WO2022093683A1 - Orthogonal il-21 receptor/cytokine systems - Google Patents
Orthogonal il-21 receptor/cytokine systems Download PDFInfo
- Publication number
- WO2022093683A1 WO2022093683A1 PCT/US2021/056439 US2021056439W WO2022093683A1 WO 2022093683 A1 WO2022093683 A1 WO 2022093683A1 US 2021056439 W US2021056439 W US 2021056439W WO 2022093683 A1 WO2022093683 A1 WO 2022093683A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- ortho
- ira
- cells
- amino acid
- candidate
- Prior art date
Links
- 102000004527 Interleukin-21 Receptors Human genes 0.000 title abstract description 18
- 108010017411 Interleukin-21 Receptors Proteins 0.000 title abstract description 18
- 102000004127 Cytokines Human genes 0.000 title description 66
- 108090000695 Cytokines Proteins 0.000 title description 66
- 210000004027 cell Anatomy 0.000 claims description 166
- 238000006467 substitution reaction Methods 0.000 claims description 87
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 65
- 102000005962 receptors Human genes 0.000 claims description 51
- 108020003175 receptors Proteins 0.000 claims description 51
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 36
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 29
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 28
- 101001010621 Homo sapiens Interleukin-21 Proteins 0.000 claims description 27
- 229920001184 polypeptide Polymers 0.000 claims description 24
- 102220282711 rs1452865935 Human genes 0.000 claims description 20
- 102220192504 rs150313549 Human genes 0.000 claims description 20
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 18
- 102220579177 SUN domain-containing protein 5_K73I_mutation Human genes 0.000 claims description 12
- 102200145956 rs138213197 Human genes 0.000 claims description 11
- 102220176693 rs886054444 Human genes 0.000 claims description 11
- 210000002865 immune cell Anatomy 0.000 claims description 8
- 210000004962 mammalian cell Anatomy 0.000 claims description 8
- 210000000130 stem cell Anatomy 0.000 claims description 8
- 230000004913 activation Effects 0.000 claims description 3
- 108010088751 Albumins Proteins 0.000 claims description 2
- 102000009027 Albumins Human genes 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 102220531737 Piwi-like protein 1_R9K_mutation Human genes 0.000 claims 1
- 108010074108 interleukin-21 Proteins 0.000 abstract description 171
- 102100030704 Interleukin-21 Human genes 0.000 abstract description 165
- 230000027455 binding Effects 0.000 abstract description 80
- 230000011664 signaling Effects 0.000 abstract description 34
- 230000001771 impaired effect Effects 0.000 abstract description 30
- 238000000034 method Methods 0.000 abstract description 19
- 208000037765 diseases and disorders Diseases 0.000 abstract description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 69
- 102000000588 Interleukin-2 Human genes 0.000 description 69
- 108090000623 proteins and genes Proteins 0.000 description 57
- 235000001014 amino acid Nutrition 0.000 description 50
- 230000004044 response Effects 0.000 description 40
- 102000004169 proteins and genes Human genes 0.000 description 39
- 235000018102 proteins Nutrition 0.000 description 38
- 239000005089 Luciferase Substances 0.000 description 34
- 108060001084 Luciferase Proteins 0.000 description 30
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 22
- 102100024040 Signal transducer and activator of transcription 3 Human genes 0.000 description 22
- 230000000694 effects Effects 0.000 description 22
- 150000001413 amino acids Chemical class 0.000 description 20
- 229940024606 amino acid Drugs 0.000 description 19
- 102220265701 rs755705607 Human genes 0.000 description 18
- 230000014509 gene expression Effects 0.000 description 17
- 206010028980 Neoplasm Diseases 0.000 description 16
- 238000003556 assay Methods 0.000 description 16
- 239000013598 vector Substances 0.000 description 16
- 102220049160 rs34395671 Human genes 0.000 description 15
- 238000012216 screening Methods 0.000 description 15
- 108010076504 Protein Sorting Signals Proteins 0.000 description 14
- 125000000539 amino acid group Chemical group 0.000 description 14
- 239000013604 expression vector Substances 0.000 description 14
- 239000006228 supernatant Substances 0.000 description 14
- 238000013459 approach Methods 0.000 description 13
- 102220367741 c.210G>A Human genes 0.000 description 13
- 201000011510 cancer Diseases 0.000 description 13
- 230000001419 dependent effect Effects 0.000 description 13
- 238000002474 experimental method Methods 0.000 description 12
- 239000003623 enhancer Substances 0.000 description 11
- 102220336001 rs1196469539 Human genes 0.000 description 11
- 230000001225 therapeutic effect Effects 0.000 description 11
- 108091026890 Coding region Proteins 0.000 description 10
- 108700019146 Transgenes Proteins 0.000 description 10
- 230000003993 interaction Effects 0.000 description 10
- 230000004048 modification Effects 0.000 description 10
- 238000012986 modification Methods 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 102000003675 cytokine receptors Human genes 0.000 description 9
- 108010057085 cytokine receptors Proteins 0.000 description 9
- 238000012217 deletion Methods 0.000 description 9
- 230000037430 deletion Effects 0.000 description 9
- 102220145630 rs886058977 Human genes 0.000 description 9
- 102220485857 Putative uncharacterized protein DHRS4-AS1_R76A_mutation Human genes 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 239000012530 fluid Substances 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 8
- 238000001727 in vivo Methods 0.000 description 8
- 238000003780 insertion Methods 0.000 description 8
- 230000037431 insertion Effects 0.000 description 8
- 150000007523 nucleic acids Chemical class 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 238000000338 in vitro Methods 0.000 description 7
- 102220287368 rs1190318676 Human genes 0.000 description 7
- 102220051014 rs141837529 Human genes 0.000 description 7
- 102220280407 rs1555280344 Human genes 0.000 description 7
- 102220130359 rs56302117 Human genes 0.000 description 7
- 238000013518 transcription Methods 0.000 description 7
- 230000035897 transcription Effects 0.000 description 7
- 102220614808 GTP-binding nuclear protein Ran_R76E_mutation Human genes 0.000 description 6
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 6
- 102000008579 Transposases Human genes 0.000 description 6
- 108010020764 Transposases Proteins 0.000 description 6
- 241000700605 Viruses Species 0.000 description 6
- 230000003292 diminished effect Effects 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 108020001507 fusion proteins Proteins 0.000 description 6
- 102000037865 fusion proteins Human genes 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 230000001404 mediated effect Effects 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 230000035755 proliferation Effects 0.000 description 6
- 238000000159 protein binding assay Methods 0.000 description 6
- 241000035537 Cypridina noctiluca Species 0.000 description 5
- 102220619487 Protein mago nashi homolog_D73K_mutation Human genes 0.000 description 5
- 230000009471 action Effects 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000012575 bio-layer interferometry Methods 0.000 description 5
- 230000001413 cellular effect Effects 0.000 description 5
- 230000001447 compensatory effect Effects 0.000 description 5
- 230000001010 compromised effect Effects 0.000 description 5
- 230000001086 cytosolic effect Effects 0.000 description 5
- 230000001613 neoplastic effect Effects 0.000 description 5
- 239000002773 nucleotide Chemical group 0.000 description 5
- 125000003729 nucleotide group Chemical group 0.000 description 5
- 238000009738 saturating Methods 0.000 description 5
- HBZBAMXERPYTFS-SECBINFHSA-N (4S)-2-(6,7-dihydro-5H-pyrrolo[3,2-f][1,3]benzothiazol-2-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid Chemical compound OC(=O)[C@H]1CSC(=N1)c1nc2cc3CCNc3cc2s1 HBZBAMXERPYTFS-SECBINFHSA-N 0.000 description 4
- AVNJFDTZJJNPKF-ZDUSSCGKSA-N 2-[3-[2-[(2S)-butan-2-yl]-3-hydroxy-6-(1H-indol-3-yl)imidazo[1,2-a]pyrazin-8-yl]propyl]guanidine Chemical compound CC[C@H](C)c1nc2c(CCCNC(N)=[NH2+])nc(cn2c1[O-])-c1c[nH]c2ccccc12 AVNJFDTZJJNPKF-ZDUSSCGKSA-N 0.000 description 4
- 208000023275 Autoimmune disease Diseases 0.000 description 4
- 241000271566 Aves Species 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 102000005720 Glutathione transferase Human genes 0.000 description 4
- 108010070675 Glutathione transferase Proteins 0.000 description 4
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 4
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 102220472894 Receptor-type tyrosine-protein phosphatase beta_R94K_mutation Human genes 0.000 description 4
- 230000002238 attenuated effect Effects 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 238000000423 cell based assay Methods 0.000 description 4
- 238000002659 cell therapy Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 239000012678 infectious agent Substances 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 238000002703 mutagenesis Methods 0.000 description 4
- 231100000350 mutagenesis Toxicity 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 102220010409 rs199422245 Human genes 0.000 description 4
- 102200082919 rs35857380 Human genes 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000004475 Arginine Substances 0.000 description 3
- 206010006187 Breast cancer Diseases 0.000 description 3
- 208000026310 Breast neoplasm Diseases 0.000 description 3
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 3
- 206010009944 Colon cancer Diseases 0.000 description 3
- 241000701022 Cytomegalovirus Species 0.000 description 3
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 239000004472 Lysine Substances 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 102220467128 Runt-related transcription factor 1_L13Y_mutation Human genes 0.000 description 3
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 3
- 239000004098 Tetracycline Substances 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- -1 antibody Proteins 0.000 description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 230000024245 cell differentiation Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 208000029742 colonic neoplasm Diseases 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 210000005220 cytoplasmic tail Anatomy 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 231100000673 dose–response relationship Toxicity 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 210000003071 memory t lymphocyte Anatomy 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 230000002688 persistence Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 102200090720 rs137852501 Human genes 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 229960002180 tetracycline Drugs 0.000 description 3
- 229930101283 tetracycline Natural products 0.000 description 3
- 235000019364 tetracycline Nutrition 0.000 description 3
- 150000003522 tetracyclines Chemical class 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 208000003174 Brain Neoplasms Diseases 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 230000006820 DNA synthesis Effects 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 2
- 108010002386 Interleukin-3 Proteins 0.000 description 2
- 108090000978 Interleukin-4 Proteins 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108700026495 N-Myc Proto-Oncogene Proteins 0.000 description 2
- 102100030124 N-myc proto-oncogene protein Human genes 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- YHIPILPTUVMWQT-UHFFFAOYSA-N Oplophorus luciferin Chemical compound C1=CC(O)=CC=C1CC(C(N1C=C(N2)C=3C=CC(O)=CC=3)=O)=NC1=C2CC1=CC=CC=C1 YHIPILPTUVMWQT-UHFFFAOYSA-N 0.000 description 2
- 241000219000 Populus Species 0.000 description 2
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 2
- 108020005091 Replication Origin Proteins 0.000 description 2
- 101150039863 Rich gene Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 102000007562 Serum Albumin Human genes 0.000 description 2
- 108010071390 Serum Albumin Proteins 0.000 description 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 238000002679 ablation Methods 0.000 description 2
- 239000000556 agonist Substances 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 108010031180 cypridina luciferase Proteins 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 102000054767 gene variant Human genes 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 2
- 230000002363 herbicidal effect Effects 0.000 description 2
- 239000004009 herbicide Substances 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 238000000099 in vitro assay Methods 0.000 description 2
- 239000012212 insulator Substances 0.000 description 2
- 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 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000000066 myeloid cell Anatomy 0.000 description 2
- 210000000822 natural killer cell Anatomy 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000003076 paracrine Effects 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 230000004983 pleiotropic effect Effects 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 210000003289 regulatory T cell Anatomy 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 230000007781 signaling event Effects 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- 239000013603 viral vector Substances 0.000 description 2
- 230000003612 virological effect Effects 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
- SGKRLCUYIXIAHR-AKNGSSGZSA-N (4s,4ar,5s,5ar,6r,12ar)-4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1=CC=C2[C@H](C)[C@@H]([C@H](O)[C@@H]3[C@](C(O)=C(C(N)=O)C(=O)[C@H]3N(C)C)(O)C3=O)C3=C(O)C2=C1O SGKRLCUYIXIAHR-AKNGSSGZSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 208000037068 Abnormal Karyotype Diseases 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 241000059559 Agriotes sordidus Species 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 206010003827 Autoimmune hepatitis Diseases 0.000 description 1
- 241000713842 Avian sarcoma virus Species 0.000 description 1
- 208000003950 B-cell lymphoma Diseases 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 206010005949 Bone cancer Diseases 0.000 description 1
- 208000018084 Bone neoplasm Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000701822 Bovine papillomavirus Species 0.000 description 1
- 241000589562 Brucella Species 0.000 description 1
- 108091033409 CRISPR Proteins 0.000 description 1
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 1
- 108091007741 Chimeric antigen receptor T cells Proteins 0.000 description 1
- 241000606161 Chlamydia Species 0.000 description 1
- 108091062157 Cis-regulatory element Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 241001445332 Coxiella <snail> Species 0.000 description 1
- 102220526128 Dihydrofolate reductase_I74V_mutation Human genes 0.000 description 1
- 102220485298 Disrupted in schizophrenia 1 protein_Y191F_mutation Human genes 0.000 description 1
- 241000605314 Ehrlichia Species 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108010003471 Fetal Proteins Proteins 0.000 description 1
- 102000004641 Fetal Proteins Human genes 0.000 description 1
- 241000700662 Fowlpox virus Species 0.000 description 1
- 241000589601 Francisella Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 241000224466 Giardia Species 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
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102220485773 Glycophorin-A_L94I_mutation Human genes 0.000 description 1
- 241000590002 Helicobacter pylori Species 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 208000009889 Herpes Simplex Diseases 0.000 description 1
- 101100232904 Homo sapiens IL2 gene Proteins 0.000 description 1
- 101001002657 Homo sapiens Interleukin-2 Proteins 0.000 description 1
- 101000638251 Homo sapiens Tumor necrosis factor ligand superfamily member 9 Proteins 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 241000701109 Human adenovirus 2 Species 0.000 description 1
- 241000701806 Human papillomavirus Species 0.000 description 1
- 108700002232 Immediate-Early Genes Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000010789 Interleukin-2 Receptors Human genes 0.000 description 1
- 108010038453 Interleukin-2 Receptors Proteins 0.000 description 1
- 102000012937 Interleukin-21 Receptor alpha Subunit Human genes 0.000 description 1
- 108010079728 Interleukin-21 Receptor alpha Subunit Proteins 0.000 description 1
- 108010002586 Interleukin-7 Proteins 0.000 description 1
- 108010002335 Interleukin-9 Proteins 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 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
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 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
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 241000589248 Legionella Species 0.000 description 1
- 208000007764 Legionnaires' Disease Diseases 0.000 description 1
- 241001137872 Leishmania sp. Species 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
- 241000186781 Listeria Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000186359 Mycobacterium Species 0.000 description 1
- 241000588653 Neisseria Species 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 108010070047 Notch Receptors Proteins 0.000 description 1
- 102000005650 Notch Receptors Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- VNQABZCSYCTZMS-UHFFFAOYSA-N Orthoform Chemical compound COC(=O)C1=CC=C(O)C(N)=C1 VNQABZCSYCTZMS-UHFFFAOYSA-N 0.000 description 1
- 102000016979 Other receptors Human genes 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 240000007019 Oxalis corniculata Species 0.000 description 1
- 108010067372 Pancreatic elastase Proteins 0.000 description 1
- 102000016387 Pancreatic elastase Human genes 0.000 description 1
- 108091093037 Peptide nucleic acid Proteins 0.000 description 1
- 102000011755 Phosphoglycerate Kinase Human genes 0.000 description 1
- 241000224016 Plasmodium Species 0.000 description 1
- 241001505332 Polyomavirus sp. Species 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 102220513478 Rab-interacting lysosomal protein_E38S_mutation Human genes 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 241000606701 Rickettsia Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 108091008874 T cell receptors Proteins 0.000 description 1
- 230000005867 T cell response Effects 0.000 description 1
- 108010092262 T-Cell Antigen Receptors Proteins 0.000 description 1
- 102220554926 Testis-expressed basic protein 1_Y36H_mutation Human genes 0.000 description 1
- 101001099217 Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8) Triosephosphate isomerase Proteins 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 241000223996 Toxoplasma Species 0.000 description 1
- 241000223104 Trypanosoma Species 0.000 description 1
- 102100032101 Tumor necrosis factor ligand superfamily member 9 Human genes 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 241000607734 Yersinia <bacteria> Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000006023 anti-tumor response Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000003118 aryl group Chemical group 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
- 238000003149 assay kit Methods 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 208000037979 autoimmune inflammatory disease Diseases 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000008901 benefit Effects 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
- 230000033228 biological regulation Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 201000006230 breast fibrosarcoma Diseases 0.000 description 1
- 102220356559 c.104A>T Human genes 0.000 description 1
- 102220367300 c.201C>G Human genes 0.000 description 1
- 239000012830 cancer therapeutic Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000005860 defense response to virus Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229910003460 diamond Inorganic materials 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 229960003722 doxycycline Drugs 0.000 description 1
- 230000005014 ectopic expression Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- 102000018146 globin Human genes 0.000 description 1
- 108060003196 globin Proteins 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
- 229940037467 helicobacter pylori Drugs 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000002390 hyperplastic effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 239000012642 immune effector Substances 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 239000002596 immunotoxin Substances 0.000 description 1
- 230000002637 immunotoxin Effects 0.000 description 1
- 231100000608 immunotoxin Toxicity 0.000 description 1
- 229940051026 immunotoxin Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 210000000661 isochromosome Anatomy 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000012804 iterative process Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000004324 lymphatic system Anatomy 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 238000010801 machine learning Methods 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 208000002780 macular degeneration Diseases 0.000 description 1
- 230000007257 malfunction Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 208000037819 metastatic cancer Diseases 0.000 description 1
- 208000011575 metastatic malignant neoplasm Diseases 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 208000030454 monosomy Diseases 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 108700043045 nanoluc Proteins 0.000 description 1
- 210000000581 natural killer T-cell Anatomy 0.000 description 1
- 210000002501 natural regulatory T cell Anatomy 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 229910052754 neon Inorganic materials 0.000 description 1
- GKAOGPIIYCISHV-UHFFFAOYSA-N neon atom Chemical compound [Ne] GKAOGPIIYCISHV-UHFFFAOYSA-N 0.000 description 1
- 210000005170 neoplastic cell Anatomy 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000000865 phosphorylative effect Effects 0.000 description 1
- 238000002428 photodynamic therapy Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000002818 protein evolution Methods 0.000 description 1
- 244000079416 protozoan pathogen Species 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 108010045647 puromycin N-acetyltransferase Proteins 0.000 description 1
- 238000007674 radiofrequency ablation Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 102220041102 rs114090343 Human genes 0.000 description 1
- 102220083823 rs863224587 Human genes 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 230000017105 transposition Effects 0.000 description 1
- 230000024540 transposon integration Effects 0.000 description 1
- 208000035408 type 1 diabetes mellitus 1 Diseases 0.000 description 1
- 108010087967 type I signal peptidase Proteins 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 238000005406 washing Methods 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
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
-
- 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
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- Cytokines are potent natural regulators of immune cell proliferation and differentiation. While this potency has made cytokines highly attractive as potential therapeutics, it has also complicated their clinical utility. This has been especially true for cytokines that have multiple cellular targets and, thus, high potential for pleiotropic effects.
- Interleukin-2 (“IL- 2”), a robust T cell mitogen whose anti-cancer activity is offset by unwanted proliferation of regulatory (suppressor) T cells and a painful vascular leak syndrome.
- protein engineering can be used to solve some of the clinical challenges: removing, for example, the cytokine’ s capacity to act preferentially on regulatory T cells.
- An alternative approach involves generating orthogonally constrained forms of cytokines and their receptors. See U.S. Patent No. 10,869,887; Sockolosky JT, TrottaE, Parisi G, et al. Selective targeting of engineered T cells using orthogonal IL-2 cytokine-receptor complexes. Science. 2018;359(6379): 1037-1042. doi: 10.1126/science.aar3246, the disclosure of each of which is incorporated by reference herein in its entirety. [0004]
- An orthogonal cytokine system is one in which a cytokine and its receptor have been mutated such that they lose compatibility with their native (parental) partners yet retain the capacity to interact productively with one another.
- Such an orthogonal cytokine: receptor pair can thus be said to demonstrate “privileged” or “private” interactions.
- the approach of generating orthogonally constrained forms of cytokines and their receptors is of value for cell therapy because it provides a way to limit the scope of a cytokine’ s activity solely to the therapeutic (i.e., adoptively transferred) cells - these being the only cells expressing the engineered receptor and, consequently, the only cells capable of responding to the engineered cytokine.
- Interleukin-21 (“IL-21”) is another pleiotropic cytokine with actions in a broad range of lymphoid, myeloid, and epithelial cells. IL-21 regulates both innate and adaptive immune responses; it not only has key roles in antitumor and antiviral responses, but also exerts major effects on inflammatory responses that promote the development of autoimmune diseases and inflammatory disorders. Spolski, R., Leonard, W. Interleukin-21 : a double-edged sword with therapeutic potential. Nat Rev Drug Discov 13, 379-395 (2014). https://doi.org/10.1038/nrd4296. The three-dimensional structure of the natural human IL-21 cytokine: receptor complex is known.
- IL-21 is of particular interest because it enhances cytotoxic T cell responses to viruses and tumors and can act in synergy with other cytokines, such as IL-2 or IL-15. IL-21 does this in part by promoting the persistence of T cells with a stem cell memory phenotype, which has been associated with beneficial outcomes in cell therapy settings. IL-21 is currently undergoing evaluation as a cancer therapeutic in multiple clinical trials. IL-21 also has significant potential utility in chimeric antigen receptor T (“CAR-T”) cell therapies, where it may help to overcome clinical failures due to poor expansion, anti-tumor efficacy, exhaustion, suppression, and persistence.
- CAR-T chimeric antigen receptor T
- an orthogonal interleukin-21 receptor alpha chain (an “ortho-IL-2 IRa” or “ortho-IL-2 IRa molecule,” or when referring to a specific ortho-IL-2 IRa constructed as provided herein, an “RV,” as in “Receptor Variant”) is provided, the ortho-IL-2 IRa comprising a modified amino acid sequence derived from SEQ ID NO: 4 that binds to an orthogonal interleukin-21 cytokine (an “ortho-IL-21” or “ortho-IL-21 molecule,” or when referring to a specific ortho-IL-21 constructed as provided herein, a “CV,” as in “Cytokine Variant”) but has impaired binding to native IL-21.
- the ortho-IL-21Ra comprises a modified amino acid sequence comprising a substitution of one or more amino acid residues of SEQ ID NO: 4 that contact IL-21, residues in the immediate vicinity of such contact residues, or residues elsewhere in IL-2 IRa that have an influence on the conformation of the IL-21 binding surface.
- the ortho-IL- 21Ra includes an amino acid substitution, numbered relative to SEQ ID NO: 6 (the human IL-21a ectodomain in mature form lacking the signal peptide), at position: Y10, Q33, Q35, Y36, E38, L39, F67, H68, F69, M70, A71, D72, D73, 174, L94, A96, E97, P126, A127, Y129, M130, K134, SI 90, Y191, or a combination thereof.
- the amino acid substitution comprises, consists essentially of, or consists of: D72E/Y129F/D73E; M70I/D73E/Q33H; D72E/L94V/Y191F; D72E/E38D/M130L; M70G/Y129F; F69L/M70L/D73I; D72K/D73K; E38K; or M70G, or a combination thereof.
- the amino acid substitution comprises, consists essentially of, or consists of: M70G/Y 129F (“RV13,” as in “Receptor Variant 13,” or SEQ ID NO: 19) or M70G (“RV22” or SEQ ID NO: 27).
- an ortho-IL-21 comprising a modified amino acid sequence derived from SEQ ID NO: 2 that binds to an ortho-IL-21Ra but has impaired binding to native IL-21Ra.
- the ortho-IL-21 comprises a modified amino acid sequence comprising a substitution of one or more amino acid residues of SEQ ID NO: 2 that contact IL-21Ra, residues in the immediate vicinity of such contact residues, or residues elsewhere in IL-21 that have an influence on the conformation of the IL-21Ra binding surface.
- the ortho-IL-21 includes an amino acid substitution, numbered relative to SEQ ID NO: 2, at position: R5, H6, 18, R9, M10, Q12, L13, K73, K75, R76, P78, G84, or P104, or a combination thereof.
- the phrase “numbered relative to SEQ ID NO: 2” means, for numbering purposes, to disregard any epitope tags and signaling peptides.
- the amino acid substitution comprises, consists essentially of, or consists of: R5Q, R5D, R5E, R5K, R5N, H6L, I8E, R9E, R9D, R9K, R9N, R9Q, M10L, Q12V, L13D, K73V, K73D, K73I, K75D, R76E, R76N, R76A, R5Q/R76E, R5Q/R76A, P78L, G84E, or P104A, or combinations thereof.
- the amino acid substitution comprises, consists essentially of, or consists of: H6L/R9K/M10L/P78L.
- an engineered human IL-21 polypeptide that comprises amino acid substitutions, numbered relative to SEQ ID NO: 2: H6L; R9K; M10L; P78L; and optionally comprises G84E or P104A or a combination thereof; and optionally comprises one of K73V or K73I.
- such an engineered human IL-21 polypeptide may include SEQ ID NO: 61 (H6L/R9K/M10L/K73V/P78L/G84E) or SEQ ID NO: 62 (H6L/R9K/M10L/K73I/ P78L/P104A).
- a system for activating IL-21 signaling in a cell comprising: an ortho-IL-2 IRa that has impaired binding to native IL-21, the ortho-IL-2 IRa comprising a modified amino acid sequence derived from SEQ ID NO: 4 comprising a substitution of one or more of the amino acid residues of SEQ ID NO: 4 that contact IL-21, residues in the immediate vicinity of such contact residues, or residues elsewhere in IL-2 IRa that have an influence on the conformation of the IL-21 binding surface; and an ortho-IL-21 that has impaired binding to native IL-2 IRa, the ortho-IL-21 comprising a modified amino acid sequence derived from SEQ ID NO: 2 comprising a substitution of one or more amino acid residues of SEQ ID NO: 2 that contact IL-2 IRa, residues in the immediate vicinity of such contact residues, or residues elsewhere in IL-21 that have an influence on the conformation of the IL-2 IRa binding surface, wherein the ortho-
- Figure 1 provides a schematic representation of an orthogonal IL-21 system.
- the cartoon at the extreme left shows the wild-type receptor and cytokine interacting productively with one another, while the adjacent cartoon depicts the impaired interaction between an ortho-IL-2 IRa and the native cytokine.
- the cartoon at the extreme right depicts the impaired interaction between ortho-IL-21 and the native (wild-type) receptor, while the adjacent cartoon shows a productive interaction between the two orthogonal molecules (ortho-IL-2 IRa and ortho-IL-21).
- Figure 2 provides a schematic representation of pathways for generating an orthogonal IL-21 system.
- Figure 3 shows the results of a representative assay in which a single (sub -saturating) concentration of IL-21-TLucl6 was tested for binding to a panel of 20 candidate ortho-IL-21Ra molecules (all of which had been bound to Streptactin-coated surfaces of wells at saturating concentrations). Eight of the 20 candidate ortho-IL-2 IRa molecules showed diminished capacity to bind IL-21-TLucl6.
- Figures 4A-E show the results of a representative assay in which a range of (subsaturating) concentrations of IL-21-TLucl6 was tested for binding to a panel of candidate ortho- IL-2 IRa molecules.
- the panel included a wild-type receptor as a control and seven of the eight candidate ortho-IL-2 IRa molecules identified in Figure 3.
- the panel also included 13 additional candidate ortho-IL-2 IRa molecules that were designed based on results such as those depicted in Figure 3; specifically, substitutions present in the eight candidate ortho-IL-2 IRa molecules were introduced alone or in combination in the additional 13 candidate ortho-IL-2 IRa molecules.
- the 21 candidate ortho-IL-2 IRa molecules were added to Streptactin-coated wells of 96-well plates at saturating concentrations.
- Figures 4A-E show luminometry data for individual plates in which, in each case, the binding of IL-21-TLucl6 to five candidate ortho-IL-21Ra molecules and a wildtype control IL-2 IRa was compared. Eleven of the additional 13 candidate ortho-IL-2 IRa molecules showed significantly diminished capacity to bind IL-21-TLucl6 relative to the wildtype control (the exceptions being receptors RV23, RV24, and RV28 carrying the single substitutions M70L, F69L, and D73E respectively).
- Figures 5A and 5B show IL-21 -induced STAT3 signaling responses by Ba/F3 cells expressing native IL-2 IRa and the eight candidate ortho-IL-2 IRa molecules identified as shown in Figure 3.
- the cells carried a STAT3 -regulated Cypridina noctiluca luciferase reporter transgene; they were exposed to different concentrations of native IL-21 overnight (approximately 20 hours) before testing the supernatant medium for luciferase activity by luminometry, with Vargulin serving as the enzyme substrate.
- Cells expressing a form of wild-type IL-21Ra lacking its cytoplasmic tail (Acyt) were included to show that STAT3 signaling in response to IL-21 depended on the cytoplasmic tail of the receptor, as expected.
- Figures 6A-6T show the capacity of native IL-21 and candidate ortho-IL-21 molecules to induce signaling via native IL-2 IRa and the eight candidate ortho-IL-2 IRa molecules identified as shown in Figure 3.
- luminometry was used to quantify luciferase produced by transfected Ba/F3 cells carrying a STAT3 -dependent luciferase reporter transgene resident on the same transposon used to confer expression of native IL-2 IRa or candidate ortho-IL-2 IRa molecules.
- the cells were exposed to increasing concentrations of the candidate ortho-IL-21 molecules overnight (approximately 20 hours) before testing the supernatant culture medium for luciferase activity.
- Figures 7A-7D show the capacity of native IL-21 and selected candidate ortho-IL-21 molecules from the collection represented in Figure 6 to induce signaling via native IL-2 IRa and selected candidate ortho-IL-2 IRa molecules.
- Ba/F3 cells expressing native IL-2 IRa or candidate ortho-IL-21 molecules from a transposon that also carried a STAT3 -luciferase reporter transgene
- STAT3 -luciferase reporter transgene were exposed to increasing concentrations of the indicated candidate ortho-IL-21 molecules (for approximately 20 hours) before recovering supernatant medium and testing it for luciferase activity by luminometry as in Figures 5 and 6.
- Figures 8A, 8B, and 8C show the results of a representative screening experiment in which a collection of 96 cytokines were tested for their capacity to induce STAT3 -dependent signaling responses in Ba/F3 cells expressing wild-type IL-21Ra ( Figure 8A) or the candidate ortho-IL-2 IRa molecules RV13 ( Figure 8B) or RV6 ( Figure 8C) comprising amino acid substitutions M70G/Y129F (SEQ ID NO: 19) or M70I/D73E/Q33H (SEQ ID NO: 12), respectively.
- luminometry was used to quantify luciferase produced by transfected Ba/F3 cells carrying a STAT3 -dependent luciferase reporter transgene resident on the same transposon used to confer expression of native IL-2 IRa or the candidate ortho-IL-2 IRa molecule.
- the cells were exposed to increasing concentrations of the candidate ortho-IL-21 molecules overnight (approximately 20 hours) before testing the supernatant culture medium for luciferase activity.
- the dotted lines show response curves for the cytokine collection, while the responses caused by five cytokines of interest (wild-type IL-21, negative control CV22, and candidate ortho-IL-21 molecules CV204, CV374, and CV388) are highlighted with solid lines and symbols.
- Figures 9A and 9B show the capacity of native IL-21 and candidate ortho-IL-21 molecules to induce signaling via wild-type IL-2 IRa ( Figure 9A) and the candidate ortho-IL- 21Ra RV13 comprising amino acid substitutions M70G/Y129F (SEQ ID NO: 19) ( Figure 9B).
- luminometry was used to quantify luciferase produced by transfected Ba/F3 cells carrying a STAT3 -dependent luciferase reporter transgene resident on the same transposon used to confer expression of native IL-2 IRa or the candidate ortho-IL-2 IRa molecule.
- the cells were exposed to increasing concentrations of the candidate ortho-IL-21 molecules overnight (approximately 20 hours) before testing the supernatant culture medium for luciferase activity.
- Figures 10A, 10B, and 10C show the capacity of native IL-21 and candidate ortho-IL-21 molecules to induce signaling via wild-type IL-2 IRa ( Figure 10A), the candidate ortho-IL-2 IRa RV13 comprising amino acid substitutions M70G/Y 129F (SEQ ID NO: 19) ( Figure 10B), and the candidate ortho-IL-21Ra RV22 comprising amino acid substitution M70G (SEQ ID NO: 27) ( Figure 10C).
- luminometry was used to quantify luciferase produced by transfected Ba/F3 cells carrying a STAT3 -dependent luciferase reporter transgene resident on the same transposon used to confer expression of native IL-2 IRa or the candidate ortho-IL-2 IRa molecule.
- the cells were exposed to increasing concentrations of the candidate ortho-IL-21 molecules overnight (approximately 20 hours) before testing the supernatant culture medium for luciferase activity.
- Orthogonal IL-21 receptors and orthogonal IL-21 cytokines are provided.
- the orthogonal IL-21 receptor: cytokine pairs may include an ortho-IL-2 IRa that has impaired binding to native IL-21 and an ortho-IL-21 that has impaired binding to native IL-2 IRa, wherein the ortho-IL-2 IRa binds to ortho-IL-21.
- the orthogonal IL-21 receptor-cytokine pair may be used to activate a signaling response in cells.
- the signaling response may be the native one normally downstream of the IL-21 receptor or an alternative one dependent on non-native IL-21 receptors and/or other non-native cellular components.
- Cells engineered to express the orthogonal IL-21 receptors are also provided, as well as methods for using such cells for treatment of various diseases and disorders.
- Treat,” “treating,” “treatment,” and the like refer to any action providing a benefit to a subject afflicted with a disease or disorder such as cancer, including improvement in the condition through lessening or suppression of at least one symptom, delay in progression of the disease, and the like.
- phrases “effective amount” and “therapeutically effective amount” refer to an amount of the composition used in the practice of the invention that is sufficient to provide effective treatment in a subject.
- WT Wild type
- WT native
- WT protein polypeptide, antibody, immunoglobulin, IgG, and the like has an amino acid sequence or a nucleotide sequence corresponding to that normally found in nature, which has not been intentionally modified.
- polynucleotide refers to oligonucleotides, nucleotides, or to a fragment of any of these; to DNA or RNA (e.g., mRNA, rRNA, tRNA) of genomic or synthetic origin, which may be single-stranded or double-stranded and may represent a sense or antisense strand; to peptide nucleic acids; or to any DNA-like or RNA-like material, whether natural or synthetic in origin.
- the term may also encompass nucleic acids, e.g., oligonucleotides, containing known analogs of natural nucleotides, as well as nucleic acid-like structures with synthetic backbones.
- polypeptide refers to an oligopeptide, peptide, or protein sequence, or to a fragment, portion, or subunit of any of these, and to naturally occurring or synthetic molecules.
- polypeptide also includes amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain any type of modified amino acids.
- polypeptide also includes peptides and polypeptide fragments, motifs, and the like.
- a protein or peptide “chain” refers to a distinct subunit of a larger protein or protein complex.
- homologs are meant that the corresponding proteins (e.g., IL-21Ra), such as those from other species, are substantially homologous at the overall protein (i.e., mature protein) level to the human protein, so long as such homologous peptides retain their respective known activities.
- Various levels of homology from 35% to 99%, can be present.
- amino acid modification includes an amino acid substitution, insertion, or deletion in a polypeptide sequence.
- amino acid substitution or “substitution” is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid.
- substitution R94K refers to a modified polypeptide in which the arginine at position 94 is replaced with a lysine.
- 94K indicates the substitution of position 94 with a lysine.
- Multiple substitutions are typically separated by a slash or a comma.
- R94K/L78V and [R94K, L78V] refer to a double variant comprising the substitutions R94K and L78V.
- amino acid insertion or “insertion” is meant the addition of an amino acid at a particular position in a parent polypeptide sequence.
- insert - 94 designates an insertion at position 94.
- amino acid deletion or “deletion” is meant the removal of an amino acid at a particular position in a parent polypeptide sequence.
- R94- designates the deletion of arginine at position 94.
- conservative modifications refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the polypeptide containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions, and deletions. Modifications can be introduced into a protein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis, or by DNA synthesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
- amino acids with basic side chains e.g., lysine, arginine, histidine
- acidic side chains e.g., aspartic acid, glutamic acid
- uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
- nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
- beta-branched side chains e.g., threonine, valine, isoleucine
- aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
- the conservative substitution variants, homologs, and analogs of the peptides will have an amino acid sequence identity to the disclosed sequences of at least about 35%, at least about 45%, at least about 55%, at least about 65%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 96% to 99%.
- Rost B Twilight zone of protein sequence alignments. Protein Eng. 1999;12(2):85-94. doi: 10.1093/protein/12.2.85.
- a gamma chain (yc) cytokine means any cytokine where the cognate cytokine receptor complex includes the common cytokine receptor gamma chain (yc). yc cytokines include IL-2, IL-4, IL-7, IL-9, IL- 15 and IL-21.
- An orthogonal cytokine refers to variants of a natural cytokine: receptor pair that interact effectively with one another (i.e., such that they can be used to initiate physiologically consequential signaling responses in cells) but are significantly impaired in their capacity to interact with their natural counterparts.
- the orthogonal cytokine can either be a mutated or otherwise modified natural cytokine (a “mutein”) or a completely synthetic protein that acts as an agonist on the orthogonal cytokine receptor (sometimes referred to as a “synthekine”).
- the orthogonal cytokine shows no, or only attenuated, agonist activity toward the wild-type cytokine receptor.
- the orthogonal cytokine: receptor pair may comprise a genetically engineered pair of proteins that are modified by amino acid changes to: (a) lack or reduce binding to the native cytokine or cognate receptor; and (b) specifically bind to the counterpart engineered (orthogonal) ligand or receptor.
- the orthogonal receptor Upon binding of the orthogonal cytokine, the orthogonal receptor activates signaling that is transduced through native cellular elements to provide for a biological activity that mimics the native response, but that is specific to an engineered cell expressing the orthogonal receptor.
- Non-native receptor or cellular elements e.g., non-native cytoplasmic domains in the receptor
- the orthogonal receptor does not bind to any endogenous cytokine or only does so with attenuated avidness, including the native counterpart of the orthogonal cytokine, while the orthogonal cytokine does not bind to any endogenous receptor or only does so with attenuated avidness, including the native counterpart of the orthogonal receptor.
- the affinity of the orthogonal cytokine for the orthogonal receptor is comparable to the affinity of the native cytokine for the native receptor, e.g., having an affinity that is at least about 1% of the native cytokine receptor pair affinity, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 100%, and may be higher, e.g. 2x, 3x, 4x, 5x, 10x or more of the affinity of the native cytokine for the native receptor.
- the phrases “does/do not bind” and “incapable of binding” refer to no detectable binding, or an insignificant binding, i.e., having a binding affinity much lower than that of the natural ligand. “Impaired binding” refers to binding that is lower than the normal level of binding between the corresponding wild-type components (e.g., cytokine and receptor).
- the present invention includes an orthogonal cytokine system based on IL-21, which is a member of the yc family of cytokines.
- IL-21 is structurally related to IL-2 and signals via a dimeric receptor comprised of IL-21Ra and yc. Whereas the IL-2 receptor promotes a STAT5-dominated signaling response, STAT3 dominates the IL-21 response.
- IL-21 promotes a form of T cell differentiation that correlates with good outcomes in adoptive cellular therapy (“ACT”), in particular ACT using CAR T cells, and it shows enhanced anti -turn or properties compared to IL- 2 in various tumor model systems. As a result, IL-21 may prove to be a better adjunct for ACT than IL-2.
- ACT adoptive cellular therapy
- an IL-21 orthogonal system comprises: 1) an ortho-IL-21 with impaired binding to native IL-21Ra; and 2) an ortho-IL-2 IRa capable of binding the ortho-IL-21 while exhibiting impaired binding to native IL-21 (Figure 1; Figure 2, path A).
- an orthogonal interleukin-21 receptor is provided.
- the orthogonal interleukin-21 receptor can include modification of either of the chains making up the overall protein.
- an ortho-IL-2 IRa is provided.
- the ortho-IL-2 IRa includes a modified amino acid sequence derived from wild-type human IL-21Ra (SEQ ID NO: 4 - human IL-21Ra in mature form lacking the signal peptide).
- the ortho-IL-2 IRa binds to an ortho-IL-21 but has impaired binding to native IL-21.
- Human IL-21Ra is represented by SEQ ID NO: 3.
- the modified amino acid sequence comprises a substitution of one or more of the amino acid residues of SEQ ID NO: 4 that contact IL-21, residues in the immediate vicinity of such contact residues, or residues elsewhere in IL-2 IRa that have an influence on the conformation of the IL-21 binding surface.
- Amino acids within the immediate vicinity are those within 1, 2, 3, 4, or 5 amino acids of the contact residues in the primary protein sequence, or amino acid residues that are similarly nearby in the tertiary protein structure.
- the ortho- IL-21Ra includes an amino acid substitution, numbered relative to SEQ ID NO: 6, at position: Y10, Q33, Q35, Y36, E38, L39, F67, H68, F69, M70, A71, D72, D73, 174, L94, A96, E97, P126, A127, Y129, M130, K134, S190, or Y191, or a combination thereof.
- One strategy for creating an orthogonal version of IL-21 involves first mutating IL-2 IRa such that it suffers reduced binding capability to IL-21. Twenty IL-2 IRa amino acids make significant direct contacts with IL-21 within a binding surface of 990 A 2 . Mutating any of these 20 residues may impair the capacity of the receptor to bind normally to IL-21. For example, the methionine residue at position 70 of IL-2 IRa has been identified as a major contributor to the binding interaction.
- the large hydrophobic side chain of this residue fits into an IL-21 pocket comprised of mostly hydrophilic residues (Arginine-9, Glutamine-12, Arginine-76, Lysine-73, and Isoleucine- 16), repositioning some of them for improved contact with IL-21Ra (notably, Arginine- 9 and Arginine-76 of IL-21 contact Aspartic acid-72 and Aspartic acid-73 of IL-21Ra, respectively).
- Changing Methionine-70 to a different residue could adjust this repositioning such that some of these contacts are weakened or lost.
- Changing Aspartic acid-72 or -73 of IL-21Ra would also likely decrease the binding free energy of the interaction such that compensatory changes in IL-21 (e.g., at positions 9 and 76) would be required to restore it.
- Helix C of IL-21 exists in two interchangeable states (one disordered, the other a-helical) in the free structure of the cytokine.
- the a-helical form is stabilized in the complex of IL-21 with IL-21Ra, as is the first part of the CD loop.
- Helix C of IL-21 contains the above-mentioned Lysine-73 and Arginine-76, which are proximal to Methionine-70 of IL-21Ra.
- the CD loop of IL-21 includes Lysine-77, Proline-79, and Serine-80, which collectively form a pocket for Tyrosine-36 of IL-21 Ra; Lysine-77 also makes an ionic contact with Glutamic acid-38 of IL- 21Ra.
- Changing the CD loop amino acid sequence to the analogous sequence found in IL-4 results in a ten-fold enhancement of IL-21 potency measured using a cellular assay. This observation suggests that significant binding energy is expended in stabilizing helix C of IL-21.
- changes to Glutamic acid-38 and Tyrosine-36 of IL-21Ra could significantly impact IL-21 binding in a manner that might be reversible by compensatory changes to IL-21 in helix C or the CD loop.
- Discrete avian sequence motifs can be used in the design of an orthogonal IL-21 system.
- 39 have significantly (six residues) shortened CD loops compared to humans and mice. Together with the absence of Tyrosine-36 in all of the 74 available avian IL-21Ra sequences, this suggests helix C of avian IL-21 likely engages its receptor in a distinct fashion to that of human IL-21. This also provides an additional basis for modifications to the binding residues mentioned above for constructing an orthogonal IL-21 system.
- candidate amino acids may be identified that can be modified to change IL-21Ra such that its binding to native IL-21 is impaired. These changes must, however, be of a character that will not be incompatible with compensatory changes in IL-21 (i.e., changes in IL-21 that would restore binding to ortho-IL-2 IRa). Changes to IL-21Ra resulting in large alterations to its conformation are not desirable because the number of compensatory changes required to restore binding may either present too great an engineering challenge or be incompatible with cytokine function (e.g., because yc binding is lost, or because the cytokine or cytokine receptor becomes unstable, difficult to express, immunogenic, or pharmacologically problematic).
- At least two kinds of assays can be used to screen ortho-IL-2 IRa for a loss of binding to IL-21.
- One is a direct binding assay, which can be carried out using purified proteins and a sensitive biophysical analytical procedure such as surface plasmon resonance (“SPR”) or biolayer interferometry (“BLI”).
- SPR surface plasmon resonance
- BLI biolayer interferometry
- Another assay is a functional assay involving an appropriate cell line.
- Ba/F3 cells have been used successfully for this purpose because they possess a number of useful traits: (i) they do not express endogenous IL-2 IRa; (ii) they express mouse yc, which substitutes effectively for human yc in signaling with human IL-2 IRa and human IL-21; (iii) they respond to IL-21R signaling by phosphorylating STAT3 and proliferating; and (iv) they permit use of a STAT3 reporter transgene (such as one expressing luciferase) as a facile and attractive means for monitoring IL-21 signaling.
- Jurkat or Molt-3 cells are alternative choices, both of which show minimal or absent expression of endogenous IL-21Ra but express yc and are IL-21R signaling- competent. HeLa cells transfected to express yc may also be used.
- the strategy for isolating candidate ortho-IL-2 IRa involves expressing the variants individually in Ba/F3 cells (or one of the alternatives just mentioned) by transfection. Flow cytometry is used to confirm the presence of the candidate ortho-IL-2 IRa molecules on the cell surface and the presence of epitopes recognized by available antibodies.
- the candidate ortho-IL-21Ra includes an amino acid substitution at position: Y10, Q33, Q35, Y36, E38, L39, F67, H68, F69, M70, A71, D72, D73, 174, L94, A96, E97, P126, A127, Y129, M130, K134, S190, or Y191, or combinations thereof (where the numbers refer to the residue position in the mature IL-21Ra ectodomain (SEQ ID NO: 6), and letters refer to amino acid identity using the single letter code, the first letter being the wild-type residue and the second, if present, the substitute residue).
- the amino acid substitution comprises, consists essentially of, or consists of: M70G, M70A, M70I, M70L, Y129F, Y129A, Y129S, Y129H, M130F, M130S, Y36H, Y36F, FMAD(69-73) to LLADI, FYM(128-130) to FHF, D72K, D73K, D72E, D73E, D73I, E38K, E38S, E38D, F67L, F67Y, F69L, S190Y, Y191F, Q35L, H68Q, I74V, L94I, L94V, E97D, A127T, Q33N, Q33H, K134R, M130I, M130L, S190T, or combinations thereof.
- the amino acid substitution comprises, consists essentially of, or consists of: D72E/Y129F/D73E; M70I/D73E/Q33H; D72E/L94V/Y191F; D72E/E38D/M130L;
- the amino acid substitution comprises, consists essentially of, or consists of: [H68Q,E38D,Q33H], [Y129F,H68Q,Y191F], [Y36F,H68Q,D73E], [Y129F,F67Y,M130L], [L94V,Q33H,M130L], [Y36F,F67Y,E38D], [D72E,E38D,M130L], [Y36F,M70I,L94V],
- the amino acid substitution comprises, consists essentially of, or consists of: M70G/Y129F or M70G.
- Ba/F3 cells expressing candidate ortho-IL-2 IRa may be incubated with native human IL- 21 before analysis for a signaling response.
- a time course may be used to improve assay sensitivity and resolution.
- Other experiments involve comparisons of doseresponse curves.
- IL-21 responsiveness is detected by monitoring proliferation of the Ba/F3 cells, tyrosine phosphorylation of STAT3 by flow cytometry or immunoblotting, or expression of a reporter (such as luciferase or secreted alkaline phosphatase) from a STAT3 -dependent reporter transgene present in the cells.
- a reporter such as luciferase or secreted alkaline phosphatase
- Ortho-IL-2 IRa molecules demonstrating this nonresponsive property are candidates for orthogonally restricted IL-21 cytokine-receptor systems.
- Another aspect provides an ortho-IL-21 having a modified amino acid sequence derived from wild-type human IL-21 (SEQ ID NO: 2 - human IL-21 in mature form lacking the signal peptide) that binds to an ortho-IL-2 IRa but has impaired binding to native IL-2 IRa.
- Human IL- 21 is represented by SEQ ID NO: 1.
- the ortho-IL-21 binds to an ortho-IL-2 IRa but has impaired binding to native IL-2 IRa.
- the modified amino acid sequence comprises a substitution of one or more amino acid residues of SEQ ID NO: 2 that contact IL-21Ra, residues in the immediate vicinity of such contact residues, or residues elsewhere in IL-21R that have an influence on the conformation of the IL-2 la binding surface.
- Arginine-5, Arginine-9, Arginine-11, Glutamine-12, Lysine-73, Arginine-76, and Lysine-77 also form significant van der Waals contacts with IL-2 IRa.
- Isoleucine-8, Isoleucine- 16, Glutamine- 19, Tyrosine-23, Isoleucine-66, Valine-69, andProline-79 make additional van der Waals contacts. Substitutions can be made to any of these residues to overcome the changes present in candidate ortho-IL-2 IRa molecules and restore binding.
- the crystal structure of IL-21 bound by IL-2 IRa can be used to identify IL-21 residues proximal to changes engineered into IL-2 IRa. If, for example, changing Methionine-70 of IL- 2 IRa is sufficient to abrogate IL-21 binding, then it is possible that compensatory changes to any of the following Methionine-70 contact residues in IL-21 may restore binding: Arginine-9, Glutamine-12, Isoleucine- 16, Lysine-73, and Arginine-76.
- IL-21Ra residues Tyrosine-10, Leucine-39, Glutamic acid-38, Phenylalanine-67, Alanine-71, Aspartic acid-72, Aspartic acid-73, Tyrosine- 129, Methionine- 130, and Tyrosine-191.
- the ten IL-21Ra residues just mentioned mediate additional contacts with IL-21 residues Arginine-5, Isoleucine-8, Glutamine- 19, Serine-70, and Lysine-77.
- Phage display or yeast display technology can permit the screening of large mutation spaces. This is typically accomplished by creating highly diverse libraries of variants wherein small numbers of residues in a protein are changed in a random (or semi-random) combinatorial fashion. The library is then screened for the desired properties. For the present invention, screening can include identifying members of a library of IL-21 variants that are capable of binding to a candidate ortho-IL-2 IRa.
- an IL-21 library can be constructed that has randomized combinatorial mutations in the ten potentially impacted contact residues (Arginine-5, Isoleucine-8, Arginine-9, Glutamine-12, Isoleucine- 16, Glutamine-19, Serine-70, Lysine-73, Arginine-76, and Lysine-77).
- the theoretical complexity of such a library could exceed 10 13 . It is typically challenging and impractical to generate libraries comprised of more than 10 8 - 10 9 variants.
- Screening of such a library could involve a selection process in which phage or yeast displaying variant forms of IL-21 attached to their surfaces are separated based on their capacity to adhere to a matrix coated with a candidate ortho-IL-2 IRa (and not to a matrix coated with wild-type IL-2 IRa). Repeated cycles of such selection are performed to enrich for the desired properties in IL-21.
- Analytical screening procedures including SPR or BLI are used to characterize the products of the selection in detail and identify those with optimal properties.
- the ortho-IL-21 comprises a modified amino acid sequence comprising a substitution of one or more amino acid residues of SEQ ID NO: 2 that contact IL-21Ra, residues in the immediate vicinity of such contact residues, or residues elsewhere in IL-21 that have an influence on the conformation of the IL-2 IRa binding surface.
- the ortho-IL-21 includes an amino acid substitution, numbered relative to SEQ ID NO: 2, at position: R5, H6, 18, R9, MIO, Q12, L13, K73, K75, R76, P78, G84, or P104, or a combination thereof.
- the amino acid substitution comprises, consists essentially of, or consists of: R5Q, R5D, R5E,
- the amino acid substitution comprises, consists essentially of, or consists of: H6L/R9K/M10L/P78L.
- An alternative approach to the identification of ortho-IL-21 molecules involves iterative cycles of mutagenesis, again focused on small numbers of residues selected from those that make direct intermolecular contacts in the IL-21 :IL-21Ra structure.
- This approach may also include residues that are near to contact points and/or residues in potentially relevant structural features.
- a broad version of this approach can involve any of the residues that are proximal to the entire area of contact with IL-21Ra and additional semi-randomly selected residues in proximal structural features.
- a more focused version involves residues - such as the ten potentially relevant for the Methionine-70 substitution - that might be directly impacted by the specific substitution(s) present in the candidate ortho-IL-2 IRa.
- the iterative process begins with a large number (e.g., 10-100) of candidate ortho-IL-21 forms.
- a large number e.g., 10-100
- double or triple mutants are included in the initial collection, but in other versions, only single point mutations of IL-21 are evaluated.
- These candidate ortho-IL-21 molecules are tested for activity using the cellular assay described above (employing, for example, Ba/F3 cells carrying a STAT3 -dependent luciferase reporter transgene).
- a minimum of two kinds of cells are used in the assay: cells expressing a candidate ortho-IL-21Ra and, as a counter- screen, cells expressing wild-type IL-21Ra.
- the likelihood of the alternative approach succeeding corresponds to the number of candidate ortho-IL-2 IRa molecules examined. Expanding this number reduces the likelihood of inadvertently selecting a candidate ortho-IL-2 IRa that does not readily allow for IL-21 binding to be restored (even partially) with small numbers (e.g., less than three) of substitutions. Expanding this number also increases the likelihood of being able to isolate parallel mutually orthogonal systems that do not demonstrate crosstalk with each other or with wild-type IL-21 or IL-2 IRa.
- the data from the initial screening round may be deconvoluted and analyzed focusing on identifying substitutions in IL-21 that in isolation promote improved binding to candidate ortho- IL-2 IRa molecules and diminished binding to native IL-2 IRa.
- a second round of screening may be performed in which positively scoring substitutions from the first round are combined in new candidate ortho-IL-21 molecules. These candidate ortho-IL-21 molecules (and, if considered desirable, additional variations in which conservative or nonconservative substitutions are made at the positively scoring positions) are tested again for improved binding to candidate ortho-IL- 2 IRa molecules and impaired binding to native IL-2 IRa.
- Additional rounds of screening may be performed involving further combinations of substitutions until at least one candidate ortho-IL-21 has been isolated with the desired properties (absence of activity with native IL-2 IRa and near-normal activity with at least one candidate ortho-IL-2 IRa).
- the alternative screening approach may, in some circumstances, be facilitated using candidate ortho-IL-2 IRa molecules that retain reduced - but not entirely absent - binding to wildtype IL-21.
- Such reduced-binding candidate ortho-IL-2 IRa molecules may prove more permissive than non-binding candidate ortho-IL-21Ra molecules (i.e., candidate ortho-IL-21Ra molecules lacking any binding to wild-type IL-21) to a restoration of some IL-21 binding activity by small numbers (e.g., less than three) of discrete substitutions in IL-21.
- candidate ortho- IL-21 molecules Once candidate ortho- IL-21 molecules have been isolated by the screening procedure outlined above, additional screening steps may be performed involving new candidate ortho-IL-2 IRa molecules in which additional substitutions are compounded with the ones already present. In some aspects, these additional mutations may entirely eliminate binding to wild-type IL-21 while retaining the capacity to bind the ortho-IL-21. Multiple rounds of this receptor mutagenesis may be performed along with subsequent refining cytokine mutagenesis rounds.
- the binding properties of the products of the screening approach may be analyzed using purified proteins and BLI or SPR.
- the products that most closely resemble wild-type IL-21 and IL-2 IRa in their binding kinetics may be chosen as candidate orthogonal IL-21 systems.
- candidate ortho-IL-21 molecules may be engineered according to the process described in one or more ofU.S. Patent Nos. 8,005,620, 8,635,029, and 8,412,461, as well as Govindarajan S, Mannervik B, Silverman JA, et al. Mapping of amino acid substitutions conferring herbicide resistance in wheat glutathione transferase. ACS Synth Biol. 2015;4(3):221 - 227. doi: 10.1021/sb500242x; Musdal Y, Govindarajan S, Mannervik B. Exploring sequencefunction space of a poplar glutathione transferase using designed information-rich gene variants. Protein Eng Des Sei.
- candidate ortho-IL-21 molecules are expressed as fusion proteins between a modified amino acid sequence derived from SEQ ID NO: 2 as described above and a second amino acid sequence that facilitates purification, increases stability and half-life of the ortho-IL- 21 molecules in vivo, or improves drug properties that are critical for successful dosing of ortho- IL-21 molecules in patients.
- Suitable second amino acid sequences are known in the art, and include, but are not limited to, serum albumin, Fc fragments of IgG, single-chain Fc antibody fragments, ABD035, and the like. Fc fragments can be modified, for example, with electrostatic steering mutations, to prevent, or at least significantly limit, the formation of homodimers.
- Another aspect provides a system for activating IL-21 signaling in a cell.
- the system includes a candidate ortho-IL-2 IRa that has impaired binding to native IL-21, the candidate ortho- IL-21Ra comprising a modified amino acid sequence derived from SEQ ID NO: 4 comprising a substitution of one or more of the amino acid residues of SEQ ID NO: 4 that contact IL-21, residues in the immediate vicinity of such contact residues, or residues elsewhere in IL-2 IRa that have an influence on the conformation of the IL-21 binding surface; and an ortho-IL-21 that has impaired binding to native IL-2 IRa, the ortho-IL-21 comprising a modified amino acid sequence derived from SEQ ID NO: 2 comprising a substitution of one or more amino acid residues of SEQ ID NO: 2 that contact IL-2 IRa, residues in the immediate vicinity of such contact residues, or residues elsewhere in IL-21 that have an influence on the conformation of the IL-2 IRa binding surface wherein the ortho-IL-2 IR
- the cell is a T cell. In further aspects, the cell is a CAR T cell.
- the orthogonal cytokine and orthogonal receptor can be any of the candidate ortho-IL-21 and candidate ortho-IL-2 IRa molecules described herein.
- the ortho-IL-21Ra includes an amino acid substitution, numbered relative to SEQ ID NO: 6, comprising, consisting essentially of, or consisting of: M70G/Y129F or M70G; and the ortho-IL-21 includes an amino acid substitution, numbered relative to SEQ ID NO: 2, comprising, consisting essentially of, or consisting of one of: (1) H6L/R9K/M10L/P78L; (2) H6L/R9K/M10L/P78L/K73V; (3) H6L/R9K/M10L/P78L/K73I; (4) H6L/R9K/M10L/P78L/K73V/G84E; (5)
- H6L/R9K/M10L/P78L/K73I/G84E H6L/R9K/M10L/P78L/K73V/P104A; and (7)
- orthogonal proteins such as ortho-IL-21 or ortho-IL-2 IRa
- Orthogonal proteins may be produced by recombinant methods.
- Ortho-IL-2 IRa may be introduced on an expression vector into a cell to be engineered.
- DNA encoding an orthogonal protein may be obtained from various sources as designed during the engineering process.
- Amino acid sequence variants may be prepared by introducing appropriate nucleotide changes into the nucleic acid coding sequence encoding the protein.
- the nucleic acid codons that encode amino acids are known to those skilled in the art. The specific codons selected may be chosen to optimize expression in the host cells being used.
- the amino acid variants may represent insertions, substitutions, and specified deletions of residues as described herein. Any combination of insertions, substitutions, and specified deletions may be made to arrive at the final construct, provided that the final construct possesses the desired biological activity as defined herein.
- the nucleic acid encodes an ortho-IL-2 IRa as described herein.
- Nucleic acids are “operably linked” when placed into a functional relationship with another nucleic acid sequence.
- DNA for a signal sequence is operably linked to DNA for a polypeptide if the DNA for a signal sequence is expressed as a preprotein that participates in the secretion of the polypeptide;
- a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the sequence;
- a ribosome binding site is operably linked to a coding sequence if the ribosome binding site is positioned so as to facilitate translation.
- operably linked means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, some sequences, such as enhancers, do not have to be contiguous to be effective.
- the nucleic acid encoding the ortho-IL-21 or ortho-IL-2 IRa may be inserted into a replicable vector for expression.
- the vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
- Vectors may include viral vectors, plasmid vectors, integrating vectors, transposons, and the like. For example, a suitable transposon/transposase-based polynucleotide vector system is described in U.S. Patent No. 10,041,077, which is incorporated by reference herein in its entirety.
- the ortho-IL-21 or ortho-IL-2 IRa may be recombinantly produced without modification or as a fusion polypeptide with a heterologous polypeptide, e.g., a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
- a heterologous polypeptide e.g., a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
- the signal sequence may be a component of the vector, or it may be a part of the coding sequence that is inserted into the vector.
- the heterologous signal sequence selected may be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.
- the native signal sequence may be used, or other mammalian signal sequences may be suitable, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders, for example, the herpes simplex gD signal.
- Selection genes typically contain a selection gene, also termed a selectable marker.
- the selection gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium.
- Typical selection genes encode proteins that: (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media.
- Expression vectors may contain a promoter that may be recognized by the host organism and may be operably linked to an orthogonal protein coding sequence. Promoters may be untranslated sequences located upstream (5’) to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of the particular nucleic acid sequence to which they are operably linked. Such promoters typically fall into two classes: inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature.
- Transcription from vectors in mammalian host cells may be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus (such as murine stem cell virus), hepatitis-B virus, and Simian Virus 40 (“SV40”), from heterologous mammalian promoters, e.g., the actin promoter, phosphoglycerate kinase (“PGK”), or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.
- the early and late promoters of SV40 are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
- the expression vector may also include an enhancer sequence.
- Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, which act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent, having been found 5’ and 3 ’ to the transcription unit, within an intron, as well as within the coding sequence itself.
- Many enhancer sequences are known from mammalian genes (e.g., globin, elastase, albumin, a- fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus.
- Examples may include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
- the enhancer may be spliced into the expression vector at a position 5’ or 3’ to the coding sequence but is preferably located at a site 5’ from the promoter.
- Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5’ and 3’ untranslated regions of eukaryotic or viral DNAs or cDNAs.
- Expression vectors might also be comprised of inducible regulatory elements for the purpose of controlling expression of a transgene (encoding, for example, ortho-IL-21 or ortho-IL- 21Ra) with small molecules or other stimulatory agents.
- regulatory elements include, but are not limited to, promoters containing tetracycline operators that render them sensitive to regulation by tetracycline or derivatives thereof (such as doxycycline).
- Promoters may also be inducibly regulated by CRISPRa (clustered regularly interspaced short palindromic repeats- activation) using fusions of transcriptional effectors and catalytically dead Cas9.
- Such promoters may in turn be downstream of other control systems such as those involving dimerizers (of an antibody-based and/or chemical nature) or components based on the Notch receptor.
- One aspect of the invention provides cells that have been engineered to express an ortho- IL-2 IRa.
- the cells may be genetically engineered to include any suitable expression vector described herein.
- the expression vector comprises a coding sequence that encodes the orthogonal receptor, the coding sequence being operably linked to a promoter active in the desired cell.
- Various vectors may be used for this purpose, e.g., transposons, viral vectors, plasmid vectors, and minicircle vectors, which can be integrated into the target cell genome or can be episomally maintained.
- the expression vector is a synthetic transposon that can be integrated into the genome by means of a transposase enzyme. Examples of transposon/transposase systems include Sleeping Beauty, PiggyBac, Leapin® from ATUM Bio, and derivatives thereof.
- the engineered cell may be a host cell for preparing recombinant protein in vitro.
- Suitable host cells for recombinant expression of orthogonal proteins include prokaryotes, yeast, and higher eukaryote cells, such as various mammalian host cell lines.
- the engineered cell is further modified beyond the expression of an ortho- IL-2 IRa. Modifications suitable for use in engineered cells are known in the art and include expression of a CAR, a T cell Receptor (“TCR”), or other receptor or receptor derivatives that recognize specific antigens on antigen presenting cells. [0080]
- the engineered cell is a cell intended for therapeutic use. Examples of therapeutic engineered cells may include stem cells, e.g., a hematopoietic stem cell, a natural killer (“NK”) cell, or a T cell. In some aspects, the engineered cell is a T cell.
- T cells refers to mammalian immune effector cells that may be characterized by expression of a CD3 and/or a T cell antigen receptor, which cells may be engineered to express an ortho-IL-2 IRa.
- the T cells are selected from naive, activated, or post-activation CD8+ T cells; cytotoxic CD8+ T cells; naive, activated, or post-activation CD4+ T cells; helper T cells, e.g., TH1, TH2, TH9, TH11, TH22, and TFH; regulatory T cells, e.g., TRI, natural TReg, and inducible TReg; and memory T cells, e.g., central memory T cells, effector memory T cells, NKT cells, and y6 T cells.
- Ortho-IL-21 may be used as an adjunct to ACT.
- T cells may be engineered to express the ortho-IL-2 IRa by gene (cDNA, minigene, or other nucleic acid construct) transfection, transduction, or transposition.
- Patients receiving the ACT may be treated (and/or pretreated) with the ortho-IL-21 and dosed repeatedly as needed to augment and sustain a desirable T cell presence and responses.
- Therapeutic cells may also be engineered to express ortho-IL-21. This could be accomplished using any of the methods appropriate for ectopic expression of ortho-IL-2 IRa.
- the ortho-IL-21 could be expressed in the same or different cells as those that express ortho-IL-2 IRa, allowing for autocrine or paracrine action, respectively.
- An example of a paracrine arrangement could be CD4+ T cells expressing the ortho-IL-21 and CD8+ T cells expressing the matched ortho- IL-2 IRa.
- ortho-IL-21 may be expressed in a membrane-tethered form. This has previously been accomplished with natural IL-21 by fusing the cytokine to the amino-terminus of an IgG4 CH2-CH3 moiety that was itself fused to a CD4 transmembrane domain. Related strategies have been employed to tether other cytokines to the membranes of cells. Such membrane tethering limits the diffusion of the cytokine and restricts its action to the immediate vicinity of the cells expressing the membrane-bound cytokine. In vivo, this approach could be exploited to ensure ortho-IL-2 IRa-expressing cells only encounter the ortho-IL-21 when they are proximal to a specific type of cell and/or location in the body. In vitro, the approach may facilitate certain kinds of selective differentiation protocols (e.g., the differentiation of NK cells from stem cells in the presence of K562 [or other] feeder cells expressing membrane-bound IL-21 and CD137L).
- selective differentiation protocols e.g., the differentiation of
- Engineered cells may be provided in pharmaceutical compositions suitable for therapeutic use, e.g., for human treatment.
- Therapeutic formulations comprising such cells can be frozen or prepared for administration with physiologically acceptable carriers, excipients, or stabilizers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of aqueous solutions.
- the cells may be formulated, dosed, and administered in a fashion consistent with good medical practice.
- a cell such as a T cell engineered to express one of the ortho-IL-2 IRa molecules described herein may be used to treat a broad range of conditions.
- Engineered properties in this therapy may allow for beneficial T cell differentiation, resistance to exhaustion, capacity for longterm persistence, anamnestic responses, and in-built safety features allowing for responses to be halted when they become pathogenic.
- Methods are provided for enhancing cellular responses by engineering cells from a recipient or donor by introduction of an ortho-IL-2 IRa of the invention and stimulating the ortho- IL-2 IRa by contacting the engineered cell with ortho-IL-21.
- the subject methods may include a step of obtaining the targeted cells, e.g., T cells, hematopoietic stem cells, etc., which may be isolated from a biological sample or may be derived in vitro from a source of progenitor cells.
- the cells may be transduced or transfected with an expression vector comprising a sequence encoding the ortho-IL-2 IRa, which step may be performed in any suitable culture medium.
- the engineered T cells may be contacted with the ortho-IL-21 in vivo, i.e., where the engineered T cells are transferred to a recipient, and an effective dose of the ortho- IL-21 is injected into the recipient and allowed to contact the engineered T cells in their native environment, e.g., in lymph nodes, etc.
- the contacting is performed in vitro.
- the contacting may be accomplished using soluble ortho-IL-21 comprised, or not, of a fusion to another protein moiety such as an immunoglobulin Fc domain.
- the contacting could be accomplished by encounter with other cells expressing secreted or membrane-tethered ortho-IL-21.
- Another aspect provides a method for treating a subject in need thereof, including introducing an engineered cell expressing an ortho-IL-2 IRa to the subject and activating the cell by contacting it with an effective amount of an ortho-IL-21.
- the cell is a T cell, while in further aspects the cell is a CAR-T cell.
- the cell is a T cell expressing a native or modified TCR.
- the cell is an NK cell.
- the cell is a macrophage or other myeloid cell or a leukocyte.
- the ortho-IL-21 is delivered as a fusion protein with a heterologous polypeptide.
- Suitable heterologous polypeptides are known in the art and include serum albumin, Fc fragments of IgG, single-chain Fc antibody fragments, ABD035, and the like. Fc fragments may be modified, for example, with electrostatic steering or other mutations, to prevent, or at least significantly limit, the formation of homodimers.
- Another aspect provides a method for treating a subject in need thereof, including introducing an engineered cell expressing an ortho-IL-2 IRa to the subject and introducing a second engineered cell expressing an ortho-IL-21.
- the cell is a T cell, while in further aspects the cell is a CAR-T cell.
- a “subject,” can be any mammal and may also be referred to as a “patient.”
- mammalian subjects include research animals (e.g., a mouse or rat), domesticated farm animals (e.g., cow, horse, pig), pets (e.g., dog, cat), and humans.
- the subject is a human.
- the subject being treated has been diagnosed as having cancer.
- cancer and “malignancy” are used as synonymous terms and refer to any of a number of diseases that are characterized by uncontrolled, abnormal proliferation of cells, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other parts of the body (i.e., metastasize), as well as any of a number of characteristic structural and molecular features.
- a “cancer cell” refers to a cell undergoing early, intermediate, or advanced stages of multi-step neoplastic progression. The features of early, intermediate, and advanced stages of neoplastic progression have been described using microscopy.
- Cancer cells at each of the three stages of neoplastic progression generally have abnormal karyotypes, including translocations, inversion, deletions, isochromosomes, monosomies, and extra chromosomes.
- Cancer cells include “hyperplastic cells,” that is, cells in the early stages of malignant progression, “dysplastic cells,” that is, cells in the intermediate stages of neoplastic progression, and “neoplastic cells,” that is, cells in the advanced stages of neoplastic progression.
- Examples of cancers are sarcoma, breast, lung, brain, bone, liver, kidney, colon, and prostate cancer.
- the engineered cells are used to treat cancer selected from the group consisting of colon cancer, brain cancer, breast cancer, fibrosarcoma, and squamous carcinoma.
- the cancer is selected from the group consisting of melanoma, breast cancer, colon cancer, lung cancer, and ovarian cancer.
- the cancer being treated is metastatic cancer.
- the method of treatment may further include the step of ablating the cancer.
- Ablating the cancer may be accomplished using a method selected from the group consisting of cryoablation, thermal ablation, radiotherapy, chemotherapy, radiofrequency ablation, electroporation, alcohol ablation, high intensity focused ultrasound, photodynamic therapy, administration of monoclonal antibodies, and administration of immunotoxins.
- the subject being treated has been diagnosed as having an infection.
- infection refers to infection of one or more cells of a subject by an infectious agent.
- Infectious agents include, but are not limited to, bacteria, viruses, protozoans, and fungi. Intracellular pathogens are of particular interest. Infectious diseases are disorders caused by infectious agents. Some infectious agents cause no recognizable symptoms or disease under certain conditions but have the potential to cause symptoms or disease under changed conditions.
- the subject methods may be used in the treatment of chronic pathogen infections, including but not limited to viral infections, e.g., retrovirus, lentivirus, hepadnavirus, herpes viruses, pox viruses, and human papilloma viruses; intracellular bacterial infections, e.g., Mycobacterium, Chlamydia, Ehrlichia, Rickettsia, Brucella, Legionella, Francisella, Listeria, Coxiella, Neisseria, Salmonella, Yersinia sp, and Helicobacter pylori; and intracellular protozoan pathogens, e.g., Plasmodium sp, Trypanosoma sp, Giardia sp, Toxoplasma sp, and Leishmania sp.
- viral infections e.g., retrovirus, lentivirus, hepadnavirus, herpes viruses, pox viruses, and human papilloma viruses
- intracellular bacterial infections
- the subject being treated has been diagnosed as having an autoimmune disease.
- Autoimmune diseases are characterized by T and B lymphocytes that aberrantly target self-proteins, -polypeptides, -peptides, or other self-molecules, causing injury and/or malfunction of an organ, tissue, or cell-type within the body.
- Autoimmune diseases include diseases such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, autoimmune hepatitis, insulin dependent diabetes mellitus, and degenerative diseases such as osteoarthritis, Alzheimer’s disease, and macular degeneration.
- one or more cells of the subject may be contacted with ortho-IL-21.
- the ortho-IL-21 may be added to the engineered cells in a dose and for a period of time sufficient to activate signaling from the ortho- IL-2 IRa, which may utilize the native cellular machinery, e.g., accessory proteins, co-receptors, and the like. Any suitable culture medium may be used.
- the cells thus activated may be used for any desired purpose, including experimental purposes relating to determination of antigen specificity, cytokine profiling, and the like, and for delivery in vivo.
- an effective dose of engineered cells expressing ortho-IL-2 IRa are infused to the recipient, in combination with or prior to administration of the ortho-IL-21.
- Dosage and frequency may vary depending on the agent, mode of administration, and the like.
- the dosage may also be varied for localized administration, e.g., intranasal, inhalation, and the like, or for systemic administration, e.g., i.m., i.p., i.v., and the like.
- at least about 10 4 engineered cells/kg are administered, at least about 10 5 engineered cells/kg, at least about 10 6 engineered cells/kg, at least about 10 7 engineered cells/kg, or more.
- Example 1 Binding Assay for Identification of Candidate Ortho-IL-21Rq Molecules with Impaired Binding to Native IL-2L
- a direct interaction assay quantifying the capacity of IL-2 IRa to bind native or mutant forms of IL-21 provides an alternative to a cell-based assay for the identification of variants of IL-21Ra with compromised IL-21 binding activity (i.e., candidate orthogonal variants).
- the feasibility of exploiting such an assay is enhanced by the fact that the native interaction (IL- 21Ra:IL-21) is avid (KD ⁇ 70pM).
- IL- 21Ra:IL-21 is avid (KD ⁇ 70pM).
- IL-21Ra:IL-21 binding assay involves attaching the receptor ectodomain to a surface, bathing the coated surface in a solution of an IL-21 -luciferase fusion protein, followed by quantitation of bound IL-21 based on luminescence when the relevant luciferase substrate is added.
- IL-21 may be immobilized, and an IL-21Ra-luciferase fusion protein may be used in solution.
- a desirable orientation of the immobilized receptor or cytokine can be accomplished through use of an affinity tag such as Twin-Strep-Tag II, which is a high affinity peptide ligand for the Streptactin protein.
- the IL-21Ra ectodomain bearing a carboxy -terminal Twin-Strep-Tag II peptide can be efficiently and selectively immobilized on the surfaces of wells of 96-well plates that have been pre-coated with Streptactin protein. In this manner, the immobilized IL-21Ra should be oriented with its cytokine-binding domain distal from the plate surface.
- IL-21 may be immobilized in a related fashion if it, too, bears an amino- or carboxy -terminal Twin-Strep-Tag II peptide tag.
- the wild-type human IL-21Ra ectodomain (mature form lacking the signal peptide) (VO (SEQ ID NO: 6)) and the 20 candidate ortho-IL-21Ra molecules shown in Table 1 (SEQ ID NOs: 7-25 & 63) (substitutions shown in the headers and by underline within the sequences) were expressed in HEK 293 cells as secreted proteins.
- Clarified supernatant fluids from the transiently transfected cells were tested for the presence of IL-21Ra with an Enzyme-Linked Immunosorbent Assay (“ELISA”) comprised of Streptactin-coated plates, dilutions of the supernatant fluids, and detection using the combination of a mouse monoclonal antibody specific for the human IL-21Ra, a horseradish peroxidase- conjugated rat antibody specific for mouse IgG, and a chromogenic substrate for the peroxidase.
- ELISA Enzyme-Linked Immunosorbent Assay
- Candidate ortho-IL-2 IRa saturated wells were incubated with a solution of IL-21-TLucl6 for 1 h (or longer in some experiments) at 4 °C (or room temperature in some experiments). The wells were washed before addition of the luciferase substrate (Coelenterazine) solution and luminometry.
- Figure 3 shows the results of a representative assay in which a single (sub -saturating) concentration of IL-21-TLucl6 was tested for binding to the panel of 20 candidate ortho-IL-21Ra molecules (SEQ ID NOs: 7-25 & 63) (all of which had been bound to the Streptactin-coated surfaces of the wells at saturating concentrations). Eight of the candidate ortho-IL-2 IRa molecules showed diminished capacity to bind IL-21-TLucl6. Repeat experiments (involving titrations of the IL-21-TLucl6) confirmed the results.
- the eight candidate IL-2 IRa molecules (the “Receptor Variants” or “RV”s) that showed impaired binding to IL-21-TLucl6 (i.e., RV2, RV6, RV7, RV10, RV13, RV15, RV18, and RV19) may be tested for their capacity to bind candidate ortho-IL-21 with the eventual goal of isolating forms of the cytokine(s) that bind productively (with respect to signaling and immunologic activity) to candidate ortho-IL-2 IRa but not to native IL-2 IRa.
- RV2 Receptor Variants
- the eight candidates from Example 1 and Figure 3 were expressed in a lymphoid cell line, namely Ba/F3 cells (a mouse pre-B cell lymphoma line).
- Ba/F3 cells are dependent on the cytokine IL-3 for growth but will also proliferate robustly in response to IL-21 if first rendered positive for expression of IL-21Ra.
- Ba/F3 cells were electroporated with Leapin Transposase® mRNA and transposons encoding wild-type or candidate ortho-IL-21Ra (RV2: D72E/Y129F/D73E; RV6: M70I/D73E/Q33H; RV7: D72E/L94V/Y191F; RV10: D72E/E38D/M130L; RV13:
- the transposons also carried a STAT- 3 -regulated gene encoding the secreted Cypridina noctiluca luciferase, a constitutively expressed cytosolic click beetle luciferase, and a constitutively expressed gene encoding puromycin N-acetyl transferase.
- the RV13 -expression construct was prepared by oligonucleotidedependent DNA synthesis by ATUM (www.atum.bio).
- This plasmid is over 13Kb in size and comprises four independent genes within a piece of DNA that is flanked first by genomic insulator sequences (from the human D4Z4 locus on one side and from the chicken locus encoding P-globin on the other), then by transposon inverted terminal repeat sequences.
- the insulator sequences are intended to protect the genes within the transposon from position effects (i.e., effects dependent on the site of transposon integration in the genome) that might reduce or variegate expression.
- the inverted terminal repeat sequences are recognized by ATUM’s proprietary Leapin® transposase enzymes that mediate integration of the transposon into genomic DNA.
- ATUM is proprietary Leapin® transposase enzymes that mediate integration of the transposon into genomic DNA.
- the four genes present inside the transposon are described in Table 3 (in the order they occur within the transposon).
- the genes and the transposon that contains them were designed according to standard molecular biology principles compatible with the construct assembly methodology used routinely by ATUM. Variants of this vector encoding wild-type or other candidate ortho-IL-2 IRa molecules were generated by making appropriate changes in the fourth gene listed in Table 3.
- the transposon vector encoding wild-type IL-2 IRa or any of the candidate ortho-IL-2 IRa molecules was co-transfected into Ba/F3 cells together with in vitro-transcribed mRNA encoding the relevant Leapin® transposase using either the MaxCyte ATx or ThermoFisher Neon instruments according to the manufacturer’s instructions. Puromycin selection (Ipg/ml or higher) was imposed at 48 hours after transfection and continued for at least a week after all the cells in an untransfected control culture had died. Flow cytometry was used to confirm that the puromycin- selected cells showed uniform expression of IL-21Ra.
- the Ba/F3 cells were first incubated for 20-24 hours in RPMI- 1640 medium lacking serum and exogenous cytokines. After washing, they were stimulated with IL-21 (in the presence of 0.4% [vol/vol] serum) for a further 20-24 hours with varying concentrations of IL-21 (or candidate ortho-IL-21) in round-bottom 96-well plates (-100,000 cells per well) before assaying secreted luciferase (from the STAT3-cLuc gene in the transposon), intracellular luciferase (from the constitutively expressed EEF2-eLuc gene in the transposon), or ATP accumulation.
- the secreted luciferase assay was used to inform on STAT3 -dependent signaling in the cells, as occurs when IL-21 engages its receptor.
- the other two assays (monitoring cytoplasmic eLuc or ATP accumulation) were used to inform on cell number (i.e., proliferation).
- Cypridina luciferase depended on the IL-21Ra cytoplasmic domain (because a tailless form of IL-21Ra [Acyt] did not induce proliferation in response to IL-21; Figure 5A); the secretion was also significantly impaired when the tyrosine residues in the cytoplasmic domain were replaced with phenylalanine residues (not shown).
- Cypridina noctiluca luciferase activity was readily detected by adding the relevant luciferase substrate (Vargulin) to samples of supernatant fluids from the cells and measuring light emission using a luminometer.
- Figures 5A and 5B show luciferase activity detected as light emission (relative light units or RLU) following admixture of 20 pL of the supernatant fluid from each of the wells with 50 pL of VLAR-2 reagent buffer (Targeting Systems) containing Vargulin at the manufacturer’s recommended concentration.
- Example 3 Screening of Candidate Ortho-IL-21 Molecules: Testing for STAT3 -Dependent Signaling Responses in Cells Expressing Candidate Ortho-IL-2 IR Molecules or Wild-Type IL- 21Rq,
- Wild-type or candidate ortho-IL-21 molecules were produced from transiently transfected HEK-293 cells according to procedures that are routinely used at ATUM (www.atum.bio).
- Expression vectors for this purpose carried the IL-21 open reading frame downstream of an optimized cytomegalovirus Immediate Early Gene 1 promoter.
- a signal peptide from the human IL-2 gene was used in place of the native one.
- Epitope tags for detection, quantitation, immobilization, or purification were fused to the amino- or carboxy -termini of the IL-21 coding sequence.
- the element fused to the amino terminus was a Twin-Strep-Tag followed by three copies of a Glycine-Glycine-Glycine-Glycine-Serine linker moiety, while the element fused to the carboxy terminus comprised two copies of the same Glycine-Glycine- Glycine-Glycine-Serine linker followed by an N-Myc epitope tag (recognized by the 9E10 monoclonal antibody).
- a second series of vectors featured no tags at the carboxy terminus but had the following element at the amino-terminus of IL-21 : Twin-Strep-Tag followed immediately by the N-Myc epitope tag then three copies of the Glycine-Glycine-Glycine-Glycine-Serine linker moiety.
- Selected candidate ortho-IL-21 molecules are shown in Table 4 (SEQ ID NOs: 39-62) (substitutions shown in the headers and by underline within the sequences).
- Ba/F3 cells expressing wild-type IL-21Ra or the eight candidate ortho-IL-2 IRa molecules from Example 1 and Figure 3 were stimulated with candidate ortho-IL-21 molecules CV1-CV22 (SEQ ID NOs: 39-59).
- the signaling responses of the Ba/F3 cells to the ortho-IL-21 molecules were monitored as above using the STAT3 -luciferase assay.
- the Ba/F3 cells differed in the form of ortho-IL-21Ra molecule they expressed (RV2: D72E/Y129F/D73E; RV6: M70I/D73E/Q33H; RV7: D72E/L94V/Y191F; RV10: D72E/E38D/M130L; RV13:
- Figures 6A-6T show activity of this luciferase detected as light emission (relative light units) following admixture of 20 pL of the supernatant fluid from each of the wells with 50 pL of VLAR-2 reagent buffer (Targeting Systems) containing the Cypridina noctiluca luciferase substrate (Vargulin) at the manufacturer’s recommended concentration.
- Selected candidate ortho-IL-21 molecules from the collection represented in Figure 6A- T and Table 4 were retested with Ba/F3 cells expressing wild-type IL-21Ra or candidate ortho-IL-2 IRa molecules RV6, RV10, RV13, or RV15 (i.e., M70I/D73E/Q33H, D72E/E38D/M130L, M70G/Y129F, or F69L/M70L/D73I respectively).
- the results of this experiment are shown in Figures 7A-D.
- Figures 8A-8C derived from the analysis of 96 cytokines, one of which comprised the wildtype form of IL-21, another comprised a negative control variant (CV22, which bears two disabling substitutions [R5Q/R76A]; SEQ ID NO:59), and 94 Infolog variants, each of which was a candidate ortho-IL-21 molecule.
- Figure 8A shows the STAT3 responses elicited in cells expressing wild-type IL-2 IRa exposed to the cytokine collection
- Figures 8B and 8C show responses made by cells expressing the candidate ortho-IL-2 IRa molecules RV13 and RV6, respectively.
- RV13 is comprised of amino acid substitutions M70G and Y129F (SEQ ID NO: 19), whereas RV6 is comprised of amino acid substitutions M70I, D73E, and Q33H (SEQ ID NO: 12).
- the highlighted curves in the three figures show responses made by the three kinds of cells to five selected cytokines, namely, wild-type IL-21, CV22, CV204 (SEQ ID NO: 60, comprised of H6L/M10L/K73V/P78L/P104A), CV374 (SEQ ID NO: 61, comprised of H6L/R9K/M10L/K73V/P78L/G84E) and CV388 (SEQ ID NO: 62, comprised of H6L/R9K/M10L/K73I/P78L/P104A).
- Candidate ortho-IL-21Ra RV13 (SEQ ID NO: 19) carries two substitutions relative to wild-type IL-21Ra, namely M70G and Y129F, whereas candidate ortho-IL-21Ra RV22 (SEQ ID NO:27) carries just M70G.
- These two variant receptors appear to be equivalently compromised in their capacity to bind native IL-21 ( Figure 4B). They also accounted for a similar pattern of reactivity to the collection of IL-21 molecules used in Figures 9A and 9B.
- RV22 mediated significantly impaired signaling responses to wild-type IL-21 (and the negative control molecule CV22) but conferred good responses to CV204, CV374, and CV388 (Figure 10C).
- both CV374 and CV388 showed impaired responses with cells expressing wild-type IL-2 IRa, while CV204 behaved similarly to wild-type IL-21 ( Figure 10A).
- RV22 may be interchangeable with RV 13. Since RV22 carries one fewer substitution than RV13, RV22 may favored for therapeutic purposes.
- in vitro assays will have only very limited predictive value of the effects of a therapeutic in vivo: many therapeutic targets are expressed in multiple cell types (often having opposing effects on the response in vivo), or the therapeutic effect of a given target is dependent on other auxiliary cells. In such situations, an in vitro model, which by its very nature is simplistic, is not a particularly good proxy for the much more complicated situation in vivo. This is not the case in the instant application: the target receptor is synthetic and will only be expressed in cells specifically engineered to do so. Given the specificity of the orthogonal cytokine-receptor system, this significantly reduces the complexity, giving an in vitro assay a better predictive value.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Ultra Sonic Daignosis Equipment (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202180073196.5A CN116529257A (en) | 2020-10-26 | 2021-10-25 | Orthogonal IL-21 receptor/cytokine systems |
CA3199086A CA3199086A1 (en) | 2020-10-26 | 2021-10-25 | Orthogonal il-21 receptor/cytokine systems |
IL302365A IL302365A (en) | 2020-10-26 | 2021-10-25 | Orthogonal il-21 receptor/cytokine systems |
JP2023549960A JP2023549293A (en) | 2020-10-26 | 2021-10-25 | Orthogonal IL-21 receptor/cytokine system |
AU2021370625A AU2021370625A1 (en) | 2020-10-26 | 2021-10-25 | Orthogonal il-21 receptor/cytokine systems |
KR1020237017723A KR20230114749A (en) | 2020-10-26 | 2021-10-25 | Orthogonal IL-21 receptor/cytokine system |
EP21887255.4A EP4232072A1 (en) | 2020-10-26 | 2021-10-25 | Orthogonal il-21 receptor/cytokine systems |
US18/304,172 US12012441B2 (en) | 2020-10-26 | 2023-04-20 | Engineered human IL-21 cytokines and methods for using the same |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202063105414P | 2020-10-26 | 2020-10-26 | |
US63/105,414 | 2020-10-26 | ||
US202163212547P | 2021-06-18 | 2021-06-18 | |
US63/212,547 | 2021-06-18 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US18/304,172 Continuation-In-Part US12012441B2 (en) | 2020-10-26 | 2023-04-20 | Engineered human IL-21 cytokines and methods for using the same |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2022093683A1 true WO2022093683A1 (en) | 2022-05-05 |
Family
ID=81383248
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2021/056439 WO2022093683A1 (en) | 2020-10-26 | 2021-10-25 | Orthogonal il-21 receptor/cytokine systems |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP4232072A1 (en) |
JP (1) | JP2023549293A (en) |
KR (1) | KR20230114749A (en) |
AU (1) | AU2021370625A1 (en) |
CA (1) | CA3199086A1 (en) |
IL (1) | IL302365A (en) |
WO (1) | WO2022093683A1 (en) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050124044A1 (en) * | 2003-06-19 | 2005-06-09 | Cunningham Mark R. | Interleukin-21 analogs |
US20190046611A1 (en) * | 2017-08-03 | 2019-02-14 | Amgen Inc. | Interleukin-21 Muteins and Methods of Treatment |
WO2019157130A1 (en) * | 2018-02-09 | 2019-08-15 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Tethered interleukin-15 and interleukin-21 |
-
2021
- 2021-10-25 WO PCT/US2021/056439 patent/WO2022093683A1/en active Application Filing
- 2021-10-25 AU AU2021370625A patent/AU2021370625A1/en active Pending
- 2021-10-25 EP EP21887255.4A patent/EP4232072A1/en active Pending
- 2021-10-25 IL IL302365A patent/IL302365A/en unknown
- 2021-10-25 KR KR1020237017723A patent/KR20230114749A/en unknown
- 2021-10-25 CA CA3199086A patent/CA3199086A1/en active Pending
- 2021-10-25 JP JP2023549960A patent/JP2023549293A/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050124044A1 (en) * | 2003-06-19 | 2005-06-09 | Cunningham Mark R. | Interleukin-21 analogs |
US20190046611A1 (en) * | 2017-08-03 | 2019-02-14 | Amgen Inc. | Interleukin-21 Muteins and Methods of Treatment |
WO2019157130A1 (en) * | 2018-02-09 | 2019-08-15 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Tethered interleukin-15 and interleukin-21 |
Also Published As
Publication number | Publication date |
---|---|
IL302365A (en) | 2023-06-01 |
CA3199086A1 (en) | 2022-05-05 |
AU2021370625A1 (en) | 2023-06-01 |
JP2023549293A (en) | 2023-11-22 |
KR20230114749A (en) | 2023-08-01 |
EP4232072A1 (en) | 2023-08-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP3558339B1 (en) | T-cell modulatory multimeric polypeptides and methods of use thereof | |
JP6995151B2 (en) | synTac polypeptide and its use | |
JP7071288B2 (en) | T cell regulatory multimeric polypeptide and its usage | |
JP6783797B2 (en) | Anti-cancer fusion polypeptide | |
US11648296B2 (en) | IL-2 orthologs and methods of use | |
US8084230B2 (en) | Trimerizing polypeptides | |
EP3402509A1 (en) | Tumor-specific ifna secretion by car t-cells to reprogram the solid tumor microenvironment | |
US10195272B2 (en) | Adoptive t-cell therapy using FcεRI-based chimeric antigen receptors for treating IgE-mediated allergic diseases | |
US8524656B2 (en) | GM-CSF and truncated CCL2 conjugates and methods and uses thereof | |
US20230027899A1 (en) | Cd122 with altered icd stat signaling | |
US12012441B2 (en) | Engineered human IL-21 cytokines and methods for using the same | |
JP7412006B2 (en) | Inducible T cell receptors and their uses | |
WO2022093683A1 (en) | Orthogonal il-21 receptor/cytokine systems | |
WO2023205738A2 (en) | Orthogonal il-21 receptor/cytokine systems | |
CN116529257A (en) | Orthogonal IL-21 receptor/cytokine systems | |
EP3864034A1 (en) | Cell | |
WO2023241653A1 (en) | Interleukin-2 (il-2) mutant and use thereof | |
WO2015197639A1 (en) | Tissue-specific cell depletion with two chimeric proteins | |
KR20230122628A (en) | Compositions and methods for site-directed mutagenesis | |
CN117247444A (en) | Interleukin-2 (IL-2) mutants and uses thereof | |
KR20220057596A (en) | Allogeneic Cell Compositions and Methods of Use |
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: 21887255 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 3199086 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2023549960 Country of ref document: JP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 202180073196.5 Country of ref document: CN |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2021370625 Country of ref document: AU Date of ref document: 20211025 Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2021887255 Country of ref document: EP Effective date: 20230526 |