WO2011130491A2 - Methods and compositions for treating hiv - Google Patents
Methods and compositions for treating hiv Download PDFInfo
- Publication number
- WO2011130491A2 WO2011130491A2 PCT/US2011/032455 US2011032455W WO2011130491A2 WO 2011130491 A2 WO2011130491 A2 WO 2011130491A2 US 2011032455 W US2011032455 W US 2011032455W WO 2011130491 A2 WO2011130491 A2 WO 2011130491A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- nucleic acid
- cells
- hiv
- acid construct
- domain
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims description 13
- 239000000203 mixture Substances 0.000 title claims description 5
- 210000004027 cell Anatomy 0.000 claims abstract description 88
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 48
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 47
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 47
- 230000001086 cytosolic effect Effects 0.000 claims abstract description 25
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 claims abstract description 24
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 claims abstract description 23
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 66
- 108020004459 Small interfering RNA Proteins 0.000 claims description 34
- 150000001413 amino acids Chemical class 0.000 claims description 33
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 25
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 25
- 229920001184 polypeptide Polymers 0.000 claims description 24
- 239000012634 fragment Substances 0.000 claims description 21
- 239000013598 vector Substances 0.000 claims description 16
- 108020001507 fusion proteins Proteins 0.000 claims description 14
- 102000037865 fusion proteins Human genes 0.000 claims description 14
- 101710149870 C-C chemokine receptor type 5 Proteins 0.000 claims description 8
- 102100035875 C-C chemokine receptor type 5 Human genes 0.000 claims description 8
- 101710135898 Myc proto-oncogene protein Proteins 0.000 claims description 7
- 102100038895 Myc proto-oncogene protein Human genes 0.000 claims description 7
- 101710150448 Transcriptional regulator Myc Proteins 0.000 claims description 7
- 239000000710 homodimer Substances 0.000 claims description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 108091008874 T cell receptors Proteins 0.000 abstract description 7
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 abstract description 7
- 230000006044 T cell activation Effects 0.000 abstract description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 53
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 20
- 235000001014 amino acid Nutrition 0.000 description 16
- 229940024606 amino acid Drugs 0.000 description 16
- 208000031886 HIV Infections Diseases 0.000 description 15
- 241000282414 Homo sapiens Species 0.000 description 15
- 108091027967 Small hairpin RNA Proteins 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 13
- 208000037357 HIV infectious disease Diseases 0.000 description 11
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 11
- 239000004055 small Interfering RNA Substances 0.000 description 11
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 8
- 208000030507 AIDS Diseases 0.000 description 7
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 7
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 7
- 241001430294 unidentified retrovirus Species 0.000 description 7
- 108010002350 Interleukin-2 Proteins 0.000 description 6
- 102000000588 Interleukin-2 Human genes 0.000 description 6
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 6
- 230000004913 activation Effects 0.000 description 5
- 239000012636 effector Substances 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 230000028327 secretion Effects 0.000 description 5
- 102000004127 Cytokines Human genes 0.000 description 4
- 108090000695 Cytokines Proteins 0.000 description 4
- 241000713340 Human immunodeficiency virus 2 Species 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- WHBIGIKBNXZKFE-UHFFFAOYSA-N delavirdine Chemical compound CC(C)NC1=CC=CN=C1N1CCN(C(=O)C=2NC3=CC=C(NS(C)(=O)=O)C=C3C=2)CC1 WHBIGIKBNXZKFE-UHFFFAOYSA-N 0.000 description 4
- 239000000539 dimer Substances 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 230000010076 replication Effects 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 238000010361 transduction Methods 0.000 description 4
- 230000026683 transduction Effects 0.000 description 4
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 3
- 108010041397 CD4 Antigens Proteins 0.000 description 3
- 210000004366 CD4-positive T-lymphocyte Anatomy 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 101100005713 Homo sapiens CD4 gene Proteins 0.000 description 3
- 108091030071 RNAI Proteins 0.000 description 3
- 230000036436 anti-hiv Effects 0.000 description 3
- 125000000151 cysteine group Chemical class N[C@@H](CS)C(=O)* 0.000 description 3
- -1 ddl) Chemical compound 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 230000009368 gene silencing by RNA Effects 0.000 description 3
- 230000001177 retroviral effect Effects 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 description 2
- BXZVVICBKDXVGW-NKWVEPMBSA-N Didanosine Chemical compound O1[C@H](CO)CC[C@@H]1N1C(NC=NC2=O)=C2N=C1 BXZVVICBKDXVGW-NKWVEPMBSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 101000922348 Homo sapiens C-X-C chemokine receptor type 4 Proteins 0.000 description 2
- 108010074328 Interferon-gamma Proteins 0.000 description 2
- 108091081021 Sense strand Proteins 0.000 description 2
- 241000713311 Simian immunodeficiency virus Species 0.000 description 2
- 206010047289 Ventricular extrasystoles Diseases 0.000 description 2
- WREGKURFCTUGRC-POYBYMJQSA-N Zalcitabine Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)CC1 WREGKURFCTUGRC-POYBYMJQSA-N 0.000 description 2
- 230000006786 activation induced cell death Effects 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000003501 co-culture Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229960002656 didanosine Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- CBVCZFGXHXORBI-PXQQMZJSSA-N indinavir Chemical compound C([C@H](N(CC1)C[C@@H](O)C[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H]2C3=CC=CC=C3C[C@H]2O)C(=O)NC(C)(C)C)N1CC1=CC=CN=C1 CBVCZFGXHXORBI-PXQQMZJSSA-N 0.000 description 2
- JTEGQNOMFQHVDC-NKWVEPMBSA-N lamivudine Chemical compound O=C1N=C(N)C=CN1[C@H]1O[C@@H](CO)SC1 JTEGQNOMFQHVDC-NKWVEPMBSA-N 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- NQDJXKOVJZTUJA-UHFFFAOYSA-N nevirapine Chemical compound C12=NC=CC=C2C(=O)NC=2C(C)=CC=NC=2N1C1CC1 NQDJXKOVJZTUJA-UHFFFAOYSA-N 0.000 description 2
- 230000036515 potency Effects 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- NCDNCNXCDXHOMX-XGKFQTDJSA-N ritonavir Chemical compound N([C@@H](C(C)C)C(=O)N[C@H](C[C@H](O)[C@H](CC=1C=CC=CC=1)NC(=O)OCC=1SC=NC=1)CC=1C=CC=CC=1)C(=O)N(C)CC1=CSC(C(C)C)=N1 NCDNCNXCDXHOMX-XGKFQTDJSA-N 0.000 description 2
- QWAXKHKRTORLEM-UGJKXSETSA-N saquinavir Chemical compound C([C@@H]([C@H](O)CN1C[C@H]2CCCC[C@H]2C[C@H]1C(=O)NC(C)(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)C=1N=C2C=CC=CC2=CC=1)C1=CC=CC=C1 QWAXKHKRTORLEM-UGJKXSETSA-N 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 238000005129 volume perturbation calorimetry Methods 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 101710149863 C-C chemokine receptor type 4 Proteins 0.000 description 1
- 102100032976 CCR4-NOT transcription complex subunit 6 Human genes 0.000 description 1
- 108010062433 CD28 Antigens Proteins 0.000 description 1
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 241001559589 Cullen Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101100166600 Homo sapiens CD28 gene Proteins 0.000 description 1
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 1
- 241000714260 Human T-lymphotropic virus 1 Species 0.000 description 1
- 241000714259 Human T-lymphotropic virus 2 Species 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 1
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108090000172 Interleukin-15 Proteins 0.000 description 1
- 108010002586 Interleukin-7 Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- NCDNCNXCDXHOMX-UHFFFAOYSA-N Ritonavir Natural products C=1C=CC=CC=1CC(NC(=O)OCC=1SC=NC=1)C(O)CC(CC=1C=CC=CC=1)NC(=O)C(C(C)C)NC(=O)N(C)CC1=CSC(C(C)C)=N1 NCDNCNXCDXHOMX-UHFFFAOYSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 101710191252 T-cell surface glycoprotein CD4 Proteins 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 101800001690 Transmembrane protein gp41 Proteins 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 238000011374 additional therapy Methods 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000000798 anti-retroviral effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000002869 basic local alignment search tool Methods 0.000 description 1
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003067 chemokine receptor CCR5 antagonist Substances 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 229940088900 crixivan Drugs 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 229960005319 delavirdine Drugs 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- QYMFNZIUDRQRSA-UHFFFAOYSA-N dimethyl butanedioate;dimethyl hexanedioate;dimethyl pentanedioate Chemical compound COC(=O)CCC(=O)OC.COC(=O)CCCC(=O)OC.COC(=O)CCCCC(=O)OC QYMFNZIUDRQRSA-UHFFFAOYSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000012137 double-staining Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 229940072253 epivir Drugs 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 229940125777 fusion inhibitor Drugs 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 235000004554 glutamine Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 102000027596 immune receptors Human genes 0.000 description 1
- 108091008915 immune receptors Proteins 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 229960001936 indinavir Drugs 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 229940124524 integrase inhibitor Drugs 0.000 description 1
- 239000002850 integrase inhibitor Substances 0.000 description 1
- 229960003130 interferon gamma Drugs 0.000 description 1
- 229940088976 invirase Drugs 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960001627 lamivudine Drugs 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000004897 n-terminal region Anatomy 0.000 description 1
- 229960000689 nevirapine Drugs 0.000 description 1
- 229940072250 norvir Drugs 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 229940063627 rescriptor Drugs 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108010056030 retronectin Proteins 0.000 description 1
- 229960000311 ritonavir Drugs 0.000 description 1
- 239000003419 rna directed dna polymerase inhibitor Substances 0.000 description 1
- 229960001852 saquinavir Drugs 0.000 description 1
- 208000011581 secondary neoplasm Diseases 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000010648 susceptibility to HIV infection Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 229960000523 zalcitabine Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
-
- 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
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/464838—Viral antigens
-
- 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
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70514—CD4
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70521—CD28, CD152
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
- A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
-
- 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
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
Definitions
- the invention features a method of treating a patient infected with HIV (e.g., HIV-1 or HIV-2) by administering a composition including any of the foregoing host cells.
- HIV e.g., HIV-1 or HIV-2
- the host cell can be isolated from the patient being treated or from another patient.
- the DNA constructs and host cells of the invention also optionally feature components to suppress HIV infection of host T-cells.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Virology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Ophthalmology & Optometry (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Tropical Medicine & Parasitology (AREA)
- AIDS & HIV (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention features nucleic acid constructs encoding chimeric immune T-cell receptors (CIRs) that are useful for treating HIV in patients. In general, the CIRs contain an extracellular domain which targets HIV or HIV infected cells (e.g., the extracellular domain of CD4), a transmembrane domain, and a cytoplasmic domain for mediating T-cell activation (e.g., CD3 zeta and/or the partial extracellular domain of CD28). The invention also features the use of host cells expressing CIRs in the treatment of HIV.
Description
METHODS AND COMPOSITIONS FOR TREATING HIV
Cross-Reference to Related Applications
This application claims benefit of U.S. Provisional Application No. 61/324,050, filed April 14, 2010, which is hereby incorporated by reference.
Statement as to Federally Funded Research
This work was supported by grant number NIH R21, 1R21AI076145-01 from the United States National Institutes of Health. The Government has certain rights in the invention.
Background of the Invention
The invention relates to the field of treating HIV with modified T-cells.
In 1984, HIV was shown to be the etiologic agent of AIDS. Since that time, the definition of AIDS has been revised a number of times with regard to what criteria should be included in the diagnosis. However, despite the fluctuation in diagnostic parameters, the simple common denominator of AIDS is the infection with HIV and subsequent development of persistent
constitutional symptoms and AIDS -defining diseases such as a secondary infections, neoplasms, and neurologic disease.
HIV is a human retrovirus of the lentivirus group. The four recognized human retroviruses belong to two distinct groups: the human T lympho tropic (or leukemia) retroviruses, HTLV-1 and HTLV-2, and the human
immunodeficiency viruses, HIV-1 and HIV-2. The former are transforming viruses whereas the latter are cytopathic viruses.
HIV-1 has been identified as the most common cause of AIDS throughout the world. Sequence identity between HIV-2 and HIV-1 is about 40%, with HIV-2 being more closely related to some members of a group of simian immunodeficiency viruses (SIV).
The main cause of the immune defect in AIDS has been identified as a quantitative and qualitative deficiency in the subset of thymus-derived (T) lymphocytes, the T4 population. This subset of cells is defined phenotypically by the presence of the CD4 surface molecule, which has been demonstrated to be the cellular receptor for HIV. Although the T4 cell is the major cell type infected with HIV, essentially any human cell that expresses the CD4 molecule on its surface is capable of binding to and being infected with HIV.
Previous attempts to treat patients with "designer" T-cells expressing chimeric immune receptors (CIRs) proved unsuccessful. There exists a need in the art for new therapies for HIV. The present invention addresses this issue and offers advantages over previous attempted therapies.
Summary of the Invention
In one aspect, the invention features a nucleic acid construct encoding a chimeric protein that includes (i) an extracellular domain of CD4 (e.g., amino acids 1-372 of SEQ ID NO: l) or a fragment thereof, (ii) a transmembrane domain, and (iii) a cytoplasmic domain that includes the cytoplasmic domain of the CD3 zeta chain (e.g., a polypeptide having the amino acids 31-142 of SEQ ID NO:3), or a fragment thereof, and the cytoplasmic domain of CD28 (e.g., a polypeptide having amino acids 127-234 of SEQ ID NO:2), or a fragment thereof. In one embodiment, the cytoplasmic domain has the amino acid sequence of SEQ ID NO: 10.
In another aspect, the invention features a nucleic acid construct encoding a chimeric protein that includes (i) an extracellular domain of CD4 (e.g., a polypeptide having amino acids 1-372 of SEQ ID NO: 1) or a fragment thereof, (ii) a transmembrane domain, and (iii) a cytoplasmic domain of the CD3 zeta chain (e.g., a polypeptide having the amino acids 31-142 of SEQ ID NO: 3), or a fragment thereof.
In either of the foregoing aspects, the chimeric protein can be capable of forming a homodimer when expressed in a T-cell, e.g., through the formation of a disulfide bond.
Also in either of the foregoing aspects, the transmembrane domain can be the transmembrane domain of the CD3 zeta chain (e.g„ a polypeptide having amino acids 7-30 of SEQ ID NO:3) or the transmembrane/partial extracellular domain of CD28.
Also in either of the foregoing aspects, the chimeric protein can include a c-myc tag (e.g., at the N-terminus).
In another aspect, the invention features a vector including any of the nucleic acid constructs described above. This vector can also include a nucleic acid construct encoding an siRNA (e.g., against CCR5 or against Tat/Rev).
In yet another aspect, the invention features a host cell (e.g., a T-cell derived from an uninfected patient or T-cell derived from a patient infected with HIV) containing any of the above nucleic acid constructs or vectors. This host cell can also include a nucleic acid construct encoding an siRNA (e.g., against CCR5 or against Tat/Rev).
In another aspect, the invention features a method of treating a patient infected with HIV (e.g., HIV-1 or HIV-2) by administering a composition including any of the foregoing host cells. In this aspect, the host cell can be isolated from the patient being treated or from another patient.
By "specifically binds" is meant an extracellular domain which recognizes and binds an HIV protein, but that does not substantially recognize and bind other molecules in a sample, e.g., a human blood sample.
By "treating" is meant ameliorating a condition or symptom(s) of the condition (e.g., the symptoms of HIV infection). To "treat HIV" or refers to administering a treatment to a subject infected with HIV to improve the subject's condition. As compared with an equivalent untreated control, such amelioration or degree of treatment is at least 5%, 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, 99%, or 100%, as measured by the subject's HIV viral load.
By "vector" is meant a DNA molecule, usually derived from a plasmid or bacteriophage, into which fragments of DNA may be inserted or cloned. A recombinant vector will contain one or more unique restriction sites, and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible. A vector contains a promoter operably-linked to a gene or coding region such that, upon transfection into a recipient cell, an RNA or an encoded protein or is expressed.
By "host T-cell" is meant a cell (e.g., a human T-cell isolated from a subject) into which one or more nucleic acid constructs is introduced.
By "chimeric immune T-cell receptor" or "CIR" is meant a fusion protein which, when expressed in a host T cell, contains an extracellular domain that specifically binds to a target protein and a cytoplasmic domain that modulates activation of the host T-cell.
By "CD4 extracellular domain" is meant a polypeptide having the N- terminal region of CD4 that is located outside the cell membrane when expressed in a T-cell, e.g., a polypeptide having the amino acid sequence of amino acids 1-372 of SEQ ID NO: l. The term "CD4 extracellular domain" is also meant to include any polypeptide fragment that binds specifically to gpl20 and is substantially identical to amino acids 1-372 of SEQ ID NO: l over the length of the polypeptide fragment.
SEQ ID NO:l Human CD4 Amino Acid sequence
LOCUS NP_000607 458 aa linear PRI
18-MAR-2010
DEFINITION T-cell surface glycoprotein CD4 precursor [Homo sapiens] . ACCESSION NP_000607
VERSION NP_000607.1 GI:10835167
DBSOURCE REFSEQ: accession NM_000616.3
1 MNRGVPFRHL LLVLQLALLP AATQGKKVVL GKKGDTVELT CTASQKKS IQ FHWKNSNQI K
6 1 ILGNQGSFLT KGPSKLNDRA DSRRSLWDQG NFPLI IKNLK IEDSDTYI CE VEDQKEEVQL
12 1 LVFGLTANSD THLLQGQSLT LTLESPPGSS PSVQCRSPRG KNIQGGKTLS VSQLELQDSG 181 TWTCTVLQNQ KKVEFKI DIV VLAFQKAS S I VYKKEGEQVE FSFPLAFTVE KLTGSGELWW
241 QAERAS SSKS WITFDLKNKE VSVKRVTQDP KLQMGKKLPL HLTLPQALPQ YAGSGNLTLA
301 LEAKTGKLHQ EVNLVVMRAT QLQKNLTCEV WGPTSPKLML SLKLENKEAK VSKREKAVWV
361 LNPEAGMWQC LLSDSGQVLL ESNIKVLPTW STPVQPMALI VLGGVAGLLL FIGLGIFFCV 421 RCRHRRRQAE RMSQIKRLLS EKKTCQCPHR FQKTCSPI
By "CD28 cytoplasmic domain" is meant a polypeptide having the C- terminal region of CD28 that is located in the cytoplasm when expressed in a T- cell, e.g., a polypeptide having the amino acid sequence of amino acids 127-234 of SEQ ID NO:2. The term "CD28 cytoplasmic domain" is also meant to include any polypeptide fragment that maintains the ability to modulate activation of T-cells (e.g., as determined using the method titled "killing of HIV infected cells by modified T-cells" below) and is substantially identical to amino acids 127-234 of SEQ ID NO:2 over the length of the polypeptide fragment.
SEQ ID NO:2 Human CD28 Amino Acid sequence:
LOCUS NP_006130 220 aa linear PRI ll-APR-2010
DEFINITION T-cell-specific surface glycoprotein CD28 precursor [Homo sapiens ] .
ACCESSION NP_006130
VERSION NP_006130 . 1 GI:5453611
DBSOURCE REFSEQ: accession NM_006139.2
1 MLRLLLALNL FPS IQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD 61 SAVEVCWYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP
21 PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVWG GVLACYSLLV TVAFIIFWVR
181 SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS
By "CD3 zeta" is meant a polypeptide having the amino acid sequence of SEQ ID NO:3. The term "CD3 zeta" is also meant to include any
polypeptide fragment that maintains the ability to modulate activation of T-cells (e.g., as determined using the method titled "killing of HIV infected cells by modified T-cells" below) and is substantially identical to SEQ ID NO:3 over the length of the protein fragment.
SEQ ID NO:3 Human CD3 zeta Amino Acid sequence
LOCUS NP_000725 163 aa linear PRI ll-APR-2010
DEFINITION T-cell receptor zeta chain isoform 2 precursor [Homo sapiens ] .
ACCESSION NP_000725
VERSION NP_000725.1 GI:4557431
DBSOURCE REFSEQ: accession NM_000734.3
1 MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD 61 APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP RRKNPQEGLY NELQKDKMAE 121 AYSEIGMKGE RRRGKGHDGL YQGLSTATKD TYDALHMQAL PPR
By "small interfering RNA" or "siRNA" is meant an isolated RNA molecule, either single- stranded or double stranded that is at least 15
nucleotides, preferably, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length and even up to 50 or 100 nucleotides in length (inclusive of all integers in between). Preferably, the siRNA is capable of mediating RNAi. As used herein the phrase "mediates RNAi" refers to (indicates) the ability to distinguish which RNAs are to be degraded by the RNAi machinery or process. siRNAs are processed from long dsRNAs and are usually double- stranded (e.g., endogenous siRNAs). siRNAs can also include short hairpin RNAs in which both strands of an siRNA duplex are included within a single RNA molecule. These terms include double- stranded RNA, single- stranded RNA, isolated RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides.
By "substantially identical" is meant a nucleic acid or amino acid sequence that, when optimally aligned, for example using the methods described below, share at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a second nucleic acid or amino acid sequence. "Substantial identity" may be used to refer to various types and lengths of sequence, such as full-length sequence, epitopes or immunogenic peptides, functional domains, coding and/or regulatory
sequences, exons, introns, promoters, and genomic sequences. Percent identity between two polypeptides or nucleic acid sequences is determined in various ways that are within the skill in the art, for instance, using publicly available computer software such as Smith Waterman Alignment (Smith and Waterman (1981) J Mol Biol 147: 195-7); "BestFit" (Smith and Waterman, Advances in Applied Mathematics, (1981) 482-489) as incorporated into GeneMatcher
Plus , Schwarz and Dayhof, Atlas of Protein Sequence and Structure, Dayhof, M.O., Ed (1979) 353-358; BLAST program (Basic Local Alignment Search Tool; (Altschul et al. (1990) J Mol Biol 215: 403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software. In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In general, for proteins or nucleic acids, the length of comparison can be any length, up to and including full length (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%).
Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and
phenylalanine, tyrosine.
Other features and advantages of the invention will be apparent from the following Detailed Description, the drawings, and the claims.
Brief Description of the Drawings
Fig. 1 is a diagram showing the structure of the indicated chimeric immune T-cell receptors (CIRs).
Fig. 2 is a diagram showing the organization of an exemplary nucleic acid construct encoding a 2nd generation CIR and siRNA construct.
Fig. 3 is a series of graphs showing the cellular surface expression of the indicated CIR. PBMCs were transduced with retrovirus and stained with anti- CD4-FITC and anti-CD8-APC antibodies after four days and analyzed by flow cytometry. % CD8+ cells expressing Hege CIR is 52%, 1st generation CIR is 44%, and 2nd generation CIR is 39%. A representative experiment of four is shown.
Fig. 4 A is a pair of graphs showing survival of the indicated cell type when incubated with HIV-infected CEM-SS cells at the indicated ratio.
Transduced PBMCs were co-cultured with HIV-infected CEM-SS cells or uninfected CEM-SS cells at an Effector to Target (E:T) ratio of 1: 1 or 1: 10. Aliquots of cells were taken from the cultures at day three and stained with anti-CD4 and anti-CD8 antibodies as described above. Data shown is % modified cells (CD8+CIR+) at each time point. A representative of two experiments is shown.
Fig. 4B is a pair of graphs showing flow cytometry analysis from day three for Hege CIR cells. Hege CIR T-cells disappear (Hege+HIV, upper right quadrant) when cultured at an E:T ratio of 1 : 10. % Modifed cells =
(Q2/Q2+Q4) is shown in each plot. (Q2 is upper right quadrant and Q4 is lower right quadrant.)
Fig. 5 is a graph showing survival of Hege CIR cells when incubated with HIV-infected CEM-SS cells at the indicated ratio in the presence and absence of AZT. Transduced PBMCs were co-cultured with HIV-infected CEM-SS cells or uninfected CEM-SS cells at an Effector to Target (E:T) ratio of 1: 1 or 1: 10. Aliquots of cells were taken from the cultures at day three and stained with anti-CD4 and anti-CD8 antibodies as described for Fig. 3. Data shown is % modified cells (CD8+CIR+ cells) from one experiment.
Fig. 6 is a series of graphs showing expression of the indicated markers on the indicated cells when exposed to HIV. Non-transduced or 1st and 2nd generation CIR transduced T-cells were co-cultured with HIV infected CEM- SS cells for two days and stained with anti-CD8 and anti-p24gag antibodies. An aliquot of unstained cells were washed and continued to culture for another nine days and stained with anti-CD8 and anti-p24gag antibodies. A
representative of two experiments is shown. At day two, infected CD8+ cells show as a distinct population (circled) in the 1st and 2nd generation T-cells compared to non-transduced cells. There is no distinct population of cells seen in non-Td CD8+ cells (day two, upper right quadrant). % modified cells for this experiment is: 1st generation = 67% and 2nd generation = 68%.
Fig. 7 is a graph showing the percent of specific killing as a function of the ratio of Effector to Target cells. Hege CIR, 1st, and 2nd generation T-cells were cultured with 51Cr labeled uninfected or chronically infected with HIV-1 IIIB CEM-SS cells. Cytotoxicity is determined from 51Cr release to the culture media after 18 hrs of co-culture at the indicated ratios of Effector to Target, and % specific killing is calculated as follows: (experimental-control)/(maximal- control) x 100. % modified cells for Hege is 47%, for 1st generation is 25%, and for 2nd generation is 47%. Data shown is representative of two
experiments. This is calculated by taking mean value of CEM-SS control as spontaneous release = (Expt-control)/(Max-control).
Fig. 8 is a pair of graphs showing the amount of secretion of the indicated cytokine in the indicate cells types. 1st and 2nd generation T-cells were assayed for IL2 or interferon gamma (IFNy) secretion by culturing for 24 hrs on anti-CD4 (5 g/ml) coated plates. Data represented as fold change over 1st generation. IL2 data shown is average +SEM of three experiments. IFN-γ data shown is average +SEM of two experiments. % modified cells are similar for 1st and 2nd generation T-cells.
Detailed Description of the Invention
The invention features nucleic acid constructs encoding chimeric immune T-cell receptors (CIRs) that are useful for treating HIV in patients. In general, the CIRs contain an extracellular domain which targets HIV or HIV- infected cells (e.g., the extracellular domain of CD4), a transmembrane domain, and a cytoplasmic domain for mediating T-cell activation (e.g., CD3 zeta and/or the partial extracellular domain of CD28). The invention also features the use of host cells expressing CIRs in the treatment of HIV. When expressed in the host cells, the CIRs can be engineered to homodimerize, thereby increasing their potency. These host cells can also contain nucleic acid constructs encoding siRNA against HIV genes in order to, e.g., disrupt HIV infection of the host T-cells. The structure of a prior art CIR and the structures of CIRs
containing the transmembrane domain of CD3 zeta and partially extracellular domain of CD28 are depicted in Fig. 1.
Extracellular Domains
The CIRs of the invention feature an extracellular domain able to specifically bind HIV and cells infected with HIV. The HIV protein gpl20 binds human CD4. Therefore, the extracellular domain of the CIRs of the invention can include the extracellular domain of CD4 (e.g., human CD4 or fragments thereof). Alternatively, the extracellular domain can include any binding moiety specific for HIV and cells infected with HIV, including, HIV specific antibodies (e.g., single-chain Fv antibody fragments that are specific to gpl20 or gp41).
The extracellular domain can optionally include a further protein tag, e.g., a c-myc tag (EQKLISEEDL (SEQ ID NO:4) of human origin, at the N-terminus. The c-myc tag does not obstruct CD4 binding to gpl20. Inclusion of c-myc in the sFv based-CIR design does not appear to affect CIR function, but can facilitate future study of the construct.
Cytoplasmic Domains
The CIRs of the invention also feature a cytoplasmic domain for signaling modulating activation of the host T-cells when bound to HIV or HIV- infected cells. Cytoplasmic domains useful for use in the CIRs of the invention include CD3 zeta, or fragments thereof, and for the cytoplasmic domain of CD28, or fragments thereof. The invention also features the fusion of polypeptides derived from multiple extracellular domains for potentiating activation of T-cells when bound to HIV or HIV-infected cells (e.g., a cytoplasmic domain that includes both active fragments of CD3 zeta and CD28).
Transmembrane Domains
The CIRs of the invention feature transmembrane domains derived from CD4, CD28, CD3 zeta, or another protein. Furthermore, the transmembrane domain (or the partial extracellular domains, "pEC") can be engineered to facilitate homodimerization of the CIRs when expressed in host T-cells. This can be accomplished, e.g., with the addition or substitution of cysteine residues capable of forming disulfide bonds with a paired molecule.
The inclusion of the transmembrane region of the zeta chain or the transmembrane and partial extracellular domain of CD28 provides the capability of intermolecular disulfide bonds. CIRs containing these
transmembrane/partial extracellular domains are predicted to form disulfide- linked dimers through a cysteine residue located in the transmembrane of zeta or in the proximal cysteine residue located in the partial extracellular domain of CD28 (position 123 of CD28), mimicking the dimer configuration of native zeta and CD28. siRNA Constructs
The DNA constructs and host cells of the invention also optionally feature components to suppress HIV infection of host T-cells. Such
components include siRNA constructs for suppression of HIV replication. These siRNA constructs can be specific for various HIV targets (reviewed in Morris (2006) Gene Ther 13:553-558; Rossi (2006) Biotechniques Suppl:25- 29; Nekhai (2006) Curr Opin Mol Ther 8:52-61; and Cullen (2005) AIDS Rev 7:22-25). One example is an siRNA targeting a highly conserved sequence in an exon common to both tat and rev, has been shown to be effective to prevent virus expression and replication (See, e.g., SEQ ID No. 1). In order to prevent HIV infection of host T-cells, the invention also features components to decrease expression of T cell coreceptors (e.g., CCR5 and CCR4). Such suppression would be expected to hinder infection of host T-cells as people with CCR5A32 mutation are resistant to HIV infection. The invention also
features the inclusion of multiple siRNA constructs (e.g., constructs against HIV genes and T-cell receptors used for HIV infection). Here, one siRNA construct can block infection and while a second siRNA construct prevents progression of infection.
Methods of designing and expressing siRNA constructs are well known in the art. For example, the siRNA constructs of the invention can utilize long- hairpin RNA (IhRNA) to express both CCR5 and Tat/Rev siRNAs. Use of a IhRNA is a viable approach in controlling HIV-1 replication since a single long transcript can in theory be processed into multiple siRNAs. Multiple targeting can be achieved from a single long-hairpin precursor, suggesting that multiple siRNAs can be processed from the long hairpins in vivo. The siRNA constructs of the invention can also include a promoter directing expression in host T- cells. Examples of such promoters are U6 and tRNA promoters. Expressing shRNAs from tRNA promoters has several advantages, compared to the more commonly used U6 and HI promoters: tRNA promoters are smaller, provide a variety of options, and are typically expressed at lower levels. Smaller promoters may be desirable in the nucleic acid constructs of the invention to facilitate inclusion in a vector including a CIR expression construct. An example of a nucleic acid construct containing a CIR and siRNA is set forth in
Fig. 2.
shRNA sequences tat/rev shRNA - sense strand SEQ ID NO:5
5 ' -GCGGAGACAGCGACGAAGAGC- 3 '
Ref: Scherer, L. J., R. Frank, and J. J. Rossi. 2007. Nucleic Acids Res 35:2620-2628. ccr5 shRNA - sense strand SEQ ID NO:6
5' - GCCUGGGAGAGCUGGGGAA - 3'
Ref: Ehsani, Mol Ther Epub ahead of print.
shRNA within CIR (SEQ ID NO:7)
-Mvc-CD4-CD28-zeta-tcaqqtqqtqqcqqttcaqqcqqaqqtqqctctqqcqqtqqcqqatcq
Generic linker (G4S)3
GCCCGGATAGCTCAGTcGGTAGAGCACAGACTTTAATCTGAGGGTCCAGGGTCAAGTCCCTGTTCGGGC GCCA
tRNA promoter
GCCTGGGAGAGCTGGGGAATTTGTACGTAGTTCCCCAGCTCTCCCAGGC
ccr5 shRNA sense shRNA loop ccr5 shRNA antisense
ggtggcagtggctccggaggttcaggaagcggcggtagtgggagc
generic linker (GGSGS)3
GCGGAGACAGCGACGAAGAGCCTTCCTGTCAGAGCGGAGACAGCGACGAAGAGCTTTTTGAA
tat /rev shRNA sense shRNA loop tat/rev shRNA antisense terminator sequence Nucleic Acid Constructs
The nucleic acid constructs of the invention are useful for expressing CIRs and siRNA constructs in host T-cells. CIRs and siRNA constructs can be included in a single nucleic acid construct or multiple nucleic acid constructs. In order to facilitate transfection of host cells, the nucleic acid construct can be included in a viral vector (e.g., a retroviral vector or adenoviral vector) or be designed to be transfected into a host cell via electroporation or chemical means (e.g., using a lipid transfection reagent).
Examples of nucleic acid constructs
Myc-CD4-zeta ( 1 st generation (dimer)) SEQ ID NO: 8
ATGAACCGGGGAGTCCCTTTTAGGCACTTGCTTCTGGTGCTGCAACTGGC
GCTCCTCCCAGCAGCCACTCAGGGAGAGCAGAAGCTGATCTCCGAGGAGG myc (underline) ACCTGAAGAAAGTGGTGCTGGGCAAAAAAGGGGATACAGTGGAACTGACC CD4
TGTACAGCTTCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAA CCAGATAAAGATTCTGGGAAATCAGGGCTCCTTCTTAACTAAAGGTCCAT CCAAGCTGAATGATCGCGCTGACTCAAGAAGAAGCCTTTGGGACCAAGGA AACTTTCCCCTGATCATCAAGAATCTTAAGATAGAAGACTCAGATACTTA CATCTGTGAAGTGGAGGACCAGAAGGAGGAGGTGCAATTGCTAGTGTTCG GATTGACTGCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACC CTGACCTTGGAGAGCCCCCCTGGTAGTAGCCCCTCAGTGCAATGTAGGAG TCCAAGGGGTAAAAACATACAGGGGGGGAAGACCCTCTCCGTGTCTCAGC TGGAGCTCCAGGATAGTGGCACCTGGACATGCACTGTCTTGCAGAACCAG AAGAAGGTGGAGTTCAAAATAGACATCGTGGTGCTAGCTTTCCAGAAGGC CTCCAGCATAGTCTATAAGAAAGAGGGGGAACAGGTGGAGTTCTCCTTCC CACTCGCCTTTACAGTTGAAAAGCTGACGGGCAGTGGCGAGCTGTGGTGG CAGGCGGAGAGGGCTTCCTCCTCCAAGTCTTGGATCACCTTTGACCTGAA GAACAAGGAAGTGTCTGTAAAACGGGTTACCCAGGACCCTAAGCTCCAGA TGGGCAAGAAGCTCCCGCTCCACCTCACCCTGCCCCAGGCCTTGCCTCAG
TATGCTGGCTCTGGAAACCTCACCCTGGCCCTTGAAGCGAAAACAGGAAA
GTTGCATCAGGAAGTGAACCTGGTGGTGATGAGAGCCACTCAGCTCCAGA AAAATTTGACCTGTGAGGTGTGGGGACCCACCTCCCCTAAGCTGATGCTG AGCTTGAAACTGGAGAACAAGGAGGCAAAGGTCTCGAAGCGGGAGAAGGC GGTGTGGGTGCTGAACCCTGAGGCGGGGATGTGGCAGTGTCTGCTGAGTG ACTCGGGACAGGTCCTGCTGGAATCCAACATCAAGGTTCTGCCCACATGG TCCACCCCGGTGCCTAGGCTGGATCCCAAACTCTGCTACCTGCTGGATGG zeta (Underline) AATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGA AGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAG CTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGA CAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGA ACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAG GCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCA CGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACG CC
Myc-CD4-CD28-zeta (2na generation) SEQ ID NO:9
ATGAACCGGGGAGTCCCTTTTAGGCACTTGCTTCTGGTGCTGCAACTGGC
GCTCCTCCCAGCAGCCACTCAGGGAGAGCAGAAGCTGATCTCCGAGGAGG myc (underline)
ACCTGAAGAAAGTGGTGCTGGGCAAAAAAGGGGATACAGTGGAACTGACC CD4
TGTACAGCTTCCCAGAAGAAGAGCATACAATTCCACTGGAAAAACTCCAA
CCAGATAAAGATTCTGGGAAATCAGGGCTCCTTCTTAACTAAAGGTCCAT
CCAAGCTGAATGATCGCGCTGACTCAAGAAGAAGCCTTTGGGACCAAGGA
AACTTTCCCCTGATCATCAAGAATCTTAAGATAGAAGACTCAGATACTTA
CATCTGTGAAGTGGAGGACCAGAAGGAGGAGGTGCAATTGCTAGTGTTCG
GATTGACTGCCAACTCTGACACCCACCTGCTTCAGGGGCAGAGCCTGACC
CTGACCTTGGAGAGCCCCCCTGGTAGTAGCCCCTCAGTGCAATGTAGGAG
TCCAAGGGGTAAAAACATACAGGGGGGGAAGACCCTCTCCGTGTCTCAGC
TGGAGCTCCAGGATAGTGGCACCTGGACATGCACTGTCTTGCAGAACCAG
AAGAAGGTGGAGTTCAAAATAGACATCGTGGTGCTAGCTTTCCAGAAGGC
CTCCAGCATAGTCTATAAGAAAGAGGGGGAACAGGTGGAGTTCTCCTTCC
CACTCGCCTTTACAGTTGAAAAGCTGACGGGCAGTGGCGAGCTGTGGTGG
CAGGCGGAGAGGGCTTCCTCCTCCAAGTCTTGGATCACCTTTGACCTGAA
GAACAAGGAAGTGTCTGTAAAACGGGTTACCCAGGACCCTAAGCTCCAGA
TGGGCAAGAAGCTCCCGCTCCACCTCACCCTGCCCCAGGCCTTGCCTCAG
TATGCTGGCTCTGGAAACCTCACCCTGGCCCTTGAAGCGAAAACAGGAAA
GTTGCATCAGGAAGTGAACCTGGTGGTGATGAGAGCCACTCAGCTCCAG
AAAAATTTGACCTGTGAGGTGTGGGGACCCACCTCCCCTAAGCTGATGC
TGAGCTTGAAACTGGAGAACAAGGAGGCAAAGGTCTCGAAGCGGGAGAAG
GCGGTGTGGGTGCTGAACCCTGAGGCGGGGATGTGGCAGTGTCTGCTGAG
TGACTCGGGACAGGTCCTGCTGGAATCCAACATCAAGGTTCTGCCCACAT
GGTCCACCCCGGTGCCTAGGAAAATTGAAGTTATGTATCCTCCTCCTTAC CD28 (underline)
CTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACA
CCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGC
TGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTG
GCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAG
TGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATT
ACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTG zeta
AAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCA
GCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGG
ACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAG
AACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGA
GGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC
ACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGAC
GCC
The amino acid sequence for the CD28 and CD3 zeta portions of the 2 nd generation construct is
KI EV Met YPPPYLDN EKSNGTI I HVKG KH LCPSPLFPG PSKP FWVLVVVGGVLACYSLLVTVAFI I FWVRSKRSRLLHSDY Met
N Met TPRRPG PTRKHYQPYAPPRDFAAYRSRVKFSRSADA PAYQQGQNQLYN ELN LG RREEYDVLD KRRG RDPE Met G G K PRRKN PQEG LYN ELQKDK Met A E A Y S E I G Met KG ERRRG KG H DG LYQG LSTATKDTYDA
(SEQ ID NO: 10) Host T-cells
The host T-cells of the invention can be isolated from, e.g., a patient infected with HIV. The host T-cells are transfected or infected with nucleic acid constructs of the invention (e.g., nucleic acid constructs encoding a CIR and, optionally, one or more siRNA constructs). Prior to administration to a patient the T-cells can be expanded in cell culture. In one embodiment, the modified T-cells are administered to the patient from whom they were originally isolated.
In one embodiment, PBMCs are isolated by standard techniques and transduced with a CIR. Cells are administered to the patient in a dose of between 109 and 1010 cells (e.g., 109, 5 x 109, or 1010 cells). Cells can be isolated once and expanded for multiple administrations or a separate isolation and transduction can be performed with each round of treatment.
Treatment can be, e.g., a single treatment, monthly treatment, semiannual treatment, or annual treatment.
Additional Agents
Additional antiviral can be, for example, a protease inhibitor, a reverse transcriptase inhibitor, an integrase inhibitor, a CCR5 antagonist, a fusion inhibitor, or a second maturation inhibitor. The additional antiviral agent can be, without limitation, azidovudine (AZT), didanosine (dideoxyinosine, ddl), d4T, zalcitabine (dideoxycytosine, ddC), nevirapine, lamivudine (epivir, 3TC), saquinavir (Invirase), ritonavir (Norvir), indinavir (Crixivan), and delavirdine (Rescriptor).
Additional Therapies
The methods of the invention can be combined with, e.g.,
lymphodepletion prior to administration of host T-cells. Furthermore, treatment can also include the administration of one or more cytokines, e.g., IL-2, IL-7, and IL-15.
Experimental Results
Construction of Retroviral Vectors
The chimeric immune T-cell receptor (CIR) of the prior anti-HIV designer T cell trials had the structure of extracellular domain of CD4 (a polypeptide corresponding to amino acids 1-372 of SEQ ID NO: l),
transmembrane domain of CD4 (a polypeptide corresponding to amino acids 373-395 of SEQ ID NO: l) and cytoplasmic domain of zeta (a polypeptide corresponding to amino acids 31-142 of SEQ ID NO:3) (Deeks et al. (2002) Mol Ther 5:788-797, Mitsuyasu et al. (2000) Blood 96:785-793) (herein "Hege CIR", Fig. 1). We also designed a signal one-only CIR that is similar to the Hege CIR except that the transmembrane domain of zeta (a polypeptide corresponding to amino acids residues 7-30 of SEQ ID NO:3) was substituted (1st generation CIR, Fig. 1). Lastly, we created a construct that integrates CD28 as well as zeta signaling in a two signal format (2nd generation CIR, Fig. 1). This employs the same extracellular domain of CD4 (a polypeptide
corresponding to amino acids 1-372 of SEQ ID NO: l) with a partial
extracellular doman/transmembrane domain/cytoplasmic domain of CD28 (a polypeptide corresponding to amino acids 127-234 of SEQ ID NO:2) and is expressed as dimer.
In addition, a c-myc tag (EQKLISEEDL) of human origin is also included in our constructs at the N-terminus of CD4.
1st and 2nd generation CIRs were constructed in the MFG retrovirus vector. Retrovirus was created by ping pong between the E+86 ecotropic and PG13 amphotropic cell lines. PG13 is a helper cell line derived from murine
fibroblasts that is used to create vector producer cells (VPC) for retroviral production. VPCs were sorted for the highest transgene expression and viral supernatants were harvested as described Beaudoin et al. ((2008) J Virol Methods 148:253-259).
Expression of CIRs
Viral supernatants from PG13 VPCs were used to transduce human PBMCs. PBMCs from normal healthy individuals were purified and activated with anti-CD3 antibody (OKT3) and 100 U/ml IL2 for two days and transduced with retrovirus by spinoculation on a retronectin coated plate. We determined the surface expression of CIRs on CD8+ T-cells by double staining for CD8 and CD4 and determined the transduction rate (as % modified cells, Fig. 3). Transduction of activated human T-cells routinely yield 40 to 70% transduction rates with these anti-HIV CIRs.
We co-cultured transduced or non-transduced T-cells with CEM-SS
HIV+ (chronically infected with HIV-1 IIIB) or HIV- cells (at an E:T ratio of 1: 1 or 1: 10). We determined the presence of transduced cells in the culture by staining with anti-CD4 and anti-CD8 antibodies. Co-culture of Hege CIR T- cells with CEM-SS HIV+ cells at an E: T ratio of 1: 10 induced cell death and all the Hege CIR cells disappeared from the culture by day 3 (Fig 4). At an E:T ratio of 1: 1, Hege CIR T cells were still present in the culture at day 13, with killing of all target cells in the culture (observed by flow cytometry analysis). Cell death observed in the Hege CIR designer T-cells could be either due to heightened sensitivity to Activation Induced Cell Death (AICD) or to HIV infection. To test this, Hege CIR cells were treated with anti-retroviral drug AZT and co-cultured with HIV infected CEM-SS cells. AZT treated Hege CIR cells did not die when co-cultured with higher target ratio (1: 10, Fig. 5). These data suggest that Hege CIR cells become infected with HIV and die by either HIV induced apoptosis or killed by other CIR containing T-cells (fratricide). These data suggest and we hypothesize that one of the reasons for the failure of
Hege CIR in the clinical trials could be due to highest susceptibility to HIV infection and elimination from the patients.
Susceptibility of Designer T-cells to HIV Infection
HIV infects CD4+ T-cells by binding to CD4 receptor and a co-receptor
(CXCR4 or CCR5). Since CIR has an extracellular CD4 domain, we postulated that this could be used by HIV to infect all CIR+ T-cells, including CD8+ T- cells. HIV infection of CIR+CD8+ cells was determined by co-culturing CIR containing T-cells with HIV+ CEM-SS cells and staining for p24-gag antigen, an indicator of productive infection. After two days of culture, cells were stained with anti-CD8 and anti-p24 gag antibodies. In contrast to non- transduced cultures, CD8+ cells are infected with HIV in transduced cultures (1st and 2nd generation) (Fig. 6). At day 11 we did not detect any HIV infected CD8+ cells in either 1st or 2nd generation CIR containing T-cells (Fig. 6).
Killing of HIV-infected Cells by Modified T-cells.
In order to compare the potencies of Hege CIR, 1st, and 2nd generation anti-HIV CIR in killing of target cells, activated T-cells were transduced as described above. Target cells (HIV-infected or uninfected CEM-SS cells) were labeled with 51Cr for 5 hrs (50 μθ for 1 x 106 cells) and co-cultured with transduced T-cells at indicated E:T ratios for 18 hrs. Hege CIR T-cells and our 1st and 2nd generation designer T-cells were all equally potent in killing HIV+ target cells (Fig. 7).
Cytokine Secretion by Modified T-cells.
Human T-cells transduced with 1st and 2nd generation CIR containing T- cells were tested for their ability to secrete cytokines upon stimulation through the CIR. Transduced or non-transduced T-cells were cultured on anti-CD4 antibody coated plates for 24 hrs. IL2 secretion was measured with an ELISA kit. 2nd generation T-cells produced more IL2 than 1st generation T-cells when stimulated with anti-CD4 antibody (Fig. 8). In contrast, IFNy secretion is
similar with anti-CD4 stimulation of and 2n generation designer T-cells, as is typical for T cell signaling.
Conferring Resistance of Designer T-cells to HIV Infection
HIV infects CD4+ T-cells by binding to CD4 receptor and a co-receptor
(CXCR4 or CCR5). As shown above, CD8+CIR+ cells (1st and 2nd generation) are susceptible to HIV infection (day two, Fig. 6). At day 11 we did not detect any HIV infected CD8+ cells in either 1st or 2nd generation T-cells (Fig. 6). These data suggest that modified T-cells could kill other HIV infected modified T-cells. Nevertheless, this is a potential source of loss of effector cells to combat HIV and provides a new reservoir to increase patient HIV load. It is therefore becomes important to eliminate or reduce the potential for HIV to infect modified T-cells.
Other Embodiments
Various modifications and variations of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific desired embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the fields of medicine, immunology, pharmacology, endocrinology, or related fields are intended to be within the scope of the invention.
All publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication was specifically and individually incorporated by reference.
What is claimed is:
Claims
1. A nucleic acid construct encoding a chimeric protein comprising
(i) an extracellular domain of CD4, or a fragment thereof,
(ii) a transmembrane domain, and
(iii) a cytoplasmic domain comprising
a) the cytoplasmic domain of the CD3 zeta chain, or a fragment thereof and b) the cytoplasmic domain of CD28, or a fragment thereof.
2. The nucleic acid construct of claim 1, wherein said chimeric protein is capable of forming a homodimer when expressed in a T cell.
3. The nucleic acid construct of claim 2, wherein the dimerized chimeric proteins are capable of forming at least one disulfide bond.
4. The nucleic acid construct of claim 1, wherein said transmembrane domain comprises a polypeptide selected from the group consisting of the transmembrane domain of the CD3 zeta chain and the transmembrane domain of CD28.
5. The nucleic acid construct of claim 4, wherein said transmembrane domain comprises amino acids 7-30 of SEQ ID NO:3.
6. The nucleic acid construct of claim 1, wherein said chimeric protein further comprises a c-myc tag.
7. The nucleic acid construct of claim 1, wherein said extracellular domain of CD4 comprises amino acids 1-372 of SEQ ID NO: l.
8. The nucleic acid construct of claim 1, wherein said cytoplasmic domain of the CD3 zeta chain comprises amino acids 31-142 of SEQ ID NO:3.
9. The nucleic acid construct of claim 1, wherein said cytoplasmic domain of CD28 comprises amino acids 127-234 of SEQ ID NO:2.
10. The nucleic acid construct of claim 1, wherein said cytoplasmic domain comprises the amino acid sequence of SEQ ID NO: 10.
11. A nucleic acid construct encoding a chimeric protein comprising
(i) an extracellular domain of CD4, or a fragment thereof,
(ii) a transmembrane domain, and
(iii) a cytoplasmic domain of the CD3 zeta chain, or a fragment thereof, wherein said chimeric protein is capable of forming a homodimer when expressed in a T cell.
12. The nucleic acid construct of claim 11, wherein the chimeric protein, when in a homodimer, is capable of forming at least one disulfide bond with the chimeric protein with which it is dimerized.
13. The nucleic acid construct of claim 11, wherein said transmembrane domain comprises the transmembrane domain of the CD3 zeta chain or the transmembrane domain of CD28.
14. The nucleic acid construct of claim 13, wherein said transmembrane domain comprises amino acids 7-30 of SEQ ID NO:3.
15. The nucleic acid construct of claim 11, wherein said chimeric protein further comprises a c-myc tag.
16. The nucleic acid construct of claim 11, wherein said extracellular domain of CD4 comprises amino acids 1-372 of SEQ ID NO: l.
17. The nucleic acid construct of claim 11, wherein said cytoplasmic domain of the CD3 zeta chain comprises amino acids 31-142 of SEQ ID NO:3.
18. A vector comprising the nucleic acid construct of any of claims 1-
17.
19. The vector of claim 18 further comprising a nucleic acid construct encoding an siRNA.
20. The vector of claim 19 wherein said siRNA is against CCR5.
21. The vector of claim 19 wherein said siRNA is against Tat/Rev.
22. A host cell comprising the nucleic acid construct of any of claims
18-21.
23. The host cell of claim 22, wherein said host cell is a T cell.
24. The host cell of claim 23, wherein said T cell is isolated from a patient infected with HIV.
25. A host cell comprising the nucleic acid construct any of claims 1-17 and a nucleic acid construct encoding an siRNA.
26. The host cell of claim 25, wherein said siRNA is against CCR5.
27. The host cell of claim 25, wherein said siRNA is against Tat/Rev.
28. A method of treating a patient infected with HIV by administering a composition comprising host cell of any of claims 22-26.
29. The method of claim 28, wherein said host cell is a T cell isolated from said patient.
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP16205421.7A EP3202410B1 (en) | 2010-04-14 | 2011-04-14 | Methods and compositions for treating hiv |
US13/641,245 US9833480B2 (en) | 2010-04-14 | 2011-04-14 | Methods and compositions for treating HIV |
EP20164927.4A EP3725319A1 (en) | 2010-04-14 | 2011-04-14 | Methods and compositions for treating hiv |
ES11769573.4T ES2620259T3 (en) | 2010-04-14 | 2011-04-14 | Methods and compositions for treating HIV |
EP11769573.4A EP2558128B1 (en) | 2010-04-14 | 2011-04-14 | Methods and compositions for treating hiv |
US15/816,454 US20180185416A1 (en) | 2010-04-14 | 2017-11-17 | Methods and compositions for treating hiv |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US32405010P | 2010-04-14 | 2010-04-14 | |
US61/324,050 | 2010-04-14 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/641,245 A-371-Of-International US9833480B2 (en) | 2010-04-14 | 2011-04-14 | Methods and compositions for treating HIV |
US15/816,454 Continuation US20180185416A1 (en) | 2010-04-14 | 2017-11-17 | Methods and compositions for treating hiv |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2011130491A2 true WO2011130491A2 (en) | 2011-10-20 |
WO2011130491A3 WO2011130491A3 (en) | 2012-01-19 |
Family
ID=44799314
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2011/032455 WO2011130491A2 (en) | 2010-04-14 | 2011-04-14 | Methods and compositions for treating hiv |
Country Status (4)
Country | Link |
---|---|
US (2) | US9833480B2 (en) |
EP (3) | EP3725319A1 (en) |
ES (1) | ES2620259T3 (en) |
WO (1) | WO2011130491A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9833480B2 (en) | 2010-04-14 | 2017-12-05 | Prospect Chartercare, Llc | Methods and compositions for treating HIV |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2018219226A1 (en) | 2017-02-07 | 2019-08-15 | Seattle Children's Hospital (dba Seattle Children's Research Institute) | Phospholipid ether (PLE) CAR T cell tumor targeting (CTCT) agents |
WO2018160622A1 (en) | 2017-02-28 | 2018-09-07 | Endocyte, Inc. | Compositions and methods for car t cell therapy |
JP2020517259A (en) | 2017-04-19 | 2020-06-18 | ボード・オブ・リージエンツ,ザ・ユニバーシテイ・オブ・テキサス・システム | Immune cells expressing engineered antigen receptors |
CN112055595A (en) | 2018-01-22 | 2020-12-08 | 恩多塞特公司 | Methods of use of CAR T cells |
KR20240128090A (en) | 2021-12-31 | 2024-08-23 | 베이징 솔로바이오 진테크놀로지 컴퍼니 리미티드 | Chimeric antigen receptor T cells targeting HIV-infected cells |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5686281A (en) | 1995-02-03 | 1997-11-11 | Cell Genesys, Inc. | Chimeric receptor molecules for delivery of co-stimulatory signals |
US20030138410A1 (en) | 1991-03-07 | 2003-07-24 | Brian Seed | Targeted cytolysis of HIV-infected cells by chimeric CD4 receptor-bearing cells |
Family Cites Families (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6506604B2 (en) * | 1993-06-11 | 2003-01-14 | Cell Genesys, Inc. | Method for production of high titer virus and high efficiency retroviral mediated transduction of mammalian cells |
US20020165360A1 (en) | 2000-11-30 | 2002-11-07 | Junghans Richard P. | Chimeric effector cell receptors against carcinoembryonic antigen |
US7919309B2 (en) * | 2001-09-13 | 2011-04-05 | California Institute Of Technology | Method for expression of small antiviral RNA molecules within a cell |
WO2003050262A2 (en) | 2001-12-10 | 2003-06-19 | California Institute Of Technology | Method for the generation of antigen-specific lymphocytes |
EP2000160A3 (en) * | 2002-10-30 | 2009-03-11 | Gambro Lundia AB | Method and apparatuses for determining the efficiency of dialysis |
US8637234B2 (en) * | 2004-04-16 | 2014-01-28 | Uab Research Foundation | Molecular scaffolds for HIV-1 epitopes |
WO2008045437A2 (en) * | 2006-10-09 | 2008-04-17 | The General Hospital Corporation | Chimeric t-cell receptors and t-cells targeting egfrviii on tumors |
WO2010025177A1 (en) | 2008-08-26 | 2010-03-04 | City Of Hope | Method and compositions for enhanced anti-tumor effector functioning of t cells |
EP2389443B1 (en) * | 2009-01-23 | 2018-11-14 | Roger Williams Hospital | Retroviral vectors encoding multiple highly homologous non-viral polypeptides and the use of same |
EP3725319A1 (en) | 2010-04-14 | 2020-10-21 | Roger Williams Medical Center | Methods and compositions for treating hiv |
US9402865B2 (en) | 2011-01-18 | 2016-08-02 | The Trustees Of The University Of Pennsylvania | Compositions and methods for treating cancer |
-
2011
- 2011-04-14 EP EP20164927.4A patent/EP3725319A1/en not_active Withdrawn
- 2011-04-14 EP EP16205421.7A patent/EP3202410B1/en active Active
- 2011-04-14 WO PCT/US2011/032455 patent/WO2011130491A2/en active Application Filing
- 2011-04-14 US US13/641,245 patent/US9833480B2/en not_active Expired - Fee Related
- 2011-04-14 ES ES11769573.4T patent/ES2620259T3/en active Active
- 2011-04-14 EP EP11769573.4A patent/EP2558128B1/en not_active Not-in-force
-
2017
- 2017-11-17 US US15/816,454 patent/US20180185416A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030138410A1 (en) | 1991-03-07 | 2003-07-24 | Brian Seed | Targeted cytolysis of HIV-infected cells by chimeric CD4 receptor-bearing cells |
US5686281A (en) | 1995-02-03 | 1997-11-11 | Cell Genesys, Inc. | Chimeric receptor molecules for delivery of co-stimulatory signals |
Non-Patent Citations (6)
Title |
---|
CULLEN, AIDS REV, vol. 7, 2005, pages 22 - 25 |
MORRIS, GENE THER, vol. 13, 2006, pages 553 - 558 |
NEKHAI, CURR OPIN MOL THER, vol. 8, 2006, pages 52 - 61 |
ROBERTS M.R. ET AL., BLOOD, vol. 84, 1994, pages 2878 - 2889 |
ROSSI, BIOTECHNIQUES, 2006, pages 25 - 29 |
See also references of EP2558128A4 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9833480B2 (en) | 2010-04-14 | 2017-12-05 | Prospect Chartercare, Llc | Methods and compositions for treating HIV |
Also Published As
Publication number | Publication date |
---|---|
EP3202410B1 (en) | 2020-03-25 |
US9833480B2 (en) | 2017-12-05 |
EP2558128A2 (en) | 2013-02-20 |
WO2011130491A3 (en) | 2012-01-19 |
EP3725319A1 (en) | 2020-10-21 |
US20180185416A1 (en) | 2018-07-05 |
EP3202410A1 (en) | 2017-08-09 |
ES2620259T3 (en) | 2017-06-28 |
US20130183276A1 (en) | 2013-07-18 |
EP2558128A4 (en) | 2013-11-06 |
EP2558128B1 (en) | 2016-12-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20180185416A1 (en) | Methods and compositions for treating hiv | |
Ghanem et al. | Bispecific chimeric antigen receptors targeting the CD4 binding site and high-mannose Glycans of gp120 optimized for anti–human immunodeficiency virus potency and breadth with minimal immunogenicity | |
Egelhofer et al. | Inhibition of human immunodeficiency virus type 1 entry in cells expressing gp41-derived peptides | |
US11591613B2 (en) | Retroviral and lentiviral vectors | |
US9193790B2 (en) | Use of antagonists of the interaction between HIV GP120 and A4B7 integrin | |
WO2019055946A1 (en) | Methods and compositions for genetically modifying and expanding lymphocytes and regulating the activity thereof | |
Bego et al. | Vpu exploits the cross-talk between BST2 and the ILT7 receptor to suppress anti-HIV-1 responses by plasmacytoid dendritic cells | |
Masiero et al. | T-cell engineering by a chimeric T-cell receptor with antibody-type specificity for the HIV-1 gp120 | |
Mwimanzi et al. | Effects of naturally-arising HIV Nef mutations on cytotoxic T lymphocyte recognition and Nef's functionality in primary macrophages | |
Schiavoni et al. | HIV-1 Nef enhances both membrane expression and virion incorporation of Env products: a model for the Nef-dependent increase of HIV-1 infectivity | |
Bouchet et al. | Single-domain antibody-SH3 fusions for efficient neutralization of HIV-1 Nef functions | |
Foster et al. | Genetic and functional diversity of human immunodeficiency virus type 1 subtype B Nef primary isolates | |
JP2020519309A (en) | Fc fusion protein derivatives with high dual HIV antiviral and immunomodulatory activity | |
Jones et al. | Induction of human T cell leukemia virus type I receptors on quiescent naive T lymphocytes by TGF-β | |
Schaefer et al. | The T-cell receptor ζ chain contains two homologous domains with which simian immunodeficiency virus Nef interacts and mediates down-modulation | |
Zhang et al. | Inhibitory effect of human TRIM5α on HIV-1 production | |
CA2484166C (en) | Improved chimeric glycoproteins and pseudotyped lentiviral vectors | |
WO2023077943A1 (en) | Bispecific chimeric antigen receptor targeting hiv-1 envelope protein, preparation method therefor and application thereof | |
Santos da Silva et al. | The envelope cytoplasmic tail of HIV-1 subtype C contributes to poor replication capacity through low viral infectivity and cell-to-cell transmission | |
Hübner et al. | Inhibition of viral assembly in murine cells by HIV-1 matrix | |
Luttge et al. | Mutations in the feline immunodeficiency virus envelope glycoprotein confer resistance to a dominant–negative fragment of Tsg101 by enhancing infectivity and cell-to-cell virus transmission | |
US20200316190A1 (en) | Psgl-1 (p-selectin glycoprotein ligand-1) to inactivate all enveloped viruses for producing live-attenuated vaccines | |
Olety et al. | AP-2 Adaptor Complex-Dependent Enhancement of HIV-1 Replication by Nef in the Absence of the Nef/AP-2 Targets SERINC5 and CD4 | |
Alsahafi | Protecting HIV-1-infected cells from ADCC: Role of Nef | |
Lodermeyer | Characterization of 90K/LGALS3BP as antiviral factor |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 11769573 Country of ref document: EP Kind code of ref document: A2 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REEP | Request for entry into the european phase |
Ref document number: 2011769573 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2011769573 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 13641245 Country of ref document: US |