WO2019222275A2 - Compositions and methods for tcr reprogramming using inducible fusion proteins - Google Patents
Compositions and methods for tcr reprogramming using inducible fusion proteins Download PDFInfo
- Publication number
- WO2019222275A2 WO2019222275A2 PCT/US2019/032298 US2019032298W WO2019222275A2 WO 2019222275 A2 WO2019222275 A2 WO 2019222275A2 US 2019032298 W US2019032298 W US 2019032298W WO 2019222275 A2 WO2019222275 A2 WO 2019222275A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- domain
- nucleic acid
- tcr
- cell
- tfp
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 89
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 37
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 36
- 230000001939 inductive effect Effects 0.000 title abstract description 17
- 239000000203 mixture Substances 0.000 title description 23
- 230000008672 reprogramming Effects 0.000 title description 2
- 108091008874 T cell receptors Proteins 0.000 claims abstract description 368
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 claims abstract description 358
- 239000000427 antigen Substances 0.000 claims abstract description 184
- 108091007433 antigens Proteins 0.000 claims abstract description 184
- 102000036639 antigens Human genes 0.000 claims abstract description 184
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 174
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 92
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 57
- 201000010099 disease Diseases 0.000 claims abstract description 51
- 201000011510 cancer Diseases 0.000 claims abstract description 41
- 210000004881 tumor cell Anatomy 0.000 claims abstract description 13
- 230000027455 binding Effects 0.000 claims description 233
- 150000007523 nucleic acids Chemical class 0.000 claims description 196
- 210000004027 cell Anatomy 0.000 claims description 190
- 102000039446 nucleic acids Human genes 0.000 claims description 183
- 108020004707 nucleic acids Proteins 0.000 claims description 183
- 241000282414 Homo sapiens Species 0.000 claims description 119
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 118
- 108090000623 proteins and genes Proteins 0.000 claims description 117
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 100
- 229920001184 polypeptide Polymers 0.000 claims description 96
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 95
- 239000013598 vector Substances 0.000 claims description 83
- 102000004169 proteins and genes Human genes 0.000 claims description 82
- 235000018102 proteins Nutrition 0.000 claims description 79
- 230000004048 modification Effects 0.000 claims description 64
- 238000012986 modification Methods 0.000 claims description 64
- -1 CD 16 Proteins 0.000 claims description 62
- 230000004068 intracellular signaling Effects 0.000 claims description 61
- 230000000903 blocking effect Effects 0.000 claims description 59
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims description 57
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims description 55
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 claims description 51
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 claims description 51
- 108020004414 DNA Proteins 0.000 claims description 49
- 125000003729 nucleotide group Chemical group 0.000 claims description 47
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 claims description 46
- 230000003834 intracellular effect Effects 0.000 claims description 44
- 108020004999 messenger RNA Proteins 0.000 claims description 42
- 239000012634 fragment Substances 0.000 claims description 41
- 230000011664 signaling Effects 0.000 claims description 40
- 235000001014 amino acid Nutrition 0.000 claims description 39
- 241000124008 Mammalia Species 0.000 claims description 38
- 230000014509 gene expression Effects 0.000 claims description 37
- 239000002773 nucleotide Substances 0.000 claims description 36
- 150000001413 amino acids Chemical class 0.000 claims description 35
- 238000000338 in vitro Methods 0.000 claims description 35
- 230000000139 costimulatory effect Effects 0.000 claims description 33
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 32
- 239000003446 ligand Substances 0.000 claims description 30
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims description 29
- 102000035195 Peptidases Human genes 0.000 claims description 27
- 108091005804 Peptidases Proteins 0.000 claims description 27
- 239000004365 Protease Substances 0.000 claims description 27
- 230000002401 inhibitory effect Effects 0.000 claims description 27
- 239000003795 chemical substances by application Substances 0.000 claims description 25
- 230000004936 stimulating effect Effects 0.000 claims description 23
- 102000003735 Mesothelin Human genes 0.000 claims description 20
- 108090000015 Mesothelin Proteins 0.000 claims description 20
- 235000019419 proteases Nutrition 0.000 claims description 20
- 108091028075 Circular RNA Proteins 0.000 claims description 19
- 230000004913 activation Effects 0.000 claims description 19
- 108010073807 IgG Receptors Proteins 0.000 claims description 18
- 102000009490 IgG Receptors Human genes 0.000 claims description 18
- 101001011906 Homo sapiens Matrix metalloproteinase-14 Proteins 0.000 claims description 17
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 claims description 17
- 102100030216 Matrix metalloproteinase-14 Human genes 0.000 claims description 17
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 claims description 17
- 230000000694 effects Effects 0.000 claims description 16
- 102100038080 B-cell receptor CD22 Human genes 0.000 claims description 15
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 claims description 15
- 102000002274 Matrix Metalloproteinases Human genes 0.000 claims description 14
- 108010000684 Matrix Metalloproteinases Proteins 0.000 claims description 14
- 108010072866 Prostate-Specific Antigen Proteins 0.000 claims description 14
- 102100038358 Prostate-specific antigen Human genes 0.000 claims description 14
- 239000013612 plasmid Substances 0.000 claims description 14
- 101000990902 Homo sapiens Matrix metalloproteinase-9 Proteins 0.000 claims description 13
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 claims description 13
- 108091036407 Polyadenylation Proteins 0.000 claims description 13
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 12
- 102000001398 Granzyme Human genes 0.000 claims description 12
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 claims description 12
- 206010033128 Ovarian cancer Diseases 0.000 claims description 12
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 12
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 claims description 12
- 206010017758 gastric cancer Diseases 0.000 claims description 12
- 201000011549 stomach cancer Diseases 0.000 claims description 12
- 102100025237 T-cell surface antigen CD2 Human genes 0.000 claims description 11
- 102000004127 Cytokines Human genes 0.000 claims description 10
- 108090000695 Cytokines Proteins 0.000 claims description 10
- 241000713666 Lentivirus Species 0.000 claims description 10
- 108091093037 Peptide nucleic acid Proteins 0.000 claims description 10
- 239000003814 drug Substances 0.000 claims description 10
- 102100027207 CD27 antigen Human genes 0.000 claims description 9
- 102000029816 Collagenase Human genes 0.000 claims description 9
- 108060005980 Collagenase Proteins 0.000 claims description 9
- 102100025012 Dipeptidyl peptidase 4 Human genes 0.000 claims description 9
- 108060005986 Granzyme Proteins 0.000 claims description 9
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 claims description 9
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 9
- 206010027406 Mesothelioma Diseases 0.000 claims description 9
- 229960002424 collagenase Drugs 0.000 claims description 9
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 9
- 206010006187 Breast cancer Diseases 0.000 claims description 8
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 claims description 8
- 102000005600 Cathepsins Human genes 0.000 claims description 8
- 108010084457 Cathepsins Proteins 0.000 claims description 8
- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 claims description 8
- 101000913074 Homo sapiens High affinity immunoglobulin gamma Fc receptor I Proteins 0.000 claims description 8
- 230000004044 response Effects 0.000 claims description 8
- 102000006942 B-Cell Maturation Antigen Human genes 0.000 claims description 7
- 108010008014 B-Cell Maturation Antigen Proteins 0.000 claims description 7
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 claims description 7
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 102100026802 72 kDa type IV collagenase Human genes 0.000 claims description 6
- 206010005003 Bladder cancer Diseases 0.000 claims description 6
- 208000026310 Breast neoplasm Diseases 0.000 claims description 6
- 102100025466 Carcinoembryonic antigen-related cell adhesion molecule 3 Human genes 0.000 claims description 6
- 108010076667 Caspases Proteins 0.000 claims description 6
- 102000011727 Caspases Human genes 0.000 claims description 6
- 108090000227 Chymases Proteins 0.000 claims description 6
- 102000005927 Cysteine Proteases Human genes 0.000 claims description 6
- 108010005843 Cysteine Proteases Proteins 0.000 claims description 6
- 108090000194 Dipeptidyl-peptidases and tripeptidyl-peptidases Proteins 0.000 claims description 6
- 102000003779 Dipeptidyl-peptidases and tripeptidyl-peptidases Human genes 0.000 claims description 6
- 102000005548 Hexokinase Human genes 0.000 claims description 6
- 108700040460 Hexokinases Proteins 0.000 claims description 6
- 101000914337 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 3 Proteins 0.000 claims description 6
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 claims description 6
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 claims description 6
- 108010073816 IgE Receptors Proteins 0.000 claims description 6
- 102000009438 IgE Receptors Human genes 0.000 claims description 6
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 claims description 6
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 6
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 6
- 102100040678 Programmed cell death protein 1 Human genes 0.000 claims description 6
- 101710089372 Programmed cell death protein 1 Proteins 0.000 claims description 6
- 206010060862 Prostate cancer Diseases 0.000 claims description 6
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 6
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 claims description 6
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 6
- 230000000735 allogeneic effect Effects 0.000 claims description 6
- 201000007270 liver cancer Diseases 0.000 claims description 6
- 208000014018 liver neoplasm Diseases 0.000 claims description 6
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 6
- 201000002528 pancreatic cancer Diseases 0.000 claims description 6
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 6
- 230000002062 proliferating effect Effects 0.000 claims description 6
- 235000019833 protease Nutrition 0.000 claims description 6
- 230000002463 transducing effect Effects 0.000 claims description 6
- 241001430294 unidentified retrovirus Species 0.000 claims description 6
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 6
- 230000003612 virological effect Effects 0.000 claims description 6
- 108010065816 zeta chain antigen T cell receptor Proteins 0.000 claims description 6
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 5
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-dimethylaminopyridine Substances CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 5
- 206010052747 Adenocarcinoma pancreas Diseases 0.000 claims description 5
- 102100037904 CD9 antigen Human genes 0.000 claims description 5
- 108090000712 Cathepsin B Proteins 0.000 claims description 5
- 102000004225 Cathepsin B Human genes 0.000 claims description 5
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 5
- 102000003858 Chymases Human genes 0.000 claims description 5
- 206010009944 Colon cancer Diseases 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 239000005977 Ethylene Substances 0.000 claims description 5
- 101000777628 Homo sapiens Leukocyte antigen CD37 Proteins 0.000 claims description 5
- 101000990915 Homo sapiens Stromelysin-1 Proteins 0.000 claims description 5
- 101000577877 Homo sapiens Stromelysin-3 Proteins 0.000 claims description 5
- 101000934346 Homo sapiens T-cell surface antigen CD2 Proteins 0.000 claims description 5
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 claims description 5
- 101000809875 Homo sapiens TYRO protein tyrosine kinase-binding protein Proteins 0.000 claims description 5
- 208000008839 Kidney Neoplasms Diseases 0.000 claims description 5
- 102100031586 Leukocyte antigen CD37 Human genes 0.000 claims description 5
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 5
- 102000000380 Matrix Metalloproteinase 1 Human genes 0.000 claims description 5
- 206010038389 Renal cancer Diseases 0.000 claims description 5
- 208000006265 Renal cell carcinoma Diseases 0.000 claims description 5
- 102100030416 Stromelysin-1 Human genes 0.000 claims description 5
- 102100028847 Stromelysin-3 Human genes 0.000 claims description 5
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 claims description 5
- 102100038717 TYRO protein tyrosine kinase-binding protein Human genes 0.000 claims description 5
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 claims description 5
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 claims description 5
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 5
- 201000010881 cervical cancer Diseases 0.000 claims description 5
- 229940088598 enzyme Drugs 0.000 claims description 5
- 201000010982 kidney cancer Diseases 0.000 claims description 5
- 201000005202 lung cancer Diseases 0.000 claims description 5
- 208000020816 lung neoplasm Diseases 0.000 claims description 5
- 230000036210 malignancy Effects 0.000 claims description 5
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical class CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 claims description 5
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 claims description 5
- 201000002094 pancreatic adenocarcinoma Diseases 0.000 claims description 5
- 208000008732 thymoma Diseases 0.000 claims description 5
- 108010046304 B-Cell Activation Factor Receptor Proteins 0.000 claims description 4
- 102000007536 B-Cell Activation Factor Receptor Human genes 0.000 claims description 4
- 208000003174 Brain Neoplasms Diseases 0.000 claims description 4
- 102100027995 Collagenase 3 Human genes 0.000 claims description 4
- 101000627872 Homo sapiens 72 kDa type IV collagenase Proteins 0.000 claims description 4
- 101000577887 Homo sapiens Collagenase 3 Proteins 0.000 claims description 4
- 101000990908 Homo sapiens Neutrophil collagenase Proteins 0.000 claims description 4
- 102100030411 Neutrophil collagenase Human genes 0.000 claims description 4
- 206010039491 Sarcoma Diseases 0.000 claims description 4
- 208000006990 cholangiocarcinoma Diseases 0.000 claims description 4
- 208000029742 colonic neoplasm Diseases 0.000 claims description 4
- 108091007504 ADAM10 Proteins 0.000 claims description 3
- 108091007507 ADAM12 Proteins 0.000 claims description 3
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 claims description 3
- 208000036832 Adenocarcinoma of ovary Diseases 0.000 claims description 3
- 208000036764 Adenocarcinoma of the esophagus Diseases 0.000 claims description 3
- 102000030431 Asparaginyl endopeptidase Human genes 0.000 claims description 3
- 108010004032 Bromelains Proteins 0.000 claims description 3
- 108010032088 Calpain Proteins 0.000 claims description 3
- 102000007590 Calpain Human genes 0.000 claims description 3
- 108010006303 Carboxypeptidases Proteins 0.000 claims description 3
- 102000005367 Carboxypeptidases Human genes 0.000 claims description 3
- 108090000397 Caspase 3 Proteins 0.000 claims description 3
- 102000003902 Cathepsin C Human genes 0.000 claims description 3
- 108090000267 Cathepsin C Proteins 0.000 claims description 3
- 102000003908 Cathepsin D Human genes 0.000 claims description 3
- 108090000258 Cathepsin D Proteins 0.000 claims description 3
- 102000004178 Cathepsin E Human genes 0.000 claims description 3
- 108090000611 Cathepsin E Proteins 0.000 claims description 3
- 108090000625 Cathepsin K Proteins 0.000 claims description 3
- 102000004171 Cathepsin K Human genes 0.000 claims description 3
- 108090000746 Chymosin Proteins 0.000 claims description 3
- 206010052360 Colorectal adenocarcinoma Diseases 0.000 claims description 3
- 102100039673 Disintegrin and metalloproteinase domain-containing protein 10 Human genes 0.000 claims description 3
- 102100031112 Disintegrin and metalloproteinase domain-containing protein 12 Human genes 0.000 claims description 3
- 108700033317 EC 3.4.23.12 Proteins 0.000 claims description 3
- 206010014733 Endometrial cancer Diseases 0.000 claims description 3
- 206010014759 Endometrial neoplasm Diseases 0.000 claims description 3
- 102100029727 Enteropeptidase Human genes 0.000 claims description 3
- 108010013369 Enteropeptidase Proteins 0.000 claims description 3
- 208000000461 Esophageal Neoplasms Diseases 0.000 claims description 3
- 108010074860 Factor Xa Proteins 0.000 claims description 3
- 101000930822 Giardia intestinalis Dipeptidyl-peptidase 4 Proteins 0.000 claims description 3
- 108050003624 Granzyme M Proteins 0.000 claims description 3
- 101000908391 Homo sapiens Dipeptidyl peptidase 4 Proteins 0.000 claims description 3
- 101000878602 Homo sapiens Immunoglobulin alpha Fc receptor Proteins 0.000 claims description 3
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 claims description 3
- 101000917826 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-a Proteins 0.000 claims description 3
- 101000917824 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-b Proteins 0.000 claims description 3
- 101000577881 Homo sapiens Macrophage metalloelastase Proteins 0.000 claims description 3
- 101000684208 Homo sapiens Prolyl endopeptidase FAP Proteins 0.000 claims description 3
- 101000577874 Homo sapiens Stromelysin-2 Proteins 0.000 claims description 3
- 102100038005 Immunoglobulin alpha Fc receptor Human genes 0.000 claims description 3
- 102000001399 Kallikrein Human genes 0.000 claims description 3
- 108060005987 Kallikrein Proteins 0.000 claims description 3
- 102100027612 Kallikrein-11 Human genes 0.000 claims description 3
- 101710180643 Leishmanolysin Proteins 0.000 claims description 3
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 claims description 3
- 102100029204 Low affinity immunoglobulin gamma Fc region receptor II-a Human genes 0.000 claims description 3
- 102100027998 Macrophage metalloelastase Human genes 0.000 claims description 3
- 108010091175 Matriptase Proteins 0.000 claims description 3
- 108010016113 Matrix Metalloproteinase 1 Proteins 0.000 claims description 3
- 102000000422 Matrix Metalloproteinase 3 Human genes 0.000 claims description 3
- NPPQSCRMBWNHMW-UHFFFAOYSA-N Meprobamate Chemical compound NC(=O)OCC(C)(CCC)COC(N)=O NPPQSCRMBWNHMW-UHFFFAOYSA-N 0.000 claims description 3
- 102000003843 Metalloendopeptidases Human genes 0.000 claims description 3
- 108090000131 Metalloendopeptidases Proteins 0.000 claims description 3
- 108010045057 Metalloexopeptidases Proteins 0.000 claims description 3
- 102000005612 Metalloexopeptidases Human genes 0.000 claims description 3
- 206010030137 Oesophageal adenocarcinoma Diseases 0.000 claims description 3
- 206010030155 Oesophageal carcinoma Diseases 0.000 claims description 3
- 206010061328 Ovarian epithelial cancer Diseases 0.000 claims description 3
- 108090000526 Papain Proteins 0.000 claims description 3
- 108090000284 Pepsin A Proteins 0.000 claims description 3
- 102000057297 Pepsin A Human genes 0.000 claims description 3
- 102000001938 Plasminogen Activators Human genes 0.000 claims description 3
- 108010001014 Plasminogen Activators Proteins 0.000 claims description 3
- 102100038946 Proprotein convertase subtilisin/kexin type 6 Human genes 0.000 claims description 3
- 108090000783 Renin Proteins 0.000 claims description 3
- 102100028255 Renin Human genes 0.000 claims description 3
- 102100028848 Stromelysin-2 Human genes 0.000 claims description 3
- 101710135785 Subtilisin-like protease Proteins 0.000 claims description 3
- 102100037942 Suppressor of tumorigenicity 14 protein Human genes 0.000 claims description 3
- 108090000190 Thrombin Proteins 0.000 claims description 3
- 201000009365 Thymic carcinoma Diseases 0.000 claims description 3
- 208000024770 Thyroid neoplasm Diseases 0.000 claims description 3
- 101710152431 Trypsin-like protease Proteins 0.000 claims description 3
- 208000023915 Ureteral Neoplasms Diseases 0.000 claims description 3
- 206010046392 Ureteric cancer Diseases 0.000 claims description 3
- 108090000350 actinidain Proteins 0.000 claims description 3
- 230000005809 anti-tumor immunity Effects 0.000 claims description 3
- 108010055066 asparaginylendopeptidase Proteins 0.000 claims description 3
- 102000012740 beta Adrenergic Receptors Human genes 0.000 claims description 3
- 108010079452 beta Adrenergic Receptors Proteins 0.000 claims description 3
- 235000019835 bromelain Nutrition 0.000 claims description 3
- 230000005754 cellular signaling Effects 0.000 claims description 3
- 229940080701 chymosin Drugs 0.000 claims description 3
- 239000013256 coordination polymer Substances 0.000 claims description 3
- 230000002901 elastaselike Effects 0.000 claims description 3
- 208000018463 endometrial serous adenocarcinoma Diseases 0.000 claims description 3
- 208000028653 esophageal adenocarcinoma Diseases 0.000 claims description 3
- 201000004101 esophageal cancer Diseases 0.000 claims description 3
- 201000008819 extrahepatic bile duct carcinoma Diseases 0.000 claims description 3
- 210000002950 fibroblast Anatomy 0.000 claims description 3
- 201000006585 gastric adenocarcinoma Diseases 0.000 claims description 3
- 201000005249 lung adenocarcinoma Diseases 0.000 claims description 3
- 108091007169 meprins Proteins 0.000 claims description 3
- 108091028606 miR-1 stem-loop Proteins 0.000 claims description 3
- GNOLWGAJQVLBSM-UHFFFAOYSA-N n,n,5,7-tetramethyl-1,2,3,4-tetrahydronaphthalen-1-amine Chemical compound C1=C(C)C=C2C(N(C)C)CCCC2=C1C GNOLWGAJQVLBSM-UHFFFAOYSA-N 0.000 claims description 3
- 208000013371 ovarian adenocarcinoma Diseases 0.000 claims description 3
- 208000016632 ovarian clear cell cancer Diseases 0.000 claims description 3
- 201000003707 ovarian clear cell carcinoma Diseases 0.000 claims description 3
- 201000006588 ovary adenocarcinoma Diseases 0.000 claims description 3
- 229940055729 papain Drugs 0.000 claims description 3
- 235000019834 papain Nutrition 0.000 claims description 3
- 229940111202 pepsin Drugs 0.000 claims description 3
- 108010020708 plasmepsin Proteins 0.000 claims description 3
- 229940012957 plasmin Drugs 0.000 claims description 3
- 229940127126 plasminogen activator Drugs 0.000 claims description 3
- 108091007196 stromelysin Proteins 0.000 claims description 3
- 229960004072 thrombin Drugs 0.000 claims description 3
- 201000002510 thyroid cancer Diseases 0.000 claims description 3
- 201000011294 ureter cancer Diseases 0.000 claims description 3
- 108090000624 Cathepsin L Proteins 0.000 claims description 2
- 102000004172 Cathepsin L Human genes 0.000 claims description 2
- 101000623901 Homo sapiens Mucin-16 Proteins 0.000 claims description 2
- 108010016183 Human immunodeficiency virus 1 p16 protease Proteins 0.000 claims description 2
- 102100023123 Mucin-16 Human genes 0.000 claims description 2
- 102100036922 Tumor necrosis factor ligand superfamily member 13B Human genes 0.000 claims description 2
- 101710181056 Tumor necrosis factor ligand superfamily member 13B Proteins 0.000 claims description 2
- 201000008274 breast adenocarcinoma Diseases 0.000 claims description 2
- 108020005345 3' Untranslated Regions Proteins 0.000 claims 2
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 claims 1
- 102000003952 Caspase 3 Human genes 0.000 claims 1
- 102100024539 Chymase Human genes 0.000 claims 1
- 102100029360 Hematopoietic cell signal transducer Human genes 0.000 claims 1
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 claims 1
- 101000990188 Homo sapiens Hematopoietic cell signal transducer Proteins 0.000 claims 1
- 238000011282 treatment Methods 0.000 abstract description 11
- 238000011467 adoptive cell therapy Methods 0.000 abstract 1
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 100
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 description 100
- 229920002477 rna polymer Polymers 0.000 description 51
- 102000053602 DNA Human genes 0.000 description 47
- 230000000259 anti-tumor effect Effects 0.000 description 44
- 230000000295 complement effect Effects 0.000 description 37
- 229940024606 amino acid Drugs 0.000 description 33
- 238000013518 transcription Methods 0.000 description 24
- 230000035897 transcription Effects 0.000 description 24
- 238000003752 polymerase chain reaction Methods 0.000 description 21
- 230000006870 function Effects 0.000 description 18
- 108060003951 Immunoglobulin Proteins 0.000 description 17
- 125000000539 amino acid group Chemical group 0.000 description 17
- 102000018358 immunoglobulin Human genes 0.000 description 17
- 108091035707 Consensus sequence Proteins 0.000 description 16
- 108091006905 Human Serum Albumin Proteins 0.000 description 16
- 102000008100 Human Serum Albumin Human genes 0.000 description 16
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 16
- 101100519207 Mus musculus Pdcd1 gene Proteins 0.000 description 16
- 108091036066 Three prime untranslated region Proteins 0.000 description 16
- 238000006467 substitution reaction Methods 0.000 description 16
- 102000040430 polynucleotide Human genes 0.000 description 14
- 108091033319 polynucleotide Proteins 0.000 description 14
- 239000002157 polynucleotide Substances 0.000 description 14
- 210000001519 tissue Anatomy 0.000 description 14
- 230000001086 cytosolic effect Effects 0.000 description 13
- 239000000047 product Substances 0.000 description 13
- 238000013519 translation Methods 0.000 description 13
- 230000028993 immune response Effects 0.000 description 11
- 102000009027 Albumins Human genes 0.000 description 10
- 108010088751 Albumins Proteins 0.000 description 10
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 9
- 230000008569 process Effects 0.000 description 9
- 239000000758 substrate Substances 0.000 description 9
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 8
- 101710163270 Nuclease Proteins 0.000 description 8
- 230000008901 benefit Effects 0.000 description 8
- 238000004422 calculation algorithm Methods 0.000 description 8
- 230000007423 decrease Effects 0.000 description 8
- 210000004443 dendritic cell Anatomy 0.000 description 8
- 239000012636 effector Substances 0.000 description 8
- 230000001976 improved effect Effects 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 7
- 108010074708 B7-H1 Antigen Proteins 0.000 description 7
- 241001529936 Murinae Species 0.000 description 7
- 238000003776 cleavage reaction Methods 0.000 description 7
- 150000001875 compounds Chemical class 0.000 description 7
- 230000001404 mediated effect Effects 0.000 description 7
- 230000006337 proteolytic cleavage Effects 0.000 description 7
- 230000007017 scission Effects 0.000 description 7
- 238000001890 transfection Methods 0.000 description 7
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 6
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 6
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 6
- 201000003793 Myelodysplastic syndrome Diseases 0.000 description 6
- 241000283984 Rodentia Species 0.000 description 6
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 6
- 230000009286 beneficial effect Effects 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
- 239000000306 component Substances 0.000 description 6
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 6
- 208000035475 disorder Diseases 0.000 description 6
- 230000003993 interaction Effects 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- 238000012546 transfer Methods 0.000 description 6
- 102100029822 B- and T-lymphocyte attenuator Human genes 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 102100031940 Epithelial cell adhesion molecule Human genes 0.000 description 5
- 101000864344 Homo sapiens B- and T-lymphocyte attenuator Proteins 0.000 description 5
- 108091092195 Intron Proteins 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 102100034256 Mucin-1 Human genes 0.000 description 5
- 108700026244 Open Reading Frames Proteins 0.000 description 5
- 108091034057 RNA (poly(A)) Proteins 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 238000007792 addition Methods 0.000 description 5
- 210000003719 b-lymphocyte Anatomy 0.000 description 5
- 230000004071 biological effect Effects 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 239000012091 fetal bovine serum Substances 0.000 description 5
- 238000010362 genome editing Methods 0.000 description 5
- 238000009169 immunotherapy Methods 0.000 description 5
- 230000008488 polyadenylation Effects 0.000 description 5
- 229920000642 polymer Polymers 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 230000002441 reversible effect Effects 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 230000019491 signal transduction Effects 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 238000011144 upstream manufacturing Methods 0.000 description 5
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 4
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 4
- 102000014914 Carrier Proteins Human genes 0.000 description 4
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 4
- 108091033380 Coding strand Proteins 0.000 description 4
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 4
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 description 4
- 102100040578 G antigen 7 Human genes 0.000 description 4
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 4
- 101000738354 Homo sapiens CD9 antigen Proteins 0.000 description 4
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 description 4
- 101000893968 Homo sapiens G antigen 7 Proteins 0.000 description 4
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 description 4
- 101001133056 Homo sapiens Mucin-1 Proteins 0.000 description 4
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 4
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 4
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 4
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 4
- 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 4
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 4
- 102100030417 Matrilysin Human genes 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 102000015636 Oligopeptides Human genes 0.000 description 4
- 108010038807 Oligopeptides Proteins 0.000 description 4
- 102000007562 Serum Albumin Human genes 0.000 description 4
- 108010071390 Serum Albumin Proteins 0.000 description 4
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 4
- 239000004473 Threonine Substances 0.000 description 4
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 4
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 4
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 4
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 4
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 4
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 210000000612 antigen-presenting cell Anatomy 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 238000006731 degradation reaction Methods 0.000 description 4
- 238000010494 dissociation reaction Methods 0.000 description 4
- 230000005593 dissociations Effects 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 4
- 230000003463 hyperproliferative effect Effects 0.000 description 4
- 210000002865 immune cell Anatomy 0.000 description 4
- 239000012642 immune effector Substances 0.000 description 4
- 210000000987 immune system Anatomy 0.000 description 4
- 229940121354 immunomodulator Drugs 0.000 description 4
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 4
- 229960000310 isoleucine Drugs 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 4
- 230000002265 prevention Effects 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 230000000638 stimulation Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 229940124597 therapeutic agent Drugs 0.000 description 4
- 239000004474 valine Substances 0.000 description 4
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 3
- 102000006306 Antigen Receptors Human genes 0.000 description 3
- 108010083359 Antigen Receptors Proteins 0.000 description 3
- 102100024263 CD160 antigen Human genes 0.000 description 3
- 102100036008 CD48 antigen Human genes 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 3
- 102100036466 Delta-like protein 3 Human genes 0.000 description 3
- 101100162296 Dictyostelium discoideum ahsa gene Proteins 0.000 description 3
- 108060002716 Exonuclease Proteins 0.000 description 3
- 102100037362 Fibronectin Human genes 0.000 description 3
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 description 3
- 108010035452 HLA-A1 Antigen Proteins 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 101000761938 Homo sapiens CD160 antigen Proteins 0.000 description 3
- 101000716130 Homo sapiens CD48 antigen Proteins 0.000 description 3
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 3
- 101000928513 Homo sapiens Delta-like protein 3 Proteins 0.000 description 3
- 101001138062 Homo sapiens Leukocyte-associated immunoglobulin-like receptor 1 Proteins 0.000 description 3
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 3
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 3
- 101001137987 Homo sapiens Lymphocyte activation gene 3 protein Proteins 0.000 description 3
- 101000831007 Homo sapiens T-cell immunoreceptor with Ig and ITIM domains Proteins 0.000 description 3
- 101000934341 Homo sapiens T-cell surface glycoprotein CD5 Proteins 0.000 description 3
- 101000946843 Homo sapiens T-cell surface glycoprotein CD8 alpha chain Proteins 0.000 description 3
- 101000801433 Homo sapiens Trophoblast glycoprotein Proteins 0.000 description 3
- 101000955999 Homo sapiens V-set domain-containing T-cell activation inhibitor 1 Proteins 0.000 description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 3
- 102100020943 Leukocyte-associated immunoglobulin-like receptor 1 Human genes 0.000 description 3
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 3
- 102100020862 Lymphocyte activation gene 3 protein Human genes 0.000 description 3
- 102000000440 Melanoma-associated antigen Human genes 0.000 description 3
- 108050008953 Melanoma-associated antigen Proteins 0.000 description 3
- 102000007474 Multiprotein Complexes Human genes 0.000 description 3
- 108010085220 Multiprotein Complexes Proteins 0.000 description 3
- 102100035486 Nectin-4 Human genes 0.000 description 3
- 101710043865 Nectin-4 Proteins 0.000 description 3
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 3
- 108700030875 Programmed Cell Death 1 Ligand 2 Proteins 0.000 description 3
- 102100024213 Programmed cell death 1 ligand 2 Human genes 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 230000006044 T cell activation Effects 0.000 description 3
- 230000005867 T cell response Effects 0.000 description 3
- 102100024834 T-cell immunoreceptor with Ig and ITIM domains Human genes 0.000 description 3
- 102100025244 T-cell surface glycoprotein CD5 Human genes 0.000 description 3
- 108010000499 Thromboplastin Proteins 0.000 description 3
- 102100030859 Tissue factor Human genes 0.000 description 3
- 102100033579 Trophoblast glycoprotein Human genes 0.000 description 3
- 102000003425 Tyrosinase Human genes 0.000 description 3
- 108060008724 Tyrosinase Proteins 0.000 description 3
- 108091023045 Untranslated Region Proteins 0.000 description 3
- 102100038929 V-set domain-containing T-cell activation inhibitor 1 Human genes 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 208000009956 adenocarcinoma Diseases 0.000 description 3
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 3
- 235000004279 alanine Nutrition 0.000 description 3
- 108091008324 binding proteins Proteins 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000002648 combination therapy Methods 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 229940127276 delta-like ligand 3 Drugs 0.000 description 3
- 102000013165 exonuclease Human genes 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 210000004602 germ cell Anatomy 0.000 description 3
- 229940072221 immunoglobulins Drugs 0.000 description 3
- 230000008676 import Effects 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 230000010354 integration Effects 0.000 description 3
- 102000006495 integrins Human genes 0.000 description 3
- 108010044426 integrins Proteins 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 229920002521 macromolecule Polymers 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 230000003211 malignant effect Effects 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 230000009870 specific binding Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 102000035160 transmembrane proteins Human genes 0.000 description 3
- 108091005703 transmembrane proteins Proteins 0.000 description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- 102100039583 116 kDa U5 small nuclear ribonucleoprotein component Human genes 0.000 description 2
- LKKMLIBUAXYLOY-UHFFFAOYSA-N 3-Amino-1-methyl-5H-pyrido[4,3-b]indole Chemical compound N1C2=CC=CC=C2C2=C1C=C(N)N=C2C LKKMLIBUAXYLOY-UHFFFAOYSA-N 0.000 description 2
- 101710151806 72 kDa type IV collagenase Proteins 0.000 description 2
- 102100021222 ATP-dependent Clp protease proteolytic subunit, mitochondrial Human genes 0.000 description 2
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical class CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 2
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 102100040069 Aldehyde dehydrogenase 1A1 Human genes 0.000 description 2
- 102100032959 Alpha-actinin-4 Human genes 0.000 description 2
- 101710115256 Alpha-actinin-4 Proteins 0.000 description 2
- 102100023635 Alpha-fetoprotein Human genes 0.000 description 2
- 102100024003 Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 1 Human genes 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- 208000010839 B-cell chronic lymphocytic leukemia Diseases 0.000 description 2
- 102100021663 Baculoviral IAP repeat-containing protein 5 Human genes 0.000 description 2
- 102100026596 Bcl-2-like protein 1 Human genes 0.000 description 2
- 108060000903 Beta-catenin Proteins 0.000 description 2
- 102000015735 Beta-catenin Human genes 0.000 description 2
- 108700012439 CA9 Proteins 0.000 description 2
- 102100038078 CD276 antigen Human genes 0.000 description 2
- 101710185679 CD276 antigen Proteins 0.000 description 2
- 101150013553 CD40 gene Proteins 0.000 description 2
- 102100035793 CD83 antigen Human genes 0.000 description 2
- 101150108242 CDC27 gene Proteins 0.000 description 2
- 238000010453 CRISPR/Cas method Methods 0.000 description 2
- 102100025588 Calcitonin gene-related peptide 1 Human genes 0.000 description 2
- 102100025570 Cancer/testis antigen 1 Human genes 0.000 description 2
- 102100024423 Carbonic anhydrase 9 Human genes 0.000 description 2
- 108010051152 Carboxylesterase Proteins 0.000 description 2
- 102000013392 Carboxylesterase Human genes 0.000 description 2
- 102100034357 Casein kinase I isoform alpha Human genes 0.000 description 2
- 102100029855 Caspase-3 Human genes 0.000 description 2
- 102100038916 Caspase-5 Human genes 0.000 description 2
- 102100026548 Caspase-8 Human genes 0.000 description 2
- 108090000538 Caspase-8 Proteins 0.000 description 2
- 102000016289 Cell Adhesion Molecules Human genes 0.000 description 2
- 108010067225 Cell Adhesion Molecules Proteins 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 108010009685 Cholinergic Receptors Proteins 0.000 description 2
- 102100039361 Chondrosarcoma-associated gene 2/3 protein Human genes 0.000 description 2
- 102000011591 Cleavage And Polyadenylation Specificity Factor Human genes 0.000 description 2
- 108010076130 Cleavage And Polyadenylation Specificity Factor Proteins 0.000 description 2
- 108050006400 Cyclin Proteins 0.000 description 2
- 102000016736 Cyclin Human genes 0.000 description 2
- 108010025464 Cyclin-Dependent Kinase 4 Proteins 0.000 description 2
- 108010009392 Cyclin-Dependent Kinase Inhibitor p16 Proteins 0.000 description 2
- 102100038111 Cyclin-dependent kinase 12 Human genes 0.000 description 2
- 102100036252 Cyclin-dependent kinase 4 Human genes 0.000 description 2
- 102100024458 Cyclin-dependent kinase inhibitor 2A Human genes 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- 241000702421 Dependoparvovirus Species 0.000 description 2
- 102100030074 Dickkopf-related protein 1 Human genes 0.000 description 2
- 102100031788 E3 ubiquitin-protein ligase MYLIP Human genes 0.000 description 2
- 101710190174 E3 ubiquitin-protein ligase MYLIP Proteins 0.000 description 2
- 108050002772 E3 ubiquitin-protein ligase Mdm2 Proteins 0.000 description 2
- 102000012199 E3 ubiquitin-protein ligase Mdm2 Human genes 0.000 description 2
- 102100026245 E3 ubiquitin-protein ligase RNF43 Human genes 0.000 description 2
- 102100037238 E3 ubiquitin-protein ligase UBR4 Human genes 0.000 description 2
- 102100031334 Elongation factor 2 Human genes 0.000 description 2
- 108010055191 EphA3 Receptor Proteins 0.000 description 2
- 102100030324 Ephrin type-A receptor 3 Human genes 0.000 description 2
- 102100028073 Fibroblast growth factor 5 Human genes 0.000 description 2
- 102100026545 Fibronectin type III domain-containing protein 3B Human genes 0.000 description 2
- 102100039699 G antigen 4 Human genes 0.000 description 2
- 101710092267 G antigen 5 Proteins 0.000 description 2
- 102100039698 G antigen 5 Human genes 0.000 description 2
- 101710092269 G antigen 6 Proteins 0.000 description 2
- 102100039713 G antigen 6 Human genes 0.000 description 2
- 102100024405 GPI-linked NAD(P)(+)-arginine ADP-ribosyltransferase 1 Human genes 0.000 description 2
- 101710144640 GPI-linked NAD(P)(+)-arginine ADP-ribosyltransferase 1 Proteins 0.000 description 2
- 101710113436 GTPase KRas Proteins 0.000 description 2
- 102100039788 GTPase NRas Human genes 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 102000010956 Glypican Human genes 0.000 description 2
- 108050001154 Glypican Proteins 0.000 description 2
- 108050007237 Glypican-3 Proteins 0.000 description 2
- 102100031493 Growth arrest-specific protein 7 Human genes 0.000 description 2
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 2
- 102100039317 HAUS augmin-like complex subunit 3 Human genes 0.000 description 2
- 102100031546 HLA class II histocompatibility antigen, DO beta chain Human genes 0.000 description 2
- 108090001101 Hepsin Proteins 0.000 description 2
- 102000004989 Hepsin Human genes 0.000 description 2
- 102100038970 Histone-lysine N-methyltransferase EZH2 Human genes 0.000 description 2
- 101000608799 Homo sapiens 116 kDa U5 small nuclear ribonucleoprotein component Proteins 0.000 description 2
- 101000750222 Homo sapiens ATP-dependent Clp protease proteolytic subunit, mitochondrial Proteins 0.000 description 2
- 101000890570 Homo sapiens Aldehyde dehydrogenase 1A1 Proteins 0.000 description 2
- 101000765923 Homo sapiens Bcl-2-like protein 1 Proteins 0.000 description 2
- 101000946856 Homo sapiens CD83 antigen Proteins 0.000 description 2
- 101000741445 Homo sapiens Calcitonin Proteins 0.000 description 2
- 101000932890 Homo sapiens Calcitonin gene-related peptide 1 Proteins 0.000 description 2
- 101000856237 Homo sapiens Cancer/testis antigen 1 Proteins 0.000 description 2
- 101000994700 Homo sapiens Casein kinase I isoform alpha Proteins 0.000 description 2
- 101000741072 Homo sapiens Caspase-5 Proteins 0.000 description 2
- 101000916489 Homo sapiens Chondroitin sulfate proteoglycan 4 Proteins 0.000 description 2
- 101000745414 Homo sapiens Chondrosarcoma-associated gene 2/3 protein Proteins 0.000 description 2
- 101000884345 Homo sapiens Cyclin-dependent kinase 12 Proteins 0.000 description 2
- 101000864646 Homo sapiens Dickkopf-related protein 1 Proteins 0.000 description 2
- 101000692702 Homo sapiens E3 ubiquitin-protein ligase RNF43 Proteins 0.000 description 2
- 101000807547 Homo sapiens E3 ubiquitin-protein ligase UBR4 Proteins 0.000 description 2
- 101001060267 Homo sapiens Fibroblast growth factor 5 Proteins 0.000 description 2
- 101001027128 Homo sapiens Fibronectin Proteins 0.000 description 2
- 101000913642 Homo sapiens Fibronectin type III domain-containing protein 3B Proteins 0.000 description 2
- 101000886678 Homo sapiens G antigen 2D Proteins 0.000 description 2
- 101000886136 Homo sapiens G antigen 4 Proteins 0.000 description 2
- 101000744505 Homo sapiens GTPase NRas Proteins 0.000 description 2
- 101000923044 Homo sapiens Growth arrest-specific protein 7 Proteins 0.000 description 2
- 101001035819 Homo sapiens HAUS augmin-like complex subunit 3 Proteins 0.000 description 2
- 101000866281 Homo sapiens HLA class II histocompatibility antigen, DO beta chain Proteins 0.000 description 2
- 101000882127 Homo sapiens Histone-lysine N-methyltransferase EZH2 Proteins 0.000 description 2
- 101001042782 Homo sapiens Inactive hydroxysteroid dehydrogenase-like protein 1 Proteins 0.000 description 2
- 101000998120 Homo sapiens Interleukin-3 receptor subunit alpha Proteins 0.000 description 2
- 101001013150 Homo sapiens Interstitial collagenase Proteins 0.000 description 2
- 101000614481 Homo sapiens Kidney-associated antigen 1 Proteins 0.000 description 2
- 101001027621 Homo sapiens Kinesin-like protein KIF20A Proteins 0.000 description 2
- 101001065550 Homo sapiens Lymphocyte antigen 6K Proteins 0.000 description 2
- 101000990912 Homo sapiens Matrilysin Proteins 0.000 description 2
- 101001011884 Homo sapiens Matrix metalloproteinase-15 Proteins 0.000 description 2
- 101001011887 Homo sapiens Matrix metalloproteinase-17 Proteins 0.000 description 2
- 101001011896 Homo sapiens Matrix metalloproteinase-19 Proteins 0.000 description 2
- 101001057156 Homo sapiens Melanoma-associated antigen C2 Proteins 0.000 description 2
- 101000628547 Homo sapiens Metalloreductase STEAP1 Proteins 0.000 description 2
- 101000972282 Homo sapiens Mucin-5AC Proteins 0.000 description 2
- 101001109503 Homo sapiens NKG2-C type II integral membrane protein Proteins 0.000 description 2
- 101000588345 Homo sapiens Nuclear transcription factor Y subunit gamma Proteins 0.000 description 2
- 101000601724 Homo sapiens Paired box protein Pax-5 Proteins 0.000 description 2
- 101000619805 Homo sapiens Peroxiredoxin-5, mitochondrial Proteins 0.000 description 2
- 101000829725 Homo sapiens Phospholipid hydroperoxide glutathione peroxidase Proteins 0.000 description 2
- 101000610208 Homo sapiens Poly(A) polymerase gamma Proteins 0.000 description 2
- 101001117317 Homo sapiens Programmed cell death 1 ligand 1 Proteins 0.000 description 2
- 101000877404 Homo sapiens Protein enabled homolog Proteins 0.000 description 2
- 101001067951 Homo sapiens Protein phosphatase 1 regulatory subunit 3B Proteins 0.000 description 2
- 101000842302 Homo sapiens Protein-cysteine N-palmitoyltransferase HHAT Proteins 0.000 description 2
- 101000620554 Homo sapiens Ras-related protein Rab-38 Proteins 0.000 description 2
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 2
- 101000591201 Homo sapiens Receptor-type tyrosine-protein phosphatase kappa Proteins 0.000 description 2
- 101001073409 Homo sapiens Retrotransposon-derived protein PEG10 Proteins 0.000 description 2
- 101000665150 Homo sapiens Small nuclear ribonucleoprotein Sm D1 Proteins 0.000 description 2
- 101000665250 Homo sapiens Small nuclear ribonucleoprotein Sm D2 Proteins 0.000 description 2
- 101001056234 Homo sapiens Sperm mitochondrial-associated cysteine-rich protein Proteins 0.000 description 2
- 101000648075 Homo sapiens Trafficking protein particle complex subunit 1 Proteins 0.000 description 2
- 101000664703 Homo sapiens Transcription factor SOX-10 Proteins 0.000 description 2
- 101000904724 Homo sapiens Transmembrane glycoprotein NMB Proteins 0.000 description 2
- 101000666896 Homo sapiens V-type immunoglobulin domain-containing suppressor of T-cell activation Proteins 0.000 description 2
- 102100021647 Inactive hydroxysteroid dehydrogenase-like protein 1 Human genes 0.000 description 2
- 102100039615 Inactive tyrosine-protein kinase transmembrane receptor ROR1 Human genes 0.000 description 2
- 102000000589 Interleukin-1 Human genes 0.000 description 2
- 108010002352 Interleukin-1 Proteins 0.000 description 2
- 102100033493 Interleukin-3 receptor subunit alpha Human genes 0.000 description 2
- 102100034872 Kallikrein-4 Human genes 0.000 description 2
- 102100040442 Kidney-associated antigen 1 Human genes 0.000 description 2
- 102100037694 Kinesin-like protein KIF20A Human genes 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- 102100031413 L-dopachrome tautomerase Human genes 0.000 description 2
- 101710093778 L-dopachrome tautomerase Proteins 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- 102100024144 Lengsin Human genes 0.000 description 2
- 101710113750 Lengsin Proteins 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 102100032129 Lymphocyte antigen 6K Human genes 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 108010010995 MART-1 Antigen Proteins 0.000 description 2
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- 102100028123 Macrophage colony-stimulating factor 1 Human genes 0.000 description 2
- 102000005727 Mammaglobin A Human genes 0.000 description 2
- 108010031030 Mammaglobin A Proteins 0.000 description 2
- 208000025205 Mantle-Cell Lymphoma Diseases 0.000 description 2
- 108090000855 Matrilysin Proteins 0.000 description 2
- 102100030201 Matrix metalloproteinase-15 Human genes 0.000 description 2
- 102100030219 Matrix metalloproteinase-17 Human genes 0.000 description 2
- 102100030218 Matrix metalloproteinase-19 Human genes 0.000 description 2
- 102100028389 Melanoma antigen recognized by T-cells 1 Human genes 0.000 description 2
- 102100027252 Melanoma-associated antigen C2 Human genes 0.000 description 2
- 241001482085 Meloe Species 0.000 description 2
- 102100026712 Metalloreductase STEAP1 Human genes 0.000 description 2
- 108010092801 Midkine Proteins 0.000 description 2
- 102100030335 Midkine Human genes 0.000 description 2
- 102100022496 Mucin-5AC Human genes 0.000 description 2
- 102000003505 Myosin Human genes 0.000 description 2
- 108060008487 Myosin Chemical class 0.000 description 2
- 102100022913 NAD-dependent protein deacetylase sirtuin-2 Human genes 0.000 description 2
- 102100022683 NKG2-C type II integral membrane protein Human genes 0.000 description 2
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 description 2
- 102100031719 Nuclear transcription factor Y subunit gamma Human genes 0.000 description 2
- 102000036673 PRAME Human genes 0.000 description 2
- 108060006580 PRAME Proteins 0.000 description 2
- 102100037504 Paired box protein Pax-5 Human genes 0.000 description 2
- 108010077519 Peptide Elongation Factor 2 Proteins 0.000 description 2
- 108010067163 Perilipin-2 Proteins 0.000 description 2
- 102000017794 Perilipin-2 Human genes 0.000 description 2
- 102100022078 Peroxiredoxin-5, mitochondrial Human genes 0.000 description 2
- 102100037419 Pituitary tumor-transforming gene 1 protein-interacting protein Human genes 0.000 description 2
- 101710199379 Pituitary tumor-transforming gene 1 protein-interacting protein Proteins 0.000 description 2
- 101710124239 Poly(A) polymerase Proteins 0.000 description 2
- 102100040153 Poly(A) polymerase gamma Human genes 0.000 description 2
- 208000006994 Precancerous Conditions Diseases 0.000 description 2
- 208000007541 Preleukemia Diseases 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- 101710120463 Prostate stem cell antigen Proteins 0.000 description 2
- 102100036735 Prostate stem cell antigen Human genes 0.000 description 2
- 102100035093 Protein enabled homolog Human genes 0.000 description 2
- 102100034504 Protein phosphatase 1 regulatory subunit 3B Human genes 0.000 description 2
- 102100022305 Ras-related protein Rab-38 Human genes 0.000 description 2
- 101001039269 Rattus norvegicus Glycine N-methyltransferase Proteins 0.000 description 2
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 2
- 102100034089 Receptor-type tyrosine-protein phosphatase kappa Human genes 0.000 description 2
- 102100037421 Regulator of G-protein signaling 5 Human genes 0.000 description 2
- 101710140403 Regulator of G-protein signaling 5 Proteins 0.000 description 2
- 102100035844 Retrotransposon-derived protein PEG10 Human genes 0.000 description 2
- 108700019345 SYT-SSX fusion Proteins 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 101710173694 Short transient receptor potential channel 2 Proteins 0.000 description 2
- 108010041216 Sirtuin 2 Proteins 0.000 description 2
- 102100038685 Small nuclear ribonucleoprotein Sm D2 Human genes 0.000 description 2
- 102100026503 Sperm mitochondrial-associated cysteine-rich protein Human genes 0.000 description 2
- 108010002687 Survivin Proteins 0.000 description 2
- 238000010459 TALEN Methods 0.000 description 2
- 108700019889 TEL-AML1 fusion Proteins 0.000 description 2
- 108010017842 Telomerase Proteins 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 102100025256 Trafficking protein particle complex subunit 1 Human genes 0.000 description 2
- 108010073062 Transcription Activator-Like Effectors Proteins 0.000 description 2
- 102100038808 Transcription factor SOX-10 Human genes 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 2
- 102000005924 Triose-Phosphate Isomerase Human genes 0.000 description 2
- 108700015934 Triose-phosphate isomerases Proteins 0.000 description 2
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 2
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- 208000002495 Uterine Neoplasms Diseases 0.000 description 2
- 102100038282 V-type immunoglobulin domain-containing suppressor of T-cell activation Human genes 0.000 description 2
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 description 2
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 description 2
- 102000040856 WT1 Human genes 0.000 description 2
- 108700020467 WT1 Proteins 0.000 description 2
- 101150084041 WT1 gene Proteins 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 102000034337 acetylcholine receptors Human genes 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 150000003838 adenosines Chemical class 0.000 description 2
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- 235000009582 asparagine Nutrition 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 108010056708 bcr-abl Fusion Proteins Proteins 0.000 description 2
- 102000004441 bcr-abl Fusion Proteins Human genes 0.000 description 2
- 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 2
- 239000011230 binding agent Substances 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 238000002659 cell therapy Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 230000001461 cytolytic effect Effects 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- IJJVMEJXYNJXOJ-UHFFFAOYSA-N fluquinconazole Chemical compound C=1C=C(Cl)C=C(Cl)C=1N1C(=O)C2=CC(F)=CC=C2N=C1N1C=NC=N1 IJJVMEJXYNJXOJ-UHFFFAOYSA-N 0.000 description 2
- 201000003444 follicular lymphoma Diseases 0.000 description 2
- 150000002270 gangliosides Chemical class 0.000 description 2
- 238000001476 gene delivery Methods 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 210000002443 helper t lymphocyte Anatomy 0.000 description 2
- 201000005787 hematologic cancer Diseases 0.000 description 2
- 230000002489 hematologic effect Effects 0.000 description 2
- 210000003630 histaminocyte Anatomy 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 108010024383 kallikrein 4 Proteins 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 210000000822 natural killer cell Anatomy 0.000 description 2
- 210000000440 neutrophil Anatomy 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 229960005489 paracetamol Drugs 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 230000002688 persistence Effects 0.000 description 2
- 108040000983 polyphosphate:AMP phosphotransferase activity proteins Proteins 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 235000002020 sage Nutrition 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000003146 transient transfection Methods 0.000 description 2
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 2
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 description 2
- 206010046766 uterine cancer Diseases 0.000 description 2
- 238000011179 visual inspection Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- SSOORFWOBGFTHL-OTEJMHTDSA-N (4S)-5-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[2-[(2S)-2-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S,3S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-5-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-5-amino-1-[[(2S)-5-carbamimidamido-1-[[(2S)-5-carbamimidamido-1-[[(1S)-4-carbamimidamido-1-carboxybutyl]amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-1,5-dioxopentan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1-oxohexan-2-yl]amino]-1-oxohexan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1,5-dioxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]carbamoyl]pyrrolidin-1-yl]-2-oxoethyl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-(1H-imidazol-4-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-4-[[(2S)-2-[[(2S)-2-[[(2S)-2,6-diaminohexanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]-5-oxopentanoic acid Chemical compound CC[C@H](C)[C@H](NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H]1CCCN1C(=O)CNC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@@H](N)CCCCN)C(C)C)C(C)C)C(C)C)C(C)C)C(C)C)C(C)C)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SSOORFWOBGFTHL-OTEJMHTDSA-N 0.000 description 1
- VGONTNSXDCQUGY-RRKCRQDMSA-N 2'-deoxyinosine Chemical group C1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC2=O)=C2N=C1 VGONTNSXDCQUGY-RRKCRQDMSA-N 0.000 description 1
- RNAMYOYQYRYFQY-UHFFFAOYSA-N 2-(4,4-difluoropiperidin-1-yl)-6-methoxy-n-(1-propan-2-ylpiperidin-4-yl)-7-(3-pyrrolidin-1-ylpropoxy)quinazolin-4-amine Chemical compound N1=C(N2CCC(F)(F)CC2)N=C2C=C(OCCCN3CCCC3)C(OC)=CC2=C1NC1CCN(C(C)C)CC1 RNAMYOYQYRYFQY-UHFFFAOYSA-N 0.000 description 1
- OGHAROSJZRTIOK-KQYNXXCUSA-O 7-methylguanosine Chemical compound C1=2N=C(N)NC(=O)C=2[N+](C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OGHAROSJZRTIOK-KQYNXXCUSA-O 0.000 description 1
- 102100031585 ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Human genes 0.000 description 1
- 108020005176 AU Rich Elements Proteins 0.000 description 1
- 208000010444 Acidosis Diseases 0.000 description 1
- 206010000830 Acute leukaemia Diseases 0.000 description 1
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- 102100034540 Adenomatous polyposis coli protein Human genes 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 208000036170 B-Cell Marginal Zone Lymphoma Diseases 0.000 description 1
- 208000032568 B-cell prolymphocytic leukaemia Diseases 0.000 description 1
- 208000025321 B-lymphoblastic leukemia/lymphoma Diseases 0.000 description 1
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 1
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 208000011691 Burkitt lymphomas Diseases 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 102100024217 CAMPATH-1 antigen Human genes 0.000 description 1
- 208000016778 CD4+/CD56+ hematodermic neoplasm Diseases 0.000 description 1
- 102100032912 CD44 antigen Human genes 0.000 description 1
- 108010058905 CD44v6 antigen Proteins 0.000 description 1
- 108010065524 CD52 Antigen Proteins 0.000 description 1
- 210000001239 CD8-positive, alpha-beta cytotoxic T lymphocyte Anatomy 0.000 description 1
- 108091033409 CRISPR Proteins 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 1
- 229940045513 CTLA4 antagonist Drugs 0.000 description 1
- 102000000905 Cadherin Human genes 0.000 description 1
- 108050007957 Cadherin Proteins 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 108090000610 Cathepsin F Proteins 0.000 description 1
- 102000004176 Cathepsin F Human genes 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 108020004638 Circular DNA Proteins 0.000 description 1
- 108091062157 Cis-regulatory element Proteins 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 101150065984 Comp gene Proteins 0.000 description 1
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 1
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 206010058314 Dysplasia Diseases 0.000 description 1
- 101150029707 ERBB2 gene Proteins 0.000 description 1
- 108010037179 Endodeoxyribonucleases Proteins 0.000 description 1
- 102000011750 Endodeoxyribonucleases Human genes 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 241000701867 Enterobacteria phage T7 Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 206010061850 Extranodal marginal zone B-cell lymphoma (MALT type) Diseases 0.000 description 1
- 102000009109 Fc receptors Human genes 0.000 description 1
- 108010087819 Fc receptors Proteins 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 208000032612 Glial tumor Diseases 0.000 description 1
- 201000010915 Glioblastoma multiforme Diseases 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- XKMLYUALXHKNFT-UUOKFMHZSA-N Guanosine-5'-triphosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XKMLYUALXHKNFT-UUOKFMHZSA-N 0.000 description 1
- 108010074032 HLA-A2 Antigen Proteins 0.000 description 1
- 102000025850 HLA-A2 Antigen Human genes 0.000 description 1
- 101000777636 Homo sapiens ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Proteins 0.000 description 1
- 101000924577 Homo sapiens Adenomatous polyposis coli protein Proteins 0.000 description 1
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 1
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 1
- 101001010541 Homo sapiens Electron transfer flavoprotein subunit alpha, mitochondrial Proteins 0.000 description 1
- 101000920667 Homo sapiens Epithelial cell adhesion molecule Proteins 0.000 description 1
- 101001103039 Homo sapiens Inactive tyrosine-protein kinase transmembrane receptor ROR1 Proteins 0.000 description 1
- 101000971538 Homo sapiens Killer cell lectin-like receptor subfamily F member 1 Proteins 0.000 description 1
- 101001013139 Homo sapiens Matrix metalloproteinase-20 Proteins 0.000 description 1
- 101000627858 Homo sapiens Matrix metalloproteinase-24 Proteins 0.000 description 1
- 101000627852 Homo sapiens Matrix metalloproteinase-25 Proteins 0.000 description 1
- 101000627854 Homo sapiens Matrix metalloproteinase-26 Proteins 0.000 description 1
- 101000627860 Homo sapiens Matrix metalloproteinase-27 Proteins 0.000 description 1
- 101001036406 Homo sapiens Melanoma-associated antigen C1 Proteins 0.000 description 1
- 101000576802 Homo sapiens Mesothelin Proteins 0.000 description 1
- 101001051490 Homo sapiens Neural cell adhesion molecule L1 Proteins 0.000 description 1
- 101001103036 Homo sapiens Nuclear receptor ROR-alpha Proteins 0.000 description 1
- 101000904196 Homo sapiens Pancreatic secretory granule membrane major glycoprotein GP2 Proteins 0.000 description 1
- 101000633784 Homo sapiens SLAM family member 7 Proteins 0.000 description 1
- 101000946860 Homo sapiens T-cell surface glycoprotein CD3 epsilon chain Proteins 0.000 description 1
- 101000795169 Homo sapiens Tumor necrosis factor receptor superfamily member 13C Proteins 0.000 description 1
- 101000801234 Homo sapiens Tumor necrosis factor receptor superfamily member 18 Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 108010009817 Immunoglobulin Constant Regions Proteins 0.000 description 1
- 102000009786 Immunoglobulin Constant Regions Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 102000012745 Immunoglobulin Subunits Human genes 0.000 description 1
- 108010079585 Immunoglobulin Subunits Proteins 0.000 description 1
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 1
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 1
- 102000004559 Interleukin-13 Receptors Human genes 0.000 description 1
- 108010017511 Interleukin-13 Receptors Proteins 0.000 description 1
- 102100021458 Killer cell lectin-like receptor subfamily F member 1 Human genes 0.000 description 1
- 208000031671 Large B-Cell Diffuse Lymphoma Diseases 0.000 description 1
- 208000031422 Lymphocytic Chronic B-Cell Leukemia Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 201000003791 MALT lymphoma Diseases 0.000 description 1
- 101000928455 Macaca fascicularis Albumin Proteins 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 102100024129 Matrix metalloproteinase-24 Human genes 0.000 description 1
- 102100024131 Matrix metalloproteinase-25 Human genes 0.000 description 1
- 102100024128 Matrix metalloproteinase-26 Human genes 0.000 description 1
- 102100024132 Matrix metalloproteinase-27 Human genes 0.000 description 1
- 102100039447 Melanoma-associated antigen C1 Human genes 0.000 description 1
- 108010061593 Member 14 Tumor Necrosis Factor Receptors Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000005741 Metalloproteases Human genes 0.000 description 1
- 108010006035 Metalloproteases Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 206010027480 Metastatic malignant melanoma Diseases 0.000 description 1
- 101150073847 Mmp23 gene Proteins 0.000 description 1
- 108010008707 Mucin-1 Proteins 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- 241000714177 Murine leukemia virus Species 0.000 description 1
- 101000930477 Mus musculus Albumin Proteins 0.000 description 1
- 101000866339 Mus musculus Transcription factor E2F6 Proteins 0.000 description 1
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 1
- 108091008877 NK cell receptors Proteins 0.000 description 1
- 208000002454 Nasopharyngeal Carcinoma Diseases 0.000 description 1
- 206010061306 Nasopharyngeal cancer Diseases 0.000 description 1
- 102000010648 Natural Killer Cell Receptors Human genes 0.000 description 1
- 102100024964 Neural cell adhesion molecule L1 Human genes 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102100024019 Pancreatic secretory granule membrane major glycoprotein GP2 Human genes 0.000 description 1
- 208000030852 Parasitic disease Diseases 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 208000007452 Plasmacytoma Diseases 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- 102000015623 Polynucleotide Adenylyltransferase Human genes 0.000 description 1
- 108010024055 Polynucleotide adenylyltransferase Proteins 0.000 description 1
- 208000009052 Precursor T-Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 1
- 208000017414 Precursor T-cell acute lymphoblastic leukemia Diseases 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 208000035416 Prolymphocytic B-Cell Leukemia Diseases 0.000 description 1
- 102000018471 Proto-Oncogene Proteins B-raf Human genes 0.000 description 1
- 108010091528 Proto-Oncogene Proteins B-raf Proteins 0.000 description 1
- 108020005161 RNA Caps Proteins 0.000 description 1
- 108010065868 RNA polymerase SP6 Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 108010006700 Receptor Tyrosine Kinase-like Orphan Receptors Proteins 0.000 description 1
- 101710100969 Receptor tyrosine-protein kinase erbB-3 Proteins 0.000 description 1
- 102100029986 Receptor tyrosine-protein kinase erbB-3 Human genes 0.000 description 1
- 241000712907 Retroviridae Species 0.000 description 1
- 102100029198 SLAM family member 7 Human genes 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 101000744436 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) Trans-acting factor D Proteins 0.000 description 1
- 102000010841 Signaling Lymphocytic Activation Molecule Family Human genes 0.000 description 1
- 108010062314 Signaling Lymphocytic Activation Molecule Family Proteins 0.000 description 1
- 108010074687 Signaling Lymphocytic Activation Molecule Family Member 1 Proteins 0.000 description 1
- 102000008115 Signaling Lymphocytic Activation Molecule Family Member 1 Human genes 0.000 description 1
- 206010041067 Small cell lung cancer Diseases 0.000 description 1
- 208000004346 Smoldering Multiple Myeloma Diseases 0.000 description 1
- 208000029052 T-cell acute lymphoblastic leukemia Diseases 0.000 description 1
- 102100035794 T-cell surface glycoprotein CD3 epsilon chain Human genes 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 108020004566 Transfer RNA Proteins 0.000 description 1
- 101800000385 Transmembrane protein Proteins 0.000 description 1
- 108060005989 Tryptase Proteins 0.000 description 1
- 102000001400 Tryptase Human genes 0.000 description 1
- 102100029690 Tumor necrosis factor receptor superfamily member 13C Human genes 0.000 description 1
- 102100028785 Tumor necrosis factor receptor superfamily member 14 Human genes 0.000 description 1
- 102100033728 Tumor necrosis factor receptor superfamily member 18 Human genes 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 208000033559 Waldenström macroglobulinemia Diseases 0.000 description 1
- 102100025093 Zinc fingers and homeoboxes protein 2 Human genes 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 239000000370 acceptor Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000007950 acidosis Effects 0.000 description 1
- 208000026545 acidosis disease Diseases 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 210000005006 adaptive immune system Anatomy 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 208000002517 adenoid cystic carcinoma Diseases 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000006023 anti-tumor response Effects 0.000 description 1
- 239000000611 antibody drug conjugate Substances 0.000 description 1
- 229940049595 antibody-drug conjugate Drugs 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 108010028263 bacteriophage T3 RNA polymerase Proteins 0.000 description 1
- 210000003651 basophil Anatomy 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 208000035269 cancer or benign tumor Diseases 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 208000024207 chronic leukemia Diseases 0.000 description 1
- 208000032852 chronic lymphocytic leukemia Diseases 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 239000000562 conjugate Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000004940 costimulation Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000011461 current therapy Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 102000003675 cytokine receptors Human genes 0.000 description 1
- 108010057085 cytokine receptors Proteins 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000003436 cytoskeletal effect Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 206010012818 diffuse large B-cell lymphoma Diseases 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000006862 enzymatic digestion Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 1
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 1
- 230000008029 eradication Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 229940014144 folate Drugs 0.000 description 1
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- 210000004475 gamma-delta t lymphocyte Anatomy 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 108010033706 glycylserine Proteins 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical class O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 1
- 229940029575 guanosine Drugs 0.000 description 1
- 201000009277 hairy cell leukemia Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000008629 immune suppression Effects 0.000 description 1
- 230000037451 immune surveillance Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 210000005007 innate immune system Anatomy 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 230000002601 intratumoral effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 210000002664 langerhans' cell Anatomy 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 201000005243 lung squamous cell carcinoma Diseases 0.000 description 1
- 210000004324 lymphatic system Anatomy 0.000 description 1
- 208000003747 lymphoid leukemia Diseases 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 210000003810 lymphokine-activated killer cell Anatomy 0.000 description 1
- 201000007919 lymphoplasmacytic lymphoma Diseases 0.000 description 1
- 230000001589 lymphoproliferative effect Effects 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 208000026037 malignant tumor of neck Diseases 0.000 description 1
- 201000007924 marginal zone B-cell lymphoma Diseases 0.000 description 1
- 208000021937 marginal zone lymphoma Diseases 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 208000021039 metastatic melanoma Diseases 0.000 description 1
- 238000012737 microarray-based gene expression Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000012243 multiplex automated genomic engineering Methods 0.000 description 1
- 210000000066 myeloid cell Anatomy 0.000 description 1
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 201000011216 nasopharynx carcinoma Diseases 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000006213 oxygenation reaction Methods 0.000 description 1
- 201000010279 papillary renal cell carcinoma Diseases 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 208000031223 plasma cell leukemia Diseases 0.000 description 1
- 208000007525 plasmablastic lymphoma Diseases 0.000 description 1
- 210000005134 plasmacytoid dendritic cell Anatomy 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 208000017426 precursor B-cell acute lymphoblastic leukemia Diseases 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 230000008521 reorganization Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 201000007416 salivary gland adenoid cystic carcinoma Diseases 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000001235 sensitizing effect Effects 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- 230000008054 signal transmission Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 208000000587 small cell lung carcinoma Diseases 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 208000010721 smoldering plasma cell myeloma Diseases 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 230000004222 uncontrolled growth Effects 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4613—Natural-killer cells [NK or NK-T]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/463—Cellular immunotherapy characterised by recombinant expression
- A61K39/4631—Chimeric Antigen Receptors [CAR]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/463—Cellular immunotherapy characterised by recombinant expression
- A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
- A61K39/464468—Mesothelin [MSLN]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16041—Use of virus, viral particle or viral elements as a vector
- C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Definitions
- Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems.
- the ability to engineer synthetic systems in T cells that are responding to multiple inputs would benefit adoptive immunotherapy using engineered T cells (Juillerat, et al., 2017; Chakravarti D. & Wong W. W., 2015).
- the tumor microenvironment is characterized by numerous factors including expression of matrix metalloproteinases, cathepsins, proteases, Indolomine 2,3-dioxygenase, low extracellular pH (acidosis) and low oxygenation (hypoxia) (Brown & Wilson, 2004; Vaupel & Mayer, 2007).
- TME tumor microenvironment
- TCR T cell receptor
- TFP T cell receptor fusion protein
- a TCR subunit comprising (i) at least a portion of a TCR extracellular domain, and (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon; (b) an antibody domain comprising a TAA binding domain; and (c) a blocking domain, wherein the TCR subunit and the antibody domain are operatively linked, the antibody domain and the blocking domain are linked by a protease -cleavable linker, and wherein the TFP incorporates into a TCR when expressed in a T cell.
- TCR T cell receptor
- TFP T cell receptor fusion protein
- TCR T cell receptor
- TFP T cell receptor fusion protein
- a TCR subunit comprising (i) at least a portion of a TCR extracellular domain, and (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 gamma; and (b) an antibody domain comprising a TAA binding domain; and (c) a blocking domain, wherein the TCR subunit and the antibody domain are operatively linked, the antibody domain and the blocking domain are linked by a protease-cleavable linker, and wherein the TFP incorporates into a TCR when expressed in a T cell.
- TCR T cell receptor
- TFP T cell receptor fusion protein
- TCR T cell receptor
- TFP T cell receptor fusion protein
- a TCR subunit comprising (i) at least a portion of a TCR extracellular domain, and (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 delta; and (b) an antibody domain comprising an antigen binding domain; and (c) a blocking domain, wherein the TCR subunit and the antibody domain are operatively linked, the antibody domain and the blocking domain are linked by a protease-cleavable linker, and wherein the TFP incorporates into a TCR when expressed in a T cell.
- TCR T cell receptor
- TFP T cell receptor fusion protein
- TCR T cell receptor
- TFP T cell receptor fusion protein
- a TCR subunit comprising (i) at least a portion of a TCR extracellular domain, and (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of TCR alpha; and (b) an antibody domain comprising an antigen binding domain; and (c) a blocking domain, wherein the TCR subunit and the antibody domain are operatively linked, the antibody domain and the blocking domain are linked by a protease-cleavable linker, and wherein the TFP incorporates into a TCR when expressed in a T cell.
- TCR T cell receptor
- TFP T cell receptor fusion protein
- TCR T cell receptor
- TFP T cell receptor fusion protein
- a TCR subunit comprising (i) at least a portion of a TCR extracellular domain, and (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of TCR beta; and (b) an antibody domain comprising an antigen binding domain; and (c) a blocking domain, wherein the TCR subunit and the antibody domain are operatively linked, the antibody domain and the blocking domain are linked by a protease-cleavable linker, and wherein the TFP incorporates into a TCR when expressed in a T cell.
- TCR T cell receptor
- TFP T cell receptor fusion protein
- the antibody domain is a human or humanized antibody.
- the encoded antigen binding domain is connected to the TCR extracellular domain by a linker sequence.
- the encoded antigen binding domain is connected to the blocking domain by a linker sequence that encodes a protease-cleavable linker.
- the linker sequence encodes a matrix metalloproteinase cleavable linker.
- the matrix metalloproteinase cleavage linker comprises a sequence selected from any of sequences in Tables 1-14.
- the linker sequence encodes a cathepsin-cleavable linker. In some embodiments, the linker sequence encodes a Urokinase plasminogen activator (uPA)-cleavable linker. In some embodiments, the encoded antigen binding domain is connected to an antibody that specifically binds a blocking domain by a linker sequence that encodes a protease-cleavable linker.
- the TCR subunit comprises a TCR extracellular domain. In some embodiments, the TCR subunit comprises a TCR transmembrane domain. In some embodiments, the TCR subunit comprises a TCR intracellular domain.
- the TCR subunit comprises (i) a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
- the TCR subunit comprises a TCR intracellular domain comprising a stimulatory domain selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having at least one modification thereto.
- the TCR subunit comprises an intracellular domain comprising a stimulatory domain selected from a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.
- the antibody domain comprises an antibody fragment.
- the antibody domain comprises a scFv or a VHH domain.
- the recombinant nucleic acid encodes (i) a light chain (LC) CDR1, LC CDR2 and LC CDR3 of an anti-TAA light chain binding domain amino acid sequence with 70-100% sequence identity to a light chain (LC) CDR1, LC CDR2 and LC CDR3 of an anti-TAA light chain binding domain provided herein, respectively, and/or (ii) a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 of an anti-TAA heavy chain binding domain amino acid sequence with 70-100% sequence identity to a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 of an anti-TAA heavy chain binding domain provided herein, respectively.
- LC light chain
- LC CDR2 and LC CDR3 of an anti-TAA light chain binding domain amino acid sequence with 70-100% sequence identity to a light chain (LC) CDR1, LC CDR2 and LC CDR3 of an anti-TAA heavy chain binding domain provided herein, respectively.
- the recombinant nucleic acid encodes a light chain variable region, wherein the light chain variable region comprises an amino acid sequence having at least one but not more than 30 modifications of a light chain variable region amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity to a light chain variable region amino acid sequence of a light chain variable region provided herein.
- the recombinant nucleic acid encodes a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least one but not more than 30 modifications of a heavy chain variable region amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to a heavy chain variable region amino acid sequence of a heavy chain variable region provided herein.
- the TFP includes an extracellular domain of a TCR subunit that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
- the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
- the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137,
- the recombinant nucleic acid molecule further comprises a sequence encoding a costimulatory domain.
- the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of DAP 10,
- DAP 12 CD30, LIGHT, 0X40, CD2, CD27, CD28, CDS, ICAM-l, LFA-l (CD 11 a/CD 18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.
- the linker sequence encodes a peptide sequence that is cleaved by at least one of a tumor cell surface protease, a carboxypeptidase, a cathepsin, a kallikrein, a hexokinase, a plasmin, a stromelysin, factor Xa, a chymotrypsin-like protease, a trypsin -like protease, a elastase-like protease, atryptase, a chymase, a subtilisin-like protease, an actinidain, a proteinase, a bromelain, a calpain, a caspase, a cysteine protease, a papain, an HIV-1 protease, an HSV protease, a CMV protease, a chymosin, a renin,
- metalloexopeptidase a metalloendopeptidase, a matrix metalloproteinase/ collagenase, a plasminogen activator, a urokinase plasminogen activator (uPA), an enterokinase, a prostate-specific antigen (PSA, hK3), an interleukin- 1 b converting enzyme, a thrombin, a fibroblast activation protein (FAP), a meprin, a granzyme, and a dipeptidyl peptidase.
- uPA urokinase plasminogen activator
- PSA prostate-specific antigen
- FAP fibroblast activation protein
- the cathepsin is cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, or cathepsin L;
- the hexokinase is hKl, hKlO, or hKl5;
- the proteinase is PR-3;
- the caspase is caspase-3;
- the cysteine protease is Mir 1 -CP or legumain;
- the matrix metalloproteinase or collagenase is MMPl/( interstitial collagenase), MMP2/type IV collagenase,
- the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP.
- the isolated nucleic acid molecule is mRNA.
- the TFP includes an immunoreceptor tyrosine-based activation motif (ITAM) of a TCR subunit that comprises an ITAM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CD5, CDl6a, CDl6b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional
- ITAM
- the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon.
- the ITAM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.
- the nucleic acid comprises a nucleotide analog.
- the nucleotide analog is selected from the group consisting of 2 -O- methyl, 2' -O-methoxyethyl (2' -O-MOE), 2' -O-aminopropyl, 2' -deoxy, T-deoxy-2' -fluoro, 2' -O- aminopropyl (2' -O-AP), 2'-0-dimethylaminoethyl (2' -O-DMAOE), 2' -O-dimethylaminopropyl (2' -O- DMAP), T-O-dimethylaminoethyloxyethyl (2' -O-DMAEOE), 2 -O-N-methylacetamido (2' -O-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a 1 ' ,5 ' - anhydrohexito
- nucleic acid molecule encoded by the nucleic acid molecule.
- a recombinant TFP molecule comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain.
- a recombinant TFP molecule comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.
- a recombinant TFP molecule comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally integrating into an endogenous TCR complex.
- the TFP molecule further comprises an antibody or antibody fragment comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain.
- the anti-TAA binding domain is a scFv or a VH domain.
- the anti-TAA binding domain comprises a heavy chain with 95-100% identity to an amino acid sequence of an anti-TAA light chain provided herein, a functional fragment thereof, or an amino acid sequence thereof having at least one but not more than 30 modifications. In some embodiments, the anti-TAA binding domain comprises a light chain with 95-100% identity to an amino acid sequence of an anti-TAA heavy chain provided herein, a functional fragment thereof, or an amino acid sequence thereof having at least one but not more than 30 modifications.
- the recombinant TFP molecule comprises a TCR extracellular domain that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
- the TCR comprises an extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma.
- the anti-TAA binding domain is connected to the TCR extracellular domain by a linker sequence.
- the recombinant TFP molecule further comprises a sequence encoding a costimulatory domain.
- the recombinant TFP molecule further comprises a sequence encoding an intracellular signaling domain.
- the recombinant TFP molecule further comprises a leader sequence.
- nucleic acid comprising a sequence encoding a TFP.
- the nucleic acid is selected from the group consisting of a DNA and a RNA.
- the nucleic acid is an mRNA.
- the nucleic acid comprises a nucleotide analog.
- the nucleotide analog is selected from the group consisting of 2' -O-methyl, V -O-methoxyethyl (2' -O-MOE), V -O- aminopropyl, 2' -deoxy, T-deoxy-2' -fluoro, V -O-aminopropyl (2' -O-AP), 2'-0-dimethylaminoethyl (2' -O-DMAOE), 2' -O-dimethylaminopropyl (2' -O-DMAP), T-O-dimethylaminoethyloxyethyl (2' -O- DMAEOE), 2' -O-N-methylacetamido (2' -O-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a 1 ' ,5 ' - anhydrohexitol
- LNA
- the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is an in vitro transcribed nucleic acid. In some embodiments, the nucleic acid further comprises a sequence encoding a poly(A) tail. In some embodiments, the nucleic acid further comprises a 3 ' UTR sequence.
- a vector comprising a nucleic acid molecule encoding a TFP.
- the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, a Rous sarcoma viral (RSV) vector, or a retrovirus vector.
- the vector further comprises a promoter.
- the vector is an in vitro transcribed vector.
- a nucleic acid sequence in the vector further comprises a poly(A) tail.
- a nucleic acid sequence in the vector further comprises a 3 ' UTR.
- a cell comprising the recombinant nucleic acid molecule described herein, the polypeptide molecule described herein, the TFP molecule described herein, and/or the vector described herein.
- the cell is a human T cell.
- the T cell is a CD8+, CD4+ or CD4+CD8+ T cell.
- the cell further comprises a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain.
- the inhibitory molecule comprise first polypeptide that comprises at least a portion of PD 1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.
- a human CD8+, CD4+ or CD4+CD8+ T cell comprising at least two TFP molecules, the TFP molecules comprising an anti- TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+, CD4+ or CD4+CD8+ T cell.
- a protein complex comprising: a TFP molecule comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and at least one endogenous TCR subunit or endogenous TCR complex.
- the TCR comprises an extracellular domain or portion thereof of a protein selected from the group consisting of TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, and a CD3 delta TCR subunit.
- the anti-TAA binding domain is connected to the TCR extracellular domain by a linker sequence.
- a protein complex comprising a TFP encoded by the recombinant nucleic acid molecule described herein, and at least one endogenous TCR subunit or endogenous TCR complex.
- a human CD8+, CD4+ or CD4+CD8+ T cell comprising at least two different TFP proteins per the protein complex.
- a human CD8+, CD4+ or CD4+CD8+ T cell comprising at least two different TFP molecules encoded by the isolated nucleic acid molecule described herein.
- a population of human CD8+, CD4+ or CD4+CD8+ T cells wherein the T cells of the population individually or collectively comprise at least two TFP molecules, the TFP molecules comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+, CD4+ T or CD4+CD8+ cell.
- a population of human CD8+, CD4+ or CD4+CD8+ T cells wherein the T cells of the population individually or collectively comprise at least two TFP molecules encoded by the isolated nucleic acid molecule.
- a method of making a cell comprising transducing a T cell with the recombinant nucleic acid molecule described herein or the vector.
- RNA-engineered cells comprising introducing an in vitro transcribed RNA, a circular RNA or synthetic RNA into a cell, where the RNA comprises a nucleic acid encoding the TFP molecule.
- provided herein is a method of providing an anti-tumor immunity in a mammal comprising administering to the mammal an effective amount of the recombinant nucleic acid molecule described herein, the polypeptide molecule described herein, a cell expressing the polypeptide molecule, the TFP molecule, the vector, or the cell.
- the cell is an autologous T cell. In some embodiments, the cell is an allogeneic T cell. In some embodiments, the mammal is a human.
- a method of treating a mammal having a disease associated with expression of mesothelin comprising administering to the mammal an effective amount of the isolated nucleic acid molecule, the polypeptide molecule, a cell expressing the polypeptide molecule, the TFP molecule, the vector, or the cell.
- the disease associated with mesothelin expression is selected from the group consisting of a proliferative disease, a cancer, a malignancy, and a non-cancer related indication associated with expression of mesothelin.
- the disease is a cancer selected from the group consisting of mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, thyroid cancer, bladder cancer, ureter cancer, kidney cancer, endometrial cancer, esophogeal cancer, gastric cancer, thymic carcinoma, cholangiocarcinoma and stomach cancer.
- the disease is a cancer selected from the group consisting of mesothelioma, papillary serous ovarian adenocarcinoma, clear cell ovarian carcinoma, mixed Mullerian ovarian carcinoma, endometroid mucinous ovarian carcinoma, pancreatic adenocarcinoma, ductal pancreatic adenocarcinoma, uterine serous carcinoma, lung adenocarcinoma, extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma, breast adenocarcinoma, a disease associated with mesothelin expression, and combinations thereof.
- the cells expressing a TFP molecule are administered in combination with an agent that increases the efficacy of a cell expressing a TFP molecule. In some embodiments, less cytokines are released in the mammal compared a mammal administered an effective amount of a T cell expressing an anti-TAA chimeric antigen receptor (CAR). In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a TFP molecule. In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that treats the disease associated with mesothelin.
- nucleic acid molecule also provided herein is the recombinant nucleic acid molecule, the isolated polypeptide molecule, a cell expressing the polypeptide molecule, the isolated TFP, the vector, the complex, or the cell, for use as a medicament.
- a method of treating a mammal having a disease associated with expression of mesothelin comprising administering to the mammal an effective amount of the isolated nucleic acid molecule described herein, the polypeptide molecule described herein, a cell expressing the polypeptide molecule described herein, the TFP molecule described herein, the vector described herein, or the cell described herein, wherein less cytokines are released in the mammal compared a mammal administered an effective amount of a T cell expressing an anti-TAA chimeric antigen receptor (CAR).
- CAR anti-TAA chimeric antigen receptor
- a method of treating a mammal having a disease associated with expression of TAA comprising administering to the mammal an effective amount of the isolated nucleic acid molecule described herein, the polypeptide molecule described herein, a cell expressing the polypeptide molecule described herein, the TFP molecule described herein, the nucleic acid described herein, the vector described herein, or the cell described herein.
- the disease associated with TAA expression is selected from the group consisting of a proliferative disease, a cancer, a malignancy, and a non-cancer related indication associated with expression of TAA.
- the disease is a cancer selected from the group consisting of mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, thyroid cancer, bladder cancer, ureter cancer, kidney cancer, endometrial cancer, esophageal cancer, gastric cancer, thymic carcinoma, cholangiocarcinoma and stomach cancer.
- the disease is a cancer selected from the group consisting of mesothelioma, papillary serous ovarian adenocarcinoma, clear cell ovarian carcinoma, mixed Mullerian ovarian carcinoma, endometroid mucinous ovarian carcinoma, pancreatic adenocarcinoma, ductal pancreatic
- adenocarcinoma uterine serous carcinoma, lung adenocarcinoma, extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma, breast
- the cells expressing a TFP molecule are administered in combination with an agent that increases the efficacy of a cell expressing a TFP molecule. In some embodiments, less cytokines are released in the mammal compared a mammal administered an effective amount of a T cell expressing an anti-TAA chimeric antigen receptor (CAR). In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a TFP molecule. In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that treats the disease associated with the antigen.
- the antigen is a tumor-associated antigen.
- the antigen is one or more selected from CD 19, B-cell maturation antigen (BCMA), mesothelin (MSLN), ILl3Rcx2, MUC16, CD22, PD-l, BAFF or BAFF receptor, and ROR-l.
- isolated nucleic acid molecule described herein is also provided herein, the isolated nucleic acid molecule described herein, the isolated polypeptide molecule described herein, a cell expressing the polypeptide molecule described herein, the isolated TFP described herein, the nucleic acid described herein, the vector described herein, the complex described herein, or the cell described herein, for use as a medicament.
- a method of treating a mammal having a disease associated with expression of TAA comprising administering to the mammal an effective amount of the isolated nucleic acid molecule described herein, the polypeptide molecule described herein, a cell expressing the polypeptide molecule described herein, the TFP molecule described herein, the nucleic acid described herein, the vector described, or the cell described herein, wherein less cytokines are released in the mammal compared a mammal administered an effective amount of a T cell expressing an anti -TAA chimeric antigen receptor (CAR).
- CAR anti -TAA chimeric antigen receptor
- the TFP comprises an anti-TAA binding domain binds to an antigen derived from antigens of alpha-actinin-4, ARTC1, BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDK12, CDKN2A, CLPP, COA-l, CSNK1A1, dek-can fusion protein, EFTUD2, Elongation factor 2, ETV6-AML1 fusion protein, FLT3-ITD, FNDC3B, FN1, GAS7, GPNMB, HAUS3, HSDL1, LDLR-fucosyltransferase AS fusion protein, HLA-A2d, HLA-A1 ld, hsp70- 2, MART2, MATN, ME1, MUM-lf, MUM-2, MUM-3, neo-PAP, Myosin class I, NFYC, OGT
- the TFP comprises one or more of an anti-CD 19 binding domain, an anti -B-cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti- IL 13Ra2 binding domain, an anti-MUCl6 binding domain, an anti-CD22 binding domain, an anti -PD- 1 binding domain, an anti-BAFF or BAFF receptor binding domain, and an anti-ROR-l binding domain.
- BCMA anti-B-cell maturation antigen
- MSLN anti-mesothelin
- FIGs. 1A-B show two drawings comprising an exemplary CAR T cell bound to HSA via HSA- binding sdAb and a protease-cleavable linker.
- FIG. 1A shows a CAR T cell fused with an anti -HSA antibody;
- FIG. IB shows the CAR T cell from FIG. 1A with HSA bound to the anti-HSA domain.
- FIGs. 2A-B show two drawings depicting the fusion of HSA -binding sdAb to the Va or Vb Domain of a TFP T cell via a protease-cleavable linker.
- FIG. 2A shows a TFP T cell fused with an anti- HSA antibody;
- FIG. 2B shows the TFP T cell from FIG. 2A with HSA bound to the anti-HSA domain.
- FIGs. 3A-B is two drawings depicting HSA binding to the sdAb of the TFP T cell. This blocks binding of the TCR to the MHC/peptide antigen complex.
- FIG. 3A shows a TFP T cell fused with an anti-HSA antibody;
- FIG. 3B shows the TFP T cell from FIG. 3A with HSA bound to the anti-HSA domain.
- FIG. 4 is a drawing depicting proteolytic cleavage of the HSA-binding sdAb at the protease - cleavage site, which enables the inducible TFP T cell to bind to its MHC/peptide complex.
- FIG. 5 is a drawing depicting direct fusion of HSA to an anti -tumor-associated antigen binding domain of a TFP T cell.
- FIG. 6 shows sequences of example anti-HSA-[cleavable-linker]-anti-MSLN constructs.
- FIG. 6A shows a uPA-cleavable fusion protein sequence.
- FIG. 6B shows an MMP9vl-cleavable fusion protein sequence.
- FIG. 6C shows an MMP9v2-cleavable fusion protein sequence.
- FIG. 6D shows a cathepsin B fusion protein sequence.
- FIG. 6E shows a non-cleavable control sequence.
- FIG. 7 depicts two graphs showing data demonstrating that inducible anti-mesothelin (MSLN) TFP T cells bind to soluble MSLN and are inhibited by their HSA blocking domains.
- FIG. 7A depicts a graph showing FACS data that illustrates the anti-HSA/anti-MSLN TFPs are able to bind to MSLN in the absence of HSA, and the constructs are able to bind HSA in the absence of MSLN.
- FIG. 7B depicts two graphs showing the ability of anti-HSA/anti-MSLN TFP T cells to induce cytokine production when expressed in Jurkat cells and co-cultured with MSTO cells that express MSLN.
- Panels show from left to right Jurkat cells alone, the anti-HSA/anti-MSLN TFP T cells alone, anti-HSA/anti-MSLN TFP cells comprising an MMP9-cleavable linker, anti-HSA/anti-MSLN TFP T cells comprising a cathepsin B- cleavable linker, and anti-HSA/anti-MSLN TFP cells comprising a non-cleavable linker, and MSTO cells alone (no effector cells, i.e., Jurkat). Each was done in the presence or absence of HSA.
- FIG. 8 is a western blot illustrating cleavability of anti-HSA -cleavable-linker-anti-MSLN ("SD1") constructs. Constructs were loaded pairwise as follows: ctHSA-sdAb-uPA-SDl sdAb digested with uPA +/- FBS; aAlb-N/C-SDl digested with uPA +/- FBS; aHSA sdAb-MMP9vl- SDlsdAb digested with MMP9 +/- FBS; aHSA sdAb-MMP9v2-SDlsdAb digested with MMP9 +/- FBS; and aHSA sdAb-cathepsin B-SD1 sdAb digested with cathepsin B +/- FBS.
- the western blot was probed with an anti-6His antibody and shows both the uncleaved constructs (around 30kDa) and the cleave
- an "inducible T cell receptor (TCR) fusion protein” includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that, when induced, is generally capable of i) binding to a surface antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on the surface of a T cell e.g., once the T cell has entered the tumor microenvironment (TME) of a solid tumor.
- TCE tumor microenvironment
- the inducible TFP T cells disclosed herein comprise a blocking domain that keeps the antigen binding domain (e.g., a tumor associated antigen binding domain) from binding until the TFP T cells reach the target site (e.g., a tumor).
- the compositions and methods disclosed herein are suitable for treating a disease wherein the target of interest is expressed on cells of a tissue other than the target tissue, as the inducible nature of the TFP T cells reduces the likelihood of off-target effects in a subject (e.g., a mammal).
- TFPs provide substantial benefits as compared to Chimeric Antigen Receptors.
- the central intracellular signaling domain of a CAR is derived from the CD3 zeta chain that is normally found associated with the TCR complex.
- the CD3 zeta signaling domain can be fused with one or more functional signaling domains derived from at least one co-stimulatory molecule such as CD2, 4-1BB (i.e., CD137), CD27 and/or CD28.
- isolated nucleic acid molecules encoding an inducible T cell Receptor (TCR) fusion protein that comprise a TCR subunit, a non-human, human or humanized antibody domain comprising an anti-tumor associated antigen (TAA) binding domain, and a blocking domain connected to the TAA via a protease-cleavable linker, e.g., a matrix metalloproteinase cleavable linker.
- the blocking domain is an antibody such as anti-albumin.
- the blocking domain is albumin, IgG, or other bulky protein that sterically hinders binding of the TAA to its target protein on the tumor cell.
- the inducible TFP comprises more than one blocking domain and more than one protease-cleavable linker.
- the TCR subunit comprises a TCR extracellular domain. In other embodiments, the TCR subunit comprises a TCR transmembrane domain. In yet other embodiments, the TCR subunit comprises a TCR intracellular domain. In further embodiments, the TCR subunit comprises (i) a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
- the TCR subunit comprises a TCR intracellular domain comprising a stimulatory domain selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having at least one, two or three modifications thereto.
- the TCR subunit comprises an intracellular domain comprising a stimulatory domain selected from a functional signaling domain of CD2 and/or 4-1BB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one, two or three modifications thereto.
- the antibody domain comprises an antibody fragment. In some embodiments, the antibody domain comprises a scFv, a sdAb or a VH domain.
- the isolated nucleic acid molecules comprise (i) a light chain (LC) CDR1, LC CDR2 and LC CDR3 of any anti-tumor-associated antigen light chain binding domain amino acid sequence provided herein, and/or (ii) a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 of any anti- tumor-associated antigen heavy chain binding domain amino acid sequence provided herein.
- LC light chain
- HC heavy chain
- the light chain variable region comprises an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity to an amino acid sequence provided herein.
- the heavy chain variable region comprises an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to an amino acid sequence provided herein.
- the TFP includes an extracellular domain of a TCR subunit that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma, or a functional fragment thereof, or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications thereto.
- the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta chain of the TCR or TCR subunits CD3 epsilon, CD3 gamma and CD3 delta, or a functional fragment thereof, or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications thereto.
- the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, functional fragment(s) thereof, and amino acid sequences thereof having at least one, two or three modifications but not more than 20 modifications thereto.
- a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamm
- the isolated nucleic acid molecule further comprises a sequence encoding a costimulatory domain.
- the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of DAP 10, DAP 12, CD30, LIGHT, 0X40, CD2, CD27, CD28, CDS, ICAM-l, LFA-l (CD 11 a/CD 18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.
- the isolated nucleic acid molecule further comprises a leader sequence.
- the isolated nucleic acid molecule is mRNA.
- the TFP includes an immunoreceptor tyrosine-based activation motif (IT AM) of a TCR subunit that comprises an IT AM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP 12), CD5, CDl6a, CDl6b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66
- IT AM immunorecept
- the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon.
- the ITAM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.
- the nucleic acid comprises a nucleotide analog.
- the nucleotide analog is selected from the group consisting of 2' -O-methyl, V -O-methoxyethyl (2' -O- MOE), 2' -O-aminopropyl, V -deoxy, T-deoxy-2' -fluoro, 2' -O-aminopropyl (2' -O-AP), 2'-0- dimethylaminoethyl (2' -O-DMAOE), 2' -O-dimethylaminopropyl (2' -O-DMAP), T-O- dimethylaminoethyloxyethyl (2' -O-DMAEOE), 2' -O-N-methylacetamido (2' -O-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (P
- isolated T cell receptor fusion protein (TFP) molecules that comprise an anti-tumor-associated antigen binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain.
- the isolated TFP molecules comprises an antibody or antibody fragment comprising an anti-tumor-associated antigen binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain.
- the anti-TAA binding domain is an antibody, Fab, scFv, a VH domain, or an sdAb, or a functional fragment thereof.
- the anti-tumor-associated antigen binding domain comprises a light chain and a heavy chain of an amino acid sequence provided herein, or a functional fragment thereof, or an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity with an amino acid sequence provided herein.
- the isolated TFP molecules comprise a TCR extracellular domain that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma, or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications thereto.
- the anti-tumor-associated antigen binding domain is connected to the TCR extracellular domain by a non-protease-cleavable linker sequence.
- the linker sequence comprises a long linker (LL) sequence.
- the linker sequence comprises a short linker (SL) sequence.
- the isolated TFP molecules further comprise a sequence encoding a costimulatory domain. In other embodiments, the isolated TFP molecules further comprise a sequence encoding an intracellular signaling domain. In yet other embodiments, the isolated TFP molecules further comprise a leader sequence.
- vectors that comprise a nucleic acid molecule encoding any of the previously described TFP molecules.
- the vector is selected from the group consisting of a DNA, an RNA, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector.
- the vector further comprises a promoter.
- the vector is an in vitro transcribed vector.
- a nucleic acid sequence in the vector further comprises a poly(A) tail.
- a nucleic acid sequence in the vector further comprises a 3 ' UTR.
- the cell is a human T cell.
- the cell is a CD8+ or CD4+ or CD4+CD8+ T cell.
- the CD8+ cell is a gamma-delta T cell.
- the CD8+ cell is an NK-T cell.
- the cells further comprise a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain.
- the inhibitory molecule comprises a first polypeptide that comprises at least a portion of PD1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.
- TFP molecules that comprise a human or humanized anti-tumor-associated antigen (TAA) binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.
- TAA anti-tumor-associated antigen
- TFP molecules that comprise an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally integrating into an endogenous TCR complex.
- human CD8+ or CD4+ or CD4+CD8+ T cells that comprise one or more inducible TFP molecules, the TFP molecules comprising a blocking domain or antibody specific to a protein suitable for a blocking domain, an antigen binding domain (e.g., an anti- tumor-associated (TAA) antigen binding domain), a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+ or CD4+ or CD4+CD8+ T cell.
- the cells comprise at least two non-identical inducible TFP molecules.
- protein complexes that comprise i) a TFP molecule comprising an anti-tumor-associated antigen binding domain, a blocking domain or an antibody specific to a blocking domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and ii) at least one endogenous TCR complex subunit.
- the TCR comprises an extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma.
- the anti-tumor-associated antigen binding domain is connected to the TCR extracellular domain by a linker sequence.
- the linker sequence comprises a long linker (LL) sequence.
- the linker sequence comprises a short linker (SL) sequence.
- human CD8+ or CD4+ or CD8+CD4+ T cells that comprise at least two different TFP molecules per any of the described protein complexes.
- a population of human CD8+ or CD4+ or CD8+CD4+ or NK T cells wherein the T cells of the population individually or collectively comprise at least two TFP molecules, the TFP molecules comprising an antigen binding domain (e.g., an anti-tumor-associated antigen binding domain), a blocking domain or an antibody to a protein suitable for a blocking domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+ or CD4+ or CD4+CD8+ T cell.
- an antigen binding domain e.g., an anti-tumor-associated antigen binding domain
- a blocking domain or an antibody to a protein suitable for a blocking domain e.g., an anti-tumor-associated antigen binding domain
- a population of human CD8+ or CD4+ T or CD8+CD4+ cells wherein the T cells of the population individually or collectively comprise at least two TFP molecules encoded by an isolated nucleic acid molecule provided herein.
- RNA-engineered cells comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, where the RNA comprises nucleic acid encoding one or more of the described TFP molecules.
- the RNA is a circular RNA.
- methods of providing an anti-tumor immunity in a mammal that comprise administering to the mammal an effective amount of a cell expressing any of the described TFP molecules.
- the cell is an autologous T cell.
- the cell is an allogeneic T cell.
- the mammal is a human.
- the disease associated with tumor-associated antigen expression is selected from a proliferative disease such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia, or is a non-cancer related indication associated with expression of tumor-associated antigen.
- the disease is a hematologic cancer selected from the group consisting of one or more acute leukemias including but not limited to B-cell acute lymphoid leukemia ("B-ALL”), T cell acute lymphoid leukemia (“T-ALL”), acute lymphoblastic leukemia (ALL); one or more chronic leukemias including but not limited to chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); additional hematologic cancers or hematologic conditions including, but not limited to B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt' s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell- follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphom
- B-ALL B-cell
- myelodysplastic syndrome non-Hodgkin' s lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom' s macroglobulinemia, and "preleukemia” which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, and to disease associated with tumor-associated antigen expression include, but not limited to atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing tumor- associated antigen; and combinations thereof.
- the disease is a solid tumor.
- the cancer is selected from the group consisting of renal cell carcinoma, breast cancer, lung cancer, mesothelioma, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, and stomach cancer.
- the tumor associated antigen is expressed on the surface of tumor cells and is suitable for treatment with a TFP T cell (comprising a T cell receptor Fusion Protein (TFP) T cell as disclosed herein, or a CAR T cell.
- TFP T cell comprising a T cell receptor Fusion Protein (TFP) T cell as disclosed herein, or a CAR T cell.
- TFP T cell receptor Fusion Protein
- the tumor associated antigen is expressed inside the cancer cell and a suitable treatment is a TCR T cell.
- the cells expressing any of the described TFP molecules are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a TFP molecule. In some embodiments, the cells expressing any of the described TFP molecules are administered in combination with an agent that treats the disease associated with tumor- associated antigen.
- an element means one element or more than one element.
- the term “about” or “approximately” can mean within an order of magnitude, within 5-fold, and more preferably within 2- fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.
- the term “about” can have the meaning as commonly understood by one of ordinary skill in the art. The term “about” can refer to +10%. The term “about” can refer to +5%.
- subject or “subjects” or “individuals” may include, but are not limited to, mammals such as humans or non-human mammals, e.g., domesticated, agricultural or wild, animals, as well as birds, and aquatic animals.
- persons are subjects suffering from or at risk of developing a disease, disorder or condition or otherwise in need of the compositions and methods provided herein.
- treating refers to any indicia of success in the treatment or amelioration of the disease or condition. Treating can include, for example, reducing, delaying or alleviating the severity of one or more symptoms of the disease or condition, or it can include reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient.
- treat or prevent is sometimes used herein to refer to a method that results in some level of treatment or amelioration of the disease or condition and
- preventing refers to the prevention of the disease or condition, e.g., tumor formation, in the patient. For example, if an individual at risk of developing a tumor or other form of cancer is treated with the methods of the present invention and does not later develop the tumor or other form of cancer, then the disease has been prevented, at least over a period of time, in that individual.
- the disease or condition e.g., tumor formation
- a “therapeutically effective amount” is the amount of a composition or an active component thereof sufficient to provide a beneficial effect or to otherwise reduce a detrimental non- beneficial event to the individual to whom the composition is administered.
- therapeutically effective dose herein is meant a dose that produces one or more desired or desirable (e.g., beneficial) effects for which it is administered, such administration occurring one or more times over a given period of time. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g. Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999))
- a "T cell receptor (TCR) fusion protein” or “TFP” includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that is generally capable of i) binding to a surface antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on the surface of a T cell.
- the portion of the TFP composition comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) derived from a murine, humanized or human antibody (Harlow et ah, 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et ah, 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et ah, 1988, Proc. Natl. Acad. Sci.
- sdAb single domain antibody fragment
- scFv single chain antibody
- the term "antigen” or “Ag” may refer to a molecule that is capable of being bound specifically by an antibody, or otherwise provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, or both.
- the term “cancer antigen” or “cancer-related antigen” may refer to any cancer cell marker expressed on the surface of a malignant or tumor cell that can be treated with the combination therapy described herein, including, but not limited to: described herein include, but are not limited to, 5T4, 8H9, o ⁇ n b Q integrin, anb6 integrin, alphafetoprotein (AFP), B7-H6, CA-125 carbonic anhydrase 9 (CA9),
- CD 19 CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD52, CD123, CD171, carcinoembryonic antigen (CEA), EpCAM (epithelial cell adhesion molecule), E-cadherin, EMA
- epithelial membrane antigen epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbBl/EGFR, ErbB2/HER2/neu/EGFR2, ErbB3/HER3, ErbB4, epithelial tumor antigen (ETA), folate binding protein (FBP), fetal acetylcholine receptor (AchR), folate rcccptor-a.
- ganglioside 2 GD2
- ganglioside 3 GD3
- HLA-A1, HLA-A2 high molecular weight melanoma-associated antigen
- IL-13 receptor «2 I L- 13Ra2).
- KDR kinase insert domain receptor
- FeY Fewis Y
- Fl cell adhesion molecule melanoma-associated antigen
- MAGE-A1 mesothelin, mucin-l (MUC1 ), mucin-l6 (MUC16), natural killer group 2 member D (NKG2D) ligands, nerve cell adhesion molecule (NCAM), NY-ESO-l, B7H4, DLL3, TROP-2, Nectin-4, tissue factor, LIV-l, CD48, cMET, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate -specific membrane antigen (PSMA), receptor-tyrosine kinase-like orphan receptor 1 (R0R1), TAA targeted by mAb IgE, tumor- associated glycoprotein-72 (TAG-72), tyrosinase, and vascular endothelial growth factor (VEGF) receptors.
- KDR kinase
- antibody refers to a protein, or polypeptide sequences derived from an immunoglobulin molecule, which specifically binds to an antigen.
- Antibodies can be intact immunoglobulins of polyclonal or monoclonal origin, or fragments thereof and can be derived from natural or from recombinant sources.
- antibody fragment refers to at least one portion of an antibody, or recombinant variants thereof, that contains the antigen binding domain, i.e., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope.
- antibody fragments include, but are not limited to, Fab, Fab' , F(ab' )2, and Fv fragments, single-chain (sc)Fv (“scFv”) antibody fragments, linear antibodies, single domain antibodies (abbreviated "sdAb”) (either VF or VH), camelid VHH domains, and multi -specific antibodies formed from antibody fragments.
- scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
- “Heavy chain variable region” or “VH” refers to the fragment of the heavy chain that contains three CDRs interposed between flanking stretches known as framework regions, these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.
- an scFv may have the VF and VH regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VF-linker- VH or may comprise VH-linker-VF.
- the portion of the TFP composition of the invention comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb) or heavy chain antibodies HCAb 242:423-426).
- the antigen binding domain of a TFP composition of the invention comprises an antibody fragment.
- the TFP comprises an antibody fragment that comprises a scFv or a sdAb.
- antibody heavy chain refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.
- antibody light chain refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (“ K ”) and lambda (“l”) light chains refer to the two major antibody light chain isotypes.
- recombinant antibody refers to an antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system.
- the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.
- antigen or “Ag” refers to a molecule that is capable of being bound specifically by an antibody, or otherwise provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, or both.
- the term "combination therapy” means a therapy strategy that embraces the administration of therapeutic compositions of the present invention (e.g., conjugates comprising one or more neoantigens) and one or more additional therapeutic agents as part of a specific treatment regimen intended to provide a beneficial (additive or synergistic) effect from the co-action of these therapeutic agents.
- Administration of these therapeutic agents in combination may be carried out over a defined time period (usually minutes, hours, days, or weeks depending upon the combination selected).
- cytotoxic T cell TC
- CTL cytotoxic T lymphocyte
- T- killer cells T- killer cells
- CD8+ T cell killer T cell
- epitope means a small peptide structure formed by contiguous amino acids, or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
- An epitope typically includes at least 3, and about 9, or about 8-15 amino acids.
- a T cell epitope means a peptide which can be bound by the MHC molecules of class I or II in the form of a peptide-presenting MHC molecule or MHC complex and then, in this form, be recognized and bound by native T cells, cytotoxic T- lymphocytes or T-helper cells, respectively.
- Immune cell refers to a cell that is capable of participating, directly or indirectly, in an immune response.
- Immune cells include, but are not limited to T cells, B-cells, antigen presenting cells, dendritic cells, natural killer (NK) cells, natural killer T (NK) cells, lymphokine- activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhans' cells, stem cells, peripheral blood mononuclear cells, cytotoxic T cells, tumor infiltrating lymphocytes (TIL), etc.
- TIL tumor infiltrating lymphocytes
- An “antigen presenting cell” is a cell that is capable of activating T cells, and includes, but is not limited to, monocytes/macrophages, B cells and dendritic cells (DCs).
- Dendritic cell or “DC” refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their distinctive morphology, high levels of surface MHC -class II expression. DCs can be isolated from a number of tissue sources. DCs have a high capacity for sensitizing MHC- restricted T cells and are very effective at presenting antigens to T cells in situ.
- the antigens may be self-antigens that are expressed during T cell development and tolerance, and foreign antigens that are present during normal immune processes.
- an "activated DC” is a DC that has been pulsed with an antigen and capable of activating an immune cell.
- T cell as used herein, is defined as a thymus-derived cell that participates in a variety of cell- mediated immune reactions, including CD8+ T cell and CD4+ T cell.
- immune response means a defensive response a body develops against "foreigner” such as bacteria, viruses and substances that appear foreign and harmful.
- An anti -cancer immune response refers to an immune surveillance mechanism by which a body recognizes abnormal tumor cells and initiates both the innate and adaptive of the immune system to eliminate dangerous cancer cells.
- the innate immune system is a non-specific immune system that comprises the cells (e.g.,
- Natural killer cells mast cells, eosinophils, basophils; and the phagocytic cells including macrophages, neutrophils, and dendritic cells
- An innate immune response can initiate the productions of cytokines, and active complement cascade and adaptive immune response.
- the adaptive immune system is specific immune system that is required and involved in highly specialized systemic cell activation and processes, such as antigen presentation by an antigen presenting cell; antigen specific T cell activation and cytotoxic effect.
- antigens can be derived from recombinant or genomic DNA.
- any DNA which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein.
- an antigen need not be encoded solely by a full-length nucleotide sequence of a gene.
- the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response.
- an antigen need not be encoded by a "gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample or might be macromolecule besides a polypeptide.
- a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components.
- Specific dissociation refers to a separation of two components that preferentially occurs in a particular environment.
- specific dissociation refers to dissociation of domains separated by proteolytic cleavage by a protease that is more highly available in the tumor microenvironment.
- specific dissociation refers to separation of components of the pharmaceutical composition upon enzymatic digestion or other enzymatic activity.
- biologically active domain refers to any protein structure able to modulate a biological activity. This can include, but is not limited to, cytokines, chemokines, antibodies, antibody-drug conjugates, T cell fusion proteins, chimeric antigen receptors (CARs), and T cell receptor subunits.
- anti -tumor effect refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, decrease in tumor cell proliferation, decrease in tumor cell survival, or amelioration of various physiological symptoms associated with the cancerous condition.
- An "anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor in the first place.
- Adoptive cellular immunotherapy' or “adoptive immunotherapy ' or “T cell immunotherapy'”, or “Adoptive T cell therapy (ACT)" are used interchangeably.
- Adoptive immunotherapy uses T cells that a natural or genetically engineered reactivity to a patient's cancer are generated in vitro and then transferred back into the cancer patient. The injection of a large number of activated tumor specific T cells can induce complete and durable regression of cancers.
- autologous refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.
- allogeneic refers to any material derived from a different animal of the same species or different patient as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.
- xenogeneic refers to a graft derived from an animal of a different species.
- cancer may refer to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include, but are not limited to, prostate cancer, breast cancer, melanoma, sarcoma, colorectal cancer, pancreatic cancer, uterine cancer, ovarian cancer, stomach cancer, gastric cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, cholangiocarcinoma, squamous cell lung cancer, mesothelioma, adrenocortico carcinoma, esophageal cancer, head & neck cancer, liver cancer, nasopharyngeal carcinoma, neuroepithelial cancer, adenoid cystic carcinoma, thymoma, chronic lymphocytic leukemia, glioma, glioblastoma multiforme, neuroblastoma, papillary
- conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. 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.
- one or more amino acid residues within a TFP of the invention can be replaced with other amino acid residues from the same side chain family and the altered TFP can be tested using the functional assays described herein.
- stimulation refers to a primary response induced by binding of a stimulatory domain or stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex.
- a stimulatory domain or stimulatory molecule e.g., a TCR/CD3 complex
- Stimulation can mediate altered expression of certain molecules, and/or reorganization of cytoskeletal structures, and the like.
- the term "stimulatory molecule” or “stimulatory domain” refers to a molecule or portion thereof expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR complex in a stimulatory way for at least some aspect of the T cell signaling pathway.
- the primary signal is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
- a primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or "ITAM".
- ITAM immunoreceptor tyrosine-based activation motif
- Examples of an ITAM containing primary cytoplasmic signaling sequence that is of particular use in the invention includes, but is not limited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS”) and CD66d.
- the term "antigen presenting cell” or “APC” refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC' s) on its surface.
- T cells may recognize these complexes using their T cell receptors (TCRs).
- APCs process antigens and present them to T cells.
- intracellular signaling domain refers to an intracellular portion of a molecule.
- the intracellular signaling domain generates a signal that promotes an immune effector function of the TFP containing cell, e.g., a TFP -expressing T cell.
- immune effector function e.g., in a TFP -expressing T cell
- examples of immune effector function, e.g., in a TFP -expressing T cell include cytolytic activity and T helper cell activity, including the secretion of cytokines.
- the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation.
- the intracellular signaling domain can comprise a costimulatory intracellular domain.
- Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.
- a primary intracellular signaling domain can comprise an ITAM ("immunoreceptor tyrosine- based activation motif").
- ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d DAP 10 and DAP 12.
- costimulatory molecule refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.
- Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response.
- Costimulatory molecules include, but are not limited to, an MHC class 1 molecule, BTLA, a Toll ligand receptor,
- a costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule.
- costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors.
- TNF receptor proteins TNF receptor proteins
- Immunoglobulin-like proteins TNF receptor proteins
- cytokine receptors cytokine receptors
- integrins signaling lymphocytic activation molecules
- activating NK cell receptors examples include CD27, CD28, 4- 1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like.
- the intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.
- 4-1BB refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Ace. No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like; and a "4-1BB costimulatory domain” is defined as amino acid residues 214-255 of GenBank Ace. No. AAA62478.2, or equivalent residues from non-human species, e.g., mouse, rodent, monkey, ape and the like.
- the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
- a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
- Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
- nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
- the phrase nucleotide sequence that encodes a protein or a RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain one or more introns.
- an effective amount or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological or therapeutic result.
- endogenous refers to any material from or produced inside an organism, cell, tissue or system.
- exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
- expression refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.
- transfer vector refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
- Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
- the term "transfer vector” includes an autonomously replicating plasmid or a virus.
- the term should also be construed to further include non plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like.
- Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
- expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
- An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
- Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
- lentivirus refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lenti viruses.
- lentiviral vector refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009).
- Other examples of lentivirus vectors that may be used in the clinic include, but are not limited to, e.g., the Lentivector® gene delivery technology from Oxford BioMedica, the
- LentiMax® vector system from Lentigen, and the like.
- Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
- homologous refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules.
- two nucleic acid molecules such as, two DNA molecules or two RNA molecules
- polypeptide molecules between two polypeptide molecules.
- a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position.
- the homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.
- Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab' , F(ab' )2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
- humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary -determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
- CDR complementary -determining region
- Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non human residues.
- a humanized antibody/antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance.
- the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence.
- the humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
- Fc immunoglobulin constant region
- Human or “fully human” refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.
- isolated means altered or removed from the natural state.
- a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
- An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
- A refers to adenosine
- C refers to cytosine
- G refers to guanosine
- T refers to thymidine
- U refers to uridine.
- operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
- a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
- a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
- Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.
- parenteral administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, intratumoral, or infusion techniques.
- nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
- DNA deoxyribonucleic acids
- RNA ribonucleic acids
- degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et ah, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et ah, J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et ak, Mol. Cell. Probes 8:91-98 (1994)).
- peptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
- a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein' s or peptide' s sequence.
- Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
- the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
- Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
- a polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
- promoter refers to a DNA sequence recognized by the transcription machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
- promoter/regulatory sequence refers to a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
- the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
- constitutive promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
- inducible promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
- tissue-specific promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
- linker and "flexible polypeptide linker” (not to be confused with “protease- cleavable linker” of the TFP molecules disclosed herein) as used in the context of a scFv refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link variable heavy and variable light chain regions together.
- the flexible polypeptide linker is a Gly/Ser linker and comprises the amino acid sequence (Gly-Gly-Gly- Ser) n , where n is a positive integer equal to or greater than 1.
- the flexible polypeptide linkers include, but are not limited to, (Gly 4 Ser) 4 or (Gly 4 Ser) 3 .
- the linkers include multiple repeats of (Gly 2 Ser), (GlySer) or (Gly 3 Ser). Also included within the scope of the invention are linkers described in
- the linker sequence comprises a long linker (LL) sequence.
- the linker sequence comprises a short linker (SL) sequence.
- a 5 ' cap (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is a modified guanine nucleotide that has been added to the "front" or 5 ' end of a eukaryotic messenger RNA shortly after the start of transcription.
- the 5 ' cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co -transcriptionally, such that each influences the other.
- RNA polymerase Shortly after the start of transcription, the 5 ' end of the mRNA being synthesized is bound by a cap -synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction.
- the capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation.
- in vitro transcribed RNA refers to RNA, preferably mRNA, which has been synthesized in vitro.
- the in vitro transcribed RNA is generated from an in vitro transcription vector.
- the in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA.
- an in vitro transcribed RNA is circularizable.
- a "poly(A)” is a series of adenosines attached by polyadenylation to the mRNA.
- the polyA is between 50 and 5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400.
- Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.
- polyadenylation refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule.
- mRNA messenger RNA
- the 3 ' poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase.
- poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal.
- Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm.
- the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase.
- the cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site.
- adenosine residues are added to the free 3 ' end at the cleavage site.
- transient refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.
- signal transduction pathway refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell.
- cell surface receptor includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.
- subject is intended to include living organisms in which an immune response can be elicited (e.g., mammals, human).
- substantially purified cell refers to a cell that is essentially free of other cell types.
- a substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state.
- a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state.
- the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.
- terapéutica means a treatment.
- a therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.
- prophylaxis means the prevention of or protective treatment for a disease or disease state.
- tumor antigen or “hyperproliferative disorder antigen” or “antigen associated with a hyperproliferative disorder” refers to antigens that are common to specific hyperproliferative disorders.
- the hyperproliferative disorder antigens of the present invention are derived from, cancers including but not limited to primary or metastatic melanoma, mesothelioma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, NHL, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.
- transfected or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
- a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
- the cell includes the primary subject cell and its progeny.
- the term “specifically binds,” refers to an antibody, an antibody fragment or a specific ligand, which recognizes and binds a cognate binding partner (e.g., BCMA, MSLN, NKG2D, ROR1, etc.) present in a sample, but which does not necessarily and substantially recognize or bind other molecules in the sample.
- a cognate binding partner e.g., BCMA, MSLN, NKG2D, ROR1, etc.
- binding ligand may generally refer to a polypeptide (e.g., a protein), a
- polynucleotide e.g., DNA, RNA, or a hybrid of DNA and RNA
- the binding ligand can comprise a polynucleotide, and the polynucleotide can be single stranded, double stranded, or a combination thereof.
- a binding ligand can comprise a biological molecule or a non -biological molecule.
- a biological molecule or non-biological molecule can be a naturally occurring molecule or an artificial molecule.
- Non-limiting examples of a binding ligand include a protein, a carbohydrate, a lipid, or a nucleic acid.
- the binding ligand may associate, bind, and/or couple with an antibody or fragment thereof (e.g., an IgA isotype antibody, an IgD isotype antibody, an IgE isotype antibody, an IgG isotype antibody, an IgM isotype antibody, an IgW isotype antibody, an IgY isotype antibody).
- the antibody or fragment thereof may be an Fc domain of the antibody (e.g., the binding ligand is an Fc receptor).
- the binding ligand is an Fc receptor
- the binding ligand can specifically bind to an IgGl antibody.
- the binding ligand may be capable of associating, capable of binding, and/or capable of coupling with an antibody or fragment thereof.
- the binding ligand can comprise multiple subunits.
- a binding ligand can comprise multiple subunits, and the subunits can be the same.
- a binding ligand can comprise multiple different subunits.
- a binding ligand can comprise multiple subunits, and at least two of the subunits can be different.
- a binding ligand can comprise a dimer, trimer, tetramer, pentamer, hexamer, heptamer, nonamer, or decamer. In some embodiments, a binding ligand can comprise greater than about ten subunits. In some embodiments, a binding ligand can comprise a polymer. In some embodiments, the binding ligand may be non-human (e.g., primate), human, or humanized.
- Ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6.
- a range such as 95-99% identity includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.
- T cell receptor (TCR) fusion proteins (TFP) T cell receptor (TCR) fusion proteins (TFP)
- the present invention encompasses recombinant DNA constructs encoding TFPs, wherein the TFP in one aspect comprises an antibody fragment that binds specifically to one or more tumor associated antigens ("TAA"), e.g., a human TAA, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
- TAA tumor associated antigens
- the TFPs comprise an intervening in-frame sequence comprising a protease -cleavable linker (e.g., between the sequence encoding the antibody fragment and the sequence encoding the TCR subunit or portion thereof, such that the expressed protein comprises a protease- cleavable site between the TCR subunit and the antibody fragment).
- the TFPs provided herein are able to associate with one or more endogenous (or alternatively, one or more exogenous, or a combination of endogenous and exogenous) TCR subunits in order to form a functional TCR complex.
- the antigen-binding domain is a humanized or human anti-TAA binding domain.
- the antibody fragment is an anti-mesothelin antibody or a fragment thereof.
- the portion of the TFP comprising the antigen binding domain comprises an antigen binding domain that targets mesothelin.
- the antigen binding domain targets human mesothelin.
- the antigen-binding domain comprises a humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment.
- the humanized or human anti-mesothelin binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain
- LC CDR3 complementary determining region 3 of a humanized or human anti-mesothelin binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-mesothelin binding domain described herein, e.g., a humanized or human anti-mesothelin binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs.
- HC CDR3 heavy chain complementary determining region 3
- the humanized or human anti-mesothelin binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-mesothelin binding domain described herein, e.g., the humanized or human anti-mesothelin binding domain has two variable heavy chain regions, each comprising a HC CDR1, a HC CDR2 and a HC CDR3 described herein.
- HC CDR1 heavy chain complementary determining region 1
- HC CDR2 heavy chain complementary determining region 2
- HC CDR3 heavy chain complementary determining region 3
- the humanized or human anti-mesothelin binding domain comprises a humanized or human light chain variable region described herein and/or a humanized or human heavy chain variable region described herein. In one embodiment, the humanized or human anti-mesothelin binding domain comprises a humanized heavy chain variable region described herein, e.g., at least two humanized or human heavy chain variable regions described herein. In one embodiment, the anti-mesothelin binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence provided herein.
- the humanized or human anti-TAA binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a humanized or human anti-TAA binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-TAA binding domain described herein, e.g., a humanized or human anti-TAA binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs.
- LC CDR1 light chain complementary determining region 1
- HC CDR2 light chain complementary determining region 2
- HC CDR3 light chain complementary determining region 3
- the humanized or human anti-TAA binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-TAA binding domain described herein, e.g., the humanized or human anti- TAA binding domain has two variable heavy chain regions, each comprising a HC CDR1, a HC CDR2 and a HC CDR3 described herein.
- the humanized or human anti-TAA binding domain comprises a humanized or human light chain variable region described herein and/or a humanized or human heavy chain variable region described herein.
- the humanized or human anti-TAA binding domain comprises a humanized heavy chain variable region described herein, e.g. , at least two humanized or human heavy chain variable regions described herein.
- the anti-TAA binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence provided herein.
- the TFP comprises an anti-TAA binding domain binds to an antigen derived from antigens of alpha-actinin-4, ARTC1, BCR-ABL fusion protein (b3a2), B- RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDK12, CDKN2A, CLPP, COA-l, CSNK1A1, dek-can fusion protein, EFTUD2, Elongation factor 2, ETV6-AML1 fusion protein, FLT3-ITD,
- FNDC3B FN1, GAS7, GPNMB, HAUS3, HSDL1, LDLR-fucosyltransferase AS fusion protein, HLA- A2d, HLA-A1 ld, hsp70-2, MART2, MATN, ME1, MUM-lf, MUM-2, MUM-3, neo-PAP, Myosin class I, NFYC, OGT, OS-9, p53, pml-RARalpha fusion protein, PPP1R3B, PRDX5, PTPRK, K-ras, N-ras, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, TGF-betaRII, triosephosphate isomerase, BAGE-l, D393-CD20n, Cyclin-Al, GAGE-l, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7
- ILl3Ralpha2 Intestinal carboxyl esterase, alpha-foetoprotein, Kallikrein 4, KIF20A, Lengsin, M-CSF, MCSP, mdm-2, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, p53, PAX5, PBF, PRAME, PSMA, RAGE-l, RGS5, RhoC, RNF43, RU2AS, secemin 1, SOX10, STEAP1, survivin, Telomerase, TPBG, VEGF, and WT1.
- the TFP comprises one or more of an anti-CDl9 binding domain, an anti-B-cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-IL 13Ra2 binding domain, an anti-MUCl6 binding domain, an anti-CD22 binding domain, an anti -PD- 1 binding domain, an anti-BAFF or BAFF receptor binding domain, and an anti-ROR-l binding domain.
- BCMA anti-B-cell maturation antigen
- MSLN anti-mesothelin
- MSLN anti-IL 13Ra2 binding domain
- an anti-MUCl6 binding domain an anti-CD22 binding domain
- an anti-PD- 1 binding domain an anti-BAFF or BAFF receptor binding domain
- an anti-ROR-l binding domain an anti-ROR-l binding domain
- the TFP constructs disclosed herein comprise a blocking domain.
- the blocking domain is tethered to the antigen binding domain via a protease-cleavable linker.
- the blocking domain is tethered to a TCR complex subunit via a protease-cleavable linker.
- the blocking domain may sterically hinder the binding of the engineered T cell to the target antigen.
- the blocking domain is albumin.
- the blocking domain is an IgG.
- the blocking domain is a peptide capable of sterically blocking the activity of a biologically active molecule, e.g., the binding of the TAA -binding domain to its target.
- the TFP does not comprise a blocking domain but rather comprises an antibody specific to a protein suitable for blocking activity of the TFP.
- the TFP comprises an anti-HSA antibody.
- Human serum albumin (molecular mass ⁇ 67 kDa) is the most abundant protein in plasma, present at about 50 mg/ml (600 mM), and has a serum half-life of around 20 days in humans. HSA serves to maintain plasma pH, contributes to colloidal blood pressure, functions as carrier of many metabolites and fatty acids, and serves as a major drug transport protein in plasma.
- the serum albumin is human; in some embodiments, the serum albumin is mouse or cynomolgus monkey albumin. In some embodiments, the serum albumin is the same serum albumin as is natively found in the subject to which the albumin-modified protein will be administered (see, e.g., U.S. Patent No. 9,920, 115).
- the TFP of the invention comprises a target -specific binding element otherwise referred to as an antigen binding domain.
- the choice of moiety depends upon the type and number of target antigen that define the surface of a target cell.
- the antigen binding domain may be chosen to recognize a target antigen that acts as a cell surface marker on target cells associated with a particular disease state.
- examples of cell surface markers that may act as target antigens for the antigen binding domain in a TFP of the invention include those associated with viral, bacterial and parasitic infections; autoimmune diseases; and cancerous diseases (e.g., malignant diseases).
- the antigen is a tumor-associated antigen.
- the blocking domain e.g., albumin or another bulky protein
- the antigen-binding domain e.g., wherein the antigen-binding domain is attached to the TFP T cell via a protease-cleavable linker.
- the albumin will be attached via a short linker (e.g., a protease-cleavable linker).
- a subject' s own albumin will be attracted to the antigen-binding domain via an albumin-binding protein, such as an antibody or fragment thereof.
- a single albumin molecule, directly attached or attracted via an albumin binding protein will be enough to block activity.
- two or more albumin molecules, directly attached or attracted via an albumin binding protein will be used.
- the TFP-mediated T cell response can be directed to an antigen of interest by way of engineering an antigen-binding domain into the TFP that specifically binds a desired antigen.
- the antigen binding domain can be any domain that binds to the antigen.
- the antigen binding domain comprises an antibody or a fragment thereof, including but not limited to: a monoclonal antibody; a polyclonal antibody; a recombinant antibody; a human antibody; a humanized antibody or a functional fragment thereof, including but not limited to a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of a camelid-derived nanobody; and to an alternative scaffold known in the art to function as antigen binding domain, such as a recombinant fibronectin domain, anticabn, DARPIN and the like.
- VH heavy chain variable domain
- VL light chain variable domain
- VHH variable domain
- a natural or synthetic ligand specifically recognizing and binding the target antigen can be used as antigen binding domain for the TFP, e.g., an NKG2D dimer or another binder comprising, e.g., a binder that participates in a receptor-ligand interaction.
- the antigen binding domain it is beneficial for the antigen binding domain to be derived from the same species in which the TFP will ultimately be used in.
- the antigen binding domain of the TFP may comprise human or humanized residues for the antigen binding domain of an antibody or antibody fragment.
- the antigen-binding domain comprises a humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment.
- the humanized or human anti-TAA binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a humanized or human anti-TAA binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-TAA binding domain described herein, e.g., a humanized or human anti-TAA binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs.
- LC CDR1 light chain complementary determining region
- the humanized or human anti-TAA binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-TAA binding domain described herein, e.g., the humanized or human anti-tumor-associated antigen binding domain has two variable heavy chain regions, each comprising a HC CDR1, a HC CDR2 and a HC CDR3 described herein.
- the humanized or human anti-TAA binding domain comprises a single domain (sdAb) antibody.
- the humanized or human anti-tumor- associated antigen binding domain comprises a humanized or human light chain variable region described herein and/or a humanized or human heavy chain variable region described herein. In one embodiment, the humanized or human anti-tumor-associated antigen binding domain comprises a humanized heavy chain variable region described herein, e.g., at least two humanized or human heavy chain variable regions described herein. In one embodiment, the anti-tumor-associated antigen binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence provided herein.
- the anti -tumor-associated antigen binding domain (e.g., an scFv or V H H) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity with an amino acid sequence provided herein; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30,
- the humanized or human anti-tumor-associated antigen binding domain is a scFv, and a light chain variable region comprising an amino acid sequence described herein, is attached to a heavy chain variable region comprising an amino acid sequence described herein, via a linker, e.g., a linker described herein.
- the humanized anti-tumor-associated antigen binding domain includes a (Gly 4 -Ser) n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4.
- the light chain variable region and heavy chain variable region of a scFv can be, e.g., in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.
- the linker sequence comprises a long linker (LL) sequence.
- the linker sequence comprises a short linker (SL) sequence.
- a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human or fragment thereof.
- the antigen binding domain is humanized.
- a humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein in its entirety by reference), veneering or resurfacing (see, e.g., European Patent Nos.
- framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, for example improve, antigen binding.
- framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entireties.)
- a humanized antibody or antibody fragment has one or more amino acid residues remaining in it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as "import” residues, which are typically taken from an “import” variable domain. As provided herein, humanized antibodies or antibody fragments comprise one or more CDRs from nonhuman
- immunoglobulin molecules and framework regions wherein the amino acid residues comprising the framework are derived completely or mostly from human germline.
- Humanized antibodies and antibody fragments substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species.
- Humanized antibodies are often human antibodies in which some CDR residues and possibly some framework (FR) residues are substituted by residues from analogous sites in rodent antibodies. Humanization of antibodies and antibody fragments can also be achieved by veneering or resurfacing
- variable domains both light and heavy
- the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity.
- sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable -domain sequences.
- the human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et ak, J. Immunol., 151:2296 (1993); Chothia et ak, J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference herein in their entirety).
- Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
- the same framework may be used for several different humanized antibodies (see, e.g., Nicholson et ak Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et ak, Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et ak, J. Immunol., 151:2623 (1993), the contents of which are incorporated herein by reference herein in their entirety).
- the framework region e.g., all four framework regions, of the heavy chain variable region are derived from a VH4-4-59 germline sequence.
- the framework region can comprise, one, two, three, four or five modifications, e.g., substitutions, e.g., from the amino acid at the corresponding murine sequence.
- the framework region e.g., all four framework regions of the light chain variable region are derived from a VK3-1.25 germline sequence.
- the framework region can comprise, one, two, three, four or five modifications, e.g., substitutions, e.g., from the amino acid at the corresponding murine sequence.
- the portion of a TFP composition of the invention that comprises an antibody fragment is humanized with retention of high affinity for the target antigen and other favorable biological properties.
- humanized antibodies and antibody fragments are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences.
- Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind the target antigen.
- FR residues can be selected and combined from the recipient and import sequences so that the desired antibody or antibody fragment characteristic, such as increased affinity for the target antigen, is achieved.
- the CDR residues are directly and most substantially involved in influencing antigen binding.
- the anti-tumor-associated antigen binding domain is a fragment, e.g., a single chain variable fragment (scFv) or a camelid heavy chain (VHH).
- the anti-tumor-associated antigen binding domain is a Fv, a Fab, a (Fab' )2, or a bi-fimctional (e.g. bi-specific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)).
- the antibodies and fragments thereof of the invention binds a tumor-associated antigen protein with wild-type or enhanced affinity.
- Also provided herein are methods for obtaining an antibody antigen binding domain specific for a target antigen comprising providing by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a V H (or V H H) domain set out herein a V H domain which is an amino acid sequence variant of the V H domain, optionally combining the V H domain thus provided with one or more V L domains, and testing the V H domain or V H /V L combination or combinations to identify a specific binding member or an antibody antigen binding domain specific for a target antigen of interest and optionally with one or more desired properties.
- VH domains, VHH domains, and scFvs can be prepared according to method known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
- scFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers.
- the scFv molecules comprise a linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition. The linker length can greatly affect how the variable regions of a scFv fold and interact.
- the linker sequence comprises a long linker (LL) sequence.
- the linker sequence comprises a short linker (SL) sequence.
- Blocking of biological activity must be reversible for the molecule to be useful.
- Blocking can be removed by removal of the sterically blocking domain.
- the blocking domain is removed by proteolytic cleavage of the linker by which the blocking domain is attached to the TFP T cell.
- the blocking domain is removed by proteolytic cleavage of the linker by which an anti-blocking domain antibody (e.g., an anti-HSA antibody) is removed by proteolytic cleavage of the linker by which the blocking domain is attached to the TFP T cell.
- the blocking domain is removed by enzymatic cleavage other than proteolytic cleavage.
- a feature of this invention is that removal of the blocking domain is enzymatic and not merely a function of elapsed time.
- the blocking domain is removed at the site of disease by an enzyme overproduced in diseased tissue.
- the domain is removed in a tumor environment by a protease produced by tumor cells.
- the protease -cleavable linker comprises a sequence recognized by a protease produced by tumor cells in the tumor microenvironment (TME).
- a "blocked" TFP T cell i.e., a TFP T cell having a blocking domain bound by a protease-cleavable linker or a TFP T cell having a blocking domain bound by an binding moiety (e.g., an antibody), will remain blocked until reaching the TME, i.e., will be in the presence of a protease that will recognize and cleave the linker that keeps the blocking domain tethered to the TFP T cell.
- an binding moiety e.g., an antibody
- the protease is at least one of a tumor cell surface protease, a
- carboxypeptidase carboxypeptidase, a cathepsin, a kallikrein, a hexokinase, a plasmin, a stromelysin, factor Xa, a chymotrypsin-like protease, a trypsin-like protease, a elastase-like protease, a tryptase, a chymase, a subtilisin-like protease, an actinidain, a proteinase, a bromelain, a calpain, a caspase, a cysteine protease, a papain, an FHV-l protease, an HSV protease, a CMV protease, a chymosin, a renin, a pepsin, a matriptase, a plasmepsin, a nepen
- the cathepsin is cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, or cathepsin F;
- the hexokinase is hKl, hKlO, or hKl5;
- the proteinase is PR-3;
- the caspase is caspase-3;
- the cysteine protease is Mir 1 -CP or legumain;
- the matrix metalloproteinase or collagenase is
- MMP l/interstitial collagenase MMP2/type IV collagenase, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP 12, MMP 13, MMP 14, MMP15, MMP 16, ADAM 10, or ADAM 12;
- the prostate -specific antigen is PSA or hK3
- the FAP is FAP-a: the granzyme is granzyme M or granzyme B; or the dipeptidyl peptidase is dipeptidyl peptidase IV (DPPIV/CD26).
- more than one proteolytic cleavage site is present.
- the linker of the blocking domain comprises a protease-cleavable linker.
- the protease-cleavable linker is engineered to comprise a sequence recognized by a protease typically present in the tumor microenvironment (TME).
- TAE tumor microenvironment
- MMP matrix metalloprotease
- MMPs examples include MMP1; MMP2; MMP3; MMP7; MMP8; MMP9; MMP 10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP 16; MMP17; MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27.
- the protease- cleavable linker is a substrate for MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19.
- the protease-cleavable linker is a substrate for MMP9.
- the protease-cleavable linker is a substrate for MMP 14.
- the protease-cleavable linker is a substrate for two or more MMPs. In some embodiments, the protease- cleavable linker is a substrate for at least MMP9 and MMP14. In some embodiments, the protease- cleavable linker comprises two or more substrates for the same MMP. In some embodiments, the protease-cleavable linker comprises at least two or more MMP9 substrates. In some embodiments, the protease-cleavable linker comprises at least two or more MMP14 substrates.
- the protease-cleavable linker is a substrate for an MMP and includes the sequence ISSGFFSS (SEQ ID NO:43); QNQAFRMA (SEQ ID NO:44); AQNLLGMV (SEQ ID NO:45); STFPFGMF (SEQ ID NO:46); PVGYTSSL (SEQ ID NO:47); DWLYWPGI (SEQ ID NO:48); MIAPVAYR (SEQ ID NO:49); RPSPMWAY (SEQ ID NO:50); WATPRPMR (SEQ ID NO:5 l); FRLLDWQW (SEQ ID NO:52); LKAAPRWA (SEQ ID NO:53); GPSHLVLT (SEQ ID NO:54); LPGGLSPW (SEQ ID NO:55);
- MGLFSEAG SEQ ID NO:56
- SPLPLRVP SEQ ID NO:57
- RMHLRSLG SEQ ID NO:58
- LAAPLGLL SEQ ID NO:59
- AVGLLAPP SEQ ID NO:60
- LLAPSHRA SEQ ID NO:6l
- PAGLWLDP (SEQ ID NO:62); and/or ISSGLSS (SEQ ID NO:63).
- X 28 is A, D, or G; and X 29 is C or Y.
- an anti-tumor-associated antigen binding domain e.g., scFv molecules (e.g., soluble scFv)
- scFv molecules e.g., soluble scFv
- biophysical properties e.g., thermal stability
- the humanized or human scFv has a thermal stability that is greater than about 0.1, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10 degrees, about 11 degrees, about 12 degrees, about 13 degrees, about 14 degrees, or about 15 degrees Celsius than a parent scFv in the described assays.
- the improved thermal stability of the anti-tumor-associated antigen binding domain e.g., scFv is subsequently conferred to the entire tumor-associated antigen-TFP construct, leading to improved therapeutic properties of the anti-tumor-associated antigen TFP construct.
- the thermal stability of the anti-tumor-associated antigen binding domain e.g., an scFv or sdAb, can be improved by at least about 2 ° C or 3 ° C as compared to a conventional antibody.
- the anti -tumor-associated antigen binding domain e.g., an scFv or sdAb, has a 1 ° C improved thermal stability as compared to a conventional antibody.
- the anti-tumor-associated antigen binding domain e.g., an scFv or sdAb
- the scFv has a 4 ° C, 5 ° C, 6 ° C, 7 ° C, 8 ° C, 9 ° C, 10 ° C, 11 ° C, 12 ° C, 13 ° C, 14 ° C, or 15 ° C improved thermal stability as compared to a conventional antibody. Comparisons can be made, for example, between the scFv molecules disclosed herein and scFv molecules or Fab fragments of an antibody from which the scFv VH and VL were derived.
- Thermal stability can be measured using methods known in the art. For example, in one embodiment, TM can be measured. Methods for measuring TM and other methods of determining protein stability are described below.
- Mutations in an scFv or an sdAb (arising through humanization or mutagenesis of the soluble scFv or sdAb) alter the stability of the scFv and improve the overall stability of the scFv and the anti- tumor-associated antigen TFP construct. Stability of the humanized scFv is compared against the murine scFv using measurements such as TM, temperature denaturation and temperature aggregation.
- the anti-tumor-associated antigen binding domain e.g., a scFv
- the anti-tumor-associated antigen binding domain comprises at least one mutation arising from the humanization process such that the mutated scFv confers improved stability to the anti-tumor-associated antigen TFP construct.
- the anti-tumor-associated antigen binding domain e.g., scFv comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations arising from the humanization process such that the mutated scFv confers improved stability to the tumor-associated antigen-TFP construct.
- the antigen binding domain of the TFP comprises an amino acid sequence that is homologous to an antigen binding domain amino acid sequence described herein, and the antigen binding domain retains the desired functional properties of the anti-tumor-associated antigen antibody fragments described herein.
- the TFP composition of the invention comprises an antibody fragment.
- that antibody fragment comprises a scFv.
- the antigen binding domain of the TFP is engineered by modifying one or more amino acids within one or both variable regions (e.g., VH and/or VL), for example within one or more CDR regions and/or within one or more framework regions.
- the TFP composition of the invention comprises an antibody fragment.
- that antibody fragment comprises a scFv.
- the antibody or antibody fragment of the invention may further be modified such that they vary in amino acid sequence (e.g., from wild-type), but not in desired activity.
- additional nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues may be made to the protein.
- a nonessential amino acid residue in a molecule may be replaced with another amino acid residue from the same side chain family.
- a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members, e.g., a conservative substitution, in which an amino acid residue is replaced with an amino acid residue having a similar side chain, may be made.
- Families of amino acid residues having similar side chains have been defined in the art, including 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
- basic side chains e.g., lysine, arginine, histidine
- acidic side chains e.g., aspartic acid
- Percent identity in the context of two or more nucleic acids or polypeptide sequences refers to two or more sequences that are the same. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 70%, 71% , 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
- the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
- sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
- test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
- sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math.
- BLAST and BLAST 2.0 algorithms Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et ah, (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et ah, (1990) J. Mol. Biol. 215:403-410, respectively.
- Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
- the present invention contemplates modifications of the starting antibody or fragment (e.g., scFv) amino acid sequence that generate functionally equivalent molecules.
- the VH or VL of an anti-tumor-associated antigen binding domain, e.g., scFv, comprised in the TFP can be modified to retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,
- the present invention contemplates modifications of the entire TFP construct, e.g., modifications in one or more amino acid sequences of the various domains of the TFP construct in order to generate functionally equivalent molecules.
- the TFP construct can be modified to retain at least about
- a short oligo- or polypeptide linker may form the linkage between the transmembrane domain and the cytoplasmic region of the TFP.
- a glycine - serine doublet provides a particularly suitable linker.
- the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 181).
- the linker is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 182).
- the linker comprises the amino acid sequence of GGGGSGGGGSGGGGSLE (SEQ ID NO: 183).
- the linker comprises the amino acid sequence of
- the linker is a long linker having the sequence AAIEVMYPPPYLGGGGSGGGGSGGGGSLE (SEQ ID NO: 185). In some embodiments, the linker is encoded by a nucleotide sequence of
- the linker is encoded by a nucleotide sequence of
- the extracellular domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any protein, but in particular a membrane- bound or transmembrane protein. In one aspect, the extracellular domain is capable of associating with the transmembrane domain.
- An extracellular domain of particular use in this invention may include at least the extracellular region(s) of e.g., the alpha, beta or zeta chain of the T cell receptor, or CD3 epsilon, CD3 gamma, or CD3 delta, or in alternative embodiments, CD28, CD45, CD2, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
- a TFP sequence contains an extracellular domain and a transmembrane domain encoded by a single genomic sequence.
- a TFP can be designed to comprise a transmembrane domain that is heterologous to the extracellular domain of the TFP.
- a transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids of the intracellular region).
- amino acid associated with the extracellular region of the protein from which the transmembrane was derived e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids of the extracellular region
- the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the extracellular region. In some cases, the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the intracellular region. In one aspect, the transmembrane domain is one that is associated with one of the other domains of the TFP is used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerization with another TFP on the TFP T cell surface. In a different aspect the amino acid sequence of the
- transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same TFP.
- the transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane -bound or transmembrane protein. In one aspect, the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TFP has bound to a target.
- a transmembrane domain of particular use in this invention may include at least the transmembrane region(s) of e.g., the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
- the transmembrane domain can be attached to the extracellular region of the TFP, e.g. , the antigen binding domain of the TFP, via a hinge, e.g. , a hinge from a human protein.
- a hinge e.g. , a hinge from a human protein.
- the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, or a CD8a hinge.
- the cytoplasmic domain of the TFP can include an intracellular signaling domain, if the TFP contains CD3 gamma, delta or epsilon polypeptides; TCR alpha and TCR beta subunits are generally lacking in a signaling domain.
- An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the TFP has been introduced.
- effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
- intracellular signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.
- intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
- intracellular signaling domains for use in the TFP of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.
- TCR T cell receptor
- naive T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen -independent manner to provide a secondary or costimulatory signal
- secondary cytoplasmic domain e.g., a costimulatory domain
- a primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
- Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine -based activation motifs (ITAMs).
- ITAMs immunoreceptor tyrosine -based activation motifs
- ITAMs containing primary intracellular signaling domains that are of particular use in the invention include those of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
- a TFP of the invention comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3-epsilon.
- a primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain.
- a primary signaling domain comprises a modified ITAM -containing primary intracellular signaling domain, e.g., an optimized and/or truncated ITAM -containing primary intracellular signaling domain.
- a primary signaling domain comprises one, two, three, four or more ITAM motifs.
- the intracellular signaling domain of the TFP can comprise the CD3 zeta signaling domain by itself or it can be combined with any other desired intracellular signaling domain(s) useful in the context of a TFP of the invention.
- the intracellular signaling domain of the TFP can comprise a CD3 epsilon chain portion and a costimulatory signaling domain.
- the costimulatory signaling domain refers to a portion of the TFP comprising the intracellular domain of a costimulatory molecule.
- a costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen.
- LFA-l lymphocyte function-associated antigen-l
- CD2, CD7, LIGHT, NKG2C, B7-H3 a ligand that specifically binds with CD83, and the like.
- CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human TFP T cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al. Blood. 2012; 119(3):696-706).
- the intracellular signaling sequences within the cytoplasmic portion of the TFP of the invention may be linked to each other in a random or specified order.
- a short oligo- or polypeptide linker for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequences.
- a glycine-serine doublet can be used as a suitable linker.
- a single amino acid e.g., an alanine, a glycine, can be used as a suitable linker.
- the TFP-expressing cell described herein can further comprise a second TFP, e.g., a second TFP that includes a different antigen binding domain, e.g., to the same target (e.g., CD22) or a different target (e.g., CD123).
- a second TFP e.g., a second TFP that includes a different antigen binding domain, e.g., to the same target (e.g., CD22) or a different target (e.g., CD123).
- the antigen binding domains of the different TFPs can be such that the antigen binding domains do not interact with one another.
- a cell expressing a first and second TFP can have an antigen binding domain of the first TFP, e.g., as a fragment, e.g., a scFv, that does not associate with the antigen binding domain of the second TFP, e.g., the antigen binding domain of the second TFP is a VHH.
- the TFP-expressing cell described herein can further express another agent, e.g., an agent which enhances the activity of a TFP-expressing cell.
- the agent can be an agent which inhibits an inhibitory molecule.
- Inhibitory molecules, e.g., PD 1 can, in some embodiments, decrease the ability of a TFP-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 and TGFR beta.
- the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein.
- the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4 and TIGIT, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 4-1BB, CD27 or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein).
- an inhibitory molecule such as PD1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4 and TIGIT
- a fragment of any of these e.g., at least a portion of an extracellular domain of any of these
- a second polypeptide which is an intracellular signal
- the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of an extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein).
- PD1 is an inhibitory member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA.
- PD-l is expressed on activated B cells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75).
- PD-L1 Two ligands for PD1, PD-L1 and PD- L2 have been shown to downregulate T cell activation upon binding to PD1 (Freeman et al. 2000 J Exp Med 192: 1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634- 43).
- PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immune suppression can be reversed by inhibiting the local interaction of PD1 with PD-L1.
- the agent comprises the extracellular domain (ECD) of an inhibitory molecule, e.g., Programmed Death 1 (PD1) can be fused to a transmembrane domain and optionally an intracellular signaling domain such as 41BB and CD3 zeta (also referred to herein as a PD1 TFP).
- the PD1 TFP when used in combinations with an anti -tumor antigen TFP described herein, improves the persistence of the T cell.
- the TFP is a PD1 TFP comprising the extracellular domain of PD 1.
- TFPs containing an antibody or antibody fragment such as a scFv that specifically binds to the Programmed Death -Ligand 1 (PD-L1) or
- the present invention provides a population of TFP -expressing T cells, e.g., TFP T cells.
- the population of TFP-expressing T cells comprises a mixture of cells expressing different TFPs.
- the population of TFP T cells can include a first cell expressing a TFP having an anti-tumor-associated antigen binding domain described herein, and a second cell expressing a TFP having a different anti-tumor-associated antigen binding domain, e.g., an anti-tumor-associated antigen binding domain described herein that differs from the anti- tumor-associated antigen binding domain in the TFP expressed by the first cell.
- the population of TFP-expressing cells can include a first cell expressing a TFP that includes an anti -tumor- associated antigen binding domain, e.g., as described herein, and a second cell expressing a TFP that includes an antigen binding domain to a target other than tumor-associated antigen (e.g., another tumor- associated antigen).
- a target other than tumor-associated antigen e.g., another tumor- associated antigen
- the present invention provides a population of cells wherein at least one cell in the population expresses a TFP having an anti-tumor-associated antigen domain described herein, and a second cell expressing another agent, e.g., an agent which enhances the activity of a TFP-expressing cell.
- the agent can be an agent which inhibits an inhibitory molecule.
- Inhibitory molecules can, in some embodiments, decrease the ability of a TFP-expressing cell to mount an immune effector response.
- inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.
- the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein.
- a first polypeptide e.g., an inhibitory molecule
- a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein.
- a method for generating mRNA for use in transfection can involve in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3 ' and 5 ' untranslated sequence ("UTR"), a 5 ' cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length.
- RNA so produced can efficiently transfect different kinds of cells.
- the template includes sequences for the TFP.
- the anti-TAA TFP is encoded by a circular RNA comprising a sequence encoding a TFP, a CAR, a TCR or combination thereof.
- circular RNA is exogenous.
- circular RNA is endogenous.
- circular RNAs with an internal ribosomal entry site (IRES) can be translated in vitro or ex vivo.
- Circular RNAs are a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. Circular RNAs are 3-5 ' covalently closed RNA rings, and circular RNAs do not display Cap or poly(A) tails. Since circular RNAs lack the free ends necessary for exonuclease-mediated degradation, rendering them resistant to several mechanisms of RNA turnover and granting them extended lifespans as compared to their linear mRNA counterparts. For this reason, circularization may allow for the stabilization of mRNAs that generally suffer from short serum half-lives and may therefore improve the overall efficacy of mRNA in a variety of applications.
- Circular RNAs are produced by the process of splicing, and circularization occurs using conventional splice sites mostly at annotated exon boundaries (Starke et ah, 2015; Szabo et ah, 2015).
- splice sites are used in reverse: downstream splice donors are "backspliced" to upstream splice acceptors (see Jeck and Sharpless, 2014; Barrett and Salzman, 2016; Szabo and Salzman, 2016; Holdt et ah, 2018 for review).
- the method for generating circular RNA comprises in vitro transcription (IVT) of a precursor linear RNA template with specially designed primers.
- IVT in vitro transcription
- Three general strategies have been reported so far for RNA circularization: chemical methods using cyanogen bromide or a similar condensing agent; enzymatic methods using RNA or DNA ligases; and ribozymatic methods using self-splicing introns.
- precursor RNA is synthesized by run-off transcription and then heated in the presence of magnesium ions and GTP to promote circularization. RNA so produced can efficiently transfect different kinds of cells.
- PCR is used to generate a template for in vitro transcription of linear precursor RNA which is used for transfection.
- Methods for performing PCR are well known in the art.
- the anti-TAA TFP is encoded by a circular RNA.
- the circular RNA encoding the anti-TAA TFP is introduced into a T cell for production of a TFP T cell.
- the in vitro transcribed RNA TFP can be introduced to a cell as a form of transient transfection.
- the circular RNA encoding the anti-TAA TFP is introduced to a subject via a targeted nanoparticle.
- the anti-TAA TFP is encoded by a messenger RNA (mRNA).
- the mRNA encoding the anti-tumor-associated antigen TFP is introduced into a T cell for production of a TFP T cell.
- the in vitro transcribed RNA TFP can be introduced to a cell as a form of transient transfection.
- the RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template.
- DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase.
- the source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.
- the desired template for in vitro transcription is a TFP of the present invention.
- the DNA to be used for PCR contains an open reading frame.
- the DNA can be from a naturally occurring DNA sequence from the genome of an organism.
- the nucleic acid can include some or all of the 5 ' and/or 3 ' untranslated regions (UTRs).
- the nucleic acid can include exons and introns.
- the DNA to be used for PCR is a human nucleic acid sequence.
- the DNA to be used for PCR is a human nucleic acid sequence including the 5 ' and 3 ' UTRs.
- the DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism.
- An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.
- PCR is used to generate a template for in vitro transcription of mRNA which is used for transfection.
- Methods for performing PCR are well known in the art.
- Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR.
- “Substantially complementary,” as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary, or one or more bases are non complementary, or mismatched. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR.
- the primers can be designed to be substantially complementary to any portion of the DNA template.
- the primers can be designed to amplify the portion of a nucleic acid that is normally transcribed in cells (the open reading frame), including 5 ' and 3 ' UTRs.
- the primers can also be designed to amplify a portion of a nucleic acid that encodes a particular domain of interest.
- the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5 ' and 3 ' UTRs. Primers useful for PCR can be generated by synthetic methods that are well known in the art.
- Forward primers are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified.
- Upstream is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand.
- reverse primers are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified.
- Downstream is used herein to refer to a location 3 ' to the DNA sequence to be amplified relative to the coding strand.
- Any DNA polymerase useful for PCR can be used in the methods disclosed herein.
- the reagents and polymerase are commercially available from a number of sources.
- the RNA preferably has 5 ' and 3 ' UTRs.
- the 5 ' UTR is between one and 3,000 nucleotides in length.
- the length of 5 ' and 3 ' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5 ' and 3 ' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.
- the 5 ' and 3 ' UTRs can be the naturally occurring, endogenous 5 ' and 3 ' UTRs for the nucleic acid of interest.
- UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template.
- the use of UTR sequences that are not endogenous to the nucleic acid of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3 ' UTR sequences can decrease the stability of mRNA. Therefore, 3 ' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
- the 5 ' UTR can contain the Kozak sequence of the endogenous nucleic acid.
- a consensus Kozak sequence can be redesigned by adding the 5 ' UTR sequence.
- Kozak sequences can increase the efficiency of translation of some RNA transcripts but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art.
- the 5 ' UTR can be 5 ' UTR of an RNA virus whose RNA genome is stable in cells.
- various nucleotide analogues can be used in the 3 ' or 5 ' UTR to impede exonuclease degradation of the mRNA.
- a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed.
- the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed.
- the promoter is a T7 polymerase promoter, as described elsewhere herein.
- Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.
- the mRNA has both a cap on the 5 ' end and a 3 ' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell.
- RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells.
- the transcription of plasmid DNA linearized at the end of the 3 ' UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription.
- phage T7 RNA polymerase can extend the 3 ' end of the transcript beyond the last base of the template (Schenbom and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270: 1485-65 (2003).
- the polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100 T tail (size can be 50-5000 Ts), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination.
- Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.
- Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly (A) polymerase, such as E. coli polyA polymerase (E-PAP).
- E-PAP E. coli polyA polymerase
- increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two fold increase in the translation efficiency of the RNA.
- the attachment of different chemical groups to the 3 ' end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds.
- ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.
- RNAs produced by the methods disclosed herein include a 5 ' cap.
- the 5 ' cap is provided using techniques known in the art and described herein (Cougot, et ah, Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et ah, RNA, 7: 1468-95 (2001); Elango, et ak, Biochim. Biophys. Res. Commun., 330:958-966 (2005)).
- RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence.
- IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.
- RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa), electroporation (Amaxa), electroporation (Amaxa), electroporation (Amaxa), electroporation (Amaxa), electroporation (Amaxa), electroporation (Amaxa), electroporation (Amaxa), electroporation (Amaxa), electroporation (Amaxa)
- Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as "gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8): 861 -70 (2001).
- the present invention also provides nucleic acid molecules encoding one or more TFP constructs described herein.
- the nucleic acid molecule is provided as a messenger RNA transcript.
- the nucleic acid molecule is provided as a DNA construct.
- nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques.
- the gene of interest can be produced synthetically, rather than cloned.
- the present invention also provides vectors in which a DNA of the present invention is inserted.
- Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
- Lentiviral vectors have the added advantage over vectors derived from onco -retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
- the vector comprising the nucleic acid encoding the desired TFP of the invention is an adenoviral vector (A5/35).
- modified T cells comprising the recombinant nucleic acid disclosed herein, or the vectors disclosed herein, wherein the modified T cell comprises a functional disruption of an endogenous TCR.
- modified T cells comprising the sequence encoding the TFP of the nucleic acid disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein, wherein the modified T cell comprises a functional disruption of an endogenous TCR.
- allogenic modified T cells comprising the sequence encoding the inducible TFP disclosed herein or an inducible TFP encoded by the sequence of the nucleic acid disclosed herein.
- the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain or a TCR alpha constant domain and a TCR beta constant domain.
- the endogenous TCR that is functionally disrupted is an endogenous TCR alpha chain, an endogenous TCR beta chain, or an endogenous TCR alpha chain and an endogenous TCR beta chain.
- the endogenous TCR that is functionally disrupted has reduced binding to MHC -peptide complex compared to that of an unmodified control T cell.
- the functional disruption is a disruption of a gene encoding the endogenous TCR.
- the disruption of a gene encoding the endogenous TCR is a removal of a sequence of the gene encoding the endogenous TCR from the genome of a T cell.
- the T cell is a human T cell.
- the T cell is a CD8+ or CD4+ T cell or CD4+CD8+ T cell.
- the T cell is an allogenic T cell.
- the modified T cells further comprise a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide comprising at least a portion of an inhibitory molecule, associated with a second polypeptide comprising a positive signal from an intracellular signaling domain.
- the inhibitory molecule comprises the first polypeptide comprising at least a portion of PD 1 and the second polypeptide comprising a costimulatory domain and primary signaling domain.
- a method of producing the modified T cell of the disclosure comprising (a) disrupting an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, or any combination thereof; thereby producing a T cell containing a functional disruption of an endogenous TCR gene; and (b) transducing the T cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid of the disclosure, or the vectors disclosed herein.
- disrupting comprises transducing the T cell with a nuclease protein or a nucleic acid sequence encoding a nuclease protein that targets the endogenous gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.
- the method comprising transducing a T cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid disclosed herein, or the vectors disclosed herein.
- the T cell containing a functional disruption of an endogenous TCR gene is a T cell containing a functional disruption of an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.
- the T cell is a human T cell. In some instances, the T cell containing a functional disruption of an endogenous TCR gene has reduced binding to MHC-peptide complex compared to that of an unmodified control T cell.
- the nuclease is a meganuclease, a zinc -finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, CRISPR/Cas nickase, or a megaTAL nuclease.
- the sequence comprised by the recombinant nucleic acid or the vector is inserted into the endogenous TCR subunit gene at the cleavage site, and wherein the insertion of the sequence into the endogenous TCR subunit gene functionally disrupts the endogenous TCR subunit.
- the nuclease is a meganuclease.
- the meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence, and wherein the second subunit binds to a second recognition half-site of the recognition sequence.
- the meganuclease is a single chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit.
- the modified T cells disclosed herein are engineered using a gene editing technique such as clustered regularly interspaced short palindromic repeats (CRISPR®, see, e.g., US
- Patent No. 8,697,359 transcription activator-like effector (TALE) nucleases
- TALE transcription activator-like effector
- TALENs transcription activator-like effector nucleases
- meganucleases endodeoxyribonucleases having large recognition sites comprising double -stranded DNA sequences of 12 to 40 base pairs
- ZFN zinc finger nuclease
- ZFN zinc finger nuclease
- megaTAL nucleases a fusion protein of a meganuclease to TAL repeats
- a chimeric construct may be engineered to combine desirable characteristics of each subunit, such as conformation or signaling capabilities. See also Sander & Joung, Nat. Biotech. (2014) v32, 347-55; and June et al., 2009 Nature Reviews Immunol. 9.10: 704- 716, each incorporated herein by reference.
- one or more of the extracellular domain, the transmembrane domain, or the cytoplasmic domain of a TFP subunit are engineered to have aspects of more than one natural TCR subunit domain (i.e., are chimeric).
- gene editing techniques are employed to disrupt an endogenous TCR gene.
- mentioned endogenous TCR gene encodes a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.
- gene editing techniques pave the way for multiplex genomic editing, which allows simultaneous disruption of multiple genomic loci in endogenous TCR gene.
- multiplex genomic editing tecniques are applied to generate gene-disrupted T cells that are deficient in the expression of endogenous TCR, and/or human leukocyte antigens (HLAs), and/or programmed cell death protein 1 (PD1), and/or other genes.
- HLAs human leukocyte antigens
- PD1 programmed cell death protein 1
- CRISPR CRISPR-associated
- DSB double-stranded DNA break
- NHEJ non -homologous end joining
- HR homologous recombination
- SSB single stranded DNA breaks
- DSBs may be repaired by single strand DNA incorporation (ssDI) or single strand template repair (ssTR), an event that introduces the homologous sequence from a donor DNA.
- Genome DNA can be performed using site -specific, rare-cutting endonucleases that are engineered to recognize DNA sequences in the locus of interest.
- Methods for producing engineered, site-specific endonucleases are known in the art.
- ZFNs zinc -finger nucleases
- ZFNs are chimeric proteins comprising a zinc finger DNA-binding domain fused to the nuclease domain of the Fokl restriction enzyme.
- the zinc finger domain can be redesigned through rational or experimental means to produce a protein that binds to a pre-determined DNA sequence -18 basepairs in length.
- TAL-effector nucleases can be generated to cleave specific sites in genomic DNA.
- a TALEN comprises an engineered, site-specific DNA-binding domain fused to the Fokl nuclease domain (reviewed in Mak et al. (2013), Curr Opin Struct Biol. 23:93-9).
- the DNA binding domain comprises a tandem array of TAL-effector domains, each of which specifically recognizes a single DNA base pair.
- Compact TALENs have an alternative
- a Compact TALEN comprises an engineered, site -specific TAL-effector DNA-binding domain fused to the nuclease domain from the I-Tevl homing endonuclease. Unlike Fokl, I-Tevl does not need to dimerize to produce a double-strand DNA break so a Compact TALEN is functional as a monomer.
- Engineered endonucleases based on the CRISPR/Cas9 system are also known in the art (Ran et al. (2013), Nat Protoc. 8:2281-2308; Mali et al. (2013), Nat Methods 10:957-63).
- the CRISPR gene editing technology is composed of an endonuclease protein whose DNA-targeting specificity and cutting activity can be programmed by a short guide RNA or a duplex crRNA/TracrRNA.
- a CRISPR endonuclease comprises two components: (1) a caspase effector nuclease, typically microbial Cas9; and (2) a short "guide RNA” or a RNA duplex comprising a 18 to 20 nucleotide targeting sequence that directs the nuclease to a location of interest in the genome.
- a caspase effector nuclease typically microbial Cas9
- a short "guide RNA” or a RNA duplex comprising a 18 to 20 nucleotide targeting sequence that directs the nuclease to a location of interest in the genome.
- CRISPR systems There are two classes of CRISPR systems known in the art (Adli (2016) Nat. Commun. 9: 191 1), each containing multiple CRISPR types. Class 1 contains type I and type III CRISPR systems that are commonly found in Archaea. And, Class II contains type II, IV, V, and VI CRISPR systems. Although the most widely used CRISPR/Cas system is the the type II CRISPR-Cas9 system, CRISPR/Cas systems have been repurposed by researchers for genome editing. More than 10 different CRISPR/Cas proteins have been remodeled within last few years (Adli (2016) Nat. Commun. 9: 1911). Among these, such as Casl2a (Cpfl) proteins from Acid- aminococcus sp (AsCpfl) and Lachnospiraceae bacterium (LbCpfl), are particularly interesting.
- Cpfl Casl2a
- AsCpfl Acid- aminococcus sp
- LbCpfl La
- Homing endonucleases are a group of naturally occurring nucleases that recognize 15-40 base- pair cleavage sites commonly found in the genomes of plants and fungi. They are frequently associated with parasitic DNA elements, such as group 1 self-splicing introns and inteins. They naturally promote homologous recombination or gene insertion at specific locations in the host genome by producing a double -stranded break in the chromosome, which recruits the cellular DNA-repair machinery (Stoddard (2006), Q. Rev. Biophys. 38: 49-95). Specific amino acid substations could reprogram DNA cleavage specificity of homing nucleases (Niyonzima (2017), Protein Eng Des Sel. 30(7): 503-522).
- Meganucleases are monomeric proteins with innate nuclease activity that are derived from bacterial homing endonucleases and engineered for a unique target site (Gersbach (2016), Molecular Therapy. 24: 430-446).
- meganuclease is engineered I-Crel homing endonuclease.
- meganuclease is engineered I-Scel homing endonuclease.
- chimeric proteins comprising fusions of meganucleases, ZFNs, and TALENs have been engineered to generate novel monomeric enzymes that take advantage of the binding affinity of ZFNs and TALENs and the cleavage specificity of meganucleases (Gersbach (2016), Molecular Therapy. 24: 430-446).
- a megaTAL is a single chimeric protein, which is the combination of the easy-to-tailor DNA binding domains from TALENs with the high cleavage efficiency of meganucleases.
- nucleases In order to perform the gene editing technique, the nucleases, and in the case of the CRISPR/ Cas9 system, a gRNA, must be efficiently delivered to the cells of interest. Delivery methods such as physical, chemical, and viral methods are also know in the art (Mali (2013). Indian J. Hum. Genet. 19: 3- 8.). In some instances, physical delivery methods can be selected from the methods but not limited to electroporation, microinjection, or use of ballistic particles. On the other hand, chemical delivery methods require use of complex molecules such calcium phosphate, lipid, or protein. In some embodiments, viral delivery methods are applied for gene editing techniques using viruses such as but not limited to adenovirus, lentivirus, and retrovirus.
- viruses such as but not limited to adenovirus, lentivirus, and retrovirus.
- the expression constructs of the present invention may also be used for nucleic acid
- the invention provides a gene therapy vector.
- the nucleic acid can be cloned into a number of types of vectors.
- the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
- Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
- the expression vector may be provided to a cell in the form of a viral vector.
- Viral vector technology is well known in the art and is described, e.g., in Sambrook et al., 2012, Molecular Cloning:
- Viruses which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
- a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
- retroviruses provide a convenient platform for gene delivery systems.
- a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
- the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
- retroviral systems are known in the art.
- adenovirus vectors are used.
- a number of adenovirus vectors are known in the art.
- lentivirus vectors are used.
- Additional promoter elements e.g., enhancers, regulate the frequency of transcriptional initiation.
- these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well.
- the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
- tk thymidine kinase
- the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
- individual elements can function either cooperatively or independently to activate transcription.
- a promoter that is capable of expressing a TFP transgene in a mammalian T cell is the EFla promoter.
- the native EFla promoter drives expression of the alpha subunit of the elongation factor-l complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome.
- the EFla promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in driving TFP expression from transgenes cloned into a lentiviral vector (see, e.g., Milone et ah, Mol. Ther. 17(8): 1453-1464 (2009)).
- CMV immediate early cytomegalovirus
- This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
- other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human
- immunodeficiency virus (HIV) long terminal repeat (LTR) promoter MoMuLV promoter
- an avian leukemia virus promoter an Epstein-Barr virus immediate early promoter
- a Rous sarcoma virus promoter as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor- la promoter, the hemoglobin promoter, and the creatine kinase promoter.
- the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention.
- an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired or turning off the expression when expression is not desired.
- inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline -regulated promoter.
- the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
- the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
- Useful selectable markers include, for example, antibiotic -resistance genes, such as neo and the like.
- Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
- a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
- Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
- Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
- the construct with the minimal 5 ' flanking region showing the highest level of expression of reporter gene is identified as the promoter.
- Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.
- the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
- the expression vector can be transferred into a host cell by physical, chemical, or biological means.
- Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art (see, e.g., Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY). One method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection
- Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
- Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.
- Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like (see, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362).
- Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
- An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
- Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system.
- an exemplary delivery vehicle is a liposome.
- lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo, or in vivo).
- the nucleic acid may be associated with a lipid.
- the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
- Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
- Lipids are fatty substances which may be naturally occurring or synthetic lipids.
- lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
- Lipids suitable for use can be obtained from commercial sources.
- DMPC dimyristyl phosphatidylcholine
- DCP dicetyl phosphate
- Choi cholesterol
- DMPG dimyristyl phosphatidylglycerol
- Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 ° C.
- Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates.
- Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium.
- Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al, 1991 Glycobiology 5: 505-10).
- compositions that have different structures in solution than the normal vesicular structure are also encompassed.
- the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
- lipofectamine -nucleic acid complexes are also contemplated.
- assays include, for example, "molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and western blots) or by assays described herein to identify agents falling within the scope of the invention.
- molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR
- biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and western blots) or by assays described herein to identify agents falling within the scope of the invention.
- the present invention further provides a vector comprising a TFP encoding nucleic acid molecule.
- a TFP vector can be directly transduced into a cell, e.g., a T cell.
- the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs.
- the vector is capable of expressing the TFP construct in mammalian T cells.
- the mammalian T cell is a human T cell. Sources of T cells
- a source of T cells is obtained from a subject.
- the term "subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
- T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain aspects of the present invention, any number of T cell lines available in the art, may be used.
- T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll® separation.
- cells from the circulating blood of an individual are obtained by apheresis.
- the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
- the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
- the cells are washed with phosphate buffered saline (PBS).
- PBS phosphate buffered saline
- the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation.
- a washing step may be accomplished by methods known to those in the art, such as by using a semi -automated "flow-through” centrifuge (for example, the COBE® 2991 cell processor, the Baxter CytoMate®, or the Haemonetics® Cell Saver® 5) according to the manufacturer' s instructions.
- a semi -automated "flow-through” centrifuge for example, the COBE® 2991 cell processor, the Baxter CytoMate®, or the Haemonetics® Cell Saver® 5
- the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte® A, or other saline solution with or without buffer.
- the undesirable components of the apheresis sample may be removed, and the cells directly resuspended in culture medium.
- T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a Percoll® gradient or by counterflow centrifugal elutriation.
- a specific subpopulation of T cells such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques.
- T cells are isolated by incubation with anti-CD3/anti-CD28 (e.g., 3x28)-conjugated beads, such as Dynabeads® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells.
- the time period is about 30 minutes. In a further aspect, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the time period is 10 to 24 hours. In one aspect, the incubation time period is 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from TIL.
- TIL tumor infiltrating lymphocytes
- Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
- One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
- a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8.
- it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+.
- T regulatory cells are depleted by anti- C25 conjugated beads or other similar method of selection.
- a T cell population can be selected that expresses one or more of IFN-g, TNF-alpha, IF-17A, IF-2, IF-3, IF-4, GM-CSF, IF-10, IF-13, granzyme B, and perforin, or other appropriate molecules, e.g., other cytokines.
- Methods for screening for cell expression can be determined, e.g., by the methods described in PCT Publication No.: WO2013/126712.
- the concentration of cells and surface can be varied.
- it may be desirable to significantly decrease the volume in which beads and cells are mixed together e.g., increase the concentration of cells, to ensure maximum contact of cells and beads.
- a concentration of 2 billion cells/mF is used.
- a concentration of 1 billion cells/mF is used.
- greater than 100 million cells/mF is used.
- a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mF is used.
- a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mF is used.
- concentrations of 125 or 150 million cells/mF can be used.
- Using high concentrations can result in increased cell yield, cell activation, and cell expansion.
- use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain.
- using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
- the concentration of cells used is 5 x l0 6 /mL. In other aspects, the concentration used can be from about 1 x l0 5 /mL to 1 x l0 6 /mL, and any integer value in between. In other aspects, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10 ° C or at room temperature.
- T cells for stimulation can also be frozen after a washing step.
- the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population.
- the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25%
- cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present invention.
- Also contemplated in the context of the invention is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed.
- the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein.
- a blood sample or an apheresis is taken from a generally healthy subject.
- a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use.
- the T cells may be expanded, frozen, and used at a later time.
- samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments.
- the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and tacrolimus (FK506), antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies, cyclophosphamide, fludarabine, cyclosporin, rapamycin, mycophenolic acid, steroids, romidepsin (formerly FR901228), and irradiation.
- agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and tacrolimus
- T cells are obtained from a patient directly following treatment that leaves the subject with functional T cells.
- the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo.
- these cells may be in a preferred state for enhanced engraftment and in vivo expansion.
- mobilization for example, mobilization with GM-CSF
- conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy.
- Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.
- T cells may be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and 7,572,631.
- the T cells of the invention may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells.
- T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore.
- a ligand that binds the accessory molecule is used for co -stimulation of an accessory molecule on the surface of the T cells.
- a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells.
- an anti-CD3 antibody and an anti-CD28 antibody can be used as can other methods commonly known in the art (Berg et ah, Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol. Meth. 227(l-2):53-63, 1999).
- T cells that have been exposed to varied stimulation times may exhibit different characteristics.
- typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8+).
- TH, CD4+ helper T cell population
- TC cytotoxic or suppressor T cell population
- Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of TC cells.
- infusing a subject with a T cell population comprising predominately of TH cells may be advantageous.
- an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.
- an anti-tumor-associated antigen TFP is constructed, various assays can be used to evaluate the activity of the molecule, such as but not limited to, the ability to expand T cells following antigen stimulation, sustain T cell expansion in the absence of re -stimulation, and anti-cancer activities in appropriate in vitro and animal models. Assays to evaluate the effects of an anti-tumor-associated antigen TFP are described in further detail below
- T cells (1: 1 mixture of CD4+ and CD8+ T cells) expressing the TFPs are expanded in vitro for more than 10 days followed by lysis and SDS-PAGE under reducing conditions. TFPs are detected by western blotting using an antibody to a TCR chain. The same T cell subsets are used for SDS-PAGE analysis under non-reducing conditions to permit evaluation of covalent dimer formation.
- TFP+ T cells following antigen stimulation can be measured by flow cytometry.
- a mixture of CD4+ and CD8+ T cells are stimulated with alphaCD3/alphaCD28 and APCs followed by transduction with lentiviral vectors expressing GFP under the control of the promoters to be analyzed.
- exemplary promoters include the CMV IE gene, EF-lalpha, ubiquitin C, or phosphoglycerokinase (PGK) promoters.
- GFP fluorescence is evaluated on day 6 of culture in the CD4+ and/or CD8+ T cell subsets by flow cytometry (see, e.g., Milone et al., Molecular Therapy 17(8): 1453 - 1464 (2009)).
- flow cytometry see, e.g., Milone et al., Molecular Therapy 17(8): 1453 - 1464 (2009).
- a mixture of CD4+ and CD8+ T cells are stimulated with
- alphaCD3/alphaCD28 coated magnetic beads on day 0 and transduced with TFP on day 1 using a bicistronic lentiviral vector expressing TFP along with eGFP using a 2A ribosomal skipping sequence.
- Sustained TFP+ T cell expansion in the absence of re -stimulation can also be measured (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Briefly, mean T cell volume (fl) is measured on day 8 of culture using a Coulter® Multisizer III particle counter following stimulation with alphaCD3/alphaCD28 coated magnetic beads on day 0, and transduction with the indicated TFP on day 1
- mice can also be used to measure a TFP T cell activity.
- a xenograft model using human BCMA-specific TFP+ T cells to treat a cancer in immunodeficient mice is described in Milone et al., Molecular Therapy 17(8): 1453-1464 (2009).
- mice are randomized as to treatment groups. Different numbers of engineered T cells are coinjected at a 1: 1 ratio into NOD/SCID/Y-/- mice bearing cancer. The number of copies of each vector in spleen DNA from mice is evaluated at various times following T cell injection. Animals are assessed for cancer at weekly intervals.
- Peripheral blood tumor-associated antigen® cancer cell counts are measured in mice that are injected with alpha tumor-associated antigen-zeta TFP+ T cells or mock-transduced T cells. Survival curves for the groups are compared using the log -rank test.
- absolute peripheral blood CD4+ and CD8+ T cell counts 4 weeks following T cell injection in NOD/SCID/Y-/- mice can also be analyzed. Mice are injected with cancer cells and 3 weeks later are injected with T cells engineered to express TFP by a bicistronic lentiviral vector that encodes the TFP linked to eGFP. T cells are nonnalized to 45-50% input GFP+ T cells by mixing with mock-transduced cells prior to injection and confirmed by flow cytometry. Animals are assessed for cancer at l-week intervals. Survival curves for the TFP+ T cell groups are compared using the log-rank test.
- Dose-dependent TFP treatment responses can be evaluated (see, e.g., Milone et ah, Molecular Therapy 17(8): 1453-1464 (2009)).
- peripheral blood is obtained 35-70 days after establishing cancer in mice injected on day 21 with TFP T cells, an equivalent number of mock- transduced T cells, or no T cells. Mice from each group are randomly bled for determination of peripheral blood + cancer cell counts and then killed on days 35 and 49. The remaining animals are evaluated on days 57 and 70.
- Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies are added to cultures with KT32-BBL cells to serve as a positive control for stimulating T cell proliferation since these signals support long-term CD8+ T cell expansion ex vivo.
- T cells are enumerated in cultures using CountBrightTM fluorescent beads (Invitrogen) and flow cytometry as described by the manufacturer.
- TFP+ T cells are identified by GFP expression using T cells that are engineered with eGFP-2A linked TFP -expressing lentiviral vectors. For TFP+ T cells not expressing GFP, the TFP+ T cells are detected with biotinylated recombinant BCMA protein and a secondary avidin-PE conjugate.
- CD4+ and CD8+ expression on T cells are also simultaneously detected with specific monoclonal antibodies (BD Biosciences). Cytokine measurements are performed on supernatants collected 24 hours following re -stimulation using the human TH1/TH2 cytokine cytometric bead array kit (BD Biosciences) according the manufacturer' s instructions. Fluorescence is assessed using a FACScaliburTM flow cytometer, and data is analyzed according to the manufacturer' s instructions.
- Cytotoxicity can be assessed by a standard 51 Cr-release assay (see, e.g., Milone et ah, Molecular Therapy 17(8): 1453-1464 (2009)).
- target cells are loaded with 51 Cr (as NaCr0 4 , New England Nuclear) at 37 ° C for 2 hours with frequent agitation, washed twice in complete RPMI and plated into microtiter plates.
- Effector T cells are mixed with target cells in the wells in complete RPMI at varying ratios of effector celktarget cell (E:T). Additional wells containing media only (spontaneous release, SR) or a 1% solution of Triton®-X 100 detergent (total release, TR) are also prepared.
- % Lysis (ER-SR)/(TR- SR), where ER represents the average 51 Cr released for each experimental condition.
- Imaging technologies can be used to evaluate specific trafficking and proliferation of TFPs in tumor-bearing animal models. Such assays have been described, e.g., in Barrett et ah, Human Gene Therapy 22: 1575-1586 (2011).
- NOD/SCID/yc-/- (NSG) mice are injected intravenously (i.v.) with cancer cells followed 7 days later with TFP T cells 4 hours after electroporation with the TFP constructs.
- the T cells are stably transfected with a lentiviral construct to express firefly luciferase, and mice are imaged for bioluminescence.
- TFP T cells in a cancer xenograft model can be measured as follows: NSG mice are injected with cancer cells transduced to stably express firefly luciferase, followed by a single tail -vein injection of T cells electroporated with BCMA TFP 7 days later. Animals are imaged at various time points post injection. For example, photon-density heat maps of firefly luciferase positive cancer in representative mice at day 5 (2 days before treatment) and day 8 (24 hours post TFP+ PBLs) can be generated.
- Patent No. 9,217,040 filed January 13, 2013; U.S. Patent No. 9,758,586, filed November 30, 2011; International Publication No. WO 2012076066, filed June 17, 2011; Mato, A. & Porter, D. (2015) Blood 126(4), 478-485; Choi, M., et al. (2015) Clinical Lymphoma, Myeloma & Leukemia 15(S 1), S167-S169; Cui, B., et al. (2015) Cancer Research 73(12), 3649-3660; Yu, J., et al. (2015) Journal of Clinical Investigation 10(1172), 1-34; Borcherding, N., et al. (2014) Protein Cell 5(7), 496-502; Zhang, S., et al.
- the invention provides methods for treating a disease associated with a TAA, e.g., ROR1 or NKG2D ligand (NKG2DL) expression.
- a disease associated with a TAA e.g., ROR1 or NKG2D ligand (NKG2DL) expression.
- the invention provides methods for treating a disease wherein part of the tumor is negative for NKG2DL and part of the tumor is positive for NKG2DL.
- the TFP is useful for treating subjects that have undergone treatment for a disease associated with elevated expression of NKG2DL, wherein the subject that has undergone treatment for elevated levels of NKG2DL exhibits a disease associated with elevated levels of NKG2DL.
- the invention pertains to a vector comprising a TAA-binding TFP operably linked to promoter for expression in mammalian T cells.
- the invention provides a recombinant T cell expressing the, e.g., NKG2D TFP for use in treating NKG2DL-expressing tumors, wherein the recombinant T cell expressing the NKG2D TFP is termed a NKG2D TFP T cell.
- the NKG2D TFP T cell is capable of contacting a tumor cell with at least one NKG2DL expressed on its surface such that the TFP T cell targets the tumor cell and growth of the tumor is inhibited.
- dual specificity TFP T cells comprise multiple blocking domains and corresponding multiple protease -cleavable linkers.
- Tumor associated antigen targets for anti-TAA inducible TFP T cells are tumor associated antigen targets for anti-TAA inducible TFP T cells
- tumor-associated antigens include, but are not limited to, oncofetal antigens (e.g., those expressed in fetal tissues and in cancerous somatic cells), oncoviral antigens (e.g., those encoded by tumorigenic transforming viruses), overexpressed/ accumulated antigens (e.g., those expressed by both normal and neoplastic tissue, with the level of expression highly elevated in neoplasia), cancer- testis antigens (e.g., those expressed only by cancer cells and adult reproductive tissues such as testis and placenta), lineage-restricted antigens (e.g., those expressed largely by a single cancer histotype), mutated antigens (e.g., those expressed by cancer as a result of genetic mutation or alteration in transcription), post-translationally altered antigens (e.g., those tumor-associated alterations in glycosylation, etc.), and idiotypic antigens (e.g., those from highly polymorph
- tumor-associated antigens include, but are not limited to, antigens of alpha- actinin-4, ARTC1, BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDK12, CDKN2A, CLPP, COA-l, CSNK1A1, dek-can fusion protein, EFTUD2, Elongation factor 2, ETV6-AML1 fusion protein, FLT3-ITD, FNDC3B, FN1, GAS7, GPNMB, HAUS3, HSDL1, LDLR-fiicosyltransferase AS fusion protein, HLA-A2d, HLA-A1 ld, hsp70-2, MART2, MATN, ME1, MUM-lf, MUM-2, MUM-3, neo-PAP, Myosin class I, NFYC, OGT, OS-9, p53, pml-RAR
- the invention provides methods for treating a disease associated with at least one tumor-associated antigen expression.
- the invention provides methods for treating a disease wherein part of the tumor is negative for the tumor associated antigen and part of the tumor is positive for the tumor associated antigen.
- the antibody or TFP of the invention is useful for treating subjects that have undergone treatment for a disease associated with elevated expression of said tumor antigen, wherein the subject that has undergone treatment for elevated levels of the tumor associated antigen exhibits a disease associated with elevated levels of the tumor associated antigen.
- the invention pertains to a vector comprising an anti-tumor-associated antigen antibody or TFP operably linked to promoter for expression in mammalian T cells.
- the invention provides a recombinant T cell expressing a tumor-associated antigen TFP for use in treating tumor-associated antigen-expressing tumors, wherein the recombinant T cell expressing the tumor- associated antigen TFP is termed a tumor-associated antigen TFP T cells.
- the tumor- associated antigen TFP T cell of the present disclosure is capable of contacting a tumor cell with at least one tumor-associated antigen TFP of the present disclosure expressed on its surface such that the TFP T cell targets the tumor cell and growth of the tumor is inhibited.
- the invention pertains to a method of inhibiting growth of a tumor-associated antigen-expressing tumor cell, comprising contacting the tumor cell with a tumor-associated antigen antibody or TFP T cell of the present disclosure such that the TFP T is activated in response to the antigen and targets the cancer cell, wherein the growth of the tumor is inhibited.
- the present disclosure pertains to a method of treating cancer in a subject.
- the method comprises administering to the subject a tumor-associated antigen antibody, bispecific antibody, or TFP T cell of the present disclosure such that the cancer is treated in the subject.
- An example of a cancer that is treatable by the tumor-associated antigen TFP T cell of the present disclosure is a cancer associated with expression of tumor-associated antigen.
- the cancer is a myeloma.
- the cancer is a lymphoma.
- the cancer is colon cancer.
- tumor-associated antigen antibodies or TFP therapy can be used in combination with one or more additional therapies.
- additional therapies comprise a chemotherapeutic agent, e.g., cyclophosphamide.
- additional therapies comprise surgical resection or radiation treatment.
- T cells are genetically modified to express a TFP and the TFP-expressing T cell is infused to a recipient in need thereof.
- the infused cell is able to kill tumor cells in the recipient.
- TFP-expressing T cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.
- the T cells administered to the patient, or their progeny persist in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty -one months, twenty-two months, twenty-three months, two years, three years, four years, or five years after administration of the T cell to the patient.
- T cells are modified, e.g., by in vitro transcribed RNA, to transiently express a TFP and the TFP-expressing T cell is infused to a recipient in need thereof.
- the infused cell is able to kill tumor cells in the recipient.
- the T cells administered to the patient is present for less than one month, e.g., three weeks, two weeks, or one week, after administration of the T cell to the patient.
- the anti -tumor immunity response elicited by the TFP-expressing T cells may be an active or a passive immune response, or alternatively may be due to a direct vs indirect immune response.
- the TFP transduced T cells exhibit specific proinflammatory cytokine secretion and potent cytolytic activity in response to human cancer cells expressing the tumor-associated antigen, resist soluble tumor-associated antigen inhibition, mediate bystander killing and/or mediate regression of an established human tumor.
- antigen-less tumor cells within a heterogeneous field of tumor-associated antigen-expressing tumor may be susceptible to indirect destruction by tumor-associated antigen-redirected T cells that has previously reacted against adjacent antigen-positive cancer cells.
- the human TFP-modified T cells of the present disclosure may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal.
- the mammal is a human.
- cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a TFP disclosed herein.
- the TFP-modified cell can be administered to a mammalian recipient to provide a therapeutic benefit.
- the mammalian recipient may be a human and the TFP-modified cell can be autologous with respect to the recipient.
- the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.
- ex vivo culture and expansion of T cells comprises: (1) collecting CD34+ hematopoietic stem and progenitor cells from a mammal from peripheral blood harvest or bone marrow explants; and (2) expanding such cells ex vivo.
- other factors such as flt3-L, IL-l, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells.
- compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient In addition to using a cell -based vaccine in terms of ex vivo immunization, the present disclosure also provides compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient.
- the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised.
- the TFP- modified T cells of the present disclosure are used in the treatment of diseases, disorders and conditions associated with expression of tumor-associated antigens.
- the cells of the present disclosure are used in the treatment of patients at risk for developing diseases, disorders and conditions associated with expression of tumor-associated antigens.
- the present disclosure provides methods for the treatment or prevention of diseases, disorders and conditions associated with expression of tumor- associated antigens comprising administering to a subject in need thereof, a therapeutically effective amount of the TFP -modified T cells of the present disclosure.
- the antibodies or TFP T cells disclosed herein may be used to treat a proliferative disease such as a cancer or malignancy or is a precancerous condition.
- a proliferative disease such as a cancer or malignancy or is a precancerous condition.
- the cancer is a myeloma.
- the cancer is a lymphoma.
- the cancer is a colon cancer.
- a disease associated with tumor-associated antigen expression includes, but is not limited to, e.g., atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing tumor-associated antigens.
- Non-cancer related indications associated with expression of tumor-associated antigens vary depending on the antigen, but are not limited to, e.g., infectious disease, autoimmune disease, (e.g., lupus), inflammatory disorders (allergy and asthma) and transplantation.
- the antibodies or TFP-modified T cells of the present disclosure may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-7, IL-12, IL-15 or other cytokines or cell populations.
- the present disclosure also provides methods for inhibiting the proliferation or reducing a tumor- associated antigen-expressing cell population, the methods comprising contacting a population of cells comprising a tumor-associated antigen-expressing cell with an anti -tumor-associated antigen TFP T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell.
- the present disclosure provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing tumor-associated antigen, the methods comprising contacting the tumor- associated antigen-expressing cancer cell population with an anti-tumor-associated antigen antibody or TFP T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell.
- the present disclosure provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing tumor-associated antigen, the methods comprising contacting the tumor- associated antigen-expressing cancer cell population with an anti-tumor-associated antigen antibody or TFP T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell.
- the anti-tumor-associated antigen antibody or TFP T cell of the present disclosure reduces the quantity, number, amount or percentage of cells and/or cancer cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% in a subject with or animal model for multiple myeloma or another cancer associated with tumor-associated antigen expressing cells relative to a negative control.
- the subject is a human.
- the present disclosure also provides methods for preventing, treating and/or managing a disease associated with tumor-associated antigen-expressing cells (e.g., a cancer expressing tumor-associated antigen), the methods comprising administering to a subject in need an anti-tumor-associated antigen antibody or TFP T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell.
- the subject is a human.
- disorders associated with tumor- associated antigen-expressing cells include autoimmune disorders (such as lupus), inflammatory disorders (such as allergies and asthma) and cancers (such as hematological cancers or atypical cancers expressing tumor-associated antigen).
- the present disclosure also provides methods for preventing, treating and/or managing a disease associated with tumor-associated antigen-expressing cells, the methods comprising administering to a subject in need an anti-tumor-associated antigen antibody or TFP T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell.
- the subject is a human.
- the present disclosure provides methods for preventing relapse of cancer associated with tumor- associated antigen-expressing cells, the methods comprising administering to a subject in need thereof an anti-tumor-associated antigen antibody and/or TFP T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell.
- the methods comprise administering to the subject in need thereof an effective amount of an anti -tumor-associated antigen antibody or TFP T cell described herein that binds to the tumor-associated antigen-expressing cell in combination with an effective amount of another therapy.
- an inducible TFP-expressing cell described herein may be used in combination with other known agents and therapies.
- Administered "in combination”, as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject' s affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons.
- the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous" or “concurrent delivery”.
- the delivery of one treatment ends before the delivery of the other treatment begins.
- the treatment is more effective because of combined administration.
- the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment or the analogous situation is seen with the first treatment.
- delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other.
- the effect of the two treatments can be partially additive, wholly additive, or greater than additive.
- the delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.
- the "at least one additional therapeutic agent” includes a TFP-expressing cell.
- T cells that express multiple TFPs, which bind to the same or different target antigens, or same or different epitopes on the same target antigen.
- populations of T cells in which a first subset of T cells expresses a first TFP and a second subset of T cells express a second TFP.
- a TFP-expressing cell described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially.
- the TFP-expressing cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.
- a TFP-expressing cell described herein may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies or other antibody therapies, cyclophosphamide, fludarabine, cyclosporin, tacrolimus (fujimycin), rapamycin, mycophenolic acid, steroids, romidepsin (also known as FR901228), cytokines, and irradiation peptide vaccine, such as that described in, e.g., Izumoto et al.
- immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies or other antibody therapies, cyclophosphamide, flu
- the subject can be administered an agent which reduces or ameliorates a side effect associated with the administration of a TFP-expressing cell.
- Side effects associated with the administration of a TFP-expressing cell include, but are not limited to, cytokine release syndrome (CRS), and hemophagocytic lymphohistiocytosis (HLH), also termed Macrophage Activation Syndrome (MAS).
- CRS cytokine release syndrome
- HHL hemophagocytic lymphohistiocytosis
- MAS Macrophage Activation Syndrome
- Symptoms of CRS include high fevers, nausea, transient hypotension, hypoxia, and the like.
- the methods described herein can comprise administering a TFP-expressing cell described herein to a subject and further administering an agent to manage elevated levels of a soluble factor resulting from treatment with a TFP-expressing cell.
- the soluble factor elevated in the subject is one or more of IFN-g, TNFc IL-2, IL-6, and IL-8. Therefore, an agent administered to treat this side effect can be an agent that neutralizes one or more of these soluble factors.
- agents include, but are not limited to a steroid, an inhibitor of TNFa, and an inhibitor of IL-6.
- An example of a TNFa inhibitor is etanercept (marketed under the name ENBREL®).
- An example of an IL-6 inhibitor is tocilizumab (marketed under the name ACTEMRA®).
- the subject can be administered an agent which enhances the activity of a TFP-expressing cell.
- the agent can be an agent which inhibits an inhibitory molecule.
- Inhibitory molecules e.g., Programmed Death 1 (PD1)
- PD1 can, in some embodiments, decrease the ability of a TFP-expressing cell to mount an immune effector response.
- Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.
- Inhibition of an inhibitory molecule e.g., by inhibition at the DNA, RNA or protein level, can optimize a TFP-expressing cell performance.
- an inhibitory nucleic acid e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA
- an inhibitory nucleic acid e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA
- the inhibitor is a shRNA.
- the inhibitory molecule is inhibited within a TFP-expressing cell.
- a dsRNA molecule that inhibits expression of the inhibitory molecule is linked to the nucleic acid that encodes a component, e.g., all of the components, of the TFP.
- the inhibitor of an inhibitory signal can be, e.g., an antibody or antibody fragment that binds to an inhibitory molecule.
- the agent can be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2 or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 and MDX-101, and marketed as YERVOY®); Bristol-Myers Squibb; tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP -675, 206)).
- ipilimumab also referred to as MDX-010 and MDX-101, and marketed as YERVOY®
- tremelimumab IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP -675, 206
- the agent is an antibody or antibody fragment that binds to T cell immunoglobulin and mucin-domain containing-3 (TIM3). In an embodiment, the agent is an antibody or antibody fragment that binds to Lymphocyte -activation gene 3 (LAG3).
- TIM3 T cell immunoglobulin and mucin-domain containing-3
- LAG3 Lymphocyte -activation gene 3
- an agent suitable for use in combination with the TFP T cells disclosed herein is an agent that modulates myeloid suppressor cells, e.g., CCR2 antibodies.
- Other therapeutics, e.g, nanoparticle therapeutics, are known in the art.
- the agent which enhances the activity of a TFP-expressing cell can be, e.g., a fusion protein comprising a first domain and a second domain, wherein the first domain is an inhibitory molecule, or fragment thereof, and the second domain is a polypeptide that is associated with a positive signal, e.g., a polypeptide comprising an intracellular signaling domain as described herein.
- the polypeptide that is associated with a positive signal can include a costimulatory domain of CD28, CD27, ICOS, e.g., an intracellular signaling domain of CD28, CD27 and/or ICOS, and/or a primary signaling domain, e.g., of CD3 zeta, e.g., described herein.
- the fusion protein is expressed by the same cell that expressed the TFP.
- the fusion protein is expressed by a cell, e.g., a T cell that does not express an anti-tumor-associated antigen TFP.
- compositions of the present disclosure may comprise a TFP-expressing cell, e.g., a plurality of TFP-expressing cells, as described herein, in combination with one or more
- compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
- buffers such as neutral buffered saline, phosphate buffered saline and the like
- carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
- proteins polypeptides or amino acids
- antioxidants e.g., antioxidants
- chelating agents such as EDTA or glutathione
- adjuvants e.g., aluminum hydroxide
- preservatives e.g., aluminum hydroxide
- compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated (or prevented).
- the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient ' s disease, although appropriate dosages may be determined by clinical trials.
- the pharmaceutical composition is substantially free of, e.g., there are no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus.
- a contaminant e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus.
- the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.
- an immunologically effective amount When “an immunologically effective amount,” “an anti-tumor effective amount,” “a tumor- inhibiting effective amount,” or “therapeutic amount” is indicated, the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 104 to 109 cells/kg body weight, in some instances 10 5 to 10 6 cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et ah, New Eng. J. of Med. 319: 1676, 1988).
- T cells can be activated from blood draws of from 10 cc to 400 cc.
- T cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.
- compositions described herein may be administered to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
- the T cell compositions of the present disclosure are administered to a patient by intradermal or subcutaneous injection.
- the T cell compositions of the present disclosure are administered by i.v. injection.
- the compositions of T cells may be injected directly into a tumor, lymph node, or site of infection.
- subjects may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate the cells of interest, e.g., T cells.
- T cell isolates may be expanded by methods known in the art and treated such that one or more TFP constructs of the present disclosure may be introduced, thereby creating a TFP -expressing T cell of the present disclosure.
- Subjects in need thereof may subsequently undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
- subjects receive an infusion of the expanded TFP T cells of the present disclosure.
- expanded cells are administered before or following surgery.
- the dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment.
- the scaling of dosages for human administration can be performed according to art-accepted practices.
- the dose for alemtuzumab (CAMPATH®) will generally be in the range 1 to about 100 mg for an adult patient, usually administered daily for a period between 1 and 30 days.
- the preferred daily dose is 1 to 10 mg per day although in some instances larger doses of up to 40 mg per day may be used (described in U.S.
- the TFP is introduced into T cells, e.g., using in vitro transcription, and the subject (e.g., human) receives an initial administration of TFP T cells of the present disclosure, and one or more subsequent administrations of the TFP T cells of the present disclosure, wherein the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration.
- more than one administration of the TFP T cells of the present disclosure are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the TFP T cells of the present disclosure are administered per week.
- the subject receives more than one administration of the TFP T cells per week (e.g., 2, 3 or 4 administrations per week) (also referred to herein as a cycle), followed by a week of no TFP T cells administrations, and then one or more additional administration of the TFP T cells (e.g., more than one administration of the TFP T cells per week) is administered to the subject.
- the subject receives more than one cycle of TFP T cells, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days.
- the TFP T cells are administered every other day for 3 administrations per week.
- the TFP T cells of the present disclosure are administered for at least two, three, four, five, six, seven, eight or more weeks.
- tumor-associated antigen TFP T cells are generated using lentiviral viral vectors, such as lentivirus. TFP T cells generated that way will have stable TFP expression.
- TFP T cells transiently express TFP vectors for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
- Transient expression of TFPs can be effected by RNA TFP vector delivery.
- the TFP RNA is transduced into the T cell by electroporation.
- a potential issue that can arise in patients being treated using transiently expressing TFP T cells is anaphylaxis after multiple treatments.
- anaphylactic response might be caused by a patient developing humoral anti-TFP response, i.e., anti-TFP antibodies having an anti-IgE isotype. It is thought that a patient' s antibody producing cells undergo a class switch from IgG isotype (that does not cause anaphylaxis) to IgE isotype when there is a ten- to fourteen-day break in exposure to antigen.
- TFP T cell infusion breaks should not last more than ten to fourteen days.
- Anti-TAA TFP constructs are engineered by cloning one or more anti-TAA scFv DNA fragment linked to a CD3 or TCR DNA fragment by either a DNA sequence encoding a short linker (SL):
- AAAGGGGSGGGGSGGGGSLE (SEQ ID NO: 188) or a long linker (LL):
- AAAIEVMYPPPYLGGGGSGGGGSGGGGSLE (SEQ ID NO: 189) into, e g., r510 vector ((System Biosciences (SBI)) at Xbal and EcoRl sites.
- CAR constructs are generated by cloning synthesized DNA encoding an anti-TAA antibody, partial CD28 extracellular domain, CD28 transmembrane domain,
- CD3 e TFP constructs disclosed herein comprise the sequence set forth in SEQ ID NO: 191, which has an N-terminal truncation in reference to the full sequence (SEQ ID NO: 192).
- an anti-tumor-associated antigen CAR construct is generated as a comparator.
- a p5 lO_antitumor-associated antigen_28 z CAR is generated by cloning synthesized DNA encoding anti-tumor-associated antigen, partial CD28 extracellular domain, CD28 transmembrane domain, CD28 intracellular domain and CD3 zeta into p510 vector at Xbal and EcoRl sites.
- An anti-HSA sdAb (SEQ ID NO:42) was genetically fused to anti-MSLN sdAb SD1 (SEQ ID NO:39) via a cleavable linker. See, e.g., FIG. 6, which shows inducible TFP construct sequences in the format 5 ' -anti-HSA sdAb— protease-cleavable linker— anti-MSLN sdAb binder-3 ' ) with a C-terminal 6His tag for purification and detection purposes. Fusion proteins were expressed in E. coli and purified to homogeneity by Ni-NTA affinity chromatography. Fusion proteins were desalted and stored and in lxPBS buffer, pH 7.4.
- antibody polypeptides fragments thereof, single domain antibodies, Fab fragments, and other antigen binding proteins that are capable of specifically binding to the human polypeptide (s), and fragments or domains thereof.
- the antigen is a tumor-associated antigen (TAA).
- TAA tumor-associated antigen
- Anti-TAA antibodies or fragments thereof can be generated using diverse technologies (see, e.g., (Nicholson et al, 1997).
- Human or humanized anti-TAA IgGs are used to generate scFv sequences for TFP constructs.
- DNA sequences coding for human or humanized VF and VH domains are obtained, and the codons for the constructs are, optionally, optimized for expression in cells from Homo sapiens.
- the order in which the VF and VH domains appear in the scFv is varied (i.e., VF-VH, or VH-VF orientation), and three copies of the "G 4 S" or "G 4 S” subunit (G 4 S) 3 connect the variable domains to create the scFv domain.
- Anti-TAA scFv plasmid constructs can have optional Flag, His or other affinity tags, and are electroporated into HEK-293 or other suitable human or mammalian cell lines and purified.
- Validation assays include binding analysis by FACS, kinetic analysis using Proteon, and staining of TAA- expressing cells.
- Subunits of the human T Cell Receptor (TCR) complex all contain an extracellular domain, a transmembrane domain, and an intracellular domain.
- a human TCR complex contains the CD3 -epsilon polypeptide, the CD3-gamma polypeptide, the CD3-delta polypeptide, the CD3-zeta polypeptide, the TCR alpha chain polypeptide and the TCR beta chain polypeptide.
- the human CD3-epsilon polypeptide canonical sequence is UniProt Accession No. P07766.
- the human CD3-gamma polypeptide canonical sequence is UniProt Accession No. P09693.
- the human CD3-delta polypeptide canonical sequence is UniProt Accession No.
- the human CD3-zeta polypeptide canonical sequence is UniProt Accession No. P20963.
- the human TCR alpha chain canonical sequence is UniProt Accession No. Q6ISU1.
- the human TCR beta chain C region canonical sequence is UniProt Accession No. P01850, a human TCR beta chain V region sequence is P04435.
- the human CD3 -epsilon polypeptide canonical sequence is:
- the human CD3-epsilon fragment used in the TFPs is
- the human CD3 -gamma polypeptide canonical sequence is:
- the human CD3-gamma fragment used in the TFPs is:
- the human CD3 -delta polypeptide canonical sequence is:
- the human CD3-zeta polypeptide canonical sequence is:
- the human CD3-zeta fragment used in the TFPs is:
- the human TCR alpha chain canonical sequence is:
- the human TCR alpha chain C region canonical sequence is:
- the human TCR alpha chain V region CTL-L17 canonical sequence is:
- the human TCR beta chain C region canonical sequence is:
- the human TCR beta chain V region YT35 canonical sequence is:
- TFPs from TCR Domains and scFvs, Fab fragments, or sdAbs
- Anti-TAA scFvs are recombinantly linked to a) CD3-epsilon or other TCR subunits, and b) a blocking domain, such as HSA, using a linker sequence, such as G4S, (G4S)2 (G4S)3 or (G4S)4.
- a linker sequence such as G4S, (G4S)2 (G4S)3 or (G4S)4.
- Various linkers and antibody (e.g., scFv or sdAb) configurations are used.
- TCR alpha and TCR beta chains are used for generation of TFPs either as full-length polypeptides or as only their constant domains (e.g., in the preparation for a construct suitable for an allogeneic immune cell product) . Any variable sequence of TCR alpha and TCR beta chains is suitable for making TFPs.
- Expression vectors include: a promoter (Cytomegalovirus (CMV) enhancer- promoter), a signal sequence to enable secretion, a polyadenylation signal and transcription terminator (Bovine Growth Hormone (BGH) gene), an element allowing episomal replication and replication in prokaryotes (e.g., SV40 origin and ColEl or others known in the art) and elements to allow selection (ampicillin resistance gene and zeocin marker).
- CMV Cytomegalovirus
- BGH Bovine Growth Hormone
- the TFP -encoding nucleic acid construct or constructs is/are cloned into one or more lentiviral expression vectors and expression validated based on the quantity and quality of the effector T cell response of transduced T cells in response to TAA+ target cells.
- Effector T cell responses include, but are not limited to, cellular expansion, proliferation, doubling, cytokine production and target cell lysis or cytolytic activity (i.e., degranulation).
- the TFP lentiviral transfer vectors are used to produce the genomic material packaged into the VSVg pseudotyped lentiviral particles.
- Lentiviral transfer vector DNA is mixed with the three packaging components of VSVg, gag/pol and rev in combination with Lipofectamine® reagent to transfect them together into 293 cells. After 24 and 48 hours, the media is collected, filtered and concentrated by ultracentrifugation. The resulting viral preparation is stored at -80° C. The number of transducing units is determined by titration on SupTl cells (T cell lymphoblastic lymphoma, (ATCC® CRL-1942TM).
- Redirected dual specificity TFP T cells are produced by activating fresh naive T cells with anti-CD3x anti-CD28 beads for 24 hrs and then adding the appropriate number of transducing units to obtain the desired percentage of transduced T cells. These modified T cells are allowed to expand until they become rested and come down in size at which point they are cryopreserved for later analysis. The cell numbers and sizes are measured using a Coulter Counter® MultisizerTM 3 (Beckman Coulter). Before
- cryopreserving percentage of cells transduced (expressing TFP.BCMA on the cell surface) and their relative fluorescence intensity of that expression are determined by flow cytometric analysis. From the histogram plots, the relative expression levels of the TFPs are examined by comparing percentage transduced with their relative fluorescent intensity.
- the vector is an adenoviral vector.
- the vector is a circular RNA or a circular RNA transfer vector.
- the vector is a lentiviral vector.
- multiple TFPs are introduced by T cell transduction with multiple viral vectors. Evaluating Cytolytic Activity. Proliferation Capabilities and Cytokine Secretion of Humanized TFP Redirected T Cells
- TFP.TAA T cells The functional abilities of TFP.TAA T cells to produce cell-surface-expressed TFPs, and to kill target tumor cells, proliferate and secrete cytokines are determined using assays known in the art.
- Cleavage of the protease-cleavable linker(s) tethering the blocking domain to the TFP T cell can be tested, for example, in vitro by proteolytic cleavage followed by western blot analysis of cleavage fragments.
- Human PBMCs e.g., blood from a normal apheresed donor whose naive T cells are obtained by negative selection for T cells, CD4+ and CD8+ lymphocytes
- IL-2 human interleukin-2
- Flow cytometry assays are utilized to confirm cell surface presence of a TFP, such as by an anti-FLAG antibody or an anti-murine variable domain antibody.
- Cytokine (e.g., IFN-g) production is measured using ELISA or other assays.
- Example 3 Human TFP T Cell Treatment in an In Vivo Solid Tumor Xenograft Mouse Model
- Tumor shrinkage in response to human inducible TFP T cell treatment can be either assessed by caliper measurement of tumor size, or by following the intensity of a GFP fluorescence signal emitted by GFP- expressing tumor cells.
- Exemplary solid cancer cells include solid tumor cell lines, such as provided in The Cancer Genome Atlas (TCGA) and/or the Broad Cancer Cell Line Encyclopedia (CCLE, see Barretina et ah, Nature 483:603 (2012)).
- Exemplary solid cancer cells include primary tumor cells isolated from mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney, endometrial, or stomach cancer.
- the cancer to be treated is selected from the group consisting of mesotheliomas, papillary serous ovarian adenocarcinomas, clear cell ovarian carcinomas, mixed Mullerian ovarian carcinomas, endometroid mucinous ovarian carcinomas, pancreatic adenocarcinomas, ductal pancreatic adenocarcinomas, uterine serous carcinomas, lung adenocarcinomas, extrahepatic bile duct carcinomas, gastric adenocarcinomas, esophageal adenocarcinomas, colorectal adenocarcinomas and breast adenocarcinomas.
- mice can be used to test the efficacy of anti-TAA TFP T cells in the human tumor xenograft models (see, e.g., Morton et ah, Nat. Procol. 2:247 (2007)).
- tumors are allowed to grow to 200-500 mm 3 prior to initiation of treatment.
- the inducible TFP is encoded by a lentivirus.
- Lentivirus encoding the appropriate constructs are prepared as follows. 5 x 10 6 HEK-293FT cells are seeded into a 100 mm dish and allowed to reach 70-90% confluency overnight. 2.5 pg of the indicated DNA plasmids and 20 pL Lentivirus Packaging Mix (ALSTEM, cat# VP100) are diluted in 0.5 mL DMEM or Opti-MEM® I Medium without serum and mixed gently.
- NanoFect® transfection reagent (ALSTEM, cat# NF100) is diluted in 0.5 mL DMEM or Opti-MEM I Medium without serum and mixed gently.
- the NanoFect/DMEM and DNA/DMEM solutions are then mixed together and vortexed for 10- 15 seconds prior to incubation of the DMEM-plasmid-NanoFect mixture at room temperature for 15 minutes.
- the complete transfection complex from the previous step is added dropwise to the plate of cells and rocked to disperse the transfection complex evenly in the plate. The plate is then incubated overnight at 37 ° C in a humidified 5% C0 2 incubator.
- the supernatant is replaced with 10 mL fresh medium and supplemented with 20 pL of ViralBoostTM (500x, ALSTEM, cat# VB100).
- ViralBoostTM 500x, ALSTEM, cat# VB100.
- the plates are then incubated at 37 ° C for an additional 24 hours.
- the lentivirus containing supernatant is then collected into a 50 mL sterile, capped conical centrifuge tube and put on ice. After centrifugation at 3000 rpm for 15 minutes at 4° C, the cleared supernatant is filtered with a low-protein binding 0.45 pm sterile filter and virus is subsequently isolated by ultracentrifugation at 25,000 rpm (Beckmann, L8-70M) for 1.5 hours, at 4° C. The pellet is removed and re-suspended in DMEM medium and lentivirus
- concentrations/titers are established by quantitative RT-PCR using the Lenti-XTM qRT-PCR Titration kit (Clontech®; catalog number 631235). Any residual plasmid DNA is removed by treatment with DNasel. The virus stock preparation is either used for infection immediately or aliquoted and stored at -80 ° C for future use.
- Lentivirus titers are established by transducing cells (e.g., Jurkat cells) with different amount of virus preparation. The DNA is then isolated from the transduced Jurkat cells 24 hours after transduction. The virus titer is determined, e.g., by quantitative real-time PCR, with in-house designed primers/probe specific for Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) as well as for an internal quantitation control.
- WP Woodchuck Hepatitis Virus
- WPRE Posttranscriptional Regulatory Element
- PBMCs Peripheral Blood Mononuclear Cells
- PBMCs Peripheral Blood Mononuclear Cells
- Whole blood is collected in 10 mL Heparin vacutainers and either processed immediately or stored overnight at 4 ° C.
- Approximately 10 mL of whole anti -coagulated blood is mixed with sterile phosphate buffered saline (PBS) buffer for a total volume of 20 mL in a 50 mL conical centrifuge tube (PBS, pH 7.4, without Ca 2+ /Mg 2+ ).
- PBS sterile phosphate buffered saline
- Buffy coat is purchased, e.g., from Research Blood Components (Boston, MA).
- LeucoSepTM tubes (Greiner bio-one) are prepared by adding 15 mL Ficoll-Paque and centrifuged at lOOOg for 1 minute.
- Buffy coat is diluted 1 :3 in PBS (pH 7.4, without Ca 2+ or Mg 2+ ).
- the diluted buffy coat is transferred to LeucoSep tube and centrifuged at lOOOg for 15 minutes with no brake application.
- the layer of cells containing PBMCs, seen at the diluted plasma/Ficoll interface, is removed carefully to minimize contamination by Ficoll®.
- Residual Ficoll, platelets, and plasma proteins are then removed by washing the PBMCs three times with 40 mL of PBS by centrifugation at 200g for 10 minutes at room temperature. The cells are then counted with a hemocytometer. CD4+ and CD8+ T cells are then frozen down in freezing medium (90% FBS+l0% DMS0 at a concentration of 30-50 x 10 6 cells per vial.
- PBMCs prepared from either whole blood or buffy coat are stimulated with anti-human CD28 and CD3 antibody -conjugated magnetic beads for 24 hours prior to viral transduction.
- Freshly isolated PBMCs are washed once in CAR T medium (AIM V-AlbuMAX BSA, Life Technologies), with 5% AB serum and 1.25 pg/mL amphotericin B (Gemini Bioproducts), 100 U/mL penicillin, and 100 pg/mL streptomycin) without huIL-2, before being re-suspended at a final concentration of 1 x 10 6 cells/mL in CAR T medium with 300 IU/mL human IL-2, IL-7, or IL-15 (from a lOOOx stock; Invitrogen).
- frozen CD4+/CD8+ T cells are thawed in pre-warmed DMEM + 10 % FBS, spun down, and then resuspended in complete T cell expansion medium supplemented with 300 IU/mL huIL2 (Thermo Fisher®) at a final concentration of 1 x 10 6 cells/mL.
- huIL2 300 IU/mL huIL2
- anti human CD28 and anti-human CD3 antibody-conjugated magnetic beads Prior to being used to activate T cells, anti human CD28 and anti-human CD3 antibody-conjugated magnetic beads (Dynabeads®, Thermo Fisher) are washed three times with sterile 1 x PBS (pH7.4), using a magnetic rack to isolate beads from the solution.
- the T cells are then mixed with the beads at a 1 : 1 ratio, by transferring 25 pL (lxlO 6 beads) of beads to 1 mL of T cell suspension.
- the beads/cells mixture is then dispensed to single wells of a non-TC treated l2-well plate, and incubated at 37 ° C with 5 % C0 2 for 24 hrs.
- anti-human CD28 and CD3 antibody-conjugated magnetic beads are washed three times with 1 mL of sterile lx PBS (pH 7.4), using a magnetic rack to isolate beads from the solution, before re -suspension in CAR T medium, with 300 IU/mL human IL-2, to a final concentration of 4 x 10 7 beads/mL.
- PBMC and beads are then mixed at a 1 : 1 bead-to-cell ratio, by transferring 25 pL (lxlO 6 beads) of beads to 1 mL of PBMC.
- the desired number of aliquots are then dispensed to single wells of a l2-well low-attachment or non-treated cell culture plate, and incubated at 37 ° C, with 5% C0 2 , for 24 hours before viral transduction.
- PBMCs peripheral blood mononuclear cells
- PBMCs peripheral blood mononuclear cells
- Lentivirus is thawed on ice and then added to activated T cells at indicated MOI in the presence of 10 pg/ml Polybrene (Sigma).
- Cells are spinoculated with the lentivirus at 200 g for 100 minutes at room temperature.
- the transduced T cells are incubated for an additional 24 hr before an additional lentivirus transduction.
- the T cells are expanded in T cell expansion medium supplemented with 300 IU/mL of hIL-2 and sub-cultured every other day at 5 x 10 5 cells/mL.
- activated PBMCs are electroporated with in vitro transcribed (IVT) mRNA.
- Human PBMCs are stimulated with Dynabeads® (Thermo Fisher®) at l-to-l ratio for 3 days in the presence of 300 IU/ml recombinant human IL-2 (R&D System). The beads are removed before electroporation. The cells are washed and re-suspended in OPTI-MEM® medium (Thermo Fisher) or AimV® medium (Invitrogen) in 5% hAB serum (Gemini Bio-Products) and 1% antibiotics at the concentration of 2.5 x 10 7 cells/mL.
- OPTI-MEM® medium Thermo Fisher
- AimV® medium Invitrogen
- the cells are transferred to fresh cell culture medium (AIM V AlbuMAX® (BSA) serum free medium + 5% human AB serum + 300 IU/ml IL-2) and incubated at 37° C.
- AIM V AlbuMAX® serum free medium + 5% human AB serum + 300 IU/ml IL-2
- linkers for an inducible TFP T cell comprising a biologically active polypeptide fusion protein e.g., the tumor-associated antigen binder
- a TAA binder is coupled to a blocking domain via a protease-cleavable linker.
- the protease-cleavable linker is engineered to comprise a sequence recognized by a protease typically present in the tumor microenvironment (TME).
- TEE tumor microenvironment
- the tumor microenvironment is characterized by numerous factors including but not limited to expression of matrix metalloproteinases, cathepsins, proteases, indolomine 2,3-dioxygenase, urokinase-type plasminogen activator (uPA), membrane-type protease 1 (MT-SPl/matriptase), legumain, low extracellular pH (acidosis) and low oxygenation (hypoxia) (Brown & Wilson, 2004; Vaupel & Mayer, 2007; Desnoyers & Vasiljeva, 2013).
- uPA urokinase-type plasminogen activator
- MT-SPl/matriptase membrane-type protease 1
- legumain low extracellular pH (a
- the protease-cleavable linker comprises a sequence recognized by matrix metalloproteinases (MMPs), which are inactive in non-cancerous cells, thereby preventing normal activity of the polypeptide fusion protein until the linker is cleaved, and the masking domain is therefore removed, by TME -expressed MMPs.
- MMPs matrix metalloproteinases
- the linker comprising the protease -cleavable sequence is covalently attached to a blocking domain that physically blocks or sterically inhibits the biologically active polypeptide from functioning in non-cancerous tissue.
- a bulky protein such as human serum albumin (HSA, or a fragment thereof) is used. Such bulky proteins can block access of an active protein to its target or prevent binding of an antigen to its receptor.
- the preparation comprising the biologically active fusion protein, protease-cleavable linker, and blocking domain remains inactive until it reaches the tumor microenvironment, at which point the linker is cleaved by proteases present and the cytokine, chemokine, or binding agent is unmasked and/or becomes active.
- a xenograft model system comprising an inoculation cell line that expresses the protease of interest and as a negative control an inoculation cell line that does not express the protease of interest is used (e.g., a similar or same cell line where the gene for the protease is knocked out).
- tumors in the two types of mice i.e., experimental mice and control mice
- a certain size e.g., about 300mm 2
- infusion of TFP T cells and subsequent measurement of tumor volume, cytokine production, and survival responses will provide a measurement of TFP efficacy and linker cleavage in protease null and positive TMEs.
- linker cleavage is tested by the harvest and co-IP of TIES expressing a TFP disclosed herein from a) a TAA+ and protease -expressing xenograft and b) TAA+ and protease null xenograft, followed by western blot analysis or intact mass spectrometry to detect linker cleavage.
- the blocking domain e.g., an anti-HSA domain
- an epitope e.g. FLAG, myc, or HA tag
- TAA+ tumors with and without the relevant protease are compared.
- the +/- protease cell line system may be achieved by genetic knockout, knock-in, null vs over-expressing lines, or by infusion of a relevant protease.
- This Example describes using linker peptide sequences to attach the blocking domain that are recognized and subsequently digested by cathepsins.
- Cathepsins are a group of serine proteases that function in acidic environments. Lysosomal cathepsins require a reducing, slightly acidic environment, such as found in the TME, in order to be optimally active.
- a polypeptide fusion protein with a blocking domain is engineered as described in Example 5.
- a linker comprising a cathepsin cleavable elastin, collagen or fibronectin is linked to the fusion protein in a fashion that prohibits binding domain recognition and/or biological activity.
- cathepsin S cleavable binding domains such as GAVVRGA (SEQ ID NO: ) may be used to link the fusion protein to the masking domain (e.g., HSA or a fragment thereof).
- the cathepsin cleavable blocking domain will be removed in the tumor microenvironment using appropriate cleavable linker sequences (e.g., cathepsin B or cathepsin S sequences).
- the biologically active fusion protein may additionally comprise a serum half-life extension element, as is evidenced by its prolonged activity or extended PK.
- the serum half- life extension element is the HSA blocking domain.
- the serum half-life extension element may extend the serum half-life of a T cell comprising a biologically active fusion protein.
- a biologically active fusion protein e.g., a chimeric antigen receptor CAR subunit
- a biologically active fusion protein e.g., a chimeric antigen receptor CAR subunit
- a protease-cleavable linker is attached to each variable domain on the antibody, and each linker is in turn covalently attached to a masking domain, such as HSA or a fragment thereof (see Figures 1A and IB).
- the masked chimeric antigen receptor is expressed in an immune cell, such as a Treg or NK cell, which is in turn administered to a patient.
- the masking domain is cleaved away and the CAR T cells are capable of immunomodulation.
- Example 8 Protease Inducible TFP T cells comprising an HSA binding domain
- a chimeric antigen receptor subunit (such as described in Example 7) comprises a tumor-associated antigen (TAA) binding protein.
- TAA tumor-associated antigen
- the TFP T cell activity is masked by a cleavable HSA binding domain, such as a single domain antibody (sdAb), Fab fragment, V H , V L , or scFv, which is fused to the TAA binder ( Figure 2A).
- the HSA binding domain is covalently attached to the TAA binder via a cleavable domain, such as those described above (e.g., MMP14, MMP9, cathepsins, etc.).
- a cleavable domain such as those described above (e.g., MMP14, MMP9, cathepsins, etc.).
- An exemplary humanized anti-HSA sdAb that may be fused to a TCR T cell that will recognize and bind HSA is
- circulating HSA in the patient will bind to the HSA -binding domain of the construct, resulting in inhibition of the tumor recognition domain.
- Masking of the TAA-binding domain by the bound HSA reduces the risk of on-target, off-tumor activity of the engineered immune cells.
- the albumin -binding domain and the bound albumin serve to both extend the serum half-life of the circulating cells and to block the function of the TAA-binding domain.
- inducible TFP T cells are prepared as described in Example 8.
- the TFP T cell activity is masked by a cleavable domain comprising a blocking moiety, e.g., HSA or a fragment thereof.
- a blocking moiety e.g., HSA or a fragment thereof.
- the HSA or fragment thereof is either fused directly to the TAA binder or is fused to another part of the TCR in a way that blocks the TAA binding site ( Figure 5).
- the HSA is covalently attached to the TFP via a cleavable domain, such as those described above.
- the TFP T cells migrate into the tumor microenvironment, and the linker is cleaved by proteases present in the tumor microenvironment, and the HSA is removed from the surface of the immune cell. In turn, the tumor recognition domain of the complex is exposed and able to recognize tumor antigen.
- the inducible TFP constructs disclosed herein can comprise an anti-mesothelin binding domain. Examples of anti-mesothelin binding domain sequences are listed in Table 15.
- the single domain antibody fusion proteins (sequences are shown in FIG. 6) were expressed in E. coli and purified by Ni-NTA affinity chromatography. Each protein was subjected to cleavage by incubation with their respective proteases (MMP9, uPA or cathepsin B) for the cleavage sequence engineered between the two sdAbs.
- MMP9, uPA or cathepsin B proteases
- Results are shown in FIG. 8. Constructs were loaded pairwise as follows: aHSA-sdAb-uPA-SD 1 sdAb digested with uPA +/- FBS; aAlb-N/C-SDl digested with uPA +/- FBS; aHSA sdAb-MMP9vl- SDlsdAb digested with MMP9 +/- FBS; aHSA sdAb-MMP9v2-SDlsdAb digested with MMP9 +/- FBS; and aHSA sdAb-cathepsin B-SD1 sdAb digested with cathepsin B +/- FBS.
- uPA and MMP9 cleavage product is visualized on the western blot.
- Cathepsin B either did not cleave or the cleavage was so inefficient that the cleavage product was at a concentration that is below the limit of detection by this method.
- the efficiency of protease cleavage is quite low, representing 5% or less of the product detected on the western blot membrane.
- the uPA protease is unable to cleave in the presence of FBS, while MMP9 seems to not be affected by the presence of FBS.
- MMP9vl cleavage sequence seems to be more efficiently cleaved compared to MMP9v2.
Abstract
Provided herein are inducible T cell receptor (TCR) fusion proteins (TFPs) having specificity for one or more tumor cell associated antigens, T cells engineered to express one or more TFPs, and methods of use thereof for the treatment of diseases, including cancer, in particular for use in adoptive cell therapy.
Description
COMPOSITIONS AND METHODS FOR
TCR REPROGRAMMING USING INDUCIBLE FUSION PROTEINS
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/671,342, filed May 14, 2018, which is entirely incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Engineered T cells expressing either T Cell Receptor Fusion Constructs (TRuC™s) or chimeric antigen receptors (CARs) at their surface have produced exciting anti-tumor responses in vitro and in vivo. Although studies have demonstrated the potential of these technologies, there have been some issues that have raised concerns associated with "on-target off-tumor" effect, especially in healthy tissues which may express low levels of the targeted antigen. As such the ability to control the functional activity of CAR or TruC™ T cells is an important objective to ensure optimal safety and efficacy of engineered T cell therapies in solid organ tumors. As such strategies that may improve engineered T cell discrimination between healthy tissue and cancer cells would provide important advantages over current therapies (Juillerat, et al., 2017).
[0003] Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. The ability to engineer synthetic systems in T cells that are responding to multiple inputs would benefit adoptive immunotherapy using engineered T cells (Juillerat, et al., 2017; Chakravarti D. & Wong W. W., 2015). In the last decade numerous approaches to spatiotemporally control engineered T cells, including those relying on the addition of exogenous small molecules or monoclonal antibodies to regulate (Juillerat A, et al., 2016; Wu CY, et al; Ma JS, et al., 2016; Rodgers DT, et al., 2016; Tamada, et al., 2012; Urbanska, et al., 2012) or terminate (Marin V, et al., 2012; Poriot L, et al; Straathof KC., 2005; Duong CP, et al., 2011) engineered T cell functions. Alternatively, to achieve optimal tuning of CAR T cell targeting and functional properties, researchers have developed novel approaches based on the use of combinatorial antigen targeting, such as trans -signaling CARs (Straathof KC., 2005; Duong CP, et al., 2011). Integration of endogenous environmental signals, in addition to antigen recognition, may represent a valuable advancement to improve the control of the CAR T cell technology. An attractive strategy to discriminate between healthy tissue and cancer cells would be to harness the idiosyncrasies of the tumor microenvironment (TME). The tumor microenvironment is characterized by numerous factors including expression of matrix metalloproteinases, cathepsins, proteases, Indolomine 2,3-dioxygenase, low extracellular pH (acidosis) and low oxygenation (hypoxia) (Brown & Wilson, 2004; Vaupel & Mayer, 2007).
[0004] Disclosed herein are approaches that take advantage of the TME to trigger the activation of engineered T cells.
SUMMARY
[0005] Disclosed herein are methods and compositions for inducible T cell therapeutics that take
advantage of the tumor microenvironment (TME) to trigger the activation of engineered T cells. Using exogenous factors or known environmental signals within the TME allows the manipulation of the engineered T cell response. In particular, engineered T cells that can only be activated in the presence of certain TME factors will only be activated in the tumor atmosphere, thereby reducing the potential for on-target off-tumor toxicities, but also potentially enhancing the activity of engineered T cells.
[0006] According to one aspect of the present disclosure, provided herein is an recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising (a) a TCR subunit comprising (i) at least a portion of a TCR extracellular domain, and (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon; (b) an antibody domain comprising a TAA binding domain; and (c) a blocking domain, wherein the TCR subunit and the antibody domain are operatively linked, the antibody domain and the blocking domain are linked by a protease -cleavable linker, and wherein the TFP incorporates into a TCR when expressed in a T cell.
[0007] According to one aspect of the present disclosure, provided herein is an recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising (a) a TCR subunit comprising (i) at least a portion of a TCR extracellular domain, and (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 gamma; and (b) an antibody domain comprising a TAA binding domain; and (c) a blocking domain, wherein the TCR subunit and the antibody domain are operatively linked, the antibody domain and the blocking domain are linked by a protease-cleavable linker, and wherein the TFP incorporates into a TCR when expressed in a T cell.
[0008] According to one aspect of the present disclosure, provided herein is an recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising (a) a TCR subunit comprising (i) at least a portion of a TCR extracellular domain, and (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 delta; and (b) an antibody domain comprising an antigen binding domain; and (c) a blocking domain, wherein the TCR subunit and the antibody domain are operatively linked, the antibody domain and the blocking domain are linked by a protease-cleavable linker, and wherein the TFP incorporates into a TCR when expressed in a T cell.
[0009] According to one aspect of the present disclosure, provided herein is an recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising (a) a TCR subunit comprising (i) at least a portion of a TCR extracellular domain, and (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of TCR alpha; and (b) an antibody domain comprising an antigen binding domain; and (c) a blocking domain, wherein the TCR subunit and the antibody domain are operatively linked, the antibody domain and the blocking domain are linked by a protease-cleavable linker, and wherein the TFP incorporates into a TCR when expressed in a T cell.
[0010] According to one aspect of the present disclosure, provided herein is an recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising (a) a TCR subunit
comprising (i) at least a portion of a TCR extracellular domain, and (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of TCR beta; and (b) an antibody domain comprising an antigen binding domain; and (c) a blocking domain, wherein the TCR subunit and the antibody domain are operatively linked, the antibody domain and the blocking domain are linked by a protease-cleavable linker, and wherein the TFP incorporates into a TCR when expressed in a T cell. In some embodiments, the antibody domain is a human or humanized antibody. In some embodiments, the encoded antigen binding domain is connected to the TCR extracellular domain by a linker sequence. In some embodiments, the encoded linker sequence comprises (G4S)n, wherein n=l to 4. In some embodiments, the encoded antigen binding domain is connected to the blocking domain by a linker sequence that encodes a protease-cleavable linker. In some embodiments, the linker sequence encodes a matrix metalloproteinase cleavable linker. In some embodiments, the matrix metalloproteinase cleavage linker comprises a sequence selected from any of sequences in Tables 1-14. In some embodiments, the linker sequence encodes a cathepsin-cleavable linker. In some embodiments, the linker sequence encodes a Urokinase plasminogen activator (uPA)-cleavable linker. In some embodiments, the encoded antigen binding domain is connected to an antibody that specifically binds a blocking domain by a linker sequence that encodes a protease-cleavable linker. In some embodiments, the TCR subunit comprises a TCR extracellular domain. In some embodiments, the TCR subunit comprises a TCR transmembrane domain. In some embodiments, the TCR subunit comprises a TCR intracellular domain. In some embodiments, the TCR subunit comprises (i) a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit. In some embodiments, the TCR subunit comprises a TCR intracellular domain comprising a stimulatory domain selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having at least one modification thereto. In some embodiments, the TCR subunit comprises an intracellular domain comprising a stimulatory domain selected from a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto. In some embodiments, the antibody domain comprises an antibody fragment. In some embodiments, the antibody domain comprises a scFv or a VHH domain. In some embodiments, the recombinant nucleic acid encodes (i) a light chain (LC) CDR1, LC CDR2 and LC CDR3 of an anti-TAA light chain binding domain amino acid sequence with 70-100% sequence identity to a light chain (LC) CDR1, LC CDR2 and LC CDR3 of an anti-TAA light chain binding domain provided herein, respectively, and/or (ii) a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 of an anti-TAA heavy chain binding domain amino acid sequence with 70-100% sequence identity to a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 of an anti-TAA heavy chain binding domain provided herein, respectively. In some embodiments, the recombinant nucleic acid encodes a light chain variable region, wherein the light chain variable region comprises an amino acid sequence having at least one but not more than 30 modifications of a light chain variable region amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity to a light chain variable region amino acid sequence of a light chain variable region
provided herein. In some embodiments, the recombinant nucleic acid encodes a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least one but not more than 30 modifications of a heavy chain variable region amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to a heavy chain variable region amino acid sequence of a heavy chain variable region provided herein. In some embodiments, the TFP includes an extracellular domain of a TCR subunit that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137,
CD 154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, the recombinant nucleic acid molecule further comprises a sequence encoding a costimulatory domain. In some embodiments, the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of DAP 10,
DAP 12, CD30, LIGHT, 0X40, CD2, CD27, CD28, CDS, ICAM-l, LFA-l (CD 11 a/CD 18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto. In some embodiments, the linker sequence encodes a peptide sequence that is cleaved by at least one of a tumor cell surface protease, a carboxypeptidase, a cathepsin, a kallikrein, a hexokinase, a plasmin, a stromelysin, factor Xa, a chymotrypsin-like protease, a trypsin -like protease, a elastase-like protease, atryptase, a chymase, a subtilisin-like protease, an actinidain, a proteinase, a bromelain, a calpain, a caspase, a cysteine protease, a papain, an HIV-1 protease, an HSV protease, a CMV protease, a chymosin, a renin, a pepsin, a matriptase, a plasmepsin, a nepenthesin, a
metalloexopeptidase, a metalloendopeptidase, a matrix metalloproteinase/ collagenase, a plasminogen activator, a urokinase plasminogen activator (uPA), an enterokinase, a prostate-specific antigen (PSA, hK3), an interleukin- 1 b converting enzyme, a thrombin, a fibroblast activation protein (FAP), a meprin, a granzyme, and a dipeptidyl peptidase. In some embodiments, the cathepsin is cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, or cathepsin L; the hexokinase is hKl, hKlO, or hKl5; the proteinase is PR-3; the caspase is caspase-3; the cysteine protease is Mir 1 -CP or legumain; the matrix metalloproteinase or collagenase is MMPl/( interstitial collagenase), MMP2/type IV collagenase,
MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM 10, or ADAM 12; the prostate-specific antigen is PSA or hK3, the FAP is FAP -a; the granzyme is granzyme M or granzyme B;
or the dipeptidyl peptidase is dipeptidyl peptidase IV (DPPIV/CD26). In some embodiments, the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP. In some embodiments, the isolated nucleic acid molecule is mRNA. In some embodiments, the TFP includes an immunoreceptor tyrosine-based activation motif (ITAM) of a TCR subunit that comprises an ITAM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CD5, CDl6a, CDl6b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications thereto. In some embodiments, the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon. In some embodiments, the ITAM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit. In some embodiments, the nucleic acid comprises a nucleotide analog. In some embodiments, the nucleotide analog is selected from the group consisting of 2 -O- methyl, 2' -O-methoxyethyl (2' -O-MOE), 2' -O-aminopropyl, 2' -deoxy, T-deoxy-2' -fluoro, 2' -O- aminopropyl (2' -O-AP), 2'-0-dimethylaminoethyl (2' -O-DMAOE), 2' -O-dimethylaminopropyl (2' -O- DMAP), T-O-dimethylaminoethyloxyethyl (2' -O-DMAEOE), 2 -O-N-methylacetamido (2' -O-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a 1 ' ,5 ' - anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2 -fluoro N3-P5 ' -phosphoramidite. In some embodiments, the recombinant nucleic acid molecule further comprises a leader sequence.
[0011] According to another aspect of the present disclosure, provided herein is a polypeptide molecule encoded by the nucleic acid molecule.
[0012] According to another aspect of the present disclosure, provided herein is a recombinant TFP molecule comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain.
[0013] According to another aspect of the present disclosure, provided herein is a recombinant TFP molecule comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.
[0014] According to another aspect of the present disclosure, provided herein is a recombinant TFP molecule comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally integrating into an endogenous TCR complex. In some embodiments, the TFP molecule further
comprises an antibody or antibody fragment comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the anti-TAA binding domain is a scFv or a VH domain. In some embodiments, the anti-TAA binding domain comprises a heavy chain with 95-100% identity to an amino acid sequence of an anti-TAA light chain provided herein, a functional fragment thereof, or an amino acid sequence thereof having at least one but not more than 30 modifications. In some embodiments, the anti-TAA binding domain comprises a light chain with 95-100% identity to an amino acid sequence of an anti-TAA heavy chain provided herein, a functional fragment thereof, or an amino acid sequence thereof having at least one but not more than 30 modifications. In some embodiments, the recombinant TFP molecule comprises a TCR extracellular domain that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, the TCR comprises an extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma. In some embodiments, the anti-TAA binding domain is connected to the TCR extracellular domain by a linker sequence. In some embodiments, the linker region comprises (G4S)n, wherein n=l to 4. In some embodiments, the recombinant TFP molecule further comprises a sequence encoding a costimulatory domain. In some embodiments, the recombinant TFP molecule further comprises a sequence encoding an intracellular signaling domain. In some embodiments, the recombinant TFP molecule further comprises a leader sequence.
[0015] According to another aspect of the present disclosure, provided herein is a nucleic acid comprising a sequence encoding a TFP. In some embodiments, the nucleic acid is selected from the group consisting of a DNA and a RNA. In some embodiments, the nucleic acid is an mRNA. In some embodiments, the nucleic acid comprises a nucleotide analog. In some embodiments, the nucleotide analog is selected from the group consisting of 2' -O-methyl, V -O-methoxyethyl (2' -O-MOE), V -O- aminopropyl, 2' -deoxy, T-deoxy-2' -fluoro, V -O-aminopropyl (2' -O-AP), 2'-0-dimethylaminoethyl (2' -O-DMAOE), 2' -O-dimethylaminopropyl (2' -O-DMAP), T-O-dimethylaminoethyloxyethyl (2' -O- DMAEOE), 2' -O-N-methylacetamido (2' -O-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a 1 ' ,5 ' - anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2 ' -fluoro N3-P5 ' - phosphoramidite. In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the nucleic acid is an in vitro transcribed nucleic acid. In some embodiments, the nucleic acid further comprises a sequence encoding a poly(A) tail. In some embodiments, the nucleic acid further comprises a 3 ' UTR sequence.
[0016] According to another aspect of the present disclosure, provided herein is a vector comprising a nucleic acid molecule encoding a TFP. In some embodiments, the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, a Rous sarcoma viral
(RSV) vector, or a retrovirus vector. In some embodiments, the vector further comprises a promoter. In some embodiments, the vector is an in vitro transcribed vector. In some embodiments, a nucleic acid sequence in the vector further comprises a poly(A) tail. In some embodiments, a nucleic acid sequence in the vector further comprises a 3 ' UTR.
[0017] Also provided herein is a cell comprising the recombinant nucleic acid molecule described herein, the polypeptide molecule described herein, the TFP molecule described herein, and/or the vector described herein. In some embodiments, the cell is a human T cell. In some embodiments, the T cell is a CD8+, CD4+ or CD4+CD8+ T cell. In some embodiments, the cell further comprises a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain. In some embodiments, the inhibitory molecule comprise first polypeptide that comprises at least a portion of PD 1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.
[0018] According to another aspect of the present disclosure, provided herein is a human CD8+, CD4+ or CD4+CD8+ T cell comprising at least two TFP molecules, the TFP molecules comprising an anti- TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+, CD4+ or CD4+CD8+ T cell.
[0019] According to another aspect of the present disclosure, provided herein is a protein complex comprising: a TFP molecule comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and at least one endogenous TCR subunit or endogenous TCR complex. In some embodiments, the TCR comprises an extracellular domain or portion thereof of a protein selected from the group consisting of TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, and a CD3 delta TCR subunit. In some embodiments, the anti-TAA binding domain is connected to the TCR extracellular domain by a linker sequence. In some embodiments, the linker region comprises (G4S)n, wherein n=l to 4.
[0020] According to another aspect of the present disclosure, provided herein is a protein complex comprising a TFP encoded by the recombinant nucleic acid molecule described herein, and at least one endogenous TCR subunit or endogenous TCR complex.
[0021] According to another aspect of the present disclosure, provided herein is a human CD8+, CD4+ or CD4+CD8+ T cell comprising at least two different TFP proteins per the protein complex.
[0022] According to another aspect of the present disclosure, provided herein is a human CD8+, CD4+ or CD4+CD8+ T cell comprising at least two different TFP molecules encoded by the isolated nucleic acid molecule described herein.
[0023] According to another aspect of the present disclosure, provided herein is a population of human CD8+, CD4+ or CD4+CD8+ T cells, wherein the T cells of the population individually or collectively comprise at least two TFP molecules, the TFP molecules comprising an anti-TAA binding domain, a
TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+, CD4+ T or CD4+CD8+ cell.
[0024] According to another aspect of the present disclosure, provided herein is a population of human CD8+, CD4+ or CD4+CD8+ T cells, wherein the T cells of the population individually or collectively comprise at least two TFP molecules encoded by the isolated nucleic acid molecule.
[0025] According to another aspect of the present disclosure, provided herein is a method of making a cell comprising transducing a T cell with the recombinant nucleic acid molecule described herein or the vector.
[0026] According to another aspect of the present disclosure, provided herein is a method of generating a population of RNA-engineered cells comprising introducing an in vitro transcribed RNA, a circular RNA or synthetic RNA into a cell, where the RNA comprises a nucleic acid encoding the TFP molecule.
[0027] According to another aspect of the present disclosure, provided herein is a method of providing an anti-tumor immunity in a mammal comprising administering to the mammal an effective amount of the recombinant nucleic acid molecule described herein, the polypeptide molecule described herein, a cell expressing the polypeptide molecule, the TFP molecule, the vector, or the cell. In some
embodiments, the cell is an autologous T cell. In some embodiments, the cell is an allogeneic T cell. In some embodiments, the mammal is a human.
[0028] According to another aspect of the present disclosure, provided herein is a method of treating a mammal having a disease associated with expression of mesothelin comprising administering to the mammal an effective amount of the isolated nucleic acid molecule, the polypeptide molecule, a cell expressing the polypeptide molecule, the TFP molecule, the vector, or the cell. In some embodiments, the disease associated with mesothelin expression is selected from the group consisting of a proliferative disease, a cancer, a malignancy, and a non-cancer related indication associated with expression of mesothelin. In some embodiments, the disease is a cancer selected from the group consisting of mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, thyroid cancer, bladder cancer, ureter cancer, kidney cancer, endometrial cancer, esophogeal cancer, gastric cancer, thymic carcinoma, cholangiocarcinoma and stomach cancer. In some embodiments, the disease is a cancer selected from the group consisting of mesothelioma, papillary serous ovarian adenocarcinoma, clear cell ovarian carcinoma, mixed Mullerian ovarian carcinoma, endometroid mucinous ovarian carcinoma, pancreatic adenocarcinoma, ductal pancreatic adenocarcinoma, uterine serous carcinoma, lung adenocarcinoma, extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma, breast adenocarcinoma, a disease associated with mesothelin expression, and combinations thereof. In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that increases the efficacy of a cell expressing a TFP molecule. In some embodiments, less cytokines are released in the mammal compared a mammal
administered an effective amount of a T cell expressing an anti-TAA chimeric antigen receptor (CAR). In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a TFP molecule. In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that treats the disease associated with mesothelin.
[0029] Also provided herein is the recombinant nucleic acid molecule, the isolated polypeptide molecule, a cell expressing the polypeptide molecule, the isolated TFP, the vector, the complex, or the cell, for use as a medicament.
[0030] According to another aspect of the present disclosure, provided herein is a method of treating a mammal having a disease associated with expression of mesothelin comprising administering to the mammal an effective amount of the isolated nucleic acid molecule described herein, the polypeptide molecule described herein, a cell expressing the polypeptide molecule described herein, the TFP molecule described herein, the vector described herein, or the cell described herein, wherein less cytokines are released in the mammal compared a mammal administered an effective amount of a T cell expressing an anti-TAA chimeric antigen receptor (CAR).
[0031] According to another aspect, provided herein is a method of treating a mammal having a disease associated with expression of TAA comprising administering to the mammal an effective amount of the isolated nucleic acid molecule described herein, the polypeptide molecule described herein, a cell expressing the polypeptide molecule described herein, the TFP molecule described herein, the nucleic acid described herein, the vector described herein, or the cell described herein.
[0032] In some embodiments, the disease associated with TAA expression is selected from the group consisting of a proliferative disease, a cancer, a malignancy, and a non-cancer related indication associated with expression of TAA. In some embodiments, the disease is a cancer selected from the group consisting of mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, thyroid cancer, bladder cancer, ureter cancer, kidney cancer, endometrial cancer, esophageal cancer, gastric cancer, thymic carcinoma, cholangiocarcinoma and stomach cancer. In some embodiments, the disease is a cancer selected from the group consisting of mesothelioma, papillary serous ovarian adenocarcinoma, clear cell ovarian carcinoma, mixed Mullerian ovarian carcinoma, endometroid mucinous ovarian carcinoma, pancreatic adenocarcinoma, ductal pancreatic
adenocarcinoma, uterine serous carcinoma, lung adenocarcinoma, extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma, breast
adenocarcinoma, a disease associated with TAA expression, and combinations thereof. In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that increases the efficacy of a cell expressing a TFP molecule. In some embodiments, less cytokines are released in the mammal compared a mammal administered an effective amount of a T cell expressing an anti-TAA chimeric antigen receptor (CAR). In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that ameliorates one or more side effects associated with
administration of a cell expressing a TFP molecule. In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that treats the disease associated with the antigen. In some embodiments, the antigen is a tumor-associated antigen. In some embodiments, the antigen is one or more selected from CD 19, B-cell maturation antigen (BCMA), mesothelin (MSLN), ILl3Rcx2, MUC16, CD22, PD-l, BAFF or BAFF receptor, and ROR-l.
[0033] Also provided herein is the isolated nucleic acid molecule described herein, the isolated polypeptide molecule described herein, a cell expressing the polypeptide molecule described herein, the isolated TFP described herein, the nucleic acid described herein, the vector described herein, the complex described herein, or the cell described herein, for use as a medicament.
[0034] According to another aspect, provided herein is a method of treating a mammal having a disease associated with expression of TAA comprising administering to the mammal an effective amount of the isolated nucleic acid molecule described herein, the polypeptide molecule described herein, a cell expressing the polypeptide molecule described herein, the TFP molecule described herein, the nucleic acid described herein, the vector described, or the cell described herein, wherein less cytokines are released in the mammal compared a mammal administered an effective amount of a T cell expressing an anti -TAA chimeric antigen receptor (CAR).
[0035] In some embodiments, the TFP comprises an anti-TAA binding domain binds to an antigen derived from antigens of alpha-actinin-4, ARTC1, BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDK12, CDKN2A, CLPP, COA-l, CSNK1A1, dek-can fusion protein, EFTUD2, Elongation factor 2, ETV6-AML1 fusion protein, FLT3-ITD, FNDC3B, FN1, GAS7, GPNMB, HAUS3, HSDL1, LDLR-fucosyltransferase AS fusion protein, HLA-A2d, HLA-A1 ld, hsp70- 2, MART2, MATN, ME1, MUM-lf, MUM-2, MUM-3, neo-PAP, Myosin class I, NFYC, OGT, OS-9, p53, pml-RARalpha fusion protein, PPP1R3B, PRDX5, PTPRK, K-ras, N-ras, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, TGF-betaRII, triosephosphate isomerase, BAGE-l, D393-CD20n, Cyclin-Al, GAGE-l, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GnTVf, HERV-K-MEL, KK-LC-l, KM-HN-l, LAGE-l, LY6K, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12 m, MAGE-C1, MAGE-C2, mucink, NA88-A, NY-ESO-l / LAGE-2, SAGE, Spl7, SSX-2, SSX-4, TAG-l, TAG-2, TRAG-3, TRP2- INT2g, XAGE- 1 b/GAGED2a, B7H4, DLL3, TROP-2, Nectin-4, tissue factor, LIV-l, CD48, cMET„ Gene / protein, CEA, gplOO / Pmell7, mammaglobin-A, Melan-A / MART-l, NY-BR-l, OA1, PAP, PSA, RAB38 / NY-MEL-l, TRP-l / gp75, TRP-2, tyrosinase, adipophilin, AIM-2, ALDH1A1, BCLX (L), BING-4, CALCA, CD45, CD274, CPSF, cyclin Dl, DKK1, ENAH (hMena), EpCAM, EphA3, EZH2, FGF5, glypican-3, HER-2/neu, HLA-DOB, Hepsin, IDOl, IGF2B3, ILl3Ralpha2, Intestinal carboxyl esterase, alpha-foetoprotein, Kallikrein 4, KIF20A, Lengsin, M-CSF, MCSP, mdm-2, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, p53, PAX5, PBF, PRAME, PSMA, RAGE-l, RGS5, RhoC, RNF43, RU2AS, secemin 1, SOX10, STEAP1, survivin, Telomerase, TPBG, VEGF, and WT1.
In some embodiments, the TFP comprises one or more of an anti-CD 19 binding domain, an anti -B-cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-
IL 13Ra2 binding domain, an anti-MUCl6 binding domain, an anti-CD22 binding domain, an anti -PD- 1 binding domain, an anti-BAFF or BAFF receptor binding domain, and an anti-ROR-l binding domain.
INCORPORATION BY REFERENCE
[0036] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIGs. 1A-B show two drawings comprising an exemplary CAR T cell bound to HSA via HSA- binding sdAb and a protease-cleavable linker. FIG. 1A shows a CAR T cell fused with an anti -HSA antibody; FIG. IB shows the CAR T cell from FIG. 1A with HSA bound to the anti-HSA domain.
[0038] FIGs. 2A-B show two drawings depicting the fusion of HSA -binding sdAb to the Va or Vb Domain of a TFP T cell via a protease-cleavable linker. FIG. 2A shows a TFP T cell fused with an anti- HSA antibody; FIG. 2B shows the TFP T cell from FIG. 2A with HSA bound to the anti-HSA domain.
[0039] FIGs. 3A-B is two drawings depicting HSA binding to the sdAb of the TFP T cell. This blocks binding of the TCR to the MHC/peptide antigen complex. FIG. 3A shows a TFP T cell fused with an anti-HSA antibody; FIG. 3B shows the TFP T cell from FIG. 3A with HSA bound to the anti-HSA domain.
[0040] FIG. 4 is a drawing depicting proteolytic cleavage of the HSA-binding sdAb at the protease - cleavage site, which enables the inducible TFP T cell to bind to its MHC/peptide complex.
[0041] FIG. 5 is a drawing depicting direct fusion of HSA to an anti -tumor-associated antigen binding domain of a TFP T cell.
[0042] FIG. 6 shows sequences of example anti-HSA-[cleavable-linker]-anti-MSLN constructs. FIG. 6A shows a uPA-cleavable fusion protein sequence. FIG. 6B shows an MMP9vl-cleavable fusion protein sequence. FIG. 6C shows an MMP9v2-cleavable fusion protein sequence. FIG. 6D shows a cathepsin B fusion protein sequence. FIG. 6E shows a non-cleavable control sequence.
[0043] FIG. 7 depicts two graphs showing data demonstrating that inducible anti-mesothelin (MSLN) TFP T cells bind to soluble MSLN and are inhibited by their HSA blocking domains. FIG. 7A depicts a graph showing FACS data that illustrates the anti-HSA/anti-MSLN TFPs are able to bind to MSLN in the absence of HSA, and the constructs are able to bind HSA in the absence of MSLN. FIG. 7B depicts two graphs showing the ability of anti-HSA/anti-MSLN TFP T cells to induce cytokine production when expressed in Jurkat cells and co-cultured with MSTO cells that express MSLN. Panels show from left to right Jurkat cells alone, the anti-HSA/anti-MSLN TFP T cells alone, anti-HSA/anti-MSLN TFP cells comprising an MMP9-cleavable linker, anti-HSA/anti-MSLN TFP T cells comprising a cathepsin B- cleavable linker, and anti-HSA/anti-MSLN TFP cells comprising a non-cleavable linker, and MSTO cells alone (no effector cells, i.e., Jurkat). Each was done in the presence or absence of HSA.
[0044] FIG. 8 is a western blot illustrating cleavability of anti-HSA -cleavable-linker-anti-MSLN ("SD1") constructs. Constructs were loaded pairwise as follows: ctHSA-sdAb-uPA-SDl sdAb digested with uPA +/- FBS; aAlb-N/C-SDl digested with uPA +/- FBS; aHSA sdAb-MMP9vl-
SDlsdAb digested with MMP9 +/- FBS; aHSA sdAb-MMP9v2-SDlsdAb digested with MMP9 +/- FBS; and aHSA sdAb-cathepsin B-SD1 sdAb digested with cathepsin B +/- FBS. The western blot was probed with an anti-6His antibody and shows both the uncleaved constructs (around 30kDa) and the cleaved products (around 14 kDa).
DETAILED DESCRIPTION
[0045] Provided herein are compositions of matter and methods of use for the treatment of a disease such as cancer, using inducible T cell receptor (TCR) fusion proteins or T cell populations. As used herein, an "inducible T cell receptor (TCR) fusion protein" includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that, when induced, is generally capable of i) binding to a surface antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on the surface of a T cell e.g., once the T cell has entered the tumor microenvironment (TME) of a solid tumor. In particular, the inducible TFP T cells disclosed herein comprise a blocking domain that keeps the antigen binding domain (e.g., a tumor associated antigen binding domain) from binding until the TFP T cells reach the target site (e.g., a tumor). Thus, the compositions and methods disclosed herein are suitable for treating a disease wherein the target of interest is expressed on cells of a tissue other than the target tissue, as the inducible nature of the TFP T cells reduces the likelihood of off-target effects in a subject (e.g., a mammal).
[0046] As provided herein, TFPs provide substantial benefits as compared to Chimeric Antigen Receptors. The term "Chimeric Antigen Receptor," or alternatively, a "CAR," refers to a recombinant polypeptide comprising an extracellular antigen binding domain in the form of a binding domain such as an scFv, sdAb, Fab, etc., a transmembrane domain, and cytoplasmic signaling domains (also referred to herein as "an intracellular signaling domains") comprising a functional signaling domain derived from a stimulatory molecule as defined below. Generally, the central intracellular signaling domain of a CAR is derived from the CD3 zeta chain that is normally found associated with the TCR complex. The CD3 zeta signaling domain can be fused with one or more functional signaling domains derived from at least one co-stimulatory molecule such as CD2, 4-1BB (i.e., CD137), CD27 and/or CD28.
[0047] In one aspect, described herein are isolated nucleic acid molecules encoding an inducible T cell Receptor (TCR) fusion protein that comprise a TCR subunit, a non-human, human or humanized antibody domain comprising an anti-tumor associated antigen (TAA) binding domain, and a blocking domain connected to the TAA via a protease-cleavable linker, e.g., a matrix metalloproteinase cleavable linker. In some embodiments, the blocking domain is an antibody such as anti-albumin. In some embodiments, the blocking domain is albumin, IgG, or other bulky protein that sterically hinders binding of the TAA to its target protein on the tumor cell. In some embodiments, the inducible TFP comprises more than one blocking domain and more than one protease-cleavable linker.
[0048] In some embodiments, the TCR subunit comprises a TCR extracellular domain. In other embodiments, the TCR subunit comprises a TCR transmembrane domain. In yet other embodiments, the TCR subunit comprises a TCR intracellular domain. In further embodiments, the TCR subunit comprises (i) a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain,
wherein at least two of (i), (ii), and (iii) are from the same TCR subunit. In yet further embodiments, the TCR subunit comprises a TCR intracellular domain comprising a stimulatory domain selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having at least one, two or three modifications thereto. In yet further embodiments, the TCR subunit comprises an intracellular domain comprising a stimulatory domain selected from a functional signaling domain of CD2 and/or 4-1BB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one, two or three modifications thereto.
[0049] In some embodiments, the antibody domain comprises an antibody fragment. In some embodiments, the antibody domain comprises a scFv, a sdAb or a VH domain.
[0050] In some embodiments, the isolated nucleic acid molecules comprise (i) a light chain (LC) CDR1, LC CDR2 and LC CDR3 of any anti-tumor-associated antigen light chain binding domain amino acid sequence provided herein, and/or (ii) a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 of any anti- tumor-associated antigen heavy chain binding domain amino acid sequence provided herein.
[0051] In some embodiments, the light chain variable region comprises an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity to an amino acid sequence provided herein. In other embodiments, the heavy chain variable region comprises an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to an amino acid sequence provided herein.
[0052] In some embodiments, the TFP includes an extracellular domain of a TCR subunit that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma, or a functional fragment thereof, or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications thereto. In other embodiments, the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta chain of the TCR or TCR subunits CD3 epsilon, CD3 gamma and CD3 delta, or a functional fragment thereof, or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications thereto.
[0053] In some embodiments, the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD2, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, functional fragment(s) thereof, and amino acid sequences thereof having at least one, two or three modifications but not more than 20 modifications thereto.
[0054] In some instances, the isolated nucleic acid molecule further comprises a sequence encoding a costimulatory domain. In some instances, the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of DAP 10, DAP 12, CD30, LIGHT, 0X40,
CD2, CD27, CD28, CDS, ICAM-l, LFA-l (CD 11 a/CD 18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto. In some instances, the isolated nucleic acid molecule further comprises a leader sequence. In some instances, the isolated nucleic acid molecule is mRNA.
[0055] In some instances, the TFP includes an immunoreceptor tyrosine-based activation motif (IT AM) of a TCR subunit that comprises an IT AM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP 12), CD5, CDl6a, CDl6b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications thereto. In some instances, the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon. In some instances, the ITAM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.
[0056] In some instances, the nucleic acid comprises a nucleotide analog. In some instances, the nucleotide analog is selected from the group consisting of 2' -O-methyl, V -O-methoxyethyl (2' -O- MOE), 2' -O-aminopropyl, V -deoxy, T-deoxy-2' -fluoro, 2' -O-aminopropyl (2' -O-AP), 2'-0- dimethylaminoethyl (2' -O-DMAOE), 2' -O-dimethylaminopropyl (2' -O-DMAP), T-O- dimethylaminoethyloxyethyl (2' -O-DMAEOE), 2' -O-N-methylacetamido (2' -O-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a 1 ' .5 ' - anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2 ' -fluoro N3-P5 ' -phosphoramidite.
[0057] Also provided herein are isolated polypeptide molecules encoded by any of the previously described nucleic acid molecules.
[0058] Also provided herein in another aspect, are isolated T cell receptor fusion protein (TFP) molecules that comprise an anti-tumor-associated antigen binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the isolated TFP molecules comprises an antibody or antibody fragment comprising an anti-tumor-associated antigen binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain.
[0059] In some embodiments, the anti-TAA binding domain is an antibody, Fab, scFv, a VH domain, or an sdAb, or a functional fragment thereof. In other embodiments, the anti-tumor-associated antigen binding domain comprises a light chain and a heavy chain of an amino acid sequence provided herein, or a functional fragment thereof, or an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity with an amino acid sequence provided herein. In
some embodiments, the isolated TFP molecules comprise a TCR extracellular domain that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma, or an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications thereto.
[0060] In some embodiments, the anti-tumor-associated antigen binding domain is connected to the TCR extracellular domain by a non-protease-cleavable linker sequence. In some instances, the linker sequence comprises (G4S)n, wherein n=l to 4. In some instances, the linker sequence comprises a long linker (LL) sequence. In some instances, the long linker sequence comprises (G4S)n, wherein n=2 to 4. In some instances, the linker sequence comprises a short linker (SL) sequence. In some instances, the short linker sequence comprises (G4S)n, wherein n=l to 3.
[0061] In some embodiments, the isolated TFP molecules further comprise a sequence encoding a costimulatory domain. In other embodiments, the isolated TFP molecules further comprise a sequence encoding an intracellular signaling domain. In yet other embodiments, the isolated TFP molecules further comprise a leader sequence.
[0062] Also provided herein are vectors that comprise a nucleic acid molecule encoding any of the previously described TFP molecules. In some embodiments, the vector is selected from the group consisting of a DNA, an RNA, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector. In some embodiments, the vector further comprises a promoter. In some embodiments, the vector is an in vitro transcribed vector. In some embodiments, a nucleic acid sequence in the vector further comprises a poly(A) tail. In some embodiments, a nucleic acid sequence in the vector further comprises a 3 ' UTR.
[0063] Also provided herein are cells that comprise any of the described vectors. In some embodiments, the cell is a human T cell. In some embodiments, the cell is a CD8+ or CD4+ or CD4+CD8+ T cell. In one embodiment, the CD8+ cell is a gamma-delta T cell. In another embodiment, the CD8+ cell is an NK-T cell. In other embodiments, the cells further comprise a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain. In some instances, the inhibitory molecule comprises a first polypeptide that comprises at least a portion of PD1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.
[0064] In another aspect, provided herein are isolated TFP molecules that comprise a human or humanized anti-tumor-associated antigen (TAA) binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.
[0065] In another aspect, provided herein are isolated TFP molecules that comprise an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally integrating into an endogenous TCR complex.
[0066] In another aspect, provided herein are human CD8+ or CD4+ or CD4+CD8+ T cells that
comprise one or more inducible TFP molecules, the TFP molecules comprising a blocking domain or antibody specific to a protein suitable for a blocking domain, an antigen binding domain (e.g., an anti- tumor-associated (TAA) antigen binding domain), a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+ or CD4+ or CD4+CD8+ T cell. In another aspect, the cells comprise at least two non-identical inducible TFP molecules.
[0067] In another aspect, provided herein are protein complexes that comprise i) a TFP molecule comprising an anti-tumor-associated antigen binding domain, a blocking domain or an antibody specific to a blocking domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and ii) at least one endogenous TCR complex subunit.
[0068] In some embodiments, the TCR comprises an extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma. In some embodiments, the anti-tumor-associated antigen binding domain is connected to the TCR extracellular domain by a linker sequence. In some instances, the linker region comprises (G4S)n, wherein n=l to 4. In some instances, the linker sequence comprises a long linker (LL) sequence. In some instances, the long linker sequence comprises (G4S)n, wherein n=2 to 4. In some instances, the linker sequence comprises a short linker (SL) sequence. In some instances, the short linker sequence comprises (G4S)n, wherein n=l to 3.
[0069] Also provided herein are human CD8+ or CD4+ or CD8+CD4+ T cells that comprise at least two different TFP molecules per any of the described protein complexes.
[0070] In another aspect, provided herein is a population of human CD8+ or CD4+ or CD8+CD4+ or NK T cells, wherein the T cells of the population individually or collectively comprise at least two TFP molecules, the TFP molecules comprising an antigen binding domain (e.g., an anti-tumor-associated antigen binding domain), a blocking domain or an antibody to a protein suitable for a blocking domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+ or CD4+ or CD4+CD8+ T cell.
[0071] In another aspect, provided herein is a population of human CD8+ or CD4+ T or CD8+CD4+ cells, wherein the T cells of the population individually or collectively comprise at least two TFP molecules encoded by an isolated nucleic acid molecule provided herein.
[0072] In another aspect, provided herein are methods of making a cell comprising transducing a T cell with any of the described vectors.
[0073] In another aspect, provided herein are methods of generating a population of RNA-engineered cells that comprise introducing an in vitro transcribed RNA or synthetic RNA into a cell, where the RNA comprises nucleic acid encoding one or more of the described TFP molecules. In some embodiments the RNA is a circular RNA.
[0074] In another aspect, provided herein are methods of providing an anti-tumor immunity in a mammal that comprise administering to the mammal an effective amount of a cell expressing any of the described TFP molecules. In some embodiments, the cell is an autologous T cell. In some embodiments, the cell is an allogeneic T cell. In some embodiments, the mammal is a human.
[0075] In another aspect, provided herein are methods of treating a mammal having a disease associated with expression of tumor-associated antigen that comprise administering to the mammal an effective amount of the cell comprising any of the described TFP molecules. In some embodiments, the disease associated with tumor-associated antigen expression is selected from a proliferative disease such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia, or is a non-cancer related indication associated with expression of tumor-associated antigen.
[0076] In some embodiments, the disease is a hematologic cancer selected from the group consisting of one or more acute leukemias including but not limited to B-cell acute lymphoid leukemia ("B-ALL"), T cell acute lymphoid leukemia ("T-ALL"), acute lymphoblastic leukemia (ALL); one or more chronic leukemias including but not limited to chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); additional hematologic cancers or hematologic conditions including, but not limited to B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt' s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell- follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, smoldering multiple myeloma, solitary plasmacytoma, lymphoplasmacytic lymphoma, plasma cell leukemia, myelodysplasia and
myelodysplastic syndrome, non-Hodgkin' s lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom' s macroglobulinemia, and "preleukemia" which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, and to disease associated with tumor-associated antigen expression include, but not limited to atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing tumor- associated antigen; and combinations thereof.
[0077] In some embodiments, the disease is a solid tumor. In some instances, the cancer is selected from the group consisting of renal cell carcinoma, breast cancer, lung cancer, mesothelioma, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, and stomach cancer. In some embodiments the tumor associated antigen is expressed on the surface of tumor cells and is suitable for treatment with a TFP T cell (comprising a T cell receptor Fusion Protein (TFP) T cell as disclosed herein, or a CAR T cell. In some embodiments the tumor associated antigen is expressed inside the cancer cell and a suitable treatment is a TCR T cell.
[0078] In some embodiments, the cells expressing any of the described TFP molecules are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a TFP molecule. In some embodiments, the cells expressing any of the described TFP molecules are administered in combination with an agent that treats the disease associated with tumor-
associated antigen.
[0079] Also provided herein are any of the described isolated nucleic acid molecules, any of the described isolated polypeptide molecules, any of the described isolated TFPs, any of the described protein complexes, any of the described vectors or any of the described cells for use as a medicament. Definitions
[0080] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.
[0081] The term "a" and "an" refers to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.
[0082] As used herein, "about" can mean plus or minus less than 1 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or greater than 30 percent, depending upon the situation and known or knowable by one skilled in the art. The term "about" or "approximately" can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e. the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term "about" or "approximately" can mean within an order of magnitude, within 5-fold, and more preferably within 2- fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed. The term "about" can have the meaning as commonly understood by one of ordinary skill in the art. The term "about" can refer to +10%. The term "about" can refer to +5%.
[0083] As used herein the specification, "subject" or "subjects" or "individuals" may include, but are not limited to, mammals such as humans or non-human mammals, e.g., domesticated, agricultural or wild, animals, as well as birds, and aquatic animals. "Patients" are subjects suffering from or at risk of developing a disease, disorder or condition or otherwise in need of the compositions and methods provided herein.
[0084] As used herein, "treating" or "treatment" refers to any indicia of success in the treatment or amelioration of the disease or condition. Treating can include, for example, reducing, delaying or alleviating the severity of one or more symptoms of the disease or condition, or it can include reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient. As used herein, "treat or prevent" is sometimes used herein to refer to a method that results in some level of treatment or amelioration of the disease or condition and
contemplates a range of results directed to that end, including but not restricted to prevention of the condition entirely.
[0085] As used herein, "preventing" refers to the prevention of the disease or condition, e.g., tumor formation, in the patient. For example, if an individual at risk of developing a tumor or other form of
cancer is treated with the methods of the present invention and does not later develop the tumor or other form of cancer, then the disease has been prevented, at least over a period of time, in that individual.
[0086] As used herein, a "therapeutically effective amount" is the amount of a composition or an active component thereof sufficient to provide a beneficial effect or to otherwise reduce a detrimental non- beneficial event to the individual to whom the composition is administered. By "therapeutically effective dose" herein is meant a dose that produces one or more desired or desirable (e.g., beneficial) effects for which it is administered, such administration occurring one or more times over a given period of time. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g. Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999))
[0087] As used herein, a "T cell receptor (TCR) fusion protein" or "TFP" includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that is generally capable of i) binding to a surface antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on the surface of a T cell.
[0088] The portion of the TFP composition comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) derived from a murine, humanized or human antibody (Harlow et ah, 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et ah, 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et ah, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et ah, 1988, Science 242:423-426). In one aspect, the antigen binding domain of a TFP composition comprises an antibody fragment. In a further aspect, the TFP comprises an antibody fragment that comprises a scFv or a sdAb.
[0089] The term "antigen" or "Ag" may refer to a molecule that is capable of being bound specifically by an antibody, or otherwise provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, or both. As used herein, the term "cancer antigen" or "cancer-related antigen" may refer to any cancer cell marker expressed on the surface of a malignant or tumor cell that can be treated with the combination therapy described herein, including, but not limited to: described herein include, but are not limited to, 5T4, 8H9, oίn b Q integrin, anb6 integrin, alphafetoprotein (AFP), B7-H6, CA-125 carbonic anhydrase 9 (CA9),
CD 19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD52, CD123, CD171, carcinoembryonic antigen (CEA), EpCAM (epithelial cell adhesion molecule), E-cadherin, EMA
(epithelial membrane antigen), EGFRvlll, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbBl/EGFR, ErbB2/HER2/neu/EGFR2, ErbB3/HER3, ErbB4, epithelial tumor antigen (ETA), folate binding protein (FBP), fetal acetylcholine receptor (AchR), folate rcccptor-a. ganglioside 2 (GD2), ganglioside 3 (GD3), HLA-A1, HLA-A2, high molecular weight melanoma-associated antigen (HMW-MAA), IL-13 receptor «2 ( I L- 13Ra2). kinase insert domain receptor (KDR), k-light chain,
Fewis Y (FeY), Fl cell adhesion molecule, melanoma-associated antigen (MAGE-A1 ), mesothelin, mucin-l (MUC1 ), mucin-l6 (MUC16), natural killer group 2 member D (NKG2D) ligands, nerve cell adhesion molecule (NCAM), NY-ESO-l, B7H4, DLL3, TROP-2, Nectin-4, tissue factor, LIV-l, CD48, cMET, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate -specific membrane antigen (PSMA), receptor-tyrosine kinase-like orphan receptor 1 (R0R1), TAA targeted by mAb IgE, tumor- associated glycoprotein-72 (TAG-72), tyrosinase, and vascular endothelial growth factor (VEGF) receptors.
[0090] The term "antibody," as used herein, refers to a protein, or polypeptide sequences derived from an immunoglobulin molecule, which specifically binds to an antigen. Antibodies can be intact immunoglobulins of polyclonal or monoclonal origin, or fragments thereof and can be derived from natural or from recombinant sources.
[0091] The terms "antibody fragment" or "antibody binding domain" refer to at least one portion of an antibody, or recombinant variants thereof, that contains the antigen binding domain, i.e., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope. Examples of antibody fragments include, but are not limited to, Fab, Fab' , F(ab' )2, and Fv fragments, single-chain (sc)Fv ("scFv") antibody fragments, linear antibodies, single domain antibodies (abbreviated "sdAb") (either VF or VH), camelid VHH domains, and multi -specific antibodies formed from antibody fragments.
[0092] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
[0093] "Heavy chain variable region" or "VH" (or, in the case of single domain antibodies, e.g., nanobodies, "VHH" or "sdAb") with regard to an antibody refers to the fragment of the heavy chain that contains three CDRs interposed between flanking stretches known as framework regions, these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.
[0094] Unless specified, as used herein an scFv may have the VF and VH regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VF-linker- VH or may comprise VH-linker-VF.
[0095] The portion of the TFP composition of the invention comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb) or heavy chain antibodies HCAb 242:423-426). In one aspect, the antigen binding domain of a TFP composition of the invention comprises an antibody fragment. In a further aspect, the TFP comprises an antibody fragment that comprises a scFv or a sdAb.
[0096] The term "antibody heavy chain," refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.
[0097] The term "antibody light chain," refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (" K ") and lambda ("l") light chains refer to the two major antibody light chain isotypes.
[0098] The term "recombinant antibody" refers to an antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art. The term "antigen" or "Ag" refers to a molecule that is capable of being bound specifically by an antibody, or otherwise provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, or both.
[0099] As used herein, the term "combination therapy" means a therapy strategy that embraces the administration of therapeutic compositions of the present invention (e.g., conjugates comprising one or more neoantigens) and one or more additional therapeutic agents as part of a specific treatment regimen intended to provide a beneficial (additive or synergistic) effect from the co-action of these therapeutic agents. Administration of these therapeutic agents in combination may be carried out over a defined time period (usually minutes, hours, days, or weeks depending upon the combination selected). In
combination therapy, combined therapeutic agent may be administered in a sequential manner, or by substantially simultaneous administration.
[0100] As used herein, the terms "cytotoxic T cell (TC)" or "cytotoxic T lymphocyte (CTL)", or "T- killer cells", or "CD8+ T cell" or "killer T cell" are used interchangeably. This type of white blood cells are T lymphocytes that can recognize abnormal cells including cancer cells, cells that are infected particularly by viruses, and cells that are damaged in other ways and induce the death of such cells.
[0101] As used herein, the term "epitope" means a small peptide structure formed by contiguous amino acids, or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and about 9, or about 8-15 amino acids. A T cell epitope means a peptide which can be bound by the MHC molecules of class I or II in the form of a peptide-presenting MHC molecule or MHC complex and then, in this form, be recognized and bound by native T cells, cytotoxic T- lymphocytes or T-helper cells, respectively.
[0102] As used herein, the term "immune cell" refers to a cell that is capable of participating, directly or indirectly, in an immune response. Immune cells include, but are not limited to T cells, B-cells, antigen
presenting cells, dendritic cells, natural killer (NK) cells, natural killer T (NK) cells, lymphokine- activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhans' cells, stem cells, peripheral blood mononuclear cells, cytotoxic T cells, tumor infiltrating lymphocytes (TIL), etc. An "antigen presenting cell" (APC) is a cell that is capable of activating T cells, and includes, but is not limited to, monocytes/macrophages, B cells and dendritic cells (DCs). "Dendritic cell" or "DC" refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their distinctive morphology, high levels of surface MHC -class II expression. DCs can be isolated from a number of tissue sources. DCs have a high capacity for sensitizing MHC- restricted T cells and are very effective at presenting antigens to T cells in situ. The antigens may be self-antigens that are expressed during T cell development and tolerance, and foreign antigens that are present during normal immune processes. As used herein, an "activated DC" is a DC that has been pulsed with an antigen and capable of activating an immune cell. "T cell" as used herein, is defined as a thymus-derived cell that participates in a variety of cell- mediated immune reactions, including CD8+ T cell and CD4+ T cell.
[0103] As used herein, the term "immune response" means a defensive response a body develops against "foreigner" such as bacteria, viruses and substances that appear foreign and harmful. An anti -cancer immune response refers to an immune surveillance mechanism by which a body recognizes abnormal tumor cells and initiates both the innate and adaptive of the immune system to eliminate dangerous cancer cells.
[0104] The innate immune system is a non-specific immune system that comprises the cells (e.g.,
Natural killer cells, mast cells, eosinophils, basophils; and the phagocytic cells including macrophages, neutrophils, and dendritic cells) and mechanisms that defend the host from infection by other organisms. An innate immune response can initiate the productions of cytokines, and active complement cascade and adaptive immune response. The adaptive immune system is specific immune system that is required and involved in highly specialized systemic cell activation and processes, such as antigen presentation by an antigen presenting cell; antigen specific T cell activation and cytotoxic effect.
[0105] The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample or might be macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with
other biological components.
[0106] "Specific dissociation," as used herein, refers to a separation of two components that preferentially occurs in a particular environment. In an aspect of the invention, specific dissociation refers to dissociation of domains separated by proteolytic cleavage by a protease that is more highly available in the tumor microenvironment. In an aspect of the invention, specific dissociation refers to separation of components of the pharmaceutical composition upon enzymatic digestion or other enzymatic activity.
[0107] As used herein, the phrase "biologically active domain" refers to any protein structure able to modulate a biological activity. This can include, but is not limited to, cytokines, chemokines, antibodies, antibody-drug conjugates, T cell fusion proteins, chimeric antigen receptors (CARs), and T cell receptor subunits.
[0108] The term "anti -tumor effect" refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, decrease in tumor cell proliferation, decrease in tumor cell survival, or amelioration of various physiological symptoms associated with the cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor in the first place.
[0109] As used herein, the terms "adoptive cellular immunotherapy'" or "adoptive immunotherapy ' or "T cell immunotherapy'", or "Adoptive T cell therapy (ACT)", are used interchangeably. Adoptive immunotherapy uses T cells that a natural or genetically engineered reactivity to a patient's cancer are generated in vitro and then transferred back into the cancer patient. The injection of a large number of activated tumor specific T cells can induce complete and durable regression of cancers.
[0110] The term "autologous" refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.
[0111] The term "allogeneic" refers to any material derived from a different animal of the same species or different patient as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.
[0112] The term "xenogeneic" refers to a graft derived from an animal of a different species.
[0113] The term "cancer" may refer to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include, but are not limited to, prostate cancer, breast cancer, melanoma, sarcoma, colorectal cancer, pancreatic cancer, uterine cancer, ovarian cancer, stomach cancer, gastric cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, cholangiocarcinoma, squamous cell lung cancer, mesothelioma, adrenocortico carcinoma, esophageal cancer, head & neck cancer, liver cancer, nasopharyngeal carcinoma, neuroepithelial cancer,
adenoid cystic carcinoma, thymoma, chronic lymphocytic leukemia, glioma, glioblastoma multiforme, neuroblastoma, papillary renal cell carcinoma, mantle cell lymphoma, lymphoblastic leukemia, acute myeloid leukemia, and the like.
[0114] The term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. 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. These families include 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) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a TFP of the invention can be replaced with other amino acid residues from the same side chain family and the altered TFP can be tested using the functional assays described herein.
[0115] The term "stimulation" refers to a primary response induced by binding of a stimulatory domain or stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex.
Stimulation can mediate altered expression of certain molecules, and/or reorganization of cytoskeletal structures, and the like.
[0116] The term "stimulatory molecule" or "stimulatory domain" refers to a molecule or portion thereof expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR complex in a stimulatory way for at least some aspect of the T cell signaling pathway. In one aspect, the primary signal is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or "ITAM". Examples of an ITAM containing primary cytoplasmic signaling sequence that is of particular use in the invention includes, but is not limited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS") and CD66d.
[0117] The term "antigen presenting cell" or "APC" refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC' s) on its surface. T cells may recognize these complexes
using their T cell receptors (TCRs). APCs process antigens and present them to T cells.
[0118] An "intracellular signaling domain," as the term is used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the TFP containing cell, e.g., a TFP -expressing T cell. Examples of immune effector function, e.g., in a TFP -expressing T cell, include cytolytic activity and T helper cell activity, including the secretion of cytokines. In an embodiment, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In an embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain.
Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.
[0119] A primary intracellular signaling domain can comprise an ITAM ("immunoreceptor tyrosine- based activation motif"). Examples of ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d DAP 10 and DAP 12.
[0120] The term "costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response. Costimulatory molecules include, but are not limited to, an MHC class 1 molecule, BTLA, a Toll ligand receptor,
0X40, CD2, CD27, CD28, CDS, ICAM-l, LFA-l (CDl la/CDl8) and 4-1BB (CD137). A costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. A
costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4- 1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like. The intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof. The term "4-1BB" refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Ace. No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like; and a "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Ace. No. AAA62478.2, or equivalent residues from non-human species, e.g., mouse, rodent, monkey, ape and the like.
[0121] The term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties
resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
[0122] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence " includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or a RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain one or more introns.
[0123] The term "effective amount" or "therapeutically effective amount" are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological or therapeutic result.
[0124] The term "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.
[0125] The term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.
[0126] The term "expression" refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.
[0127] The term "transfer vector" refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to further include non plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
[0128] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
[0129] The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene
delivery vector. HIV, SIV, and FIV are all examples of lenti viruses.
[0130] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic include, but are not limited to, e.g., the Lentivector® gene delivery technology from Oxford BioMedica, the
LentiMax® vector system from Lentigen, and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
[0131] The term "homologous" or "identity" refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.
[0132] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab' , F(ab' )2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary -determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non human residues. Furthermore, a humanized antibody/antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance. In general, the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al, Nature, 332: 323-329,
1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.
[0133] "Human" or "fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.
[0134] The term "isolated" means altered or removed from the natural state. For example, a nucleic
acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
[0135] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.
[0136] The term "operably linked" or "transcriptional control" refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.
[0137] The term "parenteral" administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, intratumoral, or infusion techniques.
[0138] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et ah, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et ah, J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et ak, Mol. Cell. Probes 8:91-98 (1994)).
[0139] The terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein' s or peptide' s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide
includes a natural peptide, a recombinant peptide, or a combination thereof.
[0140] The term "promoter" refers to a DNA sequence recognized by the transcription machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
[0141] The term "promoter/regulatory sequence" refers to a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
[0142] The term "constitutive" promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
[0143] The term "inducible" promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
[0144] The term "tissue-specific" promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
[0145] The terms "linker" and "flexible polypeptide linker" (not to be confused with "protease- cleavable linker" of the TFP molecules disclosed herein) as used in the context of a scFv refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and comprises the amino acid sequence (Gly-Gly-Gly- Ser)n, where n is a positive integer equal to or greater than 1. For example, n=l, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 and h=10. In one embodiment, the flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 or (Gly4Ser)3. In another embodiment, the linkers include multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser). Also included within the scope of the invention are linkers described in
WO2012/138475 (incorporated herein by reference). In some instances, the linker sequence comprises a long linker (LL) sequence. In some instances, the long linker sequence comprises (G4S)n, wherein n=2 to 4. In some instances, the linker sequence comprises a short linker (SL) sequence. In some instances, the short linker sequence comprises (G4S)n, wherein n=l to 3.
[0146] As used herein, a 5 ' cap (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is a modified guanine nucleotide that has been added to the "front" or 5 ' end of a eukaryotic messenger RNA shortly after the start of transcription. The 5 ' cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co -transcriptionally, such that each influences the other. Shortly after the start of transcription, the 5 ' end of the mRNA being
synthesized is bound by a cap -synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation.
[0147] As used herein, "in vitro transcribed RNA" refers to RNA, preferably mRNA, which has been synthesized in vitro. Generally, the in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA. In some embodiments, an in vitro transcribed RNA is circularizable.
[0148] As used herein, a "poly(A)" is a series of adenosines attached by polyadenylation to the mRNA. In the preferred embodiment of a construct for transient expression, the polyA is between 50 and 5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400. Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.
[0149] As used herein, "polyadenylation" refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3 ' end. The 3 ' poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm. After transcription has been terminated, the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3 ' end at the cleavage site.
[0150] As used herein, "transient" refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.
[0151] The term "signal transduction pathway" refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase "cell surface receptor" includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.
[0152] The term "subject" is intended to include living organisms in which an immune response can be elicited (e.g., mammals, human).
[0153] The term, a "substantially purified" cell refers to a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with
which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.
[0154] The term "therapeutic" as used herein means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.
[0155] The term "prophylaxis" as used herein means the prevention of or protective treatment for a disease or disease state.
[0156] In the context of the present invention, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to antigens that are common to specific hyperproliferative disorders. In certain aspects, the hyperproliferative disorder antigens of the present invention are derived from, cancers including but not limited to primary or metastatic melanoma, mesothelioma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, NHL, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.
[0157] The term "transfected" or "transformed" or "transduced" refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A "transfected" or "transformed" or "transduced" cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.
The term "specifically binds," refers to an antibody, an antibody fragment or a specific ligand, which recognizes and binds a cognate binding partner (e.g., BCMA, MSLN, NKG2D, ROR1, etc.) present in a sample, but which does not necessarily and substantially recognize or bind other molecules in the sample.
[0158] The term 'binding ligand" may generally refer to a polypeptide (e.g., a protein), a
polynucleotide (e.g., DNA, RNA, or a hybrid of DNA and RNA), a molecule, a chemical compound, a fragment thereof, and/or a hybrid thereof. In some embodiments, the binding ligand can comprise a polynucleotide, and the polynucleotide can be single stranded, double stranded, or a combination thereof. In some embodiments, a binding ligand can comprise a biological molecule or a non -biological molecule. In some embodiments, a biological molecule or non-biological molecule can be a naturally occurring molecule or an artificial molecule. Non-limiting examples of a binding ligand include a protein, a carbohydrate, a lipid, or a nucleic acid. In some embodiments, the binding ligand may associate, bind, and/or couple with an antibody or fragment thereof (e.g., an IgA isotype antibody, an IgD isotype antibody, an IgE isotype antibody, an IgG isotype antibody, an IgM isotype antibody, an IgW isotype antibody, an IgY isotype antibody). In some embodiments the antibody or fragment thereof may be an Fc domain of the antibody (e.g., the binding ligand is an Fc receptor). For example, in some
embodiments the binding ligand can specifically bind to an IgGl antibody. In some embodiments, the binding ligand may be capable of associating, capable of binding, and/or capable of coupling with an antibody or fragment thereof. In some embodiments, the binding ligand can comprise multiple subunits.
In some embodiments, a binding ligand can comprise multiple subunits, and the subunits can be the same. In some embodiments, a binding ligand can comprise multiple different subunits. In some embodiments, a binding ligand can comprise multiple subunits, and at least two of the subunits can be different. In some embodiments, a binding ligand can comprise a dimer, trimer, tetramer, pentamer, hexamer, heptamer, nonamer, or decamer. In some embodiments, a binding ligand can comprise greater than about ten subunits. In some embodiments, a binding ligand can comprise a polymer. In some embodiments, the binding ligand may be non-human (e.g., primate), human, or humanized.
[0159] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.
T cell receptor (TCR) fusion proteins (TFP)
[0160] The present invention encompasses recombinant DNA constructs encoding TFPs, wherein the TFP in one aspect comprises an antibody fragment that binds specifically to one or more tumor associated antigens ("TAA"), e.g., a human TAA, wherein the sequence of the antibody fragment is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof. In some aspects, the TFPs comprise an intervening in-frame sequence comprising a protease -cleavable linker (e.g., between the sequence encoding the antibody fragment and the sequence encoding the TCR subunit or portion thereof, such that the expressed protein comprises a protease- cleavable site between the TCR subunit and the antibody fragment). The TFPs provided herein are able to associate with one or more endogenous (or alternatively, one or more exogenous, or a combination of endogenous and exogenous) TCR subunits in order to form a functional TCR complex. In some embodiments, the antigen-binding domain is a humanized or human anti-TAA binding domain. In some embodiments, the antibody fragment is an anti-mesothelin antibody or a fragment thereof. In one aspect, the portion of the TFP comprising the antigen binding domain comprises an antigen binding domain that targets mesothelin. In one aspect, the antigen binding domain targets human mesothelin. Thus, in one aspect, the antigen-binding domain comprises a humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment. In one embodiment, the humanized or human anti-mesothelin binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain
complementary determining region 3 (LC CDR3) of a humanized or human anti-mesothelin binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining
region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-mesothelin binding domain described herein, e.g., a humanized or human anti-mesothelin binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs. In one embodiment, the humanized or human anti-mesothelin binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-mesothelin binding domain described herein, e.g., the humanized or human anti-mesothelin binding domain has two variable heavy chain regions, each comprising a HC CDR1, a HC CDR2 and a HC CDR3 described herein. In one embodiment, the humanized or human anti-mesothelin binding domain comprises a humanized or human light chain variable region described herein and/or a humanized or human heavy chain variable region described herein. In one embodiment, the humanized or human anti-mesothelin binding domain comprises a humanized heavy chain variable region described herein, e.g., at least two humanized or human heavy chain variable regions described herein. In one embodiment, the anti-mesothelin binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence provided herein.
[0161] In one embodiment, the humanized or human anti-TAA binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a humanized or human anti-TAA binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-TAA binding domain described herein, e.g., a humanized or human anti-TAA binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs. In one embodiment, the humanized or human anti-TAA binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-TAA binding domain described herein, e.g., the humanized or human anti- TAA binding domain has two variable heavy chain regions, each comprising a HC CDR1, a HC CDR2 and a HC CDR3 described herein. In one embodiment, the humanized or human anti-TAA binding domain comprises a humanized or human light chain variable region described herein and/or a humanized or human heavy chain variable region described herein. In one embodiment, the humanized or human anti-TAA binding domain comprises a humanized heavy chain variable region described herein, e.g. , at least two humanized or human heavy chain variable regions described herein. In one embodiment, the anti-TAA binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence provided herein. In some embodiments, the TFP comprises an anti-TAA binding domain binds to an antigen derived from antigens of alpha-actinin-4, ARTC1, BCR-ABL fusion protein (b3a2), B- RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDK12, CDKN2A, CLPP, COA-l, CSNK1A1,
dek-can fusion protein, EFTUD2, Elongation factor 2, ETV6-AML1 fusion protein, FLT3-ITD,
FNDC3B, FN1, GAS7, GPNMB, HAUS3, HSDL1, LDLR-fucosyltransferase AS fusion protein, HLA- A2d, HLA-A1 ld, hsp70-2, MART2, MATN, ME1, MUM-lf, MUM-2, MUM-3, neo-PAP, Myosin class I, NFYC, OGT, OS-9, p53, pml-RARalpha fusion protein, PPP1R3B, PRDX5, PTPRK, K-ras, N-ras, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, TGF-betaRII, triosephosphate isomerase, BAGE-l, D393-CD20n, Cyclin-Al, GAGE-l, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GnTVf, HERV-K-MEL, KK-LC-l, KM-HN-l, LAGE-l, LY6K, MAGE- Al, MAGE-A2, MAGE -A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12 m, MAGE- Cl, MAGE-C2, mucink, NA88-A, NY-ESO-l / LAGE-2, SAGE, Spl7, SSX-2, SSX-4, TAG-l, TAG-2, TRAG-3, TRP2-INT2g, XAGE- lb/GAGED2a, B7H4, DLL3, TROP-2, Nectin-4, tissue factor, LIV-l, CD48, cMET„ Gene / protein, CEA, gplOO / Pmell7, mammaglobin-A, Melan-A / MART-l, NY-BR-l, OA1, PAP, PSA, RAB38 / NY-MEL-l, TRP-l / gp75, TRP-2, tyrosinase, adipophilin, AIM-2,
ALDH1A1, BCLX (L), BING-4, CALCA, CD45, CD274, CPSF, cyclin Dl, DKK1, ENAH (hMena), EpCAM, EphA3, EZH2, FGF5, glypican-3, HER-2/neu, HLA-DOB, Hepsin, IDOl, IGF2B3,
ILl3Ralpha2, Intestinal carboxyl esterase, alpha-foetoprotein, Kallikrein 4, KIF20A, Lengsin, M-CSF, MCSP, mdm-2, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, p53, PAX5, PBF, PRAME, PSMA, RAGE-l, RGS5, RhoC, RNF43, RU2AS, secemin 1, SOX10, STEAP1, survivin, Telomerase, TPBG, VEGF, and WT1. In some embodiments, the TFP comprises one or more of an anti-CDl9 binding domain, an anti-B-cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-IL 13Ra2 binding domain, an anti-MUCl6 binding domain, an anti-CD22 binding domain, an anti -PD- 1 binding domain, an anti-BAFF or BAFF receptor binding domain, and an anti-ROR-l binding domain.
Blocking Domains
[0162] The TFP constructs disclosed herein comprise a blocking domain. In some embodiments, the blocking domain is tethered to the antigen binding domain via a protease-cleavable linker. In other embodiments, the blocking domain is tethered to a TCR complex subunit via a protease-cleavable linker. The blocking domain may sterically hinder the binding of the engineered T cell to the target antigen. In an embodiment, the blocking domain is albumin. In an embodiment, the blocking domain is an IgG. In an embodiment, the blocking domain is a peptide capable of sterically blocking the activity of a biologically active molecule, e.g., the binding of the TAA -binding domain to its target. In some embodiments, the TFP does not comprise a blocking domain but rather comprises an antibody specific to a protein suitable for blocking activity of the TFP. In one embodiment, the TFP comprises an anti-HSA antibody.
[0163] Human serum albumin (HSA) (molecular mass ~67 kDa) is the most abundant protein in plasma, present at about 50 mg/ml (600 mM), and has a serum half-life of around 20 days in humans. HSA serves to maintain plasma pH, contributes to colloidal blood pressure, functions as carrier of many metabolites and fatty acids, and serves as a major drug transport protein in plasma. In some embodiments, the serum albumin is human; in some embodiments, the serum albumin is mouse or cynomolgus monkey albumin. In some embodiments, the serum albumin is the same serum albumin as is natively found in the subject
to which the albumin-modified protein will be administered (see, e.g., U.S. Patent No. 9,920, 115).
[0164] In one aspect, the TFP of the invention comprises a target -specific binding element otherwise referred to as an antigen binding domain. The choice of moiety depends upon the type and number of target antigen that define the surface of a target cell. For example, the antigen binding domain may be chosen to recognize a target antigen that acts as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that may act as target antigens for the antigen binding domain in a TFP of the invention include those associated with viral, bacterial and parasitic infections; autoimmune diseases; and cancerous diseases (e.g., malignant diseases). In some embodiments, the antigen is a tumor-associated antigen.
[0165] In some embodiments, the blocking domain (e.g., albumin or another bulky protein) will be attached covalently directly to the antigen-binding domain (e.g., wherein the antigen-binding domain is attached to the TFP T cell via a protease-cleavable linker). In some embodiments, the albumin will be attached via a short linker (e.g., a protease-cleavable linker). In some embodiments, a subject' s own albumin will be attracted to the antigen-binding domain via an albumin-binding protein, such as an antibody or fragment thereof. In some embodiments, a single albumin molecule, directly attached or attracted via an albumin binding protein, will be enough to block activity. In some embodiments, two or more albumin molecules, directly attached or attracted via an albumin binding protein, will be used.
[0166] In one aspect, the TFP-mediated T cell response can be directed to an antigen of interest by way of engineering an antigen-binding domain into the TFP that specifically binds a desired antigen.
[0167] The antigen binding domain can be any domain that binds to the antigen. In some embodiments, the antigen binding domain comprises an antibody or a fragment thereof, including but not limited to: a monoclonal antibody; a polyclonal antibody; a recombinant antibody; a human antibody; a humanized antibody or a functional fragment thereof, including but not limited to a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of a camelid-derived nanobody; and to an alternative scaffold known in the art to function as antigen binding domain, such as a recombinant fibronectin domain, anticabn, DARPIN and the like. Likewise, a natural or synthetic ligand specifically recognizing and binding the target antigen can be used as antigen binding domain for the TFP, e.g., an NKG2D dimer or another binder comprising, e.g., a binder that participates in a receptor-ligand interaction. In some instances, it is beneficial for the antigen binding domain to be derived from the same species in which the TFP will ultimately be used in. For example, for use in humans, it may be beneficial for the antigen binding domain of the TFP to comprise human or humanized residues for the antigen binding domain of an antibody or antibody fragment.
[0168] Thus, in one aspect, the antigen-binding domain comprises a humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment. In one embodiment, the humanized or human anti-TAA binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a humanized or human anti-TAA binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining
region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-TAA binding domain described herein, e.g., a humanized or human anti-TAA binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs. In one embodiment, the humanized or human anti-TAA binding domain comprises one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-TAA binding domain described herein, e.g., the humanized or human anti-tumor-associated antigen binding domain has two variable heavy chain regions, each comprising a HC CDR1, a HC CDR2 and a HC CDR3 described herein. In one embodiment, the humanized or human anti-TAA binding domain comprises a single domain (sdAb) antibody. In one embodiment, the humanized or human anti-tumor- associated antigen binding domain comprises a humanized or human light chain variable region described herein and/or a humanized or human heavy chain variable region described herein. In one embodiment, the humanized or human anti-tumor-associated antigen binding domain comprises a humanized heavy chain variable region described herein, e.g., at least two humanized or human heavy chain variable regions described herein. In one embodiment, the anti-tumor-associated antigen binding domain is a scFv comprising a light chain and a heavy chain of an amino acid sequence provided herein. In an embodiment, the anti -tumor-associated antigen binding domain (e.g., an scFv or VHH) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity with an amino acid sequence provided herein; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30,
20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to an amino acid sequence provided herein. In one embodiment, the humanized or human anti-tumor-associated antigen binding domain is a scFv, and a light chain variable region comprising an amino acid sequence described herein, is attached to a heavy chain variable region comprising an amino acid sequence described herein, via a linker, e.g., a linker described herein. In one embodiment, the humanized anti-tumor-associated antigen binding domain includes a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4. The light chain variable region and heavy chain variable region of a scFv can be, e.g., in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region. In some instances, the linker sequence comprises a long linker (LL) sequence. In some instances, the long linker sequence comp rises (G4S)n, wherein n=2 to 4. In some instances, the linker sequence comprises a short linker (SL) sequence. In some instances, the short linker sequence comprises (G4S)n, wherein n=l to 3.
[0169] In some aspects, a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human or fragment
thereof. In one aspect, the antigen binding domain is humanized.
[0170] A humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein in its entirety by reference), veneering or resurfacing (see, e.g., European Patent Nos. EP592106 and EP519596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et ah, 1994, Protein Engineering, 7(6):805-8l4; and Roguska et ah, 1994, PNAS, 91:969-973, each of which is incorporated herein by its entirety by reference), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein in its entirety by reference), and techniques disclosed in, e.g., U.S. Patent Application Publication No. US20050042664, U.S. Patent Application Publication No. US20050048617, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. W09317105, Tan et ah, J. Immunol., 169: 1119-25 (2002), Caldas et al., Protein Eng., l3(5):353-60 (2000), Morea et ah, Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16): 10678-84 (1997), Roguska et al., Protein Eng., 9(l0):895-904 (1996), Couto et ak, Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8): 1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al,
J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein in its entirety by reference. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, for example improve, antigen binding. These framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entireties.)
[0171] A humanized antibody or antibody fragment has one or more amino acid residues remaining in it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. As provided herein, humanized antibodies or antibody fragments comprise one or more CDRs from nonhuman
immunoglobulin molecules and framework regions wherein the amino acid residues comprising the framework are derived completely or mostly from human germline. Multiple techniques for
humanization of antibodies or antibody fragments are well-known in the art and can essentially be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR-grafting (EP 239,400; PCT Publication No. WO 91/09967; and U.S. Patent Nos. 4,816,567;
6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference in their entirety). In such humanized antibodies and antibody fragments, substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species. Humanized antibodies are often human antibodies in which some CDR residues and possibly
some framework (FR) residues are substituted by residues from analogous sites in rodent antibodies. Humanization of antibodies and antibody fragments can also be achieved by veneering or resurfacing
(EP592106; EP519596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et ak, Protein Engineering, 7(6):805-8l4 (1994); and Roguska et ak, PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Patent No. 5,565,332), the contents of which are incorporated herein by reference in their entirety.
[0172] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable -domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et ak, J. Immunol., 151:2296 (1993); Chothia et ak, J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference herein in their entirety). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (see, e.g., Nicholson et ak Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et ak, Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et ak, J. Immunol., 151:2623 (1993), the contents of which are incorporated herein by reference herein in their entirety). In some embodiments, the framework region, e.g., all four framework regions, of the heavy chain variable region are derived from a VH4-4-59 germline sequence. In one embodiment, the framework region can comprise, one, two, three, four or five modifications, e.g., substitutions, e.g., from the amino acid at the corresponding murine sequence. In one embodiment, the framework region, e.g., all four framework regions of the light chain variable region are derived from a VK3-1.25 germline sequence. In one embodiment, the framework region can comprise, one, two, three, four or five modifications, e.g., substitutions, e.g., from the amino acid at the corresponding murine sequence.
[0173] In some aspects, the portion of a TFP composition of the invention that comprises an antibody fragment is humanized with retention of high affinity for the target antigen and other favorable biological properties. According to one aspect of the invention, humanized antibodies and antibody fragments are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind the target antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody or antibody fragment characteristic, such as increased affinity for the target antigen, is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.
[0174] In one aspect, the anti-tumor-associated antigen binding domain is a fragment, e.g., a single
chain variable fragment (scFv) or a camelid heavy chain (VHH). In one aspect, the anti-tumor-associated antigen binding domain is a Fv, a Fab, a (Fab' )2, or a bi-fimctional (e.g. bi-specific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In one aspect, the antibodies and fragments thereof of the invention binds a tumor-associated antigen protein with wild-type or enhanced affinity.
[0175] Also provided herein are methods for obtaining an antibody antigen binding domain specific for a target antigen, the method comprising providing by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a VH (or VHH) domain set out herein a VH domain which is an amino acid sequence variant of the VH domain, optionally combining the VH domain thus provided with one or more VL domains, and testing the VH domain or VH/VL combination or combinations to identify a specific binding member or an antibody antigen binding domain specific for a target antigen of interest and optionally with one or more desired properties.
[0176] In some instances, VH domains, VHH domains, and scFvs can be prepared according to method known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). scFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers. The scFv molecules comprise a linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition. The linker length can greatly affect how the variable regions of a scFv fold and interact. In fact, if a short polypeptide linker is employed (e.g., between 5-10 amino acids) intra-chain folding is prevented. Inter-chain folding is also required to bring the two variable regions together to form a functional epitope binding site. In some instances, the linker sequence comprises a long linker (LL) sequence. In some instances, the long linker sequence comprises (G4S)n, wherein n=2 to 4. In some instances, the linker sequence comprises a short linker (SL) sequence. In some instances, the short linker sequence comprises (G4S)n, wherein n=l to 3. For examples of linker orientation and size see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S.
Patent Application Publication Nos. 20050100543 and 20050175606, U.S. Patent No. 7,695,936, and PCT publication Nos. W02006020258 and W02007024715, each of which is incorporated herein by reference.
Linkers for Antibody and/or Blocking Domains
[0177] Blocking of biological activity must be reversible for the molecule to be useful. Blocking can be removed by removal of the sterically blocking domain. In one embodiment, the blocking domain is removed by proteolytic cleavage of the linker by which the blocking domain is attached to the TFP T cell. In another embodiment, the blocking domain is removed by proteolytic cleavage of the linker by which an anti-blocking domain antibody (e.g., an anti-HSA antibody) is removed by proteolytic cleavage of the linker by which the blocking domain is attached to the TFP T cell. In an aspect, the blocking domain is removed by enzymatic cleavage other than proteolytic cleavage. A feature of this invention is that removal of the blocking domain is enzymatic and not merely a function of elapsed time. In an aspect, the blocking domain is removed at the site of disease by an enzyme overproduced in diseased tissue. In some embodiments, the domain is removed in a tumor environment by a protease produced by tumor cells. In some embodiments, the protease -cleavable linker comprises a sequence recognized by a
protease produced by tumor cells in the tumor microenvironment (TME). In one embodiment, a "blocked" TFP T cell, i.e., a TFP T cell having a blocking domain bound by a protease-cleavable linker or a TFP T cell having a blocking domain bound by an binding moiety (e.g., an antibody), will remain blocked until reaching the TME, i.e., will be in the presence of a protease that will recognize and cleave the linker that keeps the blocking domain tethered to the TFP T cell.
[0178] In some embodiments, the protease is at least one of a tumor cell surface protease, a
carboxypeptidase, a cathepsin, a kallikrein, a hexokinase, a plasmin, a stromelysin, factor Xa, a chymotrypsin-like protease, a trypsin-like protease, a elastase-like protease, a tryptase, a chymase, a subtilisin-like protease, an actinidain, a proteinase, a bromelain, a calpain, a caspase, a cysteine protease, a papain, an FHV-l protease, an HSV protease, a CMV protease, a chymosin, a renin, a pepsin, a matriptase, a plasmepsin, a nepenthesin, a metalloexopeptidase, a metalloendopeptidase, a matrix metalloproteinase/ collagenase, a plasminogen activator, a urokinase plasminogen activator (uPA), an enterokinase, a prostate-specific antigen (PSA, hK3), an interleukin- 1 b converting enzyme, a thrombin, a fibroblast activation protein (FAP), a meprin, a granzyme, and a dipeptidyl peptidase. In some embodiments, the cathepsin is cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, or cathepsin F; the hexokinase is hKl, hKlO, or hKl5; the proteinase is PR-3; the caspase is caspase-3; the cysteine protease is Mir 1 -CP or legumain; the matrix metalloproteinase or collagenase is
MMP l/interstitial collagenase, MMP2/type IV collagenase, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP 12, MMP 13, MMP 14, MMP15, MMP 16, ADAM 10, or ADAM 12; the prostate -specific antigen is PSA or hK3, the FAP is FAP-a: the granzyme is granzyme M or granzyme B; or the dipeptidyl peptidase is dipeptidyl peptidase IV (DPPIV/CD26). In some embodiments, more than one proteolytic cleavage site is present.
[0179] In some embodiments, the linker of the blocking domain comprises a protease-cleavable linker. The protease-cleavable linker is engineered to comprise a sequence recognized by a protease typically present in the tumor microenvironment (TME). In some embodiments, the protease-cleavable linker is cleavable by a matrix metalloprotease (MMP). Examples of MMPs include MMP1; MMP2; MMP3; MMP7; MMP8; MMP9; MMP 10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP 16; MMP17; MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27. In some embodiments, the protease- cleavable linker is a substrate for MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19. In some embodiments, the protease-cleavable linker is a substrate for MMP9. In some embodiments, the protease-cleavable linker is a substrate for MMP 14. In some embodiments, the protease-cleavable linker is a substrate for two or more MMPs. In some embodiments, the protease- cleavable linker is a substrate for at least MMP9 and MMP14. In some embodiments, the protease- cleavable linker comprises two or more substrates for the same MMP. In some embodiments, the protease-cleavable linker comprises at least two or more MMP9 substrates. In some embodiments, the protease-cleavable linker comprises at least two or more MMP14 substrates. In some embodiments, the protease-cleavable linker is a substrate for an MMP and includes the sequence ISSGFFSS (SEQ ID NO:43); QNQAFRMA (SEQ ID NO:44); AQNLLGMV (SEQ ID NO:45); STFPFGMF (SEQ ID
NO:46); PVGYTSSL (SEQ ID NO:47); DWLYWPGI (SEQ ID NO:48); MIAPVAYR (SEQ ID NO:49); RPSPMWAY (SEQ ID NO:50); WATPRPMR (SEQ ID NO:5 l); FRLLDWQW (SEQ ID NO:52); LKAAPRWA (SEQ ID NO:53); GPSHLVLT (SEQ ID NO:54); LPGGLSPW (SEQ ID NO:55);
MGLFSEAG (SEQ ID NO:56); SPLPLRVP (SEQ ID NO:57); RMHLRSLG (SEQ ID NO:58);
LAAPLGLL (SEQ ID NO:59); AVGLLAPP (SEQ ID NO:60); LLAPSHRA (SEQ ID NO:6l);
PAGLWLDP (SEQ ID NO:62); and/or ISSGLSS (SEQ ID NO:63).
Table 1. MMP Cleavable Core Consensus Sequence 1
(SEQ ID NO: 70)
Table 2. MMP9 Cleavable Core Consensus Sequence 2
Table 3. MMP9 Cleavable Core Consensus Sequence 3
Table 5. MMP14 Cleavable Core Consensus Sequence 5
Table 6. MMP14 Cleavable Core Consensus Sequence 6
Table 6A. MMP14 Cleavable Core Consensus Sequence 6A
Table 10. MMP14 Cleavable Core Consensus Sequence 10
Table 12. MMP14 Cleavable Core Consensus Sequence 12
Table 13. MMP14 Cleavable Core Consensus Sequence 13
Stability and Mutations
[0180] The stability of an anti-tumor-associated antigen binding domain, e.g., scFv molecules (e.g., soluble scFv) can be evaluated in reference to the biophysical properties (e.g., thermal stability) of a conventional control scFv molecule or a full-length antibody. In one embodiment, the humanized or
human scFv has a thermal stability that is greater than about 0.1, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10 degrees, about 11 degrees, about 12 degrees, about 13 degrees, about 14 degrees, or about 15 degrees Celsius than a parent scFv in the described assays.
[0181] The improved thermal stability of the anti-tumor-associated antigen binding domain, e.g., scFv is subsequently conferred to the entire tumor-associated antigen-TFP construct, leading to improved therapeutic properties of the anti-tumor-associated antigen TFP construct. The thermal stability of the anti-tumor-associated antigen binding domain, e.g., an scFv or sdAb, can be improved by at least about 2 ° C or 3 ° C as compared to a conventional antibody. In one embodiment, the anti -tumor-associated antigen binding domain, e.g., an scFv or sdAb, has a 1 ° C improved thermal stability as compared to a conventional antibody. In another embodiment, the anti-tumor-associated antigen binding domain, e.g., an scFv or sdAb, has a 2 ° C improved thermal stability as compared to a conventional antibody. In another embodiment, the scFv has a 4 ° C, 5 ° C, 6 ° C, 7 ° C, 8 ° C, 9 ° C, 10 ° C, 11 ° C, 12 ° C, 13 ° C, 14 ° C, or 15 ° C improved thermal stability as compared to a conventional antibody. Comparisons can be made, for example, between the scFv molecules disclosed herein and scFv molecules or Fab fragments of an antibody from which the scFv VH and VL were derived. Thermal stability can be measured using methods known in the art. For example, in one embodiment, TM can be measured. Methods for measuring TM and other methods of determining protein stability are described below.
[0182] Mutations in an scFv or an sdAb (arising through humanization or mutagenesis of the soluble scFv or sdAb) alter the stability of the scFv and improve the overall stability of the scFv and the anti- tumor-associated antigen TFP construct. Stability of the humanized scFv is compared against the murine scFv using measurements such as TM, temperature denaturation and temperature aggregation. In one embodiment, the anti-tumor-associated antigen binding domain, e.g., a scFv, comprises at least one mutation arising from the humanization process such that the mutated scFv confers improved stability to the anti-tumor-associated antigen TFP construct. In another embodiment, the anti-tumor-associated antigen binding domain, e.g., scFv comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations arising from the humanization process such that the mutated scFv confers improved stability to the tumor-associated antigen-TFP construct.
[0183] In one aspect, the antigen binding domain of the TFP comprises an amino acid sequence that is homologous to an antigen binding domain amino acid sequence described herein, and the antigen binding domain retains the desired functional properties of the anti-tumor-associated antigen antibody fragments described herein. In one specific aspect, the TFP composition of the invention comprises an antibody fragment. In a further aspect, that antibody fragment comprises a scFv.
[0184] In various aspects, the antigen binding domain of the TFP is engineered by modifying one or more amino acids within one or both variable regions (e.g., VH and/or VL), for example within one or more CDR regions and/or within one or more framework regions. In one specific aspect, the TFP composition of the invention comprises an antibody fragment. In a further aspect, that antibody fragment
comprises a scFv.
[0185] It will be understood by one of ordinary skill in the art that the antibody or antibody fragment of the invention may further be modified such that they vary in amino acid sequence (e.g., from wild-type), but not in desired activity. For example, additional nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues may be made to the protein. For example, a nonessential amino acid residue in a molecule may be replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members, e.g., a conservative substitution, in which an amino acid residue is replaced with an amino acid residue having a similar side chain, may be made.
[0186] Families of amino acid residues having similar side chains have been defined in the art, including 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0187] Percent identity in the context of two or more nucleic acids or polypeptide sequences refers to two or more sequences that are the same. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 70%, 71% , 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
[0188] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. Nat' 1. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Brent et ah, (2003) Current Protocols in Molecular Biology). Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et ah, (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et ah, (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
[0189] In one aspect, the present invention contemplates modifications of the starting antibody or fragment (e.g., scFv) amino acid sequence that generate functionally equivalent molecules. For example, the VH or VL of an anti-tumor-associated antigen binding domain, e.g., scFv, comprised in the TFP can be modified to retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the starting VH or VL framework region of the anti -tumor-associated antigen binding domain, e.g., scFv. The present invention contemplates modifications of the entire TFP construct, e.g., modifications in one or more amino acid sequences of the various domains of the TFP construct in order to generate functionally equivalent molecules. The TFP construct can be modified to retain at least about
70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity of the starting TFP construct.
[0190] Linkers for attachment of TAA binding domains and a TCR protein
[0191] Optionally, a short oligo- or polypeptide linker, between 2 and 20 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic region of the TFP. A glycine - serine doublet provides a particularly suitable linker. For example, in one aspect, the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 181). In some embodiments, the linker is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 182). In some embodiments, the linker comprises the amino acid sequence of GGGGSGGGGSGGGGSLE (SEQ ID NO: 183). In other embodiments, the linker comprises the amino acid sequence of
AAAGGGGSGGGGSGGGGSLE (SEQ ID NO: 184). In other embodiments, the linker is a long linker having the sequence AAIEVMYPPPYLGGGGSGGGGSGGGGSLE (SEQ ID NO: 185). In some embodiments, the linker is encoded by a nucleotide sequence of
GGTGGAGGCGGTTCTGGTGGAGGCGGTTCGGATGGCGGAGGTTCA (SEQ ID NO: 186). In other embodiments, the linker is encoded by a nucleotide sequence of
GGAGAGGGTAAATCTTCCGGATCTGGTTCCGAAAGCAAGGCTAGC (SEQ ID NO: 187).
Extracellular domain
[0192] The extracellular domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any protein, but in particular a membrane- bound or transmembrane protein. In one aspect, the extracellular domain is capable of associating with the transmembrane domain. An extracellular domain of particular use in this invention may include at
least the extracellular region(s) of e.g., the alpha, beta or zeta chain of the T cell receptor, or CD3 epsilon, CD3 gamma, or CD3 delta, or in alternative embodiments, CD28, CD45, CD2, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
Transmembrane Domain
[0193] In general, a TFP sequence contains an extracellular domain and a transmembrane domain encoded by a single genomic sequence. In alternative embodiments, a TFP can be designed to comprise a transmembrane domain that is heterologous to the extracellular domain of the TFP. A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids of the intracellular region). In some cases, the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the extracellular region. In some cases, the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the intracellular region. In one aspect, the transmembrane domain is one that is associated with one of the other domains of the TFP is used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerization with another TFP on the TFP T cell surface. In a different aspect the amino acid sequence of the
transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same TFP.
[0194] The transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane -bound or transmembrane protein. In one aspect, the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TFP has bound to a target. A transmembrane domain of particular use in this invention may include at least the transmembrane region(s) of e.g., the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
[0195] In some instances, the transmembrane domain can be attached to the extracellular region of the TFP, e.g. , the antigen binding domain of the TFP, via a hinge, e.g. , a hinge from a human protein. For example, in one embodiment, the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, or a CD8a hinge.
Cytoplasmic Domain
[0196] The cytoplasmic domain of the TFP can include an intracellular signaling domain, if the TFP contains CD3 gamma, delta or epsilon polypeptides; TCR alpha and TCR beta subunits are generally
lacking in a signaling domain. An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the TFP has been introduced. The term "effector function" refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus the term "intracellular signaling domain" refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
[0197] Examples of intracellular signaling domains for use in the TFP of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.
[0198] It is known that signals generated through the TCR alone are insufficient for full activation of naive T cells and that a secondary and/or costimulatory signal is required. Thus, naive T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen -independent manner to provide a secondary or costimulatory signal
(secondary cytoplasmic domain, e.g., a costimulatory domain).
[0199] A primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine -based activation motifs (ITAMs).
[0200] Examples of ITAMs containing primary intracellular signaling domains that are of particular use in the invention include those of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, a TFP of the invention comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3-epsilon. In one embodiment, a primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain. In one embodiment, a primary signaling domain comprises a modified ITAM -containing primary intracellular signaling domain, e.g., an optimized and/or truncated ITAM -containing primary intracellular signaling domain. In an embodiment, a primary signaling domain comprises one, two, three, four or more ITAM motifs.
[0201] The intracellular signaling domain of the TFP can comprise the CD3 zeta signaling domain by itself or it can be combined with any other desired intracellular signaling domain(s) useful in the context of a TFP of the invention. For example, the intracellular signaling domain of the TFP can comprise a
CD3 epsilon chain portion and a costimulatory signaling domain. The costimulatory signaling domain refers to a portion of the TFP comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like. For example, CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human TFP T cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al. Blood. 2012; 119(3):696-706).
[0202] The intracellular signaling sequences within the cytoplasmic portion of the TFP of the invention may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequences.
[0203] In one embodiment, a glycine-serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, e.g., an alanine, a glycine, can be used as a suitable linker.
[0204] In one aspect, the TFP-expressing cell described herein can further comprise a second TFP, e.g., a second TFP that includes a different antigen binding domain, e.g., to the same target (e.g., CD22) or a different target (e.g., CD123). In one embodiment, when the TFP-expressing cell comprises two or more different TFPs, the antigen binding domains of the different TFPs can be such that the antigen binding domains do not interact with one another. For example, a cell expressing a first and second TFP can have an antigen binding domain of the first TFP, e.g., as a fragment, e.g., a scFv, that does not associate with the antigen binding domain of the second TFP, e.g., the antigen binding domain of the second TFP is a VHH.
[0205] In another aspect, the TFP-expressing cell described herein can further express another agent, e.g., an agent which enhances the activity of a TFP-expressing cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g., PD 1, can, in some embodiments, decrease the ability of a TFP-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 and TGFR beta. In one embodiment, the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4 and TIGIT, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 4-1BB, CD27 or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of an extracellular domain of PD1), and a second polypeptide of an
intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein). PD1 is an inhibitory member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA. PD-l is expressed on activated B cells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD- L2 have been shown to downregulate T cell activation upon binding to PD1 (Freeman et al. 2000 J Exp Med 192: 1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634- 43). PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immune suppression can be reversed by inhibiting the local interaction of PD1 with PD-L1.
[0206] In one embodiment, the agent comprises the extracellular domain (ECD) of an inhibitory molecule, e.g., Programmed Death 1 (PD1) can be fused to a transmembrane domain and optionally an intracellular signaling domain such as 41BB and CD3 zeta (also referred to herein as a PD1 TFP). In one embodiment, the PD1 TFP, when used in combinations with an anti -tumor antigen TFP described herein, improves the persistence of the T cell. In one embodiment, the TFP is a PD1 TFP comprising the extracellular domain of PD 1. Alternatively, provided are TFPs containing an antibody or antibody fragment such as a scFv that specifically binds to the Programmed Death -Ligand 1 (PD-L1) or
Programmed Death-Ligand 2 (PD-L2).
[0207] In another aspect, the present invention provides a population of TFP -expressing T cells, e.g., TFP T cells. In some embodiments, the population of TFP-expressing T cells comprises a mixture of cells expressing different TFPs. For example, in one embodiment, the population of TFP T cells can include a first cell expressing a TFP having an anti-tumor-associated antigen binding domain described herein, and a second cell expressing a TFP having a different anti-tumor-associated antigen binding domain, e.g., an anti-tumor-associated antigen binding domain described herein that differs from the anti- tumor-associated antigen binding domain in the TFP expressed by the first cell. As another example, the population of TFP-expressing cells can include a first cell expressing a TFP that includes an anti -tumor- associated antigen binding domain, e.g., as described herein, and a second cell expressing a TFP that includes an antigen binding domain to a target other than tumor-associated antigen (e.g., another tumor- associated antigen).
[0208] In another aspect, the present invention provides a population of cells wherein at least one cell in the population expresses a TFP having an anti-tumor-associated antigen domain described herein, and a second cell expressing another agent, e.g., an agent which enhances the activity of a TFP-expressing cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule.
Inhibitory molecules, e.g., can, in some embodiments, decrease the ability of a TFP-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. In one
embodiment, the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein.
[0209] Disclosed herein are methods for producing in vitro transcribed RNA encoding TFPs. The present invention also includes a TFP encoding RNA construct that can be directly transfected into a cell. A method for generating mRNA for use in transfection can involve in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3 ' and 5 ' untranslated sequence ("UTR"), a 5 ' cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length. RNA so produced can efficiently transfect different kinds of cells. In one aspect, the template includes sequences for the TFP.
[0210] In one aspect, the anti-TAA TFP is encoded by a circular RNA comprising a sequence encoding a TFP, a CAR, a TCR or combination thereof. In some embodiments, circular RNA is exogenous. In other embodiments, circular RNA is endogenous. In other embodiments, circular RNAs with an internal ribosomal entry site (IRES) can be translated in vitro or ex vivo.
[0211] Circular RNAs (circular RNAs) are a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. Circular RNAs are 3-5 ' covalently closed RNA rings, and circular RNAs do not display Cap or poly(A) tails. Since circular RNAs lack the free ends necessary for exonuclease-mediated degradation, rendering them resistant to several mechanisms of RNA turnover and granting them extended lifespans as compared to their linear mRNA counterparts. For this reason, circularization may allow for the stabilization of mRNAs that generally suffer from short serum half-lives and may therefore improve the overall efficacy of mRNA in a variety of applications. Circular RNAs are produced by the process of splicing, and circularization occurs using conventional splice sites mostly at annotated exon boundaries (Starke et ah, 2015; Szabo et ah, 2015). For circularization, splice sites are used in reverse: downstream splice donors are "backspliced" to upstream splice acceptors (see Jeck and Sharpless, 2014; Barrett and Salzman, 2016; Szabo and Salzman, 2016; Holdt et ah, 2018 for review).
[0212] To generate circular RNAs for subsequently transfer into cells, in vitro production of circular RNAs with autocatalytic-splicing introns can be programmed. In one embodiment, the method for generating circular RNA comprises in vitro transcription (IVT) of a precursor linear RNA template with specially designed primers. Three general strategies have been reported so far for RNA circularization: chemical methods using cyanogen bromide or a similar condensing agent; enzymatic methods using RNA or DNA ligases; and ribozymatic methods using self-splicing introns. In some embodiments, precursor RNA is synthesized by run-off transcription and then heated in the presence of magnesium ions and GTP to promote circularization. RNA so produced can efficiently transfect different kinds of cells.
In some exemplary embodiments, PCR is used to generate a template for in vitro transcription of linear precursor RNA which is used for transfection. Methods for performing PCR are well known in the art.
[0213] In one aspect the anti-TAA TFP is encoded by a circular RNA. In one embodiment, the circular RNA encoding the anti-TAA TFP is introduced into a T cell for production of a TFP T cell. In one embodiment, the in vitro transcribed RNA TFP can be introduced to a cell as a form of transient transfection. In one embodiment, the circular RNA encoding the anti-TAA TFP is introduced to a subject via a targeted nanoparticle.
[0214] In one aspect, the anti-TAA TFP is encoded by a messenger RNA (mRNA). In one aspect, the mRNA encoding the anti-tumor-associated antigen TFP is introduced into a T cell for production of a TFP T cell. In one embodiment, the in vitro transcribed RNA TFP can be introduced to a cell as a form of transient transfection. The RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA. The desired template for in vitro transcription is a TFP of the present invention. In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the nucleic acid can include some or all of the 5 ' and/or 3 ' untranslated regions (UTRs). The nucleic acid can include exons and introns. In one embodiment, the DNA to be used for PCR is a human nucleic acid sequence. In another embodiment, the DNA to be used for PCR is a human nucleic acid sequence including the 5 ' and 3 ' UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.
[0215] PCR is used to generate a template for in vitro transcription of mRNA which is used for transfection. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR. "Substantially complementary," as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary, or one or more bases are non complementary, or mismatched. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR. The primers can be designed to be substantially complementary to any portion of the DNA template. For example, the primers can be designed to amplify the portion of a nucleic acid that is normally transcribed in cells (the open reading frame), including 5 ' and 3 ' UTRs. The primers can also be designed to amplify a portion of a nucleic acid that encodes a particular domain of interest. In one embodiment, the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5 ' and 3 ' UTRs. Primers useful for PCR can be generated by synthetic methods that are well known in the art. "Forward primers" are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified. "Upstream" is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand. "Reverse primers" are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified.
"Downstream" is used herein to refer to a location 3 ' to the DNA sequence to be amplified relative to the coding strand.
[0216] Any DNA polymerase useful for PCR can be used in the methods disclosed herein. The reagents and polymerase are commercially available from a number of sources.
[0217] Chemical structures with the ability to promote stability and/or translation efficiency may also be used. The RNA preferably has 5 ' and 3 ' UTRs. In one embodiment, the 5 ' UTR is between one and 3,000 nucleotides in length. The length of 5 ' and 3 ' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5 ' and 3 ' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.
[0218] The 5 ' and 3 ' UTRs can be the naturally occurring, endogenous 5 ' and 3 ' UTRs for the nucleic acid of interest. Alternatively, UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the nucleic acid of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3 ' UTR sequences can decrease the stability of mRNA. Therefore, 3 ' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
[0219] In one embodiment, the 5 ' UTR can contain the Kozak sequence of the endogenous nucleic acid. Alternatively, when a 5 ' UTR that is not endogenous to the nucleic acid of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5 ' UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments, the 5 ' UTR can be 5 ' UTR of an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogues can be used in the 3 ' or 5 ' UTR to impede exonuclease degradation of the mRNA.
[0220] To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5 ' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one preferred embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.
[0221] In a preferred embodiment, the mRNA has both a cap on the 5 ' end and a 3 ' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of
the 3 ' UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription.
[0222] On a linear DNA template, phage T7 RNA polymerase can extend the 3 ' end of the transcript beyond the last base of the template (Schenbom and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270: 1485-65 (2003).
[0223] The conventional method of integration of polyA/T stretches into a DNA template is molecular cloning. However, polyA/T sequence integrated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes cloning procedures not only laborious and time consuming but often not reliable. That is why a method which allows construction of DNA templates with polyA/T 3 ' stretch without cloning highly desirable.
[0224] The polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100 T tail (size can be 50-5000 Ts), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.
[0225] Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly (A) polymerase, such as E. coli polyA polymerase (E-PAP). In one embodiment, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two fold increase in the translation efficiency of the RNA. Additionally, the attachment of different chemical groups to the 3 ' end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.
[0226] 5 ' caps on also provide stability to RNA molecules. In a preferred embodiment, RNAs produced by the methods disclosed herein include a 5 ' cap. The 5 ' cap is provided using techniques known in the art and described herein (Cougot, et ah, Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et ah, RNA, 7: 1468-95 (2001); Elango, et ak, Biochim. Biophys. Res. Commun., 330:958-966 (2005)).
[0227] The RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.
[0228] RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa
Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg
Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as "gene guns" (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8): 861 -70 (2001).
Nucleic Acid Constructs Encoding a TFP
[0229] The present invention also provides nucleic acid molecules encoding one or more TFP constructs described herein. In one aspect, the nucleic acid molecule is provided as a messenger RNA transcript. In one aspect, the nucleic acid molecule is provided as a DNA construct.
[0230] The nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.
[0231] The present invention also provides vectors in which a DNA of the present invention is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco -retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
[0232] In another embodiment, the vector comprising the nucleic acid encoding the desired TFP of the invention is an adenoviral vector (A5/35).
Modified T cells
[0233] Disclosed herein, in some embodiments, are modified T cells comprising the recombinant nucleic acid disclosed herein, or the vectors disclosed herein, wherein the modified T cell comprises a functional disruption of an endogenous TCR. Also disclosed herein, in some embodiments, are modified T cells comprising the sequence encoding the TFP of the nucleic acid disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein, wherein the modified T cell comprises a functional disruption of an endogenous TCR. Further disclosed herein, in some embodiments, are allogenic modified T cells comprising the sequence encoding the inducible TFP disclosed herein or an inducible TFP encoded by the sequence of the nucleic acid disclosed herein.
In some instances, the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain or a TCR alpha constant domain and a TCR beta constant domain. In some instances, the endogenous TCR that is functionally disrupted is an endogenous TCR alpha chain, an endogenous TCR beta chain, or an endogenous TCR alpha chain and an endogenous TCR beta chain. In some instances, the endogenous TCR that is functionally disrupted has reduced binding to MHC -peptide complex compared to that of an unmodified control T cell. In some instances, the functional disruption is a disruption of a gene encoding the endogenous TCR. In some instances, the disruption of a gene encoding the endogenous TCR is a removal of a sequence of the gene encoding the endogenous TCR from the genome of a T cell. In some
instances, the T cell is a human T cell. In some instances, the T cell is a CD8+ or CD4+ T cell or CD4+CD8+ T cell. In some instances, the T cell is an allogenic T cell. In some instances, the modified T cells further comprise a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide comprising at least a portion of an inhibitory molecule, associated with a second polypeptide comprising a positive signal from an intracellular signaling domain. In some instances, the inhibitory molecule comprises the first polypeptide comprising at least a portion of PD 1 and the second polypeptide comprising a costimulatory domain and primary signaling domain. Methods of Producing Modified T cells
[0234] Disclosed herein, in some embodiments, are methods of producing the modified T cell of the disclosure, the method comprising (a) disrupting an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, or any combination thereof; thereby producing a T cell containing a functional disruption of an endogenous TCR gene; and (b) transducing the T cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid of the disclosure, or the vectors disclosed herein. In some instances, disrupting comprises transducing the T cell with a nuclease protein or a nucleic acid sequence encoding a nuclease protein that targets the endogenous gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.
[0235] Further disclosed herein, in some embodiments, are methods of producing the modified T cell of the disclosure, the method comprising transducing a T cell containing a functional disruption of an endogenous TCR gene with the recombinant nucleic acid disclosed herein, or the vectors disclosed herein. In some instances, the T cell containing a functional disruption of an endogenous TCR gene is a T cell containing a functional disruption of an endogenous TCR gene encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.
[0236] In some instances, the T cell is a human T cell. In some instances, the T cell containing a functional disruption of an endogenous TCR gene has reduced binding to MHC-peptide complex compared to that of an unmodified control T cell.
[0237] In some instances, the nuclease is a meganuclease, a zinc -finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, CRISPR/Cas nickase, or a megaTAL nuclease. In some instances, the sequence comprised by the recombinant nucleic acid or the vector is inserted into the endogenous TCR subunit gene at the cleavage site, and wherein the insertion of the sequence into the endogenous TCR subunit gene functionally disrupts the endogenous TCR subunit. In some instances, the nuclease is a meganuclease. In some instances, the meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence, and wherein the second subunit binds to a second recognition half-site of the recognition sequence. In some instances, the meganuclease is a single chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit.
Gene Editing Technologies
[0238] In some embodiments, the modified T cells disclosed herein are engineered using a gene editing
technique such as clustered regularly interspaced short palindromic repeats (CRISPR®, see, e.g., US
Patent No. 8,697,359), transcription activator-like effector (TALE) nucleases (TALENs, see, e.g., U.S. Patent No. 9,393,257), meganucleases (endodeoxyribonucleases having large recognition sites comprising double -stranded DNA sequences of 12 to 40 base pairs), zinc finger nuclease (ZFN, see, e.g., Umov et al., Nat. Rev. Genetics (2010) vl 1, 636-646), or megaTAL nucleases (a fusion protein of a meganuclease to TAL repeats) methods. In this way, a chimeric construct may be engineered to combine desirable characteristics of each subunit, such as conformation or signaling capabilities. See also Sander & Joung, Nat. Biotech. (2014) v32, 347-55; and June et al., 2009 Nature Reviews Immunol. 9.10: 704- 716, each incorporated herein by reference. In some embodiments, one or more of the extracellular domain, the transmembrane domain, or the cytoplasmic domain of a TFP subunit are engineered to have aspects of more than one natural TCR subunit domain (i.e., are chimeric).
[0239] Recent developments of technologies to permanently alter the human genome and to introduce site-specific genome modifications in disease relevant genes lay the foundation for therapeutic applications. These technologies are now commonly known as "genome editing".
[0240] In some embodiments, gene editing techniques are employed to disrupt an endogenous TCR gene. In some embodiments, mentioned endogenous TCR gene encodes a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain. In some embodiments, gene editing techniques pave the way for multiplex genomic editing, which allows simultaneous disruption of multiple genomic loci in endogenous TCR gene. In some embodiments, multiplex genomic editing tecniques are applied to generate gene-disrupted T cells that are deficient in the expression of endogenous TCR, and/or human leukocyte antigens (HLAs), and/or programmed cell death protein 1 (PD1), and/or other genes.
[0241] Current gene editing technologies comprise meganucleases, zinc -finger nucleases (ZFN), TAL effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats
(CRISPR)/CRISPR-associated (Cas) system. These four major classes of gene-editing techniques share a common mode of action in binding a user-defined sequence of DNA and mediating a double-stranded DNA break (DSB). DSB may then be repaired by either non -homologous end joining (NHEJ) or -when donor DNA is present- homologous recombination (HR), an event that introduces the homologous sequence from a donor DNA fragment. Additionally, nickase nucleases generate single -stranded DNA breaks (SSB). DSBs may be repaired by single strand DNA incorporation (ssDI) or single strand template repair (ssTR), an event that introduces the homologous sequence from a donor DNA.
[0242] Genetic modification of genomic DNA can be performed using site -specific, rare-cutting endonucleases that are engineered to recognize DNA sequences in the locus of interest. Methods for producing engineered, site-specific endonucleases are known in the art. For example, zinc -finger nucleases (ZFNs) can be engineered to recognize and cut predetermined sites in a genome. ZFNs are chimeric proteins comprising a zinc finger DNA-binding domain fused to the nuclease domain of the Fokl restriction enzyme. The zinc finger domain can be redesigned through rational or experimental means to produce a protein that binds to a pre-determined DNA sequence -18 basepairs in length. By fusing this engineered protein domain to the Fokl nuclease, it is possible to target DNA breaks with
genome-level specificity. ZFNs have been used extensively to target gene addition, removal, and substitution in a wide range of eukaryotic organisms (reviewed in Durai et al. (2005), Nucleic Acids Res 33, 5978). Likewise, TAL-effector nucleases (TALENs) can be generated to cleave specific sites in genomic DNA. Like a ZFN, a TALEN comprises an engineered, site-specific DNA-binding domain fused to the Fokl nuclease domain (reviewed in Mak et al. (2013), Curr Opin Struct Biol. 23:93-9). In this case, however, the DNA binding domain comprises a tandem array of TAL-effector domains, each of which specifically recognizes a single DNA base pair. Compact TALENs have an alternative
endonuclease architecture that avoids the need for dimerization (Beurdeley et al. (2013), Nat Commun. 4: 1762). A Compact TALEN comprises an engineered, site -specific TAL-effector DNA-binding domain fused to the nuclease domain from the I-Tevl homing endonuclease. Unlike Fokl, I-Tevl does not need to dimerize to produce a double-strand DNA break so a Compact TALEN is functional as a monomer.
[0243] Engineered endonucleases based on the CRISPR/Cas9 system are also known in the art (Ran et al. (2013), Nat Protoc. 8:2281-2308; Mali et al. (2013), Nat Methods 10:957-63). The CRISPR gene editing technology is composed of an endonuclease protein whose DNA-targeting specificity and cutting activity can be programmed by a short guide RNA or a duplex crRNA/TracrRNA. A CRISPR endonuclease comprises two components: (1) a caspase effector nuclease, typically microbial Cas9; and (2) a short "guide RNA" or a RNA duplex comprising a 18 to 20 nucleotide targeting sequence that directs the nuclease to a location of interest in the genome. By expressing multiple guide RNAs in the same cell, each having a different targeting sequence, it is possible to target DNA breaks simultaneously to multiple sites in the genome (multiplex genomic editing).
[0244] There are two classes of CRISPR systems known in the art (Adli (2018) Nat. Commun. 9: 191 1), each containing multiple CRISPR types. Class 1 contains type I and type III CRISPR systems that are commonly found in Archaea. And, Class II contains type II, IV, V, and VI CRISPR systems. Although the the most widely used CRISPR/Cas system is the the type II CRISPR-Cas9 system, CRISPR/Cas systems have been repurposed by researchers for genome editing. More than 10 different CRISPR/Cas proteins have been remodeled within last few years (Adli (2018) Nat. Commun. 9: 1911). Among these, such as Casl2a (Cpfl) proteins from Acid- aminococcus sp (AsCpfl) and Lachnospiraceae bacterium (LbCpfl), are particularly interesting.
[0245] Homing endonucleases are a group of naturally occurring nucleases that recognize 15-40 base- pair cleavage sites commonly found in the genomes of plants and fungi. They are frequently associated with parasitic DNA elements, such as group 1 self-splicing introns and inteins. They naturally promote homologous recombination or gene insertion at specific locations in the host genome by producing a double -stranded break in the chromosome, which recruits the cellular DNA-repair machinery (Stoddard (2006), Q. Rev. Biophys. 38: 49-95). Specific amino acid substations could reprogram DNA cleavage specificity of homing nucleases (Niyonzima (2017), Protein Eng Des Sel. 30(7): 503-522).
Meganucleases (MN) are monomeric proteins with innate nuclease activity that are derived from bacterial homing endonucleases and engineered for a unique target site (Gersbach (2016), Molecular Therapy. 24: 430-446). In some embodiments, meganuclease is engineered I-Crel homing endonuclease.
In other embodiments, meganuclease is engineered I-Scel homing endonuclease.
[0246] In addition to mentioned four major gene editing technologies, chimeric proteins comprising fusions of meganucleases, ZFNs, and TALENs have been engineered to generate novel monomeric enzymes that take advantage of the binding affinity of ZFNs and TALENs and the cleavage specificity of meganucleases (Gersbach (2016), Molecular Therapy. 24: 430-446). For example, A megaTAL is a single chimeric protein, which is the combination of the easy-to-tailor DNA binding domains from TALENs with the high cleavage efficiency of meganucleases.
[0247] In order to perform the gene editing technique, the nucleases, and in the case of the CRISPR/ Cas9 system, a gRNA, must be efficiently delivered to the cells of interest. Delivery methods such as physical, chemical, and viral methods are also know in the art (Mali (2013). Indian J. Hum. Genet. 19: 3- 8.). In some instances, physical delivery methods can be selected from the methods but not limited to electroporation, microinjection, or use of ballistic particles. On the other hand, chemical delivery methods require use of complex molecules such calcium phosphate, lipid, or protein. In some embodiments, viral delivery methods are applied for gene editing techniques using viruses such as but not limited to adenovirus, lentivirus, and retrovirus.
[0248] The expression constructs of the present invention may also be used for nucleic acid
immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art (see, e.g., U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties). In another embodiment, the invention provides a gene therapy vector.
[0249] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
[0250] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, e.g., in Sambrook et al., 2012, Molecular Cloning:
A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
[0251] A number of virally based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.
[0252] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional
initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.
[0253] An example of a promoter that is capable of expressing a TFP transgene in a mammalian T cell is the EFla promoter. The native EFla promoter drives expression of the alpha subunit of the elongation factor-l complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome. The EFla promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in driving TFP expression from transgenes cloned into a lentiviral vector (see, e.g., Milone et ah, Mol. Ther. 17(8): 1453-1464 (2009)). Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human
immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor- la promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline -regulated promoter.
[0254] In order to assess the expression of a TFP polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic -resistance genes, such as neo and the like.
[0255] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is
assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5 ' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.
[0256] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.
[0257] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art (see, e.g., Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY). One method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection
[0258] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like (see, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362).
[0259] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system.
[0260] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a "collapsed" structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
[0261] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 ° C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. "Liposome" is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates.
Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al, 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine -nucleic acid complexes.
[0262] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and western blots) or by assays described herein to identify agents falling within the scope of the invention.
[0263] The present invention further provides a vector comprising a TFP encoding nucleic acid molecule. In one aspect, a TFP vector can be directly transduced into a cell, e.g., a T cell. In one aspect, the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs. In one aspect, the vector is capable of expressing the TFP construct in mammalian T cells. In one aspect, the mammalian T cell is a human T cell.
Sources of T cells
[0264] Prior to expansion and genetic modification, a source of T cells is obtained from a subject. The term "subject" is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain aspects of the present invention, any number of T cell lines available in the art, may be used. In certain aspects of the present invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll® separation. In one preferred aspect, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
In one aspect, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one aspect of the invention, the cells are washed with phosphate buffered saline (PBS). In an alternative aspect, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi -automated "flow-through" centrifuge (for example, the COBE® 2991 cell processor, the Baxter CytoMate®, or the Haemonetics® Cell Saver® 5) according to the manufacturer' s instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte® A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed, and the cells directly resuspended in culture medium.
[0265] In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a Percoll® gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in one aspect, T cells are isolated by incubation with anti-CD3/anti-CD28 (e.g., 3x28)-conjugated beads, such as Dynabeads® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells. In one aspect, the time period is about 30 minutes. In a further aspect, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the time period is 10 to 24 hours. In one aspect, the incubation time period is 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from
immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind
to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this invention. In certain aspects, it may be desirable to perform the selection procedure and use the "unselected" cells in the activation and expansion process. "Unselected" cells can also be subjected to further rounds of selection.
[0266] Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8. In certain aspects, it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain aspects, T regulatory cells are depleted by anti- C25 conjugated beads or other similar method of selection.
[0267] In one embodiment, a T cell population can be selected that expresses one or more of IFN-g, TNF-alpha, IF-17A, IF-2, IF-3, IF-4, GM-CSF, IF-10, IF-13, granzyme B, and perforin, or other appropriate molecules, e.g., other cytokines. Methods for screening for cell expression can be determined, e.g., by the methods described in PCT Publication No.: WO2013/126712.
[0268] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain aspects, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (e.g., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one aspect, a concentration of 2 billion cells/mF is used. In one aspect, a concentration of 1 billion cells/mF is used. In a further aspect, greater than 100 million cells/mF is used. In a further aspect, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mF is used. In yet one aspect, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mF is used. In further aspects, concentrations of 125 or 150 million cells/mF can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
[0269] In a related aspect, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be
bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In one aspect, the concentration of cells used is 5 x l06/mL. In other aspects, the concentration used can be from about 1 x l05/mL to 1 x l06/mL, and any integer value in between. In other aspects, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10 ° C or at room temperature.
[0270] T cells for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25%
Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan® and PlasmaLyte® A, the cells then are frozen to -80 ° C at a rate of 1 per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20 ° C or in liquid nitrogen. In certain aspects, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present invention.
[0271] Also contemplated in the context of the invention is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one aspect, a blood sample or an apheresis is taken from a generally healthy subject. In certain aspects, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain aspects, the T cells may be expanded, frozen, and used at a later time. In certain aspects, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further aspect, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and tacrolimus (FK506), antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies, cyclophosphamide, fludarabine, cyclosporin, rapamycin, mycophenolic acid, steroids, romidepsin (formerly FR901228), and irradiation.
[0272] In a further aspect of the present invention, T cells are obtained from a patient directly following treatment that leaves the subject with functional T cells. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system,
shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present invention to collect blood cells, including T cells, dendritic cells, or other cells of the
hematopoietic lineage, during this recovery phase. Further, in certain aspects, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.
Activation and Expansion of T Cells
[0273] T cells may be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and 7,572,631.
[0274] Generally, the T cells of the invention may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co -stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti- CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et ah, Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol. Meth. 227(l-2):53-63, 1999).
[0275] T cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8+). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of TC cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.
[0276] Further, in addition to CD4 and CD8 markers, other phenotypic markers vary significantly, but in large part, reproducibly during the course of the cell expansion process. Thus, such reproducibility
enables the ability to tailor an activated T cell product for specific purposes.
[0277] Once an anti-tumor-associated antigen TFP is constructed, various assays can be used to evaluate the activity of the molecule, such as but not limited to, the ability to expand T cells following antigen stimulation, sustain T cell expansion in the absence of re -stimulation, and anti-cancer activities in appropriate in vitro and animal models. Assays to evaluate the effects of an anti-tumor-associated antigen TFP are described in further detail below
[0278] Western blot analysis of TFP expression in primary T cells can be used to detect the presence of monomers and dimers (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). In some embodiments, T cells (1: 1 mixture of CD4+ and CD8+ T cells) expressing the TFPs are expanded in vitro for more than 10 days followed by lysis and SDS-PAGE under reducing conditions. TFPs are detected by western blotting using an antibody to a TCR chain. The same T cell subsets are used for SDS-PAGE analysis under non-reducing conditions to permit evaluation of covalent dimer formation.
[0279] In vitro expansion of TFP+ T cells following antigen stimulation can be measured by flow cytometry. For example, a mixture of CD4+ and CD8+ T cells are stimulated with alphaCD3/alphaCD28 and APCs followed by transduction with lentiviral vectors expressing GFP under the control of the promoters to be analyzed. Exemplary promoters include the CMV IE gene, EF-lalpha, ubiquitin C, or phosphoglycerokinase (PGK) promoters. GFP fluorescence is evaluated on day 6 of culture in the CD4+ and/or CD8+ T cell subsets by flow cytometry (see, e.g., Milone et al., Molecular Therapy 17(8): 1453 - 1464 (2009)). Alternatively, a mixture of CD4+ and CD8+ T cells are stimulated with
alphaCD3/alphaCD28 coated magnetic beads on day 0 and transduced with TFP on day 1 using a bicistronic lentiviral vector expressing TFP along with eGFP using a 2A ribosomal skipping sequence.
[0280] Sustained TFP+ T cell expansion in the absence of re -stimulation can also be measured (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Briefly, mean T cell volume (fl) is measured on day 8 of culture using a Coulter® Multisizer III particle counter following stimulation with alphaCD3/alphaCD28 coated magnetic beads on day 0, and transduction with the indicated TFP on day 1
[0281] Animal models can also be used to measure a TFP T cell activity. For example, a xenograft model using human BCMA-specific TFP+ T cells to treat a cancer in immunodeficient mice is described in Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Very briefly, after establishment of cancer, mice are randomized as to treatment groups. Different numbers of engineered T cells are coinjected at a 1: 1 ratio into NOD/SCID/Y-/- mice bearing cancer. The number of copies of each vector in spleen DNA from mice is evaluated at various times following T cell injection. Animals are assessed for cancer at weekly intervals. Peripheral blood tumor-associated antigen® cancer cell counts are measured in mice that are injected with alpha tumor-associated antigen-zeta TFP+ T cells or mock-transduced T cells. Survival curves for the groups are compared using the log -rank test. In addition, absolute peripheral blood CD4+ and CD8+ T cell counts 4 weeks following T cell injection in NOD/SCID/Y-/- mice can also be analyzed. Mice are injected with cancer cells and 3 weeks later are injected with T cells engineered to express TFP by a bicistronic lentiviral vector that encodes the TFP linked to eGFP. T cells are
nonnalized to 45-50% input GFP+ T cells by mixing with mock-transduced cells prior to injection and confirmed by flow cytometry. Animals are assessed for cancer at l-week intervals. Survival curves for the TFP+ T cell groups are compared using the log-rank test.
[0282] Dose-dependent TFP treatment responses can be evaluated (see, e.g., Milone et ah, Molecular Therapy 17(8): 1453-1464 (2009)). For example, peripheral blood is obtained 35-70 days after establishing cancer in mice injected on day 21 with TFP T cells, an equivalent number of mock- transduced T cells, or no T cells. Mice from each group are randomly bled for determination of peripheral blood + cancer cell counts and then killed on days 35 and 49. The remaining animals are evaluated on days 57 and 70.
[0283] Assessment of cell proliferation and cytokine production has been previously described, e.g., at Milone et ah, Molecular Therapy 17(8): 1453-1464 (2009). Briefly, assessment of TFP-mediated proliferation is performed in microtiter plates by mixing washed T cells with cells expressing BCMA or CD32 and CD137 (KT32-BBL) for a final T celkcell expressing BCMA ratio of 2: l. Cells expressing BCMA cells are irradiated with gamma-radiation prior to use. Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies are added to cultures with KT32-BBL cells to serve as a positive control for stimulating T cell proliferation since these signals support long-term CD8+ T cell expansion ex vivo. T cells are enumerated in cultures using CountBright™ fluorescent beads (Invitrogen) and flow cytometry as described by the manufacturer. TFP+ T cells are identified by GFP expression using T cells that are engineered with eGFP-2A linked TFP -expressing lentiviral vectors. For TFP+ T cells not expressing GFP, the TFP+ T cells are detected with biotinylated recombinant BCMA protein and a secondary avidin-PE conjugate. CD4+ and CD8+ expression on T cells are also simultaneously detected with specific monoclonal antibodies (BD Biosciences). Cytokine measurements are performed on supernatants collected 24 hours following re -stimulation using the human TH1/TH2 cytokine cytometric bead array kit (BD Biosciences) according the manufacturer' s instructions. Fluorescence is assessed using a FACScalibur™ flow cytometer, and data is analyzed according to the manufacturer' s instructions.
[0284] Cytotoxicity can be assessed by a standard 51Cr-release assay (see, e.g., Milone et ah, Molecular Therapy 17(8): 1453-1464 (2009)). For example, target cells are loaded with 51Cr (as NaCr04, New England Nuclear) at 37 ° C for 2 hours with frequent agitation, washed twice in complete RPMI and plated into microtiter plates. Effector T cells are mixed with target cells in the wells in complete RPMI at varying ratios of effector celktarget cell (E:T). Additional wells containing media only (spontaneous release, SR) or a 1% solution of Triton®-X 100 detergent (total release, TR) are also prepared. After 4 hours of incubation at 37 ° C, supernatant from each well is harvested. Released 51Cr is then measured using a gamma particle counter (Packard Instrument Co., Waltham, Mass.). Each condition is performed in at least triplicate, and the percentage of lysis is calculated using the formula: % Lysis=(ER-SR)/(TR- SR), where ER represents the average 51Cr released for each experimental condition.
[0285] Imaging technologies can be used to evaluate specific trafficking and proliferation of TFPs in tumor-bearing animal models. Such assays have been described, e.g., in Barrett et ah, Human Gene Therapy 22: 1575-1586 (2011). NOD/SCID/yc-/- (NSG) mice are injected intravenously (i.v.) with
cancer cells followed 7 days later with TFP T cells 4 hours after electroporation with the TFP constructs. The T cells are stably transfected with a lentiviral construct to express firefly luciferase, and mice are imaged for bioluminescence. Alternatively, therapeutic efficacy and specificity of a single injection of TFP T cells in a cancer xenograft model can be measured as follows: NSG mice are injected with cancer cells transduced to stably express firefly luciferase, followed by a single tail -vein injection of T cells electroporated with BCMA TFP 7 days later. Animals are imaged at various time points post injection. For example, photon-density heat maps of firefly luciferase positive cancer in representative mice at day 5 (2 days before treatment) and day 8 (24 hours post TFP+ PBLs) can be generated.
[0286] Other assays, including those described in the Example section herein as well as those that are known in the art can also be used to evaluate the anti-TAA TFP constructs and TFP T cells disclosed herein.
Therapeutic Applications
Tumor Antigen-Associated Diseases or Disorders
[0287] While examples and embodiments have been provided herein, additional techniques and embodiments related to TAA -associated diseases and/or anti-TAA antibodies may be found in U.S.
Patent No. 9,217,040, filed January 13, 2013; U.S. Patent No. 9,758,586, filed November 30, 2011; International Publication No. WO 2012076066, filed June 17, 2011; Mato, A. & Porter, D. (2015) Blood 126(4), 478-485; Choi, M., et al. (2015) Clinical Lymphoma, Myeloma & Leukemia 15(S 1), S167-S169; Cui, B., et al. (2015) Cancer Research 73(12), 3649-3660; Yu, J., et al. (2015) Journal of Clinical Investigation 10(1172), 1-34; Borcherding, N., et al. (2014) Protein Cell 5(7), 496-502; Zhang, S., et al. (2012) The American Journal of Pathology 181(6), 1903-1910; Hudecek, M., et al. (2010) Blood 116(22), 4532-4541; and Deniger, D., et al. (2015) PLoS ONE 10(6), 1-19, which are entirely incorporated herein by reference.
[0288] In one aspect, the invention provides methods for treating a disease associated with a TAA, e.g., ROR1 or NKG2D ligand (NKG2DL) expression. In one aspect, the invention provides methods for treating a disease wherein part of the tumor is negative for NKG2DL and part of the tumor is positive for NKG2DL. For example, the TFP is useful for treating subjects that have undergone treatment for a disease associated with elevated expression of NKG2DL, wherein the subject that has undergone treatment for elevated levels of NKG2DL exhibits a disease associated with elevated levels of NKG2DL.
[0289] In one aspect, the invention pertains to a vector comprising a TAA-binding TFP operably linked to promoter for expression in mammalian T cells. In one aspect, the invention provides a recombinant T cell expressing the, e.g., NKG2D TFP for use in treating NKG2DL-expressing tumors, wherein the recombinant T cell expressing the NKG2D TFP is termed a NKG2D TFP T cell. In one aspect, the NKG2D TFP T cell is capable of contacting a tumor cell with at least one NKG2DL expressed on its surface such that the TFP T cell targets the tumor cell and growth of the tumor is inhibited.
Dual Specificity TFP T cells
[0290] Many patients treated with cancer therapeutics that are directed to one target on a tumor cell, e.g., BCMA, CD19, CD20, CD22, CD123, mesothelin, etc., become resistant over time as escape mechanisms
such as alternate signaling pathways and feedback loops become activated. Dual specificity therapeutics attempt to address this by combining targets that often substitute for each other as escape routes.
Therapeutic T cell populations having TCRs specific to more than one tumor-associated antigen are promising combination therapeutics. In one embodiment, dual specificity TFP T cells comprise multiple blocking domains and corresponding multiple protease -cleavable linkers.
Tumor associated antigen targets for anti-TAA inducible TFP T cells
[0291] Exemplary tumor-associated antigens include, but are not limited to, oncofetal antigens (e.g., those expressed in fetal tissues and in cancerous somatic cells), oncoviral antigens (e.g., those encoded by tumorigenic transforming viruses), overexpressed/ accumulated antigens (e.g., those expressed by both normal and neoplastic tissue, with the level of expression highly elevated in neoplasia), cancer- testis antigens (e.g., those expressed only by cancer cells and adult reproductive tissues such as testis and placenta), lineage-restricted antigens (e.g., those expressed largely by a single cancer histotype), mutated antigens (e.g., those expressed by cancer as a result of genetic mutation or alteration in transcription), post-translationally altered antigens (e.g., those tumor-associated alterations in glycosylation, etc.), and idiotypic antigens (e.g., those from highly polymorphic genes where a tumor cell expresses a specific clonotype, e.g., as in B cell, T cell lymphoma/leukemia resulting from clonal aberrancies). Exemplary tumor-associated antigens include, but are not limited to, antigens of alpha- actinin-4, ARTC1, BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDK12, CDKN2A, CLPP, COA-l, CSNK1A1, dek-can fusion protein, EFTUD2, Elongation factor 2, ETV6-AML1 fusion protein, FLT3-ITD, FNDC3B, FN1, GAS7, GPNMB, HAUS3, HSDL1, LDLR-fiicosyltransferase AS fusion protein, HLA-A2d, HLA-A1 ld, hsp70-2, MART2, MATN, ME1, MUM-lf, MUM-2, MUM-3, neo-PAP, Myosin class I, NFYC, OGT, OS-9, p53, pml-RARalpha fusion protein, PPP1R3B, PRDX5, PTPRK, K-ras, N-ras, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, TGF-betaRII, triosephosphate isomerase, BAGE-l, D393-CD20n, Cyclin-Al, GAGE-l, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GnTVf, HERV-K-MEL, KK-LC- 1, KM-HN-l, LAGE-l, LY6K, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE- A9, MAGE-A10, MAGE-A12 m, MAGE-C1, MAGE-C2, mucink, NA88-A, NY-ESO-l / LAGE-2, SAGE, Spl7, SSX-2, SSX-4, TAG-l, TAG-2, TRAG-3, TRP2-INT2g, X AGE- 1 b/GAGED2a, B7H4, DLL3, TROP-2, Nectin-4, tissue factor, LIV-1, CD48, cMET„ Gene / protein, CEA, gplOO / Pmell7, mammaglobin-A, Melan-A / MART-l, NY-BR-l, OA1, PAP, PSA, RAB38 / NY-MEL-l, TRP-l / gp75, TRP-2, tyrosinase, adipophilin, AIM-2, ALDH1A1, BCLX (L), BING-4, CALCA, CD45, CD274, CPSF, cyclin Dl, DKK1, ENAH (hMena), EpCAM, EphA3, EZH2, FGF5, glypican-3, HER-2/neu, HLA- DOB, Hepsin, IDOl, IGF2B3, ILl3Ralpha2, Intestinal carboxyl esterase, alpha-foetoprotein, Kallikrein 4, KIF20A, Lengsin, M-CSF, MCSP, mdm-2, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, p53, PAX5, PBF, PRAME, PSMA, RAGE-l, RGS5, RhoC, RNF43, RU2AS, secemin 1, SOX10, STEAP1, survivin, Telomerase, TPBG, VEGF, and WT1.
[0292] In one aspect, the invention provides methods for treating a disease associated with at least one tumor-associated antigen expression. In one aspect, the invention provides methods for treating a disease
wherein part of the tumor is negative for the tumor associated antigen and part of the tumor is positive for the tumor associated antigen. For example, the antibody or TFP of the invention is useful for treating subjects that have undergone treatment for a disease associated with elevated expression of said tumor antigen, wherein the subject that has undergone treatment for elevated levels of the tumor associated antigen exhibits a disease associated with elevated levels of the tumor associated antigen.
[0293] In one aspect, the invention pertains to a vector comprising an anti-tumor-associated antigen antibody or TFP operably linked to promoter for expression in mammalian T cells. In one aspect, the invention provides a recombinant T cell expressing a tumor-associated antigen TFP for use in treating tumor-associated antigen-expressing tumors, wherein the recombinant T cell expressing the tumor- associated antigen TFP is termed a tumor-associated antigen TFP T cells. In one aspect, the tumor- associated antigen TFP T cell of the present disclosure is capable of contacting a tumor cell with at least one tumor-associated antigen TFP of the present disclosure expressed on its surface such that the TFP T cell targets the tumor cell and growth of the tumor is inhibited.
[0294] In one aspect, the invention pertains to a method of inhibiting growth of a tumor-associated antigen-expressing tumor cell, comprising contacting the tumor cell with a tumor-associated antigen antibody or TFP T cell of the present disclosure such that the TFP T is activated in response to the antigen and targets the cancer cell, wherein the growth of the tumor is inhibited.
[0295] In one aspect, the present disclosure pertains to a method of treating cancer in a subject. The method comprises administering to the subject a tumor-associated antigen antibody, bispecific antibody, or TFP T cell of the present disclosure such that the cancer is treated in the subject. An example of a cancer that is treatable by the tumor-associated antigen TFP T cell of the present disclosure is a cancer associated with expression of tumor-associated antigen. In one aspect, the cancer is a myeloma. In one aspect, the cancer is a lymphoma. In one aspect, the cancer is colon cancer.
[0296] In some embodiments, tumor-associated antigen antibodies or TFP therapy can be used in combination with one or more additional therapies. In some instances, such additional therapies comprise a chemotherapeutic agent, e.g., cyclophosphamide. In some instances, such additional therapies comprise surgical resection or radiation treatment.
[0297] In one aspect, disclosed herein is a method of cellular therapy wherein T cells are genetically modified to express a TFP and the TFP-expressing T cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient. Unlike antibody therapies, TFP-expressing T cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control. In various aspects, the T cells administered to the patient, or their progeny, persist in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty -one months, twenty-two months, twenty-three months, two years, three years, four years, or five years after administration of the T cell to the patient.
[0298] In some instances, disclosed herein is a type of cellular therapy where T cells are modified, e.g.,
by in vitro transcribed RNA, to transiently express a TFP and the TFP-expressing T cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient. Thus, in various aspects, the T cells administered to the patient, is present for less than one month, e.g., three weeks, two weeks, or one week, after administration of the T cell to the patient.
[0299] Without wishing to be bound by any particular theory, the anti -tumor immunity response elicited by the TFP-expressing T cells may be an active or a passive immune response, or alternatively may be due to a direct vs indirect immune response. In one aspect, the TFP transduced T cells exhibit specific proinflammatory cytokine secretion and potent cytolytic activity in response to human cancer cells expressing the tumor-associated antigen, resist soluble tumor-associated antigen inhibition, mediate bystander killing and/or mediate regression of an established human tumor. For example, antigen-less tumor cells within a heterogeneous field of tumor-associated antigen-expressing tumor may be susceptible to indirect destruction by tumor-associated antigen-redirected T cells that has previously reacted against adjacent antigen-positive cancer cells.
[0300] In one aspect, the human TFP-modified T cells of the present disclosure may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal. In one aspect, the mammal is a human.
[0301] With respect to ex vivo immunization, at least one of the following occurs in vitro prior to administering the cell into a mammal: i) expansion of the cells, ii) introducing a nucleic acid encoding a TFP to the cells or iii) cryopreservation of the cells.
[0302] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a TFP disclosed herein. The TFP-modified cell can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human and the TFP-modified cell can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.
[0303] The procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described, e.g., in U.S. Patent No. 5,199,942, incorporated herein by reference, can be applied to the cells of the present disclosure. Other suitable methods are known in the art; therefore, the present disclosure is not limited to any particular method of ex vivo expansion of the cells. Briefly, ex vivo culture and expansion of T cells comprises: (1) collecting CD34+ hematopoietic stem and progenitor cells from a mammal from peripheral blood harvest or bone marrow explants; and (2) expanding such cells ex vivo. In addition to the cellular growth factors described in U.S. Patent No. 5,199,942, other factors such as flt3-L, IL-l, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells.
[0304] In addition to using a cell -based vaccine in terms of ex vivo immunization, the present disclosure also provides compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient.
[0305] Generally, the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised. In particular, the TFP- modified T cells of the present disclosure are used in the treatment of diseases, disorders and conditions
associated with expression of tumor-associated antigens. In certain aspects, the cells of the present disclosure are used in the treatment of patients at risk for developing diseases, disorders and conditions associated with expression of tumor-associated antigens. Thus, the present disclosure provides methods for the treatment or prevention of diseases, disorders and conditions associated with expression of tumor- associated antigens comprising administering to a subject in need thereof, a therapeutically effective amount of the TFP -modified T cells of the present disclosure.
[0306] In one aspect, the antibodies or TFP T cells disclosed herein may be used to treat a proliferative disease such as a cancer or malignancy or is a precancerous condition. In one aspect, the cancer is a myeloma. In one aspect, the cancer is a lymphoma. In one aspect, the cancer is a colon cancer. Further, a disease associated with tumor-associated antigen expression includes, but is not limited to, e.g., atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing tumor-associated antigens. Non-cancer related indications associated with expression of tumor-associated antigens vary depending on the antigen, but are not limited to, e.g., infectious disease, autoimmune disease, (e.g., lupus), inflammatory disorders (allergy and asthma) and transplantation.
[0307] The antibodies or TFP-modified T cells of the present disclosure may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-7, IL-12, IL-15 or other cytokines or cell populations.
[0308] The present disclosure also provides methods for inhibiting the proliferation or reducing a tumor- associated antigen-expressing cell population, the methods comprising contacting a population of cells comprising a tumor-associated antigen-expressing cell with an anti -tumor-associated antigen TFP T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell. In a specific aspect, the present disclosure provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing tumor-associated antigen, the methods comprising contacting the tumor- associated antigen-expressing cancer cell population with an anti-tumor-associated antigen antibody or TFP T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell. In one aspect, the present disclosure provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing tumor-associated antigen, the methods comprising contacting the tumor- associated antigen-expressing cancer cell population with an anti-tumor-associated antigen antibody or TFP T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell. In certain aspects, the anti-tumor-associated antigen antibody or TFP T cell of the present disclosure reduces the quantity, number, amount or percentage of cells and/or cancer cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% in a subject with or animal model for multiple myeloma or another cancer associated with tumor-associated antigen expressing cells relative to a negative control. In one aspect, the subject is a human.
[0309] The present disclosure also provides methods for preventing, treating and/or managing a disease associated with tumor-associated antigen-expressing cells (e.g., a cancer expressing tumor-associated antigen), the methods comprising administering to a subject in need an anti-tumor-associated antigen antibody or TFP T cell of the present disclosure that binds to the tumor-associated antigen-expressing
cell. In one aspect, the subject is a human. Non-limiting examples of disorders associated with tumor- associated antigen-expressing cells include autoimmune disorders (such as lupus), inflammatory disorders (such as allergies and asthma) and cancers (such as hematological cancers or atypical cancers expressing tumor-associated antigen).
[0310] The present disclosure also provides methods for preventing, treating and/or managing a disease associated with tumor-associated antigen-expressing cells, the methods comprising administering to a subject in need an anti-tumor-associated antigen antibody or TFP T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell. In one aspect, the subject is a human.
[0311] The present disclosure provides methods for preventing relapse of cancer associated with tumor- associated antigen-expressing cells, the methods comprising administering to a subject in need thereof an anti-tumor-associated antigen antibody and/or TFP T cell of the present disclosure that binds to the tumor-associated antigen-expressing cell. In one aspect, the methods comprise administering to the subject in need thereof an effective amount of an anti -tumor-associated antigen antibody or TFP T cell described herein that binds to the tumor-associated antigen-expressing cell in combination with an effective amount of another therapy.
Combination Therapies
[0312] An inducible TFP-expressing cell described herein may be used in combination with other known agents and therapies. Administered "in combination", as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject' s affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons.
In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery". In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.
Other Combinations
[0313] In some embodiments, the "at least one additional therapeutic agent" includes a TFP-expressing cell. Also provided are T cells that express multiple TFPs, which bind to the same or different target antigens, or same or different epitopes on the same target antigen. Also provided are populations of T
cells in which a first subset of T cells expresses a first TFP and a second subset of T cells express a second TFP.
[0314] A TFP-expressing cell described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the TFP-expressing cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.
[0315] In further aspects, a TFP-expressing cell described herein may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies or other antibody therapies, cyclophosphamide, fludarabine, cyclosporin, tacrolimus (fujimycin), rapamycin, mycophenolic acid, steroids, romidepsin (also known as FR901228), cytokines, and irradiation peptide vaccine, such as that described in, e.g., Izumoto et al.
2008 J Neurosurg 108:963-971.
[0316] In one embodiment, the subject can be administered an agent which reduces or ameliorates a side effect associated with the administration of a TFP-expressing cell. Side effects associated with the administration of a TFP-expressing cell include, but are not limited to, cytokine release syndrome (CRS), and hemophagocytic lymphohistiocytosis (HLH), also termed Macrophage Activation Syndrome (MAS). Symptoms of CRS include high fevers, nausea, transient hypotension, hypoxia, and the like.
Accordingly, the methods described herein can comprise administering a TFP-expressing cell described herein to a subject and further administering an agent to manage elevated levels of a soluble factor resulting from treatment with a TFP-expressing cell. In one embodiment, the soluble factor elevated in the subject is one or more of IFN-g, TNFc IL-2, IL-6, and IL-8. Therefore, an agent administered to treat this side effect can be an agent that neutralizes one or more of these soluble factors. Such agents include, but are not limited to a steroid, an inhibitor of TNFa, and an inhibitor of IL-6. An example of a TNFa inhibitor is etanercept (marketed under the name ENBREL®). An example of an IL-6 inhibitor is tocilizumab (marketed under the name ACTEMRA®).
[0317] In one embodiment, the subject can be administered an agent which enhances the activity of a TFP-expressing cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g., Programmed Death 1 (PD1), can, in some embodiments, decrease the ability of a TFP-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. Inhibition of an inhibitory molecule, e.g., by inhibition at the DNA, RNA or protein level, can optimize a TFP-expressing cell performance. In embodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, can be used to inhibit expression of an inhibitory molecule in the TFP-expressing cell. In an embodiment, the inhibitor is a shRNA. In an embodiment, the inhibitory molecule is inhibited within a TFP-expressing cell. In these embodiments, a dsRNA molecule that inhibits expression of the inhibitory molecule is linked to the nucleic acid that encodes a component, e.g., all of the components, of the TFP. In one embodiment, the
inhibitor of an inhibitory signal can be, e.g., an antibody or antibody fragment that binds to an inhibitory molecule. For example, the agent can be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2 or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 and MDX-101, and marketed as YERVOY®); Bristol-Myers Squibb; tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP -675, 206)). In an embodiment, the agent is an antibody or antibody fragment that binds to T cell immunoglobulin and mucin-domain containing-3 (TIM3). In an embodiment, the agent is an antibody or antibody fragment that binds to Lymphocyte -activation gene 3 (LAG3).
[0318] In some embodiments, an agent suitable for use in combination with the TFP T cells disclosed herein is an agent that modulates myeloid suppressor cells, e.g., CCR2 antibodies. Other therapeutics, e.g, nanoparticle therapeutics, are known in the art.
[0319] In some embodiments, the agent which enhances the activity of a TFP-expressing cell can be, e.g., a fusion protein comprising a first domain and a second domain, wherein the first domain is an inhibitory molecule, or fragment thereof, and the second domain is a polypeptide that is associated with a positive signal, e.g., a polypeptide comprising an intracellular signaling domain as described herein. In some embodiments, the polypeptide that is associated with a positive signal can include a costimulatory domain of CD28, CD27, ICOS, e.g., an intracellular signaling domain of CD28, CD27 and/or ICOS, and/or a primary signaling domain, e.g., of CD3 zeta, e.g., described herein. In one embodiment, the fusion protein is expressed by the same cell that expressed the TFP. In another embodiment, the fusion protein is expressed by a cell, e.g., a T cell that does not express an anti-tumor-associated antigen TFP. Pharmaceutical Compositions
[0320] Pharmaceutical compositions of the present disclosure may comprise a TFP-expressing cell, e.g., a plurality of TFP-expressing cells, as described herein, in combination with one or more
pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present disclosure are in one aspect formulated for intravenous administration.
[0321] Pharmaceutical compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient ' s disease, although appropriate dosages may be determined by clinical trials.
[0322] In one embodiment, the pharmaceutical composition is substantially free of, e.g., there are no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus. In one
embodiment, the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.
[0323] When "an immunologically effective amount," "an anti-tumor effective amount," "a tumor- inhibiting effective amount," or "therapeutic amount" is indicated, the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 104 to 109 cells/kg body weight, in some instances 105 to 106 cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et ah, New Eng. J. of Med. 319: 1676, 1988).
[0324] In certain aspects, it may be desired to administer activated T cells to a subject and then subsequently redraw blood (or have an apheresis performed), activate T cells therefrom according to the present disclosure, and reinfuse the patient with these activated and expanded T cells. This process can be carried out multiple times every few weeks. In certain aspects, T cells can be activated from blood draws of from 10 cc to 400 cc. In certain aspects, T cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.
[0325] The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one aspect, the T cell compositions of the present disclosure are administered to a patient by intradermal or subcutaneous injection. In one aspect, the T cell compositions of the present disclosure are administered by i.v. injection. The compositions of T cells may be injected directly into a tumor, lymph node, or site of infection.
[0326] In a particular exemplary aspect, subjects may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate the cells of interest, e.g., T cells. These T cell isolates may be expanded by methods known in the art and treated such that one or more TFP constructs of the present disclosure may be introduced, thereby creating a TFP -expressing T cell of the present disclosure. Subjects in need thereof may subsequently undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain aspects, following or concurrent with the transplant, subjects receive an infusion of the expanded TFP T cells of the present disclosure. In an additional aspect, expanded cells are administered before or following surgery.
[0327] The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices. The dose for alemtuzumab
(CAMPATH®), for example, will generally be in the range 1 to about 100 mg for an adult patient, usually administered daily for a period between 1 and 30 days. The preferred daily dose is 1 to 10 mg per day although in some instances larger doses of up to 40 mg per day may be used (described in U.S.
Patent No. 6,120,766).
[0328] In one embodiment, the TFP is introduced into T cells, e.g., using in vitro transcription, and the subject (e.g., human) receives an initial administration of TFP T cells of the present disclosure, and one or more subsequent administrations of the TFP T cells of the present disclosure, wherein the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In one embodiment, more than one administration of the TFP T cells of the present disclosure are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the TFP T cells of the present disclosure are administered per week. In one embodiment, the subject (e.g., human subject) receives more than one administration of the TFP T cells per week (e.g., 2, 3 or 4 administrations per week) (also referred to herein as a cycle), followed by a week of no TFP T cells administrations, and then one or more additional administration of the TFP T cells (e.g., more than one administration of the TFP T cells per week) is administered to the subject. In another embodiment, the subject (e.g., human subject) receives more than one cycle of TFP T cells, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the TFP T cells are administered every other day for 3 administrations per week. In one embodiment, the TFP T cells of the present disclosure are administered for at least two, three, four, five, six, seven, eight or more weeks.
[0329] In one aspect, tumor-associated antigen TFP T cells are generated using lentiviral viral vectors, such as lentivirus. TFP T cells generated that way will have stable TFP expression.
[0330] In one aspect, TFP T cells transiently express TFP vectors for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15 days after transduction. Transient expression of TFPs can be effected by RNA TFP vector delivery. In one aspect, the TFP RNA is transduced into the T cell by electroporation.
[0331] A potential issue that can arise in patients being treated using transiently expressing TFP T cells (particularly with murine scFv bearing TFP T cells) is anaphylaxis after multiple treatments.
[0332] Without being bound by this theory, it is believed that such an anaphylactic response might be caused by a patient developing humoral anti-TFP response, i.e., anti-TFP antibodies having an anti-IgE isotype. It is thought that a patient' s antibody producing cells undergo a class switch from IgG isotype (that does not cause anaphylaxis) to IgE isotype when there is a ten- to fourteen-day break in exposure to antigen.
[0333] If a patient is at high risk of generating an anti-TFP antibody response during the course of transient TFP therapy (such as those generated by RNA transductions), TFP T cell infusion breaks should not last more than ten to fourteen days.
EXAMPLES
[0334] The present disclosure is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the present disclosure should in no way be construed as being
limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present disclosure and practice the claimed methods. The following working examples specifically point out various aspects of the present disclosure and are not to be construed as limiting in any way the remainder of the disclosure.
Example 1: TFP Constructs
[0335] Anti-TAA TFP constructs are engineered by cloning one or more anti-TAA scFv DNA fragment linked to a CD3 or TCR DNA fragment by either a DNA sequence encoding a short linker (SL):
AAAGGGGSGGGGSGGGGSLE (SEQ ID NO: 188) or a long linker (LL):
AAAIEVMYPPPYLGGGGSGGGGSGGGGSLE (SEQ ID NO: 189) into, e g., r510 vector ((System Biosciences (SBI)) at Xbal and EcoRl sites. CAR constructs are generated by cloning synthesized DNA encoding an anti-TAA antibody, partial CD28 extracellular domain, CD28 transmembrane domain,
CD28 intracellular domain and CD3 zeta into, e.g., a p510 vector at Xbal and EcoRl sites. CD3 e TFP constructs disclosed herein comprise the sequence set forth in SEQ ID NO: 191, which has an N-terminal truncation in reference to the full sequence (SEQ ID NO: 192).
[0336] In one embodiment, an anti-tumor-associated antigen CAR construct is generated as a comparator. A p5 lO_antitumor-associated antigen_28 z CAR is generated by cloning synthesized DNA encoding anti-tumor-associated antigen, partial CD28 extracellular domain, CD28 transmembrane domain, CD28 intracellular domain and CD3 zeta into p510 vector at Xbal and EcoRl sites.
Protease- cleavable anti-HSA/anti-MSLN Fusion Constructs
[0337] An anti-HSA sdAb (SEQ ID NO:42) was genetically fused to anti-MSLN sdAb SD1 (SEQ ID NO:39) via a cleavable linker. See, e.g., FIG. 6, which shows inducible TFP construct sequences in the format 5 ' -anti-HSA sdAb— protease-cleavable linker— anti-MSLN sdAb binder-3 ' ) with a C-terminal 6His tag for purification and detection purposes. Fusion proteins were expressed in E. coli and purified to homogeneity by Ni-NTA affinity chromatography. Fusion proteins were desalted and stored and in lxPBS buffer, pH 7.4.
Example 2: Antibody Sequences
Generation of Antibody Sequences
[0338] Provided are antibody polypeptides, fragments thereof, single domain antibodies, Fab fragments, and other antigen binding proteins that are capable of specifically binding to the human polypeptide (s), and fragments or domains thereof. In some embodiments, the antigen is a tumor-associated antigen (TAA). Anti-TAA antibodies or fragments thereof can be generated using diverse technologies (see, e.g., (Nicholson et al, 1997). Where murine anti-TAA antibodies are used as a starting material, humanization of murine anti-TAA antibodies is desired for the clinical setting, where the mouse-specific residues may induce a human-anti-mouse antigen (HAMA) response in subjects who receive T cell receptor (TCR) fusion protein (TFP) treatment, i.e., treatment with T cells transduced with the anti-TAA TFP construct. Humanization is accomplished by grafting CDR regions from murine anti-TAA antibody onto
appropriate human germline acceptor frameworks, optionally including other modifications to CDR and/or framework regions. As provided herein, antibody and antibody fragment residue numbering follows Kabat (Kabat E. A. et al, 1991; Chothia et al, 1987).
Generation of scFvs
[0339] Human or humanized anti-TAA IgGs are used to generate scFv sequences for TFP constructs. DNA sequences coding for human or humanized VF and VH domains are obtained, and the codons for the constructs are, optionally, optimized for expression in cells from Homo sapiens. The order in which the VF and VH domains appear in the scFv is varied (i.e., VF-VH, or VH-VF orientation), and three copies of the "G4S" or "G4S" subunit (G4S)3 connect the variable domains to create the scFv domain. Anti-TAA scFv plasmid constructs can have optional Flag, His or other affinity tags, and are electroporated into HEK-293 or other suitable human or mammalian cell lines and purified. Validation assays include binding analysis by FACS, kinetic analysis using Proteon, and staining of TAA- expressing cells.
Source of TCR Subunits
[0340] Subunits of the human T Cell Receptor (TCR) complex all contain an extracellular domain, a transmembrane domain, and an intracellular domain. A human TCR complex contains the CD3 -epsilon polypeptide, the CD3-gamma polypeptide, the CD3-delta polypeptide, the CD3-zeta polypeptide, the TCR alpha chain polypeptide and the TCR beta chain polypeptide. The human CD3-epsilon polypeptide canonical sequence is UniProt Accession No. P07766. The human CD3-gamma polypeptide canonical sequence is UniProt Accession No. P09693. The human CD3-delta polypeptide canonical sequence is UniProt Accession No. P043234. The human CD3-zeta polypeptide canonical sequence is UniProt Accession No. P20963. The human TCR alpha chain canonical sequence is UniProt Accession No. Q6ISU1. The human TCR beta chain C region canonical sequence is UniProt Accession No. P01850, a human TCR beta chain V region sequence is P04435.
[0341] The human CD3 -epsilon polypeptide canonical sequence is:
MQSGTHWRVFGFCFFSVGVWGQDGNEEMGGITQTPYKVSISGTTVIFTCPQYPGSEIFWQHND KNIGGDEDDKNIGSDEDHFSFKEFSEFEQSGYYVCYPRGSKPEDANFYFYFRARVCENCMEMD VMSVAT]VrVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEP IRKGQRDLYSGLNQRRI (SEQ ID NO: 193). In one embodiment, the human CD3-epsilon fragment used in the TFPs is
DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFS ELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWS KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 194)
[0342] The human CD3 -gamma polypeptide canonical sequence is:
MEOGKGT .AVT JT .ATTT J QGTT AOSTKGNHT ,VK VYPY OEPGSVT J TCDAEAKNTTWFKDGKMTGF LTEDKKKWNLGSNAKDPRGMY QCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIV SIFV LAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID
NO: 195). In one embodiment, the human CD3-gamma fragment used in the TFPs is:
QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGM YQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASD KQTLLPNDQLY QPLKDREDDQY SHLQGNQLRRN (SEQ ID NO: 196).
[0343] The human CD3 -delta polypeptide canonical sequence is:
MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDP RGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGHVTDVIATLLLALGVFCFAGHETGR LSGAADTQALLRNDQVY QPLRDRDDAQY SHLGGNWARNK (SEQ ID NO: 197). In one embodiment, the human CD3 -delta fragment used in the TFPs is:
FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQV HYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPL RDRDDAQY SHLGGNWARNK (SEQ ID NO: 198)
[0344] The human CD3-zeta polypeptide canonical sequence is:
MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 199). In one embodiment, the human CD3-zeta fragment used in the TFPs is:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNE LQKDKMAEAY SEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQ ALPPR (SEQ ID NO:300)
[0345] The human TCR alpha chain canonical sequence is:
MAGTWLLLLLALGCPALPTGVGGTPFPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPIWFS AGNGSALDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGE ASTARTCPQEPLRGTPGGALWLGVLRLLLFKLLLFDLLLTCSCLCDPAGPLPSPATTTRLRALGS HRLHPATETGGRE ATS SPRPQPRDRRW GDTPPGRKPGSP VWGEGS YLS S YPT CP AQ AW C SRS AL RAPSSSLGAFFAGDLPPPLQAGAA (SEQ ID NO:20l).
[0346] The human TCR alpha chain C region canonical sequence is:
PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGF NLLMTLRLW S S (SEQ ID NO: 202)
[0347] The human TCR alpha chain V region CTL-L17 canonical sequence is:
MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWY KKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAAKGAGTASKLT F GTGTRLQ VTL (SEQ ID NO:203).
[0348] The human TCR beta chain C region canonical sequence is:
EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLK EQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWG RADCGFTS V SYQQGVLS ATILYEILLGKATLYAVLV S ALVLMAMVKRKDF (SEQ ID NO:204).
[0349] The human TCR beta chain V region CTL-L17 canonical sequence is:
MGTSLLCWMALCLLGADHADTGVSQNPRHNITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPE FLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLAGLNQPQHFGDGT RLSIL (SEQ ID NO:205).
[0350] The human TCR beta chain V region YT35 canonical sequence is:
MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMRGLE LLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSFSTCSANYGYTFGSG TRLTVV (SEQ ID NO: 206).
Generation of TFPs from TCR Domains and scFvs, Fab fragments, or sdAbs
[0351] Anti-TAA scFvs are recombinantly linked to a) CD3-epsilon or other TCR subunits, and b) a blocking domain, such as HSA, using a linker sequence, such as G4S, (G4S)2 (G4S)3 or (G4S)4. Various linkers and antibody (e.g., scFv or sdAb) configurations are used. TCR alpha and TCR beta chains are used for generation of TFPs either as full-length polypeptides or as only their constant domains (e.g., in the preparation for a construct suitable for an allogeneic immune cell product) . Any variable sequence of TCR alpha and TCR beta chains is suitable for making TFPs.
TFP Expression Vectors
[0352] Expression vectors are provided that include: a promoter (Cytomegalovirus (CMV) enhancer- promoter), a signal sequence to enable secretion, a polyadenylation signal and transcription terminator (Bovine Growth Hormone (BGH) gene), an element allowing episomal replication and replication in prokaryotes (e.g., SV40 origin and ColEl or others known in the art) and elements to allow selection (ampicillin resistance gene and zeocin marker).
[0353] Preferably, the TFP -encoding nucleic acid construct or constructs is/are cloned into one or more lentiviral expression vectors and expression validated based on the quantity and quality of the effector T cell response of transduced T cells in response to TAA+ target cells. Effector T cell responses include, but are not limited to, cellular expansion, proliferation, doubling, cytokine production and target cell lysis or cytolytic activity (i.e., degranulation).
[0354] The TFP lentiviral transfer vectors are used to produce the genomic material packaged into the VSVg pseudotyped lentiviral particles. Lentiviral transfer vector DNA is mixed with the three packaging components of VSVg, gag/pol and rev in combination with Lipofectamine® reagent to transfect them together into 293 cells. After 24 and 48 hours, the media is collected, filtered and concentrated by ultracentrifugation. The resulting viral preparation is stored at -80° C. The number of transducing units is determined by titration on SupTl cells (T cell lymphoblastic lymphoma, (ATCC® CRL-1942™).
Redirected dual specificity TFP T cells are produced by activating fresh naive T cells with anti-CD3x anti-CD28 beads for 24 hrs and then adding the appropriate number of transducing units to obtain the desired percentage of transduced T cells. These modified T cells are allowed to expand until they become rested and come down in size at which point they are cryopreserved for later analysis. The cell numbers and sizes are measured using a Coulter Counter® Multisizer™ 3 (Beckman Coulter). Before
cryopreserving, percentage of cells transduced (expressing TFP.BCMA on the cell surface) and their
relative fluorescence intensity of that expression are determined by flow cytometric analysis. From the histogram plots, the relative expression levels of the TFPs are examined by comparing percentage transduced with their relative fluorescent intensity.
[0355] In some embodiments, the vector is an adenoviral vector. In some embodiments, the vector is a circular RNA or a circular RNA transfer vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, multiple TFPs are introduced by T cell transduction with multiple viral vectors. Evaluating Cytolytic Activity. Proliferation Capabilities and Cytokine Secretion of Humanized TFP Redirected T Cells
[0356] The functional abilities of TFP.TAA T cells to produce cell-surface-expressed TFPs, and to kill target tumor cells, proliferate and secrete cytokines are determined using assays known in the art.
Cleavage of the protease-cleavable linker(s) tethering the blocking domain to the TFP T cell can be tested, for example, in vitro by proteolytic cleavage followed by western blot analysis of cleavage fragments.
[0357] Human PBMCs (e.g., blood from a normal apheresed donor whose naive T cells are obtained by negative selection for T cells, CD4+ and CD8+ lymphocytes) are treated with human interleukin-2 (IL-2) then activated with anti-CD3x anti-CD28 beads, e.g., in 10% RPMI at 37 ° C, 5% C02 prior to transduction with the TFP -encoding lentiviral vectors. Flow cytometry assays are utilized to confirm cell surface presence of a TFP, such as by an anti-FLAG antibody or an anti-murine variable domain antibody. Cytokine (e.g., IFN-g) production is measured using ELISA or other assays.
Example 3 : Human TFP T Cell Treatment in an In Vivo Solid Tumor Xenograft Mouse Model
[0358] The efficacy of human inducible TFP T cells can also be tested in immune compromised mouse models bearing subcutaneous solid tumors derived from human TAA-expressing human cell lines.
Tumor shrinkage in response to human inducible TFP T cell treatment can be either assessed by caliper measurement of tumor size, or by following the intensity of a GFP fluorescence signal emitted by GFP- expressing tumor cells.
[0359] Primary human solid tumor cells can be grown in immune-compromised mice without having to culture the cells in vitro. Exemplary solid cancer cells include solid tumor cell lines, such as provided in The Cancer Genome Atlas (TCGA) and/or the Broad Cancer Cell Line Encyclopedia (CCLE, see Barretina et ah, Nature 483:603 (2012)). Exemplary solid cancer cells include primary tumor cells isolated from mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney, endometrial, or stomach cancer. In some embodiments, the cancer to be treated is selected from the group consisting of mesotheliomas, papillary serous ovarian adenocarcinomas, clear cell ovarian carcinomas, mixed Mullerian ovarian carcinomas, endometroid mucinous ovarian carcinomas, pancreatic adenocarcinomas, ductal pancreatic adenocarcinomas, uterine serous carcinomas, lung adenocarcinomas, extrahepatic bile duct carcinomas, gastric adenocarcinomas, esophageal adenocarcinomas, colorectal adenocarcinomas and breast adenocarcinomas. These mice can be used to test the efficacy of anti-TAA TFP T cells in the human tumor xenograft models (see, e.g., Morton et ah, Nat. Procol. 2:247 (2007)).
Following an implant or injection of 1 x 106 - 1 x 107 primary cells (collagenase -treated bulk tumor suspensions in EC matrix material) or tumor fragments (primary tumor fragments in EC matrix material) subcutaneously, tumors are allowed to grow to 200-500 mm3 prior to initiation of treatment.
Example 4: Preparation of T Cells Transduced with Inducible TFPs
Lentiviral production
[0360] In one embodiment, the inducible TFP is encoded by a lentivirus. Lentivirus encoding the appropriate constructs are prepared as follows. 5 x 106 HEK-293FT cells are seeded into a 100 mm dish and allowed to reach 70-90% confluency overnight. 2.5 pg of the indicated DNA plasmids and 20 pL Lentivirus Packaging Mix (ALSTEM, cat# VP100) are diluted in 0.5 mL DMEM or Opti-MEM® I Medium without serum and mixed gently. In a separate tube, 30 pL of NanoFect® transfection reagent (ALSTEM, cat# NF100) is diluted in 0.5 mL DMEM or Opti-MEM I Medium without serum and mixed gently. The NanoFect/DMEM and DNA/DMEM solutions are then mixed together and vortexed for 10- 15 seconds prior to incubation of the DMEM-plasmid-NanoFect mixture at room temperature for 15 minutes. The complete transfection complex from the previous step is added dropwise to the plate of cells and rocked to disperse the transfection complex evenly in the plate. The plate is then incubated overnight at 37 ° C in a humidified 5% C02 incubator. The following day, the supernatant is replaced with 10 mL fresh medium and supplemented with 20 pL of ViralBoost™ (500x, ALSTEM, cat# VB100). The plates are then incubated at 37 ° C for an additional 24 hours. The lentivirus containing supernatant is then collected into a 50 mL sterile, capped conical centrifuge tube and put on ice. After centrifugation at 3000 rpm for 15 minutes at 4° C, the cleared supernatant is filtered with a low-protein binding 0.45 pm sterile filter and virus is subsequently isolated by ultracentrifugation at 25,000 rpm (Beckmann, L8-70M) for 1.5 hours, at 4° C. The pellet is removed and re-suspended in DMEM medium and lentivirus
concentrations/titers are established by quantitative RT-PCR using the Lenti-X™ qRT-PCR Titration kit (Clontech®; catalog number 631235). Any residual plasmid DNA is removed by treatment with DNasel. The virus stock preparation is either used for infection immediately or aliquoted and stored at -80 ° C for future use.
[0361] Lentivirus titers are established by transducing cells (e.g., Jurkat cells) with different amount of virus preparation. The DNA is then isolated from the transduced Jurkat cells 24 hours after transduction. The virus titer is determined, e.g., by quantitative real-time PCR, with in-house designed primers/probe specific for Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) as well as for an internal quantitation control.
T cell isolation
[0362] Peripheral Blood Mononuclear Cells (PBMCs) are prepared from either whole blood or buffy coat. Whole blood is collected in 10 mL Heparin vacutainers and either processed immediately or stored overnight at 4 ° C. Approximately 10 mL of whole anti -coagulated blood is mixed with sterile phosphate buffered saline (PBS) buffer for a total volume of 20 mL in a 50 mL conical centrifuge tube (PBS, pH 7.4, without Ca2+/Mg2+). 20 mL of this blood/PBS mixture is then gently overlaid onto the surface of 15 mL of Ficoll-Paque® PLUS (GE Healthcare, 17-1440-03) prior to centrifugation at 400g for 30-40 min at
room temperature with no brake application.
[0363] Buffy coat is purchased, e.g., from Research Blood Components (Boston, MA). LeucoSep™ tubes (Greiner bio-one) are prepared by adding 15 mL Ficoll-Paque and centrifuged at lOOOg for 1 minute. Buffy coat is diluted 1 :3 in PBS (pH 7.4, without Ca2+ or Mg2+). The diluted buffy coat is transferred to LeucoSep tube and centrifuged at lOOOg for 15 minutes with no brake application. The layer of cells containing PBMCs, seen at the diluted plasma/Ficoll interface, is removed carefully to minimize contamination by Ficoll®. Residual Ficoll, platelets, and plasma proteins are then removed by washing the PBMCs three times with 40 mL of PBS by centrifugation at 200g for 10 minutes at room temperature. The cells are then counted with a hemocytometer. CD4+ and CD8+ T cells are then frozen down in freezing medium (90% FBS+l0% DMS0 at a concentration of 30-50 x 106 cells per vial.
T cell activation
[0364] PBMCs prepared from either whole blood or buffy coat are stimulated with anti-human CD28 and CD3 antibody -conjugated magnetic beads for 24 hours prior to viral transduction. Freshly isolated PBMCs are washed once in CAR T medium (AIM V-AlbuMAX BSA, Life Technologies), with 5% AB serum and 1.25 pg/mL amphotericin B (Gemini Bioproducts), 100 U/mL penicillin, and 100 pg/mL streptomycin) without huIL-2, before being re-suspended at a final concentration of 1 x 106 cells/mL in CAR T medium with 300 IU/mL human IL-2, IL-7, or IL-15 (from a lOOOx stock; Invitrogen).
[0365] Alternatively, frozen CD4+/CD8+ T cells are thawed in pre-warmed DMEM + 10 % FBS, spun down, and then resuspended in complete T cell expansion medium supplemented with 300 IU/mL huIL2 (Thermo Fisher®) at a final concentration of 1 x 106 cells/mL. Prior to being used to activate T cells, anti human CD28 and anti-human CD3 antibody-conjugated magnetic beads (Dynabeads®, Thermo Fisher) are washed three times with sterile 1 x PBS (pH7.4), using a magnetic rack to isolate beads from the solution. The T cells are then mixed with the beads at a 1 : 1 ratio, by transferring 25 pL (lxlO6 beads) of beads to 1 mL of T cell suspension. The beads/cells mixture is then dispensed to single wells of a non-TC treated l2-well plate, and incubated at 37 ° C with 5 % C02 for 24 hrs.
[0366] Prior to activation, anti-human CD28 and CD3 antibody-conjugated magnetic beads (available from, e.g., Invitrogen, Life Technologies) are washed three times with 1 mL of sterile lx PBS (pH 7.4), using a magnetic rack to isolate beads from the solution, before re -suspension in CAR T medium, with 300 IU/mL human IL-2, to a final concentration of 4 x 107 beads/mL. PBMC and beads are then mixed at a 1 : 1 bead-to-cell ratio, by transferring 25 pL (lxlO6 beads) of beads to 1 mL of PBMC. The desired number of aliquots are then dispensed to single wells of a l2-well low-attachment or non-treated cell culture plate, and incubated at 37 ° C, with 5% C02, for 24 hours before viral transduction.
T cell transduction and expansion
[0367] Following activation of PBMCs, cells are incubated for 24 hours at 37 ° C, 5% C02. Lentivirus is thawed on ice and then added to activated T cells at indicated MOI in the presence of 10 pg/ml Polybrene (Sigma). Cells are spinoculated with the lentivirus at 200 g for 100 minutes at room temperature. The transduced T cells are incubated for an additional 24 hr before an additional lentivirus transduction. After the 2nd round of lentivirus transduction, the T cells are expanded in T cell expansion medium
supplemented with 300 IU/mL of hIL-2 and sub-cultured every other day at 5 x 105 cells/mL.
[0368] In some instances, activated PBMCs are electroporated with in vitro transcribed (IVT) mRNA. Human PBMCs are stimulated with Dynabeads® (Thermo Fisher®) at l-to-l ratio for 3 days in the presence of 300 IU/ml recombinant human IL-2 (R&D System). The beads are removed before electroporation. The cells are washed and re-suspended in OPTI-MEM® medium (Thermo Fisher) or AimV® medium (Invitrogen) in 5% hAB serum (Gemini Bio-Products) and 1% antibiotics at the concentration of 2.5 x 107 cells/mL. 200 pL of the cell suspension (5 x 106 cells) are transferred to the 2 mm gap Electroporation Cuvettes Plus™ (Harvard Apparatus BTX) and prechilled on ice. 10 pg of IVT TFP mRNA is added to the cell suspension. The mRNA/cell mixture is then electroporated at 200 V for 20 milliseconds using ECM® 830 Electro Square Wave Porator (BTX® Harvard Apparatus).
Immediately after the electroporation, the cells are transferred to fresh cell culture medium (AIM V AlbuMAX® (BSA) serum free medium + 5% human AB serum + 300 IU/ml IL-2) and incubated at 37° C.
Example 5. Protease Inducible TFP T cells using MMP-Cleavable Blocking Domains
[0369] In this Example, linkers for an inducible TFP T cell comprising a biologically active polypeptide fusion protein (e.g., the tumor-associated antigen binder) are described; a TAA binder is coupled to a blocking domain via a protease-cleavable linker.
[0370] The protease-cleavable linker is engineered to comprise a sequence recognized by a protease typically present in the tumor microenvironment (TME). The tumor microenvironment is characterized by numerous factors including but not limited to expression of matrix metalloproteinases, cathepsins, proteases, indolomine 2,3-dioxygenase, urokinase-type plasminogen activator (uPA), membrane-type protease 1 (MT-SPl/matriptase), legumain, low extracellular pH (acidosis) and low oxygenation (hypoxia) (Brown & Wilson, 2004; Vaupel & Mayer, 2007; Desnoyers & Vasiljeva, 2013).
[0371] In one embodiment, the protease-cleavable linker comprises a sequence recognized by matrix metalloproteinases (MMPs), which are inactive in non-cancerous cells, thereby preventing normal activity of the polypeptide fusion protein until the linker is cleaved, and the masking domain is therefore removed, by TME -expressed MMPs. Non-limiting examples of MMP cleavage sites are listed in Table 14.
Table 14: Exemplary MMP-cleavable sequences
[0372] The linker comprising the protease -cleavable sequence is covalently attached to a blocking domain that physically blocks or sterically inhibits the biologically active polypeptide from functioning in non-cancerous tissue. In one embodiment, a bulky protein such as human serum albumin (HSA, or a fragment thereof) is used. Such bulky proteins can block access of an active protein to its target or prevent binding of an antigen to its receptor.
[0373] The preparation comprising the biologically active fusion protein, protease-cleavable linker, and blocking domain remains inactive until it reaches the tumor microenvironment, at which point the linker is cleaved by proteases present and the cytokine, chemokine, or binding agent is unmasked and/or becomes active.
[0374] Successful cleavage by TME proteases in vivo may be assessed in several ways. In one embodiment, a xenograft model system is used comprising an inoculation cell line that expresses the protease of interest and as a negative control an inoculation cell line that does not express the protease of interest is used (e.g., a similar or same cell line where the gene for the protease is knocked out). When tumors in the two types of mice (i.e., experimental mice and control mice) have grown to a certain size (e.g., about 300mm2), infusion of TFP T cells and subsequent measurement of tumor volume, cytokine production, and survival responses will provide a measurement of TFP efficacy and linker cleavage in protease null and positive TMEs.
[0375] In another embodiment, linker cleavage is tested by the harvest and co-IP of TIES expressing a TFP disclosed herein from a) a TAA+ and protease -expressing xenograft and b) TAA+ and protease null xenograft, followed by western blot analysis or intact mass spectrometry to detect linker cleavage.
[0376] In another embodiment, the blocking domain (e.g., an anti-HSA domain) is fused to an epitope (e.g. FLAG, myc, or HA tag) for immunoprecipitation and linker cleavage is measured by detection of the released blocking domain by western blot, ELISA, or mass spectrometry. In this system, TAA+ tumors with and without the relevant protease are compared. The +/- protease cell line system may be achieved by genetic knockout, knock-in, null vs over-expressing lines, or by infusion of a relevant protease.
Example 6. Protease Inducible TFP T cells using Cathepsin-Cleavable Blocking Domains
[0377] This Example describes using linker peptide sequences to attach the blocking domain that are recognized and subsequently digested by cathepsins. Cathepsins are a group of serine proteases that function in acidic environments. Lysosomal cathepsins require a reducing, slightly acidic environment, such as found in the TME, in order to be optimally active.
[0378] A polypeptide fusion protein with a blocking domain is engineered as described in Example 5. A linker comprising a cathepsin cleavable elastin, collagen or fibronectin is linked to the fusion protein in a
fashion that prohibits binding domain recognition and/or biological activity. In one embodiment, cathepsin S cleavable binding domains such as GAVVRGA (SEQ ID NO: ) may be used to link the fusion protein to the masking domain (e.g., HSA or a fragment thereof). The cathepsin cleavable blocking domain will be removed in the tumor microenvironment using appropriate cleavable linker sequences (e.g., cathepsin B or cathepsin S sequences).
[0379] The biologically active fusion protein may additionally comprise a serum half-life extension element, as is evidenced by its prolonged activity or extended PK. In one embodiment, the serum half- life extension element is the HSA blocking domain. In some embodiments, the serum half-life extension element may extend the serum half-life of a T cell comprising a biologically active fusion protein.
Example 7. Protease Inducible CAR T cells
[0380] In this Example, a biologically active fusion protein, e.g., a chimeric antigen receptor CAR) subunit, is engineered to comprise a blocking domain as described above. A protease-cleavable linker is attached to each variable domain on the antibody, and each linker is in turn covalently attached to a masking domain, such as HSA or a fragment thereof (see Figures 1A and IB). The masked chimeric antigen receptor is expressed in an immune cell, such as a Treg or NK cell, which is in turn administered to a patient. Upon the engineered cell reaching the tumor microenvironment, the masking domain is cleaved away and the CAR T cells are capable of immunomodulation.
Example 8. Protease Inducible TFP T cells comprising an HSA binding domain
[0381] In this Example, a chimeric antigen receptor subunit (such as described in Example 7) comprises a tumor-associated antigen (TAA) binding protein. The subunit is expressed such that it incorporates into an immune cell ' s endogenous T cell receptor complex.
[0382] The TFP T cell activity is masked by a cleavable HSA binding domain, such as a single domain antibody (sdAb), Fab fragment, VH, VL, or scFv, which is fused to the TAA binder (Figure 2A). The HSA binding domain is covalently attached to the TAA binder via a cleavable domain, such as those described above (e.g., MMP14, MMP9, cathepsins, etc.). An exemplary humanized anti-HSA sdAb that may be fused to a TCR T cell that will recognize and bind HSA is
EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSV KGRFTISRDNAKTTFYFQMNSFRPEDTAVYYCTIGGSFSRSSQGTFVTVSS (SEQ ID NO:42).
[0383] Upon infusion of engineered immune cells comprising this construct into a patient, circulating HSA in the patient will bind to the HSA -binding domain of the construct, resulting in inhibition of the tumor recognition domain. Masking of the TAA-binding domain by the bound HSA reduces the risk of on-target, off-tumor activity of the engineered immune cells. The albumin -binding domain and the bound albumin serve to both extend the serum half-life of the circulating cells and to block the function of the TAA-binding domain.
[0384] Upon migration into the tumor microenvironment, the cleavable domain between the HSA masking domain and the TAA-binding domain is cleaved, and the HSA/HSA-binder complex is removed from the surface of the immune cell. In turn, the tumor recognition domain of the complex is exposed and able to recognize tumor antigen.
Example 9. Protease Inducible TFP T cells comprising bound HSA
[0385] In this Example, inducible TFP T cells are prepared as described in Example 8.
[0386] The TFP T cell activity is masked by a cleavable domain comprising a blocking moiety, e.g., HSA or a fragment thereof. The HSA or fragment thereof is either fused directly to the TAA binder or is fused to another part of the TCR in a way that blocks the TAA binding site (Figure 5). The HSA is covalently attached to the TFP via a cleavable domain, such as those described above.
[0387] Upon infusion of engineered immune cells comprising this construct into a subject, the TFP T cells migrate into the tumor microenvironment, and the linker is cleaved by proteases present in the tumor microenvironment, and the HSA is removed from the surface of the immune cell. In turn, the tumor recognition domain of the complex is exposed and able to recognize tumor antigen.
Example 10. Anti-mesothelin inducible TFP constructs
[0388] The inducible TFP constructs disclosed herein can comprise an anti-mesothelin binding domain. Examples of anti-mesothelin binding domain sequences are listed in Table 15.
Table 15. Examples of anti-mesothelin sequences
Digestion of aAlb-linker-SDl fusion protein constructs
[0389] The single domain antibody fusion proteins (sequences are shown in FIG. 6) were expressed in E. coli and purified by Ni-NTA affinity chromatography. Each protein was subjected to cleavage by incubation with their respective proteases (MMP9, uPA or cathepsin B) for the cleavage sequence engineered between the two sdAbs. 850 pmol each of an aHSA sdAb-linker-cleavage site-linker-SD 1 sdAb-His6 construct fusion protein shown in Figure 6 was digested with 0.27 pmol MMP9, 0.22 pmol uPA or 0.18 pmol cathepsin B, corresponding to 2xlO 5U of each protease, for 16 hours at 23°C in the presence or absence of 10% fetal bovine serum (FBS). Next, 5 qg of each purified fusion protein subjected to proteolysis was analyzed by western blot using a 1:500 anti-6His HRP antibody
(ThermoFisher Cat. MA1-21315-HRP) in PBST bufferto confirm cleavage products.
[0390] Results are shown in FIG. 8. Constructs were loaded pairwise as follows: aHSA-sdAb-uPA-SD 1 sdAb digested with uPA +/- FBS; aAlb-N/C-SDl digested with uPA +/- FBS; aHSA sdAb-MMP9vl- SDlsdAb digested with MMP9 +/- FBS; aHSA sdAb-MMP9v2-SDlsdAb digested with MMP9 +/- FBS; and aHSA sdAb-cathepsin B-SD1 sdAb digested with cathepsin B +/- FBS. In this western blot, purified fusion proteins transferred to the PVDF membrane were probed with the anti-6His antibody for detection
of both the undigested fusion protein with a C-terminal 6His tag (~29 kDa MW) and the cleavage product - SD1 sdAb-His6 (~ l4kDa). As can be seen in Figure 8, the undigested product is approximately 30 kDa and the expected cleavage product is about 14 kDa.
[0391] uPA and MMP9 cleavage product is visualized on the western blot. Cathepsin B either did not cleave or the cleavage was so inefficient that the cleavage product was at a concentration that is below the limit of detection by this method. The efficiency of protease cleavage is quite low, representing 5% or less of the product detected on the western blot membrane. The uPA protease is unable to cleave in the presence of FBS, while MMP9 seems to not be affected by the presence of FBS.
[0392] MMP9vl cleavage sequence seems to be more efficiently cleaved compared to MMP9v2.
Endnotes
[0393] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
Claims
1. A recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising
(a) a TCR subunit comprising
(i) at least a portion of a TCR extracellular domain, and
(ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular
signaling domain of CD3 epsilon;
(b) an antibody domain comprising a TAA binding domain; and
(c) a blocking domain,
wherein the TCR subunit and the antibody domain are operatively linked, the antibody domain and the blocking domain are linked by a protease-cleavable linker, and wherein the TFP incorporates into a TCR when expressed in a T cell.
2. A recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP)
comprising
(a) a TCR subunit comprising
(i) at least a portion of a TCR extracellular domain, and
(ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular
signaling domain of CD3 gamma;
(b) an antibody domain comprising a TAA binding domain; and
(c) a blocking domain
wherein the TCR subunit and the antibody domain are operatively linked, the antibody domain and the blocking domain are linked by a protease-cleavable linker, and wherein the TFP incorporates into a TCR when expressed in a T cell.
3. A recombinant nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP)
comprising
(a) a TCR subunit comprising
(i) at least a portion of a TCR extracellular domain, and
(ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular
signaling domain of CD3 delta;
(b) an antibody domain comprising an antigen binding domain; and
(c) a blocking domain
wherein the TCR subunit and the antibody domain are operatively linked, the antibody domain and the blocking domain are linked by a protease-cleavable linker, and wherein the TFP incorporates into a TCR when expressed in a T cell.
4. The recombinant nucleic acid molecule of any one of claims 1-3, wherein the encoded antigen binding domain is connected to the TCR extracellular domain by a linker sequence.
5. The recombinant nucleic acid molecule of claim 4, wherein the encoded linker sequence comprises
(G4S)n, wherein n=l to 4.
6. The recombinant nucleic acid molecule of any one of claims 1-3, wherein the antibody domain is a human or humanized antibody.
7. The recombinant nucleic acid molecule of any one of claims 1-3, wherein the encoded antigen
binding domain is connected to the blocking domain by a linker sequence that encodes a protease- cleavable linker.
8. The recombinant nucleic acid molecule of claim 7, wherein the linker sequence encodes a matrix metalloproteinase cleavable linker.
9. The recombinant nucleic acid molecule of claim 8, wherein the matrix metalloproteinase cleavable linker comprises a sequence selected from any one of sequences in Tables 1-14.
10. The recombinant nucleic acid molecule of claim 7, wherein the linker sequence encodes a cathepsin- cleavable linker.
11. The recombinant nucleic acid molecule of claim 7, wherein the linker sequence encodes a Urokinase plasminogen activator (uPA)-cleavable linker.
12. The recombinant nucleic acid molecule of claim 7, wherein the encoded antigen binding domain is connected to an antibody that specifically binds a blocking domain by a linker sequence that encodes a protease-cleavable linker.
13. The recombinant nucleic acid molecule of any one of claims 1-12, wherein the TCR subunit
comprises a TCR extracellular domain.
14. The recombinant nucleic acid molecule of any one of claims 1-13, wherein the TCR subunit
comprises a TCR transmembrane domain.
15. The recombinant nucleic acid molecule of any one of claims 1-14, wherein the TCR subunit
comprises a TCR intracellular domain.
16. The recombinant nucleic acid molecule of any one of claims 1-15, wherein the TCR subunit
comprises (i) a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
17. The recombinant nucleic acid molecule of any one of claims 1-16, wherein the TCR subunit
comprises a TCR intracellular domain comprising a stimulatory domain selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having at least one modification thereto.
18. The recombinant nucleic acid molecule of any one of claims 1-17, wherein the TCR subunit
comprises an intracellular domain comprising a stimulatory domain selected from a functional signaling domain of 4-1BB, CD28, CD2, and/or CD3 zeta, or an amino acid sequence having at least one modification thereto.
19. The recombinant nucleic acid molecule of any one of claims 1-18, wherein the antibody domain comprises an antibody fragment.
20. The recombinant nucleic acid molecule of any one of claims 1-19, wherein the antibody domain comprises a VH, VL, SCFV or a VHH domain.
21. The recombinant nucleic acid molecule of any one of claims 1-20, encoding (i) a light chain (LC) CDR1, LC CDR2 and LC CDR3 of an anti-TAA light chain binding domain amino acid sequence with 70-100% sequence identity to a light chain (LC) CDR1, LC CDR2 and LC CDR3 of an anti- TAA light chain binding domain provided herein, respectively, and/or (ii) a heavy chain (HC)
CDR1, HC CDR2 and HC CDR3 of an anti-TAA heavy chain binding domain amino acid sequence with 70-100% sequence identity to a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 of an anti- TAA heavy chain binding domain provided herein, respectively.
22. The recombinant nucleic acid molecule of any one of claims 1-21, wherein the recombinant nucleic acid molecule encodes a light chain variable region, wherein the light chain variable region comprises an amino acid sequence having at least one but not more than 30 modifications of a light chain variable region amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity to a light chain variable region amino acid sequence of a light chain variable region provided herein.
23. The recombinant nucleic acid molecule of any one of claims 1-22, wherein the recombinant nucleic acid molecule encodes a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least one but not more than 30 modifications of a heavy chain variable region amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to a heavy chain variable region amino acid sequence of a heavy chain variable region provided herein.
24. The recombinant nucleic acid molecule of any one of claims 1-23, wherein the TFP includes an extracellular domain of a TCR subunit that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
25. The recombinant nucleic acid molecule of any one of claims 1-24, wherein the encoded TFP
includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
26. The recombinant nucleic acid molecule of any one of claims 1-25, wherein the encoded TFP
includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
27. The recombinant nucleic acid molecule of any one of claims 1-26, further comprising a sequence encoding a costimulatory domain.
28. The recombinant nucleic acid molecule of claim 27, wherein the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of DAP 10, DAP 12, CD30, LIGHT, 0X40, CD2, CD27, CD28, CDS, ICAM-l, LFA-l (CD1 la/CDl8), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.
29. The recombinant nucleic acid molecule of any one of claims 1-28, wherein the linker sequence
encodes a peptide sequence that is cleaved by at least one of a tumor cell surface protease, a carboxypeptidase, a cathepsin, a kallikrein, a hexokinase, a plasmin, a stromelysin, factor Xa, a chymotrypsin-like protease, a trypsin-like protease, a elastase-like protease, atryptase, a chymase, a subtilisin-like protease, an actinidain, a proteinase, a bromelain, a calpain, a caspase, a cysteine protease, a papain, an HIV-1 protease, an HSV protease, a CMV protease, a chymosin, a renin, a pepsin, a matriptase, a plasmepsin, a nepenthesin, a metalloexopeptidase, a metalloendopeptidase, a matrix metalloproteinase/ collagenase, a plasminogen activator, a urokinase plasminogen activator (uPA), an enterokinase, a prostate-specific antigen (PSA, hK3), an interleukin- 1b converting enzyme, a thrombin, a fibroblast activation protein (FAP), a meprin, a granzyme, and a dipeptidyl peptidase.
30. The recombinant nucleic acid molecule of claim 29, wherein the cathepsin is cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, or cathepsin L; the hexokinase is hKl, hKlO, or hKT5; the proteinase is PR-3; the caspase is caspase-3; the cysteine protease is Mir 1 -CP or legumain; the matrix metalloproteinase or collagenase is MMPl/( interstitial collagenase), MMP2/type IV collagenase, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM 10, or ADAM 12; the prostate-specific antigen is PSA or hK3, the FAP is FAP -a; the granzyme is granzyme M or granzyme B; or the dipeptidyl peptidase is dipeptidyl peptidase IV (DPPIV/CD26).
31. The recombinant nucleic acid molecule of any one of claims 1-28, wherein the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP.
32. The isolated nucleic acid molecule of any one of claims 1-31, wherein the isolated nucleic acid molecule is mRNA.
33. The recombinant nucleic acid molecule of any one of claims 1-32, wherein the TFP includes an immunoreceptor tyrosine-based activation motif (IT AM) of a TCR subunit that comprises an IT AM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), DAP10, CD5, CDl6a, CDl6b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20
modifications thereto.
34. The recombinant nucleic acid molecule of claim 33, wherein the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon.
35. The recombinant nucleic acid molecule of claim 33, wherein the ITAM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.
36. The recombinant nucleic acid molecule of any one of claims 1-35, wherein the nucleic acid
comprises a nucleotide analog.
37. The recombinant nucleic acid molecule of claim 36, wherein the nucleotide analog is selected from the group consisting of 2’-0-methyl, 2’-0-methoxyethyl (2’-0-M0E), 2’-0-aminopropyl, 2’-deoxy, T-deoxy-2’-fluoro, 2’-0-aminopropyl (2’-0-AP), 2'-0-dimethylaminoethyl (2’-0-DMA0E), 2 -0- dimethylaminopropyl (2’-0-DMAP), T-O-dimethylaminoethyloxyethyl (2’-0-DMAE0E), 2 -O-N- methylacetamido (2’-0-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a l’,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2’-fluoro N3-P5’- phosphoramidite.
38. The recombinant nucleic acid molecule of any one of claims 1-37, further comprising a leader
sequence.
39. A polypeptide molecule encoded by the nucleic acid molecule of any one of claims 1-38.
40. A recombinant TFP molecule comprising an anti-TAA binding domain, a TCR extracellular
domain, a transmembrane domain, and an intracellular domain.
41. An isolated recombinant TFP molecule comprising an anti-TAA binding domain, a TCR
extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide.
42. An isolated recombinant TFP molecule comprising an anti-TAA binding domain, a TCR
extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally integrating into an endogenous TCR complex.
43. The recombinant TFP molecule of claim 40, comprising an antibody or antibody fragment
comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain.
44. The recombinant TFP molecule of any one of claims 40-43, wherein the anti-TAA binding domain is a VH, VL, SCFV or a VHH domain.
45. The recombinant TFP molecule of any one of claims 40-44, wherein the anti-TAA binding domain comprises a heavy chain with 95-100% identity to an amino acid sequence of an anti-TAA light chain provided herein, a functional fragment thereof, or an amino acid sequence thereof having at
least one but not more than 30 modifications.
46. The recombinant TFP molecule of any one of claims 40-45, wherein the anti-TAA binding domain comprises a light chain with 95-100% identity to an amino acid sequence of an anti-TAA heavy chain provided herein, a functional fragment thereof, or an amino acid sequence thereof having at least one but not more than 30 modifications.
47. The recombinant TFP molecule of any one of claims 40-46, comprising a TCR extracellular domain that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
48. The recombinant TFP molecule of any one of claims 40-47, wherein the TCR comprises an
extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma.
49. The recombinant TFP molecule of any one of claims 40-48, wherein the anti-TAA binding domain is connected to the TCR extracellular domain by a linker sequence.
50. The recombinant TFP molecule of claim 49, wherein the linker region comprises (G4S)n, wherein n=l to 4.
51. The recombinant TFP molecule of any one of claims 40-50, further comprising a sequence encoding a costimulatory domain.
52. The recombinant TFP molecule of any one of claims 40-51, further comprising a sequence encoding an intracellular signaling domain.
53. The recombinant TFP molecule of any one of claims 40-52, further comprising a leader sequence.
54. A nucleic acid comprising a sequence encoding a TFP of any one of claims 40-53.
55. The nucleic acid of claim 54, wherein the nucleic acid is selected from the group consisting of a DNA and a RNA.
56. The nucleic acid of claim 54 or 55, wherein the nucleic acid is a mRNA.
57. The nucleic acid of any one of claims 54-56, wherein the nucleic acid comprises a nucleotide
analog.
58. The nucleic acid of claim 57, wherein the nucleotide analog is selected from the group consisting of 2’-0-methyl, 2’-0-methoxyethyl (2’-0-MOE), 2’-0-aminopropyl, 2’-deoxy, T-deoxy-2’-fluoro, 2’- O-aminopropyl (2’-0-AP), 2'-0-dimethylaminoethyl (2’-0-DMAOE), 2’-0-dimethylaminopropyl (2’-0-DMAP), T-O-dimethylaminoethyloxyethyl (2’-0-DMAEOE), 2’-0-N-methylacetamido (2’- O-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a G,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2’-fluoro N3-P5’-phosphoramidite.
59. The nucleic acid of any one of claims 54-58, further comprising a promoter.
60. The nucleic acid of any one of claims 54-59, wherein the nucleic acid is an in vitro transcribed nucleic acid.
61. The nucleic acid of any one of claims 54-60, wherein the nucleic acid further comprises a sequence encoding a poly (A) tail.
62. The nucleic acid of any one of claims 54-61, wherein the nucleic acid further comprises a 3’UTR sequence.
63. A vector comprising a nucleic acid molecule encoding a TFP of any one of claims 40-53.
64. The vector of claim 63, wherein the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, a Rous sarcoma viral (RSV) vector, or a retrovirus vector.
65. The vector of claim 63 or 64, further comprising a promoter.
66. The vector of any one of claims 63-65, wherein the vector is an in vitro transcribed vector.
67. The vector of any one of claims 63-66, wherein a nucleic acid sequence in the vector further
comprises a poly(A) tail.
68. The vector of any one of claims 63-67, wherein a nucleic acid sequence in the vector further
comprises a 3’UTR.
69. A cell comprising the isolated nucleic acid molecule of any one of claims 1-38, the polypeptide molecule of claim 39, the TFP molecule of any one of claims 40-53, the nucleic acid of any one of claims 54-62, the vector of any one of claims 63-68.
70. The cell of claim 69, wherein the cell is a human T cell.
71. The cell of claim 70, wherein the T cell is a CD8+ or CD4+ T cell.
72. The cell of any one of claims 69-71, further comprising a nucleic acid encoding an inhibitory
molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain.
73. The cell of claim 72, wherein the inhibitory molecule comprises a first polypeptide that comprises at least a portion of PD 1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.
74. A human CD8+ or CD4+ T cell comprising at least two TFP molecules, the TFP molecules
comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+ or CD4+ T cell.
75. A protein complex comprising:
i) a TFP molecule comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and
ii) at least one endogenous TCR subunit or endogenous TCR complex.
76. The protein complex of claim 75, wherein the TCR comprises an extracellular domain or portion thereof of a protein selected from the group consisting of TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, and a CD3 delta TCR subunit.
77. The protein complex of claim 75 or 76, wherein the anti-TAA binding domain is connected to the TCR extracellular domain by a linker sequence.
78. The protein complex of claim 77, wherein the linker region comprises (G4S)n, wherein n=l to 4.
79. A protein complex comprising
(a) a TFP encoded by the isolated nucleic acid molecule of any one of claims 1-38, and
(b) at least one endogenous TCR subunit or endogenous TCR complex.
80. A human CD8+, CD4+ or CD4+CD8+ T cell comprising at least two different TFP proteins per the protein complex of any one of claims 75-79.
81. A human CD8+, CD4+ or CD4+CD8+ T cell comprising at least two different TFP molecules encoded by the isolated nucleic acid molecule of any one of claims 1-38.
82. A population of human CD8+, CD4+ or CD4+CD8+ T cells, wherein the T cells of the population individually or collectively comprise at least two TFP molecules, the TFP molecules comprising an anti-TAA binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+, CD4+ or CD4+CD8+ T cell.
83. A population of human CD8+, CD4+ or CD4+CD8+ T cells, wherein the T cells of the population individually or collectively comprise at least two TFP molecules encoded by the isolated nucleic acid molecule of any one of claims 1-38.
84. A method of making a cell comprising transducing a T cell with the isolated nucleic acid molecule of any one of claims 1-38, the nucleic acid of any one of claims 54-62, or the vector of any one of claims 63-68.
85. A method of generating a population of RNA-engineered cells comprising introducing an in vitro transcribed RNA, a circular RNA, or a synthetic RNA into a cell, where the RNA comprises a nucleic acid encoding the TFP molecule of any one of claims 40-53.
86. A method of providing an anti-tumor immunity in a mammal comprising administering to the
mammal an effective amount of the isolated nucleic acid molecule of any one of claims 1-38, the polypeptide molecule of claim 39, a cell expressing the polypeptide molecule of claim 39, the TFP molecule of any one of claims 40-53, the nucleic acid of any one of claims 54-62, the vector of any one of claims 63-68, or the cell of any one of claims 69-74 and 80-84.
87. The method of claim 86, wherein the cell is an autologous T cell.
88. The method of claim 86, wherein the cell is an allogeneic T cell.
89. The method of any one of claims 86-88, wherein the mammal is a human.
90. A method of treating a mammal having a disease associated with expression of TAA comprising administering to the mammal an effective amount of the isolated nucleic acid molecule of any one of claims 1-38, the polypeptide molecule of claim 39, a cell expressing the polypeptide molecule of claim 39, the TFP molecule of any one of claims 40-53, the nucleic acid of any one of claims 54-62, the vector of any one of claims 63-68, or the cell of any one of claims 69-74 and 80-84.
91. The method of claim 90, wherein the disease associated with TAA expression is selected from the group consisting of a proliferative disease, a cancer, a malignancy, and a non-cancer related indication associated with expression of TAA.
92. The method of claim 90, wherein the disease is a cancer selected from the group consisting of
mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, thyroid cancer, bladder cancer, ureter cancer, kidney cancer, endometrial cancer, esophageal cancer, gastric cancer, thymic carcinoma, cholangiocarcinoma and stomach cancer.
93. The method of claim 90, wherein the disease is a cancer selected from the group consisting of
mesothelioma, papillary serous ovarian adenocarcinoma, clear cell ovarian carcinoma, mixed Mullerian ovarian carcinoma, endometroid mucinous ovarian carcinoma, pancreatic
adenocarcinoma, ductal pancreatic adenocarcinoma, uterine serous carcinoma, lung
adenocarcinoma, extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma, breast adenocarcinoma, a disease associated with TAA expression, and combinations thereof.
94. The method of claim 90, wherein the cells expressing a TFP molecule are administered in
combination with an agent that increases the efficacy of a cell expressing a TFP molecule.
95. The method of any one of claims 90-94, wherein less cytokines are released in the mammal
compared a mammal administered an effective amount of a T cell expressing an anti-TAA chimeric antigen receptor (CAR).
96. The method of any one of claims 90-95, wherein the cells expressing a TFP molecule are
administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a TFP molecule.
97. The method of any one of claims 90-96, wherein the cells expressing a TFP molecule are
administered in combination with an agent that treats the disease associated with the antigen.
98. The method of claim 97, wherein the antigen is a tumor-associated antigen.
99. The method of claim 98, wherein the antigen is one or more antigens selected from CD19, B-cell maturation antigen (BCMA), mesothelin (MSLN), ILl3Ra2, MUC16, CD22, PD-l, BAFF or BAFF receptor, and ROR-l.
100. The isolated nucleic acid molecule of any one of claims 1-38, the isolated polypeptide molecule of claim 39, a cell expressing the polypeptide molecule of claim 39, the isolated TFP of any one of claims 40-53, the nucleic acid of any one of claims 54-62, the vector of any one claims 63-68, the complex of any one of claims 75-79, or the cell of any one of claims 69-74 and 80-84, for use as a medicament.
101. A method of treating a mammal having a disease associated with expression of TAA comprising administering to the mammal an effective amount of the isolated nucleic acid molecule of any one of claims 1-38, the polypeptide molecule of claim 39, a cell expressing the polypeptide molecule of claim 39, the TFP molecule of any one of claims 40-53, the nucleic acid of any one of claims 54-62,
the vector of any one of claims 63-68, or the cell of any one of claims 69 '-74 and 80-84, wherein less cytokines are released in the mammal compared a mammal administered an effective amount of a T cell expressing an anti-TAA chimeric antigen receptor (CAR).
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201862671342P | 2018-05-14 | 2018-05-14 | |
US62/671,342 | 2018-05-14 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2019222275A2 true WO2019222275A2 (en) | 2019-11-21 |
WO2019222275A3 WO2019222275A3 (en) | 2020-08-20 |
Family
ID=68540969
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2019/032298 WO2019222275A2 (en) | 2018-05-14 | 2019-05-14 | Compositions and methods for tcr reprogramming using inducible fusion proteins |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2019222275A2 (en) |
Cited By (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020219563A1 (en) * | 2019-04-22 | 2020-10-29 | TCR2 Therapeutics Inc. | Compositions and methods for tcr reprogramming using fusion proteins |
WO2020237227A1 (en) * | 2019-05-22 | 2020-11-26 | Massachusetts Institute Of Technology | Circular rna compositions and methods |
WO2020252436A1 (en) * | 2019-06-14 | 2020-12-17 | Flagship Pioneering Innovations Vi, Llc | Circular rnas for cellular therapy |
US11028142B2 (en) | 2015-05-18 | 2021-06-08 | TCR2 Therapeutics Inc. | Compositions and methods for TCR reprogramming using fusion proteins |
US11085021B2 (en) | 2016-10-07 | 2021-08-10 | TCR2 Therapeutics Inc. | Compositions and methods for TCR reprogramming using fusion proteins |
WO2021189059A3 (en) * | 2020-03-20 | 2021-11-11 | Orna Therapeutics, Inc. | Circular rna compositions and methods |
US11203767B2 (en) | 2018-06-06 | 2021-12-21 | Massachusetts Institute Of Technology | Circular RNA for translation in eukaryotic cells |
US11242376B2 (en) | 2016-08-02 | 2022-02-08 | TCR2 Therapeutics Inc. | Compositions and methods for TCR reprogramming using fusion proteins |
WO2022056304A1 (en) * | 2020-09-10 | 2022-03-17 | TCR2 Therapeutics Inc. | Compositions and methods for tcr reprogramming using nectin-4 specific fusion proteins |
WO2022056321A1 (en) * | 2020-09-10 | 2022-03-17 | TCR2 Therapeutics Inc. | Compositions and methods for tcr reprogramming using gpc3 specific fusion proteins |
WO2022140388A1 (en) * | 2020-12-21 | 2022-06-30 | Allogene Therapeutics, Inc. | Protease-activating cd45-gate car |
WO2022187591A1 (en) | 2021-03-05 | 2022-09-09 | Go Therapeutics, Inc. | Anti-glyco-cd44 antibodies and their uses |
EP4130028A1 (en) | 2021-08-03 | 2023-02-08 | Rhazes Therapeutics Ltd | Engineered tcr complex and methods of using same |
WO2023012584A2 (en) | 2021-08-03 | 2023-02-09 | Genicity Limited | Engineered tcr complex and methods of using same |
US11679120B2 (en) | 2019-12-04 | 2023-06-20 | Orna Therapeutics, Inc. | Circular RNA compositions and methods |
WO2023023150A3 (en) * | 2021-08-18 | 2023-08-31 | Hifibio (Hk) Limited | Methods and compositions related to neutralizing antibodies against human coronavirus |
WO2023172967A3 (en) * | 2022-03-09 | 2023-11-02 | TCR2 Therapeutics Inc. | Compositions and methods for tcr reprogramming using gpc3 specific fusion proteins |
US11851491B2 (en) | 2016-11-22 | 2023-12-26 | TCR2 Therapeutics Inc. | Compositions and methods for TCR reprogramming using fusion proteins |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA3214529A1 (en) * | 2013-09-25 | 2015-04-02 | Cytomx Therapeutics, Inc. | Matrix metalloproteinase substrates and other cleavable moieties and methods of use thereof |
CN106519037B (en) * | 2015-09-11 | 2019-07-23 | 科济生物医药(上海)有限公司 | Activable Chimerical receptor |
SG11201808085WA (en) * | 2016-03-22 | 2018-10-30 | Hoffmann La Roche | Protease-activated t cell bispecific molecules |
IL302917A (en) * | 2016-10-07 | 2023-07-01 | Tcr2 Therapeutics Inc | Compositions and methods for t-cell receptors reprogramming using fusion proteins |
-
2019
- 2019-05-14 WO PCT/US2019/032298 patent/WO2019222275A2/en active Application Filing
Cited By (28)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11965012B2 (en) | 2015-05-18 | 2024-04-23 | TCR2 Therapeutics Inc. | Compositions and methods for TCR reprogramming using fusion proteins |
US11028142B2 (en) | 2015-05-18 | 2021-06-08 | TCR2 Therapeutics Inc. | Compositions and methods for TCR reprogramming using fusion proteins |
US11242376B2 (en) | 2016-08-02 | 2022-02-08 | TCR2 Therapeutics Inc. | Compositions and methods for TCR reprogramming using fusion proteins |
US11377638B2 (en) | 2016-10-07 | 2022-07-05 | TCR2 Therapeutics Inc. | Compositions and methods for TCR reprogramming using fusion proteins |
US11085021B2 (en) | 2016-10-07 | 2021-08-10 | TCR2 Therapeutics Inc. | Compositions and methods for TCR reprogramming using fusion proteins |
US11851491B2 (en) | 2016-11-22 | 2023-12-26 | TCR2 Therapeutics Inc. | Compositions and methods for TCR reprogramming using fusion proteins |
US11203767B2 (en) | 2018-06-06 | 2021-12-21 | Massachusetts Institute Of Technology | Circular RNA for translation in eukaryotic cells |
US11845950B2 (en) | 2018-06-06 | 2023-12-19 | Massachusetts Institute Of Technology | Circular RNA for translation in eukaryotic cells |
US11352640B2 (en) | 2018-06-06 | 2022-06-07 | Massachusetts Institute Of Technology | Circular RNA for translation in eukaryotic cells |
US11352641B2 (en) | 2018-06-06 | 2022-06-07 | Massachusetts Institute Of Technology | Circular RNA for translation in eukaryotic cells |
US11447796B2 (en) | 2018-06-06 | 2022-09-20 | Massachusetts Institute Of Technology | Circular RNA for translation in eukaryotic cells |
WO2020219563A1 (en) * | 2019-04-22 | 2020-10-29 | TCR2 Therapeutics Inc. | Compositions and methods for tcr reprogramming using fusion proteins |
US11603396B2 (en) | 2019-05-22 | 2023-03-14 | Orna Therapeutics, Inc. | Circular RNA compositions and methods |
US11802144B2 (en) | 2019-05-22 | 2023-10-31 | Orna Therapeutics, Inc. | Circular RNA compositions and methods |
WO2020237227A1 (en) * | 2019-05-22 | 2020-11-26 | Massachusetts Institute Of Technology | Circular rna compositions and methods |
WO2020252436A1 (en) * | 2019-06-14 | 2020-12-17 | Flagship Pioneering Innovations Vi, Llc | Circular rnas for cellular therapy |
US11766449B2 (en) | 2019-12-04 | 2023-09-26 | Orna Therapeutics, Inc. | Circular RNA compositions and methods |
US11679120B2 (en) | 2019-12-04 | 2023-06-20 | Orna Therapeutics, Inc. | Circular RNA compositions and methods |
US11771715B2 (en) | 2019-12-04 | 2023-10-03 | Orna Therapeutics, Inc. | Circular RNA compositions and methods |
WO2021189059A3 (en) * | 2020-03-20 | 2021-11-11 | Orna Therapeutics, Inc. | Circular rna compositions and methods |
WO2022056321A1 (en) * | 2020-09-10 | 2022-03-17 | TCR2 Therapeutics Inc. | Compositions and methods for tcr reprogramming using gpc3 specific fusion proteins |
WO2022056304A1 (en) * | 2020-09-10 | 2022-03-17 | TCR2 Therapeutics Inc. | Compositions and methods for tcr reprogramming using nectin-4 specific fusion proteins |
WO2022140388A1 (en) * | 2020-12-21 | 2022-06-30 | Allogene Therapeutics, Inc. | Protease-activating cd45-gate car |
WO2022187591A1 (en) | 2021-03-05 | 2022-09-09 | Go Therapeutics, Inc. | Anti-glyco-cd44 antibodies and their uses |
WO2023012584A2 (en) | 2021-08-03 | 2023-02-09 | Genicity Limited | Engineered tcr complex and methods of using same |
EP4130028A1 (en) | 2021-08-03 | 2023-02-08 | Rhazes Therapeutics Ltd | Engineered tcr complex and methods of using same |
WO2023023150A3 (en) * | 2021-08-18 | 2023-08-31 | Hifibio (Hk) Limited | Methods and compositions related to neutralizing antibodies against human coronavirus |
WO2023172967A3 (en) * | 2022-03-09 | 2023-11-02 | TCR2 Therapeutics Inc. | Compositions and methods for tcr reprogramming using gpc3 specific fusion proteins |
Also Published As
Publication number | Publication date |
---|---|
WO2019222275A3 (en) | 2020-08-20 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11851491B2 (en) | Compositions and methods for TCR reprogramming using fusion proteins | |
WO2019222275A2 (en) | Compositions and methods for tcr reprogramming using inducible fusion proteins | |
JP7262535B2 (en) | Compositions and methods for TCR reprogramming using fusion proteins | |
US11377638B2 (en) | Compositions and methods for TCR reprogramming using fusion proteins | |
US11242376B2 (en) | Compositions and methods for TCR reprogramming using fusion proteins | |
US20220362295A1 (en) | Compositions and methods for tcr reprogramming using fusion proteins | |
US20200115461A1 (en) | Compositions and methods for adoptive cell therapies | |
US20210187022A1 (en) | Engineered t cells for the treatment of cancer | |
US20210253666A1 (en) | Compositions and methods for tcr reprogramming using fusion proteins | |
WO2021035170A1 (en) | Compositions and methods for tcr reprogramming using fusion proteins | |
WO2022056321A1 (en) | Compositions and methods for tcr reprogramming using gpc3 specific fusion proteins | |
WO2022056304A1 (en) | Compositions and methods for tcr reprogramming using nectin-4 specific fusion proteins | |
WO2023086379A2 (en) | Compositions and methods for tcr reprogramming using fusion proteins | |
WO2023172967A2 (en) | Compositions and methods for tcr reprogramming using gpc3 specific fusion proteins | |
EA043737B1 (en) | COMPOSITIONS AND METHODS FOR REPROGRAMMING T-CELL RECEPTORS USING HYBRID PROTEINS |
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: 19803199 Country of ref document: EP Kind code of ref document: A2 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 19803199 Country of ref document: EP Kind code of ref document: A2 |