EP1181053A2 - Emploi de molecules de co-stimulation solubles favorisant les reponses immunitaires - Google Patents

Emploi de molecules de co-stimulation solubles favorisant les reponses immunitaires

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Publication number
EP1181053A2
EP1181053A2 EP00930437A EP00930437A EP1181053A2 EP 1181053 A2 EP1181053 A2 EP 1181053A2 EP 00930437 A EP00930437 A EP 00930437A EP 00930437 A EP00930437 A EP 00930437A EP 1181053 A2 EP1181053 A2 EP 1181053A2
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EP
European Patent Office
Prior art keywords
antigen
molecule
cells
peptide
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP00930437A
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German (de)
English (en)
Inventor
Knut Sturmhoefel
Stanley F. Wolf
Margot O'toole
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Genetics Institute LLC
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Genetics Institute LLC
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Publication of EP1181053A2 publication Critical patent/EP1181053A2/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5152—Tumor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • T cells In order for T cells to respond to foreign proteins, two signals must be provided by antigen-presenting cells (APCs) to resting T lymphocytes (Jenkins, M. and Schwartz, R. (1987) J. Exp. Med. 165, 302-319; Mueller, D.L., et al. (1990) J. Immunol. 144, 3701-3709).
  • the first signal which confers specificity to the immune response, is transduced 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 induces T cells to proliferate and become functional (Lenschow et al. 1996. Annu. Rev. Immunol.
  • 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. 140, 3324-3330; Linsley, P.S., et al. 1991 J. Exp. Med. 173, 721-730; Gimmi, CD., et al., 1991 Proc. Natl. Acad. Sci. USA. 88, 6575-6579; Young, J.W., et al. 1992 J. Clin. Invest. 90, 229-237; Koulova, L., et al. 1991 J. Exp. Med. L73, 759-762; Reiser, H., et al. 1992 Proc. Natl. Acad. Sci. USA. 89, 271-275; van-Seventer, G.A., et al.
  • CD80 and CD86 (B7-2) proteins, expressed on APCs, are critical costimulatory molecules (Freeman et al. 1991. J. Exp. Med. 174:625; Freeman et al. 1989 J. Immunol. 143:2714; Azuma et al. 1993 Nature 366:76; Freeman et al. 1993. Science 262:909).
  • B7-2 appears to play a predominant role during primary immune responses, while B7- 1 , which is upregulated later in the course of an immune response, may be important in prolonging primary T cell responses or costimulating secondary T cell responses (Bluestone. 1995. Immunity. 2:555).
  • CD28 One ligand to which B7-1 and B7-2 bind, CD28, is constitutively expressed on resting T cells and increases in expression after activation. After signaling through the T cell receptor, ligation of CD28 and transduction of a costimulatory signal induces T cells to proliferate and secrete IL-2 (Linsley, P.S., et al. 1991 J. Exp. Med. 173, 721-730; Gimmi, CD., et al. 1991 Proc. Natl. Acad. Sci. USA. 88. 6575-6579; June, C.H., et al. 1990 Immunol. Today. U, 211-6; Harding, F.A., et al. 1992 Nature. 356, 607-609).
  • CTLA4 A second ligand, termed CTLA4 (CD152) 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). In contrast to CD28, CTLA4 appears to be critical in negative regulation of T cell responses (Waterhouse et al. 1995. Science 270:985). Blockade of CTLA4 has been found to remove inhibitory signals, while aggregation of CTLA4 has been found to provide inhibitory signals that downregulate T cell responses (Allison and Krummel. 1995. Science 270:932).
  • the present invention provides methods for enhancing immune responses by manipulating the costimulatory pathway.
  • the subject methods are particularly effective in augmenting responses to tumor antigens and antigens from infectious agents. - _y
  • the present invention is based, at least in part, on the discovery that soluble forms of costimulatory molecules can prophylactically and therapeutically enhance immune responses. This enhancement is seen despite the fact that the soluble costimulatory molecules of the invention are not administered on a solid phase (e.g., are not administered on a cell) and are administered in the absence of a cross-linking agent. These findings are particularly surprising in light of the teaching that soluble forms of B7-1 and B7-2 molecules fail to generate costimulatory responses (Hayden et al. 1996. Tissue Antigens. 48:242; U.S. patent 5,580,756).
  • the present invention provides methods of prophylactically enhancing an immune response by a subject to an antigen by administering a soluble composition comprising an extracellular domain of a costimulatory molecule, such that the immune response of the subject to the antigen is enhanced.
  • the invention provides methods of therapeutically enhancing an immune response by a subject to an antigen by administering a soluble composition comprising an extracellular domain of a costimulatory molecule, such that the immune response of the subject to the antigen is enhanced.
  • the costimulatory molecule is selected from the group consisting of B7-1 and B7-2.
  • the invention provides a method of enhancing the costimulatory molecule
  • CD8+ T cell response to a class I restricted antigen in a subject by administering a first agent comprising a class I restricted antigen or fragment thereof and a soluble composition comprising an extracellular domain of a B7 molecule, such that upon administration to the subject the CD8+ T cell response to a class I restricted antigen is enhanced.
  • the methods further comprise administering a class II restricted antigen to the subject.
  • the methods further comprises administering an adjuvant to the subject.
  • the B7 molecule is a B7-1 molecule.
  • the B7 molecule is a B7-2 molecule.
  • the costimulatory molecule is monospecific.
  • the costimulatory molecule is dimeric and bivalent.
  • the soluble costimulatory molecule is monospecific and dimeric and bivalent.
  • B7 molecule is fused to a second protein or polypeptide comprising a portion of an immunoglobulin molecule.
  • the portion of the immunoglobulin molecule comprises cysteine residues.
  • the portion of the immunoglobulin molecule comprises the hinge, CH2 and CH3 regions of a human immunoglobulin molecule.
  • the portion of the immunoglobulin molecule comprises the hinge, CHI, CH2 and CH3 regions of a human immunoglobulin molecule.
  • the immunoglobulin molecule has been modified to reduce complement fixation and/or Fc receptor binding.
  • the antigen is a tumor cell antigen.
  • the subject has a cancer of a type selected from the group consisting of: colon cancer, breast cancer, prostate cancer, renal cell cancer, leukemia, lymphoma, melanoma, mastocytoma, sarcoma, and bladder carcinoma.
  • the antigen is an antigen selected from the group consisting of: a bacterial antigen, a viral antigen, and a parasite antigen.
  • the immune response is a cellular immune response.
  • the immune response is a humoral immune response.
  • Figure 1 shows antigen specific proliferative response of cells from mice immunized with Class II-restricted peptides with or without coadministration of B7-2Ig (lOO ⁇ g).
  • Figure 2 shows that cells from mice that had received a single B7-2Ig treatment at the time of primary immunization had greater proliferative responses following a second immunization than cells from mice that never received B7-2Ig.
  • the data are from replicate experiments.
  • Figure 3 shows that B7-2Ig coadministration enhances the CTL response to immunization with class I restricted peptide.
  • Figure 4 shows the CTL response of mice immunized with class I restricted peptide in the presence or absence of class II restricted peptide and B7-2 Ig treatment.
  • Figure 5 shows that B7-IgG provides a co-stimulatory signal for in vitro proliferation and lymphokine secretion in splenocytes.
  • Figure 6 shows that B7Ig is effective as an adjuvant in a prophylactic tumor vaccine model.
  • Figure 7 shows therapeutic vaccination of mice with irradiated P815 tumor cells mixed with B7-1- or B7-2-IgG induces tumor regression and prolonged survival.
  • Figure 8 shows that immunization of mice with B7-IgG as an adjuvant for a therapeutic irradiated tumor cell vaccine is effective in several different mouse tumor models.
  • Figure 9 shows the anti-tumor effect of therapeutic administration of B7-IgG alone in mice is comparable to its effect as vaccine adjuvant.
  • FIG. 10 shows that T or B cells are required for B7-IgG-mediated anti-tumor activity.
  • FIG 11 shows that CD8+, but not CD4+, T cells are required to mediate B7-IgG anti- tumor activity.
  • Figure 12 shows that B7-IgG therapy of established tumors is independent of host IFN- ⁇ .
  • the instant invention provides improved methods of enhancing immune responses by administration of soluble costimulatory molecules (e.g., an extracellular domain of a B7 molecule, or a B7 fusion protein) to thereby enhance immune responses.
  • soluble costimulatory molecules e.g., an extracellular domain of a B7 molecule, or a B7 fusion protein
  • the soluble costimulatory molecules are administered without a cross-linking agent, yet, surprisingly, stimulate T cell responses.
  • the instant methods result in an increased level of costimulation than costimulatory molecules presented on a surface, e.g.. costimulatory molecules on the surface of a cell.
  • prophylactically includes the administration of a costimulatory molecule prior to or simultaneously with exposure to the antigen against which the immune response is to be developed, augmented, and/or enhanced.
  • the term "therapeutically" includes the administration of a costimulatory molecule to treat an existing or ongoing infection or disease (e.g., cancer or a viral or bacterial infection) which would benefit by treatment with a costimulatory molecule.
  • a costimulatory molecule is administered at a point in time after exposure to the antigen against which the immune response is to be developed, augmented, and/or enhanced. It will be understood that therapeutic treatment with a costimulatory molecule may have other beneficial effects on the immune response of a subject, e.g., that are not specific for that particular antigen.
  • the term "immune cell” includes cells that are of hematopoietic origin and play a role in an immune response. Immune cells include lymphocytes, such as B cells and T cells; natural killer cells: myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • the term " immune response” includes T and/or B cell responses, i.e.. cellular and/or humoral immune responses.
  • the claimed methods can be used to reduce T helper cell responses.
  • the claimed methods can be used to reduce cytotoxic T cell responses.
  • the claimed methods can be used to reduce both primary and secondary immune responses.
  • the immune response of a subject can be determined by, for example, assaying antibody production, immune cell proliferation, the release of cytokines, the expression of cell surface markers, cytotoxicity, etc.
  • costimulate with reference to activated immune cells includes the ability of a costimulatory molecule to provide a second, non- activating receptor mediated signal (a "costimulatory signal") that induces proliferation or effector function.
  • a costimulatory signal can result in cytokine secretion, e.g., in a T cell that has received a T cell-receptor-mediated signal.
  • costimulatory molecule includes molecules which are present on antigen presenting cells (e.g., B7-1, B7-2, B7RP-1 (Yoshinaga et al. 1999. Nature 402:827), B7h (Swallow et al. 1999. Immunity.
  • B7 molecules bind to costimulatory receptors (e.g., CD28, CTLA4, ICOS (Hutloff et al. 1999. Nature 397:263), B7h ligand (Swallow et al. 1999. Immunity. 11 :423) and/or related molecules) on T cells.
  • costimulatory receptors e.g., CD28, CTLA4, ICOS (Hutloff et al. 1999. Nature 397:263), B7h ligand (Swallow et al. 1999. Immunity. 11 :423) and/or related molecules.
  • B7 or “B7 molecule” includes naturally occurring B7-1 molecules, B7-2 molecules, B7RP-1 molecules (Yoshinaga et al. 1999. Nature 402:827), B7h molecules (Swallow et al. 1999. Immunity. 11 :423), structurally related molecules, fragments of such molecules, and/or functional equivalents thereof.
  • equivalent is intended to include amino acid sequences encoding functionally equivalent costimulatory molecules having an activity of a B7 molecule, e.g., the ability to bind to the natural ligand(s) of B7 on immune cells, such as CTLA4, ICOS, and/or CD28 on T cells, and/or the ability to modulate immune cell costimulation.
  • polypeptide(s) refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds.
  • Polypeptide(s) refers to both short chains, commonly referred to as peptides. oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids.
  • Polypeptide(s) include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini.
  • Modifications include, for example, acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma- carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, se
  • Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
  • an "isolated polypeptide” or “isolated protein” refers to a polypeptide or protein that is substantially free of other polypeptides, proteins, cellular material and culture medium when isolated from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • an “isolated” or “purified” polypeptide or biologically active portion thereof is substantially free of cellular material or other contaminating polypeptides from the cell or tissue source from which the B7 polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of B7 polypeptide in which the polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of B7 polypeptide having less than about 30% (by dry weight) of non- B7 polypeptide (also referred to herein as a "contaminating polypeptide"), more preferably less than about 20% of non- B7 polypeptide, still more preferably less than about 10% of non- B7 polypeptide, and most preferably less than about 5% non- B7 polypeptide.
  • non- B7 polypeptide also referred to herein as a "contaminating polypeptide”
  • contaminating polypeptide also referred to herein as a "contaminating polypeptide”
  • the B7 polypeptide or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%. and most preferably less than about 5% of the volume of the polypeptide preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of B7 polypeptide in which the polypeptide is separated from chemical precursors or other chemicals that are involved in the synthesis of the polypeptide.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of B7 polypeptide having less than about 30% (by dry weight) of chemical precursors or non- B7 chemicals, more preferably less than about 20% chemical precursors or non- B7 chemicals, still more preferably less than about 10% chemical precursors or non- B7 chemicals, and most preferably less than about 5% chemical precursors or non- B7 chemicals.
  • B7 nucleic acid molecules and polypeptides are "naturally occurring.”
  • a "naturally-occurring" molecule refers to an B7 molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural B7 polypeptide).
  • naturally or non-naturally occurring variants of these polypeptides and nucleic acid molecules which retain the same functional activity, e.g., the ability to modulate adaptation to stress and/or virulence in a microbe.
  • Such variants can be made, e.g., by mutation using techniques that are known in the art. Alternatively, variants can be chemically synthesized.
  • variant(s) includes nucleic acid molecules or polypeptides that differ in sequence from a reference nucleic acid molecule or polypeptide, but retains its essential properties. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference nucleic acid molecule. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, and/or deletions in any combination.
  • a variant of a nucleic acid molecule or polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally.
  • Non-naturally occurring variants of nucleic acid molecules and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.
  • the B7 polypeptides described herein are also meant to include equivalents thereof.
  • variants can be made, e.g., by mutation using techniques that are known in the art.
  • variants can be chemically synthesized.
  • mutant forms of B7 polypeptides which are functionally equivalent, e.g., have the ability to bind to CTLA4 and/or CD28
  • Mutations can include, e.g., at least one of a discrete point mutation which can give rise to a substitution, or by at least one deletion or insertion.
  • random mutagenesis can be used. Mutations can also be made by random mutagenesis or using cassette mutagenesis.
  • telomere mutagenesis can be used.
  • Megaprimer PCR can be used (O.H. Landt, 1990. Gene 96:125-128).
  • the term "enhancing an immune response” includes increasing T and/or B cell responses, i.e., cellular and/or humoral immune responses, by treatment of a subject using the claimed methods.
  • the claimed methods can be used to enhance T helper cell responses.
  • the claimed methods can be used to enhance cytotoxic T cell responses.
  • the claimed methods can be used to enhance both primary and secondary immune responses.
  • the claimed methods increase the immune response by a subject when compared to the immune response by an untreated subject or a subject not treated using the claimed methods.
  • An increase in an immune response can be shown, e.g., by an increased response of immune cells from the subject to the antigen upon treatment with the claimed methods.
  • the immune response of a subject can be determined using a variety of in vitro or in vivo measurements of immune cell activation, for example, assaying antibody production, immune cell proliferation, the release of cytokines, the expression of cell surface markers, cytotoxicity, etc.
  • the term "soluble” includes molecules, e.g., costimulatory molecules, which are not cell associated. Soluble costimulatory molecules retain the function of the cell-associated molecules from which they are derived, i.e., they are capable of binding to their cognate ligands on T cells and mediating signal transduction via a CD28 and/or CTLA4 molecule on a T cell, however, they are in soluble form, i.e., are not membrane bound.
  • the soluble compositions comprise an extracellular domain of a B7 molecule.
  • extracellular domain of a costimulatory molecule includes a portion of a costimulatory molecule which, in the cell-associated form of the costimulatory molecule, is extracellular.
  • the extracellular domain of a costimulatory molecule comprises an extracellular domain of a B7 molecule.
  • a B7 extracellular domain includes the portion of a costimulatory molecule which mediates binding to CD28 and/or CTLA4.
  • the human B7-1 extracellular domain comprises from about amino acid 1 to about amino acid 208 of the mature form of B7-1 (SEQ ID NO:l) and the human B7-2 extracellular domain comprises from about amino acid 24 to about amino acid 245 of the mature form of B7- 2 (SEQ ID NO:2).
  • a soluble costimulatory molecule comprises an extracellular domain of a B7 molecule and further comprises a signal sequence.
  • class I restricted antigen includes antigens which bind to the major histocompatibility complex (MHC) class I groove and which are presented to T cells in the context of MHC class I molecules. Class I restricted antigens primarily stimulate CD8 + T cells.
  • class II restricted antigen includes antigens which bind to the MHC class II groove and are presented to T cells in the context of MHC class II molecules. Class II restricted antigens primarily stimulate CD4+ T cells.
  • the term "adjuvant” includes agents which potentiate the immune response to an antigen. Adjuvants can be administered in conjunction with costimulatory molecules to additionally augment the immune response.
  • the term “monospecific” includes soluble costimulatory molecules which have only one specificity, i.e., they specifically bind to their cognate ligand, e.g., CD28 or CTLA4 on T cells. Such monospecific agents have not been engineered to include additional specificities and, thus, do not bind in a targeted manner to other cell surface molecules.
  • oligospecific includes soluble costimulatory molecules having more than one specificity, e.g., having an additional specificity for a molecule other than a B7 ligand, e.g., a specificity for a cell surface molecule, such as a tumor cell antigen or a T cell receptor.
  • bivalent includes soluble costimulatory molecules that have two binding sites for interaction with their cognate ligand, e.g., CD28 and/or CTLA4 per soluble costimulatory molecule.
  • dimeric includes soluble forms that are present as homodimers. i.e.. as a unit comprised of two identical subunits which are joined together, e.g., by disulfide bonds.
  • multimeric includes soluble forms having more than two subunits.
  • the B7 antigens are a family of costimulatory molecules found on the surface of B lymphocytes, professional antigen presenting cells (e.g., monocytes, dendritic cells.
  • Langerhans cells and cells which present antigen to immune cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes).
  • costimulatory molecules bind either CTLA4, CD28, and/or ICOS on the surface of T cells or other known or as yet undefined receptors on immune cells.
  • the members of this family of costimulatory molecules are capable of providing costimulation to activated T cells to thereby induce T cell proliferation and/or cytokine secretion.
  • B7 genes cDNA
  • B7 genes cDNA
  • human and mouse see, for example, Freeman, G.J. et al. (1993) Science 262:909-911 ;
  • Nucleotide sequences of costimulatory molecules are known in the art and can be found in the literature or on a database such as GenBank. See, for example, B7-2 (Freeman et al. 1993 Science. 262:909 or GenBank Accession numbers P42081 or
  • DNA sequences which retain sequence identity over regions of the nucleic acid molecule which encode protein domains which are important in costimulatory molecule function, e.g., binding to other costimultory molecules, can be used to produce costimulatory proteins which can be used as immunogens.
  • nonnaturally occurring costimulatory molecules have significant (e.g., greater than 70%, preferably greater than 80%, and more preferably greater than 90-95%) amino acid identity with a naturally occurring amino acid sequence of a costimulatory molecule extracellular domain.
  • amino acid sequences comprising an extracellular domains of costimulatory molecules of different species, e.g., mouse and human, can be aligned and conserved (e.g., identical) residues noted. This can be done, for example, using any standard alignment program, such as MegAlign (DNA STAR). Such alignment programs are described in more detail below. Such conserved or identical residues are likely to be necessary for proper binding of costimulatory molecules to their receptors and are, thus, not likely to be amenable to alteration.
  • the regions of the B7-1 molecule which are important in mediating the functional interaction with CD28 and CTLA4 have been identified by mutation.
  • peptides having an activity of B7 can be produced using standard techniques.
  • Host cells transfected to express peptides can be any procaryotic or eucaryotic cell.
  • a peptide having B7 activity can be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO) and NSO cells.
  • suitable host cells and expression vectors may be found in Goeddel, (1990) supra or are known to those skilled in the art. Examples of vectors for expression in yeast S. cerivisae include pYepSecl (Baldari. et al, (1987) Embo J.
  • COS cells Gluzman, Y., (1981) Cell 23:175-182
  • pCDM8 Seed, B., (1987) Nature 329:840
  • CHO dhfr" Chinese Hamster Ovary
  • pMT2PC Kaufman et al ( 1987), EMBO J. 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. and Milstein, C.
  • 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. When used in mammalian cells, the expression vector's control functions are often provided by viral material.
  • B7 molecules for making the soluble B7 molecules for use in the instant methods can be derived from any mammalian species, and are preferably human.
  • the nucleotide sequences of B7 molecules from several sources are known in the art.
  • the complete DNA sequence of human B7-1 (CD80) has the GenBank accession number M27533 and was published by Freeman et al. in 1989 in J. Immunol. 143:2714.
  • the complete cDNA sequence of human B7-2 (CD86) has the GenBank accession number L25259 and was published by Freeman et al. in Science in 1993. 262;9090 or Azuma et al. Nature. 1993. 366:76. (See also WO 96/40915 for the sequence of both B7-1 and B7-2).
  • the nucleotide sequence of human B7-1 and human B7-2 are also shown in SEQ ID Nos: 1 and 2 (B7-1) and SEQ Nos. 3 and 4 (B7-2).
  • a sequence can be determined by isolating a B7 nucleic acid molecule from a desired source based on the ability of the sequence to hybridize to the known , e.g., human B7 sequences.
  • B7 molecules can be detected by their ability to hybridize under high or low stringency conditions to a known nucleic acid molecule which encodes a peptide having B7 activity.
  • the soluble costimulatory molecule is derived from a naturally occurring B7-1 or B7-2 molecule.
  • Polypeptides having an activity of a B7 molecule, as described herein, and having a sequence which differs from a naturally occurring B7 molecule due to degeneracy in the genetic code can also be expressed in soluble form and are also within the scope of the invention.
  • Such nucleic acids encode polypeptides which are functionally equivalent to B7, (e.g., a polypeptide having B7 activity) but differ in sequence from the sequence of B7-1 or B7-2 known in the art. For example, a number of amino acids are designated by more than one triplet.
  • 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.
  • nucleotide variations and resulting amino acid polymorphisms are also within the scope of the invention.
  • B7 molecules within the scope of the invention can be made using art recognized techniques.
  • a soluble costimulatory molecule is a modified form of B7-1 or B7-2 which retains the function of a B7 costimulatory molecule, i.e., is functionally identical.
  • the DNA sequence of a B lymphocyte antigen can be modified by genetic techniques to produce proteins or polypeptides with altered amino acid sequences. Such sequences are considered within the scope of the present invention, where the expressed polypeptide is capable of binding to CTLA4 and/or CD28 and modulating T cell mediated immune responses and immune function.
  • mutations can be introduced into a DNA molecule 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 B7-1 or B7-2 cDNA sequences 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,
  • Another example of modification of a peptide having the activity of a B7 molecule 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 activity can be chemically modified.
  • Another modification is cyclization of the peptide.
  • polypeptides having B7 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
  • Preferred B7 polypeptides have B7 activity and at least about 60% identity, preferably at least about 70% identity, and more preferably at least about 80% identity with a naturally occurring B7 amino acid sequence.
  • Polypeptides having B7 activity and at least about 90%, preferably at least about 95%, and more preferably at least about 98-99% identity with naturally occurring B7 molecule are also within the scope of the invention.
  • amino acid "identity" at a given position refers to two peptides having the same amino acids at corresponding positions when the amino acid sequences of the peptides are aligned. When a position in the compared sequences is occupied by the same amino acid, then the molecules are identical at that position.
  • a degree (or percentage) of identity between sequences is a function of the number of matching or identical positions shared by the sequences.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, 5, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5. or 6.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST See http://www.ncbi.nlm.nih.gov.
  • B7 polypeptides comprising only naturally-occurring amino acids
  • B7 peptidomimetics are also provided.
  • Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics” or “peptidomimetics” (Fauchere, J. (1986) Adv. Drug Res. 15: 29; Veber and Freidinger (1985) TINS p.392; and Evans et al. (1987) J. Med. Chem 30: 1229, which are incorporated herein by reference) and are usually developed with the aid of computerized molecular modeling.
  • peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.
  • Labeling of peptidomimetics usually involves covalent attachment of one or more labels, directly or through a spacer (e.g., an amide group), to non-interfering position(s) on the peptidomimetic that are predicted by quantitative structure-activity data and/or molecular modeling.
  • a spacer e.g., an amide group
  • non-interfering positions generally are positions that do not form direct contacts with the macromolecules(s) to which the peptidomimetic binds to produce the therapeutic effect.
  • Derivitization (e.g., labelling) of peptidomimetics should not substantially interfere with the desired biological or pharmacological activity of the peptidomimetic.
  • Systematic substitution of one or more amino acids of an B 7 amino acid sequence with a D-amino acid of the same type may be used to generate more stable peptides.
  • constrained peptides comprising an B7 amino acid sequence or a substantially identical sequence variation may be generated by methods known in the art (Rizo and Gierasch (1992) Ann. Rev. Biochem. 61 : 387, incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
  • Peptides can be produced, e.g., by direct chemical synthesis. Peptides can be produced as modified peptides, with nonpeptide moieties attached by covalent linkage to the N-terminus and/or C-terminus. In certain preferred embodiments, either the carboxy-terminus or the amino-terminus, or both, are chemically modified. The most common modifications of the terminal amino and carboxyl groups are acetylation and amidation, respectively. Amino-terminal modifications such as acylation (e.g., acetylation) or alkylation (e.g., methylation) and carboxy-terminal-modifications such as amidation, as well as other terminal modifications, including cyclization, may be incorporated into various embodiments of the invention.
  • acylation e.g., acetylation
  • alkylation e.g., methylation
  • carboxy-terminal-modifications such as amidation, as well as other terminal modifications, including cyclization
  • an B7 "chimeric polypeptide" or “fusion polypeptide” comprises an B7 polypeptide operatively linked to a non- B7 polypeptide.
  • B7 polypeptide refers to a polypeptide having an amino acid sequence corresponding to B7 polypeptide
  • a non-B7 polypeptide refers to a polypeptide having an amino acid sequence corresponding to a polypeptide which is not substantially homologous to the B7 polypeptide, e.g., a polypeptide which is different from the B7 polypeptide and which is derived from the same or a different organism.
  • the B7 polypeptide can correspond to all or a portion of an B7 polypeptide.
  • an B7 fusion polypeptide comprises at least one biologically active portion of an B7 polypeptide.
  • the term "operatively linked" is intended to indicate that the B7 polypeptide and the non-B7 polypeptide are fused in- frame to each other.
  • the non-B7 polypeptide can be fused to the N-terminus or C- terminus of the B7 polypeptide.
  • Preferred nucleic acid fragments encode B7 polypeptides of at least about
  • 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 protein fragment is synthesized and secreted. The B7 protein fragment can then readily be obtained from the culture media.
  • a nucleic acid molecule encoding at least a portion of a B7 molecule, such as an extracellular domain portion which is lacking the transmembrane portion of the molecule is placed in an expression vector and is expressed by a host cell such that the B7 molecule is not expressed on the surface of the cell.
  • cDNA encoding an extracellular domain of a B7 molecule can be synthesized using the polymerase chain reaction (U.S. patent 4,683,202) using primers derived from the published sequence of B7-1 or B7-2 (see Freeman et al., J. Immunol. 1989. 143:2714 or Science. 1993. 262:9090). The resulting cDNA sequences can then be assembled into a eukaryotic or prokaryotic expression vector and the vector can be used to direct the synthesis of an extracellular domain of B7 by appropriate host cells, for example COS or CHO cells.
  • the expression vector includes a DNA encoding a peptide having an activity of a B7 antigen and a DNA encoding a second polypeptide.
  • the second polypeptide is preferably not derived from a costimulatory molecule.
  • a B7 chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • DNA encoding a B7 molecule or portion thereof is linked in frame to DNA encoding an antigen to which an immune response is desired, e.g., a viral antigen or a tumor cell antigen.
  • DNA encoding the amino acid sequence corresponding to an extracellular domain of a B7 antigen is joined to DNA encoding the amino acid sequences corresponding to the constant region of an immunoglobulin molecule (See e.g., US patent 5,580,756 or WO 97/28267).
  • a portion of a B7 molecule is fused to the constant region of an IgM antibody or portion thereof, to allow the formation of soluble multimeric forms of B7Ig proteins.
  • Particularly preferred B7Ig fusion proteins include an extracellular domain portion or variable region-like domain of human B7-1 or B7-2 coupled to an immunoglobulin constant region.
  • the immunoglobulin constant region used in the soluble B7 molecule may contain genetic modifications which reduce or eliminate effector activity inherent in the immunoglobulin structure (see e.g., WO 97/28267).
  • DNA encoding an extracellular portion of B7-1 or B7-2 can be joined to DNA encoding the hinge, CH2 and CH3 regions of human IgC ⁇ l and/or IgC ⁇ 4 modified by site directed mutagenesis.
  • a non-human immunoglobulin constant region is used, preferably the constant region is humanized. Techniques for preparing chimeric or humanized antibodies are well known in the art (see e.g., Morrison et al., Proc. Natl. Acad. Sci. U.S.A.
  • B7 fusion proteins and polypeptides produced by recombinant techniques may be secreted and isolated from a mixture of cells and medium containing the protein or peptide. Alternatively, 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 ingredients. Suitable media for cell culture are well known in the art. Protein and polypeptides 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.
  • B7 molecules 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 to see that they maintain specific reactivity with an anti-B7 monoclonal antibody that binds to a naturally occurring B7 molecule.
  • appropriate cells such as COS cells, can be transfected with a DNA encoding a polypeptide to be tested. Production of secreted forms of B7 can be evaluated using anti-B7 monoclonal antibody or CTLA4Ig or CD28 fusion protein in an immunoprecipitation assay.
  • the ability of cells expressing a peptide of interest to bind to CTLA4 or CD28 on plates by panning can also be tested.
  • the ability of a test peptide to compete with a naturally occurring B7 molecule for binding to CD28 or CTLA4 can also be tested.
  • T cells can be provided with a first or primary activation signal by contacting them with anti-T3 monoclonal antibody (e.g. anti-CD3) or phorbol ester or. more preferably, by antigen in association with class I or class II MHC molecules.
  • T cells which have received a primary activation signal are referred to herein as activated T cells.
  • B7 function is assayed by adding a source of B7 (e.g., cells expressing a peptide having B7 activity or a secreted form of B7) and a primary activation signal such as antigen in association with class I or class II MHC to a T cell culture and assaying for a functional result, e.g., assaying the culture supernatant for interleukin-2, gamma interferon, or other known or unknown cytokine.
  • a source of B7 e.g., cells expressing a peptide having B7 activity or a secreted form of B7
  • a primary activation signal such as antigen in association with class I or class II MHC
  • a functional result e.g., assaying the culture supernatant for interleukin-2, gamma interferon, or other known or unknown cytokine.
  • any one of several conventional assays for interleukin-2 can be employed, such as the assay described in Proc
  • an antigen can be a protein, a polysaccharide, a lipopolysaccharide, a lipopeptide, or it can be a combination of any of these.
  • the antigen can include a native protein or protein fragment, of a synthetic protein or protein fragment, or peptide; it can include glycoprotein, glycopeptide, lipoprotein, lipopeptide, nucleoprotein, nucleopeptide; it can include a peptide-peptide conjugate; or it can include a recombinant nucleic acid expression product.
  • an antigenic preparation comprises a mixture of antigens, e.g., the antigen is administered in the form of irradiated cells (e.g., tumor cells or virally infected cells), viral particles, or a crude homogenate.
  • a purified preparation of an antigenic peptide or a recombinant form of an antigenic peptide is administered to the subject e.g., a viral peptide or a tumor associated antigen.
  • an antigen preparation comprises an MHC class I restricted peptide.
  • an antigen preparation comprises an MHC class II restricted peptide.
  • an antigen preparation comprises a combination of a class I restricted peptide and a class II restricted peptide for administration to the subject.
  • the antigen is administered by "genetic immunization.”
  • a DNA expression vector encoding the peptide of interest is injected into the host animal, e.g., into the skin or into a muscle of the subject.
  • the gene products are correctly synthesized and glycosylated, folded, and expressed by the subject.
  • antigens which are difficult to obtain in sufficient quantity or purity can be administered.
  • DNA is injected into muscles or delivered into the skin coated onto gold microparticles by a particle bombardment device, a "gene gun.” Genetic immunization has been shown to induce specific humoral responses and cellular immune responses (See, e.g., Mor et al. 1995. J. Immunol. 155:2039; Xu and Liew. 1995. Immunology. 84:173; Davis et al. 1994. Vaccine. 12:1503).
  • soluble B7 molecules may vary depending upon the subject to be treated.
  • soluble B7 molecules can be administered with an antigen and/or can be administered alone prior to administration of an antigen, or can be administered alone for several days after administering an antigen.
  • soluble B7 molecules can be administered "genetically" by administering a nucleic acid molecule encoding a soluble B7 molecule or portion thereof.
  • a soluble B7 molecule and an antigen are administered in the form of a conjugate. Dosage procedures of administration of soluble B7 molecules may be adjusted to provide the optimum therapeutic response for each subject without undue experimentation.
  • the active agent or composition may also be administered parenterally or intraperitoneally.
  • the agent can be administered, for example, intranasally, orally, intravenously, intramuscularly, subcutaneously or mucosally.
  • 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.
  • 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 composition or agent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization.
  • dispersions are prepared by incorporating the active compound (e.g., the costimulatory molecule and/or the antigen and any additional agent) 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., agent or composition) plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • agent or composition can be administered in a form suitable for use with a needleless injector device (such devices are known in the art (see, e.g., 5,383,851; 5,581,198; 5,846,233) for example as described in Mol Med 1998. 4:109.
  • a needleless injector device such devices are known in the art (see, e.g., 5,383,851; 5,581,198; 5,846,233) for example as described in Mol Med 1998. 4:109.
  • the agents of the invention are administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo to enhance immune responses.
  • 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 agent.
  • subject is intended to include living organisms in which an immune response can be elicited, e.g., mammals. Examples of subjects include humans, non-human primates, dogs, cats, mice, rats, and transgenic species thereof.
  • Administration of a peptide having the activity of B7 molecule as described herein can be in any pharmacological form including a therapeutically active amount of soluble B7 peptide alone, soluble B7 peptide in combination with an antigen, and soluble B7 peptide in combination with a pharmaceutically acceptable carrier.
  • Administration of a therapeutically or prophylactically active amount of the compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • the administration of a soluble B7 molecule results in an enhanced immune response to an antigen (e.g., a viral or a tumor cell antigen).
  • an antigen e.g., a viral or a tumor cell antigen
  • a therapeutically or prophylactically active amount of a polypeptide having B7 activity may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the peptide to elicit a desired response in the individual. Dosage periods may be adjusted to provide the optimum therapeutic or prophylactic 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 may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of 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.
  • the invention further pertains to the active compound in the form of a medicament for use in therapy as described herein.
  • the active compound may also be used in the manufacture of a medicament for use in therapy.
  • agents which are known adjuvants can be administered.
  • the only adjuvant widely used in humans has been alum (aluminum phosphate or aluminum hydroxide).
  • Other adjuvants e.g., saponin and its purified component Quil A, Freund's complete adjuvant and other adjuvants used in research and veterinary applications have potential use in human vaccines.
  • new chemically defined preparations such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates such as those described by Goodman- Snitkoff et al. J. Immunol.
  • lipid vesicles such as Novasome TM lipid vesicles (Micro Vescular Systems, Inc., Nashua, N. H.), to further enhance immune respones.
  • Novasome TM lipid vesicles Micro Vescular Systems, Inc., Nashua, N. H.
  • B7-1 and B7-2 are two potent co-stimulatory molecules on the surface of APCs.
  • the effects of a soluble form of B7-2 on in vivo T cell responses have been examined.
  • the soluble molecule is a chimeric protein containing the extracellular domain of B7-2 fused to the Fc region of mouse IgG2a.
  • Administration of B7- 2Ig fusion protein at the time of immunization with class II restricted peptides significantly enhanced antigen-specific T cell proliferation and cytokine responses.
  • B7- 2Ig administration also enhanced the CTL response to immunization with a class I-restricted peptide.
  • mice Female BALB/cJ mice (Jackson Labs, Bar Harbor, ME) 7 to 10 weeks of age were used throughout this study.
  • peptides used were H-2 d -restricted immunodominant epitopes from nucleoprotein (NP) of influenza virus A/PR18/34.
  • NP nucleoprotein
  • mice 100 ⁇ g of the indicated peptide or peptide mixture was mixed 1 :1 by volume with IFA (Sigma) and emulsified. Mice were immunized subcutaneously with antigen/IFA emulsion in 100 ⁇ l at the base of the tail. B7-2Ig in 100 ⁇ l was administered subcutaneously at a site proximal to the peptide immunization site. In experiments where lymph node cells were harvested, mice received peptide and B7-2Ig as described above both at the base of the tail and at the back of the neck. Treatment groups consisted of 5 mice each. B7-2Ig was administered in 0.1% aluminum hydroxide (Rehydragel, Rehies, Dublin, Ireland).
  • Lymph nodes excluding mesenteric nodes
  • spleens were collected on the indicated days and single cell suspensions prepared and cultured in flat bottom 96 well plates at 5 x 10 5 cells/well in 200 ⁇ l in RPMI 1640 (Gibco/BRL), supplemented to contain 5 x 10 "5 M 2ME, 2mM Lglutamine, 100 U/ml Penicillin and lOO. ⁇ g/ml Streptomycin (Gibco/BRL), 10% FCS. Class II-restricted peptides were added at the start of culture at the indicated concentration.
  • Proliferation was measured by 3H thymidine incorporation following an 18 hour pulse (l ⁇ C/well) with a 1450 Microbeta Plate Reader (Wallac). Comparison of proliferative responses from lymph node cells on days 3, 5, 7, and 9 post primary immunization were conducted. Secondary responses were assessed from spleens harvested three days after secondary immunization.
  • samples of blood were collected by retro-orbital bleed from mice anesthetized with Aerrane, (Ohmeda Caribe, Inc, Guayama, PR). Samples were diluted with 100 ⁇ l PBS containing 50U heparin. A 40 ⁇ l sample was removed for white blood cell counts. Differential counts on samples were not routinely performed. The percentage of lymphocytes in the white blood cell fraction was 69% + 8% by differential count of 100 samples taken at various times during the study. Cells were washed to remove heparin.
  • Cells were plated in 96 well round bottom Costar plates in lOO ⁇ l volume for a total of 8 x 10 3 while blood cells per well. A total of 80 wells were plated for each blood sample. Sixteen wells on each plate received only media with irradiated spleen cells. On day 7 of culture, supernatants were decanted by inverting the plates and cell pellets suspended by plate agitation. Antigen-positive (Ag-positive) targets were prepared by overnight pulse of P815 cells with 1 Oug/ml of the class I-restricted peptide.
  • Experimental release - medium release X 100 total release - medium release where total release was the average cpm of all wells which received targets and triton-X-100, and medium release was the average cpm + SD of all wells which received targets and media only. Mean specific percent lysis per well was calculated according to the formula:
  • mice were immunized with peptides in the presence or absence of B7-2Ig administration.
  • the class II-restricted peptides aa 55-77 and aa 206-229 from NP of A/PR/8/34 influenza virus were delivered subcutaneously as a mixture in IFA. These peptides have been shown to be immunodominant CD4+ Th cell epitopes (Brett, S. J., et al. 1991. Journal Immunology 147:1647, Brett, S. J., and J. P. Tite. 1996).
  • B7-2Ig in 0.1% alum was administered at a proximal site.
  • peptide-specific proliferative responses were measured from lymph node cells on days 3, 5, 7 and 9 post immunization. The kinetics of the response were not affected by administration of B7- 2Ig.
  • Optimal proliferative responses were observed from both B7-2Ig and control treated mice at day 7 and 9 post immunization.
  • Five mice per group were immunized with peptides alone (-0 -), treated with B7-2Ig alone (- ⁇ -), or immunized with peptides and treated with B7-2Ig (-"-).
  • both groups of peptide immunized mice were re-immunized with peptides in the absence of B7-2Ig coadministration.
  • Non-immunized mice received IFA only. Three days after re-immunization, spleen cells were restimulated in vitro with the indicated concentration of each of the two peptides used for immunization. Proliferation was assessed by 3 H-thymidine incorporation. Data are expressed as the average cpm/well of 5 mice per group ⁇ SD. Data are from one of two replicate experiments. These data indicate that B7- 2Ig, when used as an adjuvant for a primary T cell response, enhances the recall response.
  • B7-2Ig as immune adjuvant differentially promoted development of Thl or Th2 responses
  • peptide-specific cytokine responses following primary immunization were analyzed from lymph node cells of immunized mice at 3,5,7 and 9 days post immunization. Just as with proliferative responses, optimal cytokine responses were observed at day 9.
  • Antigen specific cytokine production was not observed in cultures from unimmunized mice treated with B7-2Ig.
  • B7-2Ig Enhances CTL Response To Class I-Restricted Peptide. Interaction of membrane bound B7 with CD28 has been shown to allow induction of in vitro CTL responses in the absence of CD4+ cells (Harding, F. A. et al. 1993, J Exp Med 177:1791).
  • mice were immunized with the immunodominant class I-restricted peptide in IFA with or without concomitant B7-2Ig administration.
  • Peptide specific CTL responses were measured from unfractionated peripheral blood cells using the small blood sample, CTL assay as described in Materials and Methods. This assay makes possible the analysis of large numbers of mice within a single experiment, assessment of the statistical significance of differences between groups, repeated CTL assays from individual mice during the course of an immune response, and investigation of correlation between CTL responses and in vivo results.
  • mice were immunized with an IFA emulsion containing either the class I-restricted peptide alone or a mixture of the class I-restricted peptide and the two class Il-restricted peptides described for Figures 1 and 2 and Table 1 above.
  • CTL responses were measured from unfractionated peripheral blood cells ( Figure 4).
  • Peptide immunization was done as a single emulsion in IFA. Mice were immunized and treated as indicated. Three weeks later, CTL responses were measured from peripheral blood. Data are expressed as mean specific lysis + SD. The value for the live virus immunized group is from a single pool of 2 mice, and the average mean specific lysis of this group was 52 ⁇ 12 obtained from 18 mice tested in 9 experiments.
  • mice There were five mice per group in all other groups. Mean specific lysis of the group which received Class Il-restricted peptides in IFA and control Ig in alum has been subtracted from groups shown which received control Ig. Mean specific lysis of the group which received class Il-restricted peptides in IFA and B7-2Ig in alum has been subtracted from groups shown which received B7-2Ig. The data shown are from one of four replicate experiments.
  • mice immunized with peripheral blood cells peaked at 2 to 3 weeks and waned in the fifth week in both mice immunized with live virus and mice immunized with peptides and treated with B7-2Ig, again indicating similarity of the responses. As discussed below, however, mice immunized with the peptide mixture and treated with B7-2Ig were not protected from lethal virus challenge.
  • Optimal antigen-specific activation and regulation of T cells requires the delivery of a costimulatory signal from B7 molecules on the surface of the APC to CD28 and CTLA-4 on the surface of the T cell (Boussiotis, V. A., et al. 1996, Immunol Rev 153:5; Lenschow, D. J. et al. 1996, Annual Review Immunology 14:233; Green, J. M. et al. 1994. Immunity 1:501; Tivol, E. A. et al. 1995, Immunity 3:541 and Waterhouse, P., 1995, Science 270:985). Work in a wide variety of mouse models has shown that immune responses can be enhanced by increased cell surface expression of B7.
  • B7-2Ig a soluble protein form of B7-2, B7-2Ig has been developed, consisting of the Fc of mouse IgG2a fused with an extracellular portion of B7-2 attached to each Ig chain.
  • B7-2Ig enhances antigen specific Th cell and CTL responses.
  • B7-2Ig presents a simple alternative to ex vivo B7 transduction of tumor cells or the use of viral or D ⁇ A vectors for optimizing B7-2 mediated costimulation.
  • Th2 differentiation is more dependent on B7 costimulation than is Thl differentiation (Lenschow, D. J. et al. 1995, J Exp Med 181:1145; Corry, D. B., et al. 1994, J Immunol 153:4142; Freeman, G. J. et al. 1995, Immunity 2:523 and Rulifson, I. C, A. et al. 1997, J Immunol 158:658).
  • Anti-B7 mAb have been successfully used to manipulate T cell differentiation in vivo.
  • mice were capable of a much greater anti peptide response than that which is elicited by class I-restricted peptide immunization and B7-2Ig coadministration.
  • mice immunized with class I and class Il-restricted peptides were less than that of mice immunized with live virus.
  • Addition of B7-2Ig to the protocol increased the CTL response of mice immunized with the mixture of peptides to the level of mice immunized with live virus. Responses of these two groups were comparable, but the former group was not protected from lethal virus challenge. This result is consistent with those of Lawson et al. (Lawson, C. M. et al.
  • formulation in alum results in greater enhancement than formulation in PBS.
  • Administration of control Ig in alum does not affect responses, indicating that alum alone does not enhance the response.
  • mice per group were immunized at two subcutaneous sites with an IFA emulsion containing either lOO ⁇ g per injection of each of the two class Il-restricted peptides or PBS.
  • lOO ⁇ g of B7-2IgG2a in 0.1 % alum or 0.1% alum alone was administered at sites proximal to the immunization sites.
  • lymph node cells were harvested and restimulated in culture for three days with 5 ⁇ g/ml of each peptide. Values for each mouse were obtained by averaging results from triplicate wells. Data are expressed as the average concentration of cytokine of mice within a group ⁇ SD. Results are similar to those obtained when a control mouse IgG2a mAb was used as control. Data shown are from one of three replicate experiments. b Below the limits of detection of assay.
  • Fusion proteins between an extracellular region of either B7-1 or B7-2 and IgG.2a have been evaluated for their ability to enhance anti-tumor responses in four different murine tumor models, including the poorly immunogenic melanoma B16/F10.
  • a single vaccination with irradiated tumor cells when mixed with B7-1 or B7-2-IgG protected mice against a live tumor challenge. More significantly, 7-day established tumors regressed after vaccination with irradiated tumor cells mixed with B7-IgG. Even therapeutic administration of B7-IgG alone achieved similar decreases in tumor burden. Animals that had rejected an established tumor were resistant to a rechallenge, strongly suggesting that the anti-tumor effect of B7-IgG is mediated by tumor-specific immune mechanisms.
  • the following amino acid residues in the CH2 domain were replaced by alanine: leucine at position #235, glutamic acid at #318, lysine at #320, and lysine at #322.
  • leucine at position #235 leucine at position #235
  • glutamic acid at #318 glutamic acid at #318
  • lysine at #320 lysine at #320
  • lysine at #322 lysine at #322.
  • P815 is a mastocytoma derived tumor cell line that grows as a solid tumor after intradermal (i.d.) injection of 5x 10 ⁇ cells into DBA/2 mice (Jackson Laboratories). The clone used in these experiments metastasizes spontaneously after i.d. injection and leads to the death of the mice after 25-35 days.
  • MethA a tumor cell line derived from a sarcoma in Balb/C mice, grows as solid tumor after i.d. injection of 5x10 ⁇ cells (Balb/C mice from Taconic).
  • B16/F10 a non-immunogenic tumor line derived from a melanoma in C57BL/6 mice (Taconic), grows as a solid tumor after i.d. injection of lx 10 ⁇ cells and causes death due to metastases after 25-35 days.
  • the bladder carcinoma MB49 is also derived from C57BL/6 mice and lxl 0 ⁇ cells were injected i.d. to establish solid tumors after 5-7 days in 100% of mice.
  • Spleens were collected from DBA/2 mice 10-14 days after a single immunization with irradiated P815 cells mixed with or without B7-l-IgG or B7-2IgG. After lysis of erythrocytes 20x 10° " splenocytes were restimulated in T25 flasks with 0.1 ng/ml PI A peptide and 5 U/ml IL-2 (Pharmingen) for 6 days. Then, a standard 5h Cr ⁇ l- release assay was performed with A20 cells as targets pulsed with lO ⁇ g/ml of PI A peptide or unpulsed with peptide. The percent of PlA-peptide specific lysis is expressed as the difference between the percent lysis of peptide-pulsed targets and the percent lysis of unpulsed targets.
  • Immobilized or cross-linked B7-IgG provides costimulatory signal for suboptimally stimulated murine splenocytes in vitro.
  • B7-l-IgG and B7-2- IgG fusion proteins bind to murine CD28 and CTLA-4.
  • murine splenocytes were stimulated in vitro by culturing with plate-bound anti-CD3 mAb in combination with B7-IgG immobilized on the plates. Costimulation with plate-bound anti-CD28 mAb served as a positive control.
  • B7-l-IgG and B7-2-IgG similarly induced a dose dependent increase in proliferation and IFN- ⁇ secretion (Figure 5).
  • na ' ⁇ ve splenocytes were stimulated in triplicate for 72 h with 50 ng/ml anti-CD3 monoclonal antibody and increasing amounts of immobilized anti-CD28 antibody, or B7-1 or B7-2 IgG.
  • the proliferative response was measured by incorporation of 3H-thymidine after a 6h pulse.
  • Panels B and C show the amount of IFN ⁇ or IL2 respectively, released after 72h stimulation with 50ng/ml anti-CD3 antibody and indicated amounts of immobilized B7-2-IgG. Cytokines were measured by standard ELISA. In the absence of anti-CD3 stimulation, B7-IgG did not induce proliferation.
  • B7-IgG proteins mutated in their Fc binding region were as effective in co-stimulating splenocytes when immobilized on the plate.
  • Both B7-l-IgG or B7-2-IgG enhance the protective efficacy of an irradiated tumor cell vaccine
  • the B7-IgG fusion proteins were evaluated as adjuvants in a prophylactic tumor vaccine model. Inoculation of 5x10 ⁇ live P815 tumor cells generates a solid tumor after 5-7 days in 100% of naive DBA/2 mice. 10 DBA/2 mice per group were immunized i.fp. once with 1 x 10 ⁇ irradiated P815 tumor cells. The cell vaccine was given alone or mixed with either lOO ⁇ g of B7-l-IgG, B7-2-IgG, or B7-l-IgG, B7-2-IgG mutated protein. B7-IgG was administered again on day 5.
  • mice were immunized with irradiated P815 tumor cells mixed with the mutated fusion proteins, B7-IgGmut, that do not bind Fc-receptors and do not activate complement.
  • the mutated molecules were less effective than wild type (Fig. 6, Table 2), suggesting a role for Fc binding of the B7-IgG molecules.
  • mice were injected i.d. with P815 tumor cells at a dose that generated a palpable solid tumor after five to seven days.
  • Solid P815 tumors were established on day 0 by i.d. injection of 5 x 10 ⁇ P815 cells.
  • Vaccination started on day 7 after palpable tumors had developed.
  • Figure 7 shows the results of mice injected i.fp. with either PBS as control (A, E), or with irradiated P815 tumor cells alone (B) or mixed with irrelevant mouse IgG2a Ab (F), or with irradiated P815 cells mixed with B7-1 (C)- or B7-2-IgG (G).
  • the immunization was repeated on days 7, 14, 21.
  • PBS, irrelevant Ab, or B7-IgG, respectively was administered again three days later. Tumor growth was monitored for 40 days. After 25-30 days animals in the control groups stared to die from spontaneous metastases. Panels D and H show the survival times of the different treatment groups. Data are representative for four independent experiments. The kinetics of tumor growth and survival after vaccination are shown in Figure 7, representative for four independent experiments. Beginning about one week after the first immunization, reduced tumor growth in the tumor regression in the groups of mice immunized with tumor cells mixed with B7-IgG was observed. Tumor growth was not reduced in groups treated with irradiated tumor cells alone or mixed with an irrelevant mouse IgG2a Ab.
  • Balb/c mice bearing 7-day old established MethA sarcomas were immunized with PBS, irradiated MethA cells alone, or irradiated MethA cells mixed with either B7-l-IgG or B7-2-IgG.
  • Solid MethA or B16/F10 tumors were established in Balb/c or C57BL/6 mice, respectively.
  • Figure 8 shows that ten mice per group were immunized i.fp. with either irradiated tumor cells alone (B, E), or mixed with 25 ⁇ g (C, D) or lOO ⁇ g (F, G) B7-l-IgG or B7-2-IgG, respectively.
  • One group was treated with PBS alone (A, not shown for B16/F10). Tumor growth was monitored for 35 days. Mice with MethA tumors were euthanized once the tumor reached a size of 300mm2. Mice with the B16/F10 tumor were either euthanized once the tumor reached a size of 400mm2, or the animals died from a spontaneous metastases. The percent of surviving animals is shown in panel H. Experiments were repeated at least three times.
  • mice with B7- IgGs in the absence of irradiated tumor cells did not protect mice against a tumor challenge.
  • treatment of tumor-bearing mice with B7-l-IgG or B7-2-IgG alone reduced tumor growth and increased survival (Fig. 9).
  • mice were either treated with PBS (D), irradiated tumor cells alone (0), irradiated tumor cells mixed with B7-l-IgG (A), or B7-2-IgG (O), or with B7-l-IgG (*) or B7-2-IgG alone (+).
  • the mean value of tumor size for groups of 7-10 mice has been plotted. Mice were euthanized once their tumor reached a size of 400mm2 or were assigned this value if they died from metastatic disease. In all models tested, therapeutic treatment of tumor- bearing mice with B7-IgG alone slowed tumor growth, induced tumor regression and increased survival.
  • data from the B16/F10 tumor model suggest that vaccination with irradiated tumor cells plus B7-IgG is a stronger anti-tumor treatment than therapy with B7-IgG alone, at least for poorly immunogenic tumors.
  • B7-IgG mediated tumor cure is CD8 T cell dependent but IFN- ⁇ independent
  • B7-IgG in SCID mice that lack mature T and B cells was evaluated. Solid tumors were established in SCID mice and then they were treated with either B7-IgG alone or with an irradiated cell vaccine plus B7-IgG. Neither treatment had an effect on tumor growth, demonstrating the dependence of B7-IgG mediated tumor responses on T or B cells ( Figure 10). Tumor-bearing mice were also treated after depleting CD8 or CD4 T cells. Depletion of CD8 or CD4 T cells by antibody injection was started one day prior to initiation of B7-IgG therapy. Successful depletion was verified by FACS analysis of PBL on day 28.
  • B7-IgG in anti-tumor therapy or as vaccine adjuvant is more potent than a blocking antibody to CTLA4.
  • B7-IgG treatment cured tumors or protected against tumor challenge where an anti-CTLA4 antibody had no effect despite its much higher affinity to CTLA4 and previous reports of its blocking activity.

Abstract

Cette invention concerne des méthodes propres à favoriser des réponses immunitaires, selon lesquelles on administre des formes solubles de molécules de co-stimulation, telles que des molécules B7, pour accroître les réponses immunitaires à des antigènes tels que des cellules tumorales ou des agents infectieux. Les méthodes selon l'invention conviennent à titre tant prophylactique et thérapeutique pour l'immunisation de sujets.
EP00930437A 1999-05-06 2000-05-05 Emploi de molecules de co-stimulation solubles favorisant les reponses immunitaires Withdrawn EP1181053A2 (fr)

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AU2012290306B2 (en) * 2011-07-29 2017-08-17 Selecta Biosciences, Inc. Synthetic nanocarriers that generate humoral and cytotoxic T lymphocyte (CTL) immune responses
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US20150056232A1 (en) * 2012-04-02 2015-02-26 The Arizona Board Of Regents For And On Behalf Of Arizona State University Recombinant bacterium for induction of cellular immune response
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