WO2002088304A2 - Compositions et procedes destines a supprimer des reponses immunitaires - Google Patents

Compositions et procedes destines a supprimer des reponses immunitaires Download PDF

Info

Publication number
WO2002088304A2
WO2002088304A2 PCT/US2002/011371 US0211371W WO02088304A2 WO 2002088304 A2 WO2002088304 A2 WO 2002088304A2 US 0211371 W US0211371 W US 0211371W WO 02088304 A2 WO02088304 A2 WO 02088304A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
cells
cell
molecule
megp
Prior art date
Application number
PCT/US2002/011371
Other languages
English (en)
Other versions
WO2002088304A3 (fr
Inventor
Stephen L. Eck
Original Assignee
Trustees Of The University Of Pennsylvania
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Trustees Of The University Of Pennsylvania filed Critical Trustees Of The University Of Pennsylvania
Priority to AU2002338571A priority Critical patent/AU2002338571A1/en
Publication of WO2002088304A2 publication Critical patent/WO2002088304A2/fr
Publication of WO2002088304A3 publication Critical patent/WO2002088304A3/fr

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
    • 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
    • A61K39/00113—Growth factors
    • A61K39/001131—Epidermal growth factor [EGF]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46434—Antigens related to induction of tolerance to non-self
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention is directed to the fields of immunology, molecular biology and gene therapy. More specifically, compositions and methods relating to the administration of tumor associated antigens and functional fragments thereof for modulating undesirable immune responses in a vertebrate are disclosed. The present invention also provides compositions and methods for modulating deleterious immune responses to therapeutically beneficial proteins administered directly to the patient or proteins encoded by gene therapy vectors.
  • TAA tumor-associated antigens
  • CD4+ T cells specific for tumor-associated antigens are likely activated in most cases by professional antigen presenting cells (APCs), such as B cells, dendritic cells (DCs) and macrophages, rather than by tumor cells themselves, which typically lack expression of class II and costimulatory molecules 4,8,9 APCs can internalize tumor-derived proteins, primarily by phagocytosis or macropinocytosis of tumor cell debris. These proteins are then processed and presented by major histocompatibility complex (MHC) class II molecules on the surface of the APC, permitting recognition by antigen-specific CD4+ T cells. Stimulation of these CD4+ T cells results in upregulation of CD40 ligand, which interacts with CD40 on the surface of the APC
  • MHC major histocompatibility complex
  • APCs represent a critical link between CD4+ T cells and CD8+ T cells 4 .
  • GA733-2 also known as Trop-1, KSA, CO17- IA, human epithelial glycoprotein, Ep-Cam, EGP31
  • mEGP human epithelial glycoprotein
  • mEGP bears 81% amino acid identity to GA733-2, has a similar expression pattern in normal tissues 16, 17 ⁇ anf j is similarly expressed in some murine tumor cell lines.
  • mEGP, GA733-2, and GA733-1 comprise a family of related proteins which share a few key elements.
  • Each family member is a type I transmembrane protein with a large extracellular domain and a small intracellular domain.
  • the extracellular region comprises a single thyroglobulin domain (TGD) and a single epidermal growth factor (EGF) domain, each of which appear to be important for activity.
  • the proteins appear to exist as monomers and as homodimers on the cell surface.
  • the TGD is conserved among the family members, as is a Cys-Trp-Cys-Nal motif within the TGD.
  • compositions and methods are provided for modulating an undesirable immune response in a patient.
  • the present inventor has discovered that tumor associated antigens impair immune function and may be used to advantage to suppress deleterious immune reactions.
  • the methods entail the administration of TAA molecules selected from nucleic acid sequences (SEQ ID NO: 1) encoding full length murine epithelial glycoprotein (mEGP; SEQ ID NO: 4); nucleic acid sequences (SEQ ID NO: 2) encoding full length human GA733-2 protein (SEQ ID NO: 5); and nucleic acid sequences (SEQ ID NO: 3) encoding full length GA733-1 protein (SEQ ID NO: 6) to mediate immune suppression.
  • TAA molecules selected from nucleic acid sequences (SEQ ID NO: 1) encoding full length murine epithelial glycoprotein (mEGP; SEQ ID NO: 4); nucleic acid sequences (SEQ ID NO: 2) encoding full length human GA733-2 protein (SEQ ID NO: 5); and nucleic acid sequences (SEQ ID NO: 3) encoding full length GA733-1 protein (SEQ ID NO: 6) to mediate immune suppression.
  • novel methods for the use of amino acid sequences comprising SEQ
  • the invention further provides methods for administering vectors comprising SEQ ID NO: 1, 2, or 3 to cells in vitro or in vivo. Such methods may be used to advantage to treat patients having a disorder or requiring treatment associated with deleterious immune responses and for which targeted immune suppression would be beneficial to the recovery of the patient.
  • the methods of the present invention are of utility for any condition in which a patient is mounting an undesirable immune response to a protein (endogenous or exogenous) and/or a tissue (e.g., autologous, allogeneic, xenogeneic). Such methods are also useful for the prophylactic treatment of patients at risk for a disorder associated with an autoimmune disorder.
  • the TAAs are administered in conjunction with other therapeutically beneficial proteins to suppress undesirable immune responses provoked by such proteins.
  • Gene therapy vectors encoding both the TAAs and therapeutically beneficial proteins may be administered.
  • a plurality of vectors encoding the TAAs and the therapeutically beneficial protein may be administered separately to the patient.
  • Figure 1 shows an agarose gel of RT-PCR products (A) and flow cytometry histograms (B) depicting expression of mEGP in myeloid DCs. Immature and mature DCs were examined for the expression of mEGP by RT-PCR (A) and compared to normal colon where mEGP is normally abundantly expressed. Transduction with AdmEGP or AdGA733-2 resulted in high levels of mEGP or GA733-2 expression, respectively, as detected by flow cytometry (B) without significant changes in cell surface expression of MHC Class I and ⁇ , CD80 (B7-1), CD86 (B7-2), CDl lb or CDllc.
  • FIG. 2 shows line graphs and histogram plots depicting T cell responses in mixed lymphocyte reactions (MLRs).
  • MLRs mixed lymphocyte reactions
  • Expression of mEGP in DCs impairs their ability to stimulate allogeneic T cells in a MLR compared to untransduced DCs, or DCs transduced to express GA733-2 or an empty control adenovirus (Adbgl2).
  • the impairment is reflected by decreased T cell proliferation (A), IL-2 production (B), and interferon ⁇ production (C).
  • Addition of ConA or soluble anti-CD3-antibodies as a second T cell stimulus restored the MLR proliferative response (D) in the presence of mEGP.
  • Figure 3 shows line graphs which reveal that inhibition by mEGP is dose dependent.
  • Increasing amounts of AdmEGP were incubated with DCs at a multiplicity of infection (MOI) ranging from 0 to 100 viral particles per DC resulting in incremental increases in mEGP cell surface expression (shown for MOI of 100 in Figure IB) and inversely proportional decrements in MLR activity (A).
  • MOI multiplicity of infection
  • A inversely proportional decrements in MLR activity
  • Mixtures of DCs (10 4 per well total) containing variable proportions of untransduced and transduced DCs (MOI of 100) produced MLR responses inversely proportional to the number of mEGP expressing DCs with no apparent transdominant effect (B).
  • Figure 4 shows line graphs depicting the effect of mEGP on DC-mediated stimulation of ONA and HEL specific T cells.
  • DCs from BALB/c mice were pulsed with ovalbumin protein (A) or OVA323-339-peptide (B) and used to stimulate T cells obtained from transgenic DOl 1.10 mice, that express a TCR specific for ONA323- 339-peptide presented in I-Ad.
  • DCs from C3H mice were pulsed with hen egg lysozyme (C) or HEL46-61 -peptide (D) and used to stimulate T cells from transgenic 3A9 mice that express a TCR specific for HEL46-61 -peptide presented in I-Ak.
  • C hen egg lysozyme
  • D HEL46-61 -peptide
  • FIG. 5 shows line graphs and histogram plots of T cell responses in an allogeneic MLR.
  • DCs were pulsed for 16h with different concentrations of cell debris containing mEGP (FBL-3. AdmEGP) or control cell debris (FBL-3.Adbgl2) and then used to stimulate T cells in an allogeneic MLR (A).
  • FBL-3. AdmEGP cell debris containing mEGP
  • FBL-3.Adbgl2 control cell debris
  • A allogeneic MLR
  • mEGP containing tumor debris suppressed the response of DO 11.10 T cells to ONA (B).
  • Tumor debris containing mEGP, GA733-2 or mEGPex were equally effective in inhibiting a MLR (C).
  • DCs expressing mEGP were markedly less efficient in vaccinating mice against co-expressed adeno viral antigens compared to Adbgl2 transduced control DCs.
  • FIG. 6 is a line graph showing that mEGP inhibits T cell responses to adenovirus antigens (Ag) in vivo.
  • mEGP SI, S2 & S3 adenovirus expressing mEGP
  • Bgl2 SI, S2 & S3 control adenovirus
  • SO untransfected DCs
  • Figure 7 shows line graphs revealing that antibody responses to adenoviral Ag are inhibited by mEGP in vivo.
  • the sera were assessed for antibody responses to adenoviral Ag by ELISA.
  • ELISA plates were coated with heat denatured viral particles.
  • Figure 8 shows the nucleic acid sequence of mEGP (SEQ ID NO: 1).
  • Figure 9 shows the nucleic acid sequence of GA733-2 (SEQ ID NO: 2).
  • Figure 10 shows the nucleic acid sequence of GA733-1 (SEQ ID NO: 3).
  • Figure 11 shows the amino acid sequence of mEGP (SEQ ID NO: 4) encoded by SEQ ID NO: l.
  • Figure 12 shows the amino acid sequence of GA733-2 (SEQ ID NO: 5) encoded by SEQ ID NO: 2.
  • Figure 13 shows the amino acid sequence of GA733-1 (SEQ ID NO: 6) encoded by SEQ ID NO: 3.
  • Figure 14 shows the nucleic acid sequence (SEQ TD NO: 7; Figure 14A) encoding the amino acid sequence of the terminal epidermal growth factor domain (EGF-1) of GA733-2 (SEQ ID NO: 8; Figure 14B).
  • Figure 15 shows the nucleic acid sequence (SEQ TD NO: 9; Figure 15A) encoding the amino acid sequence of the thyroglobulin domain (TGD) of GA733-2 (SEQ TD NO: 10; Figure 15B).
  • the present invention relates generally to the field of immune suppression.
  • the present inventor has made the surprising discovery that TAAs may be used to advantage as modulators of undesirable host immune responses.
  • TAAs GA733-2, mEGP, and a related human homologue GA733-1 also designated Trop-2; M1S1
  • M1S1 human homologue
  • methods are provided herein for the use of nucleic acid sequences encoding TAAs (i.e., GA733-2, mEGP, and Trop-2), and TAA polypeptides and functional fragments thereof to mediate immune suppression.
  • grafts e.g., bone marrow transplant cells, organ and tissue transplants
  • nucleic acid or a “nucleic acid molecule” as used herein refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form.
  • a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5' to 3' direction.
  • isolated nucleic acid is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated.
  • an "isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.
  • isolated nucleic acid refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above.
  • the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues).
  • An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.
  • Natural allelic variants refer to nucleic acid sequences that are closely related to a particular sequence but which may possess, either naturally or by design, changes in sequence or structure.
  • closely related it is meant that at least about 75%, but often, more than 90%, of the nucleotides of the sequence match over the defined length of the nucleic acid sequence referred to using a specific SEQ ID NO.
  • Changes or differences in nucleotide sequence between closely related nucleic acid sequences may represent nucleotide changes in the sequence that arise during the course of normal replication or duplication in nature of the particular nucleic acid sequence.
  • the present invention also includes methods of use for active portions, fragments, derivatives and functional mimetics of TAA polypeptides or proteins of the invention.
  • An "active portion" of TAA polypeptide means a peptide that is less than the full length TAA polypeptide, but which retains measurable biological activity.
  • a “fragment” or "portion” of the TAA polypeptide means a stretch of amino acid residues of at least about five to seven contiguous amino acids, often at least about seven to nine contiguous amino acids, typically at least about nine to thirteen contiguous amino acids and, most preferably, at least about twenty to thirty or more contiguous amino acids. Fragments of the TAA polypeptide sequence, antigenic determinants, or epitopes are useful for eliciting immune responses to a portion of the TAA amino acid sequence.
  • a “derivative" of the TAA polypeptide or a fragment thereof means a polypeptide modified by varying the amino acid sequence of the protein, e.g., by manipulation of the nucleic acid encoding the protein or by altering the protein itself. Such derivatives of the natural amino acid sequence may involve insertion, addition, deletion or substitution of one or more amino acids, and may or may not alter the activity of the original TAA polypeptide.
  • a TAA polypeptide or protein of the invention includes any analogue, fragment, derivative or mutant which is derived from a TAA polypeptide and which retains at least one property or other characteristic of the TAA polypeptide. Different "variants" of the TAA polypeptide exist in nature.
  • variants may be alleles characterized by differences in the nucleotide sequences of the gene coding for the protein, or may involve different RNA processing or post-translational modifications.
  • a skilled person can produce variants having single or multiple amino acid substitutions, deletions, additions or replacements.
  • variants may include inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to the TAA polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which the TAA polypeptide is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the TAA polypeptide, such as, for example, an epitope for an antibody, a polyhistidine sequence, a biotin moiety and the like.
  • TAA polypeptides of the invention include variants in which amino acid residues from one species are substituted for the corresponding residues in another species, either at the conserved or non-conserved positions. In another embodiment, amino acid residues at non-conserved positions are substituted with conservative or non-conservative residues.
  • the techniques for obtaining these variants, including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques are known to the person having ordinary skill in the art.
  • phrases "consisting essentially of" when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID NO:.
  • the phrase when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel functional characteristics of the sequence.
  • a “replicon” is any genetic element, for example, a plasmid, cosmid, bacmid, phage or virus, that is capable of replication largely under its own control.
  • a replicon may be either RNA or DNA and may be single or double stranded.
  • a "vector” is a replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element.
  • an "expression operon” refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
  • transcriptional and translational control sequences such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
  • probe refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe.
  • a probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and the method used. In diagnostic applications, for example, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the probes herein are selected to be “substantially” complementary to different strands of a particular target nucleic acid sequence. Such probes must, therefore, be sufficiently complementary so as to be able to "specifically hybridize” or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5 ' or 3' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.
  • the term “specifically hybridize” refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”).
  • the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
  • Amino acid residues described herein are preferred to be in the "L” isomeric form. However, residues in the "D” isomeric form may be substituted for any L-amino acid residue, provided the desired properties of the polypeptide are retained. All amino-acid residue sequences represented herein conform to the conventional left-to-right amino-terminus to carboxy-terminus orientation.
  • isolated protein or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form. "Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into, for example, pharmaceutically acceptable preparations.
  • substantially pure refers to a preparation comprising at least 50-60% by weight of a given material (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-95% by weight of the given compound. Purity is measured by methods appropriate for the given compound (e.g., chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).
  • tag refers to a chemical moiety, either a nucleotide, oligonucleotide, polynucleotide or an amino acid, peptide or protein or other chemical, that when added to another sequence, provides additional utility or confers useful properties, particularly in the detection or isolation, to that sequence.
  • a homopolymer nucleic acid sequence or a nucleic acid sequence complementary to a capture oligonucleotide may be added to a primer or probe sequence to facilitate the subsequent isolation of an extension product or hybridized product.
  • histidine residues may be added to either the amino- or carboxy-terminus of a protein to facilitate protein isolation by chelating metal chromatography.
  • amino acid sequences, peptides, proteins or fusion partners representing epitopes or binding determinants reactive with specific antibody molecules or other molecules (e.g., flag epitope, c-myc epitope, immunoglobulin (Ig) domains, transmembrane epitope of the influenza A virus hemaglutinin protein, protein A, cellulose binding domain, calmodulin binding protein, maltose binding protein, chitin binding domain, glutathione S-transf erase, and the like) may be attached to proteins to facilitate protein isolation by procedures such as affinity or immunoaffinity chromatography.
  • Chemical tag moieties include such molecules as biotin, which may be added to either nucleic acids or proteins and facilitate isolation or detection by interaction with avidin reagents, and the like. Numerous other tag moieties are known to, and can be envisioned by, the trained artisan, and are contemplated to be within the scope of this definition.
  • transform shall refer to any method or means by which a nucleic acid is introduced into a cell or host organism and may be used interchangeably to convey the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, PEG-fusion and the like.
  • the introduced nucleic acid may or may not be integrated (covalently linked) into nucleic acid of the recipient cell or organism.
  • the introduced nucleic acid may be maintained as an episomal element or independent replicon such as a plasmid.
  • the introduced nucleic acid may become integrated into the nucleic acid of the recipient cell or organism and be stably maintained in that cell or organism and further passed on to or inherited by progeny cells or organisms derived from the recipient cell or organism.
  • the introduced nucleic acid may exist in the recipient cell or host organism only transiently.
  • a "clone” or “clonal cell population” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell or cell population that is capable of stable growth in vitro for many generations.
  • antibody or “antibody molecule” is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen.
  • the term includes polyclonal, monoclonal, chimeric, and bispecific antibodies.
  • antibody or antibody molecule contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule such as those portions known in the art as Fab, Fab', F(ab')2 and F(v).
  • a "specific binding pair” comprises a specific binding member (sbm) and a binding partner (bp) which have a particular specificity for each other and which in normal conditions bind to each other in preference to other molecules.
  • specific binding pairs are antigens and antibodies, ligands and receptors and complementary nucleotide sequences. The skilled person is aware of many other examples and they do not need to be listed here. Further, the term "specific binding pair" is also applicable where either or both of the specific binding member and the binding partner comprise a part of a large molecule. In embodiments in which the specific binding pair are nucleic acid sequences, they will be of a length to hybridize to each other under conditions of the assay, preferably greater than 10 nucleotides long, more preferably greater than 15 or 20 nucleotides long.
  • immune suppression refers to an activity which interferes with normal immune homeostatic mechanisms.
  • immune suppression may refer to a variety of activities including, but not limited to, blocking the presentation of antigens to effector cells and blocking the function of effector cells.
  • transplantation refers to the delivery and maintenance of a functional tissue or organ into a recipient (e.g., a patient).
  • allogeneic transplantation refers to the transplantation of a functional organ or tissue from a donor to a recipient wherein both donor and recipient are the same species (e.g., transplantation from one human to another human).
  • xenotransplantation refers to the transplantation of a functional organ or tissue from a donor to a recipient wherein the donor and recipient are different species (e.g., transplantation from a pig to human).
  • antigen presenting cell refers to a specialized group of cells that express MHC class I and TJ molecules in conjunction with accessory molecules (e.g., CD 80, CD 86) that are required for activation of B and/or T cells.
  • Antigen presenting cells include, but are not limited to, dendritic cells, macrophages, Langerhans cells, B cells, Kupffer cells and potentially microglial cells.
  • type I diabetes also known as juvenile onset diabetes or insulin dependent diabetes
  • Type I diabetes is a subset of diabetes that results from the autoimmune destruction of beta cells in the pancreas.
  • Type I diabetes may also result from the surgical removal of the pancreas or a drug induced destruction of the islet cells.
  • the term "apheresis” refers to the isolation of a particular fraction of whole blood (i.e., plasma, leukocytes, and platelets) by centrifugation using a continuous extracorporeal circulation of the blood.
  • leukopheresis refers to the isolation of white blood cells by apheresis.
  • undesirable immune response refers to an immune response which impairs the recovery of a patient and/or the beneficial response of a patient to a therapeutic regime, including those involving organ transplantation and human gene therapy.
  • the term “undesirable immune response” may also refer to an immune response to an autologous molecule as observed in autoimmune disease.
  • the phrase also encompasses undesirable immune responses observed when therapeutically beneficial proteins (e.g., erythropoietin) are administered in supraphysiologic doses.
  • a "therapeutically beneficial molecule” is a molecule which may be administered to a patient and confers therapeutic benefits to the patient in need thereof.
  • such molecules are nucleic acids and encode proteins which restore wildtype function to otherwise non-functional or defective proteins.
  • the protein is not defective, but is administered in a supra- physiological dose that may elicit an undesirable immune response.
  • such molecules are the therapeutically beneficial proteins themselves.
  • the term "tolerance” refers to a biological state of unresponsiveness by immune cells to a particular antigen as a result of their interaction with that antigen. The immune response to all other immunogens is usually unaffected. Thus, there is an acquired nonresponsiveness to a specific antigen.
  • TAAs Full length cDNA sequences encoding each member of a family of tumor- associated proteins, designated herein as TAAs, are provided herein.
  • the TAA family of proteins comprises three members, mEGP, GA733-2, and GA733-1, which appear to be involved in cellular adhesion and whose expression is up-regulated in a number of carcinomas.
  • the above TAAs play a role in immune suppression and are thus of utility for methods in which they are used to modulate immune responses.
  • Nucleic acid sequences encoding full length mEGP are provided herein as SEQ TD NO: 1 ( Figure 8).
  • the amino acid sequence of full length mEGP is provided in Figure 9 and is referred to herein as SEQ TD NO: 4.
  • Nucleic acid sequences encoding full length human GA733-2 are provided herein as SEQ TD NO: 2 ( Figure 10).
  • the amino acid sequence of full length GA733-2 is provided in Figure 11 and is referred to herein as SEQ ID NO: 5.
  • Nucleic acid sequences encoding full length human GA733-1 are provided herein as SEQ ID NO: 3 ( Figure 12).
  • the amino acid sequence of full length GA733-1 is provided in Figure 13 and is referred to herein as SEQ TD NO: 6.
  • TAA thyroglobulin domain
  • EGF epidermal growth factor
  • Nucleic acid molecules encoding any of the above TAA proteins may be prepared by two general methods: (1) they may be synthesized from appropriate nucleotide triphosphates, or (2) they may be isolated from biological sources. Both methods utilize protocols well known in the art.
  • nucleotide sequence information such as full length cDNA having any of the SEQ ID NOS herein enables preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis.
  • Synthetic oligonucleotides may be prepared by the phosphoramadite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices.
  • the resultant construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • a large double-stranded DNA molecule may be synthesized as several smaller segments of appropriate complementarity. Complementary segments thus produced may be annealed such that each segment possesses appropriate cohesive termini for attachment of an adjacent segment. Adjacent segments may be ligated by annealing cohesive termini in the presence of DNA ligase to construct the entire protein encoding sequence. A synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector.
  • Nucleic acid sequences encoding a TAA of the present invention may be isolated from appropriate biological sources using methods known in the art.
  • a cDNA clone is isolated from an expression library of human origin.
  • genomic clones encoding TAA may be isolated.
  • cDNA or genomic clones having homology to a TAA protein may be isolated from other species, such as other vertebrates or mammals, using oligonucleotide probes corresponding to predetermined sequences within the TAA gene.
  • nucleic acids having the appropriate level of sequence homology with the protein coding region of SEQ ID NO: 1, 2 or 3 may be identified using hybridization and washing conditions of appropriate stringency.
  • hybridizations may be performed, according to the method of Sambrook et al., (supra) using a hybridization solution comprising: 5X SSC, 5X Denhardt's reagent, 0.5-1.0% SDS, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42°C for at least six hours.
  • filters are washed as follows: (1) 5 minutes at room temperature in 2 X SSC and 0.5-1% SDS; (2) 15 minutes at room temperature in 2 X SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37°C in 1 X SSC and 1% SDS; (4) 2 hours at 42-65°C in 1 X SSC and 1% SDS, changing the solution every 30 minutes.
  • T m 81.5°C + 16.6Log [Na+] + 0.41(% G+C) - 0.63 (% formamide) - 600/#bp in duplex
  • the T m is 57°C.
  • the T m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology.
  • targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C.
  • Such a sequence would be considered substantially homologous to the nucleic acid sequence of the present invention.
  • Nucleic acids of the present invention may be maintained as DNA in any convenient cloning vector.
  • clones are maintained in plasmid cloning/expression vector, such as pBluescript (Stratagene, La Jolla, CA), which is propagated in a suitable E. coli host cell.
  • TAA-encoding nucleic acid molecules of the invention include cDNA, genomic DNA, RNA, and fragments thereof which may be single- or double-stranded.
  • this invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing with at least one sequence of a nucleic acid molecule of the present invention, such as selected segments of a cDNA having SEQ TD NO: 1, 2, or 3.
  • Such oligonucleotides are useful as agents to modulate TAA activity in cells or tissues.
  • the present invention describes the use of TAA encoding nucleic acids in immune assays such mixed lymphocyte reactions (MLR), wherein the effects of test agents on modulating and/or restoring TAA expression can be assessed.
  • MLR mixed lymphocyte reactions
  • the present invention describes the use of TAA encoding nucleic acids to express a TAA protein (e.g., mEGP, GA733-2, or GA733-1) in the context of a transplanted cell (e.g., pancreatic beta islet cell, bone marrow graft cell), wherein the effect of TAA expression may be measured by assaying the immune response elicited in reaction to the TAA expressing transplant cell.
  • a transplanted cell e.g., pancreatic beta islet cell, bone marrow graft cell
  • nucleic acid molecules encoding antibodies or fragments thereof may be utilized in the compositions of the invention.
  • Intact antibodies may be monoclonal or polyclonal.
  • Nucleic acid sequences encoding a variety of antibody fragments comprising Fab, Fab', F(ab')2, F(v) and Sfv fragment are known to those skilled in the art.
  • Nucleic acid sequences encoding antibodies and/or antibody fragments may be used in conjunction with the TAA molecules of the present invention.
  • expression vectors comprising nucleic acid sequences encoding a TAA and nucleic acid sequences encoding an antibody or fragment thereof may be used to express both encoded polypeptides either as independent proteins or as a fusion protein.
  • TAA polypeptides of the present invention may be cross-linked to antibodies or fragments thereof by chemical means. Such means are of common knowledge and described in a variety of laboratory manuals referenced herein (e.g., see Sambrook et al., supra). The incorporation of antibodies into the constructs of the invention facilitates tissue or cell type specific targeting of the TAAs.
  • TAA polypeptides of the present invention may be prepared in a variety of ways, according to known methods.
  • a TAA protein may be purified from appropriate sources, e.g., human or animal cultured cells or tissues, by immunoaffinity purification. However, this is not a preferred method due to the small amounts of protein likely to be present in a given cell type at any time.
  • a cDNA or gene may be cloned into an appropriate in vitro transcription vector, such a pSP64 or pSP65 for in vitro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocytes.
  • in vitro transcription and translation systems are commercially available (e.g., from Promega Biotech, Madison, Wisconsin or BRL, Rockville, Maryland).
  • larger quantities of a TAA protein may be produced by expression in a suitable prokaryotic or eukaryotic system.
  • a DNA molecule such as a cDNA having SEQ TD NO: 1, 2 or 3
  • a plasmid vector adapted for expression in a bacterial cell, such as E. coli, or into a baculovirus vector for expression in an insect cell.
  • Such vectors comprise the regulatory elements necessary for expression of the DNA in the host cell, positioned in such a manner as to permit expression of the DNA in the host cell.
  • regulatory elements required for expression include promoter sequences, transcription initiation sequences and, optionally, enhancer sequences.
  • a TAA polypeptide produced by gene expression in a recombinant prokaryotic or eukaryotic system may be purified according to methods known in the art.
  • a commercially available expression/secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium.
  • an alternative approach involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein. Such methods are commonly used by skilled practitioners.
  • TAA proteins of the invention prepared by the aforementioned methods, may be analyzed according to standard procedures. For example, such proteins may be subjected to amino acid sequence analysis, according to known methods.
  • TAA proteins of the present invention are involved in antigen recognition and immune suppression.
  • recombinant cells expressing the TAA sequences (SEQ ID NOs: 4, 5, and 6) are provided herein for use in screening assays to assess agents which modulate TAA mediated regulation of antigen recognition and immune suppression.
  • TAA nucleic acids and proteins may be used as research tools to identify other proteins that are intimately involved in antigen presentation by MHC Class II molecules. Biochemical elucidation of proteins that interact physically and/or biochemically with a TAA of the present invention may facilitate the identification and characterization of agents of utility in modulating TAA activity.
  • TAA Nucleic acids encoding TAA may be used for a variety of purposes in accordance with the present invention. As discussed above, the TAA of the invention may be used alone to suppress the immune response of a patient. The TAA may also be used in conjunction with additional molecules to restore defective or impaired protein function.
  • Nucleic acid molecules, or fragments thereof, encoding a TAA of the present invention may also be utilized to control the expression of a TAA (e.g., mEGP, GA733-2, or GA733-1), thereby regulating the amount of TAA protein available to participate in immune responses.
  • a TAA e.g., mEGP, GA733-2, or GA733-1
  • alterations in the physiological amount of TAA act to modulate the immune response of Class II restricted T cells to APCs.
  • the nucleic acid molecules of the invention may be used to create recombinant cell lines for use in assays to identify agents that modulate a TAA protein-mediated suppression of T cell immune responses.
  • TAA-encoding nucleic acids are also used to advantage to produce large quantities of substantially pure TAA protein, or selected portions thereof.
  • the full-length protein or a selected domain may be used for research, diagnostic and therapeutic purposes, as described below.
  • a wide variety of expression vectors are available that may be modified to express DNA sequences of this invention.
  • the specific vectors exemplified herein are merely illustrative, and are not intended to limit the scope of the invention. Expression methods are described by Sambrook et al. Molecular Cloning: A Laboratory Manual or Current Protocols in Molecular Biology 16.3-17.44 (1989). Expression methods in Saccharomyces are also described in Current Protocols in Molecular Biology (1989).
  • Suitable vectors for use in practicing the invention include prokaryotic vectors such as the pNH vectors (Stratagene Inc., 11099 N. Torrey Pines Rd., La Jolla, Calif.
  • eukaryotic vectors useful in practicing the present invention include the vectors pRc/CMV, pRc/RSN, and pREP (Invitrogen, 11588 Sorrento Valley Rd., San Diego, Calif.
  • pcD ⁇ A3.1/V5&His Invitrogen
  • baculovirus vectors such as PNL1392, PNL1393, or pAC360 (Invitrogen)
  • yeast vectors such as YRP17, YTJP5, and YEP24 (New England Biolabs, Beverly, Mass.), as well as pRS403 and pRS413 Stratagene Inc.) and Picchia vectors such as pHJL-Dl (Phillips Petroleum Co., Bartlesville, OK 74004).
  • viral vectors both viral vectors and plasmid vectors are known in the art, see US Patent No. 5,252,479 and WO 93/07282.
  • viruses have been used as gene transfer vectors, including papovaviruses, such as SN40, vaccinia virus, herpes viruses including HSN and EBN, and retroviruses.
  • papovaviruses such as SN40, vaccinia virus, herpes viruses including HSN and EBN, and retroviruses.
  • retroviruses including papovaviruses, such as SN40, vaccinia virus, herpes viruses including HSN and EBN, and retroviruses.
  • Many gene therapy protocols in the prior art have employed disabled murine retroviruses.
  • Table 1 provides a list of adenoviral vector constructs comprising exemplary nucleic acid sequences encoding TAA and hybrid TAA proteins of utility in the methods of the present invention.
  • ICD intracellular domain
  • TROP-2 murine murine variant of mEGP
  • ex extracellular region
  • in intracellular region
  • del- del- (deleted in)
  • mut- mutated in
  • the adenoviral constructs exemplified in Table 1 may be prepared by PCR cloning into an adenoviral shuttle plasmid from which the virus may be prepared as previously described (Smith and Eck, 1999, Cancer Gene Therapy 6:475-481).
  • the mEGP transmembrane domain is frequently used in chimeric constructs.
  • the constructs may be characterized by western blot assay using anti-mEGP or anti-GA733-2 monoclonal antibodies (mAbs) to demonstrate that the adenoviral constructs express a protein of the expected length.
  • mAbs monoclonal antibodies
  • Each of the adenoviral vectors may be used to transfect, for example, DCs and FBL3 tumor cells.
  • Transfected DCs may be used in MLR assays as described in Example I.
  • Graded amounts (mg/ml protein) of transfected FBL3 lysate, for example, may also be added to MLRs comprising untransfected DCs as described in Example I.
  • the mEGPex-LAMP construct fuses the mEGP-ECD to LAMP, which is a known endosomal targeting sequence (Lin et al., 1996, Cancer Res 56:21-26; Ji et al., 1998, Int J Cancer Res 78:41-45).
  • the TROP2 construct expresses a known murine variant of mEGP that has only 42 % overall sequence homology but retains the overall structure including the intracellular domain (ICD) and TGD.
  • Trop-2 is expressed on placental trophoblasts and in the eye (Lipinski et al., 1981, Proc Natl Acad Sci 78:5147-5150; Ripani et al., 1998, Int J Cancer 76:671-676; Boxhorn et al., 1998, Cancer Immunol Immunother 46:283-292), tissues which exhibit unusual properties with regard to immune cell recognition.
  • Promoters for use in expression vectors of this invention include promoters that are operable in prokaryotic or eukaryotic cells. Promoters that are operable in prokaryotic cells include lactose (lac) control elements, bacteriophage lambda (pL) control elements, arabinose control elements, tryptophan (trp) control elements, bacteriophage T7 control elements, and hybrids thereof.
  • lac lactose
  • pL bacteriophage lambda
  • trp tryptophan
  • Promoters that are operable in eukaryotic cells include Epstein Barr virus promoters, adenovirus promoters, SN40 promoters, Rous Sarcoma Virus promoters, cytomegalovirus (CMN) promoters, baculovirus promoters such as AcM ⁇ PN polyhedrin promoter, Picchia promoters such as the alcohol oxidase promoter, and Saccharomyces promoters such as the gal4 inducible promoter and the PGK constitutive promoter.
  • Epstein Barr virus promoters include Epstein Barr virus promoters, adenovirus promoters, SN40 promoters, Rous Sarcoma Virus promoters, cytomegalovirus (CMN) promoters, baculovirus promoters such as AcM ⁇ PN polyhedrin promoter, Picchia promoters such as the alcohol oxidase promoter, and Saccharomyces promoters such as the gal4 inducible promoter and the PGK constitutive promote
  • a vector of this invention may contain any one of a number of various markers facilitating the selection of a transformed host cell.
  • markers include genes associated with temperature sensitivity, drug resistance, or enzymes associated with phenotypic characteristics of the host organisms.
  • the vector may be used to transform a host cell.
  • the host cell may comprise any cell including a prokaryotic cell or eukaryotic cell that is capable of being transformed with the vector comprising D ⁇ A encoding a TAA polypeptide.
  • Techniques of transforming and transfecting cells are well known in the art and may be found in such general references as Sambrook et al. (1989) or Current Protocols in Molecular Biology (1989).
  • the present invention is not limited to use in a particular host cell.
  • the vectors of the invention may be transformed into and expressed in many host cells.
  • Transformed host cells of this invention may be cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants or amplifying genes.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the choice of a particular host cell depends to some extent on the particular expression vector used to drive expression of TAA. After transformation of a vector of the invention into a host cell one can select transformants on the basis of a selectable phenotype. This selectable phenotype can be conferred by a selectable marker present on the expression vector.
  • Suitable host cells include, for example, prokaryotic cells such as Escherichia coli and Bacillus subtilis; eukaryotic cells such as Mardin Darby canine kidney (MDCK) cells (American Type Culture Collection (ATCC CCL-34), Cos 7 cells (ATCC CRL-1651), 293 cells (ATCC CRL-1573), Chinese hamster ovary cells CHO-DHFR- (ATCC CRL-9096), Chinese hamster ovary cells CHO-K1 (ATCC CCL-61), Syrian Hamster cells AN12 (ATCC CRL 1573), human lymphocyte CCRF-CEM cells, FBL-3 murine erythroleukemia cells (a mEGP-negative cell line), human pancreatic beta islet cells isolated from cadavers, pig and other large animal pancreatic beta islet cells, pancreatic beta islet cell lines, and cells derived from kidney, liver, brain, skin, and muscle; yeast cells, including Saccharomyces cerevisiae and Picchia pastoris; insect
  • the use of cells and cell lines that naturally express mEGP or GA733-2 may be desirable (e.g., screening for agents which modulate the activity of mEGP and/or GA733-2).
  • Such cells and cell lines include, but are not limited to, human cell lines SW480, MCF7, 293 (see above), and SICBR3 (which express GA733-2), and murine SCK cells, mammary tumor cell lines, hybridomas, and plasma cells (which express mEGP).
  • TAA proteins of the present invention provides reagents having a high degree of homogeneity, which are readily used in the methods described herein. Purified full length GA733-2 protein, for example, may be prepared using an immuno-affinity chromatography method.
  • a preferred method for the expression and purification of the TAA proteins of the present invention involves baculovirus expression systems.
  • Baculovirus expression systems provide several advantages including high expression levels which afford purification of milligram quantities of protein, eukaryotic post-translational modification machinery which enables glycosylation of expressed proteins (e.g., native mEGP is heavily glycosylated). Such modifications may affect the biological activity, stability, and localization of TAA proteins.
  • full-length mEGP FL-mEGP
  • mEGP-ECD mEGP extracellular domain
  • mEGP-Ig Fc Ig domain
  • TAA proteins produced in a baculovirus expression system as exemplified by FL-mEGP, EGP-ECD, and mEGP-Ig, may be purified utilizing a number of different protocols well known to those of skill in the art.
  • mEGP proteins having protein tags may be purified by virtue of the physical properties imbued by the tag. As described herein, a variety of protein tags are available for such purposes and are known to those skilled in the art.
  • the mEGP-Ig fusion protein may be purified by virtue of its Fc component, which facilitates affinity purification of the fusion protein by column chromatography over an appropriate matrix (e.g., Protein A-sepharose beads).
  • the Fc component of a fusion protein also facilitates formation of fusion protein dimers.
  • the mEGP-Ig fusion protein may thus also be used to advantage for applications in which dimeric forms of mEGP possess higher levels of immune modulating activity (e.g., T cell inhibition) than monomeric forms.
  • the mEGP-Ig fusion protein may bind directly to cells (e.g., DCs) expressing a compatible Fc receptor (FCR) on their cell surface.
  • FCR Fc receptor
  • mEGP-IG fusion protein bound to a cell surface via an FCR may be of utility for suppressing class TJ mediated T cell recognition.
  • adenoviral constructs (as exemplified in Table 1) may be used to transduce mammalian cells, which provide a system for the expression of large quantities of the TAA proteins of the present invention.
  • Graded amounts of the TAA proteins of the present invention may be used in the assays and methods of the present invention to determine the optimal protein concentration range required for each protein to achieve maximal immune modulatory activity.
  • the present invention provides methods for measuring the ability of a TAA protein to inhibit an immune response in a cellular context. Such methods are also applicable to the screening of compounds to test the ability of an individual compound or combination of compounds to modulate TAA activity in the above assays.
  • one embodiment of this invention provides a method for assaying TAA activity in a cell having the following steps: a) contacting the compound with a cell that is transformed with a recombinant DNA expression vector which provides for expression of a TAA activity, and b) assaying for modulation of TAA mediated suppression of immune response in cellular assays.
  • TAA-encoding nucleic acids and TAA proteins of the invention can be used to modulate TAA gene expression and protein activity for the purposes of assessing the impact of TAA modulation on antigen recognition, T cell activation, and immune response.
  • compositions of the invention have been described with respect to human therapeutics, it will be apparent to one skilled in the art that these tools will also be useful in animal and cultured cell experimentation with respect to suppression of immune responses.
  • therapeutics they can be used either alone or as adjuncts to other drugs for improving the effectiveness of such agents.
  • These molecules can also be used therapeutically in patients for the inhibition of undesirable immune responses.
  • TAA proteins of the present invention are involved in antigen recognition by Class TJ restricted T cells, methods for identifying agents that modulate their activity are highly desirable. Such agents should have utility for the treatment of a variety of diseases
  • a TAA polypeptide or fragment thereof employed in drug screening assays may either be free in solution, affixed to a solid support or within a cell.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably , transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays.
  • One may determine, for example, formation of complexes between a TAA polypeptide or fragment and the agent being tested, or examine the degree to which the formation of a complex between a TAA polypeptide or fragment and a known compound is interfered with by the agent being tested.
  • TAA polypeptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the peptide test compounds are reacted with a TAA polypeptide and washed. Bound TAA polypeptide may then detected by methods well known in the art.
  • Another approach entails the use of phage display libraries engineered to express fragments of TAA on the phage surface. Such libraries are then contacted with a combinatorial chemical library under conditions wherein binding affinity between the TAA peptides and the components of the chemical library may be detected.
  • US Patents 6,057,098 and 5,965,456 provide methods and apparatus for performing such assays.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo. See, e.g., Hodgson, (1991) Bio/Technology 9:19-21.
  • the three-dimensional structure of a protein of interest or, for example, of the protein-substrate complex is solved by x-ray crystallography, by nuclear magnetic resonance, by computer modeling or most typically, by a combination of approaches.
  • peptides e.g., a TAA polypeptide
  • an amino acid residue is replaced by Ala, and its effect on the peptide' s activity is determined.
  • Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.
  • anti-ids anti- idiotypic antibodies
  • the binding site of the anti-ids would be expected to be an analog of the original molecule.
  • the anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced peptides. Selected peptides would then act as the pharmacore.
  • drugs which have, e.g., improved TAA polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of TAA polypeptide activity.
  • TAA polypeptide By virtue of the identification of a full length TAA clone as described herein, sufficient amounts of the TAA polypeptide may be made available to perform such analytical studies as x-ray crystallography. In addition, knowledge of a TAA protein sequence will guide those employing computer modeling techniques in place of, or in addition to x-ray crystallography.
  • Suitable peptide targets for identifying specific TAA binding and modulating agents are provided herein.
  • functional fragments of each TAA comprising the extracellular domain are provided.
  • Critical domains of a functional fragment of GA733-2 include the terminal epidermal growth factor- 1 (EGF-1) domain (SEQ ID NO: 8), which is encoded by nucleic acid sequences comprising SEQ TD NO: 7; Figure 14), and the thyroglobulin domain (TGD; SEQ TD NO: 10), which is encoded by nucleic acid sequences comprising SEQ TD NO: 9; Figure 15).
  • the TGD is conserved among mEGP, GA733-2, and GA733-1, as is a Cys-Trp-Cys-Val motif within the TGD.
  • the Cys-Trp-Cys-Val motif may be used to advantage as a candidate peptide for drug design studies.
  • TAA activity-modulating drugs can be optimized for both the timing of delivery and maximal uptake in, for example, cells of the pancreas (e.g., beta islet cells).
  • These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the carrier or other material may depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes. Whether it is a polypeptide, nucleic acid molecule, small molecule or other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a "prophylactically effective amount” or a "therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual.
  • routes of administration e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
  • administration is preferably in a "prophylactically effective amount” or a "therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual.
  • Organ transplantation is a standard treatment for many life-threatening diseases.
  • the immune response of the recipient to foreign cell surface antigens expressed on an organ transplant or graft generally precludes successful transplantation of tissues and organs unless the transplant tissues are derived from a compatible donor and/or the normal immune response of the recipient is suppressed.
  • the immune response of the recipient is predominantly directed against cell surface proteins on the donor graft, which are encoded by the MHC genes. Since MHC proteins are expressed on the majority of mammalian somatic cells, MHC compatibility of donor and host is a major concern with virtually all organ transplants. Donors are, therefore, selected based on the degree of identity at MHC loci of the host and donor.
  • MHC identical sibling donors or MHC identical unrelated cadaver donors provide the best degree of compatibility and, therefore, transplants from such donors provide a recipient with the best chance for successful, long term engraftment.
  • the host immune response to an organ allograft involves a complex series of cellular interactions involving T and B lymphocytes and antigen presenting cells (APCs) that recognize and are activated by a foreign antigen (Strom, 1989, supra; Strom, 1990, Clinical Aspects of Autoimmunity 4:8-19).
  • APCs antigen presenting cells
  • Specific cell-cell interactions, provided by activated APCs or accessory cells such as macrophages and dendritic cells, and stimulatory factors (e.g., cytokines) are essential for T cell proliferation.
  • Macrophages and dendritic cells may activate T cells directly via cell- cell interactions mediated by specific adhesion proteins or indirectly via secretion of cytokines (e.g., IL-1 and TL-6) that stimulate T cells in trans (Strom, 1989, In: Organ Transplantation: Current Clinical and Immunological Concepts: 4:8-19; Strom, 1990, Clinical Aspects of Autoimmunity 4:8-19).
  • cytokines e.g., IL-1 and TL-6
  • a cascade of cytokine expression by both T cells and APCs results in activation of cytotoxic T cells, which in turn activate cytodestructive properties of macrophages (Farrar, et al., 1981 J. Immunol. 126: 1120-1125).
  • Some of the cytokines released also induce an increased expression of MHC class II proteins in the transplanted organ, which renders the grafted organ more immunogenic (Pober, et al., 1983, J. Exp. Med., 157:1339; Kelley, et al., 1984 J. Immunol, 132:240-245). Similar mechanisms are involved in the development of many autoimmune diseases, including type I diabetes.
  • insulin-dependent diabetes mellitus results from a spontaneous T-cell dependent autoimmune destruction of insulin-producing pancreatic ⁇ cells that intensifies with age. The process is preceded by infiltration of the pancreatic islets with mononuclear cells (insulitis), wherein the infiltrate is predominantly comprised of T lymphocytes (Bottazzo et al., 1985, J. Engl. J. Med., 113:353; Miyazaki et al., 1985, Clin. Exp. Immunol., 60:622).
  • a delicate balance between autoaggressive T- cells and suppressor-type immune activity determines whether the clinical manifestation of such autoimmunity is limited to insulitis or progresses to insulin dependent diabetes mellitus (IDDM).
  • immunosuppressive therapies include administration of immunosuppressive compounds such as cyclosporine A, FK506 and rapamycin (First, 1992, Transplantation, 53:1-11). Because these agents inhibit proliferation of T cells generally, systemic treatment of patients leads to systemic immunosuppression which carries with it potential complications, including increased risk of infections and cancer (Wilkinson et al., 1989, Transplant, 47:293-296; Penn, 1991 Transplant Proc, 23: 1101 ; Beveridge, et al., 1984, Lancet, 1 :788).
  • immunosuppressive agents cause considerable side effects, including nephrotoxicity, hepatotoxicity, hypertension, hirsutism, and neurotoxicity (Strom, 1989, supra; Strom, 1990, Clinical Aspects of Autoimmunity, 4:8-19; Tilney, et al., 1991 Ann Surg., 214:42-49; Myers, et al., 1984 N. Engl. J. Med., 311:699).
  • nucleic acid sequences encoding TAA polypeptides and TAA polypeptides of the present invention may be used to advantage to suppress immune responses.
  • methods are provided wherein nucleic acid sequences encoding TAA polypeptides and TAA polypeptides may be used to prevent immune cell mediated rejection of transplanted organs and tissues, and prevent tissue destruction due to autoimmune disease.
  • the invention is directed to methods for the use of nucleic acid sequences encoding TAA polypeptides and TAA polypeptides to specifically suppress a local immune response at the tissue or organ which is targeted for autoimmune destruction or at the transplantation site. Localized and specific i munosuppression provides a significant improvement over existing clinical therapies, all of which involve general suppression of the immune response as described above. To utilize the methods for treatment of the varied conditions described herein modifications of the general technique may be employed.
  • the methods of the present invention may be used to prevent rejection of a transplanted organ or tissue.
  • nucleic acid sequences encoding a TAA e.g., mEGP, GA733-1, or GA733-2, or functional fragments thereof or TAA fusion proteins
  • a recipient e.g., a mammal. This may be accomplished, for example, by ex vivo transfection of isolated cells of the tissue/organ, or by transfection of the tissue/organ itself.
  • introduction of expression vectors comprising nucleic acid sequences encoding TAA polypeptides may also be accomplished by transfection of the tissue/organ in vivo, for example, by introducing suitably prepared expression vectors directly into the tissue/organ of a mammal following transplantation.
  • Another method for the introduction of DNA into an organ involves the generation of transgenic animals in which the appropriate genes are expressed in the organ to be transplanted. Organs that express TAA polypeptides may be harvested from such transgenic animals and used as donor transplants.
  • transfected cells may be introduced into the area surrounding the transplant, but not be included in the actual transplant.
  • Cultured myoblasts or immortalized renal epithelial cells may be intermixed with islet allografts, or may be injected into or near organ allografts (i.e., under the renal capsule) to facilitate localized immune suppression.
  • the transfected cells may express a soluble form of a TAA polypeptide, the secretion of which provides a localized high concentration of the TAA polypeptide which may be engulfed by DCs in the region of the transplant. Engulfment of soluble TAA polypeptide would thus suppress the ability of such DCs to present antigen to class II restricted T cells.
  • the TAA proteins of the present invention are expressed in vivo as either cell surface or soluble TAA polypeptides (as described above) and act to inhibit rejection of the transplanted tissue/organ.
  • methods are provided for the prophylactic and/or therapeutic treatment of autoimmune disease.
  • expression constructs comprising nucleic acid sequences encoding a TAA polypeptide (e.g., mEGP, GA733-1, or GA733-2, or functional fragments thereof) may be introduced into the tissues or surrounding tissues that are targeted for immune destruction in a particular autoimmune disease.
  • a TAA polypeptide e.g., mEGP, GA733-1, or GA733-2, or functional fragments thereof
  • such expression constructs may be administered prior to onset of the disease phenotype.
  • pre-diabetic patients it may be preferable to introduce the expression construct encoding a TAA polypeptide into beta-islet cells of the pancreas prior to onset of the disease.
  • Preferred target tissues for treating rheumatoid arthritis patients are the synovial tissues of the joints, whereas the brain and myelin sheath are preferred targets in patients suffering from multiple sclerosis (MS).
  • MS multiple sclerosis
  • cells such as myoblasts and renal epithelial cells may be transplanted into an appropriate locale after transduction to express the desired TAA.
  • Transfection of dispersed cells from a tissue /organ may be accomplished by any number of known methods, including calcium phosphate precipitation, DEAE dextran, electroporation, and liposome mediated transfection.
  • the choice of a particular method may be made based upon a number of different factors, including , but not limited to the cell type to be transfected. Each of these methods is well known to those skilled in the art.
  • Retroviral vectors may be used to advantage to provide a gene transfer delivery system for the methods of the invention.
  • a viral vector may be selected based on the cellular tropism of a particular vector and the cells for which therapeutic delivery of TAAs is intended. Numerous vectors useful for such purposes have been described (Miller, 1990, Human Gene Therapy, pp. 15-14; Friedman, 1989, Science, 244:1275-1281; Eglitis and Anderson,
  • Retroviral vectors are particularly well developed and have been used in a clinical setting (Rosenberg, et al., 1990, N. Engl. J. Med., 323:370).
  • Retroviral constructs, packaging cell lines and delivery systems which may be useful for this purpose include, but are not limited to, one, or a combination of, the following: Moloney murine leukemia viral vector types; self inactivating vectors; double copy vectors; and selection marker vectors.
  • adenoviral vectors may be utilized to deliver nucleic acid sequences encoding TAA polypeptides. See Table 1 for a list of exemplary adenoviral vectors comprising nucleic acid sequences encoding TAA polypeptides and Example I for construction of such adenoviral vectors.
  • Nucleic acid encoding the TAA polypeptides of the invention under the regulation of the appropriate promoter, and including the appropriate sequences required for insertion into genomic DNA of the patient, or autonomous replication, may be administered to the patient using the following gene transfer techniques: microinjection (Wolff et al., 1990, Science, 247:1465); calcium phosphate transfer (Graham and Van der Eb, 1973, Virology, 52:456; Wigler et al, 1978, Cell, 14:725; Feigner et al, 1987, Proc. Natl. Acad. Sci. USA, 84:7413); lipofection (Feigner et al., 1987, Proc. Natl. Acad. Sci.
  • Targeted delivery of TAA molecules to specific cells may also be achieved by utilizing antibody-studded liposomes comprising, for example, sfv-encoding antibodies immunologically specific for a cell surface receptor expressed on the target cells.
  • Methods for the production of such antibody-studded liposomes and administration of targeted liposomes to patients have been previously described in Maulik et al., 1997, Molecular Biotechnology: Therapeutic Applications and Strategies, Wiley-Liss, Inc.; and U.S. Patent Nos. 5,709,879 (Barchfeld et al.); and 6,025,193 (Weiss), the entire contents of which are incorporated herein by reference.
  • Organs or tissue such as pancreatic islets, may be transfected using either electroporation (BioRad) as described by Welsh et al. (1990, Proc. Natl. Acad. Sci USA. 87:5807-5811), lipofectin reagent (GIBCO-BRL), pH-sensitive liposomes as described by Welsh et al. (Welsh, et al., 1990, Biomed. Biochim. Acta, 49:1157-1164) or microparticle bombardment with the biolistic PDS-1000/He system (BioRad) (Yang, et al., 1990, Proc. Natl. Acad. Sci. USA, 87:9568-9572).
  • TAA expression suppresses MHC class U restricted T cell responses to the transplanted organ.
  • One of the expression vector constructs described above, or the like may be used to express a TAA polypeptide in a transplanted organ or tissue (e.g., pancreatic islets).
  • TAA polypeptides For production of transgenic animals in which TAA polypeptides are expressed, expression vector constructs may be linearized and injected into oocytes according to standard protocols (Sarvetnick, et al., 1988, Cell, 52:773-782). Expression of TAA polypeptides may be evaluated by sacrificing a second generation animal and isolating mRNA from different organs and from multiple hematopoietic cell types. Since constitutive expression of TAA genes in transgenic animals might lead to developmental defects or other pathological changes, the transgenic animals preferably contain TAA genes which are under the transcriptional control of, for example, inducible promoters or cell type specific promoters. Such promoters are described herein and are known to those skilled in the art.
  • DNA constructs employed in the invention may be tested in vitro for their general immunosuppressive effects using an in vitro MLR assay. See Example I Constructs may also be readily tested in vivo using mouse islet transplant assays (see, for example, U.S. Patent No. 5,958,403) or other transplant and autoimmune models readily available to those skilled in the art. The methods of the invention may be used alone for the treatment of autoimmune disease or for the prevention of graft rejection or they may be used in combination with other known local or general immunosuppressive therapies.
  • Constructs of the invention which are immunosuppressive are those constructs which prolong engraftment beyond the 50% rejection period for the control animal similarly treated, but lacking the transgene.
  • constructs which encode TAA polypeptides may be tested in the pancreatic islet cell transplant model described in Example UI and U.S. Patent No. 5,958,403.
  • Constructs for use in the treatment of autoimmune disease may be evaluated by examination of tissue injury in an appropriate model. For example, non-obese diabetic (NOD) mice (an animal model for type I diabetes) and experimental autoimmune encephalomyelitis (EAE) mice (an animal model for MS) may be utilized. In NOD mice, tissue injury in the pancreas may be evaluated by immunopathologic techniques.
  • NOD mice receiving pancreatic beta islet cells which produce a TAA polypeptide may be compared to genetically identical mice receiving beta islet cells that have not been transduced to express the TAA polypeptide.
  • constructs which confer increased tissue survival relative to that of the untreated control are useful in the methods described herein.
  • Useful constructs may also be identified following an assessment of their ability to inhibit T cell activation in MLRs as described hereinbelow.
  • pancreatic beta islet cells may be isolated from a variety of sources, including but not limited to, human cadavers, human beta islet pancreatic cell lines, mammalian beta pancreatic cell lines, pigs, and other large animals. Methods for the isolation of islet cells and islet tissue have been previously described in Scharp et al.[1973, Transplantation 16:686-689] and van der Vliet et al., [1988, Transplantation 45:493-495], the entire contents of which are incorporated herein by reference.
  • freshly isolated beta islet cells or islets may be transduced with an adenoviral vector.
  • Methods for the transduction of beta islet cells with adenoviral constructs are known to those skilled in the art and have been previously described (Csete et al., 1995, Transplantation
  • beta islet cells or cultured islets may be counted by means known in the art (e.g., trypan blue exclusion) and allowed to pellet at room temperature in a minimal volume of appropriate islet cell medium.
  • An islet may be expected to comprise approximately 1000 cells.
  • An adenoviral vector (see Table 1 for examples) may be added to the islet cell pellet at a multiplicity of infection (MOI) of 10 plaque forming units (pfu):target cell for 1 hr at room temperature. Islets may then be washed and recultured at 37°C until assayed for expression of adenoviral encoded proteins.
  • MOI multiplicity of infection
  • pfu plaque forming units
  • an adenoviral vector comprising nucleic acid sequences encoding a TAA polypeptide (e.g., mEGP, GA733-2, or GA733-1 or functional fragments thereof) of the present invention may be used to transduce the beta islet cells or cultured islets.
  • a TAA polypeptide e.g., mEGP, GA733-2, or GA733-1 or functional fragments thereof
  • Exemplary adenoviral vectors encoding TAA polypeptides of the present invention are provided in Table 1. Islet cells transduced to express a TAA or functional fragment thereof are thus rendered less immunogenic by virtue of the immunosuppressive properties of the above TAAs. Specifically, expression of TAA on islet cells inactivates class TJ MHC restricted T cell recognition of such cells.
  • islet cells and/or islets transduced to express a TAA may be used as modified grafts or transplants.
  • modified beta islet grafts provide a renewable resource of beta islet cell transplants which may be used to advantage in the treatment of patients having disorders in which their beta islet cells have been destroyed or rendered non-functional. Such disorders include, for example, type I diabetes.
  • modified beta islet grafts expressing a TAA of the present invention are not recognized by MHC class II restricted T cells to same extent as non- modified beta islet grafts.
  • T cell responses to such modified beta islet grafts are completely inhibited by the immunosuppressive properties of the TAA expressed therein.
  • modified beta islet grafts may be injected into the liver or implanted under the renal capsule of a patient.
  • a TAA protein, functional fragment thereof, or fusion protein comprising a TAA polypeptide of the present invention may be purified to homogeneity using conventional techniques.
  • a purified TAA polypeptide may be mixed with a pharmaceutical carrier to form a composition and administered intravenously or by some other standard method, or used to perfuse a graft to be transplanted.
  • a TAA polypeptide may be used to treat autoimmune diseases such as type I diabetes and rheumatoid arthritis, as well as other disease states involving the immune system, such as graft versus host disease.
  • the appropriate dose of a purified TAA polypeptide used to treat a patient to effect immune suppression is based on a number of factors, including, for example, the type of disease/disorder manifested, the patient' s condition, history of previous treatments, and the judgment of the attending physician.
  • Purified TAA polypeptides may be administered to a patient by any means known to those of skill in the art. In a preferred embodiment, purified TAA polypeptides may be administered at or in the immediate vicinity of tissue destruction. TAA polypeptides may be administered into the synovial fluid of a patient with rheumatoid arthritis, for example, so as to suppress the immune response in the localized environment of a joint.
  • a purified TAA polypeptide may be administered to a patient in need thereof to effect a systemic state of immune tolerance.
  • Purified GA733-2 mEGP
  • LAIR-1 a putative cell surface lymphocyte protein having phosphatase activity that acts as an inhibitor of T, B and natural killer (NK) cell function
  • TAA polypeptides of the present invention may be administered in a systemic and/ or a localized fashion.
  • a fusion protein comprising a TAA polypeptide and a therapeutically beneficial molecule (e.g., an immunoglobulin domain) may be generated and purified by methods discussed herein and known to those of skill in the art.
  • a therapeutically beneficial molecule e.g., an immunoglobulin domain
  • purified TAA fusion proteins may also be administered to a patient in need thereof to effect a systemic state of immune tolerance.
  • purified TAA-Ig fusion proteins will bind to cells via interactions conferred by either or both the TAA and the Ig component of the fusion protein.
  • the TAA component will mediate binding to cells via LAIR-1.
  • the Ig component of the fusion protein will bind to cells via cell surface receptors for Ig (e.g., FcR), if the Ig component used was, for example, the Fc domain, or via cell surface antigens recognized by, for example an Fab component of an Ig, if an antigen recognition domain of the Ig was used.
  • FcR cell surface receptors for Ig
  • fusion proteins therefore, provide agents that may be used to advantage to induce immune tolerance in a patient in need thereof and have multiple, diverse cellular targets.
  • a fusion protein comprising a TAA polypeptide and a therapeutically beneficial molecule may be generated and purified by methods discussed herein and known to those of skill in the art.
  • TAA fusion proteins may be used to advantage for therapeutic regimens in which it is desirable to inhibit the immune response to the therapeutically beneficial molecule (e.g., a protein) or enhance tolerance of a recipient to such a therapeutically beneficial molecule. Accordingly, a TAA fusion protein comprising a therapeutic protein may be administered to a patient in a therapeutically effective amount.
  • the therapeutically beneficial molecule e.g., a protein
  • a TAA fusion protein comprising a therapeutic protein may be administered to a patient in a therapeutically effective amount.
  • a patient with hemophilia B is deficient for Factor IX (FIX) activity and therefore requires administration of exogenous FIX in order to efficiently form blood clots following tissue damage or hemorrhage.
  • FIX Factor IX
  • Some patients receiving exogenous FIX mount a deleterious immune response to the exogenous FIX, which results in premature clearance of FIX from the bloodstream of the patient.
  • deleterious immune responses minimally diminish and may eliminate the therapeutic benefit of exogenous FIX administration to a patient.
  • TAA-FIX fusion protein administered to a patient with hemophilia B inhibits the ability of the patient's immune response to recognize the FIX component of the fusion protein and thus prevents premature clearance of the therapeutic fusion protein.
  • the appropriate dose and route of administration for delivery of such TAA fusion proteins to a patient may be determined based on a number of factors, including, for example, the type of disease/disorder manifested, the patient's condition, history of previous treatments, and the judgment of the attending physician.
  • the methods of the present invention may be used to advantage to treat a patient with hemophilia A, who is deficient for Factor
  • VTJI activity A patient with hemophilia A who is treated with exogenous Factor VUI (recombinant or native purified forms) may mount a deleterious immune response to the exogenous Factor VIII, which results in premature clearance of Factor VTJI (FNQI) from the bloodstream.
  • exogenous Factor VUI recombinant or native purified forms
  • FNQI premature clearance of Factor VTJI
  • the administration of a TAA-FNIII fusion protein to a patient with hemophilia A inhibits the ability of the patient's immune response to recognize the FVUI component of the fusion protein and thus prevents premature clearance of the therapeutic fusion protein.
  • the appropriate dose and route of administration for delivery of a TAA-FVITI fusion protein to such a patient may be determined based on a number of factors, as exemplified hereinabove.
  • constructs comprising nucleic acid sequences encoding fusion proteins comprised of TAA polypeptides and therapeutic polypeptides may be administered to a patient to effect expression of such fusion proteins in vivo.
  • Such constructs and methods for their delivery are known to those skilled in the art and are described herein.
  • such constructs would comprise a promoter to drive the expression of a TAA polypeptide and a first termination signal for the TAA polypeptide and a second promoter to drive expression of a therapeutic polypeptide and a second termination signal for the therapeutic polypeptide.
  • an expression vector comprising a first nucleic acid sequence encoding a TAA molecule operably linked to a first regulatory element and a second nucleic acid sequence encoding a therapeutically beneficial molecule operably linked to a second regulatory element may be introduced into a cell.
  • Such an expression vector may be introduced into a cell in vitro, in vivo, and/or ex vivo.
  • Table 2 provides a partial list of therapeutically beneficial molecules which may be utilized to treat the indicated human disorder using the methods and compositions of the invention. All nucleic acid sequences are human in origin unless otherwise specified. Other disorders which may be treated according to the present invention include without limitation, ornithine transcarbamylase deficiency, glutaric aciduria and maple syrup urine disease.
  • TAA polypeptides of the present invention may be used in conjunction with gene therapy.
  • the expression of a TAA polypeptide of the present invention may be used to advantage to suppress the immune response of a patient treated with a gene therapy vector.
  • a gene therapy vector may be, for example, a viral vector comprising nucleic acid sequences encoding a therapeutically beneficial molecule (e.g., a protein which is missing or defective in a patient).
  • a therapeutically beneficial molecules e.g., a protein which is missing or defective in a patient.
  • An exemplary list of such therapeutically beneficial molecules (including the GenBank Accession numbers) and the human disorders associated with deficiencies and/or defects in such molecules is provided in Table 2.
  • Patients treated with gene therapy vectors may mount an immune response to antigenic epitopes of a viral vector and/or the expressed protein, which hinders the efficacy of such treatment. In some circumstances, an immune response to such vectors can lead to serious physiological complications, even death.
  • vectors comprising nucleic acid sequences encoding TAA polypeptides of the present invention may be administered in conjunction with gene therapy vectors comprising nucleic acid sequences encoding a therapeutically beneficial or desired protein.
  • Methods for the production of vectors encoding TAA polypeptides are described herein and known to skilled artisans. Methods for the production and use of gene therapy vectors have been previously described. See, for example, Current Protocols in Human Genetics (eds., Dracopoli et al., John Wiley & Sons, Inc, 1996), the entire contents of which is incorporated herein by reference.
  • Vectors encoding TAA polypeptides may be administered before, during, or after administration of a gene therapy vector. Administration of vectors encoding TAA polypeptides and gene therapy vectors may be performed in accordance with administration protocols described for delivery of the gene therapy vector alone. The appropriate dose and administration route for such treatment may be determined by a clinician, in view of a number of criteria, which include, for example, the type of disease/disorder manifested, the patient's condition, and history of previous treatments.
  • Table 3 provides an exemplary list of disorders associated with undesirable immune responses treatable by the methods and compositions of the invention.
  • CFTDS Chronic Fatigue Immune Dysfunction Syndrome
  • TIP Idiopathic Thrombocytopenia Purpura
  • the present inventor has made the surprising discovery that bone marrow derived dendritic cells (DCs) that have been transfected to express mEGP lose their ability to stimulate T cells. Further studies revealed that mEGP specifically interferes with antigen presentation by MHC class II molecules on DCs, and thereby blocks recognition by CD4+ T cells. The results presented herein, therefore, demonstrate that a TAA directs down-regulation and evasion of anti-tumor immune responses. Accordingly, the present invention is directed to novel methods of use for TAAs, wherein TAAs may be used to induce immune tolerance.
  • DCs bone marrow derived dendritic cells
  • Bone marrow dendritic cells were obtained following published protocols 43,44 j n brief, bone marrow cells were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS), 100 U/ml Penicillin G, 100 ⁇ g/ml Streptomycin, 1000 U/ml GM-CSF, 1000 U/ml IL-4 (both from R&D systems, Minneapolis, MN), 1.5 ⁇ g/ml indomethacin (Cayman, Ann Arbor, MI), and 50 ⁇ M NG-monomethyl-L-arginine (Calbiochem, San Diego, CA). Cells were washed with PBS and supplied with fresh media on days 3 and 5 of culture.
  • FBS fetal bovine serum
  • Penicillin G 100 ⁇ g/ml Streptomycin
  • 1000 U/ml GM-CSF 1000 U/ml IL-4
  • IL-4 both from R&D systems, Minneapolis, MN
  • 1.5 ⁇ g/ml indomethacin
  • mice Female Balb/c, C3H, and C57BL/6 mice (6-8 weeks old) were purchased from Jackson Laboratories, Bar Harbor, Maine. D011.10 mice are also commercially available (Jackson Laboratories, Bar Harbor, Maine). 3A9 mice were kindly obtained from Dr. R. Eisenberg (Univ. of Pennsylvania).
  • Flow cytometry To prevent non-specific staining (mediated via FcR interactions), cells were pre-incubated with mAbs to CD 16 and CD32, specific for Fc ⁇ RIII and RII, according to the manufacturer's instructions (Pharmingen, San Diego, CA). For flow cytometry, the cells were adjusted to a concentration of 5xl0 6 cells/ml in RPMI 1640 with 3% FBS, 0.15 M HEPES and 0.1% sodium azide, stained with saturating antibody concentrations for 30 min at 4»C, washed, and then analyzed on a FACScan (Becton Dickinson, Mountain View, CA) using CellQuest software (v. 3.1).
  • FACScan Becton Dickinson, Mountain View, CA
  • anti-MHC class I H-2D d : clone 34-2-12
  • anti-MHC class ⁇ I-A d /I-E d , clone 2G9; I-A k , clone 11-5.2
  • anti-CD80/ B7-1 clone 16-10A1
  • anti-CD86/B7-2 clone GL1
  • anti-CDllc clone HL3
  • anti-CDllb clone Ml/70.
  • the antibodies were directly conjugated with either fluorescein isothiocyanate or phycoerytnrin.
  • the G8.8 hybridoma which produces an antibody specific for mEGP, was kindly provided by Dr. D. Herlyn (Wistar Institute, Philadelphia, PA) and FITC-conjugated (Molecular Probes, Eugene, OR) according to the manufacturer's instructions.
  • mEGP cDNA was provided by Dr. W.M. Kuehl (NCI, Bethesda, MD). GA733-2 cDNA was a gift of Dr. D. Herlyn (Wistar Institute, Philadelphia, PA). The cDNA was subcloned into pAdCMVlink, which was used to prepare El and E3 deleted adenoviruses (AdmEGP and AdGA733-2, respectively) as previously described 45.
  • AdmEGP and AdGA733-2 An adenoviral vector without a transgene served as a control (Adbgl2).
  • MLR Mixed lymphocyte reaction
  • ELISA ELISA.
  • the following antibody pairs were purchased from Pharmingen (San Diego, CA) and used according to the manufacturer's instructions: IL-2, clones JES6-1A12 and JES6-5H4; interferon ⁇ , clones R4-6A2 and XMG1.2; TL-10, clones JES5-2A5 and SXC-1 ; IL-4, clones 1 IB 11 and B VD6-24G2. All ELIS As had a detection limit of 60 pg/ml. Optical density readings were performed on a BioRad Microplate Reader Model 3550.
  • T cells express a T cell receptor recognizing HEL46-61 -peptide presented in I-A k .
  • DCs from C3H mice were pulsed with either hen egg lysozyme protein (Sigma) or HEL46-61 peptide (University of Illinois).
  • DO 11.10 T cells express a T cell receptor recognizing OVA323-339-peptide presented in I-A d .
  • DCs from Balb/c mice were pulsed with either ovalbumin protein (Sigma) or ONA323-339-peptide (University of Pennsylvania). The pulse was done with 0.5mg/ml protein for 16 hrs or with l ⁇ g/ml peptide for 2 hours.
  • DCs were washed extensively and treated with mitomycin C (100 ⁇ g/ml for 30 min.) and co- cultured with the antigen-specific T cells.
  • DCs can present peptides from exogenously supplied ovalbumin protein in association with MHC class I and class II molecules 46.
  • cell hybridomas RF3370 and M22D9 (kindly provided by Dr. K.L. Rock, University of Massachusetts, Worchester, Massachusetts) recognize ovalbumin peptides presented by MHC class I (H-2K b , RF3370) or MHC class II (I-A b , M22D9).
  • DCs from C57BL/6 mice were used as stimulators and pulsed with ovalbumin peptide (0.5 mg/ml for 16 hours).
  • lxlO 5 hybridoma cells were co-cultured with 2xl0 4 DCs for 24 hours, after which culture supernatants were harvested and assayed for TL-2 concentration by ELISA.
  • the naturally mEGP-negative FBL-3 murine erythroleukemia cell line (kindly provided by Dr. W. Chen, The Cleveland Clinic Foundation, Cleveland, Ohio) was infected with AdmEGP or control adenovirus. Cells were harvested 48 h post-infection and subjected to three cycles of freeze-thaw to lyse the cells, thus generating cellular debris. Increasing amounts of the cell debris were incubated with dendritic cells for 16 hours, after which pre-incubated dendritic cells were used as stimulators in an allogeneic MLR.
  • Myeloid DCs do not express mEGP as determined by flow cytometry and RT- PCR (Fig. la) 18.
  • murine DCs were transduced with a control replication-deficient adenoviral vector (Adbgl2) or with vectors encoding mEGP (AdmEGP) or GA733-2 (AdGA733-2). Expression of mEGP or GA733-2 at the surface of AdmEGP and AdGA733-2 transfected cells, respectively, was verified by flow cytometry (Fig 1).
  • murine DCs expressing mEGP showed a marked decrease in T cell stimulatory capacity in mixed lymphocyte reactions (MLR), relative to DCs expressing GA733-2, the human homologue of mEGP, and control DCs (Fig. 2a).
  • MLR mixed lymphocyte reactions
  • Fig. 2b A significant decrease in production of TL-2 (Fig. 2b) and interferon- ⁇ (Fig. 2c) paralleled the decrease in T cell stimulation.
  • TL-4 production was not detectable in these assays, and TL-10 production was similar for all groups (100-200 pg/ml in supernatants). Similar results were obtained using infected DCs derived from Balb/c, C3H, or C57B1/6 mice as stimulators and T cells from C3H or Balb/c mice as responders.
  • the MLR inhibitory effect of mEGP was observed when using DCs matured for 48 hrs with LPS (200 ng/ml; Fig. 2a) or TNF ⁇ (200 Units/ml; not shown).
  • LPS 200 ng/ml
  • TNF ⁇ 200 Units/ml
  • TCR T cell receptor
  • Con A concanavalin A
  • SEB staphylococcal super antigen
  • T cells were isolated from transgenic mice expressing either the DOl 1.10 TCR specific for OVA peptide 333-339 (I-A d ) l9 or the 3A9 TCR specific for the HEL peptide 46-61 (I-A k ) 20 .
  • the T cells were stimulated with untransfected or mEGP-transfected DCs, which were pulsed with either the respective full length protein or the antigenic peptide.
  • the stimulatory capacity of DCs pulsed with either protein or peptide was markedly reduced in the presence of mEGP relative to the control DCs (Fig. 4).
  • DCs were pulsed with OVA protein and co-cultured with either RF3370 or MF22D9 hybridoma cells.
  • EL-2 secretion was assessed by ELISA and used as an indicator of antigen T cell activation. Results were normalized to untransfected DC (% control).
  • AdmEGP or control- transfected DCs were pulsed with ONA protein and co-cultured with a T cell hybridoma (RF3370) specific for an ONA-derived peptide presented by MHC class I (H-2K b ).
  • T cell hybridoma RF3370
  • H-2K b T cell hybridoma
  • the above DCs were co-cultured with a second T cell hybridoma (MF22D9) specific for an ONA peptide presented by MHC class TJ (I-A b ).
  • EL-2 secretion by the hybridomas was used to assess antigen-specific T cell stimulation.
  • DCs expressing mEGP inhibited the secretion of IL-2 by the MHC class Il-restricted MF22D9 cells (p ⁇ 0.05), but did not significantly alter stimulation of the MHC class I-restricted RF3370 cells (Table 4). These results demonstrate that expression of mEGP diminishes antigen presentation by MHC class II molecules and has a minimal effect on MHC class I presentation.
  • DCs within the tumor microenvironment are able to engulf tumor cell debris and process the resident proteins for presentation by MHC class I or class JJ molecules 22-24 ⁇ h e observed inhibition of antigen presentation by MHC class II molecules in DCs that express mEGP suggested that the presence of mEGP in internalized tumor cell debris might also inhibit T cell proliferation.
  • the stimulatory capacity of DCs incubated with control cell debris was compared to that of DCs incubated with cell debris having augmented levels of mEGP.
  • DCs incubated with control cell debris showed slightly enhanced MLR stimulatory capacity (Fig. 5a).
  • mEGPex did not inhibit a MLR when transfected into DCs (not shown), but did inhibit a MLR when tumor cell debris was added from mEGPex expressing cells (Fig. 5c).
  • Transfection of DCs with an adenovirus expressing mEGPin, a truncated form of mEGP that lacks most of the extracellular domain of mEGP but retains the intracellular and transmembrane domains revealed that these subdomains did not mediate inhibition of T cell proliferation when expressed in DCs or added as tumor cell debris (not shown).
  • DCs expressing mEGP were functional in vivo
  • the immune response to adenoviral antigens following intravenous vaccination was determined.
  • Untransduced DCs and DCs transduced with AdmEGP or control vector Adbgl2 were injected into the tail vein of adenovirus naive mice.
  • DCs transduced with AdmEGP express mEGP and would, therefore, present adenoviral antigens in the presence of mEGP, whereas DCs transduced with control vector Adbgl2 present adenoviral vectors in the absence of mEGP.
  • Seven days later, splenocytes were isolated and allowed to proliferate in co-culture with DCs transfected with Adbgl2.
  • mice vaccinated with mEGP transfected DCs had a significantly diminished T cell proliferative response to adenoviral antigens compared to the mice vaccinated with Adbgl2 transfected DCs (Fig. 5d). These results demonstrate that mEGP can exert its inhibitory effects when T cell stimulation takes place in vivo.
  • the natural immune response to most tumors is either absent or very inefficient due to several mechanisms that allow tumor cells to regularly escape immune surveillance.
  • One mechanism for interfering with immune surveillance is direct inhibition of T cell activity through production of immunosuppressive factors, such as IL-10 25,26 ⁇ or through stimulation of the secretion of immunosuppressive factors by macrophages 27.
  • immunosuppressive factors such as IL-10 25,26 ⁇
  • macrophages 27 Many tumors down-regulate gene expression of particular MHC class I loci, ⁇ 2-microglobulin (reviewed in 28,29) 9 G r molecules required for MHC class I-associated antigen processing such as TAP1/ TAP2 30 as a means to evade cytotoxic CD8+ T cell responses.
  • viruses e.g., adenovirus, herpesvirus, and cytomegalovirus
  • MHC class I molecule expression and antigen processing are blocked in infected cells 1,32 j n contrast to these well characterized mechanisms to evade antigen presentation to CD8+ T cells, much less is known regarding mechanisms by which tumor cells interfere with MHC class ⁇ antigen presentation.
  • Tumor infiltrating DCs in human breast cancer 33 nd n a rat colon cancer model 34 have been shown to be inefficient in antigen presentation.
  • This defect in antigen presentation may be due to the suppressive effects of IL-10 on MHC class U expression and antigen processing 35 ; but other more direct mechanisms, like those described for antigen processing by MHC class I molecules, may exist.
  • the human cytomegalovirus protein US2 was found to cause degradation of HLA-DR- alpha and DM-alpha, both of which are essential proteins in the MHC class JJ antigen presentation pathway 6.
  • ⁇ n ⁇ s mechanism prevents activation of CD4+ T cells and allows the virus to go undetected by the imm ⁇ ne system.
  • the effect of mEGP on T cell proliferation can be seen following in vivo vaccination with DCs carrying both mEGP and adenoviral antigens.
  • the data presented herein provide the first evidence of a tumor-associated antigen interfering with MHC class JJ-dependent antigen presentation by antigen presenting cells. The data may potentially explain why women with breast cancers expressing GA733-2 have a poorer prognosis than women with tumors that lack GA733-2 expression 37.
  • One potential mechanism would be by a direct interaction between mEGP and either MHC class II components or the invariant chain (Ii). Such an interaction could be mediated by oligomerization of thyroglobulin-like domains present in both mEGP 6 and the p41 form of Ii 40-42 ⁇ w hich could prevent Ii from assembling class II complexes. Indirect effects of mEGP on MHC class II complex assembly, however, cannot be ruled out. While defects in the assembly of the MHC class II ⁇ li complex may contribute to mEGP inhibition within the MHC class JJ antigen presentation pathway, this is not likely to be the only mechanism given mEGP's striking inhibition of antigen presentation in the face of persistent MHC class TJ surface expression.
  • MHC class Il-associated antigen presentation by DCs was inhibited not only by expression of endogenously synthesized mEGP, but also after mEGP was internalized following exposure to cell debris derived from mEGP-expressing cells (Fig. 5).
  • DCs are known to effectively internalize cellular fragments from apoptotic or necrotic cells and process these proteins for presentation by MHC class I and class TJ molecules 17,18. Should tumor infiltrating DCs internalize such fragments from mEGP-expressing tumor cells in vivo, DC presentation of tumor-associated antigens would likely be inhibited by the internalized mEGP.
  • tumor infiltrating DCs have been shown to be ineffective at antigen presentation 33,34 Although this defect has been attributed to a loss of inducible expression of costimulatory molecules 34 ⁇ inhibition of antigen presentation by tumor cell-expressed mEGP provides a compelling alternative explanation for this defect in antigen presentation observed for tumor infiltrating DCs. Accordingly, mEGP inhibited, tumor infiltrating DCs would be unable to activate CD4+ T cells specific for TAA. In this manner, mEGP expression by tumor cells could potentially disable an essential component in the establishment and maintenance of an anti-tumor immune response 4 and permit escape from immune surveillance. EXAMPLE II In Vitro Human Systems for Detecting TAA-mediated Immune Suppression
  • GA733-2 inhibits MLR in murine systems when added as tumor debris.
  • the effects of GA733-2 may also be examined in human DC-T cell MLR experiments.
  • the ability of human DCs transfected with GA733-2 may be compared to the ability of untransfected control human DCs to elicit proliferation of allogeneic human T cells in human DC-T cell MLRs.
  • human DCs exposed to GA733-2 expressing human breast cancer cells may be evaluated in human DC-T cell MLRs.
  • PBMCs Peripheral blood monocytic cells
  • 1 cc of blood comprises only approximately 10 6 PBMCs, from which only ⁇ 8xl0 4 DCs may be isolated.
  • a single leukopheresis will yield about 10 10 PBMCs from which ⁇ 10 7 DCs may be isolated.
  • PBMCs are cultured in RPMI 1640 containing 1% human AB + plasma and antibiotics at a density of ⁇ 50xl0 6 cells per ml in 75cm 2 tissue culture flasks. After 2 hours of culturing, the non-adherent cells are removed by aspiration, and the adherent cells (DCs) are gently washed.
  • T cells may be isolated from the non-adherent cell population, which is mainly comprised of lymphocytes, by negative-selection using magnetic bead coupled antibodies that remove B-lymphocytes, NK cells, macrophages and granulocytes leaving an eluate that contains 99+% T cells.
  • the T cells may be used fresh or cryo-preserved for future use as the responding cells in T cell stimulation assays (e.g., MLR).
  • Adherent cells may be further cultured in medium containing final concentrations of 100 ng/ml GM-CSF (Immunex) and 20 ⁇ g/ml IL-4 (Genzyme). On day 5 of culture, cells may be harvested, washed and treated with AdGA733-2, AdGA733-l, or controls (no adenovirus, AdBgl2 or AdmEGP; MOI of 100). After transfection, cells may be re-cultured for another 48 hours in media containing cytokines. On day 7, DCs may be used as stimulators (ranging from 10 4 to 5xl0 5 ) in T cell proliferation assays (e.g., MLR) using T cells (10 5 cells) isolated from a different donor.
  • T cell proliferation assays e.g., MLR
  • DC surface markers CDllc ⁇ HLA-DR + , MHC class I&IT, CD25+, CD40 + , CD80 + , CD86 + ) and demonstrate that they express the relevant proteins (mEGP, GA733-2, or GA733-1). See Example I for protocol.
  • the optimal range of GA733-2 or GA733-1 expression that inhibits T cell responses in MLRs may be determined by varying the amount of vector used to transduce DCs (e.g., MOI of 1 , 50, 100, 500) and by mixing varying proportions of GA733 + -DCs (Ad.GA733-2 or Ad.GA733-2, MOI -500) with control transduced DCs (Ad.Bgl2, MOI -500). Such experiments may be performed as described in Example I.
  • the effects of GA733-2 or GA733-1 expression in a "human MLR system" may be assessed as described in Example I wherein T cell proliferation and activation are assessed using a variety of assays, including cytokine production.
  • chimeric GA733-2/mEGPconstructs may also be evaluated in human MLR systems.
  • Human DCs exposed to human cancer cells which express either GA733-1 or GA733-2 may also be evaluated for their ability to stimulate T cells in a human MLR. Such experiments may be performed as described in Example I. Graded amounts of tumor cell debris may be added to isolated human DCs, which are very efficient at capturing antigens through macropinocytosis or by mannose receptor-mediated uptake. Hela cells (GA733 and mEGP negative), for example, may be untransfected or transfected PBS containing AdGA733-2, AdGA733-l, AdmEGP or AdBgl2 to serve as negative and positive controls. Cells may be pelleted by centrifugation and lysed by repetitive freeze thawing.
  • the protein concentration of a lysate may be determined by means known to those skilled in the art (e.g., BioRad assay). As determined by the protein concentration, increasing amounts of HeLa cell debris may be added to different MLRs. Tumor cell debris from human breast cancer cell lines that naturally express GA733-1 or GA733-2 may also be tested in parallel MLR assays. MCF-7 (breast cancer), SW480 (colon cancer), BT-20 (breast cancer), and 293 cell lines (El A transformed human embryonic kidney cell) may be used in these assays. Human tumor cells that do not express GA733 may be used as negative controls. Cell lines appropriate for use as negative controls include, but are not limited to, Hela cells (cervical cancer), WM9 cells (melanoma), WM35 cells (melanoma), and LNCaP cells (prostate cancer).
  • recombinant full length GA733-1 or GA733-2 protein, or truncated proteins thereof, produced in baculovirus or mammalian cells may be added to human MLR assays to determine the effect of soluble GA733-1 or GA733-2 polypeptides on T cell activation.
  • the human homologue of mEGP, GA733-2 also blocked the activation of human T cell responses to adenoviral proteins in human MLR systems (data not shown).
  • the response of human T cells to adenoviral antigens is categorized as an antigen recall response.
  • the inhibitory effect of GA733-2 on human T cells appears to involve the up-regulation of CD69 (124% of control after one stimulation), CD25 (120% of control after one stimulation) and CD152 (182% of control after one stimulation). This pattern of changes in T cell expression of surface proteins establishes two points. The first point is that the absence of T cell responses in the above assays is not the result of failure to activate the T cells. If this were the case, CD69 levels would be less than those of controls (in principle, zero).
  • the up- regulation of CD69 establishes that GA733-2 has a direct effect on the T cells resulting in their failure to respond to the specific stimuli (i.e., adenoviral antigens). It effectively rules out the possibility that GA733-2 exercises its inhibitory effect by depriving T cells of any stimulation (e.g., ignorance). That is, the T cells appear to have been activated in a non-productive manner, which failed to induce cellular proliferation and cytokine (e.g., TL-2 or interferon) production.
  • cytokine e.g., TL-2 or interferon
  • the methods of the present invention may be used to advantage in the treatment of patients having a variety of disorders.
  • the methods of the present invention are well suited for therapeutic regimens in which immune suppression is beneficial to the patient.
  • the methods of the present invention provide significant advantages over other available means of immunosuppression.
  • Systemic immunosuppression which may be mediated by pharmacologic agents, frequently leads to complications including, but not limited to, opportunistic infections, myelotoxicity, gastrointestinal toxicity, hypertension, hyperlipidemia, and secondary malignancies.
  • Type I diabetes is provided herein as an exemplary disease for which the methods of the present invention may be used to improve the prognosis of a patient.
  • Type I diabetes results from an immune-mediated destruction of pancreatic beta cells.
  • the effector mechanisms leading to type I diabetes have been studied extensively.
  • Autoreactive T cells clearly play a pivotal role in disease etiology as evidenced by the identification of T cells that are reactive to islet autoantigens in both diabetic mice and humans (Roep et al., 1991, Lancet 337:1439-41; Roep et al., 1995, Diabetes 44:278- 83).
  • type I diabetes has been observed in a male patient with X-linked agammaglobulinemia, a disorder with severely decreased B cell numbers in the peripheral blood and very low serum levels of all immunoglobulins (Martin et al.,
  • B cells play a secondary role in the development of diabetes.
  • Activated B cells which produce auto-antibodies specific for pancreatic beta-cell antigens and are able to present autoantigen to T cells have also been identified (Roll et al., 2000, Diabetologia 43:598-608; Reijonen et al., 2000, Diabetes 49:1621-6). B cells might, therefore, influence disease progression by providing antibody-facilitated T cell responses.
  • Islet cell transplantation remains the most attractive means of achieving sustained normal glycemia in type I diabetic patients.
  • Two major obstacles facing islet cell transplantation are preventing allograft rejection and increasing the availability of islet cells suitable for transplantation.
  • Gene transfer of a cDNA encoding a therapeutic protein may potentially be employed to specifically modify islets to enhance their viability and suppress allograft rejection. Such an approach would obviate the need for systemic immunosuppression and the complications that accompany pharmacologic immune suppression.
  • a successful gene transfer approach could pave the way for transgenic animals constitutively expressing the therapeutic protein in islet cells. This would enhance the availability of beta cells or islets for transplantation by making xenotransplantation more feasible.
  • CTLA4Ig administration decreases the immune response by blocking T cell activation and have been successfully used in some transplantation models (Uchikoshi et al., 1999, Diabetes 48:652-7; Boiling et al., 1994, Journal of Surgical Research. 57:60-4; Judge et al., 1999, J. Immunology. 162:1947-51).
  • This later approach is intrinsically more appealing since it functions at the level of antigen presentation and therefore has the potential to engage tolerogenic pathways at this pivotal juncture in immune activation.
  • this approach may also suppress the elaboration of inflammatory cytokines thought to play a role in autoimmune diabetes (Holz et al., 2001, J. Clin. Invest.108: 1749-58; Gysemans et al., 2001, Diabetologia. 44:567-74; Christen et al., 2001, J. Immunology 166:7023-32).
  • GA733-2 is the ligand for LAIR-1, a putative cell surface lymphocyte protein with phosphatase activity that acts as an inhibitor of T, B and NK cell function
  • mEGP a putative cell surface lymphocyte protein with phosphatase activity that acts as an inhibitor of T, B and NK cell function
  • Islets may be isolated from C57BL/6 mice, for example, using established methods (Shewade et al., 1999, Transplantation Proceedings 31:1721-3) and exposed to AdmEGP, or control AdlacZ.
  • the fraction and distribution of mEGP expressing cells may be determined using a combination of immunohistochemical staining for mEGP in histologic sections of transfected islets and by flow cytometry using disaggregated AdmEGP-transfected islets.
  • Islets expressing mEGP (mEGP-islets) may be stained with dithiazone (Garnuszek et al., 1998, Applied Radiation & Isotopes 49:1563-71; Fiedor et al., 1998, Transplantation Proceedings 30:533; Fiedor et al., 1998, Ann. Transplantation 3:21-30) to demonstrate that they retain their characteristic zinc-dependent staining.
  • Insulin production may be determined by immunochemical assays (Webster et al., 1990, J. Immunological Methods 134:95-100) using commercially available reagents (Insulin ELISA Kit, Crystal Chem Inc,) for samples where an immediate determination is needed. Routine determinations of insulin levels may be done by radioimmunoassay (RIA) methods. For these experiments AdlacZ-transduced and untransduced islets may serve as controls.
  • RIA radioimmunoassay
  • BALB/c DCs and C3H T cells (“directly primed”MLR) may be incubated with mEGP-islets from BALB/c mice. Additionally, antigens from donor (islet) cells may be acquired by bone marrow derived dendritic cells (DMBC) and presented in host MHC molecules (see Albert et al, 1998, Nature 392:86-9). Such "cross-priming" provides a convenient in vitro model to demonstrate the immunosuppressive effect of mEGP-islets. Using the models described above in Example I, BALB/c DCs may be used as stimulators and BALB/c T cells as responders in such "cross-priming experiments".
  • DMBC bone marrow derived dendritic cells
  • Lysates from freeze-thawed cells derived from C57BL/6 mEGP-islets may also be added in increasing amounts to demonstrate that mEGP in this context can block cross-priming to alloantigens as measured by T cell proliferation.
  • Controls may consist of lysates derived from untransduced C57BL/6 islets (+ proliferative response) and/or syngeneic (BALB/c) islets (- control).
  • a conditioned dendritic cell can be a temporal bridge between a CD4+ T- helper and a T-killer cell. Nature 393, 474-478 (1998).
  • gp40 the murine homologue of human epithelial cell adhesion molecule (Ep-CAM), by murine dendritic cells.
  • Ep-CAM human epithelial cell adhesion molecule
  • Dendritic cells acquire antigen from apoptotic cells and induce class I- restricted CTLs. Nature 392, 86-89 (1998).
  • Interleukin- 10 is a growth factor for human melanoma cells and down- regulates HLA class-I, HLA class-II and ICAM-1 molecules. Int J Cancer 71, 630-637 (1997).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Oncology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Dermatology (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Toxicology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne des procédés destinés à supprimer la réponse immunitaire de vertébrés. De manière plus spécifique, la présente invention concerne des procédés destinés à administrer des antigènes associés aux tumeurs de manière à supprimer des réponses immunitaires indésirables.
PCT/US2002/011371 2001-04-11 2002-04-11 Compositions et procedes destines a supprimer des reponses immunitaires WO2002088304A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002338571A AU2002338571A1 (en) 2001-04-11 2002-04-11 Compositions and methods for suppressing immune responses

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US28294601P 2001-04-11 2001-04-11
US60/282,946 2001-04-11

Publications (2)

Publication Number Publication Date
WO2002088304A2 true WO2002088304A2 (fr) 2002-11-07
WO2002088304A3 WO2002088304A3 (fr) 2005-02-17

Family

ID=23083813

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/011371 WO2002088304A2 (fr) 2001-04-11 2002-04-11 Compositions et procedes destines a supprimer des reponses immunitaires

Country Status (2)

Country Link
AU (1) AU2002338571A1 (fr)
WO (1) WO2002088304A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014179771A1 (fr) * 2013-05-03 2014-11-06 Selecta Biosciences, Inc. Associations de dosages destinées à réduire les réponses immunitaires à médiation humorale non souhaitées
CN105307641A (zh) * 2013-05-03 2016-02-03 西莱克塔生物科技公司 用于降低不期望体液免疫应答的给药组合
US9987354B2 (en) 2011-04-29 2018-06-05 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for antigen-specific deletion of T effector cells
US10046064B2 (en) 2014-09-07 2018-08-14 Selecta Biosciences, Inc. Methods and compositions for attenuating exon skipping anti-viral transfer vector immune responses
US11426451B2 (en) 2017-03-11 2022-08-30 Selecta Biosciences, Inc. Methods and compositions related to combined treatment with antiinflammatories and synthetic nanocarriers comprising an immunosuppressant

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996039176A1 (fr) * 1995-06-05 1996-12-12 Brigham & Women's Hospital Utilisation de la tolerance orale pour supprimer les reponses immunitaires de th1 et de th2 et pour supprimer la production d'anticorps
EP1264598A1 (fr) * 2001-06-05 2002-12-11 Universitair Medisch Centrum Utrecht Modification d'une réponse immunitaire par intervention dans l'interaction de LAIR avec son ligand

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996039176A1 (fr) * 1995-06-05 1996-12-12 Brigham & Women's Hospital Utilisation de la tolerance orale pour supprimer les reponses immunitaires de th1 et de th2 et pour supprimer la production d'anticorps
EP1264598A1 (fr) * 2001-06-05 2002-12-11 Universitair Medisch Centrum Utrecht Modification d'une réponse immunitaire par intervention dans l'interaction de LAIR avec son ligand

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CIRULLI ET AL.: 'KSA Antigen Ep-CAM mediates cell-cell adhesion of pancreatic epithelial cells: morphoregulatory roles in pancreatic islet development' vol. 140, 23 March 1998, pages 1519 - 1534, XP002983500 *
DATABASE CAPLUS [Online] 1999 XP002984286 Retrieved from STN Database accession no. 2000:435379 & LI ET AL.: 'Immunohisotchemical study of the expressions of EGF and EGFR during tolerization' *
TAGUCHI ET AL.: 'Abnormal thymic expression of epithelial cell adhesion molecule (EP-CAM) in new England Black (NZB) Mice' JOURNAL OF AUTOIMMUNITY vol. 13, December 1999, pages 393 - 404, XP002983499 *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11235057B2 (en) 2011-04-29 2022-02-01 Selecta Biosciences, Inc. Methods for providing polymeric synthetic nanocarriers for generating antigen-specific tolerance immune responses
US9987354B2 (en) 2011-04-29 2018-06-05 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for antigen-specific deletion of T effector cells
US11779641B2 (en) 2011-04-29 2023-10-10 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for allergy therapy
US11717569B2 (en) 2011-04-29 2023-08-08 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers
US10420835B2 (en) 2011-04-29 2019-09-24 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for antigen-specific deletion of T effector cells
US10039822B2 (en) 2011-04-29 2018-08-07 Selecta Biosciences, Inc. Method for providing polymeric synthetic nanocarriers for generating antigen-specific tolerance immune responses
US10441651B2 (en) 2011-04-29 2019-10-15 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for generating CD8+ regulatory T cells
US9993548B2 (en) 2011-04-29 2018-06-12 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for inducing regulatory B cells
US10004802B2 (en) 2011-04-29 2018-06-26 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers for generating CD8+ regulatory T cells
US11298342B2 (en) 2013-05-03 2022-04-12 Selecta Biosciences, Inc. Methods providing a therapeutic macromolecule and synthetic nanocarriers comprising immunosuppressant locally and concomitantly to reduce both type I and type IV hypersensitivity
US10357482B2 (en) 2013-05-03 2019-07-23 Selecta Biosciences, Inc. Methods providing a therapeutic macromolecule and synthetic nanocarriers comprising immunosuppressant locally and concomitantly to reduce both type I and type IV hypersensitivity
US10335395B2 (en) 2013-05-03 2019-07-02 Selecta Biosciences, Inc. Methods of administering immunosuppressants having a specified pharmacodynamic effective life and therapeutic macromolecules for the induction of immune tolerance
US10668053B2 (en) 2013-05-03 2020-06-02 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers to reduce or prevent anaphylaxis in response to a non-allergenic antigen
WO2014179771A1 (fr) * 2013-05-03 2014-11-06 Selecta Biosciences, Inc. Associations de dosages destinées à réduire les réponses immunitaires à médiation humorale non souhaitées
CN105307641A (zh) * 2013-05-03 2016-02-03 西莱克塔生物科技公司 用于降低不期望体液免疫应答的给药组合
US10434088B2 (en) 2013-05-03 2019-10-08 Selecta Biosciences, Inc. Methods related to administering immunosuppressants and therapeutic macromolecules at a reduced pharmacodynamically effective dose
US10357483B2 (en) 2013-05-03 2019-07-23 Selecta Biosciences, Inc. Methods comprising dosing combinations for reducing undesired humoral immune responses
US10071114B2 (en) 2014-09-07 2018-09-11 Selecta Biosciences, Inc. Methods and compositions for attenuating gene expression modulating anti-viral transfer vector immune responses
US10046064B2 (en) 2014-09-07 2018-08-14 Selecta Biosciences, Inc. Methods and compositions for attenuating exon skipping anti-viral transfer vector immune responses
US11633422B2 (en) 2014-09-07 2023-04-25 Selecta Biosciences, Inc. Methods and compositions for attenuating anti-viral transfer vector immune responses
US11426451B2 (en) 2017-03-11 2022-08-30 Selecta Biosciences, Inc. Methods and compositions related to combined treatment with antiinflammatories and synthetic nanocarriers comprising an immunosuppressant

Also Published As

Publication number Publication date
AU2002338571A1 (en) 2002-11-11
WO2002088304A3 (fr) 2005-02-17

Similar Documents

Publication Publication Date Title
AU2002341266B2 (en) Use of HMGB1 for the activation of dendritic cells
Carapito et al. Genetics, genomics, and evolutionary biology of NKG 2D ligands
AU2001255326B2 (en) Targeted vaccine delivery systems
AU2008328183B2 (en) System for delivery into a XCR1 positive cell and uses thereof
CA2314397C (fr) Nouveau peptide apoep1.b, compositions correspondantes et utilisations
KR102319839B1 (ko) 항원-특이적 tcr의 신규 생성
AU2002341266A1 (en) Use of HMGB1 for the activation of dendritic cells
US20210023191A1 (en) Vaccine compositions comprising tryptophan 2,3-dioxygenase or fragments thereof
CN110799206A (zh) 使用可溶性cd24治疗癌症疗法中免疫相关不良事件的方法
JP2021511030A (ja) Ｃ３ｂ結合ポリペプチド
JP2023052647A (ja) 標的治療免疫調節のためのＭＨＣクラスＩｂ分子及びペプチドの組合せ
JP2017503810A (ja) 癌治療のための改善された細胞組成物および方法
CA2410736A1 (fr) Recepteur de macroglobuline alpha (2) utilise comme recepteur de la proteine de choc thermique et ses utilisations
Fedoseyeva et al. CD4+ T cell responses to self-and mutated p53 determinants during tumorigenesis in mice
WO2002088304A2 (fr) Compositions et procedes destines a supprimer des reponses immunitaires
JP6363996B2 (ja) 免疫原性が低下した凝固因子ｖｉｉｉ
JP2002506618A (ja) 腫瘍自己抗原に対する免疫誘導
AU2016322506A1 (en) Vaccine compositions comprising C-C motif chemokine 22 (CCL22) or fragments thereof
CA2301169A1 (fr) Genes de classe i mhc porcins et utilisations de ces genes
De Leo p53-based immunotherapy of cancer
Ruiz-Cabello et al. Tumor evasion of immune system
EA041619B1 (ru) Способ продуцирования человеческих антигенспецифических т-лимфоцитов

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP