WO2000066764A1 - Procédés visant à accroître la prolifération de lymphocytes t - Google Patents

Procédés visant à accroître la prolifération de lymphocytes t Download PDF

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WO2000066764A1
WO2000066764A1 PCT/US2000/012118 US0012118W WO0066764A1 WO 2000066764 A1 WO2000066764 A1 WO 2000066764A1 US 0012118 W US0012118 W US 0012118W WO 0066764 A1 WO0066764 A1 WO 0066764A1
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cells
indoleamine
dioxygenase
tryptophan
cell
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PCT/US2000/012118
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Benoit Van Den Eynde
Janine Bilsborough
Thierry Boon-Falleur
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Ludwig Institute For Cancer Research
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Priority to EP00928796A priority Critical patent/EP1185687A1/fr
Priority to AU46976/00A priority patent/AU4697600A/en
Publication of WO2000066764A1 publication Critical patent/WO2000066764A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90241—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)

Definitions

  • T lymphocyte culture is a difficult procedure. For reasons which are not understood, only a limited number of cytolytic T lymphocyte (CTL) clones can grow in vitro and most of those clones will do so only for a limited number of divisions, or at a very slow pace.
  • CTL cytolytic T lymphocyte
  • the methods include growing the cells in the presence of an effective amount of one or more tryptophan enhancing agents to increase the proliferation of the cells.
  • Proliferation of cells is determined preferably by measuring the number of cells in a population before and after growing the cells.
  • Increasing cell proliferation means increasing the number of cells by at least about 10% relative to the number of cells that are present in a parallel control population of cells that are subjected to the same conditions as the tryptophan enhancing agent treated population with the exception that the control population is not contacted with the tryptophan enhancing agents.
  • the number of cells are increased at least about 50%, more preferably 2-fold, yet more preferably 4-fold, still more preferably, 10-fold and, most preferably, at least 20-fold relative to the number of cells that are present in the parallel control population.
  • the cells are not macrophages or trophoblasts.
  • the cells are T cells.
  • growing the T cells means proliferating the T cells in vivo, e.g., by administering a population of T cells to a subject.
  • In vivo methods for increasing proliferation of T cells can be tested in animal models to establish effective amounts of tryptophan enhancing agents for increasing T cell proliferation.
  • growing the T cells means proliferating the T cells in vitro, e.g., by culturing according to standard cell culture methods.
  • In vitro proliferation of T cells is readily tested by standard methods of measuring cell proliferation including cell enumeration and metabolism tests.
  • Preferred tryptophan enhancing agents are IDO inhibitors, including 1-methyl-DL- tryptophan, ⁇ -(3-benzofuranyl)-DL-alanine and ⁇ -[3-benzo(b)thienyl]-DL-alanine, most preferably 1-methyl-DL-tryptophan.
  • the tryptophan enhancing agent preferably is not tryptophan.
  • methods for increasing activity of T cells include culturing the T cells in the presence of an effective amount of one or more tryptophan enhancing agents to increase the activity of the T cells.
  • T cell activity is increased at least about 10%, preferably at least about 25%, more preferably at least about 50%, and most preferably at least about 100%.
  • an apparatus for practicing the methods of the invention.
  • the apparatus includes a container, an inhibitor of IDO contained therein or attached thereto and at least one T cell.
  • the container is a sterile conventional cell culture container, known to those of ordinary skill in the art, including e.g., stirring flasks, stirred tank reactors, air lift reactors, suspension cell reactors, cell adsorption reactors and cell entrapment reactors, petri dishes, multi well plates, micro titer plates, test tubes, culture flasks, and bags.
  • the inhibitor of IDO is contained in the container in soluble or immobilized form or directly attached to the internal surface of the container.
  • the container optionally includes one or more growth media components for cell culture.
  • growth media components for cell culture.
  • Such components are known to those of ordinary skill in the art.
  • tryptophan is contained in the culture container.
  • tryptophan and one or more inhibitors of IDO are contained in the culture container.
  • Additional cell culture components such as growth medium, serum and other cell types including feeder cells and target cells or antigen presenting cells also can be included.
  • the container does not contain macrophages or trophoblasts.
  • kits for stimulating the proliferation of T cells in culture contains the apparatus described above and instructions for using the apparatus to stimulate proliferation of T cells in vitro.
  • the invention provides a growth medium for the culture of cells including a tryptophan enhancing agent.
  • the growth medium contains nutrients for the culture of T cells.
  • a T cell culture is provided.
  • the T cell culture includes at least one T cell, growth medium and a tryptophan enhancing agent. In some embodiments the culture does not contain macrophages or trophoblasts.
  • the invention also provides adjuvant compositions comprising the tryptophan enhancing agents described herein and a pharmaceutically acceptable carrier. Use of the compositions described herein in the preparation of a medicament for increasing T cell proliferation and/or activity in a subject also is provided.
  • an immune response modulation composition in accordance with the invention.
  • the composition includes an amount of a tryptophan enhancing agent effective to increase local tryptophan concentrations in the presence of constitutively expressed indoleamine 2,3-dioxygenase.
  • the composition also includes a pharmaceutically acceptable carrier.
  • the tryptophan enhancing agent is an inhibitor of indoleamine 2,3-dioxygenase, preferably 1 -methyl-DL-tryptophan.
  • the immune response modulation composition further includes one or more immune modulators selected from the group consisting of antigen-specific T lymphocytes, peptide antigens, antigenic proteins and nucleic acids encoding peptide antigens.
  • These immune response modulation compositions preferably also include an adjuvant.
  • Preferred adjuvants include monophosphoryl lipid A (MPL); saponins including QS21, DQS21, QS-7, QS-17, QS-18, and QS-L1; DQS21/MPL; incomplete Freund's adjuvant; complete Freund * s adjuvant; montanide; and water-in-oil emulsions prepared from biodegradable oils.
  • the methods include administering to a subject in need of such treatment an amount of a tryptophan enhancing agent effective to increase T cell-mediated cytolysis of the cancer cell.
  • the cancer cells constitutively expresses indoleamine 2,3-dioxygenase.
  • Other methods include contacting the cancer cells with an amount of a tryptophan enhancing agent effective to increase T cell-mediated cytolysis of the cancer cell.
  • Preferred tryptophan enhancing agents for use in these methods are set forth above.
  • methods for determining a condition characterized by the ability of cancer cells to resist or evade T cell-mediated cytolysis are provided.
  • the methods include monitoring a sample of cancer cells from a patient who has or is suspected of having the condition for constitutive expression of indoleamine 2,3- dioxygenase, as a determination of the condition. Additional methods for determining whether to treat a cancer patient with an inhibitor of indoleamine 2,3-dioxygenase also are provided. The methods include determining the expression of indoleamine 2,3-dioxygenase by the cancer cells of the patient; the expression of indoleamine 2,3-dioxygenase by the cancer cells determines that the patient can be treated with an inhibitor of indoleamine 2,3- dioxygenase to increase the susceptibility of the cancer cells to T cell attack.
  • expression can be monitored by any method, including measuring the amount of IDO protein (e.g., by immunoassay methods, by measuring IDO enzymatic activity) or nucleic acid encoding the protein (e.g., by nucleic acid hybridization or amplification) that is expressed by the cancer cells.
  • IDO protein e.g., by immunoassay methods, by measuring IDO enzymatic activity
  • nucleic acid encoding the protein e.g., by nucleic acid hybridization or amplification
  • the invention also provides pharmaceutical preparations containing any one or more of the compositions described herein.
  • Such pharmaceutical preparations can include pharmaceutically acceptable diluents, carriers or excipients.
  • the use of such compositions in the preparation of medicaments, particularly medicaments for the treatment of cancer and for increasing T cell proliferation also is provided.
  • Fig. 1 shows the growth in culture of CTL clone MZ2-CTL-82/30 with or without the addition of 1-methyl-tryptophan.
  • Fig. 2 shows the growth in culture of CTL clone CTL-361 A/17 with or without the addition of 1-methyl-tryptophan.
  • Fig. 3 shows the growth in culture of CTL clone CTL-361 A/21 with or without the addition of 1 -methyl-tryptophan.
  • Figs. 4A, 4B and 4C depict the lytic activity of CTL clones MZ2-CTL-82/30, CTL- 361A/17 and CTL-361 A/21 respectively on the autologous tumor cells (-•-) or NK-target cells K562 (- ⁇ -) after culture with or without the addition of 1-methyl-tryptophan.
  • the autologous tumor cells were melanoma cells MZ2-MEL-43 in the case of CTL 82/30 and renal carcinoma cells LE9211-RCC in the case of CTLs 361 A/17 and 361 A/21.
  • Fig. 5 shows the tumor size and growth rate of tumors for groups of animals treated either with the IDO inhibitor 1 -methyl tryptophan or PBS.
  • T cell refers to T lymphocytes including CD8 + T lymphocytes and CD4 + T lymphocytes.
  • IDO indoleamine 2,3-dioxygenase
  • EC 1.13.11.17 an enzyme responsible for the first step of tryptophan catabolism. which is the oxygenation of tryptophan into N'-formyl-kynurenine. This enzyme is expressed at very high levels in placenta.
  • the report suggested that IDO activity in placental cells suppressed cytolytic T lymphocyte responses, thereby permitting gestation of allogeneic embryos. When IDO activity of the placental cells was inhibited, cytolytic T lymphocyte responses were no longer inhibited and allogeneic embryos were rejected.
  • the compound 1-methyl-DL-tryptophan is a tryptophan enhancing agent that increases T cell proliferation.
  • a "tryptophan enhancing agent” is a compound or a molecule added to a cell or cell culture that increases the concentration of tryptophan in a cell culture and increases proliferation of cells in the cell culture in the absence of macrophages or trophoblasts.
  • Exemplary tryptophan enhancing agents include IDO inhibitors (e.g., competitive inhibitors of IDO such as 1-methyl-DL-tryptophan, antisense nucleic acids that inhibit expression of IDO, anti-IDO antibodies that inhibit IDO activity) and compounds which directly increase tryptophan (e.g., tryptophan and analogs thereof which can be metabolized by T cells).
  • IDO inhibitors e.g., competitive inhibitors of IDO such as 1-methyl-DL-tryptophan, antisense nucleic acids that inhibit expression of IDO, anti-IDO antibodies that inhibit IDO activity
  • compounds which directly increase tryptophan e.g., tryptophan and analogs thereof which can be metabolized by T cells.
  • Small molecule IDO inhibitors such as those described above, are preferred.
  • Other small molecule IDO inhibitors include 6-chloro-DL-tryptophan (Saito et al., Neurosci. Lett. 178(2):211-215, 1994), nitric oxide (NO) gas or the NO-generating compound, diethylamine dinitric oxide adduct (Thomas et al., J Biol. Chem.
  • IDO inhibitors that inhibit the induction of IDO by interferon- ⁇ , such as indomethacin, phenylbutazone and aspirin (Sayama et al., Proc. Natl Acad. Sci. USA 78(12):7327-30, 1981) also may be used as IDO inhibitors.
  • Inhibitors of tryptophan 2,3- dioxygenase such as 540C91 [(E)-3-[2-(4'-pyridyl)-vinyl]-lH-indole] (Reinhard et al., Biochem Pharmacol. 51(2):159-63, 1996) and 680C91 [(E)-6-fluoro-3-[2-(3- pyridyl)vinyl]-lH-indole] (Salter et al., Biochem. Pharmacol. 49(10):1435-42, 1995) also may be used as tryptophan enhancing agents.
  • IDO inhibitors are isolated polypeptides which selectively bind IDO and reduce its activity.
  • Isolated binding polypeptides include antibodies and fragments of antibodies (e.g. Fab, F(ab) 2 , Fd and antibody fragments which include a CDR3 region which binds selectively to IDO).
  • Preferred isolated binding polypeptides are those that bind to an epitope that is at or near the catalytic site of IDO.
  • the invention therefore, in some embodiments involves the use of antibodies or fragments of antibodies which have the ability to selectively bind to IDO and stimulate T cell proliferation and/or activity under the conditions disclosed herein.
  • Antibodies include polyclonal and monoclonal antibodies, prepared according to conventional methodology. Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W.R. (1986) 77ze Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford).
  • the pFc' and Fc regions are effectors of the complement cascade but are not involved in antigen binding.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule.
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd
  • CDRs complementarity determining regions
  • FRs framework regions
  • CDR1 through CDR3 complementarity determining regions
  • non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody.
  • This is most clearly manifested in the development and use of "chimeric” or “humanized” antibodies in which non-human variable regions or CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. See, e.g., U.S. patents 4,816,567, 5,225,539, 5,585,089, 5,693,762 and 5,859,205.
  • the present invention also provides for F(ab') 2 , Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab') 2 fragment antibodies in which the FR and/or CDR1 and or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences.
  • the present invention also includes so-called single chain antibodies.
  • the invention involves polypeptides of numerous size and type that bind specifically to IDO and inhibit its functional activity.
  • polypeptides may be derived also from sources other than antibody technology.
  • polypeptide binding agents can be provided by degenerate peptide libraries which can be readily prepared in solution, in immobilized form or as phage display libraries.
  • Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptides and non-peptide synthetic moieties.
  • Other antibodies that bind IDO inducers such as interferon- ⁇ also are useful as IDO inhibitors in accordance with the invention.
  • Still other tryptophan enhancing agents include antisense nucleic acids.
  • Antisense nucleic acids include short oligonucleotides as well as longer nucleic acids.
  • the antisense nucleic acids are complementary to and bind to portions of the IDO coding sequence or 5' nontranslated sequence, thereby inhibiting translation of functional IDO protein.
  • Other antisense nucleic acids which reduce or block IDO transcription are also useful as tryptophan enhancing agents.
  • antisense oligonucleotides that selectively bind to a nucleic acid molecule encoding IDO, to reduce the expression (transcription or translation) of IDO.
  • antisense oligonucleotide describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide.
  • modified oligoribonucleotide or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular IDO gene (e.g., human IDO such as the cDNA sequences referenced in GenBank accession numbers M34455, AH002828, or XI 7668) or to an mRNA transcript of that gene and, thereby, inhibits the transcription of that gene and/or the translation of that mRNA.
  • a particular IDO gene e.g., human IDO such as the cDNA sequences referenced in GenBank accession numbers M34455, AH002828, or XI 7668
  • the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence. It is preferred that the antisense oligonucleotide be constructed and arranged so as to bind selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions. Based upon the sequences of IDO nucleic acids, including allelic or homologous genomic and/or cDNA sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention.
  • antisense oligonucleotides should comprise at least 10 and, more preferably, at least 15 consecutive bases which are complementary to the target, although in certain cases modified oligonucleotides as short as 7 bases in length have been used successfully as antisense oligonucleotides (Wagner et al., Nature Biotechnol 14:840-844, 1996). Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases.
  • oligonucleotides may be chosen which are antisense to any region of the gene or mRNA transcripts, in preferred embodiments the antisense oligonucleotides correspond to N-terminal or 5' upstream sites such as translation initiation, transcription initiation or promoter sites. In addition, 3 '-untranslated regions may be targeted. Targeting to mRNA splicing sites has also been used in the art but may be less preferred if alternative mRNA splicing occurs. In addition, the antisense is targeted, preferably, to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al., Cell Mol Neurobiol.
  • IDO sequences referenced above are cDNA sequences, one of ordinary skill in the art may easily derive the genomic DNA corresponding to those cDNAs.
  • the present invention also provides for antisense oligonucleotides which are complementary to the genomic DNA corresponding to nucleic acids encoding IDO.
  • antisense to allelic or homologous cDNAs and genomic DNAs are enabled without undue experimentation.
  • the antisense oligonucleotides of the invention may be composed of "natural" deoxyribonucleotides, ribonucleotides, or any combination thereof. That is. the 5' end of one native nucleotide and the 3' end of another native nucleotide may be covalently linked, as in natural systems, via a phosphodiester internucleoside linkage.
  • These oligonucleotides may be prepared by art recognized methods which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors.
  • the antisense oligonucleotides of the invention also may include "modified" oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not prevent them from hybridizing to their target but which enhance their stability or targeting or which otherwise enhance their therapeutic effectiveness.
  • modified oligonucleotide as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide.
  • a synthetic internucleoside linkage i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide
  • Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.
  • modified oligonucleotide also encompasses oligonucleotides with a covalently modified base and/or sugar.
  • modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position.
  • modified oligonucleotides may include a 2'-O- alkylated ribose group.
  • modified oligonucleotides may include sugars such as arabinose instead of ribose.
  • the present invention contemplates in vitro use of IDO antisense molecules as well as in vivo pharmaceutical preparations containing modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acids encoding IDO enzyme, together with pharmaceutically acceptable carriers.
  • the antisense nucleic acids of the invention may be produced by expression in cells by expression vectors introduced therein.
  • expression vectors introduced therein.
  • the choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
  • Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al.,
  • cells are genetically engineered by the introduction into the cells of heterologous DNA (RNA) encoding antisense IDO nucleic acid.
  • RNA heterologous DNA
  • the antisense IDO nucleic acid is placed under operable control of transcriptional elements to permit the expression of the antisense IDO nucleic acid in the host cell.
  • Preferred systems for mRNA expression in mammalian cells are those such as pRc/CMV (available from Invitrogen, Carlsbad, CA) that contain a selectable marker such as a gene that confers G418 resistance (which facilitates the selection of stably transfected cell lines) and the human cytomegalovirus (CMV) enhancer-promoter sequences.
  • pCEP4 vector Invitrogen
  • EBV Epstein Barr Virus
  • Another expression vector is the pEF- BOS plasmid containing the promoter of polypeptide Elongation Factor l ⁇ , which stimulates efficiently transcription in vitro.
  • the plasmid is described by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), and its use in transfection experiments is disclosed by, for example, Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996).
  • Additional vectors for delivery of antisense IDO nucleic acid will be known to one of ordinary skill in the art.
  • Various techniques may be employed for introducing antisense IDO nucleic acids into cells in accordance with the invention, depending on whether the nucleic acids are introduced in vitro or in vivo in a host.
  • Such techniques include transfection of nucleic acid-CaPO 4 precipitates, transfection of nucleic acids associated with DEAE, transfection or infection with viruses including the nucleic acid of interest, liposome mediated transfection, and the like.
  • a vehicle used for delivering a nucleic acid of the invention into a cell e.g., a retrovirus, adenovirus or other virus; a liposome
  • a targeting molecule attached thereto.
  • a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be bound to or incorporated within the nucleic acid delivery vehicle.
  • proteins which bind to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation for targeting and/or to facilitate uptake.
  • proteins include capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like.
  • Polymeric delivery systems also have been used successfully to deliver nucleic acids into cells, as is known by those skilled in the art. Such systems even permit oral delivery of nucleic acids. Expansion of T cells can be carried out in a variety of different culture vessels and under different culture conditions.
  • the same culturing conditions that are used for culturing T cells using the prior art methods are used herein, with the exception that one or more IDO inhibitors (and/or tryptophan) are added to the culture.
  • An exemplary protocol for culturing T cells according to the invention is provided in the Examples below.
  • Other standard T cell culture protocols which differ in components or conditions (e.g., serum-free culture, addition of different cytokines, growth factors or nutrients, etc.) also can be modified in accordance with the invention.
  • T cells in addition to T cells can benefit from being grown in the presence of tryptophan enhancing agents, e.g. in growth medium.
  • Most growth media contain fixed amounts of tryptophan as an essential amino acid for cell metabolism.
  • T cells have an unexpected requirement for additional tryptophan for increased proliferation, which can be provided in growth medium by adding tryptophan to the medium or, as shown herein, by inhibiting enzymes that catabolize tryptophan.
  • the proliferation of many kinds of cells that cannot synthesize tryptophan will benefit from the inclusion of tryptophan enhancing agents in growth media in addition to the standard nutrients which are well known in the art.
  • Increasing the proliferation of cells in vitro means increasing the number of cells by at least about 10% relative to the number of cells that are present in a parallel control population of cells that are subjected to the same conditions as the IDO inhibitor-treated population with the exception that such control population is not contacted with the IDO inhibitors.
  • the number of cells are increased at least about 50%, more preferably 2-fold, yet more preferably 4-fold, still more preferably 10-fold and, most preferably, at least 20-fold relative to the number of cells that are present in the parallel control population. It will be seen from the Examples that the increase in T cell numbers cultured according to the invention can be even greater (see, e.g., Figure 2B, growth curve of CTL-361A/17).
  • Increasing the proliferation of cells in vivo similarly means increasing the number of a particular kind of cells in the body, by similar amounts as above.
  • cytolytic T cells that recognize a cancer associated antigen can be administered to a subject for the treatment of cancer cells that express the cancer associated antigen.
  • the in vivo proliferation of the cytolytic T cells can be increased by the coadministration of a tryptophan enhancing agent.
  • the proliferation of T cells in vivo can be determined, for example, by measuring T cells in the subject's peripheral blood. Additional methods to measuring T cell proliferation in vivo will be known to one of ordinary skill in the art.
  • tryptophan enhancing agents to enhance T cell culture also can increase the effector properties of the T cells, e.g., cytolytic activity, at least for some T cell clones cultured in the presence of one or more tryptophan enhancing agents.
  • cytolytic activity As is shown in the Examples and depicted in Figs. 4A, 4B and 4C, the specific lysis of target tumor cells by certain cytolytic T cell clones was increased after culture in the presence of 1-methyl-DL- tryptophan.
  • the invention includes methods for increasing activity of T cells by culturing the T cells in the presence of an effective amount of tryptophan enhancing agents to increase the activity of the T cells. In these methods, T cell activity is increased at least about 10%, preferably at least about 25%, more preferably at least about 50%, and most preferably at least about 100%.
  • the time period in which the number of T cells are increased can be adjusted according to the needs of the person culturing the T cells, and can be, at least in part, a function of the cell type (e.g., T cell clone) and the specific culture conditions used (growth medium, serum, cytokines, culture vessel, etc.) and/or the desired outcome (e.g., increased proliferation, increased activity, etc.). In general, this time period ranges from about 7 days (for short term expansion of T cells) to several weeks. Routine procedures known to those of ordinary skill in the art can be used to determine the number of cells in culture as a function of increasing incubation time of the cultured cells with the IDO inhibitor (and/or tryptophan).
  • the IDO inhibitor and/or tryptophan
  • expansion of the T cells in culture is measured by counting the cell numbers according to standard methods, for example, determining the actual cell numbers using a hematocytometer or cell counter or measuring incorporation of a specific dye.
  • T cells also can be labeled using specific labeled antibodies and counted using an automated devices such as a fluorescence activated cell sorter (FACS).
  • FACS fluorescence activated cell sorter
  • Such routine experimentation involves, for example, (i) varying the amount of a tryptophan enhancing agent at constant incubation time; (ii) varying the incubation time at constant amounts of tryptophan enhancing agent; (iii) applying the foregoing optimization experiments to determine the particular conditions necessary to achieve a pre-selected fold increase in T cell number; and (iv) varying other factors including, for example, the identity or the state of the tryptophan enhancing agent (e.g., soluble or immobilized), to optimize the culture conditions to achieve the desired results. Similar routine experimental studies can be carried out using animal models for optimization of in vivo use of tryptophan enhancing agents.
  • T cells that are grown in accordance with the invention can be used in a variety of in vitro and in vivo applications. For example, generating larger numbers of T cells will find application in the field of drug testing and for in vitro study of T cell biology.
  • T cells cultured according to the invention also can be used for therapeutic purposes in vivo.
  • T cells isolated from a subject can be cultured in vitro for expansion and eventual return to the subject.
  • Some therapeutic approaches using T cells are premised on a response by a subject's immune system, leading to lysis of antigen presenting cells, such as cancer cells which present one or more cancer associated antigens.
  • CTLs autologous cytolytic T lymphocytes
  • HLA major histocompatibility complex
  • HLA human leukocyte antigen
  • CTL clones Specific production of CTL clones is well known in the art.
  • An example of a method for T cell differentiation is presented in International Application number PCT/US96/05607.
  • a sample of cells taken from a subject such as blood cells, are contacted with a cell presenting the complex and capable of provoking CTLs to proliferate (e.g., cancer cells, dendritic cells).
  • the target cell can be a transfectant, such as a COS cell transfected with nucleic acids encoding an antigen and a HLA molecule capable of presenting the antigen. These transfectants present the desired HLA/antigen complex on their surface and, when combined with a CTL of interest, stimulate its proliferation.
  • COS cells are widely available, as are other suitable host cells.
  • the clonally expanded autologous CTLs then are administered to the subject.
  • Other methods for selecting antigen-specific CTL clones include the use of fluorogenic tetramers of MHC class I molecule/peptide complexes which are used to detect specific CTL clones (Altman et al., Science 274:94-96, 1996; Dunbar et al., Curr. Biol 8:413-416, 1998).
  • T cell clones that are specific for antigens expressed on cells infected by a pathogen also can be prepared and administered as described above.
  • Adoptive transfer or other methods premised on in vitro expansion of T cells are not the only forms of therapy that is available in accordance with the invention.
  • CTLs can also be provoked in vivo, using a number of approaches.
  • One approach is the use of non-proliferative cells expressing the complex.
  • T cells expanded according to the invention can be used to "immunize” subjects or as “vaccines".
  • immunize or “vaccination” means increasing or activating an immune response against an antigen. It does not require elimination or eradication of a condition but rather contemplates the clinically favorable enhancement of an immune response toward an antigen.
  • Generally accepted animal models can be used for testing of immunization against cancer using a T cells expanded according to the invention. For example, human cancer cells can be introduced into a mouse to create a tumor, and T cells that were expanded in cultures including tryptophan enhancing agents by the methods described herein can be administered to the mouse.
  • the effect on the cancer cells can be assessed as a measure of the effectiveness of the cancer associated antigen nucleic acid immunization.
  • Methods for increasing an immune response with T cells including formulation of a T cell composition and selection of doses, route of administration and the schedule of administration are well known in the art.
  • the tests also can be performed in humans, where the end point can be to test for the presence of enhanced levels of circulating CTLs against cells bearing the antigen.
  • an immune response modulation composition is a composition administered to a subject to increase an immune response mediated by T cells.
  • the immune response modulation composition can include T cells, antigens (e.g., peptides, proteins), nucleic acids encoding antigens, etc. which stimulate an immune response.
  • the T cell mediated immune response induced or increased by any of these immune response modulation composition will be favorably modulated by inclusion of one or more tryptophan enhancing agents as part of the immune response modulation composition.
  • Adjuvants also can be added to the immune response modulation compositions.
  • Many kinds of adjuvants are well known in the art. Specific examples of adjuvants include monophosphoryl lipid A (MPL. SmithKline Beecham), a congener obtained after purification and acid hydrolysis of Salmonella minnesota Re 595 lipopolysaccharide; saponins including QS21 (SmithKline Beecham), a pure QA-21 saponin purified from Quillja saponaria extract; DQS21, described in PCT application WO96/33739 (SmithKline Beecham); combinations of DQS21/MPL mixed in ratios of about l :10 to 10: 1; QS-7, QS-17, QS-18, and QS-L1 (So et al., Mol.
  • MPL. SmithKline Beecham monophosphoryl lipid A
  • saponins including QS21 (SmithKline Beecham), a pure QA-21 saponin purified from Quillja saponaria extract
  • cytokines are also useful in vaccination protocols as a result of their lymphocyte regulatory properties.
  • cytokines useful for such purposes will be known to one of ordinary skill in the art, including interleukin-12 (IL-12) which has been shown to enhance the protective effects of vaccines (see, e.g., Science 268: 1432-1434, 1995), GM-CSF and IL- 18.
  • IL-12 interleukin-12
  • GM-CSF GM-CSF
  • IL- 18 IL-12
  • cytokines can be administered in conjunction with T cells and adjuvants to increase the immune response to the antigens.
  • costimulatory molecules provided in either protein or nucleic acid form.
  • costimulatory molecules include the B7-1 and B7-2 (CD80 and CD86 respectively) molecules which are expressed on dendritic cells (DC) and interact with the CD28 molecule expressed on the T cell.
  • Another costimulatory molecule is the ICOS protein. These interactions provide costimulation (signal 2) to an antigen/MHC/TCR (T cell receptor) stimulated (signal 1) T cell, increasing T cell proliferation and effector function.
  • certain cancers unexpectedly have been found to constitutively express indoleamine 2,3-dioxygenase. These cancer cells would be expected to reduce the local concentration of tryptophan and disable T cell mediated immune responses to the cancer.
  • the recognition of this unexpected property of cancer cells permits treatment of the cells, which may have evaded or have the potential to evade T cell-mediated cytolysis, to increase immune recognition and destruction of the cancer cells.
  • the methods include administering to a subject in need of such treatment an amount of a tryptophan enhancing agent effective to increase T cell-mediated cytolysis of the cancer cell.
  • Other methods include contacting the cancer cells with an amount of a tryptophan enhancing agent effective to increase T cell-mediated cytolysis of the cancer cell.
  • Preferred tryptophan enhancing agents are described elsewhere herein.
  • cancer cells can constitutively express indoleamine 2,3- dioxygenase also permits one of ordinary skill in the art to determine a condition characterized by the ability of cancer cells to resist or evade T cell-mediated cytolysis. For example, one can monitor a sample of cancer cells from a patient who has or is suspected of having the condition for constitutive expression of indoleamine 2,3-dioxygenase, as a determination of the condition. Once it is established that the subject has a cancer that expresses indoleamine 2,3-dioxygenase, the skilled artisan can determine whether to treat the cancer patient with an inhibitor of indoleamine 2,3-dioxygenase.
  • expression of indoleamine 2,3-dioxygenase by the cancer cells indicates that the patient is a good candidate to be treated with an inhibitor of indoleamine 2,3-dioxygenase to increase the susceptibility of the cancer cells to T cell attack.
  • expression of indoleamine 2,3-dioxygenase by the cancer cells of the patient can be monitored by any method, including measuring the amount of IDO protein or nucleic acid encoding the protein that is expressed by the cancer cells.
  • IDO protein can be measured directly, such as by standard immunoassays, or indirectly, such as by measuring IDO enzymatic activity in accordance with known methods.
  • IDO nucleic acid can be measured by nucleic acid hybridization or amplification, such as PCR.
  • the compositions of the present invention can be administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.
  • the therapeutics of the invention such as tryptophan enhancing agents and/or T cells expanded in vitro in the presence of tryptophan enhancing agents can be administered in vivo by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal.
  • antibodies are used therapeutically (e.g., antibodies that bind IDO)
  • a preferred route of administration is by pulmonary aerosol.
  • compositions of the invention are added to cell cultures in effective amounts or administered to a subject in effective amounts.
  • An "effective amount” is that amount of a tryptophan enhancing agent composition that alone, or together with further doses, produces the desired response, e.g. increases proliferation and/or activity of T cells. This can be monitored by routine methods known to one of ordinary skill in the art.
  • the amount added to a cell culture in vitro may depend on the particular T cell clone and/or cell culture conditions used.
  • the amounts administered in vivo may depend, of course, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner.
  • a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.
  • compositions used in the foregoing methods preferably are sterile and contain an effective amount of one or more tryptophan enhancing agents for producing the desired response in a unit of weight or volume suitable for addition to a cell culture or administration to a subject.
  • a subject is a human or non-human veterinary animal, including non-human primates, mice, rats, cows, pigs, horses, sheep, goats, dogs, cats, etc.
  • the subject is a human.
  • tryptophan enhancing agents When added to cell cultures, tryptophan enhancing agents are sterile and formulated for compatibility with cells, cell culture media and other cell culture components. Sterilization may be carried out by filtration, irradiation or any other method compatible with cell culture components.
  • the pharmaceutical preparations of the invention When administered to a subject, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutical ly-acceptable compositions.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.
  • the salts When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like.
  • pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
  • a tryptophan enhancing agent composition may be combined, if desired, with a pharmaceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • the pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
  • suitable buffering agents including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
  • the pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
  • suitable preservatives such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
  • the pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or di-glycerides.
  • Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
  • Example 1 Expression of IDO in tumor cells.
  • IDO Indoleamine 2,3-dioxygenase
  • IDO expression in a variety of mouse and human tumor cell lines was tested by RT- PCR in the absence of IFN ⁇ to avoid induction of IDO expression by IFN ⁇ .
  • the tumor cell lines that were tested were tumor lines growing in vitro; the cells were not in contact with IFN ⁇ .
  • the PCR reactions for mouse IDO expression were performed as described by Munn et al. (Science 281 :1 191-1 193, 1998).
  • PCR amplification primers were selected based on the human IDO nucleotide sequence (GenBank accession number M34455).
  • the sense primer was 5'-ggt cat gga gat gtc cgt aa-3' (SEQ ID NO: 1).
  • the antisense primer was 5'-ctt tea cac agg cgt cat aa-3' (SEQ ID NO:2).
  • the PCR was run as follows: 5 min at 94°C; 30 cycles of 1 min at 94°C, 2 min at 59°C and 3 min at 72°C; and a final elongation step of 15 min at 72°C.
  • the size of the PCR product was 690 bp.
  • expression of IDO was determined by PCR in placenta and in human tumor cells treated with IFN ⁇ . High levels of expression were found in placenta and in induced tumor cells as expected according to previously published results. Unexpectedly, two out of twelve mouse tumors were positive (one very strongly).
  • IDO inhibitors could be used in vivo in patients bearing tumors that express IDO constitutively. This possibility was tested in mice bearing tumors and the results are presented below in Example 4. These patients are more likely to benefit from IDO inhibition in the framework of cancer immunotherapy, as their tumors, by expressing IDO constitutively, are capable of downregulating the tumoricidal effects of T cells which recognize the tumor. Therefore IDO inhibitors can be useful as a therapeutic agent for increasing the effectiveness of immunotherapy.
  • Example 2 Effect of IDO inhibitor on T cell proliferation and activity in vitro.
  • IDO indoleamine 2,3-dioxygenase
  • 1-methyl-DL-tryptophan purchased from Sigma Chemical Co., St. Louis, MO
  • MZ2-CTL-82/30 which recognizes a MAGE-1 peptide presented by HLA-A1 (Traversari, J. Exp.
  • CTL 361A/17 which recognizes a RAGE-1 peptide presented by HLA-B7 (Gaugler et al., Immunogenetics 44(5):323-30,1996)
  • CTL 361A/21 which recognizes a RUR-1 peptide presented by HLA-B7 (U.S. serial number 60/085,318).
  • a 4mM stock solution of 1 -methyl-tryptophan was prepared in PBS containing 5mM HC1 and was sterilized by irradiation.
  • the autologous tumor cells were melanoma cells MZ2-MEL.43 in the case of MZ2-CTL-82/30, and renal carcinoma cells LE9211-RCC in the case of CTL 361 A/17 and 361 A/21. Every week, the CTL were harvested, washed and restimulated in the same conditions with or without fresh inhibitor. CTL were counted weekly at the time of restimulation, and the cumulative growth curves calculated for each CTL clone are shown in Figs. 1, 2 and 3.
  • the test was performed after 4 weeks of culture with or without the inhibitor for CTL 82/30, and after 6 weeks of culture for CTL 361A/17 and 361 A/21.
  • the data showed that the CTL treated with 1-methyl-tryptophan were still active and specific.
  • the lytic activity of treated CTLs 82/30 and 361 A/17 was even higher than that of the untreated ones.
  • 1-methyl-tryptophan can be used to increase the proliferation of T cells in vitro without any loss of activity or specificity, and for certain clones improves the activity (see Figs. 4A, 4B and 4C).
  • CTL 82/30, MZ2-MEL.43 and LG2-EBV were tested for IDO expression by PCR and all were negative.
  • LE-9211-RCC was also tested and is positive (although not as strong as placenta).
  • the tumor cells quickly disappear in the culture since they are lysed by the CTL. Therefore there are possibly IDO expressing cells in the culture at the beginning, but these are removed by lysis and therefore cannot account for the effect of the IDO inhibitor that was observed.
  • IDO inhibitors such as 1-methyl-tryptophan can have effects in the absence of cells that constitutively express high levels of IDO, such as trophoblasts or macrophages, and outside of the placenta.
  • IDO inhibitors could also be used in vivo as an adjuvant for improving T cell responses against antigens, including cancer associated antigens or antigens expressed on cells infected by a pathogen.
  • IDO Indoleamine 2, 3-dioxygenase
  • PCR primers used for amplification were, as above, 5'-ggtcatggagatgtccgtaa-3' (sense; SEQ ID NO:l) and 5'-ctttcacacaggcgtcataa-3' (antisense;
  • the DNA was denatured at 94°C for 5 min followed by 30 cycles of 94°C (1 min), 59°C (2 min), 72°C (3 min).
  • the expected size for the amplified product was 690 bp.
  • Table 1 summarizes the results of the amplifications, showing 29% (77/268) of all tumors tested constitutively express IDO. In some tumor groups the constitutive expression of IDO was found in 80% of the tumors tested.
  • Example 4 Treatment with IDO inhibitor retards in vivo growth of an IDO expressing murine tumor
  • T cell responses may be inhibited in vivo by tumors that express IDO
  • the effect on tumor growth by treating tumor-laden mice with the IDO inhibitor, 1-methyl- tryptophan was investigated.
  • mice Three groups of 20 female Balb/c mice were injected subcutaneously in the left flank with either 100,000, 200,000 or 500,000 live cells of tumor line WEHI 3B which expresses high levels of the T cell inhibitor, IDO.
  • 10 mice from each of these groups underwent therapeutic treatment with an intraperitoneal injection of 10 mg of 1 -methyl-tryptophan.
  • the remaining 10 mice from each of these groups were treated with phosphate buffered saline (PBS) as a control.
  • PBS phosphate buffered saline
  • Fig. 5 summarizes the tumor size and growth rate over a period of 30 days for the three groups of animals treated either with 1-methyl-tryptophan or PBS.
  • the data indicate that treatment with 1-methyl-tryptophan in the group of mice injected with 100,000 tumor cells retarded the development of the tumor compared to the rate of growth of tumors in mice treated with PBS.

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Abstract

L'invention concerne des procédés et des compositions permettant d'accroître la prolifération de lymphocytes T au moyen d'agents facilitants à base de tryptophane. On accroît la prolifération de lymphocytes T in vitro en ajoutant des agents facilitants à base de tryptophane à une culture de lymphocytes T, ou in vivo par l'administration d'agents facilitants à base de tryptophane. L'invention concerne aussi des procédés utiles pour diagnostiquer et traiter des affections caractérisées par l'expression constitutive d'indoleamine-2,3-dioxygénase. L'invention concerne également des compositions et un dispositif associés aux procédés.
PCT/US2000/012118 1999-05-03 2000-05-03 Procédés visant à accroître la prolifération de lymphocytes t WO2000066764A1 (fr)

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