgV domains; positions 157 and 217, IgC domain) which the Ig superfamily domains and by many other common amino acids.
  • hB7-l and mB7 have been shown to bind to both human CTLA4 and human CD28, the amino acids in common between these two related proteins will be those necessary to comprise a CTLA4 or CD28 binding sequence.
  • An example of such a sequence would be the KYMGRTSFD (position 81-89, hB7-2) (SEQ ID NO: 17) or KSQDNVTELYDVS (position 188-200, hB7-2) (SEQ ID NO: 18). Additional related sequences are evident from the sequence comparison and others can be inferred by considering homologous related amino acids such as aspartic acid and glutamic acid, alanine and glycine and other recognized functionally related amino acids.
  • the B7 sequences share a highly positive charged domain with the cytoplasmic portion WKWKKKKRPRNSYKC (position 269-282, hB7-2) (SEQ ID NO: 19) which is probably involved in intracellular signaling.
  • B7-2 Binds CTLA4Ig and Not Anti-B7-1 and Anti-B7-3 Monoclonal Antibodies
  • COS cells transfected with either vector DNA (pCDNAI), or an expression plasmid containing B7-1 (B7-1) or B7-2 (B7-2) were prepared. After 72 hours, the transfected COS cells were detached by incubation in PBS containing 0.5 mM EDTA and 0.02% Na azide for 30 min. at 37°C Cells were analyzed for cell surface expression by indirect immunofluorescence and flow cytometric analysis using fluoroscein isothiocyanate conjugated (FITC) goat-anti-mouse Ig or goat-anti-human IgG FITC ( Figure 9).
  • FITC fluoroscein isothiocyanate conjugated
  • B7-1 Cell surface expression of B7-1 was detected with mAbs 133 (anti-B7-l) and BB-1 (anti-B7-l and anti- B7-3) and with CTLA4Ig, whereas B7-2 reacted only with CTLA4Ig.
  • Neither of the B7 transfectants showed any staining with the isotype controls (IgM or control Ig).
  • the vector transfected COS cells showed no staining with any of the detection reagents.
  • none of the cells showed any staining with the FITC labeled detection reagents and alone. This demonstrates that B7-2 encodes a protein that is a CTLA4 counter-receptor but is distinct from B7-1 and B7-3.
  • RNA Blot Analysis of B7-2 Expression in Unstimulated and Activated Human B Cells Cell Lines, and Myelomas Human splenic B cells were isolated by removing T cells and monocytes as previously described (Freedman, A.S., Freeman, G.J., Horowitz, J.C, Daley, J., Nadler, L.M., J. Immunol. (1987) 112:3260-3267). Splenic B cells were activated using anti-Ig beads and cells were harvested at the indicated times (Freedman et al., (1987), cited supra).
  • Figure 10 panel a shows RNA blot analysis of unstimulated and anti-Ig activated human splenic B cells and of cell lines including Raji (B cell Burkitts lymphoma), Daudi (B cell Burkitt's lymphoma), RPMI 8226 (myeloma), K562 (erythroleukemia), and REX (T cell acute lymphoblastic leukemia).
  • Figure 10, panel b shows RNA blot analysis of human myeloma specimens.
  • RNA blot analysis demonstrated that B7-2 mRNA is expressed in unstimulated human splenic B cells and increases 4-fold following activation ( Figure 10, panel a).
  • B7-2 mRNA was expressed in B cell neoplastic lines (Raji, Daudi) and a myeloma (RPMI 8226) but not in the erythroleukemia K562 and the T cell line REX.
  • B7-1 mRNA is not expressed in resting B cells and is transiently expressed following activation (G.J. Freeman et al. (1989) supra).
  • Human CD28 + T cells were isolated by immunomagnetic bead depletion using monoclonal antibodies directed against B cells, natural killer cells and macrophages as previously described (Gimmi, CD., et al. (1993) Proc. Natl. Acad. Sci. USA ___., 6586-6590).
  • B7-1, B7-2 and vector transfected COS cells were harvested 72 hours after transfection, incubated with 25 ⁇ g/ml of mitomycin-C for 1 hour, and then extensively washed.
  • 10-5 CD28 + and T cells were incubated with 1 ng/ml of phorbol myristic acid (PMA) and the indicated number of COS transfectants (Figure 11).
  • PMA phorbol myristic acid
  • panel a T cell proliferation was measured by 3H-thymidine (1 ⁇ Ci) incorporated for the last 12 hours of a 72 hour incubation.
  • Panel b of Figure 11 shows IL-2 production by T cells as measured by ELISA (Biosource, CA) using supernatants harvested 24 hours after the initiation of culture.
  • Human CD28 + T cells were isolated by immunomagnetic bead depletion using mAbs directed against B cells, natural killer cells, and macrophages as previously described (Gimmi, CD., Freeman, G.J., Gribben, J.G., Gray, G., Nadler, L.M. (1993) Proc. Natl. Acad. Sci USA __2, 6586-6590).
  • B7-1, B7-2, and vector transfected COS cells were harvested 72 hours after transfection, incubated with 25 ⁇ g/ml of mitomycin-C for 1 hour, and then extensively washed.
  • Blocking agents are indicated on the left side of Figure 12 and include: 1) no monoclonal antibody (no blocking agents), 2) mAb 133 (anti-B7-l mAb), 3) mAb BBl (anti-B7-l and anti-B7-3 mAb), 4) mAb B5 (control IgM mAb), 5) anti-CD28 Fab (mAb 9.3), 6) CTLA-Ig, and 7) control Ig.
  • Panel a of Figure 12 shows proliferation measured by -1H-thymidine (l ⁇ Ci) incorporation for the last 12 hours of a 72 hour incubation.
  • Figure 12, panel b shows IL-2 production as measured by ELISA (Biosource, CA) using supernatants harvested 24 hours after the initiation of culture.
  • B7-1 and B7-2 transfected COS cells costimulated equivalent levels of T cell proliferation when tested at various stimulator to responder ratios (Figure 11). Like B7-1 , B7-2 transfected COS cell costimulation resulted in the production of IL-2 over a wide range of stimulator to responder cell ratios ( Figure 11). In contrast, vector transfected COS cells did not costimulate T cell proliferation or IL-2 production.
  • B7-2 Costimulation is not Blocked by Anti-B7-1 and Anti-B7-3 mAbs but is Blocked by CTLA4- ⁇ g and Anti-CD28 Fab
  • Human CD28 + T cells were isolated by immunomagnetic bead depletion using mAbs directed against B cells, natural killer cells, and macrophages as previously described (Gimmi, CD., Freeman, G.J., Gribben, J.G., Gray, G., Nadler, L.M. (1993) Proc. Natl. Acad. Sci USA ___, 6586-6590).
  • B7-1, B7-2, and vector transfected COS cells were harvested 72 hours after transfection, incubated with 25 ⁇ g/ml of mitomycin-C for 1 hour, and then extensively washed.
  • Blocking agents are indicated on the left side of Figure 12 and include: 1) no monoclonal antibody (no blocking agents), 2) mAb 133 (anti-B7-l mAb), 3) mAb BBl (anti-B7-l and anti-B7-3 mAb), 4) mAb B5 (control IgM mAb), 5) anti-CD28 Fab (mAb 9.3), 6) CTLA-Ig, and 7) control Ig.
  • Panel a of Figure 12 shows proliferation measured by -1H-thymidine (l ⁇ Ci) incorporation for the last 12 hours of a 72 hour incubation.
  • Figure 12, panel b shows IL-2 production as measured by ELISA (Biosource, CA) using supernatants harvested 24 hours after the initiation of culture.
  • ELISA Biosource, CA
  • mAbs directed against B7-1 and B7-3 were used to inhibit proliferation and IL-2 production of submitogenically activated human CD28 + T cells.
  • Both B7-1 and B7-2 COS transfectants costimulated T cell proliferation and IL-2 production (Figure 12).
  • MAbs 133 Freedman, A.S. et al.
  • B7-2 is a counter-receptor for the CD28 and CTLA4 T cell surface molecules. Both proteins are similar in that they are: 1) expressed on the surface of APCs; 2) structurally related to the Ig supergene family with an IgV and IgC domain which share 26% amino acid identity, and 3) capable of costimulating T cells to produce IL-2 and proliferate.
  • B7-1 and B7-2 differ in several fundamental ways. First, B7-2 mRNA is constitutively expressed in unstimulated B cells, whereas B7-1 mRNA does not appear until 4 hours and cell surface protein is not detected until 24 hours (Freedman, A.S., et al. (1987) supra; Freeman, G.J., et al.
  • B7-1 and B7-2 in cell lines and human B cell neoplasms substantially differs.
  • B7-2 protein contains a longer cytoplasmic domain than B7-1 and this could play a role in signaling B-cell differentiation. These phenotypic and functional differences suggest that these homologous molecules may have biologically distinct functions.
  • a cDNA library was constructed in the pCDM8 vector (Seed, Nature, 329:840
  • Ml 2 cells were cultured at lxl cells/ml in complete culture media, ⁇ RPMI 1640 with 10% heat inactivated fetal calf serum (FCS), 2mM glutamine, 1 mM sodium pyruvate, penicillin (100 units/ml), streptomycin sulfate (lOO ⁇ g/ml) and gentamycin sulfate (5 ⁇ g/ml) ⁇ , in tissue culture flasks and were activated by 300 ⁇ g/ml dibutryl cAMP (Nabavi, N., et al.
  • RNA was prepared by homogenizing activated Ml 2 cells in a solution of 4M guanidine thiocyanate, 0.5% sarkosyl, 25mM EDTA, pH 7.5, 0.13% Sigma anti-foam A, and 0.7% mercaptoethanol. RNA was purified from the homogenate by centrifugation for 24 hour at 32,000 rpm through a solution of 5.7M CsCl, lOmM EDTA, 25mM Na acetate, pH 7.
  • RNA was dissolved in 5% sarkosyl, ImM EDTA, lOmM Tris, pH 7.5 and extracted with two volumes of 50% phenol, 49% chloroform, 1% isoamyl alcohol. RNA was ethanol precipitated twice. Poly (A) + RNA used in cDNA library construction was purified by two cycles of oligo (dT)-cellulose selection
  • Complementary DNA was synthesized from 5.5 ⁇ g of dibutryl cAMP activated murine M12 cell poly(A) + RNA in a reaction containing 50mM Tris, pH 8.3, 75mM KC1, 3mM MgCl2, 10mM dithiothreitol, 500 ⁇ M dATP, dCTP, dGTP, dTTP, 50 ⁇ g/ml oligo(dT)i2-18, 180 units/ml RNasin, and 10,000 units/ml Moloney-MLV reverse transcriptase in a total volume of 55 ⁇ l at 37 C for 1 hr.
  • the cDNA was converted to double-stranded DNA by adjusting the solution to 25mM Tris, pH 8.3, 1 OOmM KC1, 5mM MgCl2, 250 ⁇ M each dATP, dCTP, dGTP, dTTP, 5mM dithiothreitol, 250 units/ml DNA polymerase I, 8.5 units/ml ribonuclease H and incubating at
  • the cDNA was converted to double-stranded DNA by adding 320 ⁇ l H2O and 80 ⁇ l of a solution of 0.1M Tris, pH 7.5, 25mM MgCl2, 0.5M KC1, 250 ⁇ g/ml bovine serum albumin, and 50mM dithiothreitol, and adjusting the solution to 200 ⁇ M each dATP, dCTP, dGTP, dTTP, 50 units/ml DNA polymerase I, 8 units/ml ribonuclease H and incubating at 16 C for 2 hours.
  • EDTA was added to 18 mM and the solution was extracted with an equal volume of 50% phenol, 49% chloroform, 1% isoamyl alcohol. DNA was precipitated with two volumes of ethanol in the presence of 2.5M ammonium acetate and with 4 micrograms of linear polyacrylamide as carrier.
  • DNA larger than 600bp was selected as follows: The adaptored DNA was redissolved in lOmM Tris, pH 8, ImM EDTA, 600mM NaCl, 0.1% sarkosyl and chromatographed on a Sepharose CL-4B column in the same buffer. DNA in the void volume of the column (containing DNA greater than 600bp) was pooled and ethanol precipitated.
  • the pCDM8 vector was prepared for cDNA cloning by digestion with BstXI and purification on an agarose gel. Adaptored DNA from 5.5 ⁇ g of poly(A) + RNA was ligated to 2.25 ⁇ g of BstXI cut pCDM8 in a solution containing 6mM Tris, pH 7.5, 6mM MgCl2, 5mM NaCl, 350 ⁇ g/ml bovine serum albumin, 7mM mercaptoethanol, O.lmM ATP, 2mM dithiothreitol, ImM spermidine, and 600 units T4 DNA ligase in a total volume of 1.5ml at 15° for 24 hr. The ligation reaction mixture was transformed into competent E.coli MC1061/P3 and a total of 200 x 10 ⁇ independent cDNA clones were obtained.
  • Plasmid DNA was prepared from a 500 ml culture of the original transformation of the cDNA library. Plasmid DNA was purified by the alkaline lysis procedure followed by twice banding in CsCl equilibrium gradients (Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1987)).
  • the detached cells were treated with 10 ⁇ g/ml/human CTLA4Ig and murine CD28Ig for 45 minutes at 4°C Cells were washed and distributed into panning dishes coated with affinity-purified Goat anti- human IgG antibody and allowed to attach at room temperature. After 3 hours, the plates were gently washed twice with PBS/0.5mM EDTA, pH 7.4/0.02% Na azide, 5% FCS and once with 0.15M NaCl, 0.01 M Hepes, pH 7.4, 5% FCS. Episomal DNA was recovered from the panned cells and transformed into E. coli DH10B/P3.
  • the plasmid DNA was re- introduced into COS cells via spheroplast fusion as described (Seed et al, Proc. Natl. Acad. Sci. USA, 84:3365 (1987)) and the cycle of expression and panning was repeated twice.
  • the detached COS cells were first incubated with ⁇ -murine B7-1 mAb (16-10A1, 10 ⁇ g/ml), and COS cells expressing B7-1 were removed by ⁇ -mouse IgG and IgM coated magnetic beads.
  • COS cells were then treated with lO ⁇ g/ml of human CTLA4Ig and murine CD28Ig and murine B7-2 expressing COS cells were selected by panning on dishes coated with goat anti-human IgG antibody.
  • plasmid DNA was prepared from individual colonies and transfected into COS cells by the DEAE-Dextran method. Expression of B7-2 on transfected COS cells was analyzed by indirect immunofluorescence with CTLA4Ig.
  • plasmid DNA was prepared from individual colonies. A total of 6 of 8 candidate clones contained a cDNA insert of approximately 1.2 kb. Plasmid DNA from these eight clones was transfected into COS cells. All six clones with the 1.2 Kb cDNA insert were strongly positive for B7-2 expression by indirect immunofluorescence using CTLA4Ig and flow cytometric analysis.
  • the B7-2 cDNA insert in clone4 was sequenced in the pCDM8 expression vector employing the following strategy.
  • Initial sequencing was performed using sequencing primers T7, CDM8R (Invitrogen) homologous to pCDM8 vector sequences adjacent to the cloned B7-2 cDNA (see Table II). Sequencing was performed using dye terminator chemistry and an ABI automated DNA sequencer. (ABI, Foster City, CA). DNA sequence obtained using these primers was used to design additional sequencing primers (see Table II). This cycle of sequencing and selection of additional primers was continued until the murine B7-2 cDNA was completely sequenced on both strands.
  • CDM8(R) (SEQ ID NO:4) 5'd[TAAGGTTCCTTCACAAAG]3' MBX4-lF (SEQ ID NO:24) 5'd[ACATAAGCCTGAGTGAGCTGG]3'
  • MBX4-2R (SEQ ID NO:25) 5'd[ATGATGAGCAGCATCACAAGG]3'
  • MBX4-14 (SEQ ID NO:26) 5'd[TGGTCGAGTGAGTCCGAATAC]3'
  • MBX4-2F (SEQ ID NO:27) 5*d[GACGAGTAGTAACATACAGTG]3'
  • a murine B7-2 clone (mB7-2, clone 4) was obtained containing an insert of 1 , 163 base pairs with a single long open reading frame of 927 nucleotides and approximately 126 nucleotides of 3' noncoding sequences ( Figure 14, SEQ ID NO:22).
  • the predicted amino acid sequence encoded by the open reading frame of the protein is shown below the nucleotide sequence in Figure 14.
  • the encoded murine B7-2 protein is predicted to be 309 amino acid residues in length (SEQ ID NO:23). This protein sequence exhibits many features common to other type I Ig superfamily membrane proteins.
  • Protein translation is predicted to begin at the methionine codon (ATG, nucleotides 111 to 113) based on the DNA homology in this region with the consensus eucaryotic translation initiation site (see Kozak, M. (1987) Nucl. Acids Res. 15:8125-8148).
  • the amino terminus of the murine B7-2 protein (amino acids 1 to 23) has the characteristics of a secretory signal peptide with a predicted cleavage between the alanine at position 23 and the valine at position 24 (von Heijne (1987) Nucl. Acids Res. 14:4683).
  • This protein would consist of an approximate extracellular Ig superfamily V and C like domains of from about amino acid residue 24 to 246, a hydrophobic transmembrane domain of from about amino acid residue 247 to 265, and a long cytoplasmic domain of from about amino acid residue 266 to 309.
  • the homologies to the Ig superfamily are due to the two contiguous Ig-like domains in the extracellular region bound by the cysteines at positions 40 to 110 and 157 to 216.
  • the extracellular domain also contains nine potential N-linked glycosylation sites and, like murine B7-1, is probably glycosylated.
  • Glycosylation of the murine B7-2 protein may increase the molecular weight to about 50-70 kDa.
  • the cytoplasmic domain of murine B7-2 contains a common region which has a cysteine followed by positively charged amino acids which presumably functions as signaling or regulatory domain within an APC
  • Comparison of both the nucleotide and amino acid sequences of murine B7-2 with the GenBank and EMBL databases yielded significant homology (about 26% amino acid sequence identity) with human and murine B7-1.
  • Murine B7-2 exhibits about 50% identity and 67% similarity with its human homologue, hB7-2.
  • coli DH106/p3 transfected with a vector (plasmid pmBx4) containing a cDNA insert encoding murine B7-2 (clone 4) was deposited with the American Type Culture Collection (ATCC) on August 18, 1993 as Accession No. 69388.
  • ATCC American Type Culture Collection
  • CD4 + murine T cells were purified by first depleting red blood cells by treatment with Tris-NH4C1. T cells were enriched by passage over a nylon wool column. CD4 + T cells were purified by two-fold treatment with a mixture of anti-MHC class II and anti-CD28 mAbs and rabbit complement.
  • Murine B7-1 obtained from Dr. Gordon Freeman, Pana- Farber Cancer Institute, Boston, MA; see also, Freeman, G.J. et al (1991) J. Exp. Med. 174.
  • the extracellular portion of human B7-2 was prepared as a fusion protein coupled to an immunoglobulin constant region.
  • the immunoglobulin constant region may contain genetic modifications including those which reduce or eliminate effector activity inherent in the immunoglobulin structure. Briefly, DNA encoding the extracellular portion of hB7-2 was joined to DNA encoding the hinge, CH2 and CH3 regions of human IgC ⁇ l or IgC ⁇ 4 modified by directed mutagenesis. This was accomplished as described in the following subsections.
  • DNA fragments corresponding to the DNA sequences of interest were prepared by polymerase chain reaction (PCR) using primer pairs described below.
  • PCR reactions were prepared in 100 ⁇ l final volume composed of Taq, polymerase buffer (Gene Amp PCR Kit, Perkin-Elmer/Cetus, Norwalk, CT) containing primers (1 ⁇ M each), dNTPs (200 ⁇ M each) 1 ng of template DNA, and Taq, polymerase (Saiki, R.K., et al. (1988) Science 239:487-491).
  • PCR DNA amplifications were run on a thermocycler (Ericomp, San Diego, CA) for 25 to 30 cycles each composed of a denaturation step (1 minute at 94°C), a renaturation step (30 seconds at 54°C), and a chain elongation step (1 minute at 72°C).
  • the structure of each hB7-2 Ig genetic fusion consisted of a signal sequence to facilitate secretion coupled to the extracellular domain of B7-2 and the hinge, CH2 and CH3 domains of human IgC ⁇ l or IgC ⁇ 4.
  • the IgC gamma 1 and IgC gamma 4 sequences contained nucleotide changes within the hinge region to replace cysteine residues available for disulfide bond formation with serine residues and may contain nucleotide changes to replace amino acids within the CH2 domain thought to be required for IgC binding to Fc receptors and complement activation.
  • the Ig signal sequence was prepared from a plasmid containing the murine IgG heavy chain gene (Orlandi. R. et al. (1989) Proc. Natl. Acad. Sci. USA.
  • oligonucleotide 5'- GGCACTAGGTCTCCAGCTTGAGATCACAGTTCTCTCTAC-3' (#01)
  • SEQ ID NO: oligonucleotide 5'- GCTTGAATCTTCAGAGGAGCGGAGTGGACACCTGTGG-3'
  • SEQ ID NO: The forward PCR primer (SEQ ID NO: ) contains recognition sequences for restriction enzymes Bsal and is homologous to sequences 5' to the initiating methionine of the Ig signal sequence.
  • the reverse PCR primer (SEQ ID NO: ) is composed of sequences derived from the 5' end of the extracellular domain of hB7-2 and the 3' end of the Ig signal sequence. PCR amplification of the murine Ig signal template DNA using these primers resulted in a 224 bp product which is composed of Bsal restriction sites followed by the sequence of the Ig signal region fused to the first 20 nucleotides of the coding sequence of the extracellular domain of hB7-2. The junction between the signal sequence and hB7-2 is such that protein translation beginning at the signal sequence will continue into and through hB7-2 in the correct reading frame.
  • the extracellular domain of the hB7.2 gene was prepared by PCR amplification of plasmid containing the hB7-2 cDNA inserted into expression vector pCDNAI (Freeman et al., Science 262:909-11 (1994)):
  • the extracellular domain of hB7-2 was prepared by PCR amplification using oligonucleotide 5*-GCTCCTCTGAAGATTCAAGC-3' (#03) (SEQ ID NO: ) as the forward primer and oligonucleotide 5*-GGCACTATGATCAGGGGGAGGCTGAGGTCC-3' (#04) (SEQ ID NO: ) as the reverse primer.
  • the forward PCR primer contained sequences corresponding to the first 20 nucleotides of the B7-2 extracellular domain and the reverse PCR primer contained sequences corresponding to the last 22 nucleotides of the B7-2 extracellular domain followed by a Bel I restriction site and 7 noncoding nucleotides.
  • PCR amplification with primer #03 and #04 yields a 673 bp product corresponding to the extracellular IgV and IgC like domains of hB7-2 followed by a unique Bel I restriction site.
  • the signal sequence was attached to the extracellular portion of hB7-2 by PCR as follows. DNA-PCR products obtained above corresponding to the signal sequence and the hB7-2 extracellular domain were mixed in equimolar amounts, denatured by heating to 100°C, held at 54°C for 30°C to allow the complementary ends to anneal and the strands were filled in using dNTPs and Toq polymerase.
  • PCR primers #01 and #04 were added and the entire fragment produced by PCR amplification to yield a -880 fragment composed of a Bsal restriction site followed by the signal sequence fused to the extracellular domain of hB7- 2, followed by a Bel I restriction site.
  • (3). Cloning and Modification of Immunoglobulin Fusion Domain Plasmid pSP721gGl was prepared by cloning the 2000 bp segment of human IgGl heavy chain genomic DNA (Ellison, J.W., et al. (1982) Nucl. Acids. Res. 10:4071-4079) into the multiple cloning site of cloning vector pSP72 (Promega, Madison, Wl).
  • Plasmid pSP721gGl contained genomic DNA encoding the CHI, hinge, CH2 and CH3 domains of the heavy chain human IgC ⁇ l gene.
  • PCR primers designed to amplify the hinge-CH2-CH3 portion of the heavy chain along with the intervening DNA were prepared as follows.
  • the forward PCR primer 5'-GCATTTTAAGCTTTTTCCTGATCAGGAGCCCAAATCTTCT GACAAAACTCACACATCTCCACCGTCTCCAGGTAAGCC-3' contained Hindlll and Bel I restriction sites and was homologous to the hinge domain sequence except for five nucleotide substitutions which would change the three cysteine residues to serines.
  • the reverse PCR primer 5 AATACGACTCACTATAGGG-3' was identical to the commercially available T7 primer (Promega, Madison, Wl). Amplification with these primers yielded a 1050 bp fragment bounded on the 5' end by Hindlll and Bell restriction sites and on the 3' end by BamHl, Smal, Kpnl, Sacl, EcoRl, Clal, EcoR5 and Bglll restriction sites. This fragment contained the IgC hinge domain in which the three cysteine codons had been replaced by serine codons followed by an intron, the CH2 domain, an intron, the CH3 domain and additional 3' sequences.
  • DNA 1: 11 -18 was used as a template for PCR amplification using oligonucleotide 5'GAGCATTTTCCTGATCAGGA GTCCAAATATGGTCCCCCATCCCATCATCCCCAGGTAAGCCAACCC-3' (SEQ ID NO: ) as the forward PCR primer and oligonucleotide 5 * GCAGAGGAATCGAGCTCGGTACCCGGGGATCCCCAGTGTGGGGACAGTGGGA CCGCTCTGCCTCCC-3' (SEQ ID NO: ) as the reverse PCR primer.
  • the forward PCR primer contains a Bel 1 restriction site followed by the coding sequence for the hinge domain of IgCgamma4.
  • the reverse PCR primer contains a PspAI restriction site (5'CCCGGG-3 ! ). PCR amplification with these primers results in a 1179 bp DNA fragment.
  • the PCR product was digested with Bell and PspAI and ligated to pNRDSH/IgGl digested with the same restriction enzymes to yield plasmid pNRDSH/IgG4. In this reaction, the IgC ⁇ 4 domain replaced the IgC ⁇ l domain present in pNRDSH/IgGl.
  • Plasmid pNRDSH/IgGl served as template for modifications of the IgC ⁇ l CH2 domain and plasmid pNRDSH/IgG4 served as template for modifications of the IgC ⁇ 4 CH2 domain.
  • Plasmid pNRDSH/IgGl was PCR amplified using a forward PCR primer (SEQ ID NO: ) and oligonucleotide 5'-GGGTTTT GGGGGGAAGAGGAAGACTGACGGTGCCCCC TCGGCTTCAGGTGCTGAGGAAG-3' (SEQ ID NO: ) as the reverse PCR primer.
  • the forward PCR primer (SEQ ID NO: ) has been previously described and the reverse PCR primer (SEQ ID NO: ) was homologous to the amino terminal portion of the CH2 domain of IgGl except for five nucleotide substitutions designed to change amino acids 234, 235, and 237 (Canfield, S. M. and Morrison, S. L. (1991) J. Exp. Med. 173: 1483-1491.) from Leu to Ala, Leu to Glu, and Gly to Ala, respectively. Amplification with these PCR primers will yield a 239 bp DNA fragment consisting of a modified hinge domain, an intron and modified portion of the CH2 domain.
  • Plasmid pNRDSH/IgGl was also PCR amplified with the oligonucleotide 5'- CATCTCTTCCTCAGCACCTGAAGCCGAGGGGGCACCGTCAGTCTTCCTCTTCCC CC-3' (SEQ ID NO: ) as the forward primer and oligonucleotide (SEQ ID NO: ) as the reverse PCR primer.
  • the forward PCR primer (SEQ ID NO: ) is complementary to primer (SEQ ID NO: ) and contains the five complementary nucleotide changes necessary for the CH2 amino acid replacements.
  • the reverse PCR primer (SEQ ID NO: ) has been previously described.
  • Amplification with these primes yields a 875 bp fragment consisting of the modified portion of the CH2 domain, an intron, the CH3 domain, and 3' additional sequences.
  • the complete IgC ⁇ l segment consisting of modified hinge domain, modified CH2 domain and CH3 domain was prepared by an additional PCR reaction.
  • the purified products of the two PCR reactions above were mixed, denatured (95°C,1 minute) and then renatured (54°C, 30 seconds) to allow complementary ends of the two fragments to anneal.
  • the strands were filled in using dNTP and Taq polymerase and the entire fragment amplified using forward PCR primer (SEQ ID NO: ) and reverse PCR primer (SEQ ID NO: ).
  • the resulting fragment of 1050 bp was purified, digested with Hindlll and EcoRl and ligated to pNRDSH previously digested with the same restriction enzymes to yield plasmid pNRDSHIgGl m.
  • Plasmid pNRDSH/IgG4 was PCR amplified using the forward primer (SEQ ID NO: ) and the oligonucleotide 5'-
  • CGCACGTGACCTCAGGGGTCCGGGAGATCATGAGAGTGTCCTTGGGTTTTGGGG GGAACAGGAAGACTGATGGTGCCCCCTCGAACTCAGGTGCTGAGG-3 * (SEQ ID NO: ) as the reverse primer.
  • the forward primer has been previously described and the reverse primer was homologous to the amino terminal portion of the CH2 domain, except for three nucleotide substitutions designed to replace the amino acids described above.
  • This primer also contained a Pmll restriction site for subsequent cloning. Amplification with these primers yields a 265 bp fragment composed of the modified hinge region, and intron, and the modified 5' portion of the CH2 domain.
  • Plasmid pNRDSH/lgG4 was also PCR amplified with the oligonucleotide 5 '-CCTCAGCACCTGAGTTCGAGGGGGCACCATCAGTCTCCTGTTCCCCCC AAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCG-3 ' (SEQ ID NO: ) as the forward primer and oligonucleotide (SEQ ID NO: ) as the reverse PCR primer.
  • the forward PCR primer (SEQ ID NO: ) is complementary to primer (SEQ ID NO: ) and contains the three complementary nucleotide changes necessary for the CH2 amino acid replacements.
  • the reverse PCR primer (SEQ ID NO: ) has been previously described.
  • Amplification with these primes yields a 1012 bp fragment consisting of the modified portion of the CH2 domain, an intron, the CH3 domain, and 3' additional sequences.
  • the complete IgC ⁇ 4 segment consisting of modified hinge domain, modified CH2 domain and CH3 domain was prepared by an additional PCR reaction.
  • the purified products of the two PCR reactions above were mixed, denatured (95°C,1 minute) and then renatured (54°C, 30 seconds) to allow complementary ends of the two fragments to anneal.
  • the strands were filled in using dNTP and Taq polymerase and the entire fragment amplified using forward PCR primer (SEQ ID NO: ) and reverse PCR primer (SEQ ID NO: ).
  • the resulting fragment of 1179 bp was purified, digested with Bell and PspAI and ligated to pNRDSH previously digested with the same restriction enzymes to yield plasmid pNRDSH/IgG4m.
  • the PCR fragment corresponding to the Ig signal-hB7-2 gene fusion prepared above was digested with Bsal and Bel 1 restriction enzymes and ligated to pNRDSH/IgGl, pNRDSH/IgGlm, pNRDSH/IgG4, and pNRDSH/IgG4m previously digested with Hind III and Bell.
  • the ligated plasmids were transformed into E. coli JM109 using CaC12 competent cells and transformants were selected on L-agar containing ampicillin (50 ⁇ g/ml; Molecular Cloning: A Laboratory Manual (1982) Eds. Maniatis, T., Fritsch, E. E., and Sambrook, J. Cold Spring Harbor Laboratory).
  • Plasmids isolated from the transformed E. coli were analyzed by restriction enzyme digestion. Plasmids with the expected restriction plasmid were sequenced to verify all portions of the signal-hB7-2-IgG gene fusion segments. D. Expression Cloning of hB7-2V-IgGl and hB7-2C IgGl
  • variable and constant domains of human B7-2 were separately cloned into pNRDSH/IgGl . These clonings were accomplished using PCR. The portions of hB7-2 corresponding to the variable and constant regions were determined from intron exon mapping and previously published gene structure analysis.
  • the hB7-2V domain Ig sequence was assembled using a PCR strategy similar to that shown above.
  • the signal sequence was derived from the onco M gene by PCR amplification of a plasmid containing the onco M gene using oligonucleotide 5'- GCAACCGGAAGCTTGCCACCATGGGGGTACTGCTCACACAGAGGACG-3' (#05) (SEQ ID NO: ) as the forward PCR primer and 5'-
  • the reverse primer contains a Hind III restriction site and the amino terminal portion of the onco M signal sequence.
  • the reverse PCR contains the sequence corresponding to the 3' portion of the onco M signal sequence fused to the 5' end of the hB7-2 IgV like domain.
  • the hB7-2 IgV like domain was obtained by PCR amplification of the hB7-2 cDNA using oligonucleotide 5'-CTCCTGTTTCCAAGCATGGCCAGCATGGCTCCTCTGAA GATTCAGGCTTATTTCAATGAGAC-3' (#07) (SEQ ID NO: ) as the forward and oligonucleotide 5'- TGTGTGTGGAATTCTCATTACTGATCAAGCACTGACAGTTCAGAATTCATC-3' (#08) (SEQ ID NO : ) as the reverse PCR primer.
  • PCR amplification with these primers yields the hB7-2 IgV domain with a portion of the 3' end of the onco M signal sequence on the 5' end and a Bel I restriction site on the 3' end.
  • the signal and IgV domain were linked together in a PCR reaction in which equimolar amounts of the onco M signal and IgV domain DNA fragments were mixed, denatured, annealed, and the strands filled in.
  • Subsequent PCR amplification using forward primer #05 and reverse primer #08 yielded a DNA fragment containing a Hind III restriction site, followed by the onco M signal fused to the B7-2 IgV domain followed by a Bel I restriction site.
  • This PCR fragment was digested with Hind II and Bel I and cloned into expression vector pNRDSH/IgGl digested with the same restriction enzymes to yield pNRDSH/B7-2CIg.
  • the expression plasmid for hB7-2IgC domain was prepared as described above for the IgV domain except for using PCR primers specific for the IgC domain.
  • the onco M signal sequence was prepared using oligonucleotide #05 as the forward PCR primer and ' oligonucleotide 5'- AGAAATTGGTACTATTTCAGGTTGACTGAAGTTAGCCATGCTGGCC ATGCTTGGA AACAGGAG-3' (#09) (SEQ ID NO: ) as the reverse PCR primer.
  • the hB7-2 IgC domain was prepared using oligonucleotide 5'-
  • the two PCR products were mixed and amplified with primers #05 and #11 to assemble the onco M signal sequence with the hB7-2IgC domain.
  • the PCR product was subsequently digested with Hind III and Bell and ligated to pNRDSH/IgGl digested with similar restriction enzymes to yield the final expression plasmid pNRDSH/hB7-2CIgGl.
  • B7 family-Ig fusion proteins The ability of various B7 family-Ig fusion proteins to competitively inhibit the binding of biotinylated-CTLA4Ig to immobilized B7-2Ig was determined. Competition binding assays were done as follows and analysed according to McPherson (McPherson, G.A. (1985) J. Pharmacol. Methods 14:213-228). Soluble hCTLA4Ig was labelled with 125 l to a specific activity of approximately 2 x 10" cpm/pmol. hB7-2-Ig fusion protein was coated overnight onto microtiter plates at lO ⁇ g/ml in 10 mM Tris-HCl, pH8.0, 50 ⁇ l /well.
  • the wells were blocked with binding buffer (DMEM containing 10% heat-inactivated FBS, 0.1% BSA, and 50 mM BES, pH 6.8) for 2 h at room temperature.
  • the labeled CTLA4-Ig (4nM) was added to each well in the presence or absence of unlabeled competing Ig fusion proteins, including full-length B7-2 (hB7-2Ig), full-length B7- 1 (hB7- 1 Ig), the variable region-like domain of B7-2 (hB7-2VIg) and the constant region-like domain of B7-2 (hB7- 2CIg) and allowed to bind for 2.5 h at room temperature.
  • Biotinylated hCTLA4IgGl (ref, MFGR;1 ⁇ g/ml serially diluted in twofold steps to 15.6 ng/mL; 50 ⁇ L) was added to each well and incubated for 2.5 hours at room temperature. The wells were washed as above. The bound biotinylated CTLA4Ig was detected by the addition of 50 1/1 of a 1 :2000 dilution of streptavidin-HRP (Pierce Chemical Co., Rockford, IL) for 30 minutes at room temperature. The wells were washed as above and 50 ⁇ L of ABTS (Zymed, California) added and the developing blue color monitored at 405 nm after 30 min.
  • ABTS Zamed, California
  • FIG. 16 A graphic representation of a typical binding assay is shown in Figure 16.
  • the ability of the various forms of CTLA4 to compete with biotinylated CTLA4IgGl was assessed by mixing varying amounts of the competing protein with a quantity of biotinylated CTLA4IgGl shown to be non-saturating (i.e., 70 ng/mL; 1.5nM) and performing the binding assays as described above ( Figure 15).
  • mice obtained from Taconic Labs, Germantown, NY were immunized intraperitoneally with 50 ⁇ g human B7.2-Ig emulsified in complete Freund's adjuvant (Sigma Chemical Co., St. Louis, MO) or 10 ⁇ CHO-human B7.2 cells per mouse. The mice were given two booster immunizations with 10-25 ⁇ g human B7.2-Ig emulsified in incomplete Freund's adjuvant (Sigma Chemical Co., St. Louis, MO) or CHO-human B7.2 cells at fourteen day intervals following the initial immunization for the next two months.
  • mice were bled by retro-orbital bleed and the sera assayed for the presence of antibodies reactive to the immunogen by ELISA against human B7.2-Ig.
  • ELISA against hCTLA4-Ig was also used to control for Ig tail directed antibody responses.
  • Mice showing a strong serological response were boosted intravenously via the tail vein with 25 ⁇ g human hB7.2-Ig diluted in phosphate- buffered saline (PBS), pH 7.2 (GIBCO, Grand Island, NY). Three to four days following this boost, the spleens from these mice were fused 5:1 with SP 2/0 myeloma cells (American Type Culture Collection, Rockville, MD, No.
  • the wells were washed three times with PBS and 50 ⁇ l of hybridoma supernatant was added per well and incubated for 1.5 hours at room temperature. Following this incubation, the wells were washed three times with PBS and then incubated for 1.5 hours at room temperature with 50 ⁇ l per well of a 1 :4000 dilution of horseradish peroxidase-conjugated, affinity purified, goat anti-mouse IgG or IgM heavy and light chain-specific antibodies (HRP; Zymed Laboratories, San Francisco, CA).
  • HRP horseradish peroxidase-conjugated, affinity purified, goat anti-mouse IgG or IgM heavy and light chain-specific antibodies
  • Hybidoma cells were deposited with the American Type Culture Collection, which meets the requirements of the Budapest Treaty, on July 19, 1994 as ATCC Accession No. (hybridoma HA3.1F9), ATCC Accession No. (HA5.2B7) and
  • Supernatants from the hybridomas HA3.1F9, HA5.2B7 and HF2.3D1 were further characterized by competitive ELISA, in which the ability of the monoclonal antibodies to inhibit the binding of biotinylated hCTLA4Ig to immobilized hB7-2 immunoglobulin fusion proteins was examined.
  • Biotinylation of hCTLA4Ig was performed using Pierce Immunopure NHS-LC Biotin (Cat. No. 21335).
  • B7-2 immunoglobulin fusion proteins used were: hB7.2-Ig (full-length hB7-2), hB7.2-VIg (hB7-2 variable domain only) and hB7.2-CIg (B7-2 constant domain only).
  • a hB7.1-Ig fusion protein was used as a control.
  • 96 well plates were coated with the Ig fusion protein (50 ⁇ l/well of a 20 ⁇ g/ml solution) overnight at room temperature. The wells were washed three times with PBS, blocked with 10 % fetal bovine serum (FBS), 0.1 % bovine serum albumin (BSA) in PBS for 1 hour at room temperature, and washed again three times with PBS.
  • FBS fetal bovine serum
  • BSA bovine serum albumin
  • Control antibodies were an anti-B7.1 mAb (EW3.5D12) and the anti-hB7-2 mAb B70 (IgG2b ⁇ , obtained from Pharmingen).
  • the wells were washed again and streptavidin-conjugated horse radish peroxidase (from Pierce, Cat. No. 21126; 1 :2000 dilution, 50 ⁇ l/well) was added and incubated for 30 minutes at room temperature.
  • the wells were washed again, followed by a 30 minute incubation in 50 ⁇ l per well of ABTS in 0.1 M Na-Citrate, pH 4.2 to which a 1 :1000 dilution of 30 % hydrogen peroxide had been added as a substrate for HRP to detect bound antibody.
  • Supernatants from the hybridomas HA3.1F9, HA5.2B7 and HF2.3P1 were also characterized by flow cytometry.
  • Supernatants collected from the clones were screened by flow cytometry on CHO and 3T3 cells transfected to express hB7.2 (CHO-hB7.2 and 3T3- hB7.2, respectively) or control transfected 3T3 cells (3T3-Neo).
  • Flow cytometry was performed as follows: 1 x 10 ⁇ cells were washed three times in 1 % BSA in PBS, then the cells were incubated in 50 ⁇ l hybridoma supernatant or culture media per 1 x 10° cells for 30 minutes at 4 °C Following the incubation, the cells were washed three times with 1 % BSA in PBS, then incubated in 50 ⁇ l fluorescein-conjugated goat anti-mouse IgG or IgM antibodies (Zymed Laboratories, San Francisco, CA) at 1 :50 dilution per 1 x 10 ⁇ cells for 30 minutes at 4 °C The cells were then washed three times in 1 % BSA in PBS and fixed with 1 % paraformaldehyde solution.
  • Hybridoma supernatants containing anti-human B7-2 mAbs were tested for their ability to inhibit hB7-2 costimulation of human T cells.
  • purified CP28 + human T cells were treated with submitogenic amounts of PMA (lng/ml) to deliver the primary signal and with CHO cells expressing hB7-2 on their surface to deliver the costimulatory signal.
  • Proliferation of the T cells was measured after three days in culture by the addition of H-thymidine for the remaining 18 hours. As shown in Table V, resting T cells show little proliferation as measured by 3 H-thymidine incorporation (510 pm).
  • J558 plasmacytoma cells obtained from the American Type Culture Collection,
  • Rockville, MO; # TIB 6) were transfected with an expression vector containing cPNA encoding either mouse B7-2 (pAWNE03) or B7-1 (pNRPSH or pAWNE03) and a neomycin- resistance gene. Stable transfectants were selected based upon their neomycin resistance and cell surface expression of B7-2 or B7-1 on the tumor cells was confirmed by FACS analysis using either an anti-B7-2 or anti-B7-l antibody.
  • mice in groups of 5-10 mice/set, were used in experiments designed to determine whether cell-surface expression of B7-2 on tumor cells would result in regression of the implanted tumor cells.
  • Untransfected and transfected J558 cells were cultured in vitro, collected, washed and resuspended in Hank's buffered salt solution (GIBCO, Grand Island, New York) at a concentration of 10 - * cells/ml.
  • a patch of skin on the right flank of each mouse was removed of hair with a depilatory and, 24 hours later, 5 x 10 ⁇ tumor cells/mouse were implanted intradermally or subdermally.
  • This example demonstrates that cell-surface expression of B7-2 on tumor cells, such as by transfection of the tumor cells with a B7-2 cPNA, induces an anti -tumor response in naive animals that is sufficient to cause rejection of the tumor cells.
  • NAME Mandragouras, Amy E.
  • ATC GAG TAT GAT GGT ATT ATG CAG AAA TCT CAA GAT AAT GTC ACA GAA 691 lie Glu Tyr Asp Gly lie Met Gin Lys Ser Gin Asp Asn Val Thr Glu 180 185 190 195 CTG TAC GAC GTT TCC ATC AGC TTG TCT GTT TCA TTC CCT GAT GTT ACG 739 Leu Tyr Asp Val Ser lie Ser Leu Ser Val Ser Phe Pro Asp Val Thr 200 205 210
  • AGC AAT ATG ACC ATC TTC TGT ATT CTG GAA ACT GAC AAG ACG CGG CTT 787 Ser Asn Met Thr lie Phe Cys lie Leu Glu Thr Asp Lys Thr Arg Leu 215 220 225
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:11: CTCAGGCTTT GGTTTTGAGA G 21 (2) INFORMATION FOR SEQ ID NO:12 :
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE peptide
  • SEQUENCE DESCRIPTION SEQ ID NO:18: Lys Ser Gin Asp Asn Val Thr Glu Lys Tyr Asp Val Ser
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • CAC CCG AAA CCT AAG AAG ATG TAT TTT CTG ATA ACT AAT TCA ACT AAT 644 His Pro Lys Pro Lys Lys Met Tyr Phe Leu He Thr Asn Ser Thr Asn 165 170 175
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE oligonucleotide
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL no
  • ANTI-SENSE no
  • ORIGINAL SOURCE
  • NAME/KEY Alternate polyadenylation signal
  • B LOCATION: 1474 to 1479 bp
  • GGA CAT TTA AGA GTG AAT CAG ACC TTC AAC TGG AAT ACA ACC AAG CAA 1025 Gly His Leu Arg Val Asn Gin Thr Phe Asn Trp Asn Thr Thr Lys Gin 190 195 200
  • MOLECULE TYPE protein
  • DESCRIPTION B cell activation antigen; natural ligand for CD28 T cell surface antigen,- transmembrane protein

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PCT/US1994/008423 1993-07-26 1994-07-26 B7-2: ctl a4/cd 28 counter receptor WO1995003408A1 (en)

Priority Applications (10)

Application Number Priority Date Filing Date Title
AU74052/94A AU7405294A (en) 1993-07-26 1994-07-26 B7-2: ctl a4/cd 28 counter receptor
EP94924017A EP0711345A1 (de) 1993-07-26 1994-07-26 B7-2: ctl a4/cd 28 "gegen rezepter"
JP7505397A JPH09500788A (ja) 1993-07-26 1994-07-26 Ｂ７−２：ｃｔｌａ４／ｃｄ２８カウンターレセプター
US08/479,744 US6084067A (en) 1993-07-26 1995-06-07 CTLA4/CD28 ligands and uses therefor
US09/206,132 US6723705B1 (en) 1993-08-19 1998-12-07 Tumor cells modified to express B7-2 with increased immunogenicity and uses therefor
US09/425,762 US6605279B2 (en) 1993-07-26 1999-10-22 Therapeutic compositions for inhibiting the interactions of B7-1 and B7-2 with their natural ligands
AU72086/00A AU772061B2 (en) 1993-07-26 2000-12-07 B7-2:CTL A4/CD 28 counter receptor
US10/429,079 US7459544B2 (en) 1993-07-26 2003-05-02 Nucleic acids encoding B7-2 fusion proteins
US10/767,561 US20050129670A1 (en) 1993-07-26 2004-01-28 Tumor cells modified to express B7-2 with increased immunogenicity and uses therefor
AU2004200586A AU2004200586C1 (en) 1993-07-26 2004-02-13 Novel CTLA4/CD28 ligands and uses therefor

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US08/101,624 US5942607A (en) 1993-07-26 1993-07-26 B7-2: a CTLA4/CD28 ligand
US08/101,624 1993-07-26
US10939393A 1993-08-19 1993-08-19
US08/109,393 1993-08-19
US14777393A 1993-11-03 1993-11-03
US08/147,773 1993-11-03

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US08/280,757 Continuation-In-Part US6130316A (en) 1993-07-26 1994-07-26 Fusion proteins of novel CTLA4/CD28 ligands and uses therefore

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US5811097A (en) * 1995-07-25 1998-09-22 The Regents Of The University Of California Blockade of T lymphocyte down-regulation associated with CTLA-4 signaling
US5855887A (en) * 1995-07-25 1999-01-05 The Regents Of The University Of California Blockade of lymphocyte down-regulation associated with CTLA-4 signaling
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US6319906B1 (en) 1996-12-31 2001-11-20 Isis Pharmaceuticals Oligonucleotide compositions and methods for the modulation of the expression of B7 protein
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US6113901A (en) * 1989-10-27 2000-09-05 Arch Development Corporation Methods of stimulating or enhancing the immune system with anti-CD3 antibodies
US6406696B1 (en) 1989-10-27 2002-06-18 Tolerance Therapeutics, Inc. Methods of stimulating the immune system with anti-CD3 antibodies
US6143297A (en) * 1989-10-27 2000-11-07 Arch Development Corporation Methods of promoting immunopotentiation and preparing antibodies with anti-CD3 antibodies
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