EP1697502A2 - Methods for the identification and preparation of regulator/suppressor t lymphocytes, compositions and uses thereof - Google Patents
Methods for the identification and preparation of regulator/suppressor t lymphocytes, compositions and uses thereofInfo
- Publication number
- EP1697502A2 EP1697502A2 EP04816487A EP04816487A EP1697502A2 EP 1697502 A2 EP1697502 A2 EP 1697502A2 EP 04816487 A EP04816487 A EP 04816487A EP 04816487 A EP04816487 A EP 04816487A EP 1697502 A2 EP1697502 A2 EP 1697502A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- cells
- lymphocytes
- thy
- suppressor
- pts
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 210000001744 T-lymphocyte Anatomy 0.000 title claims abstract description 255
- 238000000034 method Methods 0.000 title claims abstract description 63
- 239000000203 mixture Substances 0.000 title claims abstract description 56
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 210000004027 cell Anatomy 0.000 claims abstract description 298
- 210000004698 lymphocyte Anatomy 0.000 claims abstract description 68
- 239000002243 precursor Substances 0.000 claims abstract description 41
- 238000011282 treatment Methods 0.000 claims abstract description 34
- 230000000694 effects Effects 0.000 claims abstract description 31
- 210000000056 organ Anatomy 0.000 claims abstract description 28
- 238000001727 in vivo Methods 0.000 claims abstract description 20
- 230000001225 therapeutic effect Effects 0.000 claims abstract description 20
- 210000001519 tissue Anatomy 0.000 claims abstract description 19
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 17
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 15
- 201000010099 disease Diseases 0.000 claims abstract description 15
- 208000023275 Autoimmune disease Diseases 0.000 claims abstract description 14
- 238000002054 transplantation Methods 0.000 claims abstract description 11
- 206010020751 Hypersensitivity Diseases 0.000 claims abstract description 9
- 230000007815 allergy Effects 0.000 claims abstract description 9
- 230000003612 virological effect Effects 0.000 claims abstract description 9
- 241000124008 Mammalia Species 0.000 claims abstract description 7
- 208000009329 Graft vs Host Disease Diseases 0.000 claims abstract description 6
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 claims abstract description 6
- 208000024908 graft versus host disease Diseases 0.000 claims abstract description 6
- 210000000130 stem cell Anatomy 0.000 claims abstract description 5
- 208000035143 Bacterial infection Diseases 0.000 claims abstract description 4
- 208000036142 Viral infection Diseases 0.000 claims abstract description 4
- 208000022362 bacterial infectious disease Diseases 0.000 claims abstract description 4
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 claims description 123
- 239000003446 ligand Substances 0.000 claims description 62
- 101150052863 THY1 gene Proteins 0.000 claims description 61
- 239000003550 marker Substances 0.000 claims description 50
- 108010031650 Thy-1 Antigens Proteins 0.000 claims description 37
- 239000000427 antigen Substances 0.000 claims description 25
- 108091007433 antigens Proteins 0.000 claims description 24
- 102000036639 antigens Human genes 0.000 claims description 24
- 230000003321 amplification Effects 0.000 claims description 15
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 15
- 238000000684 flow cytometry Methods 0.000 claims description 14
- 238000000338 in vitro Methods 0.000 claims description 13
- 239000012634 fragment Substances 0.000 claims description 12
- 239000002671 adjuvant Substances 0.000 claims description 11
- 230000005291 magnetic effect Effects 0.000 claims description 11
- 238000011084 recovery Methods 0.000 claims description 10
- 239000012472 biological sample Substances 0.000 claims description 9
- 239000008194 pharmaceutical composition Substances 0.000 claims description 7
- 238000000926 separation method Methods 0.000 claims description 7
- 210000004369 blood Anatomy 0.000 claims description 6
- 239000008280 blood Substances 0.000 claims description 6
- 230000003750 conditioning effect Effects 0.000 claims description 6
- 210000004700 fetal blood Anatomy 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 5
- 239000000523 sample Substances 0.000 claims description 5
- 210000001541 thymus gland Anatomy 0.000 claims description 5
- 230000000890 antigenic effect Effects 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 210000001185 bone marrow Anatomy 0.000 claims description 4
- 238000004113 cell culture Methods 0.000 claims description 4
- 238000002955 isolation Methods 0.000 claims description 4
- 239000013603 viral vector Substances 0.000 claims description 4
- 238000001042 affinity chromatography Methods 0.000 claims description 3
- 208000026935 allergic disease Diseases 0.000 claims description 3
- 230000000779 depleting effect Effects 0.000 claims description 3
- 230000002757 inflammatory effect Effects 0.000 claims description 3
- 210000004185 liver Anatomy 0.000 claims description 3
- 230000002285 radioactive effect Effects 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 108091023037 Aptamer Proteins 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 230000002255 enzymatic effect Effects 0.000 claims description 2
- 238000002372 labelling Methods 0.000 claims description 2
- 230000002093 peripheral effect Effects 0.000 claims description 2
- 210000000952 spleen Anatomy 0.000 claims description 2
- 238000002560 therapeutic procedure Methods 0.000 claims description 2
- 238000002826 magnetic-activated cell sorting Methods 0.000 claims 2
- KZMAWJRXKGLWGS-UHFFFAOYSA-N 2-chloro-n-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-n-(3-methoxypropyl)acetamide Chemical compound S1C(N(C(=O)CCl)CCCOC)=NC(C=2C=CC(OC)=CC=2)=C1 KZMAWJRXKGLWGS-UHFFFAOYSA-N 0.000 claims 1
- 239000004744 fabric Substances 0.000 claims 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 claims 1
- 230000024664 tolerance induction Effects 0.000 claims 1
- 229960005486 vaccine Drugs 0.000 claims 1
- 239000012636 effector Substances 0.000 abstract description 11
- 208000015181 infectious disease Diseases 0.000 abstract description 8
- 208000027866 inflammatory disease Diseases 0.000 abstract description 6
- 230000002159 abnormal effect Effects 0.000 abstract description 5
- 208000032839 leukemia Diseases 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 230000001575 pathological effect Effects 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 3
- 230000006698 induction Effects 0.000 abstract description 3
- 208000030852 Parasitic disease Diseases 0.000 abstract description 2
- 230000001580 bacterial effect Effects 0.000 abstract description 2
- 238000002659 cell therapy Methods 0.000 abstract description 2
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 93
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 91
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 91
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 88
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 description 40
- 108090000623 proteins and genes Proteins 0.000 description 25
- 230000007170 pathology Effects 0.000 description 21
- 102000039446 nucleic acids Human genes 0.000 description 19
- 108020004707 nucleic acids Proteins 0.000 description 19
- 150000007523 nucleic acids Chemical class 0.000 description 19
- 210000003162 effector t lymphocyte Anatomy 0.000 description 18
- 230000014509 gene expression Effects 0.000 description 18
- 241000700605 Viruses Species 0.000 description 17
- 230000006870 function Effects 0.000 description 17
- 210000004443 dendritic cell Anatomy 0.000 description 15
- 230000035755 proliferation Effects 0.000 description 14
- 239000002609 medium Substances 0.000 description 13
- 108090000765 processed proteins & peptides Proteins 0.000 description 13
- 102000000588 Interleukin-2 Human genes 0.000 description 12
- 108010002350 Interleukin-2 Proteins 0.000 description 12
- 102000004196 processed proteins & peptides Human genes 0.000 description 12
- 102000004127 Cytokines Human genes 0.000 description 11
- 108090000695 Cytokines Proteins 0.000 description 11
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 11
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 11
- 230000028993 immune response Effects 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 210000004962 mammalian cell Anatomy 0.000 description 10
- 235000018102 proteins Nutrition 0.000 description 10
- 230000004913 activation Effects 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 8
- 238000011161 development Methods 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- 239000011324 bead Substances 0.000 description 7
- 229920001184 polypeptide Polymers 0.000 description 7
- 102000003814 Interleukin-10 Human genes 0.000 description 6
- 108090000174 Interleukin-10 Proteins 0.000 description 6
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 6
- 229960004308 acetylcysteine Drugs 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 6
- 230000005764 inhibitory process Effects 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 230000002588 toxic effect Effects 0.000 description 6
- 241000713813 Gibbon ape leukemia virus Species 0.000 description 5
- 241000282412 Homo Species 0.000 description 5
- -1 IL 10 Proteins 0.000 description 5
- 102000003812 Interleukin-15 Human genes 0.000 description 5
- 108090000172 Interleukin-15 Proteins 0.000 description 5
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 5
- 230000000735 allogeneic effect Effects 0.000 description 5
- 230000004069 differentiation Effects 0.000 description 5
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 5
- 230000001506 immunosuppresive effect Effects 0.000 description 5
- 238000012544 monitoring process Methods 0.000 description 5
- 201000006417 multiple sclerosis Diseases 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 231100000331 toxic Toxicity 0.000 description 5
- 231100000419 toxicity Toxicity 0.000 description 5
- 230000001988 toxicity Effects 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 4
- 101000924577 Homo sapiens Adenomatous polyposis coli protein Proteins 0.000 description 4
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 description 4
- 102000000704 Interleukin-7 Human genes 0.000 description 4
- 108010002586 Interleukin-7 Proteins 0.000 description 4
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 4
- 239000013566 allergen Substances 0.000 description 4
- 210000000612 antigen-presenting cell Anatomy 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 238000012239 gene modification Methods 0.000 description 4
- 230000005017 genetic modification Effects 0.000 description 4
- 235000013617 genetically modified food Nutrition 0.000 description 4
- 210000000987 immune system Anatomy 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 238000002255 vaccination Methods 0.000 description 4
- IQFYYKKMVGJFEH-OFKYTIFKSA-N 1-[(2r,4s,5r)-4-hydroxy-5-(tritiooxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound C1[C@H](O)[C@@H](CO[3H])O[C@H]1N1C(=O)NC(=O)C(C)=C1 IQFYYKKMVGJFEH-OFKYTIFKSA-N 0.000 description 3
- 201000001320 Atherosclerosis Diseases 0.000 description 3
- 229920001917 Ficoll Polymers 0.000 description 3
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 3
- 101100005713 Homo sapiens CD4 gene Proteins 0.000 description 3
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 3
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- 108010032774 Interleukin-2 Receptor alpha Subunit Proteins 0.000 description 3
- 102000006601 Thymidine Kinase Human genes 0.000 description 3
- 108020004440 Thymidine kinase Proteins 0.000 description 3
- 108091023040 Transcription factor Proteins 0.000 description 3
- 102000040945 Transcription factor Human genes 0.000 description 3
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 3
- 108700019146 Transgenes Proteins 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 230000001684 chronic effect Effects 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 230000006735 deficit Effects 0.000 description 3
- 230000002939 deleterious effect Effects 0.000 description 3
- 206010012601 diabetes mellitus Diseases 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 208000006454 hepatitis Diseases 0.000 description 3
- 210000004408 hybridoma Anatomy 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 238000010348 incorporation Methods 0.000 description 3
- 239000012678 infectious agent Substances 0.000 description 3
- 229940100994 interleukin-7 Drugs 0.000 description 3
- 238000001990 intravenous administration Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 210000005087 mononuclear cell Anatomy 0.000 description 3
- 210000005259 peripheral blood Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 206010039073 rheumatoid arthritis Diseases 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 208000011580 syndromic disease Diseases 0.000 description 3
- 239000003053 toxin Substances 0.000 description 3
- 231100000765 toxin Toxicity 0.000 description 3
- 108700012359 toxins Proteins 0.000 description 3
- 208000035473 Communicable disease Diseases 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- 241000713666 Lentivirus Species 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 108010026552 Proteome Proteins 0.000 description 2
- 239000012979 RPMI medium Substances 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 108091008874 T cell receptors Proteins 0.000 description 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 2
- 206010052779 Transplant rejections Diseases 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 230000000961 alloantigen Effects 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000002269 analeptic agent Substances 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 230000006023 anti-tumor response Effects 0.000 description 2
- 230000001363 autoimmune Effects 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 238000010322 bone marrow transplantation Methods 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000004163 cytometry Methods 0.000 description 2
- 230000000994 depressogenic effect Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 230000008030 elimination Effects 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 230000008029 eradication Effects 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000003394 haemopoietic effect Effects 0.000 description 2
- 231100000283 hepatitis Toxicity 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 208000026278 immune system disease Diseases 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 210000004153 islets of langerhan Anatomy 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 238000004091 panning Methods 0.000 description 2
- 208000005987 polymyositis Diseases 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 210000001179 synovial fluid Anatomy 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- 238000010600 3H thymidine incorporation assay Methods 0.000 description 1
- 241000272522 Anas Species 0.000 description 1
- 102000006306 Antigen Receptors Human genes 0.000 description 1
- 108010083359 Antigen Receptors Proteins 0.000 description 1
- 108091008875 B cell receptors Proteins 0.000 description 1
- 108010035053 B7-1 Antigen Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102100037084 C4b-binding protein alpha chain Human genes 0.000 description 1
- 101710159767 C4b-binding protein alpha chain Proteins 0.000 description 1
- 108010063916 CD40 Antigens Proteins 0.000 description 1
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 1
- 229940045513 CTLA4 antagonist Drugs 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- 102000000311 Cytosine Deaminase Human genes 0.000 description 1
- 108010080611 Cytosine Deaminase Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 208000005176 Hepatitis C Diseases 0.000 description 1
- 101001018097 Homo sapiens L-selectin Proteins 0.000 description 1
- 101000933607 Homo sapiens Protein BTG3 Proteins 0.000 description 1
- 101001072237 Homo sapiens Protocadherin-16 Proteins 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 102000007351 Interleukin-2 Receptor alpha Subunit Human genes 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 102100033467 L-selectin Human genes 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- 108010074338 Lymphokines Proteins 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 102100035384 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex assembly factor 2 Human genes 0.000 description 1
- 101710098657 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex assembly factor 2 Proteins 0.000 description 1
- 108091061960 Naked DNA Proteins 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010004729 Phycoerythrin Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 235000004443 Ricinus communis Nutrition 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000713675 Spumavirus Species 0.000 description 1
- 101150057615 Syn gene Proteins 0.000 description 1
- 238000002679 ablation Methods 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 208000037919 acquired disease Diseases 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 230000003172 anti-dna Effects 0.000 description 1
- 210000003293 antilymphocyte serum Anatomy 0.000 description 1
- 230000008106 antitumoral immune reaction Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 210000004507 artificial chromosome Anatomy 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000006472 autoimmune response Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 229940112129 campath Drugs 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 201000003278 cryoglobulinemia Diseases 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 210000001808 exosome Anatomy 0.000 description 1
- 210000000943 fetal thymocyte Anatomy 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000008348 humoral response Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000002134 immunopathologic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229940076144 interleukin-10 Drugs 0.000 description 1
- 210000003535 interstitial dendritic cell Anatomy 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000007885 magnetic separation Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000011325 microbead Substances 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 230000001400 myeloablative effect Effects 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 210000004967 non-hematopoietic stem cell Anatomy 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000005298 paramagnetic effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 230000003836 peripheral circulation Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 125000002467 phosphate group Chemical class [H]OP(=O)(O[H])O[*] 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102100029526 rRNA 2'-O-methyltransferase fibrillarin Human genes 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 239000000021 stimulant Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 1
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- 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
- A61K39/462—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
- A61K39/464—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
- A61K39/464402—Receptors, cell surface antigens or cell surface determinants
-
- 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
- A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
Definitions
- the present invention relates to the fields of biology, genetics and medicine.
- the invention describes methods and compositions for the identification, production and ex vivo and in vivo manipulation of suppressor T cells or lymphocytes (Ts, including their pTs precursors), also called regulatory T (or Treg), and use of said suppressor lymphocytes to control various pathological conditions, including diseases associated with abnormal activity of effector lymphocytes and / or regulatory / suppressor T cells.
- Ts suppressor T cells or lymphocytes
- Treg regulatory T
- the invention relates to the preparation of such compositions based on Ts and pTs lymphocytes, and their use in the context of cellular and / or gene therapies.
- compositions or cell populations based on Ts and pTs lymphocytes obtained in the context of the invention are in particular suitable for the treatment of genetic or acquired diseases, in particular tumors, autoimmune diseases, allergies, graft disease against the host, transplant against infection (GNI) or transplant against leukemia (GNL) effects, inflammatory diseases including, for example, atherosclerosis, diabetes, viral, bacterial or parasitic infections, immune reconstitution or induction of tolerance in the event of transplantation of stem cells, tissues or organ in a mammal.
- diseases including, for example, atherosclerosis, diabetes, viral, bacterial or parasitic infections, immune reconstitution or induction of tolerance in the event of transplantation of stem cells, tissues or organ in a mammal.
- Ts cells express a T receptor specific for the antigen, like other T lymphocytes, but their overall action is partly non-specific with the possibility of recruiting other additional suppressor T lymphocytes by a phenomenon called "infectious suppression".
- CD4 + / CD25 + regulatory cell population which represents less than 5% of CD4 + T cells, has also been described.
- Ts cells play a major role in the control of autoimmune diseases such as type I diabetes or graft versus host disease (GNH) induced by allogenic T cells.
- Addition of Ts cells to grafts containing allogeneic hematopoietic stem cells and effector T cells can control the onset or emergence of GNH.
- Injection of Ts cells can reduce the autoimmune response in autoimmune polymyositis (unpublished).
- Ts cells also play a major role in establishing or inducing tolerance during the transplantation of tissues or organs and / or in the presence of immunogenic molecules such as transgenes.
- Ts cells also play an important role in modulating the response to infectious agents, including intracellular bacteria and viruses.
- Ts cells play a role in several inflammatory diseases such as atherosclerosis. In this case, the absence or reduction of the number of Ts cells leads to an acceleration of the development of the disease and an increase in its severity (unpublished results).
- Ts cells prevent the development of effector anti-tumor responses, which can otherwise lead to the eradication of tumors.
- depletion of Ts cells leads in many cancer models to the eradication of tumors by an effector immune response.
- Ts are associated with reduced survival.
- the drug modulation of Ts improves treatments based on Tumor Infiltrating Lymphocytes.
- Ts cells are also important during vaccination since they can suppress the development of a specific immune response. Likewise, depletion or reduction in the number of Ts cells very significantly improves the effects of an anti-cancer vaccination.
- Ts cells The characterization of Ts cells is therefore of major importance. Although few elements are known concerning the homeostasis and the regulation of this population of Ts lymphocytes, it appears that the transcription factor Foxp3 plays an important role in the development and the functioning of suppressor T lymphocytes CD4 + / CD25 +. It is not established that Foxp3 is expressed on all Ts cells but the absence of expression of Foxp3 in mice is correlated with a dramatic loss of functions of Ts cells, while the forced expression of Foxp3 in lymphocytes T effectors transform these into Ts cells.
- CD4 and CD25 characterize a population of cells which contains the suppressor T lymphocytes, it appears in fact that the suppressive functions are not entirely carried by the CD4 + / CD25 + cells and especially that all the CD4 + / CD25 + cells are not suppressor.
- CD25 is a marker also expressed by activated effector T cells. The identification and purification of Ts cells on the basis of such a marker is a major problem since they present the risk of identifying and actually purifying activated effector T cells.
- activated T lymphocytes expressing CD4 and CD25 have a high probability of precisely containing the effector T against which a therapeutic intervention is desirable.
- the use of CD4 and CD25 in a diagnostic setting (identification) would not be reliable, and in a therapeutic setting (purification, injection) would run the risk of being ineffective or even increasing the pathology.
- the best marker currently known to be able to differentiate Ts cells from activated effector T lymphocytes is the expression of the transcription factor Foxp3.
- This intracellular transcription factor cannot be used in the context of simple immunophenotypic identification and purification methods.
- Other markers such as CD62L allow better characterization of Ts cells but they are far from allowing perfect identification.
- suppressive activities exist in the CD4 + / CD25- population as well as in certain CD8 + cells. It therefore appears that the diagnostic and therapeutic use of Ts cells is clearly dependent on their own identification and that current knowledge has so far described no specific marker for suppressor T lymphocytes.
- Ts lymphocytes In addition, if the differentiation of certain Ts lymphocytes seems to be carried out in the thymus (they are often called “natural” Ts), other Ts lymphocytes could be generated at the periphery and nothing is known about the ontogenic development of Ts at from the T progenitors
- the present invention offers for the first time the possibility of identifying, isolating, analyzing (transcriptome, proteome, etc.) and of manipulating (culture, activation, depletion, genetic modifications, etc.) of T cell populations. suppressive, in particular human, and in particular (i) populations of Ts precursors, and (ii) populations of pure Ts among CD4 + and CD8 + cells.
- CD90 also called THY-1
- THY-1 represents a characteristic marker of human Ts cells CD4 + and / or CD8 +, and of their precursors and can be used effectively to identify these cell populations.
- the THY-1 antigen (Seki et al., 1985; Planelles et al., 1995) corresponds to a well-characterized surface glycoprotein anchored to the membrane by a phosphatidyl-inositol bridge. This protein belongs to the immunoglobulin superfamily and contains around 140 amino acids (25-30 kDa). This antigen was first identified as a differentiation marker expressed in the thymus and the mouse brain. In humans, THY-1 is expressed on a small percentage of fetal thymocytes, on immature hematopoietic progenitors CD34 + and on less than 1% of CD3 + lymphocytes present in the peripheral circulation. THY-1 is also expressed on mesenchymal cells, endothelial cells as well as on several continuous cell lines. The function of THY-1 is not known.
- THY-1 is expressed at the level of the thymus on the progenitors and precursors T. It is also expressed on regulatory cells (Mukasa et al., Clin. Exp. Immunol. 96 (1994) 138; Torre-Amione and al., Cell. Immunol. 124 (1989) 50; Sakatsume et al., Int. Immunol. 3 (1991) 377) as well as on all circulating T lymphocytes. Therefore, it cannot constitute a discriminating marker of a particular cell type. Furthermore, two isoforms Thy-1.1 and Thy-1.2 have been described in mice.
- the inventors have now discovered that, surprisingly, the expression of the THY-1 molecule in humans is very closely correlated with Ts activity, and that the THY-1 molecule constitutes a specific marker for suppressor T lymphocytes, allowing in particular the identification, selection, expansion or depletion in vitro or in vivo of precursors of Ts and / or pure Ts populations among CD4 + or CD8 + lymphocytes.
- a first aspect of the present invention therefore relates to a method for obtaining, preparing or producing suppressor T lymphocytes (and / or their precursors), comprising a step of selection, separation, and / or isolation of T lymphocytes expressing the THY-1 molecule. This step can be carried out from any biological samples comprising lymphocytes.
- a more particular subject of the present invention relates to a method for obtaining, preparing or producing suppressor T lymphocytes (and / or their precursors) comprising:
- the T lymphocytes expressing the THY-1 antigen are preferably selected, separated, isolated, recovered or eliminated by means of a specific ligand for THY-1.
- the ligand is chosen from an antibody or an antibody fragment.
- the ligand can for example be immobilized on a support or placed in solution. Such a ligand is more fully defined in the following description of the invention.
- step (b) can be preceded and / or followed by a step of amplification of the T lymphocytes and / or of a step of purification of subpopulation (s) of lymphocytes, such as for example the CD4 + lymphocytes. or CD8 +, or even lymphocytes specific for a given antigen.
- Another subject of the invention relates to a method of identifying and / or quantifying suppressor T lymphocytes (and / or their precursors) in a cell population, comprising the exposure of said cell population to a specific ligand for THY-1 and determining and / or quantifying the formation of a complex between the ligand and the cells, the formation of such complexes indicating the presence and / or the amount of suppressor T lymphocytes (and / or their precursors) within the cell population.
- Ligand-binding cells can be separated from non-ligand-binding cells.
- Another subject of the invention relates to the use of a specific ligand for the THY-1 antigen for enriching or eliminating ex vivo the suppressor T lymphocytes (and / or their precursors) of a cell population.
- the THY-1 antigen can itself be used as a marker for the selection of Ts or pTs lymphocytes within a cell population.
- Another object of the invention resides in a method of diagnosing a patient, comprising determining the presence, number or state of activity of Ts cells in this patient using a specific ligand for THY-1 .
- Such a diagnosis can be carried out in vitro, ex vivo or in vivo, and allow the detection of a pathological condition linked to the activity of the immune system, or the monitoring of the effectiveness of a treatment, or the selection of a patient for inclusion in a particular therapeutic program.
- Another object of the invention resides in the use of a specific ligand for the THY-1 antigen for selecting, identifying, sorting or preparing (in vitro or ex vivo) Ts or pTs lymphocytes.
- the invention further relates to suppressor T lymphocytes (and / or their precursors) expressing the THY-1 antigen capable of being obtained by a method according to the invention.
- Another object of the invention resides in the use of a specific ligand for the THY-1 antigen for preparing a diagnostic composition intended for selecting, identifying or quantifying in vivo suppressor T lymphocytes (including their precursors).
- Another subject of the invention relates to the use of a specific ligand for the THN -1 antigen for preparing a therapeutic composition intended to modify, stimulate or suppress suppressor T lymphocytes in vivo.
- a particular object of the invention relates to the use of a specific THY-1 ligand for enriching or eliminating ex vivo or in vivo suppressor T lymphocytes (including their precursors) from a cell population.
- THY-1 antigen as a selection marker for enriching or eliminating, in vivo, in vitro or ex vivo, Ts or pTs lymphocytes within a cell population.
- the invention further relates to suppressor T lymphocytes (including their precursors) expressing the THY-1 antigen capable of being obtained by a method as defined above, as well as a population of cells enriched in Ts or pTs cells, in which at least 30%, preferably at least 50%, even more preferably at least 65% of the T cells express the THY-1 antigen.
- Particularly preferred compositions or cell populations according to the present invention comprise at least 75%, preferably at least 80% of Ts or pTs cells expressing THY-1, more preferably at least 85, 90 or 95%.
- the invention also relates to an isolated human T lymphocyte, characterized in that it exhibits suppressive activity and in that it expresses the markers CD8 or CD4 and THY-1, as well as a population of cells comprising suppressive T cells CD8 + THY-1 + or CD4 + THY-1 +, preferably a population comprising at least 50, 60, 70, 80, 85, 90 or 95% of CD8 + THY-1 + T cells.
- Such cells can, in part, also express the CD25 antigen.
- the T lymphocytes present in the mammalian cell population or the Ts or pTs lymphocytes (carrying the THY-1 marker) can be genetically modified so as to express biological products of interest , allowing in particular to improve their efficiency and / or their safety of use.
- the invention also relates to a pharmaceutical composition
- a pharmaceutical composition comprising cells or cell populations as defined above, typically in combination with a pharmaceutically acceptable vehicle or excipient.
- Another particular subject of the invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising suppressor T cells (and / or their precursors) amplified ex vivo and an adjuvant or a pharmaceutically acceptable medium, said amplified cells being enriched in cells expressing the THY-1 antigen and, possibly, in specific cells of a particular antigen, such as allergens, auto-antigens, allo-antigens or antigens of infectious agents.
- the antigen is involved in or specific to a pathological condition chosen from an immune disease, in particular autoimmune diseases, inflammatory diseases, graft versus host disease, allergy and rejection of 'transplant.
- the invention also relates to the preparation of a composition consisting of at least one such suppressor T lymphocyte, of a population enriched in Ts cells and / or pTs as defined above or on the contrary of a population depleted in cells.
- Ts and / or pTs and an adjuvant or a pharmaceutically acceptable medium as well as the composition itself intended for the implementation of a therapeutic method.
- a particular subject of the invention thus also relates to a method of producing a pharmaceutical composition, comprising:
- Another particular object of the invention thus also relates to a method for producing a pharmaceutical composition, comprising: (a) obtaining a biological sample comprising T lymphocytes, (b) depletion of T lymphocytes expressing the THY-1 antigen within this biological sample, and (c) the conditioning of said T lymphocytes obtained which do not express the THY-1 antigen in an adjuvant or a pharmaceutically acceptable medium.
- the invention further relates to a kit for isolating or characterizing Ts cells comprising a specific ligand for THY-1, optionally placed on a support or placed in solution as well as, possibly, reagents for detecting the ligand.
- the ligand is typically placed in a container, such as a plate, syringe, tube, pipette, vial, etc.
- a kit can also be used to diagnose the presence of such Ts cells and pTs from a biological sample taken from the individual to be tested or directly in vivo.
- the invention further relates to a kit or a composition for eliminating Ts cells and pTs in vivo, in vitro or ex vivo, comprising a specific ligand for THY-1, optionally placed in solution or on a support, and coupled to a product. toxic (radioactive, toxins ).
- the invention also relates to the use of a Thy-1 ligand to specifically target a viral or non-viral vector in Ts and pTs in order to express genes.
- the invention further relates to a kit or a composition for activating Ts and pTs cells in vivo, ex vivo or in vitro, comprising a specific ligand for THY-1, optionally placed in solution or on a support, coupled to a product capable activate T cells (eg cytokme, such as IL2, IL7, IL 10, IL15).
- a product capable activate T cells eg cytokme, such as IL2, IL7, IL 10, IL15.
- the invention also relates to the use of a THY-1 ligand to specifically target a viral or non-viral vector in Ts in order to express activator genes or all therapeutic genes.
- Ts cells the compositions made up of isolated or amplified Ts and pTs cells and the compositions enriched in Ts and pTs cells obtained within the framework of the present invention can be advantageously used within the framework of experimental or therapeutic applications.
- the cells used in the context of the present invention are mammalian cells, typically human.
- the invention can also be used in particular in primates, and therefore also relates to primate suppressor T cells, in particular monkey.
- a particular subject of the invention thus also relates to methods of analysis and of obtaining genetic sequences specifically expressed in suppressor T lymphocytes (or their precursors), a method comprising the isolation of RNA from a population of T lymphocytes expressing the Thy-1 antigen, comparison of said RNA to RNA extracted from a population of non-suppressor T cells and recovery of RNA specific for suppressor T cells.
- the invention also relates to a method as described above, further comprising the manufacture of a probe from the RNA specific for suppressor T lymphocytes (or their precursors) and the screening of a population of nucleic acids intended to be hybrid to said probe.
- One method also corresponds to the analysis of the transcriptome by RNA hybridization on biochips in order to establish expression profiles.
- a particular object of the invention thus relates to a method for obtaining proteins specifically expressed in suppressor T lymphocytes (or their precursors).
- One method includes isolating proteins from a population of T cells expressing the Thy-1 antigen, comparing these proteins to those extracted from a population of non-suppressor T cells.
- a particular object of the invention thus relates to a method of identifying new molecules specifically expressed in suppressor T lymphocytes (or their precursors) by immunization using Ts lymphocytes (or pTs) expressing the Thy- antigen. 1, or cell or protein fractions of these same cells.
- Ts cells or pTs
- compositions made up of isolated or amplified Ts cells or pTs
- compositions enriched in Ts cells obtained in the context of the present invention, for example for treating numerous subjects, for example human patients suffering from or at risk of developing an immune disease, in particular a disease caused by a abnormal T response.
- Ts cells are thus suitable for the treatment of various pathologies or conditions caused by a disorder affecting T lymphocytes and in particular a tumor, an autoimmune disease, an allergy, graft versus host disease, a inflammatory disease, type I diabetes, viral or bacterial infection, etc.
- Treatment can be preventive or curative. It can also be combined with other treatments.
- suppressor T lymphocytes designates a population of T cells which are characterized by their capacity to suppress or reduce the immune reactions mediated by effector T cells, such as CD4 + T cells or CD8 +.
- This term includes conventional Ts cells, which strongly express the CD25 marker, as well as their precursors, designated pTs, which are endowed with suppressive activity and can give rise, in culture, to conventional Ts cells.
- the invention indeed demonstrates the existence of a population of suppressive T cells, designated pTs, expressing the markers THY-1 and CD25, endowed with suppressive property, and capable of giving rise in culture to conventional Ts cells.
- suppressor T lymphocytes also includes Ts lymphocytes originating from total lymphocyte populations (or CD25-), of the CD4 + or CD8 + type, expressing THY-1.
- CD4 and CD25 characterize suppressor T lymphocytes (or their precursors) on an immunophenotypic level, it appears in fact that the suppressive functions are not entirely carried by CD4 + cells / CD25 + and that all CD4 + / CD25 + cells are not suppressive.
- CD25 is indeed a marker also expressed by activated effector T cells.
- THY-1 represents a characteristic marker human Ts and pTs cells and can be used effectively to identify this cell population.
- the present invention thus relates, as indicated above, to a method for obtaining, preparing, selecting or producing human suppressor T lymphocytes (including their precursors) comprising:
- the cell population can be obtained, in the context of step (a), from biological samples comprising lymphocytes, in particular from samples of a tissue chosen from bone marrow, spleen, liver, thymus , blood that may or may not have been previously enriched for T lymphocytes, umbilical cord blood, peripheral fetal blood, newborns or adults, plasma, a lymph node, a tumor, a inflammatory site, a transplanted organ, or a cell culture established with either of these tissues. Lymphocytes are typically isolated or collected from peripheral blood.
- the T lymphocytes expressing THY-1 can be recovered, selected, isolated, removed or sorted, in particular during step (b), using any specific ligands for THY-1, that is to say typically of all molecules capable of selectively binding Thy-1 to the surface of a cell.
- the ligand is preferably chosen from an antibody, preferably an anti-THY-1 antibody, an analog or a fragment of such an antibody.
- THY-1 is a molecule devoid of an intra-cytoplasmic domain which interacts with the cell membrane via a glycophosphatidylinositol (GPI) which attaches to the membrane by its C-terminal end.
- GPI glycophosphatidylinositol
- the sequence of Thy-1 has been determined and is accessible in the literature, such as for example the nucleotide sequence (no NM 006288 (gi: 199 233 61)) and the amino acid sequence (no P 006279 (gi: 199 233 62)) of human protein.
- a specific ligand according to the invention is preferably a molecule capable of selectively binding a polypeptide comprising all or part of the sequence of the human Thy-1 protein, preferably a molecule comprising an epitope of the human Thy-1 protein.
- Such ligands are naturally chosen from recognized molecules and / or capable of interacting with the extracellular part of THY-1.
- a preferred ligand for THY-1 which can be used in the context of the present invention, is an anti-THY-1 antibody (that is to say an antibody specific for THY-1).
- the antibody can be polyclonal or monoclonal. It can also be fragments and derivatives of a fragment or derivative of antibodies having substantially the same antigenic specificity, in particular fragments of antibodies (eg, Fab, Fab'2, CDRs), of humanized antibodies, human antibodies, polyfunctional, single-stranded (ScFv), or multimeric antibodies (C4bp coupling for example), etc.
- the antibodies, and therefore the recognition sites of the THY-1 molecule which can be used to generate a specific ligand can be produced using conventional methods, comprising the immunization of a non-human animal with a THY-1 polypeptide or a fragment thereof comprising an epitope, and the recovery of its serum (polyclonal) or of spleen cells (so as to produce hybridomas by fusion with appropriate cell lines).
- a non-human animal with a THY-1 polypeptide or a fragment thereof comprising an epitope
- serum polyclonal
- spleen cells so as to produce hybridomas by fusion with appropriate cell lines.
- Various methods of producing polyclonal antibodies from various species have been described in the prior art.
- the antigen is combined with an adjuvant (eg Freund's adjuvant) and administered to an animal, for example by subcutaneous injection. Repeated injections can be given. Blood samples are collected and immunoglobulin or serum are separated.
- an adjuvant e
- monoclonal antibodies include immunizing a non-human animal with an antigen, followed by the recovery of spleen cells which are then fused with immortalized cells, such as myeloma cells.
- the resulting hybridomas produce monoclonal antibodies and can be selected by limiting dilutions so as to isolate the individual clones.
- the Fab or F (ab ') 2 fragments can be produced by digestion using a protease according to conventional techniques.
- the preferred antibodies are antibodies specific for the THY-1 protein, that is to say having a higher affinity for this protein than for other antigens, even if a non-specific or less affine bond cannot be excluded.
- the term “specific” or “selective” indicates in particular that the binding of the ligand to the THY-1 protein can be discriminated from the possible binding of the ligand to other molecules.
- Ts and pTs cells can thus be isolated, in the context of step (b), by bringing the cell population into contact with specific ligands, such as those defined above.
- specific ligands such as those defined above.
- specific ligands according to the invention are in particular the monoclonal antibodies produced by the hybridomas Kl 7 (ATCC n ° HB- 8553), the clones 5E10, F15-42-1, Thy-1/310, FIB1 (clone AS02 ), as well as any fragments or derivatives of these antibodies.
- ligands according to the invention are for example artificial ligands, having a particular affinity for THY-1.
- Such ligands can be of varied nature, such as nucleic acids (for example aptamers) or synthetic chemical molecules.
- Such molecules can be generated for example on the basis of the sequences of the recognition sites of the THY-1 molecule by the specific antibodies defined above.
- Ts and pTs cells can thus be isolated, in the context of step (b), by bringing the cell population into contact with one or more specific ligands, such as those defined above.
- the invention uses a combination of a specific ligand for THY-1 and a specific ligand for CD25.
- the second ligand can be specific for any other marker of T cells, in particular of suppressor T cells, for example markers identified by the genomics and proteomics techniques described in the present application.
- the ligand (s) can (can) be immobilized on a support, for example a column or a ball (in particular a magnetic ball), or placed (s) in solution.
- the ligand can optionally be labeled.
- the labeling can be carried out using a fluorescent, radioactive, luminescent, phosphorescent, chemical or enzymatic detection marker.
- the detection marker is preferably chosen from fluorescein, Texas red, rhodamine, phycoerythrin, dlophycocyanin, biotin and streptavidin, Cyanine.
- the complexes formed by the ligand and the labeled cells can then be used to visualize, detect, quantify, sort, isolate and / or deploy the cells, according to various techniques which are known per se to those skilled in the art.
- the cells can be recovered, selected, sorted, separated, isolated, depleted for example by a method chosen from flow cytometry, affinity chromatography, FACS (fluorescent activated cell sorting "Fluorescent Activated Cell Sorting"), MACS (magneticbead cell sorting "Magnetic bead Cell Sorting"), D / MACS (double magnetic bead cell sorting), affinity chromatography "Double Magnetic bead Cell Sorting”), a selection method on solid surface (“panning ”), An ELIS A test, an RIA test, etc.
- the MACS procedure is described in detail by Miltenyi et al., "High Gradient Magnetic Cell Separation ith MACS,” Cytometry 11: 231-238 (1990).
- the cells labeled with magnetic beads pass through a paramagnetic separation column.
- the separation column is placed near a magnet, thereby creating a magnetic field inside the column.
- the magnetically marked cells are trapped in the column, the others cross it.
- the cells trapped inside the column are then eluted.
- a sample of cells is labeled using magnetic beads comprising antibodies, and the cells are harvested or sorted by application of a magnetic field.
- the cells for example peripheral blood
- the cells are incubated sequentially with saturating amounts of functionalized anti-THY-1 antibody (eg, labeled with biotin) and with a solid support (for example microbeads) functionalized (eg, coated with streptavidin).
- the cells are then purified by recovery of the support, eg, by magnetic separation of the cells.
- the cells of the positive fraction can be subsequently separated on another column.
- the purification is generally carried out in a phosphate salt buffer, although other suitable media can be used.
- the cells can be cultured or maintained in any suitable buffer or medium, such as a saline solution, a buffer, a culture medium, in particular DMEM, RPMI etc. They can be frozen or kept in a cold situation. They can be formulated in any suitable device or apparatus, such as a tube, a flask, a vial, a dish, a syringe, a bag, etc., preferably under sterile conditions suitable for pharmaceutical use.
- step (b) of the method described above can advantageously be preceded and / or followed by a step of purifying a subpopulation of T lymphocytes (CD4 + and or CD8 + for example) and / or a lymphocyte amplification step (which can be carried out ex vivo or in vitro.).
- a step of purifying a subpopulation of T lymphocytes CD4 + and or CD8 + for example
- a lymphocyte amplification step which can be carried out ex vivo or in vitro.
- Amplification can be obtained by activation of lymphocytes.
- This activation can be non-specific (obtained for example by anti-CD3 and / or anti-CD28 antibodies, with or in the presence of an interleukin, for example IL2) or specific (obtained by antigens or allo-antigens presented adequately to Ts or pTs cells, for example by antigen presenting cells (dendritic cells, B lymphocytes, macrophage monocytes, genetically modified cells capable of presenting the antigen and activating lymphocytes), exosomes, dexosomes, artificial structures, etc.).
- antigen presenting cells dendritic cells, B lymphocytes, macrophage monocytes, genetically modified cells capable of presenting the antigen and activating lymphocytes, exosomes, dexosomes, artificial structures, etc.
- the amplification step makes it possible to increase the number of T lymphocytes present in the starting T lymphocyte population (which includes effector T lymphocytes and suppressor T lymphocytes) before proceed to the selection of Ts and pTs lymphocytes, and / or to increase the number of Ts and pTs lymphocytes after having selected the T lymphocytes expressing the THY-1 antigen. It is also possible to carry out two amplification stages, one concerning the general T lymphocyte population present in the population of mammalian cells, the other concerning the population of Ts and pTs lymphocytes.
- the amplification step is carried out under conditions which favor the Ts (or pTs), thus allowing their enrichment.
- the present invention shows that culture in the absence of N-acetyl cysteine promotes the proliferation of Ts ( Figure 1).
- the cells are amplified by culture in a medium devoid of N-acetyl cysteine.
- the use of certain populations of natural or modified (in particular genetically modified) antigen presenting cells can promote the proliferation of Ts (or pTs).
- the examples show that the dendritic cells derived from CD34 + hematopoietic progenitors and having an interstitial DC type phenotype promote the proliferation of Ts (or pTs) (FIG. 2).
- the cells are amplified by culture in the presence of dendritic cells, in particular interstitial dendritic cells.
- the population obtained at the end of step (a) can also be enriched in T cells belonging to the general population of T cells, ie, comprising effector T lymphocytes and suppressor T lymphocytes or their precursors.
- the population of step (a) can thus be enriched in T cells, possibly in one or more specific subpopulations of lymphocytes (for example CD4 + and / or CD8 +). It can also be rid of certain subpopulations of lymphocytes, if necessary.
- the population obtained at the end of step (a) then, optionally amplified and / or sorted, thus preferably comprises at least 30%, preferably at least 50%, even more preferably at least 65% of cells. T.
- compositions enriched in T cells capable of being used in the context of step (b) comprise at least 75%, preferably at least 80% of T cells.
- the population of T lymphocytes expressing the THY-1 antigen can also be amplified. It is also possible, as indicated above, to carry out two amplification steps, one concerning the general T lymphocyte population, the other concerning the Ts or pTs lymphocyte population.
- a particular object of the present invention thus relates to a method for obtaining suppressor T lymphocytes (and / or their precursors) comprising. : (a) obtaining a population of mammalian cells comprising T lymphocytes, (a ') amplifying T lymphocytes within said population of cells, and (b) recovering T lymphocytes expressing the THY-1 antigen.
- Another particular object of the present invention relates to a method for obtaining suppressor T lymphocytes (and / or their precursors) comprising: (a) obtaining a population of mammalian cells comprising T lymphocytes, (b) recovery of T lymphocytes expressing the THY-1 antigen, and (b ') amplification of said T lymphocytes expressing the THY-1 antigen.
- Cellular amplification of lymphocytes belonging to the general population of T lymphocytes is preferably carried out by culturing the cells in the presence of a cytokine and optionally a stimulating agent.
- a cytokine and optionally a stimulating agent.
- the culture is maintained for a period sufficient to obtain the amplification of said cell population within CD4 + and / or CD8 + T lymphocyte populations.
- Activation usually involves culture in the presence of a cytokine, such as, for example, interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-10 (IL-10 ) or Interleukin-15 (IL-15), preferably of human origin.
- the stimulating agent can be an antigen presenting cell (“APC”), Le., Any antigen presenting cell or any cell promoting activation of T cells, in particular Ts cells.
- APCs are preferably irradiated before their use in order to avoid their amplification.
- CPA can be cells isolated from a donor or from the patient himself. They can be chosen to produce Ts and pTs cells with a chosen activity profile.
- CPA peripheral blood mononuclear cells, dendritic cells, splenocytes, umbilical cord blood cells, tissue or organ samples, etc.
- Ts and pTs cell stimulants include MHC polymers, lectins (such as PHA), antibodies (such as anti-CD3 and / or anti-CD28 antibodies) or fragments thereof, autoantigens (including tissues, cells, cell fragments or debris, purified polypeptides or peptides, etc., preferably in combination with CPA), etc.
- Ts and pTs cells can be amplified in different ways, whether or not they are specific antigens.
- large quantities of the complete repertoire of T cells are preferably used (e.g., injected).
- This technique is, in particular, suitable for patients who have an overall deficit (quantitative or functional) in Ts and pTs cells.
- the Ts and pTs cells are preferably amplified, for example, using autologous CPA and PHA cells or anti-CD3 and / or anti-CD25 antibodies (or any other activator of T cells or Ts) in the presence of cytokines of identical or different nature.
- Ts and pTs lymphocytes in humans and in mice, can be cultured and amplified in vitro in the presence of a culture medium containing interleukin 2, anti-CD3 and anti-CD28 antibodies.
- Specific Ts and pTs lymphocytes can also be isolated, generated for example by stimulation in the presence of cells presenting allogenic antigens, followed by a culture by interleukin 2.
- a more specific amplification can be envisaged, in particular when suppression of specific effector T cells is desired, such as in the context of autoimmune diseases, allergies, transplant rejection, GNHD, etc.
- the cells are preferably amplified in the presence of CPA presenting particular antigens, for example allogenic or of infectious origin, in order to favor the amplification of Ts cells preferentially active against pathogenic effector T cells.
- the antigens are presented in peptide form or after transfer of AR ⁇ or DNA.
- Ts and pTs cells preferably come from the patient and are stimulated using autologous CPA and autoantigens from the target tissue, in the presence of cytokines.
- Autoantigens can be from tissues, cells, cell fragments, purified proteins, peptides, nucleic acids, etc.
- Ts cells preferably come from the patient and are stimulated by APCs or tissues from the donor, in the presence of cytokines. Ts cells from the patient can also be stimulated by autologous APCs in the presence of tissues, cells, cell fragments, purified proteins, or peptides from the donor and cytokines.
- Ts cells typically come from the patient and are stimulated by CPA and allergens in the presence of cytokines.
- the cytokines preferentially used are 1TL-2, IL-10 and / or IL-15.
- the Ts and pTs cells used to treat various pathologies are preferably autologous, Le. , they come from the subject to be treated. Syngene cells can also be used. In other situations, for example in the treatment of GNHD or other pathologies, Ts and pTs cells are typically allogenic, ie, they come from a different human being. In these cases, it is preferable to use Ts and pTs cells originating from a donor subject (eg, the donor subject of effector cells). Genetic modification of T cells and in particular of Ts and pTs cells
- the T lymphocytes (general population of T lymphocytes) present in the population of mammalian cells or the suppressor T lymphocytes (carrying the THY-1 marker) can be genetically modified so as to express organic products of interest.
- the expression "genetically modified” indicates that the cells comprise a nucleic acid molecule which is not naturally present in unmodified T cells, or which is present in these cells when they are not in their natural state ( eg, when amplified).
- the nucleic acid molecule may have been introduced into said cells or into a parent or progenitor cell.
- a particular subject of the invention thus relates to a method for obtaining or producing suppressor T lymphocytes (and / or their precursors) comprising: (a) obtaining a population of mammalian cells comprising T lymphocytes, ( b) recovery of the T lymphocytes expressing the THY-1 antigen, and (c) the genetic modification of said T lymphocytes expressing the THY-1 antigen by bringing said lymphocytes into contact with a recombinant nucleic acid molecule.
- Another particular object of the invention relates to a method for obtaining or producing suppressor T lymphocytes (or their precursors) comprising: (a) obtaining a population of mammalian cells comprising T lymphocytes, (b) the genetic modification of said T lymphocytes by bringing said population of cells into contact with a recombinant nucleic acid molecule, and (c) the recovery of T lymphocytes expressing the THY-1 antigen.
- Several approaches can be used to genetically modify the T cells belonging to the mammalian cell population [comparable to the general population of T lymphocytes (effector T cells and Ts cells)] or Ts and pTs lymphocytes, such as, for example, gene delivery via virus, naked DNA, physical treatments, etc.
- the nucleic acid is generally incorporated into a vector, such as a recombinant virus, a plasmid, a phage, an episome, an artificial chromosome, etc.
- the T cells as defined in the preceding paragraph are genetically modified using a viral vector (or a recombinant virus).
- the heterologous nucleic acid is, for example, introduced into a recombinant virus which is then used to infect T lymphocytes.
- Different types of recombinant viruses can be used, in particular recombinant refroviruses or ANAs.
- the T lymphocytes are preferably modified using a recombinant refrovirus. The use of refrovirus is particularly appreciated insofar as refroviral infection allows stable integration of the nucleic acid into the genome of the cells.
- refractive viruses susceptible of being used come from the family of oncoviruses, lentiviruses or spumaviruses.
- Particular examples of the oncovirus family are MoMLN, ALN, BLN or MMTN but also RSN, etc.
- Examples of the lentivirus family are HIN, SIN, FIN, ELAN or CAEN, etc.
- the recombinant refrovirus comprises the envelope of the GALN virus (refrovirus pseudotyped with GALN).
- T cells can be infected using recombinant viruses and various protocols, such as incubation with a virus supernatant, with purified viruses, by coculture of T cells with viral packaging cells, Transwell techniques, etc.
- a particularly effective method comprising a centrifugation step has been described by Movassagh et al. (Movassagh M, Desmyter C, Baillou C, Chapel-Fernandes S, Guigon M, Klatzmann D, Lemoine FM. Hum Gène Ther. 1998; 9: 225-234).
- Non-viral techniques include the use of cationic lipids, polymers, peptides, synthetic agents, etc.
- Alternative methods use the “gun gene” technique, electric fields, bombardment, precipitation, etc.
- Different selection techniques can be used, including the use of antibodies recognizing specific markers present on the surface of the modified cells, the use of resistance genes (such as the neomycin resistance gene and the G418 molecule), or the use of compounds that are toxic to cells that do not express fransgene (ie, thymidine kinase).
- the selection is preferably carried out using a marker gene expressing a membrane protein. The presence of this protein allows selection using techniques separation techniques such as separation using magnetic beads, the use of columns or flow cytometry.
- the nucleic acid used to genetically modify T cells can be a therapeutic fransgene and can code for various active biological products, including polypeptides (e.g., proteins, peptides, etc.), RNAs, etc.
- the nucleic acid encodes a polypeptide having immunosuppressive activity.
- the nucleic acid encodes a polypeptide that is toxic or has conditional toxicity to cells.
- Preferred examples include thymidine kinase (which confers toxicity in the presence of nucleoside analogs), such as HSN-1 TK, cytosine deaminase, gprt, etc.
- nucleic acids can also be a non-toxic polypeptide but which can allow the elimination of the injected cells if necessary (such as for example a molecule expressed at the cell membrane and a monoclonal antibody fixing the complement).
- nucleic acids are those for targeting. They may be nucleic acids encoding a T or B cell receptor or a subunit or a functional equivalent thereof. For example, the expression within Ts cells of a specific recombinant TCR of an autoantigen produces Ts cells and pTs which can act more specifically on effector T cells which destroy tissue in a subject.
- T-bodies ie, hybrid receptors between T cell receptors and an immunoglobulin. Such "T-bodies” allow targeting of antigenic complexes, for example.
- the suppressor T lymphocytes are genetically modified and comprise a recombinant nucleic acid coding for a product having a conditional toxicity for these cells, such as thymidine kinase.
- the genetically modified Ts and pTs cells comprise a recombinant nucleic acid molecule encoding a T cell receptor or a subunit or a functional equivalent thereof.
- the nucleic acid which is introduced into the T cells according to the invention typically comprises, in addition to the coding region, regulatory sequences, such as a promoter and a polyadenylation sequence.
- a particular object of this invention is a composition comprising at least one suppressor T lymphocyte according to the invention, eg isolated, genetically modified and / or amplified ex vivo, or a population enriched in suppressor T cells as defined above or, on the contrary, a population depleted in suppressor T cells, as well as a pharmaceutically acceptable adjuvant or medium.
- Another particular object of the invention is a composition comprising suppressor T lymphocytes (including their precursors) transduced using a first suicide gene and effector T cells which are transplanted using a second suicide gene, different from the first.
- compositions may include other cell types, without significantly affecting the therapeutic benefit of said compositions.
- the cells are packaged in a composition comprising between approximately 10 5 and 10 10 suppressor T cells depending on the pathology to be treated, more generally between 10 5 and approximately 10 9 suppressor T cells.
- a particular composition within the meaning of the invention comprises a population of THY-1 positive human lymphocytes, endowed with suppressive properties with regard to effector T cells.
- the medium or adjuvant can be any culture medium, defined medium, aqueous solution or suspension, buffered, optionally supplemented with preservatives.
- the compositions according to the invention can be administered by any suitable route, such as intravenous, intraarterial, subcutaneous, fransdermal, etc. Repeated administrations of such compositions can be implemented.
- compositions according to the invention comprise a specific Thy-1 ligand coupled or conjugated to an effector molecule, for example a molecule exhibiting a toxicity (conditional or not, for example "a TK, castor toxin, etc.) or a stimulating activity for T lymphocytes (for example a cytokine, in particular IL-2, IL-7, IL-15, etc.).
- an effector molecule for example a molecule exhibiting a toxicity (conditional or not, for example "a TK, castor toxin, etc.) or a stimulating activity for T lymphocytes (for example a cytokine, in particular IL-2, IL-7, IL-15, etc.).
- a cytokine for example a cytokine, in particular IL-2, IL-7, IL-15, etc.
- the administration of a conjugate comprising a toxic molecule can make it possible to inactivate or reduce the suppressor T cell activity in a subject, and therefore to increase the activity
- the administration of a conjugate comprising an activating molecule can make it possible to stimulate the activity of suppressor T cells in a subject, and therefore to reduce the activity of effector cells. be covalent or not.
- compositions of the invention include a transfection agent coupled to a specific Thy-1 ligand.
- the transfection agent can be a viral particle (for example recombinant, defective, attenuated, synthetic, etc.) or a non-viral transfection agent, such as a liposome, a canonical lipid, a polymer, etc.
- the coupling can be covalent or not.
- Such compositions make it possible to modify in a targeted manner the suppressor T cells of a subject, for example to confer on them new properties. uses
- the present invention makes it possible to obtain cell populations which can be used for the treatment of various pathologies associated with the activity of T cells, as indicated above. Treatment can be preventive or curative.
- suppressor T cells including pTs
- cell populations enriched in suppressor T cells including pTs
- the compositions according to the invention can be used in combination with other agents or active ingredients. , such as other cell populations, immunosuppressive molecules or conditions, radiation, gene therapy products, etc.
- treatment means the reduction of symptoms or causes of a disease, the regression of a disease, the delay of a disease, the improvement of the condition of patients, the reduction of their suffering, the increase in their lifespan, etc.
- Suppressor T cells including pTs cells
- cell populations enriched in suppressor T cells including pTs cells
- the compositions according to the invention are particularly suitable for delaying or preventing graft versus host disease (GNHD) in subjects who have undergone an allogeneic organ transplant, in particular bone marrow (or hematopoietic stem cells or non-hematopoietic stem cells).
- GNHD graft versus host disease
- GNHD and the frequent complications caused by transplantation of hematopoietic stem cells are due to the presence of mature donor T cells in the fransplant.
- the elimination of these cells before the transplant leads to a failure of the latter, prolongs immunosuppression and the recurrence of leukemia.
- Ts cells according to the invention at the time of the graft delays or even prevents GNHD.
- This depletion of THY-1 cells may or may not be associated with a depletion using other antibodies such as those specific for CD25.
- Suppressor T cells including pTs
- cell populations enriched in suppressor T cells including pTs
- compositions according to the invention are also suitable for the treatment of autoimmune diseases (including chronic inflammatory diseases ), such as systemic lupus erythematosus, rheumatoid arthritis, polymyositis, multiple sclerosis, diabetes, atherosclerosis etc.
- autoimmune diseases including chronic inflammatory diseases
- Autoimmune diseases have an immunological component as numerous biological and histological investigations have been able to demonstrate. For these diseases, the central element is an inadequate immune response.
- the present invention can be used to prevent, treat, reduce or alleviate such pathologies by administering to the subject an effective amount of suppressor T cells (including pTs cells) to suppress or reduce the activity of these deleterious T cells. Repeated administrations can be carried out if necessary.
- suppressor T cells including pTs cells
- Suppressor T cells including pTs cells
- cell populations enriched in suppressor T cells including pTs cells
- the compositions according to the invention can also be used in the context of the treatment of infectious diseases and in particular pathologies immune systems induced by viruses.
- the immune response against infectious agents can have immunopathological consequences which can lead to death.
- One example is the response to certain viruses responsible for hepatitis. These viruses replicate in hepatocytes and the destruction of these infected hepatocytes by the immune system causes hepatitis, which is sometimes fatal.
- the progression of this chronic hepatitis is accompanied by biological signs and an abnormal immune response (for example, the presence of anti-DNA antibodies or cryoglobulinemia).
- the suppressor T cells (including the pTs), the cell populations enriched in suppressor T cells (including the pTs) and the compositions according to the invention make it possible to eliminate, delete or reduce the active T lymphocytes responsible for the pathology and thus reduce the consequences of immune pathologies induced by viruses.
- Suppressor T cells (including pTs), cell populations enriched in suppressor T cells (including pTs) and compositions according to the invention can also be used for the treatment or prevention of rejection of transplanted organs such as heart, liver, kidneys, lungs, pancreas, etc.
- the usual treatment for a certain number of disorders affecting the organs consists, when it becomes necessary, in replacing the organ by a healthy organ coming from a deceased donor (or from a living donor in certain cases, or even from a donor of another species). This is also the case for the treatment of insulin-dependent diabetes, through the transplantation of cells or of an insulin-producing organ, such as the pancreas or the pancreatic islets. Even if great care is taken in the selection of organ donors with maximum compatibility with histocompatibility antigens, the transplanted organ always leads, with the exception of transplants made between homozygous twins, to development of an immune response directed against the antigens specifically expressed by this organ.
- transplanted organ This is the main cause of failure of allogenic transplants.
- rejection of the transplanted organ is, in the majority of cases, essentially mediated by effector T lymphocytes.
- the present invention now makes it possible to envisage treatment (eg reduction or delay) of organ rejection using suppressor T cells (including pTs cells).
- suppressor T cells including pTs cells.
- Such cells can be prepared from patient cells, stimulated with donor antigens and re-administered to the patient, before or during organ transplantation. Repeated administrations can be carried out if necessary.
- Ts cells are amplified and activated by culture in the presence of autoantigens from the donor tissue. These cells can be produced for example by culture in the presence of dendritic cells autologous to the graft. These amplified and activated suppressor T cells (including pTs cells) can be injected into the patient before, during and / or after organ transplantation, thereby reducing the destructive activity of effector T cells.
- Suppressor T cells including pTs
- cell populations enriched in suppressor T cells including pTs
- compositions according to the invention are also suitable for treating allergies, which are mediated by directed immune responses against specific antigens called allergens.
- suppressor T cells including pTs
- Another subject of the invention relates to a method of decreasing the activity (and or the amount of effector T lymphocytes) in a host mammal, said method comprising the administration to the mammal of suppressor T lymphocytes (or their precursors) according to the invention compatible with said host mammal, preferably autologous.
- suppressor T lymphocytes may also be sought (for example in the context of the treatment of cancer).
- treatment methods can be used.
- a first approach consists in preparing ex vivo cells with activity (for example anti-cancer), depleted in suppressor T cells (including pTs cells). Such depletion can be carried out ex vivo in accordance with a method according to the invention as described above. Depletion can be carried out ex vivo without a prior culture phase and / or after a culture phase in a medium containing N-acetyl cysteine which decreases the proliferation of Ts lymphocytes (including pTs cells) (see FIG. 1).
- the treatment then consists in the re-administration to the patient of a population of T lymphocytes or of a composition comprising such a population lacking T suppressor cells (including the pTs cells), and having or not having been activated ex vivo.
- This treatment may be accompanied by one or more vaccinations (for example anti-tumor), combined or not with chemotherapy and / or radiotherapy, in a patient who has possibly received conditioning.
- This conditioning can in particular comprise lympho-ablative treatments, myeloablative or not, intended to eliminate T lymphocytes, in particular dividing T lymphocytes, and which include suppressor T cells (including pTs cells) responsible for the absence of an effective immune response (like for example the Ts which prevent the development of an effective anti-tumor response).
- Another modality consists in depleting the T suppressors in vivo by the use of ligand and of any adequate toxic activity or molecule (such as for example radioactivity or toxin).
- the treatment of all these pathologies can also be carried out by in vivo modulation (suppression or activation) of suppressor T cells (including ⁇ Ts cells) using any molecule having these activities, and in particular anti-THYl antibodies, or all molecules modulating the activity of suppressor T (including pTs cells) whose discovery results from knowledge of the franscriptome and proteome of suppressor T (including pTs cells).
- Suppressor T lymphocytes can also be activated in vivo, for example by thy-1 ligands coupled to lymphocyte activation molecules (IL2, IL10 for example).
- the treatment can then be administered by general route (intravenous for example) or on the site where the action is sought (in the synovial fluid during the treatment of rheumatoid arthritis for example).
- a particular object of the present application corresponds to the use, in the context of vaccination, of a suppressor T cell (including pTs cells), of a cell population enriched in suppressor T cells (including pTs cells) ) or of a composition according to the invention.
- Various routes of administration and protocols can be implemented in the context of the present invention. They can be adapted by a person skilled in the art depending on the pathology to be treated. Generally, systemic or local administrations can be envisaged and take the intravenous, infra-arterial, infra-peritoneal, infra-muscular or subcutaneous route, etc.
- Cells can be injected during the surgical operation or by any appropriate means, for example using a syringe.
- the cell composition can be administered before, during or after bone marrow transplantation bone (or organ).
- additional administrations may be performed after transplantation, in order to prevent or delay the pathology.
- CD3 + purified T lymphocytes were cultured in RPMI medium containing 10% human serum, interleukin 2, anti-CD3 antibodies and presence or absence of N-acetyl Cysteine (NAC). The percentage of CD3 + T cells expressing the CD90 marker was studied over time by flow cytometry.
- Figure 2 Effect of dendritic cells on the preferential expansion or not of human CD4 + CD90 + T cells.
- Dendritic cells derived from CD34 + cells and enriched on the CD la marker (Langerhansian DC) or on the CD 14 marker (interstitial DC) were cultured with allogenic T lymphocytes in a 1/5 ratio for 5 days. The percentage of CD3 + T cells expressing the CD90 marker was studied over time by flow cytometry.
- FIG. 3 Expression of CD25 and CD90 antigens by human CD4 + (A) and CD8 + (B) T cells.
- T cells were labeled with antibodies recognizing the CD4, CD8, CD25 and CD90 antigens. The expression of these different markers was carried out by flow cytometry.
- CD4 + / CD90 + and CD8 + / CD90 + T cells have a suppressive function
- the purified populations CD4CD90 +, CD4CD25 ++ and CD8CD90 + were irradiated with 15 grays, cultured at equal ratio for 4 days with autologous T lymphocytes depressed in CD25 (CD25-) cells stimulated (A) by a mixture of OKT3 / CD28 antibodies. , (B) by allogenic B lymphocytes transformed by EBN, (C) by allogenic dendritic cells (DC). CD25 cells stimulated alone under the same conditions represent the positive control. The proliferation is evaluated after 4 days by incorporation of tritiated thymidine.
- CD4, IL-10, CTLA-4 and FoxP3 genes was carried out by RT-PCR on the different CD4 + CD25-, CD4 + CD25 ++, CD4 + CD90 +, CD8 + CD90 + cell populations.
- CD90 identifies the precursors of suppressor lymphocytes CD4CD25 ++
- the cell populations CD4CD90 +, CD4CD25 +, CD4CD25 ++ were highly purified by cell sorting, cultured in RPMI medium containing human serum AB, 1TL-2 and a mixture of antibodies OKT3 / CD28 for 7 days. Analysis of the CD90 and CD25 markers was carried out at different culture times. On day 7, the populations were enriched by cell sorting, irradiated with 15 grays and tested for their suppressive capacity by co-cultivating them in equal ratio with allogenic T lymphocytes doubly depressed in CD25 and CD90 (CD25-CD90-) cells stimulated. by mixing OKT3 / CD28 antibodies.
- CD90 identifies suppressor CD4 + / CD90 + and CD8 + / CD90 + lymphocytes after 6 days of culture
- CD3 T cells were cultured in the presence of IL-2 and a mixture of OKT3 / CD28 antibodies for 6 days. Analysis of the markers CD25 and CD90 on the populations CD4 and CD8 makes it possible to determine the percentages CD25 + and CD90 + (A).
- the CD4CD25 ++, CD4CD90 + and CD8CD90 + cells were then sorted, irradiated and tested for their suppressive capacity by co-culturing them in equal ratio with allogenic T lymphocytes depleted in CD25 (CD25-) cells stimulated by mixing of OKT3 / CD28 antibodies ( B). The proliferation is evaluated after 4 days by incorporation of tritiated thymidine.
- CD90 allows identification from CD25-CD90 T cell culture - the appearance of precursor cells and suppressor lymphocytes
- T lymphocytes doubly depleted in CD25 and CD90 cells were cultured in the presence of IL-2 and a mixture of OKT3 / CD28 antibodies for 7 days. Analysis of the CD90 and CD25 markers was carried out at different culture times. On day 7, the CD25 + CD90 + population was enriched by cell sorting, irradiated with 15 grays and tested for its suppressive capacity by co-cultivating it in equal ratio with allogenic T lymphocytes doubly depleted in CD25 and CD90 cells (CD25-CD90 -) stimulated by mixing OKT3 / CD28 antibodies. The figures indicate the percentage of inhibition of proliferation compared to the control (CD25-CD90- cells cultured and stimulated alone).
- Figure 9 Use of the CD90 marker in human pathology: example of multiple sclerosis.
- Figure 10 Use of the CD90 marker in human pathology: example of a patient suffering from LPEX syndrome.
- Mononuclear cells obtained after Ficoll from samples from healthy donors, from patients with LPEX syndrome confirmed by sequencing of the FoXP3 gene were labeled with monoclonal antibodies CD4, CD25, CD90.
- the percentage of CD4 + T cells expressing the marker CD25 and CD90 was studied by flow cytometry. The results of an LPEX patient are shown.
- the CD90 marker is expressed by human CD4 + / CD25 + T cells and by human CD8 + / CD25 + lymphocytes
- CD90 marker compared to CD25 on the CD4 + and CD8 + T lymphocyte populations
- mononuclear cells from adult peripheral blood were obtained by Ficoll gradient and then labeled with the following antibodies directly to fluorochromes: anti-CD4 , anti-CD8, anti-CD25 and anti-CD90.
- the cells were analyzed by flow cytometry (FACscalibur), the events being re-analyzed on the Cellquest and FlowJo software.
- FIG. 3A shows the expression of the markers CD25 and CD90 on the CD4 + T lymphocytes as well as the co-expression of CD25 and CD90 within the CD4 + cells.
- 6% and 1.2% of the CD4 + T lymphocytes express, respectively, the markers CD25 and CD90.
- the majority (> 80%) of CD4 + / CD90 + cells express CD25 + at an intermediate level while approximately 5% and 15% of CD4 + / CD90 + cells are CD25 ++ and CD25- respectively.
- FIG. 3B shows the expression of the markers CD25 and CD90 on the CD8 + T lymphocytes as well as the coexpression of CD25 and CD90 within the CD8 +. As can be seen, 7% and 0.2% of CD8 + T cells express, respectively, the markers
- CD25 and CD90 are CD25 and CD90. It should be noted that no CD8 + cell strongly expresses CD25 unlike CD4 + cells. The majority of CD8 + / CD90 + cells (75%) are CD25- while about 25% of CD8 + / CD90- cells are CD25 +.
- the CD90 marker identifies human suppressor T lymphocytes in the CD4 + and CD8 + populations.
- CD4 + / CD90 + cells have a suppressive function
- autologous CD4 + T lymphocytes which have been screened in CD4 + / CD25 + cells (CD25-) were stimulated by a mixture of OKT3 / CD28 antibodies previously immobilized on the bottom of the culture wells.
- CD25- cells were cultured alone or in the presence of an equal number of CD4 + / CD90 + or CD4 + / CD25 + cells (positive control) for 4 days then the proliferation was measured by incorporation test for tritiated thymidine, the reading being made on ⁇ counter.
- Figure 4A shows the inhibition exerted populations CD4 + / CD90 + and CD4 + / CD25 ++ on the proliferation of T cells autologous CD25 ".
- CD8 + / CD90 + cells have a suppressive function
- these were brought into contact with an equal number of CD25- cells stimulated by mixing with a mixture of OKT3 / CD28 antibodies, the cell proliferation being evaluated four days later late by tritiated thymidine incorporation test.
- the results presented FIG. 4A show that the CD8 + / CD90 + population exerts a suppressive function on the proliferation of CD25- cells.
- CD4 + / 90 + and CD8 + CD90 + lymphocytes express Foxp3, CTLA4 and TGF ⁇
- CD90 makes it possible to identify a population of lymphocytes that are precursors of CD4 + / CD25-I- lymphocytes.
- the CD4 + / CD90 + cells were highly purified by flow cytometry (purity greater than 98%) and then cultured in a liquid medium in the presence of a mixture of antibodies OKT3 / CD28 and interleukin 2. cells were analyzed by flow cytometry sequentially between 1 day in culture and the 7th day of culture at markers CD4 / CD90 and CD4 / CD25.
- the sorted populations CD4 + / CD25 ++ and CD4 + / CD25 + / CD90- which represent respectively the conventional suppressor T cells and the activated T cells were cultivated in parallel under the same conditions. The results presented in FIG.
- CD4 + / CD90 + cells gradually lose the CD90 marker and become strongly positive for the CD25 marker.
- the CD4 + / CD25 + / CD90- population strongly acquires the CD25 marker without acquiring the CD90 marker.
- the cells were sorted, irradiated with 15 grays and placed in the presence of CD25-allogenic cells.
- CD4 + / CD90 + cells are capable of giving rise to CD4 + / CD25 ++ cells with suppressive activity; 2) CD4 + / CD25 ++ cells retain their suppressive activity; 3) CD4 + / CD25 + / CD90- give rise to CD25 ++ cells without suppressive activity.
- CD4 + / CD90 + cells are capable of giving rise to suppressive CD4 + CD25 ++ cells and can be considered as precursor cells (pTs) of suppressor lymphocytes (Ts).
- CD90 makes it possible to identify the CD4 + / CD90 + and CD8 + / CD90 + suppressor lymphocytes after culture.
- CD90 marker makes it possible, after activation and culture of T lymphocytes, to identify suppressor T lymphocytes unlike the CD25 marker also expressed by activated T lymphocytes.
- FIG. 7A shows the expression of the markers CD25 and CD90 on the T lymphocytes CD4 + and CD8 + after 6 days of culture. As can be seen, 6.9% and 10% of the CD4 + and CD8 + T lymphocytes express the CD90 marker respectively, while 84% and 94.3% of the CD4 + and CD8 + T lymphocytes express the CD25 marker.
- FIG. 7B shows the inhibition exerted by the CD4 + / CD90 + and CD8 + / CD90 + populations on the proliferation of autologous CD25 T lymphocytes, while the CD4 + / CD25 + lymphocytes no longer have any suppressing effect on CD25 cells after culture.
- CD90 + cells become CD90 + CD25 ++ while CD25 + CD90- cells become CD25 ++ CD90-.
- the sorting and the functional study of CD90 + CD25 ++ cells show that these cells inhibit the proliferation of autologous CD25-CD90 T lymphocytes stimulated by OKT3 / CD28 antibodies. These results show that it is possible to generate identifiable suppressor T lymphocytes using the CD90 marker from CD25- CD90- T lymphocytes.
- Ts tissue or organ of interest
- a biological fluid of interest blood, CSF, synovial fluid for example
- tissue or organ of interest tissue or organ of interest
- FIG. 9 shows an increase in CD4 + CD25 + T lymphocytes (activated T lymphocytes) and on the contrary a decrease in CD4 + CD90 + cells during MS in relapses compared to MS in chronic phase or in the control group.
- FIG. 10 shows the virtual absence in the blood of CD4 + / CD90 + cells in an LPEX patient compared to a healthy donor, while the use of the CD25 marker which also recognizes activated T lymphocytes does not make such a difference.
- Ts play an important role in the control of GNH and that it is possible to prepare specific Ts by allo-activation (Cohen et al. JEM 2003, Trénado et al., JCI 2003).
- the Ts can be obtained from blood, cord blood, bone marrow of all tissues containing T lymphocytes. In these applications, they may or may not be genetically modified.
- the Ts are obtained from the patient or from a compatible donor, preferably genome-identical. They are purified by immunomagnetic beads, flow cytometry, by adhesion to a solid support covered with specific antibodies (panning) and possibly frozen.
- the patient is the subject of biological monitoring to assess his number of Ts cells. The appearance of clinical signs indicating the beginning of a surge or fall in the number of Ts leads to the injection of Ts which are either prepared for this occasion, or those prepared previously.
- Ts prevent the development of effective anti-tumor immune reactions.
- the depletion of these cells allows these responses to develop.
- preparation of anti-tumor T cells ex vivo encounters the problem of their contamination by Ts.
- the principle of the treatment is therefore to eliminate the Ts in vivo, in particular using a CD90 ligand coupled to a toxic. It may also involve depleting all of the T lymphocytes by conventional treatments (anti-lymphocyte serum, anti-CD3, Campath antibody, irradiation, etc., for example). This treatment can be supplemented by the administration of lymphocytes activated ex vivo against tumor antigens after having been depleted in Ts.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Oncology (AREA)
- Wood Science & Technology (AREA)
- Diabetes (AREA)
- Hematology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Communicable Diseases (AREA)
- Cardiology (AREA)
- Obesity (AREA)
- Rheumatology (AREA)
- Surgery (AREA)
- Heart & Thoracic Surgery (AREA)
- Transplantation (AREA)
- Tropical Medicine & Parasitology (AREA)
- Vascular Medicine (AREA)
- Virology (AREA)
Abstract
The invention relates to the fields of biology, genetics and medicine. The invention describes methods and compositions enabling (1) the identification of suppressor T cells or lymphocytes (Ts) or the precursors thereof (pTs) for diagnostic and therapeutical purposes and for carrying out genomic and proteomic studies, i.e. for the identification of new markers and/or therapeutical targets for said cells; (2) the production of of suppressor T cells or lymphocytes (Ts) or the precursors thereof (pTs) and/or the manipulations thereof in vivo or ex vivo for controlling various pathological conditions, including diseases associated with abnormal activity of effector and/or regulator lymphocytes. The invention relates to the preparation of said compositions based on Ts lymphocytes and pTS and to the use thereof in cell therapies. The compositions or cell populations based on the Ts lymphocytes and pTs obtained according to the invention are particularly suitable for the treatment of tumours, auto-immune diseases, allergies, graft-versus-host disease, graft versus infection effects (GVI) or graft-versus-leukemia effects (GVL), inflammatory diseases, type 1 diabetes, viral, bacterial or parasitic infections, for immune reconstruction or induction of tolerance in the event of transplantation of stem cells, tissue or organs in mammals.
Description
Méthodes d'identification et de préparation de lymphocytes T régulateurs/suppresseurs, compositions et utilisations. Methods of identifying and preparing regulatory / suppressor T cells, compositions and uses.
La présente invention concerne les domaines de la biologie, de la génétique et de la médecine. L'invention décrit des méthodes et compositions permettant l'identification, la production et la manipulation ex vivo et in vivo de cellules ou lymphocytes T suppresseurs (Ts, y compris leurs précurseurs pTs), aussi appelés T régulateurs (ou Treg), et l'utilisation desdits lymphocytes suppresseurs pour contrôler des conditions pathologiques variées, incluant des maladies associées à une activité anormale des lymphocytes effecteurs et/ou des T régulateurs/suppresseurs. L'invention concerne la préparation de telles compositions à base de lymphocytes Ts et pTs, et leur utilisation dans le cadre de thérapies cellulaires et/ou géniques. Les compositions ou populations cellulaires à base de lymphocytes Ts et pTs obtenues dans le cadre de l'invention sont en particulier adaptées au traitement de maladies génétique ou acquises, notamment des tumeurs, des maladies auto-immunes, des allergies, de la maladie du greffon contre l'hôte, des effets greffe contre infection (GNI) ou greffe contre leucémie (GNL), des maladies inflammatoires incluant par exemple l'athérosclérose, du diabète, des infections virales, bactériennes ou parasitaires, à la reconstitution immune ou à l'induction d'une tolérance en cas de transplantation de cellules souches, tissus ou organe chez un mammifère.The present invention relates to the fields of biology, genetics and medicine. The invention describes methods and compositions for the identification, production and ex vivo and in vivo manipulation of suppressor T cells or lymphocytes (Ts, including their pTs precursors), also called regulatory T (or Treg), and use of said suppressor lymphocytes to control various pathological conditions, including diseases associated with abnormal activity of effector lymphocytes and / or regulatory / suppressor T cells. The invention relates to the preparation of such compositions based on Ts and pTs lymphocytes, and their use in the context of cellular and / or gene therapies. The compositions or cell populations based on Ts and pTs lymphocytes obtained in the context of the invention are in particular suitable for the treatment of genetic or acquired diseases, in particular tumors, autoimmune diseases, allergies, graft disease against the host, transplant against infection (GNI) or transplant against leukemia (GNL) effects, inflammatory diseases including, for example, atherosclerosis, diabetes, viral, bacterial or parasitic infections, immune reconstitution or induction of tolerance in the event of transplantation of stem cells, tissues or organ in a mammal.
L'existence dans le système immunitaire de cellules capables d'avoir des fonctions régulatrices/suppressives a été suspectée depuis longtemps. Dans les années 80, de nombreuses publications scientifiques ont montré l'existence d'activités suppressives au sein de la population lymphocytaire T. Cependant, l'impossibilité de caractériser et d'isoler des cellules portant une telle fonction à partir de la population lymphocytaire globale, qui possède par ailleurs de nombreuses autres fonctions incluant notamment celles de fonction effectrice, n'a pas permis de mieux comprendre ce phénomène. À partir de 1995, une sous-population de lymphocytes T CD4+ exprimant de façon constitutive le marqueur CD25 a été identifiée chez les rongeurs comme jouant un rôle majeur dans le contrôle des réponses immunes et des maladies auto-immunes. Ces cellules T CD4+/CD25+ appelées cellules T régulatrices ou suppressives (Ts)
représentent chez la souris environ 5-10 % des cellules T CD4+. Les cellules Ts expriment un récepteur T spécifique pour l'antigène, comme les autres lymphocytes T, mais leur action globale est en partie non-spécifique avec la possibilité de recruter d'autres lymphocytes T suppresseurs additionnels par un phénomène nommé " infectious suppression ". Chez l'homme, une population cellulaire régulatrice CD4+/CD25+, qui représente moins de 5 % des cellules T CD4+, a également été décrite.The existence in the immune system of cells capable of having regulatory / suppressive functions has been suspected for a long time. In the 1980s, numerous scientific publications showed the existence of suppressive activities within the T lymphocyte population. However, the impossibility of characterizing and isolating cells carrying such a function from the overall lymphocyte population , which also has many other functions including notably those of effector function, did not allow a better understanding of this phenomenon. From 1995, a sub-population of CD4 + T lymphocytes constitutively expressing the CD25 marker was identified in rodents as playing a major role in the control of immune responses and autoimmune diseases. These CD4 + / CD25 + T cells called regulatory or suppressive T cells (Ts) represent in mice about 5-10% of CD4 + T cells. Ts cells express a T receptor specific for the antigen, like other T lymphocytes, but their overall action is partly non-specific with the possibility of recruiting other additional suppressor T lymphocytes by a phenomenon called "infectious suppression". In humans, a CD4 + / CD25 + regulatory cell population, which represents less than 5% of CD4 + T cells, has also been described.
Plusieurs expériences ont maintenant clairement établi le potentiel thérapeutique des lymphocytes T suppresseurs CD4+/CD25+ dans de nombreuses maladies.Several experiments have now clearly established the therapeutic potential of CD4 + / CD25 + suppressor T cells in many diseases.
Ainsi, les cellules Ts jouent un rôle majeur dans le contrôle des maladies auto-immunes telles que le diabète de type I ou la maladie du greffon contre l'hôte (GNH) induites par les lymphocytes T allogéniques. L'addition de cellules Ts à des greffons contenant des cellules souches hématopoïétiques allogéniques et des lymphocytes T effecteurs peut contrôler la survenue ou l'émergence de la GNH. L'injection de cellules Ts peut réduire la réponse auto-immune dans les polymyosites auto-immunes (non publié). Les cellules Ts jouent aussi un rôle majeur pour l'établissement ou l'induction d'une tolérance lors de la transplantation de tissus ou d'organes et/ou en présence de molécules immunogéniques telles que des transgènes. Les cellules Ts jouent également un rôle important dans la modulation de la réponse aux agents infectieux, et notamment aux bactéries intra-cellulaires et aux virus.Thus, Ts cells play a major role in the control of autoimmune diseases such as type I diabetes or graft versus host disease (GNH) induced by allogenic T cells. Addition of Ts cells to grafts containing allogeneic hematopoietic stem cells and effector T cells can control the onset or emergence of GNH. Injection of Ts cells can reduce the autoimmune response in autoimmune polymyositis (unpublished). Ts cells also play a major role in establishing or inducing tolerance during the transplantation of tissues or organs and / or in the presence of immunogenic molecules such as transgenes. Ts cells also play an important role in modulating the response to infectious agents, including intracellular bacteria and viruses.
Les cellules Ts jouent un rôle dans plusieurs maladies inflammatoires telles que l'athérosclérose. Dans ce cas, l'absence ou la réduction du nombre de cellules Ts conduit à une accélération du développement de la maladie et à une augmentation de sa sévérité (résultats non publiés).Ts cells play a role in several inflammatory diseases such as atherosclerosis. In this case, the absence or reduction of the number of Ts cells leads to an acceleration of the development of the disease and an increase in its severity (unpublished results).
Il est maintenant bien établi que les cellules Ts empêchent le développement des réponses anti-tumorales effectrices, qui sans cela peuvent aboutir à l'éradication des tumeurs. Chez la souris, la déplétion des cellules Ts aboutit dans de nombreux modèles de cancer à l'éradication des tumeurs par une réponse immunitaire effectrice. Chez l'homme, une corrélation entre évolution défavorable et Ts a été décrite dans plusieurs
pathologies malignes. Les Ts tumorales sont associées à une survie réduite. De plus, la modulation médicamenteuse des Ts améliore les traitements à base de Lymphocytes Infiltrant les Tumeurs.It is now well established that Ts cells prevent the development of effector anti-tumor responses, which can otherwise lead to the eradication of tumors. In mice, depletion of Ts cells leads in many cancer models to the eradication of tumors by an effector immune response. In humans, a correlation between unfavorable course and Ts has been described in several malignant pathologies. Tumor Ts are associated with reduced survival. In addition, the drug modulation of Ts improves treatments based on Tumor Infiltrating Lymphocytes.
Les cellules Ts sont également importantes au cours de la vaccination puisqu'elles peuvent supprimer le développement d'une réponse immune spécifique. De même, la déplétion ou la réduction du nombre de cellules Ts améliore très sensiblement les effets d'une vaccination anti-cancéreuse.Ts cells are also important during vaccination since they can suppress the development of a specific immune response. Likewise, depletion or reduction in the number of Ts cells very significantly improves the effects of an anti-cancer vaccination.
Finalement, de nombreuses publications font maintenant état de la présence d'un nombre ou d'un pourcentage anormal de cellules Ts qui se manifestent dans le cadre de pathologies variées, et lors de l'évolution d'une maladie donnée.Finally, numerous publications now report the presence of an abnormal number or percentage of Ts cells which manifest themselves in the context of various pathologies, and during the course of a given disease.
Tous ces arguments montrent que l'identification, la sélection, l'expansion ou la déplétion des lymphocytes T régulateurs CD4+/CD25+ in vitro ou in vivo représentent un énorme potentiel diagnostic et thérapeutique pour de nombreuses pathologies et en particulier pour les maladies auto-immunes, les maladies inflammatoires, les maladies infectieuses, le cancer et les rejets de greffe.All these arguments show that the identification, selection, expansion or depletion of CD4 + / CD25 + regulatory T lymphocytes in vitro or in vivo represent an enormous diagnostic and therapeutic potential for many pathologies and in particular for autoimmune diseases. , inflammatory diseases, infectious diseases, cancer and transplant rejection.
La caractérisation des cellules Ts revêt ainsi une importance majeure. Bien que peu d'éléments soient connus concernant l'homéostasie et la régulation de cette population de lymphocytes Ts, il apparaît que le facteur de transcription Foxp3 joue un rôle important dans le développement et le fonctionnement des lymphocytes T suppresseurs CD4+/CD25+. Il n'est pas établi que Foxp3 est exprimé sur toutes les cellules Ts mais l'absence d'expression de Foxp3 chez la souris est corrélée à une perte dramatique des fonctions des cellules Ts, tandis que l'expression forcée de Foxp3 dans des lymphocytes T effecteurs transforme ces derniers en cellules Ts.The characterization of Ts cells is therefore of major importance. Although few elements are known concerning the homeostasis and the regulation of this population of Ts lymphocytes, it appears that the transcription factor Foxp3 plays an important role in the development and the functioning of suppressor T lymphocytes CD4 + / CD25 +. It is not established that Foxp3 is expressed on all Ts cells but the absence of expression of Foxp3 in mice is correlated with a dramatic loss of functions of Ts cells, while the forced expression of Foxp3 in lymphocytes T effectors transform these into Ts cells.
Bien que les marqueurs CD4 et CD25 caractérisent une population de cellules qui contient les lymphocytes T suppresseurs, il apparaît en fait que les fonctions suppressives ne sont pas entièrement portées par les cellules CD4+/CD25+ et surtout que tontes les cellules CD4+/CD25+ ne sont pas suppressives. En effet, CD25 est un
marqueur également exprimé par les cellules T effectrices activées. L'identification et la purification des cellules Ts sur la base d'un tel marqueur est un problème majeur puisqu'elles présentent le risque d'identifier et de purifier en réalité des cellules T effectrices activées. Dans le cadre d'une pathologie immunologique donnée, les lymphocytes T activés exprimant CD4 et CD25 ont de grande probabilité de contenir justement les T effecteurs contre lesquels une intervention thérapeutique est souhaitable. Ainsi l'utilisation de CD4 et CD25 dans un cadre diagnostique (identification) ne serait pas fiable, et dans un cadre thérapeutique (purification, injection) ferait encourir le risque d'être inefficace voire de majorer la pathologie.Although the markers CD4 and CD25 characterize a population of cells which contains the suppressor T lymphocytes, it appears in fact that the suppressive functions are not entirely carried by the CD4 + / CD25 + cells and especially that all the CD4 + / CD25 + cells are not suppressor. Indeed, CD25 is a marker also expressed by activated effector T cells. The identification and purification of Ts cells on the basis of such a marker is a major problem since they present the risk of identifying and actually purifying activated effector T cells. In the context of a given immunological pathology, activated T lymphocytes expressing CD4 and CD25 have a high probability of precisely containing the effector T against which a therapeutic intervention is desirable. Thus the use of CD4 and CD25 in a diagnostic setting (identification) would not be reliable, and in a therapeutic setting (purification, injection) would run the risk of being ineffective or even increasing the pathology.
Le meilleur marqueur actuellement connu comme susceptible de différencier les cellules Ts des lymphocytes T effecteurs activés est l'expression du facteur de transcription Foxp3. Cependant, ce facteur de transcription intra-cellulaire n'est pas utilisable dans le cadre de simples méthodes d'identification et de purification immunophénotypiques. D'autres marqueurs tels que CD62L permettent de mieux caractériser les cellules Ts mais ils sont loin de permettre une identification parfaite. De plus, plusieurs publications ont montré qu'il existait des activités suppressives au sein de la population CD4+/CD25- de même que dans certaines cellules CD8+. Il apparaît donc que l'utilisation diagnostique et thérapeutique des cellules Ts est clairement dépendante de leur identification propre et que les connaissances actuelles n'ont jusqu'à ce jour décrit aucun marqueur spécifique des lymphocytes T suppresseurs.The best marker currently known to be able to differentiate Ts cells from activated effector T lymphocytes is the expression of the transcription factor Foxp3. However, this intracellular transcription factor cannot be used in the context of simple immunophenotypic identification and purification methods. Other markers such as CD62L allow better characterization of Ts cells but they are far from allowing perfect identification. In addition, several publications have shown that suppressive activities exist in the CD4 + / CD25- population as well as in certain CD8 + cells. It therefore appears that the diagnostic and therapeutic use of Ts cells is clearly dependent on their own identification and that current knowledge has so far described no specific marker for suppressor T lymphocytes.
En outre, si la différentiation de certains lymphocytes Ts semble réalisée dans le thymus (ils sont souvent appelés Ts « naturels »), d'autres lymphocytes Ts pourraient être générés à la périphérie et rien n'est connu sur le développement ontogénique des Ts à partir des progéniteurs T.In addition, if the differentiation of certain Ts lymphocytes seems to be carried out in the thymus (they are often called "natural" Ts), other Ts lymphocytes could be generated at the periphery and nothing is known about the ontogenic development of Ts at from the T progenitors
La présente invention offre pour la première fois la possibilité d'identifier, d'isoler, d'analyser (transcriptome, protéome...) et de manipuler (culture, activation, déplétion, modifications génétiques...) des populations de cellules T suppressives, en particulier humaines, et notamment (i) des populations de précurseurs des Ts, et (ii) des populations de Ts pures parmi les cellules CD4+ et CD8+. La présente invention
découle de la découverte que la molécule CD90, encore appelée THY-1, représente un marqueur caractéristique des cellules Ts humaines CD4+ et/ou CD8+, et de leurs précurseurs et peut être utilisée efficacement pour identifier ces populations cellulaires.The present invention offers for the first time the possibility of identifying, isolating, analyzing (transcriptome, proteome, etc.) and of manipulating (culture, activation, depletion, genetic modifications, etc.) of T cell populations. suppressive, in particular human, and in particular (i) populations of Ts precursors, and (ii) populations of pure Ts among CD4 + and CD8 + cells. The present invention follows from the discovery that the molecule CD90, also called THY-1, represents a characteristic marker of human Ts cells CD4 + and / or CD8 +, and of their precursors and can be used effectively to identify these cell populations.
L'antigène THY-1 (Seki et al., 1985 ; Planelles et al., 1995) correspond à une glycoprotéine de surface bien caractérisée et ancrée à la membrane par un pont phosphatidyl-inositol. Cette protéine appartient à la superfamille des immunoglobulines et comprend environ 140 acides aminés (25-30 kDa). Cet antigène a été tout d'abord identifié comme un marqueur de différenciation exprimé dans le thymus et le cerveau de souris. Chez l'homme, THY-1 est exprimé sur un faible pourcentage de thymocytes fœtaux, sur les progéniteurs hématopoïétiques CD34+ immatures et sur moins de 1 % des lymphocytes CD3+ présents dans la circulation périphérique. THY-1 est aussi exprimé sur les cellules mésenchymateuses, les cellules endothéliales ainsi que sur plusieurs lignées cellulaires continues. La fonction de THY-1 n'est pas connue.The THY-1 antigen (Seki et al., 1985; Planelles et al., 1995) corresponds to a well-characterized surface glycoprotein anchored to the membrane by a phosphatidyl-inositol bridge. This protein belongs to the immunoglobulin superfamily and contains around 140 amino acids (25-30 kDa). This antigen was first identified as a differentiation marker expressed in the thymus and the mouse brain. In humans, THY-1 is expressed on a small percentage of fetal thymocytes, on immature hematopoietic progenitors CD34 + and on less than 1% of CD3 + lymphocytes present in the peripheral circulation. THY-1 is also expressed on mesenchymal cells, endothelial cells as well as on several continuous cell lines. The function of THY-1 is not known.
Chez la souris, THY-1 est exprimé au niveau du thymus sur les progéniteurs et précurseurs T. Il est également exprimé sur des cellules régulatrices (Mukasa et al., Clin. Exp. Immunol. 96 (1994) 138 ; Torre-Amione et al., Cell. Immunol. 124 (1989) 50 ; Sakatsume et al., Int. Immunol. 3 (1991) 377) ainsi que sur l'ensemble des lymphocytes T circulants. De ce fait, il ne peut constituer un marqueur discriminant d'un type cellulaire particulier. Par ailleurs, deux isoformes Thy-1.1 et Thy-1.2 ont été décrites chez la souris.In mice, THY-1 is expressed at the level of the thymus on the progenitors and precursors T. It is also expressed on regulatory cells (Mukasa et al., Clin. Exp. Immunol. 96 (1994) 138; Torre-Amione and al., Cell. Immunol. 124 (1989) 50; Sakatsume et al., Int. Immunol. 3 (1991) 377) as well as on all circulating T lymphocytes. Therefore, it cannot constitute a discriminating marker of a particular cell type. Furthermore, two isoforms Thy-1.1 and Thy-1.2 have been described in mice.
Les inventeurs ont maintenant découvert que, de manière surprenante, l'expression de la molécule THY-1 chez l'homme est très étroitement corrélée à l'activité Ts, et que la molécule THY-1 constitue un marqueur spécifique de lymphocytes T suppresseurs, permettant notamment l'identification, la sélection, l'expansion ou la déplétion in vitro ou in vivo de précurseurs des Ts et/ou de populations Ts pures parmi les lymphocytes CD4+ ou CD8+.The inventors have now discovered that, surprisingly, the expression of the THY-1 molecule in humans is very closely correlated with Ts activity, and that the THY-1 molecule constitutes a specific marker for suppressor T lymphocytes, allowing in particular the identification, selection, expansion or depletion in vitro or in vivo of precursors of Ts and / or pure Ts populations among CD4 + or CD8 + lymphocytes.
Un premier aspect de la présente l'invention concerne donc une méthode d'obtention, de préparation ou de production de lymphocytes T suppresseurs (et/ou leurs
précurseurs), comprenant une étape de sélection, de séparation, et/ou d'isolement des lymphocytes T exprimant la molécule THY-1. Cette étape peut être réalisée à partir de tous échantillons biologiques comprenant des lymphocytes.A first aspect of the present invention therefore relates to a method for obtaining, preparing or producing suppressor T lymphocytes (and / or their precursors), comprising a step of selection, separation, and / or isolation of T lymphocytes expressing the THY-1 molecule. This step can be carried out from any biological samples comprising lymphocytes.
Un objet plus particulier de la présente invention concerne une méthode d'obtention, de préparation ou de production de lymphocytes T suppresseurs (et/ou leurs précurseurs) comprenant :A more particular subject of the present invention relates to a method for obtaining, preparing or producing suppressor T lymphocytes (and / or their precursors) comprising:
(a) l'obtention d'une population de cellules de mammifère comprenant des lymphocytes T, et (b) la récupération des lymphocytes T exprimant l'antigène THY-1.(a) obtaining a population of mammalian cells comprising T lymphocytes, and (b) recovering T lymphocytes expressing the THY-1 antigen.
Les lymphocytes T exprimant l'antigène THY-1 sont de préférence sélectionnés, séparés, isolés, récupérés ou éliminés au moyen d'un ligand spécifique de THY-1. Avantageusement, le ligand est choisi parmi un anticorps ou un fragment d'anticorps. Le ligand peut être par exemple immobilisé sur un support ou placé en solution. Un tel ligand est plus amplement défini dans la description qui suit de l'invention. En outre, l'étape (b) peut être précédée et/ou suivie d'une étape d'amplification des lymphocytes T et/ou d'une étape de purification de sous population(s) de lymphocytes, comme par exemple les lymphocytes CD4+ ou CD8+, voire de lymphocytes spécifiques d'un antigène donné.The T lymphocytes expressing the THY-1 antigen are preferably selected, separated, isolated, recovered or eliminated by means of a specific ligand for THY-1. Advantageously, the ligand is chosen from an antibody or an antibody fragment. The ligand can for example be immobilized on a support or placed in solution. Such a ligand is more fully defined in the following description of the invention. In addition, step (b) can be preceded and / or followed by a step of amplification of the T lymphocytes and / or of a step of purification of subpopulation (s) of lymphocytes, such as for example the CD4 + lymphocytes. or CD8 +, or even lymphocytes specific for a given antigen.
Un autre objet de l'invention concerne une méthode d'identification et/ou de quantification de lymphocytes T suppresseurs (et/ou leurs précurseurs) dans une population cellulaire, comprenant l'exposition de ladite population cellulaire à un ligand spécifique de THY-1 et la détermination et/ou la quantification de la formation d'un complexe entre le ligand et les cellules, la formation de tels complexes indiquant la présence et/ou la quantité de lymphocytes T suppresseurs (et/ou leur précurseurs) au sein de la population cellulaire. Les cellules liant le ligand peuvent être séparées des cellules ne liant pas le ligand.Another subject of the invention relates to a method of identifying and / or quantifying suppressor T lymphocytes (and / or their precursors) in a cell population, comprising the exposure of said cell population to a specific ligand for THY-1 and determining and / or quantifying the formation of a complex between the ligand and the cells, the formation of such complexes indicating the presence and / or the amount of suppressor T lymphocytes (and / or their precursors) within the cell population. Ligand-binding cells can be separated from non-ligand-binding cells.
Un autre objet de l'invention concerne l'utilisation d'un ligand spécifique de l'antigène THY-1 pour enrichir ou éliminer ex vivo les lymphocytes T suppresseurs (et/ou leurs
précurseurs) d'une population cellulaire. L'antigène THY-1 peut lui-même être utilisé comme marqueur de sélection des lymphocytes Ts ou pTs au sein d'une population cellulaire. Un autre objet de l'invention réside dans une méthode de diagnostic d'un patient, comprenant la détermination de la présence, du nombre ou de l'état d'activité de cellules Ts chez ce patient en utilisant un ligand spécifique de THY-1. Un tel diagnostic peut être réalisé in vitro, ex vivo ou in vivo, et permettre la détection d'un état pathologique lié à l'activité du système immunitaire, ou le suivi de l'efficacité d'un traitement, ou la sélection d'un patient en vue de l'inclure dans un programme thérapeutique particulier.Another subject of the invention relates to the use of a specific ligand for the THY-1 antigen for enriching or eliminating ex vivo the suppressor T lymphocytes (and / or their precursors) of a cell population. The THY-1 antigen can itself be used as a marker for the selection of Ts or pTs lymphocytes within a cell population. Another object of the invention resides in a method of diagnosing a patient, comprising determining the presence, number or state of activity of Ts cells in this patient using a specific ligand for THY-1 . Such a diagnosis can be carried out in vitro, ex vivo or in vivo, and allow the detection of a pathological condition linked to the activity of the immune system, or the monitoring of the effectiveness of a treatment, or the selection of a patient for inclusion in a particular therapeutic program.
Un autre objet de l'invention réside dans l'utilisation d'un ligand spécifique de l'antigène THY-1 pour sélectionner, identifier, trier ou préparer (in vitro ou ex vivo) des lymphocytes Ts ou pTs .Another object of the invention resides in the use of a specific ligand for the THY-1 antigen for selecting, identifying, sorting or preparing (in vitro or ex vivo) Ts or pTs lymphocytes.
L'invention concerne en outre les lymphocytes T suppresseurs (et/ou leur précurseurs) exprimant l'antigène THY-1 susceptibles d'être obtenus grâce à une méthode selon l'invention.The invention further relates to suppressor T lymphocytes (and / or their precursors) expressing the THY-1 antigen capable of being obtained by a method according to the invention.
Un autre objet de l'invention réside dans l'utilisation d'un ligand spécifique de l'antigène THY-1 pour préparer une composition diagnostique destinée à sélectionner, identifier ou quantifier in vivo des lymphocytes T suppresseurs (y compris leurs précurseurs).Another object of the invention resides in the use of a specific ligand for the THY-1 antigen for preparing a diagnostic composition intended for selecting, identifying or quantifying in vivo suppressor T lymphocytes (including their precursors).
Un autre objet de l'invention concerne l'utilisation d'un ligand spécifique de l'antigène THN -1 pour préparer une composition thérapeutique destinée à modifier, stimuler ou supprimer in vivo des lymphocytes T suppresseurs. A cet égard, un objet particulier de l'invention concerne l'utilisation d'un ligand spécifique de THY-1 pour enrichir ou éliminer ex vivo ou in vivo les lymphocytes T suppresseurs, (y compris leurs précurseurs) d'une population cellulaire.Another subject of the invention relates to the use of a specific ligand for the THN -1 antigen for preparing a therapeutic composition intended to modify, stimulate or suppress suppressor T lymphocytes in vivo. In this regard, a particular object of the invention relates to the use of a specific THY-1 ligand for enriching or eliminating ex vivo or in vivo suppressor T lymphocytes (including their precursors) from a cell population.
Un autre aspect de l'invention réside dans l'utilisation de l'antigène THY-1 comme marqueur de sélection pour enrichir ou éliminer, in vivo, in vitro ou ex vivo, les lymphocytes Ts ou pTs au sein d'une population cellulaire.
L'invention concerne en outre les lymphocytes T suppresseurs (y compris leurs précurseurs) exprimant l'antigène THY-1 susceptibles d'être obtenus par une méthode telle que définie précédemment, ainsi qu'une population de cellules enrichie en cellules Ts ou pTs, dans laquelle au moins 30%, de préférence au moins 50%, de manière encore plus préférée au moins 65% des cellules T expriment l'antigène THY-1. Des compositions ou populations cellulaires particulièrement préférées selon la présente invention comprennent au moins 75%, de préférence au moins 80% de cellules Ts ou pTs exprimant THY-1, plus préférentiellement au moins 85, 90 ou 95%.Another aspect of the invention lies in the use of the THY-1 antigen as a selection marker for enriching or eliminating, in vivo, in vitro or ex vivo, Ts or pTs lymphocytes within a cell population. The invention further relates to suppressor T lymphocytes (including their precursors) expressing the THY-1 antigen capable of being obtained by a method as defined above, as well as a population of cells enriched in Ts or pTs cells, in which at least 30%, preferably at least 50%, even more preferably at least 65% of the T cells express the THY-1 antigen. Particularly preferred compositions or cell populations according to the present invention comprise at least 75%, preferably at least 80% of Ts or pTs cells expressing THY-1, more preferably at least 85, 90 or 95%.
L'invention concerne encore un lymphocyte T humain isolé, caractérisé en ce qu'il présente une activité suppressive et en ce qu'il exprime les marqueurs CD8 ou CD4 et THY-1, ainsi qu'une population de cellules comprenant des cellules T suppressives CD8+THY-1+ ou CD4+THY-1+, de préférence une population comprenant au moins, 50, 60, 70, 80, 85, 90 ou 95% de cellules T CD8+THY-1+. De telles cellules peuvent, en partie exprimer également l'antigène CD25.The invention also relates to an isolated human T lymphocyte, characterized in that it exhibits suppressive activity and in that it expresses the markers CD8 or CD4 and THY-1, as well as a population of cells comprising suppressive T cells CD8 + THY-1 + or CD4 + THY-1 +, preferably a population comprising at least 50, 60, 70, 80, 85, 90 or 95% of CD8 + THY-1 + T cells. Such cells can, in part, also express the CD25 antigen.
Dans un mode de réalisation particulier de l'invention, les lymphocytes T présents dans la population de cellules de mammifère ou les lymphocytes Ts ou pTs (porteurs du marqueur THY-1) peuvent être modifiés génétiquement de manière à exprimer des produits biologiques d'intérêt, permettant notamment d'améliorer leur efficacité et/ou leur sécurité d'utilisation.In a particular embodiment of the invention, the T lymphocytes present in the mammalian cell population or the Ts or pTs lymphocytes (carrying the THY-1 marker) can be genetically modified so as to express biological products of interest , allowing in particular to improve their efficiency and / or their safety of use.
L'invention concerne également une composition pharmaceutique comprenant des cellules ou populations cellulaires telles que définies précédemment, typiquement en association avec un véhicule ou excipient acceptable sur le plan pharmaceutique.The invention also relates to a pharmaceutical composition comprising cells or cell populations as defined above, typically in combination with a pharmaceutically acceptable vehicle or excipient.
Un autre objet particulier de l'invention concerne une composition pharmaceutique comprenant des cellules T suppresseurs (et/ou leurs précurseurs) humaines amplifiées ex vivo et un adjuvant ou un milieu acceptable sur le plan pharmaceutique, lesdites cellules amplifiées étant enrichies en cellules exprimant l'antigène THY-1 et, éventuellement, en cellules spécifiques d'un antigène particulier, tels que des allergènes,
des auto-antigènes, des allo-antigènes ou des antigènes d'agents infectieux. De manière préférée, l'antigène est impliqué dans ou spécifique d'une condition pathologique choisie parmi une maladie immunitaire, en particulier les maladies auto-immunes, les maladies inflammatoires, la maladie du greffon contre l'hôte, une allergie et le rejet d'une greffe.Another particular subject of the invention relates to a pharmaceutical composition comprising suppressor T cells (and / or their precursors) amplified ex vivo and an adjuvant or a pharmaceutically acceptable medium, said amplified cells being enriched in cells expressing the THY-1 antigen and, possibly, in specific cells of a particular antigen, such as allergens, auto-antigens, allo-antigens or antigens of infectious agents. Preferably, the antigen is involved in or specific to a pathological condition chosen from an immune disease, in particular autoimmune diseases, inflammatory diseases, graft versus host disease, allergy and rejection of 'transplant.
L'invention concerne également la préparation d'une composition constituée d'au moins un tel lymphocyte T suppresseur, d'une population enrichie en cellules Ts et/ou pTs telle que définie ci-dessus ou au contraire d'une population déplétée en cellules Ts et/ou pTs et d'un adjuvant ou d'un milieu acceptable sur le plan pharmaceutique ainsi que la composition elle-même destinée à la mise en œuvre d'une méthode thérapeutique.The invention also relates to the preparation of a composition consisting of at least one such suppressor T lymphocyte, of a population enriched in Ts cells and / or pTs as defined above or on the contrary of a population depleted in cells. Ts and / or pTs and an adjuvant or a pharmaceutically acceptable medium as well as the composition itself intended for the implementation of a therapeutic method.
Un objet particulier de l'invention concerne ainsi également une méthode de production d'une composition pharmaceutique, comprenant :A particular subject of the invention thus also relates to a method of producing a pharmaceutical composition, comprising:
(a) l'obtention d'un échantillon biologique comprenant des lymphocytes T,(a) obtaining a biological sample comprising T lymphocytes,
(b) la sélection des lymphocytes T exprimant l'antigène THY-1 au sein de cet échantillon biologique, et(b) the selection of T lymphocytes expressing the THY-1 antigen within this biological sample, and
(c) le conditionnement desdits lymphocytes T exprimant l'antigène THY-1 dans un adjuvant ou un milieu acceptable sur le plan pharmaceutique.(c) conditioning said T lymphocytes expressing the THY-1 antigen in an adjuvant or a pharmaceutically acceptable medium.
Un autre objet particulier de l'invention concerne ainsi également une méthode de production d'une composition pharmaceutique, comprenant : (a) l'obtention d'un échantillon biologique comprenant des lymphocytes T, (b) la déplétion des lymphocytes T exprimant l'antigène THY-1 au sein de cet échantillon biologique, et (c) le conditionnement desdits lymphocytes T obtenus n'exprimant pas l'antigène THY- 1 dans un adjuvant ou un milieu acceptable sur le plan pharmaceutique.Another particular object of the invention thus also relates to a method for producing a pharmaceutical composition, comprising: (a) obtaining a biological sample comprising T lymphocytes, (b) depletion of T lymphocytes expressing the THY-1 antigen within this biological sample, and (c) the conditioning of said T lymphocytes obtained which do not express the THY-1 antigen in an adjuvant or a pharmaceutically acceptable medium.
L'invention concerne par ailleurs un kit pour isoler ou caractériser des cellules Ts comprenant un ligand spécifique de THY-1, éventuellement disposé sur un support ou placé en solution ainsi que, éventuellement, des réactifs pour la détection du ligand. Le
ligand est typiquement disposé dans un conteneur, tel qu'une plaque, seringue, tube, pipette, flacon, etc. Un tel kit peut en outre être utilisé pour diagnostiquer la présence de telles cellules Ts et pTs à partir d'un échantillon biologique prélevé sur l'un individu à tester ou directement in vivo.The invention further relates to a kit for isolating or characterizing Ts cells comprising a specific ligand for THY-1, optionally placed on a support or placed in solution as well as, possibly, reagents for detecting the ligand. The ligand is typically placed in a container, such as a plate, syringe, tube, pipette, vial, etc. Such a kit can also be used to diagnose the presence of such Ts cells and pTs from a biological sample taken from the individual to be tested or directly in vivo.
L'invention concerne par ailleurs un kit ou une composition pour éliminer les cellules Ts et pTs in vivo, in vitro ou ex vivo, comprenant un ligand spécifique de THY-1, éventuellement placé en solution ou sur un support, et couplé à un produit toxique (radioactif, toxines...). L'invention vise également l'utilisation d'un ligand de Thy-1 pour cibler spécifiquement un vecteur viral ou non viral dans les Ts et pTs afin d'exprimer des gènes.The invention further relates to a kit or a composition for eliminating Ts cells and pTs in vivo, in vitro or ex vivo, comprising a specific ligand for THY-1, optionally placed in solution or on a support, and coupled to a product. toxic (radioactive, toxins ...). The invention also relates to the use of a Thy-1 ligand to specifically target a viral or non-viral vector in Ts and pTs in order to express genes.
L'invention concerne par ailleurs un kit ou une composition pour activer les cellules Ts et pTs in vivo, ex vivo ou in vitro, comprenant un ligand spécifique de THY-1, éventuellement placé en solution ou sur un support, couplé à un produit capable d'activer les lymphocytes T (par exemple une cytokme, telle que IL2, IL7, IL 10, IL15). L'invention vise également l'utilisation d'un ligand de THY-1 pour cibler spécifiquement un vecteur viral ou non viral dans les Ts afin d'exprimer des gènes activateurs ou tous gènes thérapeutiques.The invention further relates to a kit or a composition for activating Ts and pTs cells in vivo, ex vivo or in vitro, comprising a specific ligand for THY-1, optionally placed in solution or on a support, coupled to a product capable activate T cells (eg cytokme, such as IL2, IL7, IL 10, IL15). The invention also relates to the use of a THY-1 ligand to specifically target a viral or non-viral vector in Ts in order to express activator genes or all therapeutic genes.
Les cellules Ts, les compositions constituées de cellules Ts et pTs isolées ou amplifiées et les compositions enrichies en cellules Ts et pTs obtenues dans le cadre de la présente invention peuvent être avantageusement utilisées dans le cadre d'applications expérimentales ou thérapeutiques. Les cellules utilisées dans le cadre de la présente invention sont des cellules de mammifère, typiquement humaines. L'invention est également utilisable notamment chez les primates, et concerne donc également des cellules T suppresseurs de primates, notamment de singe.Ts cells, the compositions made up of isolated or amplified Ts and pTs cells and the compositions enriched in Ts and pTs cells obtained within the framework of the present invention can be advantageously used within the framework of experimental or therapeutic applications. The cells used in the context of the present invention are mammalian cells, typically human. The invention can also be used in particular in primates, and therefore also relates to primate suppressor T cells, in particular monkey.
Un objet particulier de l'invention a ainsi également trait à des méthodes d'analyse et d'obtention de séquences génétiques spécifiquement exprimées dans des lymphocytes T suppresseurs (ou leurs précurseurs), une méthode comprenant l'isolement de l'ARN d'une population de lymphocytes T exprimant l'antigène Thy-1, la comparaison dudit
ARN à l'ARN extrait d'une population de lymphocytes T non suppresseurs et la récupération de l'ARN spécifique des lymphocytes T suppresseurs. L'invention concerne également une méthode telle que décrite précédemment comprenant en outre la fabrication d'une sonde à partir de l'ARN spécifique des lymphocytes T suppresseurs (ou leur précurseurs) et le criblage d'une population d'acides nucléiques destinés à être hybrides à ladite sonde. Une méthode correspond également à l'analyse du transcriptome par hybridation d'ARN sur des biopuces afin d'établir des profils d'expression. Ces différentes méthodes aboutissent à caractériser l'expression des gènes importants pour la différentiation, la maturation, la régulation et la fonction des Ts et pTs, permettant ainsi de définir de nouveaux marqueurs et/ou cibles thérapeutiques potentielles.A particular subject of the invention thus also relates to methods of analysis and of obtaining genetic sequences specifically expressed in suppressor T lymphocytes (or their precursors), a method comprising the isolation of RNA from a population of T lymphocytes expressing the Thy-1 antigen, comparison of said RNA to RNA extracted from a population of non-suppressor T cells and recovery of RNA specific for suppressor T cells. The invention also relates to a method as described above, further comprising the manufacture of a probe from the RNA specific for suppressor T lymphocytes (or their precursors) and the screening of a population of nucleic acids intended to be hybrid to said probe. One method also corresponds to the analysis of the transcriptome by RNA hybridization on biochips in order to establish expression profiles. These different methods result in characterizing the expression of genes important for the differentiation, maturation, regulation and function of Ts and pTs, thus making it possible to define new markers and / or potential therapeutic targets.
Un objet particulier de l'invention a ainsi trait à une méthode d'obtention de protéines spécifiquement exprimées dans des lymphocytes T suppresseurs (ou leurs précurseurs). Une méthode comprend l'isolement des protéines d'une population de lymphocytes T exprimant l'antigène Thy-1, la comparaison de ces protéines à celles extraites d'une population de lymphocytes T non suppresseurs. Ces différentes méthodes aboutissent à caractériser l'expression de protéines importantes pour la différentiation, la maturation, la régulation et la fonction des Ts et pTs, permettant de définir ainsi de nouveaux marqueurs et/ou cibles thérapeutiques potentielles.A particular object of the invention thus relates to a method for obtaining proteins specifically expressed in suppressor T lymphocytes (or their precursors). One method includes isolating proteins from a population of T cells expressing the Thy-1 antigen, comparing these proteins to those extracted from a population of non-suppressor T cells. These different methods result in characterizing the expression of proteins important for the differentiation, maturation, regulation and function of Ts and pTs, thus making it possible to define new markers and / or potential therapeutic targets.
Un objet particulier de l'invention a ainsi trait à une méthode d'identification de nouvelles molécules spécifiquement exprimées dans des lymphocytes T suppresseurs (ou leurs précurseurs) par immunisation à l'aide de lymphocytes Ts (ou pTs) exprimant l'antigène Thy-1, ou de fractions cellulaires ou protéiques de ces mêmes cellules.A particular object of the invention thus relates to a method of identifying new molecules specifically expressed in suppressor T lymphocytes (or their precursors) by immunization using Ts lymphocytes (or pTs) expressing the Thy- antigen. 1, or cell or protein fractions of these same cells.
Un autre objet particulier de l'invention concerne en outre l'utilisation, dans un cadre, thérapeutique, des cellules Ts (ou pTs), des compositions constituées de cellules Ts (ou pTs) isolées ou amplifiées et des compositions enrichies en cellules Ts (ou pTs) obtenues dans le cadre de la présente invention, par exemple pour traiter de nombreux sujets, par exemple des patients humains souffrant de ou présentant un risque de développer une maladie immunitaire, en particulier une maladie provoquée par une
réponse T anormale. Les cellules Ts (ou pTs) sont ainsi adaptées au traitement de diverses pathologies ou affections provoquées par un désordre affectant les lymphocytes T et en particulier une tumeur, une maladie auto-immune, une allergie, la maladie du greffon contre l'hôte, une maladie inflammatoire, un diabète de type I, une infection virale ou bactérienne, etc. Elles favorisent également la reconstitution immune et l'induction d'une tolérance en cas de greffe ou de transplantation de cellules souches, tissus ou organe chez un mammifère. C'est le cas,. par exemple suite à une greffe de moelle osseuse ou de cellules souches hématopoïétiques. Le traitement peut être préventif ou curatif. Il peut également être combiné à d'autres traitements.Another particular object of the invention also relates to the use, in a therapeutic context, of Ts cells (or pTs), compositions made up of isolated or amplified Ts cells (or pTs) and compositions enriched in Ts cells ( or pTs) obtained in the context of the present invention, for example for treating numerous subjects, for example human patients suffering from or at risk of developing an immune disease, in particular a disease caused by a abnormal T response. Ts cells (or pTs) are thus suitable for the treatment of various pathologies or conditions caused by a disorder affecting T lymphocytes and in particular a tumor, an autoimmune disease, an allergy, graft versus host disease, a inflammatory disease, type I diabetes, viral or bacterial infection, etc. They also promote immune reconstitution and the induction of tolerance in the event of transplantation or transplantation of stem cells, tissues or organs in a mammal. It's the case,. for example following a bone marrow or hematopoietic stem cell transplant. Treatment can be preventive or curative. It can also be combined with other treatments.
Cellules T suppresseurs (ou leur précurseurs^ humainesSuppressor T cells (or their human precursors ^
Dans le contexte de la présente invention, le terme lymphocytes (ou cellules) T suppresseurs désigne une population de cellules T qui sont caractérisées par leur capacité à supprimer ou diminuer les réactions immunitaires médiées par les cellules T effectrices, telles que les cellules T CD4+ ou CD8+. Ce terme inclut les cellules Ts conventionnelles, qui expriment fortement le marqueur CD25, de même que leurs précurseurs, désignés pTs, qui sont doués d'activité suppressive et peuvent donner naissance, en culture, aux cellules Ts conventionnelles. L'invention démontre en effet l'existence d'une population de cellules T suppressives, désignées pTs, exprimant les marqueurs THY-1 et CD25, douée de propriété suppressive, et capable de donner naissance en culture aux cellules Ts conventionnelles. Le terme lymphocytes T suppresseurs inclut également des lymphocytes Ts issus de populations lymphocytaires totales (ou CD25-), de type CD4+ ou CD8+, exprimant THY-1.In the context of the present invention, the term suppressor T lymphocytes (or cells) designates a population of T cells which are characterized by their capacity to suppress or reduce the immune reactions mediated by effector T cells, such as CD4 + T cells or CD8 +. This term includes conventional Ts cells, which strongly express the CD25 marker, as well as their precursors, designated pTs, which are endowed with suppressive activity and can give rise, in culture, to conventional Ts cells. The invention indeed demonstrates the existence of a population of suppressive T cells, designated pTs, expressing the markers THY-1 and CD25, endowed with suppressive property, and capable of giving rise in culture to conventional Ts cells. The term suppressor T lymphocytes also includes Ts lymphocytes originating from total lymphocyte populations (or CD25-), of the CD4 + or CD8 + type, expressing THY-1.
Comme indiqué dans l'introduction de la présente demande, bien que les marqueurs CD4 et CD25 caractérisent les lymphocytes T suppresseurs (ou leurs précurseurs) sur un plan immunophénotypique, il apparaît en fait que les fonctions suppressives ne sont pas entièrement portées par les cellules CD4+/CD25+ et que toutes les cellules CD4+/CD25+ ne sont pas suppressives. CD25 est en effet un marqueur également exprimé par les cellules T effectrices activées. La présente invention découle de la mise en évidence que la molécule antigénique THY-1 représente un marqueur caractéristique
des cellules Ts et pTs humaines et peut être utilisée efficacement pour identifier cette population cellulaire.As indicated in the introduction to the present application, although the markers CD4 and CD25 characterize suppressor T lymphocytes (or their precursors) on an immunophenotypic level, it appears in fact that the suppressive functions are not entirely carried by CD4 + cells / CD25 + and that all CD4 + / CD25 + cells are not suppressive. CD25 is indeed a marker also expressed by activated effector T cells. The present invention follows from the demonstration that the antigenic molecule THY-1 represents a characteristic marker human Ts and pTs cells and can be used effectively to identify this cell population.
La présente invention concerne ainsi, comme indiqué précédemment, une méthode d'obtention, de préparation, de sélection ou de production de lymphocytes T suppresseurs (y compris leurs précurseurs) humains comprenant :The present invention thus relates, as indicated above, to a method for obtaining, preparing, selecting or producing human suppressor T lymphocytes (including their precursors) comprising:
(a) l'obtention d'une population de cellules d'origine humaine comprenant des lymphocytes T, et(a) obtaining a population of cells of human origin comprising T lymphocytes, and
(b) la récupération des lymphocytes T exprimant THY-1.(b) recovery of T lymphocytes expressing THY-1.
La population cellulaire peut être obtenue, dans le cadre de l'étape (a), à partir d'échantillons biologiques comprenant des lymphocytes, notamment d'échantillons d'un tissu choisi parmi la moelle osseuse, la rate, le foie, le thymus, du sang ayant ou non été préalablement enrichi en lymphocytes T, du sang de cordon ombilical, du sang périphérique fœtal, de nouveaux-nés ou d'adulte, du plasma, d'un ganglion lymphatique, d'une tumeur, d'un site inflammatoire, d'un organe transplanté ou d'une culture de cellules établie avec l'un ou l'autre desdits tissus. Les lymphocytes sont typiquement isolés ou collectés à partir du sang périphérique.The cell population can be obtained, in the context of step (a), from biological samples comprising lymphocytes, in particular from samples of a tissue chosen from bone marrow, spleen, liver, thymus , blood that may or may not have been previously enriched for T lymphocytes, umbilical cord blood, peripheral fetal blood, newborns or adults, plasma, a lymph node, a tumor, a inflammatory site, a transplanted organ, or a cell culture established with either of these tissues. Lymphocytes are typically isolated or collected from peripheral blood.
Les lymphocytes T exprimant THY-1 peuvent être récupérés, sélectionnés, isolés, dépiétés ou triés, notamment lors de l'étape (b), à l'aide de tous ligands spécifiques de THY-1, c'est-à-dire typiquement de toutes molécules capables de lier sélectivement Thy-1 à la surface d'une cellule. Le ligand est de préférence choisi parmi un anticorps, de préférence un anticorps anti-THY-1, un analogue ou un fragment d'un tel anticorps.The T lymphocytes expressing THY-1 can be recovered, selected, isolated, removed or sorted, in particular during step (b), using any specific ligands for THY-1, that is to say typically of all molecules capable of selectively binding Thy-1 to the surface of a cell. The ligand is preferably chosen from an antibody, preferably an anti-THY-1 antibody, an analog or a fragment of such an antibody.
THY-1 est une molécule dépourvue de domaine intra-cytoplasmique qui interagit avec la membrane cellulaire par l'intermédiaire d'un glycophosphatidylinositol (GPI) qui s'attache à la membrane par son extrémité C-terminale. La séquence de Thy-1 a été déterminée et est accessible dans la littérature, comme par exemple la séquence nucléotidique ( n° NM 006288 (gi : 199 233 61)) et la séquence en acides aminés (n° P 006279 (gi : 199 233 62)) de la protéine humaine. Un ligand spécifique selon l'invention est de préférence une molécule capable de lier sélectivement un polypeptide
comprenant tout ou partie de la séquence de la protéine Thy-1 humaine, de préférence une molécule comprenant un épitope de la protéine Thy-1 humaine. De tels ligands sont naturellement choisis parmi les molécules reconnues et/ou capables d'interagir avec la partie extra-cellulaire de THY-1.THY-1 is a molecule devoid of an intra-cytoplasmic domain which interacts with the cell membrane via a glycophosphatidylinositol (GPI) which attaches to the membrane by its C-terminal end. The sequence of Thy-1 has been determined and is accessible in the literature, such as for example the nucleotide sequence (no NM 006288 (gi: 199 233 61)) and the amino acid sequence (no P 006279 (gi: 199 233 62)) of human protein. A specific ligand according to the invention is preferably a molecule capable of selectively binding a polypeptide comprising all or part of the sequence of the human Thy-1 protein, preferably a molecule comprising an epitope of the human Thy-1 protein. Such ligands are naturally chosen from recognized molecules and / or capable of interacting with the extracellular part of THY-1.
Un ligand préféré de THY-1, utilisable dans le cadre de la présente invention, est un anticorps anti-THY-1 (c'est-à-dire un anticorps spécifique de THY-1). L'anticorps peut être polyclonal ou monoclonal. Il peut également s'agir de fragments et dérivés d'un fragment ou dérivé d'anticorps présentant substantiellement la même spécificité antigénique, en particulier des fragments d'anticorps (e.g., Fab, Fab'2, CDRs), d'anticorps humanisés, d'anticorps humains, d'anticorps poly-fonctionnels, monocaténaires (ScFv), ou multimériques (couplage C4bp par exemple), etc. Les anticorps, et donc les sites de reconnaissance de la molécule THY-1 utilisables pour générer un ligand spécifique, peuvent être produits à l'aide de méthodes conventionnelles, comprenant Pirnmunisation d'un animal non-humain avec un polypeptide THY-1 ou un fragment de celui-ci comportant un épitope, et la récupération de son sérum (polyclonal) ou de cellules spléniques (de manière à produire des hybridomes par fusion avec des lignées cellulaires appropriées). Diverses méthodes de production d'anticorps polyclonaux à partir d'espèces variées ont été décrites dans l'art antérieur. Typiquement, l'antigène est combiné avec un adjuvant (par exemple l'adjuvant de Freund) et administré à un animal, par exemple par injection sous-cutanée. Des injections répétées peuvent être réalisées. Les échantillons sanguins sont collectés et l'immunoglobuline ou le sérum sont séparés.A preferred ligand for THY-1, which can be used in the context of the present invention, is an anti-THY-1 antibody (that is to say an antibody specific for THY-1). The antibody can be polyclonal or monoclonal. It can also be fragments and derivatives of a fragment or derivative of antibodies having substantially the same antigenic specificity, in particular fragments of antibodies (eg, Fab, Fab'2, CDRs), of humanized antibodies, human antibodies, polyfunctional, single-stranded (ScFv), or multimeric antibodies (C4bp coupling for example), etc. The antibodies, and therefore the recognition sites of the THY-1 molecule which can be used to generate a specific ligand, can be produced using conventional methods, comprising the immunization of a non-human animal with a THY-1 polypeptide or a fragment thereof comprising an epitope, and the recovery of its serum (polyclonal) or of spleen cells (so as to produce hybridomas by fusion with appropriate cell lines). Various methods of producing polyclonal antibodies from various species have been described in the prior art. Typically, the antigen is combined with an adjuvant (eg Freund's adjuvant) and administered to an animal, for example by subcutaneous injection. Repeated injections can be given. Blood samples are collected and immunoglobulin or serum are separated.
Les méthodes classiques de production d'anticorps monoclonaux comprennent l'immunisation d'un animal non-humain avec un antigène, suivie de la récupération des cellules spléniques qui sont ensuite fusionnées avec, des cellules immortalisées, telles que des cellules de myélome. Les hybridomes résultant produisent des anticorps monoclonaux et peuvent être sélectionnés par dilutions limites de manière à isoler les clones individuels. Les fragments Fab ou F(ab')2 peuvent être produits par digestion à l'aide d'une protéase selon les techniques conventionnelles.
Les anticorps préférés sont des anticorps spécifiques de la protéine THY-1, c'est-à-dire ayant une affinité supérieure pour cette protéine que pour d'autres antigènes, même si une liaison non-spécifique ou moins affine ne peut être exclue. Le terme « spécifique » ou « sélectif» indique notamment que la liaison du ligand à la protéine THY-1 peut être discriminée de la liaison éventuelle du ligand à d'autres molécules.Conventional methods of producing monoclonal antibodies include immunizing a non-human animal with an antigen, followed by the recovery of spleen cells which are then fused with immortalized cells, such as myeloma cells. The resulting hybridomas produce monoclonal antibodies and can be selected by limiting dilutions so as to isolate the individual clones. The Fab or F (ab ') 2 fragments can be produced by digestion using a protease according to conventional techniques. The preferred antibodies are antibodies specific for the THY-1 protein, that is to say having a higher affinity for this protein than for other antigens, even if a non-specific or less affine bond cannot be excluded. The term “specific” or “selective” indicates in particular that the binding of the ligand to the THY-1 protein can be discriminated from the possible binding of the ligand to other molecules.
Les cellules Ts et pTs peuvent ainsi être isolées, dans le cadre de l'étape (b), par mise en contact de la population cellulaire avec des ligands spécifiques, tels que ceux définis précédemment. Des exemples particuliers de ligands spécifiques selon l'invention sont notamment les anticorps monoclonaux produits par les hybridomes Kl 7 (ATCC n° HB- 8553), les clones 5E10, F15-42-1, Thy-1/310, FIB1 (clone AS02), ainsi que tous fragments ou dérivés de ces anticorps.Ts and pTs cells can thus be isolated, in the context of step (b), by bringing the cell population into contact with specific ligands, such as those defined above. Particular examples of specific ligands according to the invention are in particular the monoclonal antibodies produced by the hybridomas Kl 7 (ATCC n ° HB- 8553), the clones 5E10, F15-42-1, Thy-1/310, FIB1 (clone AS02 ), as well as any fragments or derivatives of these antibodies.
D'autres ligands spécifiques selon l'invention sont par exemple des ligands artificiels, présentant une affinité particulière pour THY-1. De tels ligands peuvent être de nature variée, comme des acides nucléiques (par exemple des aptamères) ou des molécules chimiques de synthèse. De telles molécules peuvent être générées par exemple sur la base des séquences des sites de reconnaissance de la molécule THY-1 par les anticorps spécifiques définis précédemment.Other specific ligands according to the invention are for example artificial ligands, having a particular affinity for THY-1. Such ligands can be of varied nature, such as nucleic acids (for example aptamers) or synthetic chemical molecules. Such molecules can be generated for example on the basis of the sequences of the recognition sites of the THY-1 molecule by the specific antibodies defined above.
Les cellules Ts et pTs peuvent ainsi être isolées, dans le cadre de l'étape (b), par mise en contact de la population cellulaire avec un ou plusieurs ligands spécifiques, tels que ceux définis précédemment.Ts and pTs cells can thus be isolated, in the context of step (b), by bringing the cell population into contact with one or more specific ligands, such as those defined above.
II est possible d'utiliser, dans le cadre de la présente invention, un ou plusieurs ligands spécifiques de THY-1, éventuellement en combinaison avec d'autres ligands spécifiques d'autres marqueurs de cellules T, comme CD25. notamment. Ainsi, dans un mode particulier de réalisation, l'invention utilise une combinaison d'un ligand spécifique de THY-1 et d'un ligand spécifique de CD25. Le second ligand peut être spécifique de tout autre marqueur de cellules T, notamment de cellules T suppresseurs, par exemple des marqueurs identifiés par les techniques de génomique et protéomique décrites dans la présente demande.
Le (ou les) ligand(s) peut (peuvent) être immobilisé(s) sur un support, par exemple une colonne ou une bille (notamment une bille magnétique), ou placé(s) en solution. En outre, ou en variante, le ligand peut éventuellement être marqué. Le marquage peut être réalisé à l'aide d'un marqueur de détection fluorescent, radioactif, luminescent, phosphorescent, chimique ou enzymatique. Le marqueur de détection est de préférence choisi parmi la fluorescéine, le rouge Texas, la rhodamine, la phycoérythrine, l'dlophycocyanine, la biotine et la streptavidine, la Cyanine.It is possible to use, in the context of the present invention, one or more ligands specific for THY-1, optionally in combination with other ligands specific for other T cell markers, such as CD25. especially. Thus, in a particular embodiment, the invention uses a combination of a specific ligand for THY-1 and a specific ligand for CD25. The second ligand can be specific for any other marker of T cells, in particular of suppressor T cells, for example markers identified by the genomics and proteomics techniques described in the present application. The ligand (s) can (can) be immobilized on a support, for example a column or a ball (in particular a magnetic ball), or placed (s) in solution. In addition, or alternatively, the ligand can optionally be labeled. The labeling can be carried out using a fluorescent, radioactive, luminescent, phosphorescent, chemical or enzymatic detection marker. The detection marker is preferably chosen from fluorescein, Texas red, rhodamine, phycoerythrin, dlophycocyanin, biotin and streptavidin, Cyanine.
Les complexes formés par le ligand et les cellules marquées peuvent ensuite être utilisés pour visualiser, détecter, quantifier, trier, isoler et/ou dépléter les cellules, selon des techniques variées et connues en soi de l'homme du métier. Ainsi, les cellules peuvent être récupérées, sélectionnées, triées, séparées, isolées, déplétées par exemple par une méthode choisie parmi la cytométrie de flux, la chromatographie d'affinité, le FACS (fluorescent activated cell sorting« Fluorescent Activated Cell Sorting »), le MACS (magneticbead cell sorting« Magnetic bead Cell Sorting »), le D/MACS (double magnetic bead cell sorting), une chromatographie d'affinité« Double Magnetic bead Cell Sorting »), une méthode de sélection sur surface solide (« panning »), un test ELIS A, un test RIA, etc.The complexes formed by the ligand and the labeled cells can then be used to visualize, detect, quantify, sort, isolate and / or deploy the cells, according to various techniques which are known per se to those skilled in the art. Thus, the cells can be recovered, selected, sorted, separated, isolated, depleted for example by a method chosen from flow cytometry, affinity chromatography, FACS (fluorescent activated cell sorting "Fluorescent Activated Cell Sorting"), MACS (magneticbead cell sorting "Magnetic bead Cell Sorting"), D / MACS (double magnetic bead cell sorting), affinity chromatography "Double Magnetic bead Cell Sorting"), a selection method on solid surface ("panning ”), An ELIS A test, an RIA test, etc.
La procédure MACS est décrite en détails par Miltenyi et al.,"High Gradient Magnetic Cell Séparation ith MACS," Cytometry 11: 231-238 (1990). Afin de récupérer les cellules, les cellules marquées avec des billes magnétiques traversent une colonne de séparation paramagnétique. La colonne de séparation est placée à proximité d'un aimant, créant ainsi un champ magnétique à l'intérieur de la colonne. Les cellules marquées magnétiquement sont piégées dans la colonne, les autres traversent cette dernière. Les cellules piégées à l'intérieur de la colonne sont ensuite éluées.The MACS procedure is described in detail by Miltenyi et al., "High Gradient Magnetic Cell Separation ith MACS," Cytometry 11: 231-238 (1990). In order to recover the cells, the cells labeled with magnetic beads pass through a paramagnetic separation column. The separation column is placed near a magnet, thereby creating a magnetic field inside the column. The magnetically marked cells are trapped in the column, the others cross it. The cells trapped inside the column are then eluted.
Dans la procédure D/MACS, un échantillon de cellules est marqué à l'aide de billes magnétiques comprenant des anticorps, et les cellules sont récoltées ou triées par application d'un champ magnétique.
Selon un mode de réalisation préféré, les cellules (par exemple du sang périphérique) sont incubées de façon séquentielle avec des quantités saturantes d'anticorps anti-THY- 1 fonctionnalisé (e.g., marqué à la biotine) et avec un support solide (par exemple des microbilles) fonctionnalisé (e.g., recouvert de streptavidine). Les cellules sont ensuite purifiées par récupération du support, e.g., par séparation magnétique des cellules. De manière à augmenter la purification des cellules, les cellules de la fraction positive peuvent être ultérieurement séparées sur une autre colonne. La purification est généralement réalisée dans un tampon de sels phosphate, bien que d'autres milieux adaptés puissent être utilisés.In the D / MACS procedure, a sample of cells is labeled using magnetic beads comprising antibodies, and the cells are harvested or sorted by application of a magnetic field. According to a preferred embodiment, the cells (for example peripheral blood) are incubated sequentially with saturating amounts of functionalized anti-THY-1 antibody (eg, labeled with biotin) and with a solid support (for example microbeads) functionalized (eg, coated with streptavidin). The cells are then purified by recovery of the support, eg, by magnetic separation of the cells. In order to increase the purification of the cells, the cells of the positive fraction can be subsequently separated on another column. The purification is generally carried out in a phosphate salt buffer, although other suitable media can be used.
Les cellules peuvent être cultivées ou maintenues dans tous tampons ou milieux adaptés, telle qu'une solution saline, un tampon, un milieu de culture, en particulier le DMEM, le RPMI etc. Elles peuvent être congelées ou maintenues dans une situation froide. Elles peuvent être formulées dans tous dispositifs ou appareils appropriés, tel qu'un tube, une flasque, une ampoule, un plat, une seringue, une poche, etc., de préférence dans des conditions stériles adaptées à une utilisation pharmaceutique.The cells can be cultured or maintained in any suitable buffer or medium, such as a saline solution, a buffer, a culture medium, in particular DMEM, RPMI etc. They can be frozen or kept in a cold situation. They can be formulated in any suitable device or apparatus, such as a tube, a flask, a vial, a dish, a syringe, a bag, etc., preferably under sterile conditions suitable for pharmaceutical use.
Comme indiqué précédemment, l'étape (b) de la méthode décrite ci-dessus peut avantageusement être précédée et/ou suivie d'une étape de purification d'une sous population de lymphocytes T (CD4+ et ou CD8+ par exemple) et/ou d'une étape d'amplification des lymphocytes (qui peut être réalisée ex vivo ou in vitro.).As indicated above, step (b) of the method described above can advantageously be preceded and / or followed by a step of purifying a subpopulation of T lymphocytes (CD4 + and or CD8 + for example) and / or a lymphocyte amplification step (which can be carried out ex vivo or in vitro.).
L'amplification peut être obtenue par activation des lymphocytes. Cette activation peut être non spécifique (obtenue par exemple par des anticorps anti-CD3 et/ou anti-CD28, avec ou en présence d'une interleukine, par exemple l'IL2) ou spécifique (obtenue par des antigènes ou allo-antigènes présentés de façon adéquate au lymphocytes Ts ou pTs, par exemple par des cellules présentatrices de l'antigènes (cellules dendritiques, lymphocytes B, monocytes macrophages, cellules génétiquement modifiées capable de présenter l'antigène et d'activer les lymphocytes), des exosomes, des dexosomes, des structures artificielles, etc.). L'étape d'amplification permet d'augmenter le nombre de lymphocytes T présents au sein de la population lymphocytaire T de départ (qui comprend des lymphocytes T effecteurs et des lymphocytes T suppresseurs) avant de
procéder à la sélection des lymphocytes Ts et pTs, et/ou d'augmenter le nombre de lymphocytes Ts et pTs après avoir sélectionné les lymphocytes T exprimant l'antigène THY-1. Il est également possible de procéder à deux étapes d'amplification, l'une concernant la population lymphocytaire T générale présente dans la population de cellules de mammifère, l'autre concernant la population de lymphocyte Ts et pTs.Amplification can be obtained by activation of lymphocytes. This activation can be non-specific (obtained for example by anti-CD3 and / or anti-CD28 antibodies, with or in the presence of an interleukin, for example IL2) or specific (obtained by antigens or allo-antigens presented adequately to Ts or pTs cells, for example by antigen presenting cells (dendritic cells, B lymphocytes, macrophage monocytes, genetically modified cells capable of presenting the antigen and activating lymphocytes), exosomes, dexosomes, artificial structures, etc.). The amplification step makes it possible to increase the number of T lymphocytes present in the starting T lymphocyte population (which includes effector T lymphocytes and suppressor T lymphocytes) before proceed to the selection of Ts and pTs lymphocytes, and / or to increase the number of Ts and pTs lymphocytes after having selected the T lymphocytes expressing the THY-1 antigen. It is also possible to carry out two amplification stages, one concerning the general T lymphocyte population present in the population of mammalian cells, the other concerning the population of Ts and pTs lymphocytes.
Dans un mode de mise en œuvre préféré, l'étape d'amplification est réalisée dans des conditions qui favorisent les Ts (ou pTs), permettant ainsi leur enrichissement. Ainsi, la présente invention montre que la culture en absence de N-acetyl cystéine favorise la prolifération des Ts (figure 1). Dans un mode de réalisation particulier de l'invention, les cellules sont amplifiées par culture dans un milieu dépourvu de N-acetyl cystéine. D'autre part, l'utilisation de certaines populations de cellules présentatrices d'antigène naturelles ou modifiées (notamment génétiquement) peunt favoriser la prolifération des Ts (ou pTs). Ainsi, les exemples montrent que les cellules dendritiques dérivées de progéniteurs hématopoïétiques CD34+ et ayant un phénotype de type DC interstitielles favorisent la prolifération des Ts (ou pTs) (Figure 2). Dans un mode de réalisation particulier de l'invention, les cellules sont amplifiées par culture en présence de cellules dendritiques, notamment de cellules dendritiques interstitielles.In a preferred embodiment, the amplification step is carried out under conditions which favor the Ts (or pTs), thus allowing their enrichment. Thus, the present invention shows that culture in the absence of N-acetyl cysteine promotes the proliferation of Ts (Figure 1). In a particular embodiment of the invention, the cells are amplified by culture in a medium devoid of N-acetyl cysteine. On the other hand, the use of certain populations of natural or modified (in particular genetically modified) antigen presenting cells can promote the proliferation of Ts (or pTs). Thus, the examples show that the dendritic cells derived from CD34 + hematopoietic progenitors and having an interstitial DC type phenotype promote the proliferation of Ts (or pTs) (FIG. 2). In a particular embodiment of the invention, the cells are amplified by culture in the presence of dendritic cells, in particular interstitial dendritic cells.
La population obtenue à l'issue de l'étape (a) peut également être enrichie en cellules T appartenant à la population générale des cellules T, i.e., comprenant des lymphocytes T effecteurs et des lymphocytes T suppresseurs ou leurs précurseurs. La population de l'étape (a) peut ainsi être enrichie en cellules T, éventuellement en une ou des sous-populations spécifiques de lymphocytes (par exemple CD4+ et/ou CD8+). Elle peut également être débarrassée de certaines sous-populations de lymphocytes, le cas échéant. La population obtenue à l'issue de l'étape (a) puis, éventuellement amplifiée et/ou triée, comprend ainsi de préférence au moins 30%, de préférence au moins 50%, de manière encore plus préférée au moins 65% de cellules T. Des compositions enrichies en cellules T particulièrement préférées susceptibles d'être utilisées dans le cadre de l'étape (b) comprennent au moins 75%, de préférence au moins 80% de cellules T.
La population de lymphocytes T exprimant l'antigène THY-1 peut également être amplifiée. Il est par ailleurs possible, comme indiqué ci-dessus, de procéder à deux étapes d'amplification, l'une concernant la population lymphocytaire T générale, l'autre concernant la population de lymphocyte Ts ou pTs.The population obtained at the end of step (a) can also be enriched in T cells belonging to the general population of T cells, ie, comprising effector T lymphocytes and suppressor T lymphocytes or their precursors. The population of step (a) can thus be enriched in T cells, possibly in one or more specific subpopulations of lymphocytes (for example CD4 + and / or CD8 +). It can also be rid of certain subpopulations of lymphocytes, if necessary. The population obtained at the end of step (a) then, optionally amplified and / or sorted, thus preferably comprises at least 30%, preferably at least 50%, even more preferably at least 65% of cells. T. Particularly preferred compositions enriched in T cells capable of being used in the context of step (b) comprise at least 75%, preferably at least 80% of T cells. The population of T lymphocytes expressing the THY-1 antigen can also be amplified. It is also possible, as indicated above, to carry out two amplification steps, one concerning the general T lymphocyte population, the other concerning the Ts or pTs lymphocyte population.
Un objet particulier de la présente invention concerne ainsi une méthode d'obtention de lymphocytes T suppresseurs (et/ou leurs précurseurs) comprenant. : (a) l'obtention d'une population de cellules de mammifère comprenant des lymphocytes T, (a') l'amplification des lymphocytes T au sein de ladite population de cellules, et (b) la récupération des lymphocytes T exprimant l'antigène THY-1.A particular object of the present invention thus relates to a method for obtaining suppressor T lymphocytes (and / or their precursors) comprising. : (a) obtaining a population of mammalian cells comprising T lymphocytes, (a ') amplifying T lymphocytes within said population of cells, and (b) recovering T lymphocytes expressing the THY-1 antigen.
Un autre objet particulier de la présente invention concerne une méthode d'obtention de lymphocytes T suppresseurs (et/ou leurs précurseurs) comprenant : (a) l'obtention d'une population de cellules de mammifère comprenant des lymphocytes T, (b) la récupération des lymphocytes T exprimant l'antigène THY-1, et (b') l'amplification desdits lymphocytes T exprimant l'antigène THY-1.Another particular object of the present invention relates to a method for obtaining suppressor T lymphocytes (and / or their precursors) comprising: (a) obtaining a population of mammalian cells comprising T lymphocytes, (b) recovery of T lymphocytes expressing the THY-1 antigen, and (b ') amplification of said T lymphocytes expressing the THY-1 antigen.
L'amplification cellulaire des lymphocytes appartenant à la population générale des lymphocytes T (cellules T effectrices, cellules Ts et pTs) est de préférence réalisée par culture des cellules en présence d'une cytokine et éventuellement d'un agent de stimulation. Dans le cas des lymphocytes exprimant l'antigène THY-1 (lymphocytes Ts et pTs), la culture est maintenue pendant une période suffisante pour obtenir l'amplification de ladite population cellulaire au sein des populations lymphocytaires T CD4+ et/ou CD8+. L'activation implique généralement la culture en présence d'une cytokine, telle que par exemple, l'interleukine-2 (IL-2), l'interleukine-7 (IL-7), l'interleukine-10 (IL-10) ou Pinterleukine-15 (IL- 15), de préférence d'origine humaine. L'agent de stimulation peut être une cellule présentatrice d'antigène (« CPA »), Le., toute cellule présentatrice d'antigènes ou toute cellule favorisant l'activation des cellules T, en particulier des cellules Ts. Les CPA sont de préférence irradiées avant leur utilisation afin d'éviter leur amplification. Les CPA peuvent être des cellules
isolées d'un donneur ou du patient lui-même. Elles peuvent être choisies pour produire des cellules Ts et pTs présentant un profil d'activité choisi. Des exemples caractéristiques de telles CPA incluent les cellules mononucléaires du sang périphérique, des cellules dendritiques, des splénocytes, des cellules du sang de cordon ombilical, des échantillons de tissu ou d'organe, etc. D'autres agents de stimulation des cellules Ts et pTs adaptés incluent les polymères MHC, les lectines (telles que PHA), les anticorps (tels que les anticorps anti-CD3 et/ou anti-CD28) ou des fragments de ces derniers, les auto-antigènes (incluant les tissus, cellules, fragments cellulaires ou débris, les polypeptides ou peptides purifiés, etc., de préférence en combinaison avec les CPA), etc.Cellular amplification of lymphocytes belonging to the general population of T lymphocytes (effector T cells, Ts cells and pTs) is preferably carried out by culturing the cells in the presence of a cytokine and optionally a stimulating agent. In the case of lymphocytes expressing the THY-1 antigen (Ts lymphocytes and pTs), the culture is maintained for a period sufficient to obtain the amplification of said cell population within CD4 + and / or CD8 + T lymphocyte populations. Activation usually involves culture in the presence of a cytokine, such as, for example, interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-10 (IL-10 ) or Interleukin-15 (IL-15), preferably of human origin. The stimulating agent can be an antigen presenting cell (“APC”), Le., Any antigen presenting cell or any cell promoting activation of T cells, in particular Ts cells. The APCs are preferably irradiated before their use in order to avoid their amplification. CPA can be cells isolated from a donor or from the patient himself. They can be chosen to produce Ts and pTs cells with a chosen activity profile. Typical examples of such CPA include peripheral blood mononuclear cells, dendritic cells, splenocytes, umbilical cord blood cells, tissue or organ samples, etc. Other suitable Ts and pTs cell stimulants include MHC polymers, lectins (such as PHA), antibodies (such as anti-CD3 and / or anti-CD28 antibodies) or fragments thereof, autoantigens (including tissues, cells, cell fragments or debris, purified polypeptides or peptides, etc., preferably in combination with CPA), etc.
Selon l'utilisation envisagée, les cellules Ts et pTs peuvent êfre amplifiées de différentes façons, qu'elles soient antigènes spécifiques ou non. En particulier, pour certaines utilisations, des quantités élevées du répertoire complet des cellules T sont, de préférence, utilisées (e.g., injectées). Cette technique est, en particulier, adaptée aux patients qui présentent un déficit global (quantitatif ou fonctionnel) en cellules Ts et pTs. Dans de telles indications, les cellules Ts et pTs sont de préférence amplifiées par exemple, à l'aide de cellules CPA autologues et PHA ou d'anticorps anti-CD3 et/ou anti-CD25 (ou de tout autre activateur de cellules T ou Ts) en présence de cytokines de nature identique ou différente.Depending on the intended use, Ts and pTs cells can be amplified in different ways, whether or not they are specific antigens. In particular, for certain uses, large quantities of the complete repertoire of T cells are preferably used (e.g., injected). This technique is, in particular, suitable for patients who have an overall deficit (quantitative or functional) in Ts and pTs cells. In such indications, the Ts and pTs cells are preferably amplified, for example, using autologous CPA and PHA cells or anti-CD3 and / or anti-CD25 antibodies (or any other activator of T cells or Ts) in the presence of cytokines of identical or different nature.
Généralement, la spécificité des cellules Ts et pTs est importante à prendre en compte. En effet, bien qu'il soit possible d'utiliser des cellules Ts non-spécifiques pour contrôler des réponses immunes spécifiques, l'utilisation de Ts spécifiques apparaît plus efficace. Ainsi, les lymphocytes Ts et pTs, chez l'homme et chez la souris, peuvent être cultivés et amplifiés in vitro en présence d'un milieu de culture contenant de l'interleukine 2, des anticorps anti-CD3 et anti-CD28. Des lymphocytes Ts et pTs spécifiques peuvent être aussi isolés, générés par exemple par stimulation en présence de cellules présentatrices d'antigènes allogéniques, suivie d'une culture par interleukine 2. Selon un autre mode de réalisation, une amplification plus spécifique peut être envisagée, en particulier lorsqu'une suppression de cellules T effectrices spécifiques est souhaitée, comme dans le cadre des maladies auto-immunes, des allergies, des rejets de greffe, de
la GNHD, etc. Dans de telles indications, les cellules sont de préférence amplifiées en présence de CPA présentant des antigènes particuliers, par exemple allogéniques ou d'origine infectieuse, afin de favoriser l'amplification des cellules Ts préférentiellement actives à l'encontre de cellules T effectrices pathogènes. Les antigènes sont présentés sous forme peptidiques ou après transfert d'ARΝ ou d'ADN.Generally, the specificity of Ts and pTs cells is important to take into account. Indeed, although it is possible to use non-specific Ts cells to control specific immune responses, the use of specific Ts appears to be more effective. Thus, Ts and pTs lymphocytes, in humans and in mice, can be cultured and amplified in vitro in the presence of a culture medium containing interleukin 2, anti-CD3 and anti-CD28 antibodies. Specific Ts and pTs lymphocytes can also be isolated, generated for example by stimulation in the presence of cells presenting allogenic antigens, followed by a culture by interleukin 2. According to another embodiment, a more specific amplification can be envisaged, in particular when suppression of specific effector T cells is desired, such as in the context of autoimmune diseases, allergies, transplant rejection, GNHD, etc. In such indications, the cells are preferably amplified in the presence of CPA presenting particular antigens, for example allogenic or of infectious origin, in order to favor the amplification of Ts cells preferentially active against pathogenic effector T cells. The antigens are presented in peptide form or after transfer of ARΝ or DNA.
Pour traiter les maladies auto-immunes, les cellules Ts et pTs proviennent de préférence du patient et sont stimulées à l'aide de CPA autologues et d'auto-antigènes provenant du tissu cible, en présence de cytokines. Les auto-antigènes peuvent êfre des tissus, cellules, fragments cellulaires, protéines purifiées, peptides, acides nucléiques, etc.To treat autoimmune diseases, Ts and pTs cells preferably come from the patient and are stimulated using autologous CPA and autoantigens from the target tissue, in the presence of cytokines. Autoantigens can be from tissues, cells, cell fragments, purified proteins, peptides, nucleic acids, etc.
Pour le traitement des allogreffes ou xénogreffes, les cellules Ts proviennent de préférence du patient et sont stimulées par des CPA ou des tissus provenant du donneur, en présence de cytokines. Les cellules Ts provenant du patient peuvent aussi être stimulées par des CPA autologues en présence de tissus, cellules, fragments cellulaires, protéines purifiées ou peptides provenant du donneur et de cytokines.For the treatment of allografts or xenografts, Ts cells preferably come from the patient and are stimulated by APCs or tissues from the donor, in the presence of cytokines. Ts cells from the patient can also be stimulated by autologous APCs in the presence of tissues, cells, cell fragments, purified proteins, or peptides from the donor and cytokines.
Pour traiter les allergies, les cellules Ts proviennent typiquement du patient et sont stimulées par des CPA et des allergènes, en présence de cytokines.To treat allergies, Ts cells typically come from the patient and are stimulated by CPA and allergens in the presence of cytokines.
Comme indiqué ci-dessus, les cytokines préférentiellement utilisées sont 1TL-2, l'IL-10 et/ou l'LL-15.As indicated above, the cytokines preferentially used are 1TL-2, IL-10 and / or IL-15.
Comme indiqué précédemment, les cellules Ts et pTs utilisées pour traiter diverses pathologies telles que le rejet d'un organe transplanté, des maladies auto-immune, les allergies, les pathologies induites par un virus, etc., sont de préférence autologues, Le., elles proviennent du sujet à traiter. Les cellules syngéniques peuvent également être utilisées. Dans d'autres situations, par exemple dans le cadre du traitement de la GNHD ou d'autres pathologies, les cellules Ts et pTs sont typiquement allogéniques, i.e., elles proviennent d'un être humain différent. Dans ces cas, il est préférable d'utiliser des cellules Ts et pTs provenant d'un sujet donneur (e.g., le sujet donneur des cellules effectrices).
Modification génétique des cellules T et notamment des cellules Ts et pTsAs indicated previously, the Ts and pTs cells used to treat various pathologies such as rejection of a transplanted organ, autoimmune diseases, allergies, pathologies induced by a virus, etc., are preferably autologous, Le. , they come from the subject to be treated. Syngene cells can also be used. In other situations, for example in the treatment of GNHD or other pathologies, Ts and pTs cells are typically allogenic, ie, they come from a different human being. In these cases, it is preferable to use Ts and pTs cells originating from a donor subject (eg, the donor subject of effector cells). Genetic modification of T cells and in particular of Ts and pTs cells
Dans un mode de réalisation particulier de l'invention, les lymphocytes T (population générale des lymphocytes T) présents dans la population de cellules de mammifère ou les lymphocytes T suppresseurs (porteurs du marqueur THY-1) peuvent êfre modifiés génétiquement de manière à exprimer des produits biologiques d'intérêt.In a particular embodiment of the invention, the T lymphocytes (general population of T lymphocytes) present in the population of mammalian cells or the suppressor T lymphocytes (carrying the THY-1 marker) can be genetically modified so as to express organic products of interest.
L'expression « génétiquement modifiées » indique que les cellules comprennent une molécule d'acide nucléique qui n'est pas naturellement présente dans les cellules T non modifiées, ou qui est présente dans ces cellules lorsqu'elles ne sont pas dans leur état naturel (e.g., lorsqu'elles sont amplifiées). La molécule d'acide nucléique peut avoir été introduite dans lesdites cellules ou dans une cellule parente ou progénitrice.The expression "genetically modified" indicates that the cells comprise a nucleic acid molecule which is not naturally present in unmodified T cells, or which is present in these cells when they are not in their natural state ( eg, when amplified). The nucleic acid molecule may have been introduced into said cells or into a parent or progenitor cell.
Un objet particulier de l'invention concerne ainsi une méthode d'obtention ou de production de lymphocytes T suppresseurs (et/ou leurs précurseurs) comprenant : (a) l'obtention d'une population de cellules de mammifère comprenant des lymphocytes T, (b) la récupération des lymphocytes T exprimant l'antigène THY-1, et (c) la modification génétique desdits lymphocytes T exprimant l'antigène THY-1 par mise en contact desdits lymphocytes avec une molécule d'acide nucléique recombinant.A particular subject of the invention thus relates to a method for obtaining or producing suppressor T lymphocytes (and / or their precursors) comprising: (a) obtaining a population of mammalian cells comprising T lymphocytes, ( b) recovery of the T lymphocytes expressing the THY-1 antigen, and (c) the genetic modification of said T lymphocytes expressing the THY-1 antigen by bringing said lymphocytes into contact with a recombinant nucleic acid molecule.
Un autre objet particulier de l'invention concerne une méthode d'obtention ou de production de lymphocytes T suppresseurs (ou leurs précurseurs) comprenant : (a) l'obtention d'une population de cellules de mammifère comprenant des lymphocytes T, (b) la modification génétique desdits lymphocytes T par mise en contact de ladite population de cellules avec une molécule d'acide nucléique recombinant, et (c) la récupération des lymphocytes T exprimant l'antigène THY-1.
Plusieurs approches peuvent être utilisées pour modifier sur le plan génétique les cellules T appartenant à la population de cellules de mammifère [assimilable à la population générale des lymphocytes T (cellules T effectrices et cellules Ts)] ou les lymphocytes Ts et pTs, telles que, par exemple, la délivrance de gène par l'intermédiaire d'un virus, l'ADN nu, des traitements physiques, etc. A cette fin, l'acide nucléique est généralement incorporé dans un vecteur, tel qu'un virus recombinant, un plasmide, un phage, un épisome, un chromosome artificiel, etc.Another particular object of the invention relates to a method for obtaining or producing suppressor T lymphocytes (or their precursors) comprising: (a) obtaining a population of mammalian cells comprising T lymphocytes, (b) the genetic modification of said T lymphocytes by bringing said population of cells into contact with a recombinant nucleic acid molecule, and (c) the recovery of T lymphocytes expressing the THY-1 antigen. Several approaches can be used to genetically modify the T cells belonging to the mammalian cell population [comparable to the general population of T lymphocytes (effector T cells and Ts cells)] or Ts and pTs lymphocytes, such as, for example, gene delivery via virus, naked DNA, physical treatments, etc. To this end, the nucleic acid is generally incorporated into a vector, such as a recombinant virus, a plasmid, a phage, an episome, an artificial chromosome, etc.
Selon un mode de réalisation particulier de l'invention, les cellules T telles que définies au paragraphe précédant, sont modifiées génétiquement à l'aide d'un vecteur viral (ou d'un virus recombinant). L'acide nucléique hétérologue est, par exemple, introduit dans un virus recombinant qui est ensuite utilisé pour infecter des lymphocytes T. Différents types de virus recombinants peuvent êfre utilisés, en particulier des réfrovirus recombinants ou des AAN. Les lymphocytes T sont de préférence modifiés à l'aide d'un réfrovirus recombinant. L'utilisation de réfrovirus est particulièrement appréciée dans la mesure où l'infection réfrovirale permet une intégration stable de l'acide nucléique dans le génome des cellules. Cette propriété est particulièrement importante, dans la mesure où l'amplification des lymphocytes, qu'elle soit réalisée in vitro ou in vivo après l'injection chez le sujet, requiert que le transgène soit maintenu stable durant la division cellulaire. Des exemples de réfrovirus susceptibles d'êfre utilisés proviennent de la famille des oncovirus, des lentivirus ou des spumavirus. Des exemples particuliers de la famille des oncovirus sont MoMLN, ALN, BLN ou MMTN mais également RSN, etc. Des exemples de la famille des lentivirus sont HIN, SIN, FIN, ELAN ou CAEN, etc.According to a particular embodiment of the invention, the T cells as defined in the preceding paragraph are genetically modified using a viral vector (or a recombinant virus). The heterologous nucleic acid is, for example, introduced into a recombinant virus which is then used to infect T lymphocytes. Different types of recombinant viruses can be used, in particular recombinant refroviruses or ANAs. The T lymphocytes are preferably modified using a recombinant refrovirus. The use of refrovirus is particularly appreciated insofar as refroviral infection allows stable integration of the nucleic acid into the genome of the cells. This property is particularly important, insofar as the amplification of lymphocytes, whether carried out in vitro or in vivo after injection into the subject, requires that the transgene be kept stable during cell division. Examples of refractive viruses susceptible of being used come from the family of oncoviruses, lentiviruses or spumaviruses. Particular examples of the oncovirus family are MoMLN, ALN, BLN or MMTN but also RSN, etc. Examples of the lentivirus family are HIN, SIN, FIN, ELAN or CAEN, etc.
Des techniques permettant de construire des réfrovirus recombinants ont été largement décrites dans la littérature (WO 89/07150, WO 90/02806 et WO 94/19478, dont les enseignements sont intégralement incorporés dans la présente demande). Ces techniques comprennent généralement l'introduction d'un vecteur rétroviral comprenant le transgène dans une lignée de cellules d'empaquetage appropriée, suivie de la récupération des virus produits, lesdits virus comprenant le fransgène dans leur génome. Dans un mode de réalisation particulier de l'invention, le réfrovirus recombinant comprend l'enveloppe du virus GALN (réfrovirus pseudotypé avec GALN). Il a été
démontré que l'infection des cellules hématopoïétiques par un réfrovirus recombinant est plus efficace lorsque l'enveloppe du réfrovirus provient du réfrovirus GALN (Gibbon Ape Leukemia Virus). En utilisant cette enveloppe réfrovirale, un taux de fransduction de plus de 95% des lymphocytes a pu être obtenu avant la sélection des cellules transduites.Techniques allowing the construction of recombinant refroviruses have been widely described in the literature (WO 89/07150, WO 90/02806 and WO 94/19478, the teachings of which are fully incorporated in the present application). These techniques generally include the introduction of a retroviral vector comprising the transgene into an appropriate packaging cell line, followed by the recovery of the viruses produced, said viruses comprising fransgene in their genome. In a particular embodiment of the invention, the recombinant refrovirus comprises the envelope of the GALN virus (refrovirus pseudotyped with GALN). He was demonstrated that infection of hematopoietic cells with a recombinant refrovirus is more effective when the envelope of the refrovirus comes from the GALN (Gibbon Ape Leukemia Virus) refrovirus. By using this refroviral envelope, a fransduction rate of more than 95% of the lymphocytes could be obtained before the selection of the transduced cells.
Les lymphocytes T peuvent êfre infectés à l'aide de virus recombinants et au moyen de divers protocoles, tels que l'incubation avec un surnageant de virus, avec des virus purifiés, par coculture de lymphocytes T avec les cellules virales d'empaquetage, par des techniques Transwell, etc. Une méthode particulièrement efficace comprenant une étape de centrifugation a été décrite par Movassagh et al. (Movassagh M, Desmyter C, Baillou C, Chapel-Fernandes S, Guigon M, Klatzmann D, Lemoine FM. Hum Gène Ther. 1998;9:225-234).T cells can be infected using recombinant viruses and various protocols, such as incubation with a virus supernatant, with purified viruses, by coculture of T cells with viral packaging cells, Transwell techniques, etc. A particularly effective method comprising a centrifugation step has been described by Movassagh et al. (Movassagh M, Desmyter C, Baillou C, Chapel-Fernandes S, Guigon M, Klatzmann D, Lemoine FM. Hum Gène Ther. 1998; 9: 225-234).
Les techniques non virales incluent l'utilisation de lipides cationiques, de polymères, de peptides, d'agents synthétiques, etc. Des méthodes alternatives utilisent la technique du « gène gun », les champs électriques, le bombardement, la précipitation, etc. En réalisant la présente invention, il n'est pas nécessaire, que toutes les cellules Ts et pTs soient modifiées génétiquement. H est ainsi possible d'utiliser une population de lymphocytes T comprenant au moins 50%, de préférence au moins 65%, encore plus préférentiellement au moins 80% de lymphocytes modifiés génétiquement. Des niveaux plus élevés (e.g., jusqu'à 100%) peuvent êfre obtenus in vitro ou ex vivo ; par exemple en utilisant l'enveloppe GALN et/ou des conditions d'infection particulières (Movassagh et al.) et/ou en sélectionnant les cellules qui ont effectivement été modifiées génétiquement. Différentes techniques de sélection peuvent être utilisées, incluant l'utilisation d'anticorps reconnaissant des marqueurs spécifiques présents à la surface des cellules modifiées, l'utilisation de gènes de résistance (tel que le gène de résistance à la néomycine et la molécule G418), ou l'utilisation de composés toxiques pour les cellules n'exprimant pas le fransgène (i.e., la thymidine kinase). La sélection est de préférence réalisée à l'aide d'un gène marqueur exprimant une protéine membranaire. La présence de cette protéine permet la sélection à l'aide de techniques
conventionnelles de séparation telles que la séparation à l'aide de billes magnétiques, l'utilisation de colonnes ou la cytométrie de flux.Non-viral techniques include the use of cationic lipids, polymers, peptides, synthetic agents, etc. Alternative methods use the “gun gene” technique, electric fields, bombardment, precipitation, etc. In carrying out the present invention, it is not necessary that all the Ts and pTs cells be genetically modified. It is thus possible to use a population of T lymphocytes comprising at least 50%, preferably at least 65%, even more preferably at least 80% of genetically modified lymphocytes. Higher levels (eg, up to 100%) can be obtained in vitro or ex vivo; for example by using the GALN envelope and / or particular infection conditions (Movassagh et al.) and / or by selecting the cells which have actually been genetically modified. Different selection techniques can be used, including the use of antibodies recognizing specific markers present on the surface of the modified cells, the use of resistance genes (such as the neomycin resistance gene and the G418 molecule), or the use of compounds that are toxic to cells that do not express fransgene (ie, thymidine kinase). The selection is preferably carried out using a marker gene expressing a membrane protein. The presence of this protein allows selection using techniques separation techniques such as separation using magnetic beads, the use of columns or flow cytometry.
L'acide nucléique utilisé pour modifier les cellules T sur le plan génétique peut êfre un fransgène thérapeutique et peut coder pour des produits biologiques actifs variés, incluant des polypeptides (e.g., protéines, peptides, etc.), des ARNs, etc. Dans un mode de réalisation préféré, l'acide nucléique code pour un polypeptide présentant une activité immunosuppressive. Dans un autre mode de réalisation, l'acide nucléique code pour un polypeptide toxique ou présentant une toxicité conditionnelle pour les cellules. Des exemples préférés incluent la thymidine kinase (qui confère la toxicité en présence d'analogues de nucléoside), telles que HSN-1 TK, une cytosine déaminase, gprt, etc. Il peut aussi s'agir d'un polypeptide non toxique mais pouvant permettre l'élimination des cellules injectées en cas de besoin (comme par exemple une molécule exprimée à la membrane cellulaire et un anticorps monoclonal fixant le complément). Une autre catégorie préférée d'acides nucléiques sont ceux permettant le ciblage. Il peut s'agir d'acides nucléiques codant pour un récepteur de cellule T ou B ou une sous-unité ou un équivalent fonctionnel de celui-ci. Par exemple, l'expression au sein des cellules Ts d'un TCR recombinant spécifique d'un auto-antigène produit des cellules Ts et pTs qui peuvent agir plus spécifiquement sur des cellules T effectrices qui détruisent un tissu chez un sujet. D'autres types de molécules actives sur le plan biologique incluent des facteurs de croissance, des lymphokines (comprenant diverses cytokines qui activent les cellules Ts), des cytokines immunosuppressives (telles que IL-10 ou TGF- β), des molécules accessoires, des molécules présentatrices d'antigènes, des récepteurs d'antigènes, etc. L'acide nucléique peut coder pour des « T-bodies », i.e., des récepteurs hybrides entre des récepteurs de cellule T et une immunoglobuline. De tels « T-bodies » permettent le ciblage de complexes antigéniques, par exemple.The nucleic acid used to genetically modify T cells can be a therapeutic fransgene and can code for various active biological products, including polypeptides (e.g., proteins, peptides, etc.), RNAs, etc. In a preferred embodiment, the nucleic acid encodes a polypeptide having immunosuppressive activity. In another embodiment, the nucleic acid encodes a polypeptide that is toxic or has conditional toxicity to cells. Preferred examples include thymidine kinase (which confers toxicity in the presence of nucleoside analogs), such as HSN-1 TK, cytosine deaminase, gprt, etc. It can also be a non-toxic polypeptide but which can allow the elimination of the injected cells if necessary (such as for example a molecule expressed at the cell membrane and a monoclonal antibody fixing the complement). Another preferred category of nucleic acids are those for targeting. They may be nucleic acids encoding a T or B cell receptor or a subunit or a functional equivalent thereof. For example, the expression within Ts cells of a specific recombinant TCR of an autoantigen produces Ts cells and pTs which can act more specifically on effector T cells which destroy tissue in a subject. Other types of biologically active molecules include growth factors, lymphokines (including various cytokines that activate Ts cells), immunosuppressive cytokines (such as IL-10 or TGF-β), accessory molecules, antigen presenting molecules, antigen receptors, etc. The nucleic acid can code for "T-bodies", ie, hybrid receptors between T cell receptors and an immunoglobulin. Such "T-bodies" allow targeting of antigenic complexes, for example.
De manière préférée, les lymphocytes T suppresseurs (ou leurs précurseurs) sont génétiquement modifiés et comprennent un acide nucléique recombinant codant pour un produit présentant une toxicité conditionnelle pour ces cellules, tel que la thymidine kinase. Selon un autre mode de réalisation préféré de l'invention, les cellules Ts et pTs génétiquement modifiées comprennent une molécule d'acide nucléique recombinant
codant pour un récepteur de cellule T ou pour une sous-unité ou pour un équivalent fonctionnel de celui-ci.Preferably, the suppressor T lymphocytes (or their precursors) are genetically modified and comprise a recombinant nucleic acid coding for a product having a conditional toxicity for these cells, such as thymidine kinase. According to another preferred embodiment of the invention, the genetically modified Ts and pTs cells comprise a recombinant nucleic acid molecule encoding a T cell receptor or a subunit or a functional equivalent thereof.
Dans certaines indications, comme notamment la greffe de moelle osseuse allogénique, on peut être amené à réaliser des préparations séparées de lymphocytes Ts (et pTs) et T effecteurs, chacun exprimant un gène différent codant un produit présentant une toxicité conditionnelle, et permettant ainsi d'éliminer si nécessaire l'une ou l'autre des populations cellulaires.In certain indications, such as in particular allogeneic bone marrow transplantation, it may be necessary to carry out separate preparations of effector Ts (and pTs) and T lymphocytes, each expressing a different gene coding for a product having conditional toxicity, and thus making it possible to '' If necessary, eliminate either of the cell populations.
L'acide nucléique qui est introduit dans les cellules T selon l'invention comprend typiquement, en plus de la région codante, des séquences de régulation, telle qu'un promoteur et une séquence de polyadénylation.The nucleic acid which is introduced into the T cells according to the invention typically comprises, in addition to the coding region, regulatory sequences, such as a promoter and a polyadenylation sequence.
Compositionscompositions
Un objet particulier de cette invention est une composition comprenant au moins un lymphocyte T suppresseur selon l'invention, e.g. isolé, génétiquement modifié et/ou amplifié ex vivo, ou une population enrichie en cellules T suppresseurs telle que définie précédemment ou au contraire une population déplétée en cellules T suppresseurs, ainsi qu'un adjuvant ou un milieu acceptable sur le plan pharmaceutique.A particular object of this invention is a composition comprising at least one suppressor T lymphocyte according to the invention, eg isolated, genetically modified and / or amplified ex vivo, or a population enriched in suppressor T cells as defined above or, on the contrary, a population depleted in suppressor T cells, as well as a pharmaceutically acceptable adjuvant or medium.
Un autre objet particulier de l'invention est une composition comprenant des lymphocytes T suppresseurs (y compris leurs précurseurs) transduits à l'aide d'un premier gène suicide et des cellules T effectrices fransduites à l'aide d'un second gène suicide, différent du premier.Another particular object of the invention is a composition comprising suppressor T lymphocytes (including their precursors) transduced using a first suicide gene and effector T cells which are transplanted using a second suicide gene, different from the first.
Les compositions peuvent comprendre d'autres types cellulaires, sans affecter de façon significative le bénéfice thérapeutique desdites compositions.The compositions may include other cell types, without significantly affecting the therapeutic benefit of said compositions.
Selon un mode de réalisation préféré, les cellules sont conditionnées dans une composition comprenant entre environ 105 et 1010 cellules T suppresseurs selon la pathologie à traiter, plus généralement entre 105 et environ 109 cellules T suppresseurs.
Une composition particulière au sens de l'invention comprend une population de lymphocytes humains THY-1 positifs, doués de propriétés suppressives vis-à-vis de cellules T effectrices.According to a preferred embodiment, the cells are packaged in a composition comprising between approximately 10 5 and 10 10 suppressor T cells depending on the pathology to be treated, more generally between 10 5 and approximately 10 9 suppressor T cells. A particular composition within the meaning of the invention comprises a population of THY-1 positive human lymphocytes, endowed with suppressive properties with regard to effector T cells.
Le milieu ou adjuvant peut êfre tout milieu de culture, milieu défini, soluté ou suspension aqueuse, tamponnée, éventuellement supplémentée par des agents conservateurs. Les compositions selon l'invention peuvent être administrées par toute voie appropriée, telle que intraveineuse, intraartérielle, sous-cutanée, fransdermique, etc. Des administrations répétées de telles compositions peuvent être mises en œuvre.The medium or adjuvant can be any culture medium, defined medium, aqueous solution or suspension, buffered, optionally supplemented with preservatives. The compositions according to the invention can be administered by any suitable route, such as intravenous, intraarterial, subcutaneous, fransdermal, etc. Repeated administrations of such compositions can be implemented.
D'autres compositions particulières selon l'invention comprennent un ligand spécifique de Thy-1 couplé ou conjugué à une molécule effectrice, par exemple une molécule présentant une toxicité (conditionnelle ou non, par exemple "une TK, toxine de ricin, etc.) ou une activité stimulante pour les lymphocytes T (par exemple une cytokine, notamment IL-2, IL-7, IL-15, etc.). De telles compositions peuvent êfre utilisées in vivo (ou ex vivo) pour moduler le répertoire ou l'activité des cellules T suppresseurs chez un sujet. Ainsi, l'administration d'un conjugué comprenant une molécule toxique peut permetfre d'inactiver ou de réduire l'activité de cellules T suppresseurs chez un sujet, et donc d'augmenter l'activité de cellules effectrices. Inversement, l'administration d'un conjugué comprenant une molécule activatrice peut permettre de stimuler l'activité de cellules T suppresseurs chez un sujet, et donc de réduire l'activité de cellules effectrices. Le couplage peut être covalent ou non.Other particular compositions according to the invention comprise a specific Thy-1 ligand coupled or conjugated to an effector molecule, for example a molecule exhibiting a toxicity (conditional or not, for example "a TK, castor toxin, etc.) or a stimulating activity for T lymphocytes (for example a cytokine, in particular IL-2, IL-7, IL-15, etc.). Such compositions can be used in vivo (or ex vivo) to modulate the repertoire or the suppressor T cell activity in a subject. Thus, the administration of a conjugate comprising a toxic molecule can make it possible to inactivate or reduce the suppressor T cell activity in a subject, and therefore to increase the activity Conversely, the administration of a conjugate comprising an activating molecule can make it possible to stimulate the activity of suppressor T cells in a subject, and therefore to reduce the activity of effector cells. be covalent or not.
D'autres compositions particulières de l'invention comprennent un agent de transfection couplé à un ligand spécifique de Thy-1. Un tel couplage permet de cibler ou de favoriser l'interaction entre l'agent de transfection et les cellules Ts. L'agent de transfection peut êfre une particule virale (par exemple recombinante, défective, atténuée, synthétique, etc.) ou un agent de transfection non-viral, tel qu'un liposome, un lipide canonique, un polymère, etc. Le couplage peut être covalent ou non. De telles compositions permettent de modifier de manière cibler les cellules T suppresseurs d'un sujet, par exemple pour leur conférer de nouvelles propriétés.
UtilisationsOther particular compositions of the invention include a transfection agent coupled to a specific Thy-1 ligand. Such coupling makes it possible to target or promote the interaction between the transfection agent and the Ts cells. The transfection agent can be a viral particle (for example recombinant, defective, attenuated, synthetic, etc.) or a non-viral transfection agent, such as a liposome, a canonical lipid, a polymer, etc. The coupling can be covalent or not. Such compositions make it possible to modify in a targeted manner the suppressor T cells of a subject, for example to confer on them new properties. uses
La présente invention permet d'obtenir des populations cellulaires utilisables pour le traitement de pathologies variées, associées à l'activité des cellules T, comme indiqué ci-dessus. Le traitement peut êfre préventif ou curatif. De plus, les cellules T suppresseurs (y compris les cellules pTs), les populations cellulaires enrichies en cellules T suppresseurs (y compris les cellules pTs) et les compositions selon l'invention peuvent êfre utilisées en combinaison avec d'aufres agents ou principes actifs, tels que d'autres populations cellulaires, des molécules ou des conditions immunosuppressives, des irradiations, des produits de thérapie génique, etc.The present invention makes it possible to obtain cell populations which can be used for the treatment of various pathologies associated with the activity of T cells, as indicated above. Treatment can be preventive or curative. In addition, suppressor T cells (including pTs), cell populations enriched in suppressor T cells (including pTs) and the compositions according to the invention can be used in combination with other agents or active ingredients. , such as other cell populations, immunosuppressive molecules or conditions, radiation, gene therapy products, etc.
Le terme traitement signifie la diminution des symptômes ou des causes d'une maladie, la régression d'une maladie, le retardement d'une maladie, l'amélioration de l'état de patients, la réduction de leur souffrance, l'augmentation de leur durée de vie, etc.The term treatment means the reduction of symptoms or causes of a disease, the regression of a disease, the delay of a disease, the improvement of the condition of patients, the reduction of their suffering, the increase in their lifespan, etc.
Les cellules T suppresseurs (y compris les cellules pTs), les populations cellulaires enrichies en cellules T suppresseurs (y compris les cellules pTs)et les compositions selon l'invention sont particulièrement adaptées pour retarder ou prévenir la maladie du greffon contre l'hôte (GNHD) chez les sujets ayant subi une transplantation d'organe allogénique, en particulier de moelle osseuse (ou de cellules souches hématopoïetiques ou de cellules souches non hématopoïetiques). La GNHD et les fréquentes complications provoquées par la transplantation de cellules souches hématopoïetiques sont dues à la présence de cellules T donneuses matures dans le fransplant. Cependant, l'élimination de ces cellules avant la greffe conduit à un échec de cette dernière, prolonge l'immunosuppression et la réapparition d'une leucémie. L'administration de cellules Ts selon l'invention au moment de la greffe retarde ou même prévient la GNHD. A l'inverse, il peut être avantageux de dépleter les cellules T suppresseurs (y compris les cellules pTs)d'un greffon pour augmenter la réactivité des cellules injectées contre les cellules leucémiques résiduelles. Cette déplétion des cellules THY-1 peut êfre ou non associée à une déplétion à l'aide d'aufres anticorps comme par exemple ceux spécifiques de CD25.
Les cellules T suppresseurs (y compris les cellules pTs), les populations cellulaires enrichies en cellules T suppresseurs (y compris les cellules pTs)et les compositions selon l'invention sont également adaptées au traitement des maladies auto-immunes (incluant les maladies inflammatoires chroniques), telles que le lupus systémique érythémateux, l'arthrite rhumatoïde, la polymyosite, la sclérose multiple, le diabète, l'athérosclérose etc. Les maladies auto-immunes ont une composante imnrunologique comme de nombreuses investigations biologiques et histologiques ont pu le démontrer. Pour ces maladies, l'élément central, est une réponse immunitaire inadaptée. De plus, il est souvent possible d'identifier l'auto-antigène dans le cadre de ces maladies et de définir la période durant laquelle les cellules T délétères sont activées. La présente invention peut êfre utilisée pour prévenir, traiter, réduire ou atténuer de telles pathologies en administrant au sujet une quantité efficace de cellules T suppresseurs (y compris les cellules pTs)pour supprimer ou réduire l'activité de ces cellules T délétères. Des administrations répétées peuvent êfre réalisées si besoin.Suppressor T cells (including pTs cells), cell populations enriched in suppressor T cells (including pTs cells) and the compositions according to the invention are particularly suitable for delaying or preventing graft versus host disease ( GNHD) in subjects who have undergone an allogeneic organ transplant, in particular bone marrow (or hematopoietic stem cells or non-hematopoietic stem cells). GNHD and the frequent complications caused by transplantation of hematopoietic stem cells are due to the presence of mature donor T cells in the fransplant. However, the elimination of these cells before the transplant leads to a failure of the latter, prolongs immunosuppression and the recurrence of leukemia. The administration of Ts cells according to the invention at the time of the graft delays or even prevents GNHD. Conversely, it may be advantageous to deplete suppressor T cells (including pTs cells) from a graft to increase the reactivity of the injected cells against the residual leukemia cells. This depletion of THY-1 cells may or may not be associated with a depletion using other antibodies such as those specific for CD25. Suppressor T cells (including pTs), cell populations enriched in suppressor T cells (including pTs) and the compositions according to the invention are also suitable for the treatment of autoimmune diseases (including chronic inflammatory diseases ), such as systemic lupus erythematosus, rheumatoid arthritis, polymyositis, multiple sclerosis, diabetes, atherosclerosis etc. Autoimmune diseases have an immunological component as numerous biological and histological investigations have been able to demonstrate. For these diseases, the central element is an inadequate immune response. In addition, it is often possible to identify the autoantigen in the context of these diseases and to define the period during which the deleterious T cells are activated. The present invention can be used to prevent, treat, reduce or alleviate such pathologies by administering to the subject an effective amount of suppressor T cells (including pTs cells) to suppress or reduce the activity of these deleterious T cells. Repeated administrations can be carried out if necessary.
Les cellules T suppresseurs (y compris les cellules pTs), les populations cellulaires enrichies en cellules T suppresseurs (y compris les cellules pTs)et les compositions selon l'invention peuvent également êfre utilisées dans le cadre du traitement des maladies infectieuses et notamment des pathologies immunitaires induites par des virus. La réponse immune dirigée contre les agents infectieux peut avoir des conséquences immunopathologiques qui peuvent conduire à la mort. Un exemple est la réponse à certains virus responsables de l'hépatite. Ces virus se répliquent dans les hépatocytes et la destruction de ces hépatocytes infectés par le système immunitaire provoque une hépatite, qui est parfois mortelle. L'évolution de cette hépatite chronique est accompagnée de signes biologiques et d'une réponse immunitaire anormale (par exemple, la présence d'anticorps anti-ADN ou d'une cryoglobulinémie). Les cellules T suppresseurs (y compris les cellules pTs), les populations cellulaires enrichies en cellules T suppresseurs (y compris les cellules pTs)et les compositions selon, l'invention permettent d'éliminer, supprimer ou réduire les lymphocytes T actifs responsables de la pathologie et ainsi de réduire les conséquences des pathologies immunitaires induites par des virus.
Les cellules T suppresseurs (y compris les cellules pTs), les populations cellulaires enrichies en cellules T suppresseurs (y compris les cellules pTs)et les compositions selon l'invention peuvent également être utilisées pour le traitement ou la prévention du rejet d'organes transplantés tels que le cœur, le foie, les reins, les poumons, le pancréas, etc. Le traitement habituel d'un certain nombre de désordres affectant les organes consiste, lorsque cela devient nécessaire, en un remplacement de l'organe par un organe sain provenant d'un donneur décédé (ou d'un donneur vivant dans certains cas, ou même d'un donneur d'une autre espèce). C'est également le cas pour le traitement des diabètes insulino-dépendants, à travers la greffe de cellules ou d'un organe producteur d'insuline, tels que le pancréas ou les îlots pancréatiques. Même si un grand soin est pris dans la sélection de donneurs d'organes présentant un maximum de compatibilité vis-à- vis des antigènes d'histocompatibilité, l'organe transplanté conduit toujours, à l'exception des transplants réalisés entre jumeaux homozygotes, au développement d'une réponse immune dirigée contre les antigènes spécifiquement exprimés par cet organe. En dépit des traitements immunosuppresseurs menés, cette réaction aboutit souvent au rejet de l'organe transplanté (il s'agit de la principale cause d'échec des transplants allogéniques). A l'exception de certains rejet super-aigus ou aigus qui impliquent essentiellement des réponses humorales le rejet de l'organe transplanté est, dans la majorité des cas, essentiellement médié par les lymphocytes T effecteurs. La présente invention permet désormais d'envisager un fraitement (e.g. la diminution ou le retardement) du rejet d'organe à l'aide de cellules T suppresseurs (y compris les cellules pTs). De telles cellules peuvent être préparées à partir de cellules du patient, stimulées avec les antigènes du donneur et ré-administrées au patient, avant ou pendant la transplantation d'organe. Des administrations répétées peuvent être réalisées si besoin. Cette approche est particulièrement adaptée au fraitement des diabètes, i.e., pour réduire, retarder ou prévenir le rejet de cellules productrices d'insuline, de tissus ou d'organes (en particulier les îlots pancréatiques) transplantés. Typiquement, les cellules Ts sont amplifiées et activées par culture en présence d'auto-antigènes provenant du tissu donneur. Ces cellules peuvent être produites par exemple par culture en présence de cellules dendritiques autologues par rapport à la greffe. Ces cellules T suppresseurs (y compris les cellules pTs)amplifiées et activées peuvent être injectées au patient avant,
pendant et/ou après la transplantation d'organe, réduisant ainsi l'activité destructrice des cellules T effectrices.Suppressor T cells (including pTs cells), cell populations enriched in suppressor T cells (including pTs cells) and the compositions according to the invention can also be used in the context of the treatment of infectious diseases and in particular pathologies immune systems induced by viruses. The immune response against infectious agents can have immunopathological consequences which can lead to death. One example is the response to certain viruses responsible for hepatitis. These viruses replicate in hepatocytes and the destruction of these infected hepatocytes by the immune system causes hepatitis, which is sometimes fatal. The progression of this chronic hepatitis is accompanied by biological signs and an abnormal immune response (for example, the presence of anti-DNA antibodies or cryoglobulinemia). The suppressor T cells (including the pTs), the cell populations enriched in suppressor T cells (including the pTs) and the compositions according to the invention make it possible to eliminate, delete or reduce the active T lymphocytes responsible for the pathology and thus reduce the consequences of immune pathologies induced by viruses. Suppressor T cells (including pTs), cell populations enriched in suppressor T cells (including pTs) and compositions according to the invention can also be used for the treatment or prevention of rejection of transplanted organs such as heart, liver, kidneys, lungs, pancreas, etc. The usual treatment for a certain number of disorders affecting the organs consists, when it becomes necessary, in replacing the organ by a healthy organ coming from a deceased donor (or from a living donor in certain cases, or even from a donor of another species). This is also the case for the treatment of insulin-dependent diabetes, through the transplantation of cells or of an insulin-producing organ, such as the pancreas or the pancreatic islets. Even if great care is taken in the selection of organ donors with maximum compatibility with histocompatibility antigens, the transplanted organ always leads, with the exception of transplants made between homozygous twins, to development of an immune response directed against the antigens specifically expressed by this organ. Despite the immunosuppressive treatments carried out, this reaction often results in rejection of the transplanted organ (this is the main cause of failure of allogenic transplants). With the exception of certain super-acute or acute rejection which essentially involve humoral responses, rejection of the transplanted organ is, in the majority of cases, essentially mediated by effector T lymphocytes. The present invention now makes it possible to envisage treatment (eg reduction or delay) of organ rejection using suppressor T cells (including pTs cells). Such cells can be prepared from patient cells, stimulated with donor antigens and re-administered to the patient, before or during organ transplantation. Repeated administrations can be carried out if necessary. This approach is particularly suited to the treatment of diabetes, ie, to reduce, delay or prevent the rejection of transplanted insulin-producing cells, tissues or organs (in particular the pancreatic islets). Typically, Ts cells are amplified and activated by culture in the presence of autoantigens from the donor tissue. These cells can be produced for example by culture in the presence of dendritic cells autologous to the graft. These amplified and activated suppressor T cells (including pTs cells) can be injected into the patient before, during and / or after organ transplantation, thereby reducing the destructive activity of effector T cells.
Les cellules T suppresseurs (y compris les cellules pTs), les populations cellulaires enrichies en cellules T suppresseurs (y compris les cellules pTs)et les compositions selon l'invention sont également adaptées au fraitement des allergies, qui sont médiées par des réponses immunes dirigées contre des antigènes particuliers appelés allergènes. En administrant aux patients les cellules T suppresseurs (y compris les cellules pTs), éventuellement activées ex vivo avec de tels allergènes, il est possible de réduire ces réponses immunitaires délétères.Suppressor T cells (including pTs), cell populations enriched in suppressor T cells (including pTs) and the compositions according to the invention are also suitable for treating allergies, which are mediated by directed immune responses against specific antigens called allergens. By administering suppressor T cells (including pTs) to patients, possibly activated ex vivo with such allergens, it is possible to reduce these deleterious immune responses.
Un autre objet de l'invention concerne une méthode de diminuer l'activité (et ou la quantité de lymphocytes T effecteurs) chez un mammifère hôte, ladite méthode comprenant l'administration au mammifère de lymphocytes T suppresseurs (ou leurs précurseurs) selon l'invention compatibles avec ledit mammifère hôte, de préférence autologues.Another subject of the invention relates to a method of decreasing the activity (and or the amount of effector T lymphocytes) in a host mammal, said method comprising the administration to the mammal of suppressor T lymphocytes (or their precursors) according to the invention compatible with said host mammal, preferably autologous.
La diminution des lymphocytes T suppresseurs (ou leurs précurseurs) peut également êfre recherchée (par exemple dans le cadre du traitement d'un cancer). Plusieurs modalités de fraitement peuvent êfre utilisées.The decrease in suppressor T lymphocytes (or their precursors) may also be sought (for example in the context of the treatment of cancer). Several treatment methods can be used.
Une première approche consiste à préparer ex vivo des cellules ayant une activité (par exemple anti-cancéreuse), déplétées en cellules T suppresseurs (y compris les cellules pTs). Une telle déplétion peut êfre réalisée ex vivo conformément à une méthode selon l'invention telle que décrite précédemment. La déplétion peut êfre réalisée ex vivo sans phase préalable de culture et/ou après une phase de culture dans un milieu contenant de la N-acetyl cystéine qui diminue la prolifération des lymphocytes Ts (y compris les cellules pTs) (voir figure 1). Le traitement consiste alors en la ré-administration au patient d'une population de lymphocytes T ou d'une composition comprenant une telle population dépourvue de cellules T suppresseurs (y compris les cellules pTs), et ayant ou non été activé ex vivo. Ce fraitement peut être accompagné d'une ou plusieurs vaccinations (par exemple anti-tumorales), combinées ou non à de la chimiothérapie et/ou radiothérapie, chez un patient ayant éventuellement reçu un conditionnement. Ce
conditionnement peut notamment comprendre des traitements lympho-ablatifs, myeloablatifs ou non, destinés à éliminer les lymphocytes T , notamment les lymphocytes T en division, et qui comprennent les T suppresseurs (y compris les cellules pTs)responsables de l'absence de réponse immunitaire efficace (comme par exemple les Ts qui empêchent le développement d'une réponse anti-tumorale efficace). Une autre modalité consiste à dépléter les T suppresseurs in vivo par l'utilisation se ligand et de tout activité ou molécule toxique adéquate (comme par exemple radioactivité ou toxine).A first approach consists in preparing ex vivo cells with activity (for example anti-cancer), depleted in suppressor T cells (including pTs cells). Such depletion can be carried out ex vivo in accordance with a method according to the invention as described above. Depletion can be carried out ex vivo without a prior culture phase and / or after a culture phase in a medium containing N-acetyl cysteine which decreases the proliferation of Ts lymphocytes (including pTs cells) (see FIG. 1). The treatment then consists in the re-administration to the patient of a population of T lymphocytes or of a composition comprising such a population lacking T suppressor cells (including the pTs cells), and having or not having been activated ex vivo. This treatment may be accompanied by one or more vaccinations (for example anti-tumor), combined or not with chemotherapy and / or radiotherapy, in a patient who has possibly received conditioning. This conditioning can in particular comprise lympho-ablative treatments, myeloablative or not, intended to eliminate T lymphocytes, in particular dividing T lymphocytes, and which include suppressor T cells (including pTs cells) responsible for the absence of an effective immune response (like for example the Ts which prevent the development of an effective anti-tumor response). Another modality consists in depleting the T suppressors in vivo by the use of ligand and of any adequate toxic activity or molecule (such as for example radioactivity or toxin).
Le traitement de toutes ces pathologies peut également être réalisé par une modulation (suppression ou activation) in vivo des T suppresseurs (y compris les cellules ρTs)à l'aide de toutes molécules ayant ces activités, et notamment des anticorps anti-THYl, ou toutes molécules modulant l'activité des T suppresseurs (y compris les cellules pTs)dont la découverte résulte de la connaissance du franscriptome et protéome des T suppresseurs (y compris les cellules pTs).The treatment of all these pathologies can also be carried out by in vivo modulation (suppression or activation) of suppressor T cells (including ρTs cells) using any molecule having these activities, and in particular anti-THYl antibodies, or all molecules modulating the activity of suppressor T (including pTs cells) whose discovery results from knowledge of the franscriptome and proteome of suppressor T (including pTs cells).
Les lymphocytes T suppresseurs (y compris les cellules pTs)peuvent aussi êfre activés in vivo, par exemple par des ligands de thy-1 couplé a des molécules d'activation des lymphocytes (IL2, IL10 par exemple). Le traitement peut alors être administré par voie générale (infra-veineuse par exemple) ou sur le site ou l'action est recherchée (dans le liquide synovial lors du traitement de la Polyarthrite rhumatoïde par exemple).Suppressor T lymphocytes (including pTs cells) can also be activated in vivo, for example by thy-1 ligands coupled to lymphocyte activation molecules (IL2, IL10 for example). The treatment can then be administered by general route (intravenous for example) or on the site where the action is sought (in the synovial fluid during the treatment of rheumatoid arthritis for example).
Un objet particulier de la présente demande correspond à l'utilisation, dans le cadre de la vaccination, d'une cellule T suppresseur (y compris les cellules pTs), d'une population cellulaire enrichie en cellules T suppresseurs (y compris les cellules pTs)ou d'une composition selon l'invention.A particular object of the present application corresponds to the use, in the context of vaccination, of a suppressor T cell (including pTs cells), of a cell population enriched in suppressor T cells (including pTs cells) ) or of a composition according to the invention.
Des voies d'administrations et des protocoles variés peuvent êfre mis en œuvre dans le cadre de la présente invention. Ils peuvent êfre adaptés par l'homme du métier en fonction de la pathologie à fraiter. Généralement, des administrations systémiques ou locales peuvent êfre envisagées et empruntent la voie intraveineuse, infra-artérielle, infra-péritonéal, infra-musculaire ou sous-cutanée, etc. Les cellules peuvent êfre
injectées durant l'opération chirurgicale ou par tout moyen approprié, à l'aide par exemple, d'une seringue. Pour contrôler des maladies telles que la GNHD, les effets greffe contre infection (GNI) ou greffe conte leucémie (GNL) ou encore le rejet d'un organe transplanté, la composition cellulaire peut être administrée avant, pendant ou après la transplantation de la moelle osseuse (ou de l'organe). De plus, des administrations additionnelles peuvent êfre réalisées après la transplantation, de façon à prévenir ou retarder la pathologie.Various routes of administration and protocols can be implemented in the context of the present invention. They can be adapted by a person skilled in the art depending on the pathology to be treated. Generally, systemic or local administrations can be envisaged and take the intravenous, infra-arterial, infra-peritoneal, infra-muscular or subcutaneous route, etc. Cells can be injected during the surgical operation or by any appropriate means, for example using a syringe. To control diseases such as GNHD, transplant against infection (GNI) or leukemia transplant (GNL) effects or rejection of a transplanted organ, the cell composition can be administered before, during or after bone marrow transplantation bone (or organ). In addition, additional administrations may be performed after transplantation, in order to prevent or delay the pathology.
Il est entendu que la présente invention n'est pas limitée aux modes spécifiques de réalisation décrits ci-après, mais s'étend également aux variantes d'exécution entrant dans les connaissance normales de l'homme du métier.It is understood that the present invention is not limited to the specific embodiments described below, but also extends to variant embodiments which fall within the normal knowledge of those skilled in the art.
LEGENDES DES FIGURESLEGENDS OF FIGURES
Figure 1 : Effet de la N-acetyl Cystéine sur l'expansion préférentielle ou non des lymphocytes T humains CD90+Figure 1: Effect of N-acetyl Cysteine on the preferential expansion or not of human CD90 + T lymphocytes
Des lymphocytes T purifiés CD3+ ont été mis en culture en milieu RPMI contenant 10% de sérum humain , de l'interleukine 2, des anticorps anti-CD3 et présence ou non de N-acetyl Cystéine (NAC). Le pourcentage de cellules T CD3+ exprimant le marqueur CD90 a été étudié au cours du temps par cytométrie en flux.CD3 + purified T lymphocytes were cultured in RPMI medium containing 10% human serum, interleukin 2, anti-CD3 antibodies and presence or absence of N-acetyl Cysteine (NAC). The percentage of CD3 + T cells expressing the CD90 marker was studied over time by flow cytometry.
Figure 2 : Effet des cellules dendritiques sur l'expansion préférentielle ou non des lymphocytes T humains CD4+ CD90+.Figure 2: Effect of dendritic cells on the preferential expansion or not of human CD4 + CD90 + T cells.
Des cellules dendritiques (DC) dérivées de cellules CD34+ et enrichies sur le marqueur CD la (DC langerhansiennes) ou sur le marqueur CD 14 (DC intersticielles) ont été cultivées avec des lymphocytes T allogéniques dans un rapport 1/5 pendant 5 jours. Le pourcentage de cellules T CD3+ exprimant le marqueur CD90 a été étudié au cours du temps par cytométrie en flux.Dendritic cells (DC) derived from CD34 + cells and enriched on the CD la marker (Langerhansian DC) or on the CD 14 marker (interstitial DC) were cultured with allogenic T lymphocytes in a 1/5 ratio for 5 days. The percentage of CD3 + T cells expressing the CD90 marker was studied over time by flow cytometry.
Figure 3 : Expression des antigènes CD25 et CD90 par les lymphocytes T humains CD4+ (A) et CD8+ (B).
Des cellules T ont été marquées avec des anticorps reconnaissant les antigènes CD4, CD8, CD25 et CD90. L'expression de ces différents marqueurs a été réalisée par cytométrie en flux.Figure 3: Expression of CD25 and CD90 antigens by human CD4 + (A) and CD8 + (B) T cells. T cells were labeled with antibodies recognizing the CD4, CD8, CD25 and CD90 antigens. The expression of these different markers was carried out by flow cytometry.
Figure 4 : Les lymphocytes T CD4+/CD90+ et CD8+/CD90+ont une fonction suppressiveFigure 4: CD4 + / CD90 + and CD8 + / CD90 + T cells have a suppressive function
Les populations purifiées CD4CD90+, CD4CD25++ et CD8CD90+ ont été irradiées à 15 grays, mis en culture à rapport égal pendant 4 jours avec des lymphocytes T autologues dépiétés en cellules CD25 (CD25-) stimulés (A) par un mélange d'anticorps OKT3/CD28, (B) par des lymphocytes B allogéniques transformés par EBN, (C) par des cellules dendritiques (DC) allogéniques. Les cellules CD25- stimulées seules dans les mêmes conditions représentent le contrôle positif. La prolifération est évaluée après 4 jours par incorporation de thymidine tritiée.The purified populations CD4CD90 +, CD4CD25 ++ and CD8CD90 + were irradiated with 15 grays, cultured at equal ratio for 4 days with autologous T lymphocytes depressed in CD25 (CD25-) cells stimulated (A) by a mixture of OKT3 / CD28 antibodies. , (B) by allogenic B lymphocytes transformed by EBN, (C) by allogenic dendritic cells (DC). CD25 cells stimulated alone under the same conditions represent the positive control. The proliferation is evaluated after 4 days by incorporation of tritiated thymidine.
Figure 5 expression du gène FoxP3 par les lymphocytes T CD4+/CD90+ et CD8+/CD90+Figure 5 Expression of the FoxP3 gene by CD4 + / CD90 + and CD8 + / CD90 + T lymphocytes
L'expression des gènes CD4, IL-10, CTLA-4 et FoxP3 a été réalisée par RT-PCR sur les différentes populations cellulaires CD4+CD25-, CD4+CD25++, CD4+CD90+, CD8+CD90+ purifiées.The expression of the CD4, IL-10, CTLA-4 and FoxP3 genes was carried out by RT-PCR on the different CD4 + CD25-, CD4 + CD25 ++, CD4 + CD90 +, CD8 + CD90 + cell populations.
Figure 6 : CD90 identifie les précurseurs des lymphocytes suppresseurs CD4CD25++ Les populations cellulaires CD4CD90+, CD4CD25+, CD4CD25++ ont été hautement purifiées par tri cellulaire, cultivées en milieu RPMI contenant du sérum humain AB, de 1TL-2 et un mélange d'anticorps OKT3/CD28 pendant 7 jours. L'analyse des marqueurs CD90 et CD25 a été réalisée à différents temps de culture. Au jour 7, les populations ont été enrichies par tri cellulaire, irradiées à 15 grays et testées pour leur capacité suppressive en les co-cultivant à rapport égale avec des lymphocytes T allogéniques doublement dépiétés en cellules CD25 et CD90 (CD25-CD90-) stimulés par mélange d'anticorps OKT3/CD28. Les chiffres indiquent le pourcentage d'inhibition de la prolifération comparativement au témoin (cellules CD25-CD90- cultivées et stimulées seules).
Figure 7 : CD90 identifie les lymphocytes CD4+/CD90+ et CD8+/CD90+ suppresseurs après 6 jours de cultureFigure 6: CD90 identifies the precursors of suppressor lymphocytes CD4CD25 ++ The cell populations CD4CD90 +, CD4CD25 +, CD4CD25 ++ were highly purified by cell sorting, cultured in RPMI medium containing human serum AB, 1TL-2 and a mixture of antibodies OKT3 / CD28 for 7 days. Analysis of the CD90 and CD25 markers was carried out at different culture times. On day 7, the populations were enriched by cell sorting, irradiated with 15 grays and tested for their suppressive capacity by co-cultivating them in equal ratio with allogenic T lymphocytes doubly depressed in CD25 and CD90 (CD25-CD90-) cells stimulated. by mixing OKT3 / CD28 antibodies. The figures indicate the percentage of inhibition of proliferation compared to the control (CD25-CD90- cells cultured and stimulated alone). Figure 7: CD90 identifies suppressor CD4 + / CD90 + and CD8 + / CD90 + lymphocytes after 6 days of culture
Des lymphocytes T CD3 ont été cultivés en présence d'IL-2 et d'un mélange d'anticorps OKT3/CD28 pendant 6 jours. L'analyse des marqueurs CD25 et CD90 sur les populations CD4 et CD8 permet de déterminer les pourcentages CD25+ et CD90+ (A). Les cellules CD4CD25++, CD4CD90+ et CD8CD90+ ont ensuite été triées, irradiées et testées pour leur capacité suppressive en les co-cultivant à rapport égale avec des lymphocytes T allogéniques dépiétés en cellules CD25 (CD25-) stimulés par mélange d'anticorps OKT3/CD28 (B). La prolifération est évaluée après 4 jours par incorporation de thymidine tritiée.CD3 T cells were cultured in the presence of IL-2 and a mixture of OKT3 / CD28 antibodies for 6 days. Analysis of the markers CD25 and CD90 on the populations CD4 and CD8 makes it possible to determine the percentages CD25 + and CD90 + (A). The CD4CD25 ++, CD4CD90 + and CD8CD90 + cells were then sorted, irradiated and tested for their suppressive capacity by co-culturing them in equal ratio with allogenic T lymphocytes depleted in CD25 (CD25-) cells stimulated by mixing of OKT3 / CD28 antibodies ( B). The proliferation is evaluated after 4 days by incorporation of tritiated thymidine.
Figure 8 : CD90 permet d'identifier à partir de culture de lymphocytes T CD25-CD90- l'apparition de cellules précurseurs et de lymphocytes suppresseursFigure 8: CD90 allows identification from CD25-CD90 T cell culture - the appearance of precursor cells and suppressor lymphocytes
Des lymphocytes T doublement dépiétés en cellules CD25 et CD90 (CD25-CD90-) ont été cultivés en présence d'IL-2 et d'un mélange d'anticorps OKT3/CD28 pendant 7 jours. L'analyse des marqueurs CD90 et CD25 a été réalisée à différents temps de culture. Au jour 7, la population CD25+CD90+ a été enrichie par tri cellulaire, irradiée à 15 grays et testée pour sa capacité suppressive en la co-cultivant à rapport égale avec des lymphocytes T allogéniques doublement dépiétés en cellules CD25 et CD90 (CD25- CD90-) stimulés par mélange d'anticorps OKT3/CD28. Les chiffres indiquent le pourcentage d'inhibition de la prolifération comparativement au témoin (cellules CD25- CD90- cultivées et stimulées seules).T lymphocytes doubly depleted in CD25 and CD90 cells (CD25-CD90-) were cultured in the presence of IL-2 and a mixture of OKT3 / CD28 antibodies for 7 days. Analysis of the CD90 and CD25 markers was carried out at different culture times. On day 7, the CD25 + CD90 + population was enriched by cell sorting, irradiated with 15 grays and tested for its suppressive capacity by co-cultivating it in equal ratio with allogenic T lymphocytes doubly depleted in CD25 and CD90 cells (CD25-CD90 -) stimulated by mixing OKT3 / CD28 antibodies. The figures indicate the percentage of inhibition of proliferation compared to the control (CD25-CD90- cells cultured and stimulated alone).
Figure 9 : Utilisation du marqueur CD90 en pathologie humaine : exemple de la sclérose en plaques.Figure 9: Use of the CD90 marker in human pathology: example of multiple sclerosis.
Des cellules mononucléées obtenues après Ficoll à partir de prélèvements de donneurs sains (n=6), de patients atteints de sclérose en plaques en phase chronique (multiple sclerosis MS n=5), de patients atteints de sclérose en plaques en poussées évolutives (activ MS n= 3) ont été marquées par anticorps monoclonaux CD4, CD25, CD90. Le pourcentage de cellules T CD4+ exprimant le marqueur CD25 et CD90 a été étudié par cytométrie en flux.
Figure 10 : Utilisation du marqueur CD90 en pathologie humaine : exemple d'un patient atteint du syndrome LPEX.Mononuclear cells obtained after Ficoll from samples from healthy donors (n = 6), from patients with chronic multiple sclerosis (multiple sclerosis MS n = 5), from patients with active multiple sclerosis (activ MS n = 3) were labeled with monoclonal antibodies CD4, CD25, CD90. The percentage of CD4 + T cells expressing the marker CD25 and CD90 was studied by flow cytometry. Figure 10: Use of the CD90 marker in human pathology: example of a patient suffering from LPEX syndrome.
Des cellules mononucleées obtenues après Ficoll à partir de prélèvement de donneurs sains, de patients atteints du syndrome LPEX confirmé par séquençage du gène FoXP3 ont été marquées par anticorps monoclonaux CD4, CD25, CD90. Le pourcentage de cellules T CD4+ exprimant le marqueur CD25 et CD90 a été étudié par cytométrie en flux. Les résultats d'un patient LPEX sont montré.Mononuclear cells obtained after Ficoll from samples from healthy donors, from patients with LPEX syndrome confirmed by sequencing of the FoXP3 gene were labeled with monoclonal antibodies CD4, CD25, CD90. The percentage of CD4 + T cells expressing the marker CD25 and CD90 was studied by flow cytometry. The results of an LPEX patient are shown.
EXEMPLESEXAMPLES
1. Le marqueur CD90 est exprimé par les lymphocytes T CD4+/CD25+ humains et par les lymphocytes CD8+/CD25+ humains1. The CD90 marker is expressed by human CD4 + / CD25 + T cells and by human CD8 + / CD25 + lymphocytes
Afin d'étudier l'expression du marqueur CD90 comparativement à CD25 sur les populations lymphocytaires T CD4+ et CD8+, des cellules mononucleées du sang périphérique adulte ont été obtenues par gradient de Ficoll puis marquées avec les anticorps suivants directement à des fluorochromes : anti-CD4, anti-CD8, anti-CD25 et anti-CD90. Pour l'analyse immunophénotypique, les cellules ont été analysées par cytométrie en flux (FACscalibur), les événements étant ré-analysés sur les logiciels Cellquest et FlowJo.In order to study the expression of the CD90 marker compared to CD25 on the CD4 + and CD8 + T lymphocyte populations, mononuclear cells from adult peripheral blood were obtained by Ficoll gradient and then labeled with the following antibodies directly to fluorochromes: anti-CD4 , anti-CD8, anti-CD25 and anti-CD90. For the immunophenotypic analysis, the cells were analyzed by flow cytometry (FACscalibur), the events being re-analyzed on the Cellquest and FlowJo software.
La figure 3 A montre l'expression des marqueurs CD25 et CD90 sur les lymphocytes T CD4+ ainsi que la co-expression de CD25 et CD90 au sein des cellules CD4+. Comme on peut le voir, 6 % et 1.2 % des lymphocytes T CD4+ expriment, respectivement, les marqueurs CD25 et CD90. La majorité (>80%) des cellules CD4+/CD90+ expriment CD25+ à un niveau intermédiaire tandis qu'environ 5% et 15% des cellules CD4+/CD90+ sont respectivement CD25++ et CD25-.FIG. 3A shows the expression of the markers CD25 and CD90 on the CD4 + T lymphocytes as well as the co-expression of CD25 and CD90 within the CD4 + cells. As can be seen, 6% and 1.2% of the CD4 + T lymphocytes express, respectively, the markers CD25 and CD90. The majority (> 80%) of CD4 + / CD90 + cells express CD25 + at an intermediate level while approximately 5% and 15% of CD4 + / CD90 + cells are CD25 ++ and CD25- respectively.
La figure 3 B montre l'expression des marqueurs CD25 et CD90 sur les lymphocytes T CD8+ ainsi que la co-expression de CD25 et CD90 au sein des CD8+. Comme on peut le voir, 7% et 0,2 % des lymphocytes T CD8+ expriment, respectivement, les marqueursFIG. 3B shows the expression of the markers CD25 and CD90 on the CD8 + T lymphocytes as well as the coexpression of CD25 and CD90 within the CD8 +. As can be seen, 7% and 0.2% of CD8 + T cells express, respectively, the markers
CD25 et CD90. Il faut noter qu'aucune cellule CD8+ n'exprime fortement CD25
contrairement aux cellules CD4+. La majorité des cellules CD8+/CD90+ (75%) sont CD25- tandis qu'environ 25% des cellules CD8+/CD90- sont CD25+.CD25 and CD90. It should be noted that no CD8 + cell strongly expresses CD25 unlike CD4 + cells. The majority of CD8 + / CD90 + cells (75%) are CD25- while about 25% of CD8 + / CD90- cells are CD25 +.
2. Le marqueur CD90 identifie des lymphocytes T suppresseurs humains au sein des populations CD4+ et CD8+.2. The CD90 marker identifies human suppressor T lymphocytes in the CD4 + and CD8 + populations.
Afin de montrer que les cellules CD4+/CD90+ ont une fonction suppressive, des lymphocytes T autologues CD4+ dépiétés en cellules CD4+/CD25+ (CD25-) ont été stimulés par un mélange d'anticorps OKT3/CD28 préalablement immobilisé sur le fond des puits de culture. Les cellules CD25- ont été cultivées seules ou en présence d'un nombre égal de cellules CD4+/CD90+ ou CD4+/CD25+ (contrôle positif) pendant 4 jours puis la prolifération a été mesurée par test d'incorporation de thymidine tritiéé, la lecture étant faite sur compteur β. Dans ces expériences les cellules CD4+/CD90+ ou CD4+/CD25+ ont été préalablement irradiées à 15 grays. Les résultats sont exprimés en cpm. Le pourcentage d'inhibition est calculé selon la formule suivant : %inhibition = Nbre cpm (1 - Nbre cpm (CD25- + Ts)/ Nbre cpm (CD25-) X 100.In order to show that the CD4 + / CD90 + cells have a suppressive function, autologous CD4 + T lymphocytes which have been screened in CD4 + / CD25 + cells (CD25-) were stimulated by a mixture of OKT3 / CD28 antibodies previously immobilized on the bottom of the culture wells. . CD25- cells were cultured alone or in the presence of an equal number of CD4 + / CD90 + or CD4 + / CD25 + cells (positive control) for 4 days then the proliferation was measured by incorporation test for tritiated thymidine, the reading being made on β counter. In these experiments, the CD4 + / CD90 + or CD4 + / CD25 + cells were previously irradiated with 15 grays. The results are expressed in cpm. The percentage of inhibition is calculated according to the following formula:% inhibition = No. cpm (1 - No. cpm (CD25- + Ts) / No. cpm (CD25-) X 100.
La figure 4A montre l'inhibition qu'exercent les populations CD4+/CD90+ et CD4+/CD25++ sur la prolifération de lymphocytes T autologues CD25". Les résultats obtenus monfrent que les cellules CD4+/CD90+ inhibent de plus de 75% la prolifération des cellules CD25", révélant ainsi leur fonction suppressive.Figure 4A shows the inhibition exerted populations CD4 + / CD90 + and CD4 + / CD25 ++ on the proliferation of T cells autologous CD25 ". The results obtained monfrent that CD4 + / CD90 + cells inhibit more than 75% the proliferation of CD25 " , thus revealing their suppressive function.
Des expériences utilisant des cellules EBN allogéniques ou des cellules dendritiques (DC) allogéniques comme stimuli de prolifération des cellules CD25- ont également été effectuées. En ajoutant des cellules CD4+/CD90+ ou CD4+/CD25+, il a été montré que ces dernières exerçaient une action suppressive sur la prolifération cellulaire des cellules CD25-. Les figures 4B et 4C montrent ces résultats.Experiments using allogeneic EBN cells or allogeneic dendritic cells (DC) as stimuli for CD25- cell proliferation were also carried out. By adding CD4 + / CD90 + or CD4 + / CD25 + cells, it has been shown that the latter exert a suppressive action on the cell proliferation of CD25- cells. Figures 4B and 4C show these results.
Afin de montrer que les cellules CD8+/CD90+ ont une fonction suppressive, celles-ci ont été mises en présence à nombre égal de cellules CD25- stimulées par mélange par un mélange d'anticorps OKT3/CD28, la prolifération cellulaire étant évaluée quatre jours plus tard par test d'incorporation de thymidine tritiée. Les résultats présentés
figure 4A montrent que la population CD8+/CD90+ exerce une fonction suppressive sur la prolifération des cellules CD25-.In order to show that the CD8 + / CD90 + cells have a suppressive function, these were brought into contact with an equal number of CD25- cells stimulated by mixing with a mixture of OKT3 / CD28 antibodies, the cell proliferation being evaluated four days later late by tritiated thymidine incorporation test. The results presented FIG. 4A show that the CD8 + / CD90 + population exerts a suppressive function on the proliferation of CD25- cells.
3. Les lymphocytes CD4+/90+ et CD8+CD90+expriment Foxp3, CTLA4 et TGFβ3. CD4 + / 90 + and CD8 + CD90 + lymphocytes express Foxp3, CTLA4 and TGFβ
L'analyse de l'expression des gènes Foxp3, CTLA4, CD4, CD25, TGFβ a été réalisée par PCR nestée après transcription inverse de l'ARN extrait de 1000 ou 5000 cellules issues des différentes populations lymphocytaires étudiées. Les résultats obtenus (Figure 5) montrent que les lymphocytes CD4+/90+ et CD8+CD90+expriment Foxp3, CTLA4 et TGFβ comme les cellules CD4+CD25++.The analysis of the expression of the Foxp3, CTLA4, CD4, CD25, TGFβ genes was carried out by nested PCR after reverse transcription of the RNA extracted from 1000 or 5000 cells from the different lymphocyte populations studied. The results obtained (Figure 5) show that the CD4 + / 90 + and CD8 + CD90 + lymphocytes express Foxp3, CTLA4 and TGFβ like CD4 + CD25 ++ cells.
4. CD90 permet d'identifier une population de lymphocytes précurseurs des lymphocytes CD4+/CD25-I-.4. CD90 makes it possible to identify a population of lymphocytes that are precursors of CD4 + / CD25-I- lymphocytes.
Afin d'étudier si la population CD4+/CD90+ a un lien avec les cellules CD4+/CD25++, les cellules CD4+/CD90+ ont été hautement purifiées par cytométrie en flux (pureté supérieure à 98 %) puis mises en culture en milieu liquide en présence d'un mélange d'anticorps OKT3/CD28 et d' Interleukine 2. Les cellules ont été analysées par cytométrie en flux de façon séquentielle entre le 1er jour de culture et le 7eme jour de culture au niveau des marqueurs CD4/CD90 et CD4/CD25. Les populations triées CD4+/CD25++ et CD4+/CD25+/CD90- qui représentent respectivement les lymphocytes T suppresseurs conventionnels et les lymphocytes T activés ont été cultivées en parallèle dans les mêmes conditions. Les résultats présentés figure 6 indiquent que les cellules CD4+/CD90+ perdent progressivement le marqueur CD90 et deviennent fortement positives pour le marqueur CD25. L'évolution immunophénotypique des cellules CD4+/CD25++, qui étaient initialement CD90-, indique que cette population surexprime encore plus fortement après quelques jours de culture le marqueur CD25 sans acquisition du marqueur CD90. La population CD4+/CD25+/CD90- acquiert fortement le marqueur CD25 sans acquisition du marqueur CD90. Afin d'étudier la fonction suppressive de ces différentes populations après culture, les cellules ont été triées, irradiées à 15 grays et mises en présence de cellules CD25- allogéniques.
Les résultats indiquent que 1) les cellules CD4+/CD90+ sont capables de donner naissance à des cellules CD4+/CD25++ à activité suppressive ; 2) les cellules CD4+/CD25++ conservent leur activité suppressive ; 3) les CD4+/CD25+/CD90- donnent naissance à des cellules CD25++ sans activité suppressive. Ces résultats monfrent que les cellules CD4+/CD90+ sont capables de donner naissance à des cellules CD4+CD25++ suppressives et peuvent être considérées comme des cellules précurseurs (pTs) de lymphocytes suppresseurs (Ts).In order to study whether the CD4 + / CD90 + population has a link with CD4 + / CD25 ++ cells, the CD4 + / CD90 + cells were highly purified by flow cytometry (purity greater than 98%) and then cultured in a liquid medium in the presence of a mixture of antibodies OKT3 / CD28 and interleukin 2. cells were analyzed by flow cytometry sequentially between 1 day in culture and the 7th day of culture at markers CD4 / CD90 and CD4 / CD25. The sorted populations CD4 + / CD25 ++ and CD4 + / CD25 + / CD90- which represent respectively the conventional suppressor T cells and the activated T cells were cultivated in parallel under the same conditions. The results presented in FIG. 6 indicate that the CD4 + / CD90 + cells gradually lose the CD90 marker and become strongly positive for the CD25 marker. The immunophenotypic evolution of CD4 + / CD25 ++ cells, which were initially CD90-, indicates that this population overexpressed even more strongly after a few days of culture the CD25 marker without acquisition of the CD90 marker. The CD4 + / CD25 + / CD90- population strongly acquires the CD25 marker without acquiring the CD90 marker. In order to study the suppressive function of these different populations after culture, the cells were sorted, irradiated with 15 grays and placed in the presence of CD25-allogenic cells. The results indicate that 1) CD4 + / CD90 + cells are capable of giving rise to CD4 + / CD25 ++ cells with suppressive activity; 2) CD4 + / CD25 ++ cells retain their suppressive activity; 3) CD4 + / CD25 + / CD90- give rise to CD25 ++ cells without suppressive activity. These results show that CD4 + / CD90 + cells are capable of giving rise to suppressive CD4 + CD25 ++ cells and can be considered as precursor cells (pTs) of suppressor lymphocytes (Ts).
5. CD90 permet d'identifier les lymphocytes CD4+/CD90+ et CD8+/CD90+ suppresseurs après culture.5. CD90 makes it possible to identify the CD4 + / CD90 + and CD8 + / CD90 + suppressor lymphocytes after culture.
Afin d'étudier si le marqueur CD90 permet, après activation et culture de lymphocytes T, d'identifier les lymphocytes T suppresseurs confrairement au marqueur CD25 également exprimé par les lymphocytes T activés, nous avons cultivé, en milieu liquide RPMI 1640 en présence 10% de sérum humain AB et 5μ d'anticorps OKT3, des lymphocytes T totaux dont les populations CD4+/CD90+, CD8+/CD90+ et CD4+/CD25+ ont été purifiées par cytométrie en flux après 6 jours de culture. Après culture, les différents types de cellules ont été analysés par cytométrie et testés pour leurs fonctions suppressives.In order to study whether the CD90 marker makes it possible, after activation and culture of T lymphocytes, to identify suppressor T lymphocytes unlike the CD25 marker also expressed by activated T lymphocytes, we cultured, in RPMI 1640 liquid medium in the presence of 10% human serum AB and 5 μ of OKT3 antibody, total T lymphocytes whose populations CD4 + / CD90 +, CD8 + / CD90 + and CD4 + / CD25 + were purified by flow cytometry after 6 days of culture. After culture, the different types of cells were analyzed by cytometry and tested for their suppressive functions.
La figure 7A montre l'expression des marqueurs CD25 et CD90 sur les lymphocytes T CD4+ et CD8+ après 6 jours de culture. Comme on peut le voir, 6,9 % et 10 % des lymphocytes T CD4+ et CD8+ expriment respectivement le marqueur CD90, tandis que 84 % et 94,3 % des lymphocytes T CD4+ et CD8+ expriment le marqueur CD25.FIG. 7A shows the expression of the markers CD25 and CD90 on the T lymphocytes CD4 + and CD8 + after 6 days of culture. As can be seen, 6.9% and 10% of the CD4 + and CD8 + T lymphocytes express the CD90 marker respectively, while 84% and 94.3% of the CD4 + and CD8 + T lymphocytes express the CD25 marker.
La figure 7B montre l'inhibition qu'exercent les populations CD4+/CD90+ et CD8+/CD90+ sur la prolifération de lymphocytes T autologues CD25- tandis que les lymphocytes CD4+/CD25+ n'ont plus aucun effet suppresseur sur les cellules CD25- après culture. Ces résultats monfrent que le marqueur CD90 reste un marqueur spécifique, confrairement au marqueur CD25, de populations suppressives au sein des lymphocytes T CD4+ et CD8+ cultivés.
6. CD90 permet d'identifier à partir de culture de lymphocytes T CD25-CD90- l'apparition de cellules précurseurs et de lymphocytes suppresseurs.FIG. 7B shows the inhibition exerted by the CD4 + / CD90 + and CD8 + / CD90 + populations on the proliferation of autologous CD25 T lymphocytes, while the CD4 + / CD25 + lymphocytes no longer have any suppressing effect on CD25 cells after culture. These results show that the CD90 marker remains a specific marker, unlike the CD25 marker, in suppressive populations within cultured CD4 + and CD8 + T cells. 6. CD90 makes it possible to identify, from T-cell culture CD25-CD90- the appearance of precursor cells and suppressor lymphocytes.
Afin d'étudier si le marqueur CD90 permet, après activation et culture de lymphocytes T doublement dépiétés en cellules CD25 et CD90 (cellules CD25-CD90-), d'identifier encore des lymphocytes T suppresseurs, nous avons cultivé, en milieu liquide RPMI 1640 en présence 10% de sérum humain AB et 5microgr d'anticorps OKT3 des lymphocytes T CD25-CD90-, L'analyse des marqueurs CD25 et CD90 a été réalisée à différents temps de culture par cytométrie en flux. Les résultats (Figure 8) monfrent l'apparition de deux voies de différenciation dès 24 heures de culture. Ainsi, il est possible de détecter des cellules CD90+CD25- concomitamment avec l'apparition de cellules CD25+CD90-. A partir de 2-3 jours, les cellules CD90+ deviennent CD90+CD25++ tandis que les cellules CD25+CD90- deviennent CD25++CD90-. Le tri et l'étude fonctionnelle des cellules CD90+CD25++ monfrent que ces cellules inhibent la prolifération de lymphocytes T autologues CD25-CD90- stimulées par des anticorps OKT3/CD28. Ces résultats monfrent qu'il est possible de générer des lymphocytes T suppresseurs identifiables grâce au marqueur CD90 à partir de lymphocytes T CD25- CD90-.In order to study whether the marker CD90 makes it possible, after activation and culture of T lymphocytes doubly depleted in CD25 and CD90 cells (CD25-CD90- cells), to further identify suppressor T lymphocytes, we cultivated, in liquid medium RPMI 1640 in the presence of 10% human serum AB and 5 microgr of antibody OKT3 of the T lymphocytes CD25-CD90-, The analysis of the markers CD25 and CD90 was carried out at different culture times by flow cytometry. The results (Figure 8) show the appearance of two differentiation pathways after 24 hours of culture. Thus, it is possible to detect CD90 + CD25- cells concomitantly with the appearance of CD25 + CD90- cells. From 2-3 days, CD90 + cells become CD90 + CD25 ++ while CD25 + CD90- cells become CD25 ++ CD90-. The sorting and the functional study of CD90 + CD25 ++ cells show that these cells inhibit the proliferation of autologous CD25-CD90 T lymphocytes stimulated by OKT3 / CD28 antibodies. These results show that it is possible to generate identifiable suppressor T lymphocytes using the CD90 marker from CD25- CD90- T lymphocytes.
7. Identification et suivi diagnostique7. Identification and diagnostic follow-up
Nous avons montré que les patients présentant des complications auto-immunes d'une hépatite C présentent un déficit en lymphocytes CD4+CD25+ (Boyer et al. Blood, in press). D'aufres auteurs ont montré un tel déficit pour le diabète de type I. Le diagnostique de ces pathologies et le suivi biologique et clinique sera plus spécifique en suivant les Ts par un marquage CD90 qui identifie notamment une population précurseurs des Ts. Ce suivi sera d'autant plus important pour les maladies évoluant par poussées, comme la polyarthrite rhumatoïde ou la SEP par exemple. Le choix et la date de l'intervention thérapeutique, qui pourra notamment êfre une injection de Ts, sera défini grâce au suivi des Ts par le marqueur CD90. L'identification des Ts est réalisée dans tout liquide biologique d'intérêt (sang, LCR, liquide synovial par exemple) ou dans tout tissu ou organe d'intérêt (tumeur, organe transplanté, etc.).
A titre d'exemple nous avons étudié des patients atteints de SEP en phase chronique (MS) et en poussées évolutives (activ MS). La figure 9 montre une augmentation des lymphocytes T CD4+CD25+ (lymphocytes T activées) et au contraire une diminution des cellules CD4+CD90+ au cours des SEP en poussées comparativement aux SEP en phase chronique ou au groupe contrôle. Ces résultats montrent l'intérêt du marqueur CD90 pour évaluer la diminution de lymphocytes T suppresseurs au cours d'une poussée de maladie auto-immune en les distinguant notamment de lymphocytes T activés.We have shown that patients with autoimmune complications of hepatitis C have a deficit of CD4 + CD25 + lymphocytes (Boyer et al. Blood, in press). Other authors have shown such a deficit for type I diabetes. The diagnosis of these pathologies and the biological and clinical monitoring will be more specific by following the Ts by a CD90 marking which identifies in particular a population that is a precursor of the Ts. This monitoring will be all the more important for diseases evolving by flare-ups, such as rheumatoid arthritis or MS for example. The choice and the date of the therapeutic intervention, which could in particular be an injection of Ts, will be defined thanks to the monitoring of Ts by the marker CD90. The identification of Ts is carried out in any biological fluid of interest (blood, CSF, synovial fluid for example) or in any tissue or organ of interest (tumor, transplanted organ, etc.). By way of example, we have studied patients with MS in chronic phase (MS) and in progressive flares (activ MS). FIG. 9 shows an increase in CD4 + CD25 + T lymphocytes (activated T lymphocytes) and on the contrary a decrease in CD4 + CD90 + cells during MS in relapses compared to MS in chronic phase or in the control group. These results show the interest of the CD90 marker for evaluating the decrease in suppressor T lymphocytes during an attack of autoimmune disease by distinguishing them in particular from activated T lymphocytes.
A titre d'exemple nous avons étudié des patients atteints du syndrome LPEX. La figure 10 montre la quasi-absence dans le sang de cellules CD4+/CD90+ chez un patient LPEX comparativement à un donneur sain tandis que l'utilisation du marqueur CD25 qui reconnaît également des lymphocytes T activés ne permet pas de faire une telle différence.As an example, we have studied patients with LPEX syndrome. FIG. 10 shows the virtual absence in the blood of CD4 + / CD90 + cells in an LPEX patient compared to a healthy donor, while the use of the CD25 marker which also recognizes activated T lymphocytes does not make such a difference.
8. Injection Thérapeutique de Ts pour le contrôle de la GVB8. Therapeutic Ts injection for the control of GVB
Nous avons montré que les Ts jouent un rôle important dans le contrôle de la GNH et qu'il est possible de préparer des Ts spécifiques par allo-activation (Cohen et al. JEM 2003, Trénado et al., JCI 2003). Pour ces applications les Ts peuvent êtres obtenues à partir du sang, du sang de cordon, de la moelle osseuse de tous tissus contenant des lymphocytes T. Dans ces applications, elles peuvent être ou non modifiées génétiquement.We have shown that Ts play an important role in the control of GNH and that it is possible to prepare specific Ts by allo-activation (Cohen et al. JEM 2003, Trénado et al., JCI 2003). For these applications, the Ts can be obtained from blood, cord blood, bone marrow of all tissues containing T lymphocytes. In these applications, they may or may not be genetically modified.
9. Injection Thérapeutique de Ts pour le contrôle de la SEP.9. Therapeutic injection of Ts for the control of MS.
Les Ts sont obtenues du patient ou d'un donneur compatible, de préférence géno- identique. Elle sont purifiées par billes immunomagnétiques, cytométrie en flux, par adhérence sur support solide recouvert d'anticorps spécifiques (panning) et éventuellement congelées. Le patient est l'objet d'un suivi biologique pour évaluer son nombre de cellules Ts. L'apparition de signes cliniques indiquant le début d'une
poussée ou d'une chute du nombre de Ts conduit à l'injection des Ts qui sont soit préparées à cette occasion, soit celles préparées précédemment.The Ts are obtained from the patient or from a compatible donor, preferably genome-identical. They are purified by immunomagnetic beads, flow cytometry, by adhesion to a solid support covered with specific antibodies (panning) and possibly frozen. The patient is the subject of biological monitoring to assess his number of Ts cells. The appearance of clinical signs indicating the beginning of a surge or fall in the number of Ts leads to the injection of Ts which are either prepared for this occasion, or those prepared previously.
10. Ablation des Ts pour le traitement de tumeurs10. Ablation of Ts for the treatment of tumors
Nous avons monfré que les Ts empêchent le développement de réactions immunitaires anti-tumorales efficaces. La déplétion de ces cellules permet à ces réponses de se développer. De plus, la préparation de lymphocytes T anti-tumoraux ex vivo se heurte au problème de leur contamination par les Ts. Le principe du traitement est donc d'éliminer les Ts in vivo, notamment à l'aide d'un ligand de CD90 couplé à un toxique. Il peut également s'agir de dépléter l'ensemble des lymphocytes T par des traitements classiques (sérum anti-lymphocytaires, anti-CD3, anticorps Campath, irradiation, etc., par exemple). Ce traitement peut être complété par une administration de lymphocytes activés ex vivo contre les antigènes tumoraux après avoir été dépiétés en Ts.
We have shown that Ts prevent the development of effective anti-tumor immune reactions. The depletion of these cells allows these responses to develop. In addition, the preparation of anti-tumor T cells ex vivo encounters the problem of their contamination by Ts. The principle of the treatment is therefore to eliminate the Ts in vivo, in particular using a CD90 ligand coupled to a toxic. It may also involve depleting all of the T lymphocytes by conventional treatments (anti-lymphocyte serum, anti-CD3, Campath antibody, irradiation, etc., for example). This treatment can be supplemented by the administration of lymphocytes activated ex vivo against tumor antigens after having been depleted in Ts.
Claims
1. Méthode d'obtention, de préparation ou de production de lymphocytes T suppresseurs humains (et/ou leurs précurseurs), comprenant une étape de sélection, de séparation ou d'isolement in vitro ou ex vivo des lymphocytes T humains exprimant la molécule THY-1.1. Method for obtaining, preparing or producing human suppressor T lymphocytes (and / or their precursors), comprising a step of selection, separation or isolation in vitro or ex vivo of human T lymphocytes expressing the THY molecule -1.
2. Méthode selon la revendication 1, comprenant : (a) l'obtention d'une population de cellules d'origine humaine comprenant des lymphocytes T, et (b) la récupération des lymphocytes T exprimant l'antigène THY-1.2. Method according to claim 1, comprising: (a) obtaining a population of cells of human origin comprising T lymphocytes, and (b) recovering T lymphocytes expressing the THY-1 antigen.
3. Méthode selon la revendication 1, caractérisée en ce que l'étape (b) est précédée ou suivie d'une étape d'amplification des lymphocytes T.3. Method according to claim 1, characterized in that step (b) is preceded or followed by a step of amplification of T lymphocytes.
4. Méthode selon l'une quelconque des revendications précédentes,, caractérisée en ce que les lymphocytes T exprimant l'antigène THY-1 sont sélectionnés, séparés, isolés ou récupérés au moyen d'un ligand spécifique de THY-1.4. Method according to any one of the preceding claims, characterized in that the T lymphocytes expressing the THY-1 antigen are selected, separated, isolated or recovered by means of a specific ligand for THY-1.
5. Méthode selon la revendication 4, caractérisée en ce que le ligand spécifique est un anticorps spécifique, de THY-lou un fragment ou dérivé d'un tel anticorps ayant substantiellement la même spécificité antigéήique.5. Method according to claim 4, characterized in that the specific ligand is a specific antibody, of THY-lou a fragment or derivative of such an antibody having substantially the same antigenic specificity.
6. Méthode selon la revendication 5, caractérisée en ce que le ligand spécifique est un anticorps monoclonal ou polyclonal spécifique de THY- 1.6. Method according to claim 5, characterized in that the specific ligand is a monoclonal or polyclonal antibody specific for THY-1.
7. Méthode selon la revendication 5, caractérisée en ce que le ligand spécifique est un anticorps polyfonctionnel, monocaténaire ou multimérique, spécifique de THY-1.7. Method according to claim 5, characterized in that the specific ligand is a polyfunctional, single-stranded or multimeric antibody, specific for THY-1.
8. Méthode selon la revendication 4, caractérisée en ce que le ligand spécifique est un aptamère. 8. Method according to claim 4, characterized in that the specific ligand is an aptamer.
9. Méthode selon l'une des revendications 4 à 8, caractérisée en ce que le ligand est immobilisé sur un support ou placé en solution.9. Method according to one of claims 4 to 8, characterized in that the ligand is immobilized on a support or placed in solution.
10. Méthode selon la revendication 9, caractérisée en ce que le support est une colonne ou une bille, de préférence une bille magnétique.10. Method according to claim 9, characterized in that the support is a column or a ball, preferably a magnetic ball.
11. Méthode selon l'une des revendications 4 à 10, caractérisée en ce que le ligand est marqué.11. Method according to one of claims 4 to 10, characterized in that the ligand is labeled.
12. Méthode selon la revendication 11, caractérisée en ce que le marquage est réalisé à l'aide d'un marqueur de détection fluorescent, radioactif, luminescent, phosphorescent, chimique ou enzymatique.12. Method according to claim 11, characterized in that the labeling is carried out using a fluorescent, radioactive, luminescent, phosphorescent, chemical or enzymatic detection marker.
13. Méthode selon l'une quelconque des revendications précédentes, caractérisée en ce que l'étape de récupération, sélection ou d'isolement est réalisée par cytométrie de flux, chromatographie d'affinité, FACS, MACS ou D/MACS.13. Method according to any one of the preceding claims, characterized in that the recovery, selection or isolation step is carried out by flow cytometry, affinity chromatography, FACS, MACS or D / MACS.
14. Méthode selon l'une quelconque des revendications précédentes, caractérisée en ce que la population cellulaire provient d'un tissu choisi parmi la .moelle osseuse, la rate, le foie, le thymus, du sang ayant ou non été préalablement enrichi en lymphocytes T, du sang de cordon ombilical, du sang périphérique fœtal, de nouveaux-nés ou d'adulte, d'une tumeur, d'un site inflammatoire, d'un organe transplanté ou d'une culture de cellules établie avec l'un ou l'autre desdits tissus.14. Method according to any one of the preceding claims, characterized in that the cell population comes from a tissue chosen from bone marrow, spleen, liver, thymus, blood which may or may not have been previously enriched with lymphocytes T, umbilical cord blood, peripheral fetal blood, newborns or adults, a tumor, an inflammatory site, a transplanted organ, or an established cell culture with one either of said fabrics.
15. Méthode d'identification et/ou de quantification de lymphocytes T suppresseurs humains dans une population cellulaire, comprenant l'exposition de ladite population cellulaire à un ligand spécifique de THY-1 et la détermination et/ou la quantification de la formation d'un complexe entre le ligand et les cellules, la formation de tels complexes indiquant la présence et/ou la quantité de lymphocyte T suppresseurs au sein de la population cellulaire. 15. A method of identifying and / or quantifying human suppressor T cells in a cell population, comprising exposing said cell population to a specific ligand for THY-1 and determining and / or quantifying the formation of a complex between the ligand and the cells, the formation of such complexes indicating the presence and / or the quantity of suppressor T lymphocytes within the cell population.
16. Méthode de production d'une composition pharmaceutique, comprenant : (a) l'obtention d'un échantillon biologique comprenant des lymphocytes T humains, (b) la sélection des lymphocytes T exprimant l'antigène THY-1 au sein de cet échantillon biologique, et (c) le conditionnement desdits lymphocytes T exprimant l'antigène THY-1 dans un adjuvant ou un milieu acceptable sur le plan pharmaceutique.16. A method of producing a pharmaceutical composition, comprising: (a) obtaining a biological sample comprising human T lymphocytes, (b) selecting the T lymphocytes expressing the THY-1 antigen within this sample biological, and (c) conditioning said T lymphocytes expressing the THY-1 antigen in an adjuvant or a pharmaceutically acceptable medium.
17. Méthode de production d'une composition pharmaceutique, comprenant : (a) l'obtention d'un échantillon biologique comprenant des lymphocytes T humains, (b) la déplétion des lymphocytes T exprimant l'antigène THY-1 au sein de cet échantillon biologique, et (c) le conditionnement desdits lymphocytes T obtenus n'exprimant pas l'antigène THY-1 dans un adjuvant ou un milieu acceptable sur le plan pharmaceutique.17. A method of producing a pharmaceutical composition, comprising: (a) obtaining a biological sample comprising human T lymphocytes, (b) depleting T lymphocytes expressing the THY-1 antigen within this sample biological, and (c) conditioning said T lymphocytes obtained which do not express the THY-1 antigen in an adjuvant or a pharmaceutically acceptable medium.
18. Utilisation d'un ligand spécifique de l'antigène THY-1 pour sélectionner, identifier, trier ou préparer, in vitro ou ex vivo, des lymphocytes T suppresseurs humains.18. Use of a specific ligand for the THY-1 antigen to select, identify, sort or prepare, in vitro or ex vivo, human suppressor T lymphocytes.
19. Utilisation d'un ligand spécifique de l'antigène THY-1 pour préparer une composition diagnostique destinée à sélectionner, identifier ou quantifier in vivo des lymphocytes T suppresseurs humains.19. Use of a specific ligand for the THY-1 antigen to prepare a diagnostic composition intended to select, identify or quantify in vivo human suppressor T lymphocytes.
20. Utilisation d'un ligand spécifique de l'antigène THY- 1 ..pour préparer une composition thérapeutique destinée à modifier, stimuler ou supprimer in vivo des lymphocytes T suppresseurs humains.20. Use of a specific ligand for the THY-1 .. antigen to prepare a therapeutic composition intended to modify, stimulate or suppress in vivo human suppressor T lymphocytes.
21. Lymphocyte T humain isolé, caractérisé en ce qu'il présente une activité suppressive et en ce qu'il exprime les marqueurs CD8 ou CD4 et THY-1. 21. Isolated human T lymphocyte, characterized in that it exhibits suppressive activity and in that it expresses the markers CD8 or CD4 and THY-1.
22. Composition cellulaire comprenant au moins, 50, 60, 70, 80, 85, 90 ou 95% de cellules T humaines CD8+THY-1+ ou CD4+THY-1+.22. Cell composition comprising at least 50, 60, 70, 80, 85, 90 or 95% of human T cells CD8 + THY-1 + or CD4 + THY-1 +.
23. Lymphocyte T selon la revendication 21, caractérisé en ce qu'il est modifié génétiquement à l'aide d'un vecteur viral.23. T lymphocyte according to claim 21, characterized in that it is genetically modified using a viral vector.
24. Composition pharmaceutique caractérisée en ce qu'elle comprend au moins un lymphocyte T suppresseur selon l'une des revendications 21 à 23 et un adjuvant ou un milieu acceptable sur le plan pharmaceutique.24. Pharmaceutical composition characterized in that it comprises at least one suppressor T lymphocyte according to one of claims 21 to 23 and an adjuvant or a pharmaceutically acceptable medium.
25. Utilisation d'un lymphocyte T ou d'une population de lymphocytes T selon l'une des revendications 21 à 23 pour la préparation d'une composition destinée à la mise en œuvre d'une méthode thérapeutique.25. Use of a T lymphocyte or a population of T lymphocytes according to one of claims 21 to 23 for the preparation of a composition intended for the implementation of a therapeutic method.
26. Utilisation selon la revendication 25, caractérisée en ce que la composition est destinée à être utilisée comme vaccin.26. Use according to claim 25, characterized in that the composition is intended to be used as a vaccine.
27. Utilisation selon la revendication 25 pour la préparation d'une composition destinée au traitement d'une tumeur, d'une maladie auto-immune, d'une allergie, d'une maladie du greffon contre l'hôte, d'une maladie inflammatoire, d'un diabète de type I, d'une infection virale ou bactérienne, à la reconstitution immune ou à l'induction d'une tolérance en cas de transplantation de cellules souches, tissus ou organe chez un mammifère.27. Use according to claim 25 for the preparation of a composition intended for the treatment of a tumor, an autoimmune disease, an allergy, a graft versus host disease, a disease inflammatory, type I diabetes, viral or bacterial infection, immune reconstitution or tolerance induction in case of transplantation of stem cells, tissues or organ in a mammal.
28. Kit pour isoler ou caractériser des lymphocytes T suppresseurs, humains comprenant un ligand spécifique de THY-1, placé ou solution ou sur un support, ainsi que, éventuellement, des réactifs pour la détection du ligand. 28. Kit for isolating or characterizing suppressor T lymphocytes, human comprising a specific ligand for THY-1, placed or solution or on a support, as well as, possibly, reagents for the detection of the ligand.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR0315360A FR2864546A1 (en) | 2003-12-24 | 2003-12-24 | METHOD OF IDENTIFYING AND PREPARING T REGULATORY / SUPPRESSOR T CELLS, COMPOSITIONS AND USES |
PCT/FR2004/003374 WO2005063969A2 (en) | 2003-12-24 | 2004-12-23 | Methods for the identification and preparation of regulator/suppressor t lymphocytes, compositions and uses thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1697502A2 true EP1697502A2 (en) | 2006-09-06 |
Family
ID=34639584
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP04816487A Withdrawn EP1697502A2 (en) | 2003-12-24 | 2004-12-23 | Methods for the identification and preparation of regulator/suppressor t lymphocytes, compositions and uses thereof |
Country Status (6)
Country | Link |
---|---|
US (1) | US20070128670A1 (en) |
EP (1) | EP1697502A2 (en) |
JP (1) | JP2007521803A (en) |
CA (1) | CA2549394A1 (en) |
FR (1) | FR2864546A1 (en) |
WO (1) | WO2005063969A2 (en) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2898275B1 (en) * | 2006-03-10 | 2012-12-14 | Genethon | CD4 + CD25 + REGULATORY T CELLS SPECIFIC FOR HEMATOPOIETIC CELL TRANSPLANT AND IMMUNE TOLERANCE |
EP3858347A3 (en) | 2007-08-17 | 2021-12-01 | Purdue Research Foundation | Psma binding ligand-linker conjugates and methods for using |
ITMI20080865A1 (en) * | 2008-05-13 | 2009-11-14 | Istituto Naz Di Genetica Molecolare Ingm | HEMATOPOIETAL CELLS EXPRESSING SUSD3 PROTEIN AND BINDERS FOR SUSD3 PROTEIN |
US9951324B2 (en) | 2010-02-25 | 2018-04-24 | Purdue Research Foundation | PSMA binding ligand-linker conjugates and methods for using |
US8232059B2 (en) | 2010-06-14 | 2012-07-31 | Alsultan Abdulrahman A | Method of identifying A. baumannii with OXA-131-like drug resistance in diabetic patients |
MX2015006109A (en) | 2012-11-15 | 2016-02-05 | Endocyte Inc | Conjugates for treating diseases caused by psma expressing cells. |
RS65324B1 (en) | 2013-10-18 | 2024-04-30 | Novartis Ag | Labeled inhibitors of prostate specific membrane antigen (psma), their use as imaging agents and pharmaceutical agents for the treatment of prostate cancer |
US10188759B2 (en) | 2015-01-07 | 2019-01-29 | Endocyte, Inc. | Conjugates for imaging |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001032841A2 (en) * | 1999-10-29 | 2001-05-10 | The Government Of The United States Of America, Represented By The Secretary, Dept. Of Health And Human Services | Method of in vitro t cell differentiation of cd34+ progenitor cells |
EP1241249A1 (en) * | 2001-03-12 | 2002-09-18 | Gerold Schuler | CD4+CD25+regulatory T cells from human blood |
-
2003
- 2003-12-24 FR FR0315360A patent/FR2864546A1/en not_active Withdrawn
-
2004
- 2004-12-23 US US10/584,728 patent/US20070128670A1/en not_active Abandoned
- 2004-12-23 WO PCT/FR2004/003374 patent/WO2005063969A2/en not_active Application Discontinuation
- 2004-12-23 JP JP2006546253A patent/JP2007521803A/en active Pending
- 2004-12-23 CA CA002549394A patent/CA2549394A1/en not_active Abandoned
- 2004-12-23 EP EP04816487A patent/EP1697502A2/en not_active Withdrawn
Non-Patent Citations (1)
Title |
---|
See references of WO2005063969A2 * |
Also Published As
Publication number | Publication date |
---|---|
WO2005063969A2 (en) | 2005-07-14 |
WO2005063969A3 (en) | 2005-10-13 |
JP2007521803A (en) | 2007-08-09 |
CA2549394A1 (en) | 2005-07-14 |
US20070128670A1 (en) | 2007-06-07 |
FR2864546A1 (en) | 2005-07-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8053235B2 (en) | Methods of generating antigen-specific CD4+CD25+regulatory T cells, compositions and methods of use | |
Li et al. | Mechanism and localization of CD8 regulatory T cells in a heart transplant model of tolerance | |
ES2328650T3 (en) | DIRECT SELECTION METHOD OF ANTIGEN T SPECIFIC CELLS. | |
ES2686593T3 (en) | CD127 expression that correlates inversely with FoxP3 and the Treg CD4 + suppressor function | |
JP7254128B2 (en) | New Subpopulations of CD8+CD45RClow Tregs and Their Use | |
JP2021518121A (en) | How to Enhance the Persistence of Adopted T Cells | |
JP2004529631A (en) | CD4 + CD25 + regulatory T cells from human blood | |
JP2004512030A (en) | Compositions and methods for inducing a specific cytolytic T cell response | |
US20080175830A1 (en) | Culture-expanded T suppressor cells and methods of use thereof | |
CN115747156A (en) | NKT cell subsets for in vivo persistence and therapeutic activity and propagation thereof | |
EP1697502A2 (en) | Methods for the identification and preparation of regulator/suppressor t lymphocytes, compositions and uses thereof | |
WO2009145238A1 (en) | Immunomodulatory agent and use thereof | |
AU2018250926B2 (en) | Methods to produce peptides, polypeptides or cells for modulating immunity | |
KR20060040671A (en) | Autologous self-tolerance inducing cells of monocytic origin and their use in pharmaceutical preparations | |
EP2201103B1 (en) | Treatment of tumours using t lymphocyte preparations | |
EP3319625B1 (en) | Immunogenic preprocalcitonin peptides | |
EP4347794A1 (en) | Person-tailored t cell composition targeting merkel cell carcinoma | |
CN114891741A (en) | Tumor antigen/MHC-I compound and preparation method and application thereof | |
Deniz | Subsets of human natural killer cells and their regulatory effect | |
WO2000044880A1 (en) | Means for inducing or increasing the immunogenicity of myeloid acute leukemia cells | |
Blais | Étude de la fonction et de la régulation homéostatique des lymphocytes T extrathymiques | |
Sirkiä | Small-molecules as regulatory T cell modulators: A pilot screen | |
Bischoff | Chemokine-mediated modulation of autoimmunity in type 1 diabetes | |
WO1997018232A2 (en) | Peptides derived from the oncogene proteine bcr-abl expressed by leukemic cells and utilisation thereof for the therapy of human leukemiae | |
FR2960882A1 (en) | Use of mononucleated cells activated in presence of mesenchymal cells to obtain interleukin-17 producing cells, and to treat infection, cancer or autoimmune diseases such as rheumatoid arthritis, psoriasis and multiple sclerosis |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20060612 |
|
AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU MC NL PL PT RO SE SI SK TR |
|
17Q | First examination report despatched |
Effective date: 20061004 |
|
DAX | Request for extension of the european patent (deleted) | ||
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20070417 |