US20220296690A1 - Engineered t cells and uses therefor - Google Patents
Engineered t cells and uses therefor Download PDFInfo
- Publication number
- US20220296690A1 US20220296690A1 US17/664,731 US202217664731A US2022296690A1 US 20220296690 A1 US20220296690 A1 US 20220296690A1 US 202217664731 A US202217664731 A US 202217664731A US 2022296690 A1 US2022296690 A1 US 2022296690A1
- Authority
- US
- United States
- Prior art keywords
- cell
- cells
- domain
- modified
- hngal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000001744 T-lymphocyte Anatomy 0.000 title claims abstract description 137
- 102000019298 Lipocalin Human genes 0.000 claims abstract description 23
- 108050006654 Lipocalin Proteins 0.000 claims abstract description 23
- 230000000139 costimulatory effect Effects 0.000 claims abstract description 9
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims description 86
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 31
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 29
- 229920001184 polypeptide Polymers 0.000 claims description 28
- 101001023833 Homo sapiens Neutrophil gelatinase-associated lipocalin Proteins 0.000 claims description 27
- 230000004913 activation Effects 0.000 claims description 15
- 230000004068 intracellular signaling Effects 0.000 claims description 12
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 claims description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 10
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 claims description 8
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 claims description 8
- 230000011664 signaling Effects 0.000 claims description 7
- 102100027207 CD27 antigen Human genes 0.000 claims description 4
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 claims description 4
- 239000003937 drug carrier Substances 0.000 claims description 4
- 101150013553 CD40 gene Proteins 0.000 claims description 3
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 claims description 3
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 claims description 3
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 claims description 3
- 150000001413 amino acids Chemical class 0.000 claims description 3
- 239000008194 pharmaceutical composition Substances 0.000 claims description 3
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 3
- 230000003248 secreting effect Effects 0.000 claims description 3
- 230000009870 specific binding Effects 0.000 claims description 3
- 102000047202 human LCN2 Human genes 0.000 claims 26
- 238000006467 substitution reaction Methods 0.000 claims 4
- 125000000539 amino acid group Chemical group 0.000 claims 2
- 230000001086 cytosolic effect Effects 0.000 claims 2
- 108020001756 ligand binding domains Proteins 0.000 claims 2
- 101000946124 Homo sapiens Lipocalin-1 Proteins 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 19
- 230000028327 secretion Effects 0.000 abstract description 18
- 238000000034 method Methods 0.000 abstract description 17
- 102000004169 proteins and genes Human genes 0.000 abstract description 13
- 210000004881 tumor cell Anatomy 0.000 abstract description 12
- 230000008901 benefit Effects 0.000 abstract description 10
- 239000000427 antigen Substances 0.000 abstract description 8
- 102000036639 antigens Human genes 0.000 abstract description 8
- 108091007433 antigens Proteins 0.000 abstract description 8
- 230000001225 therapeutic effect Effects 0.000 abstract description 7
- 230000002519 immonomodulatory effect Effects 0.000 abstract description 6
- 102000037982 Immune checkpoint proteins Human genes 0.000 abstract description 4
- 108091008036 Immune checkpoint proteins Proteins 0.000 abstract description 4
- 239000012634 fragment Substances 0.000 abstract description 3
- 239000012528 membrane Substances 0.000 abstract description 3
- 108010083359 Antigen Receptors Proteins 0.000 abstract description 2
- 102000006306 Antigen Receptors Human genes 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 102
- 229940045513 CTLA4 antagonist Drugs 0.000 description 37
- 206010028980 Neoplasm Diseases 0.000 description 23
- 230000014509 gene expression Effects 0.000 description 21
- 238000013459 approach Methods 0.000 description 18
- 239000006228 supernatant Substances 0.000 description 18
- 238000002965 ELISA Methods 0.000 description 15
- 230000001177 retroviral effect Effects 0.000 description 15
- 238000002474 experimental method Methods 0.000 description 14
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 12
- 238000012512 characterization method Methods 0.000 description 12
- 210000005259 peripheral blood Anatomy 0.000 description 12
- 239000011886 peripheral blood Substances 0.000 description 12
- 238000010361 transduction Methods 0.000 description 12
- 239000013598 vector Substances 0.000 description 12
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 11
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 11
- 102000004127 Cytokines Human genes 0.000 description 11
- 108090000695 Cytokines Proteins 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 10
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 10
- 230000006044 T cell activation Effects 0.000 description 10
- 230000027455 binding Effects 0.000 description 10
- 230000003013 cytotoxicity Effects 0.000 description 10
- 231100000135 cytotoxicity Toxicity 0.000 description 10
- 238000010186 staining Methods 0.000 description 10
- 230000026683 transduction Effects 0.000 description 10
- 102100037850 Interferon gamma Human genes 0.000 description 9
- 108010074328 Interferon-gamma Proteins 0.000 description 9
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 8
- 241000283973 Oryctolagus cuniculus Species 0.000 description 8
- 239000012228 culture supernatant Substances 0.000 description 8
- 238000010166 immunofluorescence Methods 0.000 description 8
- 230000001939 inductive effect Effects 0.000 description 8
- 102000005962 receptors Human genes 0.000 description 8
- 108020003175 receptors Proteins 0.000 description 8
- 108010065805 Interleukin-12 Proteins 0.000 description 7
- 102000013462 Interleukin-12 Human genes 0.000 description 7
- 108010002350 Interleukin-2 Proteins 0.000 description 7
- 229960000074 biopharmaceutical Drugs 0.000 description 7
- 238000013461 design Methods 0.000 description 7
- 230000004927 fusion Effects 0.000 description 7
- 239000013642 negative control Substances 0.000 description 7
- 239000002245 particle Substances 0.000 description 7
- 102100032530 Glypican-3 Human genes 0.000 description 6
- 101001014668 Homo sapiens Glypican-3 Proteins 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 210000004986 primary T-cell Anatomy 0.000 description 6
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 5
- 239000000556 agonist Substances 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 5
- 108091008915 immune receptors Proteins 0.000 description 5
- 102000027596 immune receptors Human genes 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- 210000004962 mammalian cell Anatomy 0.000 description 5
- 210000000822 natural killer cell Anatomy 0.000 description 5
- 230000000770 proinflammatory effect Effects 0.000 description 5
- 241001430294 unidentified retrovirus Species 0.000 description 5
- 239000003981 vehicle Substances 0.000 description 5
- 102000003752 Lipocalin 1 Human genes 0.000 description 4
- 108010057281 Lipocalin 1 Proteins 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 4
- 239000005557 antagonist Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 4
- 238000000099 in vitro assay Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 230000000670 limiting effect Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 4
- 230000009257 reactivity Effects 0.000 description 4
- 230000002269 spontaneous effect Effects 0.000 description 4
- 230000004936 stimulating effect Effects 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 3
- -1 CD44v7/8 Proteins 0.000 description 3
- 241000283707 Capra Species 0.000 description 3
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 3
- 102100035405 Neutrophil gelatinase-associated lipocalin Human genes 0.000 description 3
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 3
- 230000001270 agonistic effect Effects 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 3
- 108700004026 gag Genes Proteins 0.000 description 3
- 102000043321 human CTLA4 Human genes 0.000 description 3
- 230000003308 immunostimulating effect Effects 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 238000005462 in vivo assay Methods 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 108700004029 pol Genes Proteins 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 3
- 229930182490 saponin Natural products 0.000 description 3
- 150000007949 saponins Chemical class 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 108010078373 tisagenlecleucel Proteins 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 2
- 102100034608 Angiopoietin-2 Human genes 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 108700022150 Designed Ankyrin Repeat Proteins Proteins 0.000 description 2
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 2
- 241000713813 Gibbon ape leukemia virus Species 0.000 description 2
- 101000924533 Homo sapiens Angiopoietin-2 Proteins 0.000 description 2
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 description 2
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 description 2
- 102000014158 Interleukin-12 Subunit p40 Human genes 0.000 description 2
- 108010011429 Interleukin-12 Subunit p40 Proteins 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 108010051335 Lipocalin-2 Proteins 0.000 description 2
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 2
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 description 2
- 102000009524 Vascular Endothelial Growth Factor A Human genes 0.000 description 2
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 2
- 230000003042 antagnostic effect Effects 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- 108091008324 binding proteins Proteins 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 230000022534 cell killing Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000000975 co-precipitation Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 108091008034 costimulatory receptors Proteins 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 239000006223 plastic coating Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 230000002463 transducing effect Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- 230000004614 tumor growth Effects 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 1
- 102100029822 B- and T-lymphocyte attenuator Human genes 0.000 description 1
- 208000010839 B-cell chronic lymphocytic leukemia Diseases 0.000 description 1
- 102100038080 B-cell receptor CD22 Human genes 0.000 description 1
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 1
- 108010074708 B7-H1 Antigen Proteins 0.000 description 1
- 108700012439 CA9 Proteins 0.000 description 1
- 102100024263 CD160 antigen Human genes 0.000 description 1
- 102100038077 CD226 antigen Human genes 0.000 description 1
- 102100024423 Carbonic anhydrase 9 Human genes 0.000 description 1
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 1
- 102000005598 Chondroitin Sulfate Proteoglycans Human genes 0.000 description 1
- 108010059480 Chondroitin Sulfate Proteoglycans Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 101150076616 EPHA2 gene Proteins 0.000 description 1
- 101150029707 ERBB2 gene Proteins 0.000 description 1
- 102100030340 Ephrin type-A receptor 2 Human genes 0.000 description 1
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 description 1
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 description 1
- 102100031940 Epithelial cell adhesion molecule Human genes 0.000 description 1
- 241001663880 Gammaretrovirus Species 0.000 description 1
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 description 1
- 102100034458 Hepatitis A virus cellular receptor 2 Human genes 0.000 description 1
- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 description 1
- 101000864344 Homo sapiens B- and T-lymphocyte attenuator Proteins 0.000 description 1
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 description 1
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 1
- 101000761938 Homo sapiens CD160 antigen Proteins 0.000 description 1
- 101000884298 Homo sapiens CD226 antigen Proteins 0.000 description 1
- 101000920667 Homo sapiens Epithelial cell adhesion molecule Proteins 0.000 description 1
- 101100066427 Homo sapiens FCGR1A gene Proteins 0.000 description 1
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 description 1
- 101001068133 Homo sapiens Hepatitis A virus cellular receptor 2 Proteins 0.000 description 1
- 101001138062 Homo sapiens Leukocyte-associated immunoglobulin-like receptor 1 Proteins 0.000 description 1
- 101001137987 Homo sapiens Lymphocyte activation gene 3 protein Proteins 0.000 description 1
- 101001133056 Homo sapiens Mucin-1 Proteins 0.000 description 1
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 1
- 101001109501 Homo sapiens NKG2-D type II integral membrane protein Proteins 0.000 description 1
- 101001136592 Homo sapiens Prostate stem cell antigen Proteins 0.000 description 1
- 101000831007 Homo sapiens T-cell immunoreceptor with Ig and ITIM domains Proteins 0.000 description 1
- 101000934346 Homo sapiens T-cell surface antigen CD2 Proteins 0.000 description 1
- 101000801234 Homo sapiens Tumor necrosis factor receptor superfamily member 18 Proteins 0.000 description 1
- 101000679903 Homo sapiens Tumor necrosis factor receptor superfamily member 25 Proteins 0.000 description 1
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 1
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 1
- 102100029567 Immunoglobulin kappa light chain Human genes 0.000 description 1
- 101710189008 Immunoglobulin kappa light chain Proteins 0.000 description 1
- 102000014154 Interleukin-12 Subunit p35 Human genes 0.000 description 1
- 108010011301 Interleukin-12 Subunit p35 Proteins 0.000 description 1
- 108090000172 Interleukin-15 Proteins 0.000 description 1
- 102000003812 Interleukin-15 Human genes 0.000 description 1
- 102000003810 Interleukin-18 Human genes 0.000 description 1
- 108090000171 Interleukin-18 Proteins 0.000 description 1
- 108010065637 Interleukin-23 Proteins 0.000 description 1
- 102000013264 Interleukin-23 Human genes 0.000 description 1
- 108010066979 Interleukin-27 Proteins 0.000 description 1
- 102000017578 LAG3 Human genes 0.000 description 1
- 102100020943 Leukocyte-associated immunoglobulin-like receptor 1 Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108010061593 Member 14 Tumor Necrosis Factor Receptors Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000003735 Mesothelin Human genes 0.000 description 1
- 108090000015 Mesothelin Proteins 0.000 description 1
- 102100034256 Mucin-1 Human genes 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 101000859077 Mus musculus Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 1
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 1
- 102100031789 Myeloid-derived growth factor Human genes 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 102100022680 NKG2-D type II integral membrane protein Human genes 0.000 description 1
- 108010012255 Neural Cell Adhesion Molecule L1 Proteins 0.000 description 1
- 102100024964 Neural cell adhesion molecule L1 Human genes 0.000 description 1
- 240000007019 Oxalis corniculata Species 0.000 description 1
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 1
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 description 1
- 102100036735 Prostate stem cell antigen Human genes 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 230000010799 Receptor Interactions Effects 0.000 description 1
- 108010074687 Signaling Lymphocytic Activation Molecule Family Member 1 Proteins 0.000 description 1
- 102100029215 Signaling lymphocytic activation molecule Human genes 0.000 description 1
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 1
- 108091008874 T cell receptors Proteins 0.000 description 1
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 1
- 102100024834 T-cell immunoreceptor with Ig and ITIM domains Human genes 0.000 description 1
- 102100025237 T-cell surface antigen CD2 Human genes 0.000 description 1
- 101150056647 TNFRSF4 gene Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 108010028230 Trp-Ser- His-Pro-Gln-Phe-Glu-Lys Proteins 0.000 description 1
- 108060008683 Tumor Necrosis Factor Receptor Proteins 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
- 102100022203 Tumor necrosis factor receptor superfamily member 25 Human genes 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000008484 agonism Effects 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 108091008033 coinhibitory receptors Proteins 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 210000005220 cytoplasmic tail Anatomy 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 230000007783 downstream signaling Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 108700004025 env Genes Proteins 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
- 230000001747 exhibiting effect Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 239000012997 ficoll-paque Substances 0.000 description 1
- 229940014144 folate Drugs 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 229940117681 interleukin-12 Drugs 0.000 description 1
- 238000010212 intracellular staining Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000010172 mouse model Methods 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
- 238000005457 optimization Methods 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 239000008191 permeabilizing agent Substances 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 229920001481 poly(stearyl methacrylate) Polymers 0.000 description 1
- 230000004492 positive regulation of T cell proliferation Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000000018 receptor agonist Substances 0.000 description 1
- 229940044601 receptor agonist Drugs 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 231100000057 systemic toxicity Toxicity 0.000 description 1
- 101150047061 tag-72 gene Proteins 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 231100000440 toxicity profile Toxicity 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 1
Images
Classifications
-
- 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
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
-
- 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
- A61K39/463—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
- A61K39/4636—Immune checkpoint inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464402—Receptors, cell surface antigens or cell surface determinants
- A61K39/464403—Receptors for growth factors
- A61K39/46441—Hepatocyte growth factor receptor [HGFR or c-met]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464402—Receptors, cell surface antigens or cell surface determinants
- A61K39/464416—Receptors for cytokines
- A61K39/464417—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
-
- 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
- A61K39/464452—Transcription factors, e.g. SOX or c-MYC
-
- 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
- A61K39/464474—Proteoglycans, e.g. glypican, brevican or CSPG4
-
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/5434—IL-12
-
- 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
-
- 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
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70521—CD28, CD152
-
- 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
- C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5156—Animal cells expressing foreign proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
-
- 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
- C07K2318/00—Antibody mimetics or scaffolds
- C07K2318/20—Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- 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
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/22—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a Strep-tag
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
-
- 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
- C12N2510/00—Genetically modified cells
Definitions
- CARs chimeric antigen receptors
- CLL chronic lymphocytic leukemia
- CARs are transmembrane proteins stably anchored in the T-cell plasma membrane.
- a CAR is an artificial fusion of multiple parts: the extracellular part consists of a recognition domain binding to a cancer cell target; the intracellular part contains the signaling domain(s) of one or more (co-)stimulatory immunoreceptors; and the two are fused via a linker- and transmembrane domain.
- the recognition domain is an antibody single chain fragment (scFv) specific for CD19
- the linker- and transmembrane regions are grafted from the membrane protein CD8, and the intracellular signaling part consists of the complete intracellular domains of CD137 and CD3zeta fused in tandem.
- CTL019 is an example for a “second generation” CAR, identified by the presence of two immunostimulatory domains.
- “first generation” CARs contain only a single immunostimulatory domain—usually that of CD3zeta—while in “third generation” CARs, overall three intracellular immunostimulatory domains are fused in tandem, for example those of CD3zeta, CD28 and CD137.
- First generation CARs have failed to show efficacy in the clinic—potentially due to the lack of activity and long-term persistence of the CAR-T cells in patients (Kershaw et al. 2006)—while third generation CARs have yet to show their utility in a clinical setting.
- the extracellular recognition domains of CARs are almost exclusively scFv's (with the few exceptions instead utilizing the extracellular domains of ligands or receptors), which is due to the fact that a single-chain functional protein (or monomer) is required to facilitate a straightforward fusion design. Accordingly, a tetrameric mAb or even a dimeric Fab would present severe challenges as an extracellular recognition domain.
- a mutein of a lipocalin that binds to a desired tumor target is an example of a suitable alternative.
- a lipocalin mutein, or Anticalin® is an engineered binding protein based on a lipocalin (preferably of human origin) such as Tear Lipocalin (TLc) or NGAL.
- TLc Tear Lipocalin
- an Anticalin is a single-domain protein; and unlike an scFv, an Anticalin is single-domain by nature, therefore per se a well-behaved monomer from a biophysical property standpoint.
- Anticalins provide tunable affinities from nanomolar to low picomolar range, while having a demonstrated ability to engage targets across a broad spectrum, including immune checkpoints.
- Another application of a CAR-T involves engineering these cells with the capacity to secrete proinflammatory cytokines.
- the engineered CAR-T can lead to accumulation of a proinflammatory cytokine in the tumor micronenvironment where the CAR-T traffics.
- a proinflammatory cytokine may be desired to facilitate recruiting a second wave of immune cells in a locally restricted fashion to initiate a more complete and potentially target-independent attack of the cells of the tumor.
- This approach as recently reviewed (Chmielewski et al. 2014), has been described in particular utilizing an engineered single-chain variant of Interleukin 12, thereafter called scIL-12.
- secretion of scIL-12 can either be constitutive or be coupled to activation of the T-cell via the CAR.
- TRUCK T-cells redirected for universal cytokine killing
- TRUCK-like TRUCK-like approach with constitutive scIL-12 expression has already been brought to the clinic in two separate trials, which currently are suspended (NCT01236573) or terminated (NCT01457131), respectively. Potentially, constitutive expression of scIL-12 was not sufficiently localized to lead to the desired effects, or led to intolerable toxicity.
- CAR-T cells have been transduced with the scIL-12 construct expressed under control of the nuclear-factor of the activated T-cell (NFAT)-derived minimal promoter to initiate IL-12 transcription upon signaling of the TCR/CD3 pathway (Chmielewski et al. 2011b; Zhang et al. 2011).
- NFAT activated T-cell
- Other promoters sensitive to signals of the same pathway like the IL-2 promoter (Jaalouk et al. 2006) may also be feasible and could allow tuning the produced amount and the selectivity of cytokine production.
- cytokines such as IL15, IL-18, IL-23, IL-27 and IFN-gamma, and other producer cells, such as NK cells, have been proposed for the approach. (Chmielewski et al. 2014).
- T-Cell The ability to engineer a T-Cell to secrete yet other types of therapeutic proteins upon T-Cell activation would be desirable, further expanding the repertoire to therapeutic modalities available in the tumor microenvironment.
- Particularly desirable would be specific binding proteins, e.g. a monoclonal antibody, that could bind to an immunomodulatory target, such as an immune checkpoint like CTLA4 or PD1, or to a costimulatory target such as CD137 or OX40.
- an antibody There are at least two drawbacks to using an antibody in this setting, however.
- a smaller, more stable monomeric polypeptide having a shorter half-life could overcome these two shortcomings of antibodies.
- An example for such a molecule would be a mutein of a lipocalin that is specific for an immunomodulatory target and is monomeric, stable and has a half-life tunable from hours to days in man and a molecular weight as low as 18 kilodalton.
- the disclosure provides a CAR that contains a polypeptide defining a naturally occurring monomeric motif (e.g. a lipocalin mutein) specific to a predetermined antigen (e.g., a target differentially expressed on the surface of a tumor cell) that is transduced into a T cell membrane to serve as an antigen receptor, offering benefits over conventionally deployed antibody-derived protein moieties such as a single chain variable fragment (scFv). Benefits include a more stable structure, leading to superior target engagement, for example.
- a naturally occurring monomeric motif e.g. a lipocalin mutein
- a predetermined antigen e.g., a target differentially expressed on the surface of a tumor cell
- scFv single chain variable fragment
- a “naturally occurring motif” is hereby defined as a motif that can be found endogenously in a reference species, such as human; for example, a lipocalin is a naturally occurring motif (as is a lipocalin mutein that maintains the supersecondary or tertiary structure of a lipocalin), whereas a polypeptide such as a scFv is not a naturally occurring motif.
- “motif” when used herein in the context of a “naturally occurring motif” is preferably a naturally-occurring structure, preferably a polypeptide. Said structure preferably exists endogenously in a reference species. The skilled worker is able to readily determine whether an engineered polypeptide defines such a naturally occurring motif.
- a polypeptide defining a naturally occurring monomeric motif is not a scFv.
- a polypeptide defining a naturally occurring monomeric motif may comprise a scFv, however, in addition to a further monomeric polypeptide which is not a scFv.
- a polypeptide defining a naturally occurring monomeric motif may comprise a scFv and a lipocalin, e.g. fused by a spacer, such as a linker amino acid sequence.
- Both the scFv and the further monomeric polypeptide which is not a scFV may be specific to the same predetermined antigen or may be specific for different predetermined targets. This means that the scFv binds to a predetermined target that is different from the predetermined target to which the further monomeric polypeptide which, however, is not a scFv, binds to.
- a “polypeptide that defines a naturally occurring motif” includes that such a polypeptide may be modified (engineered) to be able to bind to a target associated with a tumor.
- a lipocalin that is within the context of the present invention a polypeptide that defines a naturally occurring motif, binds to its natural target such as lysozyme in case of tear lipocalin (TLC) or iron in case of LCN2/NGAL.
- TLC tear lipocalin
- iron in case of LCN2/NGAL.
- lipocalins can be modified such that they bind to a target other than their natural target.
- the term “naturally occurring motif” includes, that a polypeptide defining such a naturally occurring motif that can be found endogenously in a reference species, can preferably be modified or engineered to bind to a target associated with a tumor. Yet, the modification is preferably not a modification resulting in the generation of a scFv.
- the linker region and transmembrane domain have the function of stably anchoring the CAR in the cell membrane, while providing for a certain distance between the membrane and the binding element to allow for an effective and activatory formation of the CAR/target complex upon an encounter of the T-cell and the target cell.
- Typical linker regions described in published examples lead to dimerization of the CAR.
- the cytoplasmic tail of the dimeric CAR therefore contains two copies of the respective intracellular signaling regions, copying what is found in nature for the T-cell receptor (2 copies of CD3zeta) and the homodimeric receptor CD28.
- SEQ ID NO: 4 represents the amino acid sequence of one such example, based on a linker region and the transmembrane domain of CD8 alpha (cf. WO2014011988 A2; for all SEQ ID NO's of this application, one of the possible DNA sequences encoding the respective amino acid sequence is provided below as detailed in Table 1).
- a CAR may contain up to three intracellular signaling domains in tandem, one of which being a full intracellular signaling domain of CD3zeta (SEQ ID NO: 5) and located at the C-terminus.
- an additional costimulatory signaling domain is inserted between the transmembrane domain and the CD3zeta intracellular domain; for example, the full intracellular domain of CD28 (SEQ ID NO: 6) or that of CD137 (SEQ ID NO: 7).
- the intracellular signaling domain of ICOS or of other costimulatory receptors from the TNFR family including but not limited to Ox-40, CD27 or CD40, may also be employed.
- third-generation CARs three of these costimulatory domains can be combined, with a typical example consisting of the combination CD137, CD28 and CD3zeta.
- a mutein of a polypeptide that, endogenously, defines a monomeric polypeptide (such as a lipocalin) can be employed in a first, second, third or future generation CAR.
- the tumor-binding moiety may, for example, be an Anticalin specific for a target selected from the group consisting of c-Met and GPC-3.
- the sequence of such an Anticalin binding c-Met is provided by SEQ ID NO: 1, while the sequence of an Anticalin binding GPC-3 is provided by SEQ ID NO: 2.
- the linker and transmembrane region the CD8 linker and transmembrane region (SEQ ID NO: 4) may be utilized.
- the intracellular signaling domain of CD3zeta SEQ ID NO: 5
- CD28 SEQ ID NO: 6
- CD137 SEQ ID NO: 7
- the resulting four constructs accordingly, could be: c-Met-CD28-CD3zeta (SEQ ID NO: 8), c-Met-CD137-CD3zeta (SEQ ID NO: 9), GPC3-CD28-CD3zeta (SEQ ID NO: 10) and GPC3-CD137-CD3zeta (SEQ ID NO: 11).
- Retroviral vectors are established and currently widely used, allowing for permanent and heritable CAR expression due to their integration into genomic DNA.
- retroviral vectors derived from gamma-retrovirus have been utilized for lymphocyte gene transfer in clinical applications since 1990 (Rosenberg et al. 1990).
- an HIV-based lentiviral vector may provide advantages such as higher and more stable expression of the transgene, and potentially increased safety compared to gamma-retroviral vectors.
- Other possible methods for gene transfer include electroporation of mRNA constructs, if CAR expression is desired to be transient only (Birkholz et al.
- T-cells can be employed to test the functionality of a CAR, using standard methods that serve to demonstrate the activation of T-cells. Investigations include the responses of T-cells to activation, namely proliferation and prolonged survival, the production of cytokines such as IL-2 and IFN-gamma, and the capacity to kill target cells. The latter can be determined, e.g., by direct observation of target cell killing, but also by indirect methods that show for example the release of intracellular components of the target cell or the generation of cytotoxic molecules by the T-cells.
- mouse models may be employed.
- the disclosure provides a CAR that secretes a polypeptide, which may or may not define a naturally occurring monomeric motif (e.g. a lipocalin mutein) specific to a predetermined antigen (e.g. an immunomodulatory target such as an immune checkpoint or costimulatory molecule) that is transduced into a T cell for secretion thereby, bringing an added therapeutic benefit.
- a predetermined antigen e.g. an immunomodulatory target such as an immune checkpoint or costimulatory molecule
- the secreted polypeptide preferably is monomeric; further, the secreted polypeptide preferably is specific for an immunomodulatory target.
- This construct can be based on T-cells with a defined specificity (either without genetic manipulation or by transduction with a CAR or a recombinant TCR) that are equipped with the capacity to secrete therapeutically relevant biologics.
- TRUCK-like approaches which are based on the secretion of proinflammatory cytokines
- the present disclosure provides for a T cell that secretes a specific binding polypeptide that can be antagonistic or agonistic vis-h-vis its target.
- tumor-specific T cells that secrete biologics that are antagonistic inhibitors of the interaction of co-inhibitory immune receptors with their ligands also known as “checkpoint inhibitors” may be employed.
- non-limiting target examples include PD-1 and CTLA-4.
- constructs that allow the agonistic activation of stimulatory and/or costimulatory immune receptors can be utilized, with examples for the respective targets being CD3 and CD137.
- antagonists to anti-inflammatory cytokines such as IL-10 or TGF-also can be employed. This approach can be termed T-cell activation-induced localized secretion (TAILS).
- TILS T-cell activation-induced localized secretion
- NK cells NK cells or B-cells.
- an analogous approach can be applied to auto-inflammatory conditions; however, in this case antagonists to stimulatory receptors or proinflammatory cytokines or agonists of coinhibitory immune receptors may be applied, and vehicle cells that have no cytotoxic capacity may be employed.
- TAILS can be viewed as an extension of the CAR-T/Recombinant TCR approaches with an improved efficiency.
- TAILS can also be viewed as an alternative to traditional biologics, where production of the therapeutic occurs in vivo.
- This firstly provides the advantage of highly localized (ant-)agonism with concomitant minimal unwanted systemic toxicity, in particular if biologics with a low systemic half-life are employed, and secondly facilitates efficient delivery of biologics to any site of action within the human body, including tissues which would otherwise not be reachable by biologics drugs that are systemically applied, such as the brain.
- local delivery of biologic therapeutics has the potential to not only have a more benign toxicity profile, but also to display an improved efficacy as it is well known that local and systemic effects of agonists or antagonists can be profoundly different.
- both TRUCK-like or TAILS approaches can be implemented using proteins consisting of multiple subunits (i.e. multiple polypeptide chains); for example, a heterodimeric cytokine such as IL-12 consisting of the IL-12p40 and the IL-12p35 subunits, or an IgG antibody consisting of two heavy chains and two light chains, as such multimeric proteins might be assembled within a vehicle cell that is transduced with all the subunits endcoded on suitable vectors.
- biologics used for TAILS should preferably define a monomer, with examples including but not limited to Affibody molecules, Affilins, Affimers, Affitins, Anticalins, Avimers, DARPins, Fynomers, Kunitz domain peptides, Monobodies or single-chain variants of Antibodies such as scFv and their derivatives, camelid single-chain Antibodies, nanobodies or domain Antibodies.
- the targets recognized by the T-cell include, but are not limited to the targets described above.
- targets that are tumor-associated but not necessarily tumor cell-associated such as VEGF-A, Ang2 and others
- a CAR is employed in the approach, the overall design will follow the principles described herein for the Anticalin-based CAR.
- Potential targets of the secreted biologic include coinhibitory receptors or their ligands such as CTLA-4, B7-1, PD-1, PD-L1, LAG3, BTLA, TIM3, TIGIT, CD160 or LAIR1.
- the secreted biologic must be capable of interfering with the ligand/receptor interaction or must otherwise block downstream signaling.
- constructs that allow the agonistic activation of stimulatory and/or costimulatory immune receptors can be utilized, with examples for receptors being the TCR (e.g. via CD3), CD28, ICOS, CD137, Ox40, GITR, HVEM, CD27, CD30, DR3, SLAM, CD2 or CD226.
- TCR e.g. via CD3
- CD28 CD28
- ICOS CD137
- Ox40 GITR
- HVEM CD27
- CD30 CD30
- DR3, SLAM CD2 or CD226.
- T-, B- and NK cells and, where applicable, their subtypes are in principle amenable to this task. How this would be done in practice is well known in the art for T-cells (cf. CAR-T approach), and could analogously be implemented for the other cell types.
- the assays used to characterize engineered lymphocytes capable of TAILS can be essentially the same as the ones described for CAR- or TCR-transduced T-cells above. Additionally, assays can be used to measure successful secretion of the therapeutic produced by the cells, with ELISA assays being a straightforward possibility.
- a representative example, below, is based on a CAR-T cell generated by transduction of T-cells with the construct GPC3-CD137-CD3zeta (SEQ ID NO: 11) described herein.
- the T-cell may be co-transduced with a suitable vector encoding the secreted biologic; for this purpose, an Anticalin-based CTLA-4 antagonist (SEQ ID NO: 3), which binds both human and mouse CTLA-4, can be employed.
- composition comprising a T cell as described herein and optionally a pharmaceutically acceptable carrier.
- the present invention provides a T cell as described herein for use in a method of treatment of cancer.
- the present invention provides a method of treatment of cancer comprising administering to a subject in need thereof a pharmaceutically efficient amount or numbers of T cells as described herein.
- the treatment of cancer includes the treatment of a tumor.
- the subject is preferably a mammal, more preferably a human.
- a pharmaceutically acceptable carrier also includes a pharmaceutically acceptable excipient.
- FIG. 1 Inducible expression of CTLA-4 antagonist in primary T cells analyzed by FACS.
- Human peripheral blood T cells were transduced with pSIN-(NFAT)6- ⁇ CTLA-4 and incubated for 48 h at 37° C. under activatory conditions by utilizing plates precoated with anti-CD3 mAb and anti-CD28 mAb.
- Cells were stained extracellularly using a FITC-labeled anti-CD3 antibody, and intracellulary—after fixation and permeabilization—for SEQ ID NO: 28 utilizing a primary polyclonal anti-NGAL rabbit antibody, followed by secondary staining with a Dylight594-conjugated anti-rabbit IgG antibody. Immunofluorescence was analyzed using a FACS instrument. A detailed protocol of the experiment is provided in Example 1.
- FIG. 2 Detection of functional T-cell secreted CTLA-4 antagonist by ELISA.
- the supernatant of human peripheral blood T cells, mock-transduced or transduced with pSIN-(NFAT)6- ⁇ CTLA-4 and cultured under activatory conditions (cf. Example 1 and FIG. 1 ) was analyzed for the presence of functionally active CTLA-4 antagonist SEQ ID NO: 28 by ELISA.
- Recombinant human CTLA-4 was coated on an ELISA plate as described in Example 1, and culture supernatants of transduced and mock-transduced T cells were added.
- Plate-bound SEQ ID NO: 28 was detected by primary staining with an anti-NGAL polyclonal antibody from the rabbit, secondary staining using goat anti rabbit IgG-HRP and addition of a chromogenic HRP substrate as described in Example 1.
- FIG. 3 Detection of functional T-cell secreted CTLA-4 antagonist by FACS of CTLA-4-positive CHO cells.
- the supernatant of Jurkat cells transduced with pSIN-(NFAT)6- ⁇ CTLA-4 and cultured under activatory conditions (cf. Example 2) was analyzed for the presence of functionally active CTLA-4 antagonist SEQ ID NO: 28 by incubation of the supernatant with CHO cells stably expressing CTLA-4 on their surface (CHO::CTLA-4).
- CHO::CTLA-4 were incubated with isotype control.
- Cell-bound SEQ ID NO: 28 that had been excreted by the Jurkat cells was then detected using anti-NGAL polyclonal antibody followed by secondary staining with a fluorescently labeled anti-rabbit Antibody as described in Example 2.
- FIG. 4 Inducible expression of Fc fusion of CTLA-4 antagonist in primary T cells analyzed by FACS.
- Human peripheral blood T cells were transduced with pSIN-(NFAT)6- ⁇ CTLA-4-Fc and incubated for 48 h at 37° C. under activatory conditions by utilizing plates precoated with anti-CD3 mAb and anti-CD28 mAb.
- Cells were stained extracellularly using a FITC-labeled anti-CD3 antibody, and intracellulary—after fixation and permeabilization—for SEQ ID NO: 29 utilizing a primary polyclonal anti-NGAL antibody, followed by secondary staining with a Dylight594-conjugated anti-rabbit IgG antibody. Immunofluorescence was analyzed using a FACS instrument. Cf. Example 3 for details.
- FIG. 5 Detection of functional T-cell secreted Fc fusion of a CTLA-4 antagonist by ELISA.
- the supernatant of human peripheral blood T cells, mock-transduced or transduced with pSIN-(NFAT)6- ⁇ CTLA-4-Fc and cultured under activatory conditions (cf. Example 3 and FIG. 4 ) was analyzed for the presence of the functionally active Fc fusion of the CTLA-4 antagonist SEQ ID NO: 29 by ELISA.
- Recombinant human CTLA-4 was coated on an ELISA Plate as described in Example 3, and culture supernatants of transduced and mock-transduced T cells were added.
- Plate-bound SEQ ID NO: 29 was detected by primary staining with an anti-NGAL polyclonal rabbit antibody, secondary staining using goat anti rabbit IgG-HRP and addition of a chromogenic HRP substrate as described in Example 3.
- the coding DNA corresponding to the SEQ ID NOs: 8-11 is generated by gene synthesis.
- a set of codons that is compatible with mammalian expression needs to be employed.
- the DNA stretches encoding the CARs are equipped with suitable restriction sites at the N- and C-termini to allow cloning into the retroviral expression vector pBullet (Willemsen et al. 2000), or, alternatively, pSTITCH (Weijtens et al. 1998).
- the gene synthesis is straightforwardly performed using a commercial provider, and the resulting DNA is cloned into pBullet using standard molecular biology methods.
- DNA of the respective retroviral vector (pBullet or pSTITCH) containing the Anticalin-based CAR (6 mg DNA) is co-transfected into 293T cells by calcium phosphate co-precipitation with the retroviral helper plasmid DNA pHIT60 and pCOLT (each 6 mg DNA) encoding the MuLV gag and pol genes (pHIT60) and the GALV envelope gene (pCOLT), respectively, under control of the CMV promotor/enhancer (Weijtens et al. 1998).
- This procedure results in transient production of high titers of infectious retrovirus.
- Peripheral blood lymphocytes from healthy donors are isolated by density centrifugation and cultured for 48 h in RPMI-1640 medium supplemented with 10% FCS in the presence of IL-2 (400 U/ml) and OKT3 MAb (100 ng/ml).
- Cells are harvested, washed, resuspended in medium with IL-2 (400 U/ml), and co-cultured for 48 h with 293T cells that are transiently transfected as described above and therefore produce and excrete the retroviral transducing particles into the medium.
- T cells are harvested and subsequently tested for successful CAR expression and functional assays as described further below.
- the latter are either T-cells not expressing a CAR, or CAR-transduced T-cells incubated in wells coated with the control IgG1.
- a second step which is more akin to the desired situation in-vivo, different amounts of receptor-grafted T cells (1.25 to 10 ⁇ 10 4 cells/well) are co-cultured for 48 h with GPC-3+ or c-Met+ immortal tumor cell lines.
- Target-positive cell lines that can be used are HepG2 (GPC-3) and H441 (c-Met), and examples for GPC-3/c-Met-negative control cells are SK-HEP-1 (GPC-3) and A2870 (c-Met).
- GPC-3+ or c-Met+ immortal tumor cell lines examples for GPC-3/c-Met-negative control cells are SK-HEP-1 (GPC-3) and A2870 (c-Met).
- LDH lactate dehydrogenase
- human T cells are isolated from peripheral blood by magnetic activated cell sorting, using human CD3+ beads.
- the transduction-competent retroviri encoding the c-Met- and GPC3-specific CARs are generated and used for transduction of human T-cells as described above.
- NIH-Ill mouse Chargeles River
- MC38 cells stably transfected with human GPC-3 or c-Met (9 ⁇ 10 5 cells/mouse) are s.c.
- tumors are induced by s.c. injection of MC38 cells stably transfected with human GPC-3 or c-Met and matched CAR-T cells are applied by i.v. injection at day 6.
- the respective experiment using CAR-T cells and the mismatched transfected tumor cell line serves as a negative control. Tumor growth is monitored daily by external measurement with a digital caliper.
- GPC3-CD137-CD3zeta (SEQ ID NO: 11) is generated and cloned into a suitable retroviral vector as described above.
- Retroviral particles suitable for peripheral blood T-cell transduction are obtained by co-transfection with retroviral helper particles into 293T cells, also as described above.
- a vector suitable to facilitate retroviral expression of the T-cell activation inducible Anticalin-based CTLA4-antagonist (“ ⁇ CTLA-4”) is obtained by exchanging the mature IL-12 sequence in pSIN-(NFAT)6-IL-12 (Chmielewski et al. 2011b) by the Anticalin sequence (SEQ ID NO: 3) using standard molecular biology methods, to obtain pSIN-(NFAT)6- ⁇ CTLA-4. Note that the leader sequence of IL-12 remains in the construct to serve as a secretion signal.
- T cells are retrovirally transduced with the ⁇ CTLA-4 expression cassette and positively selected in the presence of 0.5 mg/mL Geneticin (G418) and IL-2 (500 IU/mL) on plates precoated with anti-CD3 mAb OKT-3 and anti-CD28 mAb 15E8 as previously described (Chmielewski et al. 2011b). Geneticin-resistant clones are transduced with the GPC-3-specific CAR GPC3-CD137-CD3zeta.
- the latter are either T-cells not expressing a CAR, but transduced with ⁇ CTLA-4 only (SEQ ID NO: 3), or doubly transduced CAR/TAILS T-cells incubated in wells coated with the control IgG1.
- a second step which is more akin to the desired situation in-vivo, different amounts of receptor-grafted T cells (1.25 to 10 ⁇ 10 4 cells/well) are co-cultured for 48 h with GPC-3+ immortal tumor cell lines.
- the GPC3-positive cell line used is HepG2
- the GPC-3-negative control cell line is SK-HEP-1.
- T-cell CAR specificity we co-incubate with GPC-3+ cells with T-cells not expressing a CAR, but transduced with ⁇ CTLA-4 (SEQ ID NO: 3) only. In all cases, cells are coincubated for 48 h, and supernatants are subsequently analysed for IFN- ⁇ expression and the expression of the anti-CTLA-4 Anticalin.
- LDH lactate dehydrogenase
- tumors are induced by s.c. injection of AT3 cells stably transfected with human GPC-3 and CAR/TAILS-T cells are applied by i.v. injection at day 6.
- Negative controls include a vehicle injection control, the respective experiment using CAR/TAILS-T and mock-transfected AT3 cells, and the respective experiment utilizing CAR-T cells that do not secrete ⁇ -CTLA-4 Anticalin. Tumor growth is monitored daily by external measurement with a digital caliper.
- a vector suitable to facilitate retroviral expression of the T-cell activation inducible Anticalin-based CTLA4-antagonist (“ ⁇ CTLA-4”) was obtained by replacing the IL-12 sequence in pSIN-(NFAT)6-IL-12 (Chmielewski et al. 2011b) by the Anticalin sequence (SEQ ID NO: 3) with a Strep-tag II (SEQ ID NO: 27) using standard molecular biology methods, to obtain pSIN-(NFAT)6- ⁇ CTLA-4.
- the leader sequence of the mouse immunoglobulin kappa light chain (GenBank Acc #CAB46127.1) instead of the IL-12 leader was employed in the construct to serve as a secretion signal.
- the final amino acid sequence corresponds to SEQ ID NO: 23, encoded by the DNA sequence SEQ ID NO: 24.
- DNA of pSIN-(NFAT)6- ⁇ CTLA-4 (6 mg DNA) was co-transfected into 293T cells by calcium phosphate co-precipitation with the retroviral helper plasmid DNA pHIT60 and pCOLT (each 6 mg DNA) encoding the MuLV gag and pol genes (pHIT60) and the GALV envelope gene (pCOLT), respectively, under control of the CMV promotor/enhancer (Weijtens et al. 1998). This procedure resulted in the production of high titers of infectious retrovirus.
- peripheral blood lymphocytes from healthy donors were isolated by density centrifugation in a Ficoll-Paque (GE Healthcare) density gradient and cultured for 48 h in RPM11640 medium supplemented with 10% (v/v) FCS in the presence of IL-2 (400 U/ml) and OKT3 mAb (100 ng/ml).
- Cells were harvested, washed, resuspended in medium with IL-2 (400 U/ml), and co-cultured for 48 h with 293T cells that were transiently transfected as described above and therefore released the retrovirus particles into the medium.
- T cells were harvested. The same procedure was also carried out using mock-transduction, i.e. transducing the gag, pol and env genes of the retrovirus without the expression cassette.
- SEQ ID NO: 28 the mature processed form of SEQ ID NO:24
- Intracellular staining of SEQ ID NO: 28 was then achieved by thoroughly resuspending the fixed and permeabilized cells in 50 ⁇ L of a saponin-containing buffer (BD Perm/WashTM buffer) containing 0.5 ⁇ g anti-NGAL mAb per 1 million cells, washing twice with saponin-containing BD Perm/WashTM buffer, and incubation for 30 minutes at 4° C. with 1 ⁇ g of the Dylight594-conjugated anti-rabbit IgG antibody per 1 million cells. Finally, cells were washed twice with PBS buffer and immunofluorescence was analyzed using a BD FACS Canto II instrument.
- BD Perm/WashTM buffer containing 0.5 ⁇ g anti-NGAL mAb per 1 million cells
- transduced cells labeled in this manner showed a clearly positive anti-NGAL reactivity ( FIG. 1A ) compared to the mock-transduced T-cell control ( FIG. 1B ).
- FIG. 1A we found that according to a threshold set based on the mock-transduced T-cell control ( FIG. 1B ), 52% of primary T cells had been transduced and were found to be SEQ ID NO: 28-positive.
- rabbit anti-NGAL polyclonal antibody at a concentration of 1 ⁇ g/mL in PBS/0.1% (w/v) Tween20/2% (w/v) BSA was added, and bound antibody was detected using goat anti-rabbit IgG-HRP in a dilution of 1:5,000 in PBS/0.1% (w/v) Tween20/2% (w/v) BSA.
- Chromogenic 3,3′, 5,5;-tetramethylbenzidine (TMB) substrate was used as a detection agent according to the manufacturer's instructions (Life Technologies). Fluorescence signals in RFU (relative fluorescence units) were measured using a plate fluorescence reader.
- the experiment of this example was carried out in analogy to Example 1, but using the Jurkat T cell line instead of primary T cells.
- Jurkat T cells were transduced with pSIN-(NFAT)6- ⁇ CTLA-4 as described in Example 1, and incubated in medium on plates precoated with anti-CD3 mAb OKT-3 and anti-CD28 mAb 15E8 as previously described (Chmielewski et al. 2011b).
- the culture supernatant containing the anti-CTLA-4 Anticalin (SEQ ID NO: 28) was obtained from the Jurkat cells by centrifugation and added to Chinese Hamster Ovary cells stably transfected with CTLA-4 (CHO::CTLA-4).
- CHO::CTLA-4 Chinese Hamster Ovary cells stably transfected with CTLA-4
- CHO::CTLA-4 were incubated with an isotype control antibody.
- Cell-bound SEQ ID NO: 28 was detected using anti-NGAL rabbit polyclonal antibody followed by staining with anti-rabbit antibody labeled with the fluorescent dye Dylight594.
- FACS fluorescence histogram FIG.
- SEQ ID NO: 25 is a fusion of the Anticalin-based CTLA-4 Antagonist (SEQ ID NO: 23) to the Fc fragment of a human IgG1 antibody.
- SEQ ID NO: 26 The DNA sequence encoding SEQ ID NO: 25 is provided by SEQ ID NO: 26.
- the corresponding inducible expression vector pSIN-(NFAT)6- ⁇ CTLA-4-Fc was generated by standard molecular biology methods.
Abstract
Lipocalin muteins specific to a predetermined antigen can be transduced into a T cell to bring therapeutic benefits to patients in need. In one example, a lipocalin mutein specific to a predetermined antigen (e.g., a target differentially expressed on the surface of a tumor cell) can be transduced into a T cell membrane to serve as an antigen receptor, offering benefits over conventionally deployed antibody-derived protein moieties such as a single chain variable fragment (scFv). Benefits include a more stable structure, leading to superior target engagement, for example. Further, lipocalin muteins specific to a predetermined antigen (e.g. an immunomodulatory target such as an immune checkpoint or costimulatory molecule) can be transduced into a T cell for secretion thereby, bringing an added therapeutic benefit. Specific examples of such modified T cells and methods of making and using the same are provided herein.
Description
- This application is a continuation of U.S. application Ser. No. 15/542,915, filed Jul. 11, 2017 as a National Stage application of PCT/EP2016/050326, filed Jan. 11, 2016, which claims priority from European application EP 15000047.9, filed Jan. 12, 2015. The entire contents of each of the aforementioned applications are incorporated by reference in their entireties.
- T-cells transduced with artificial chimeric antigen receptors (CARs), also called CAR-T cells, are being deployed for the treatment of cancer (Shi et al. 2014). The CAR-T approach has provided positive clinical results in hematologic cancers such as acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). In a recent trial, for example, 30 patients with relapsed or refractory ALL were infused with autologous T cells transduced with the CD19-directed chimeric antigen receptor CTL019 (formerly called CART-19). Of these, 27 patients (90%) achieved complete remission (Maude et al. 2014). Sustained remission was achieved with a 6-month event-free survival rate of 67% and an overall survival rate of 78%.
- CARs are transmembrane proteins stably anchored in the T-cell plasma membrane. A CAR is an artificial fusion of multiple parts: the extracellular part consists of a recognition domain binding to a cancer cell target; the intracellular part contains the signaling domain(s) of one or more (co-)stimulatory immunoreceptors; and the two are fused via a linker- and transmembrane domain. In the case of CTL019, for example, the recognition domain is an antibody single chain fragment (scFv) specific for CD19, the linker- and transmembrane regions are grafted from the membrane protein CD8, and the intracellular signaling part consists of the complete intracellular domains of CD137 and CD3zeta fused in tandem. When a T-cell transduced with this construct encounters a CD19-positive target cell, the chimeric antigen receptor is clustered, which results in activation of the signaling pathways downstream of CD3zeta and of the costimulatory receptor CD137, in turn leading to activation of T-cell proliferation, cytokine secretion, survival and the capacity to kill.
- With this design, CTL019 is an example for a “second generation” CAR, identified by the presence of two immunostimulatory domains. In contrast, “first generation” CARs contain only a single immunostimulatory domain—usually that of CD3zeta—while in “third generation” CARs, overall three intracellular immunostimulatory domains are fused in tandem, for example those of CD3zeta, CD28 and CD137. First generation CARs have failed to show efficacy in the clinic—potentially due to the lack of activity and long-term persistence of the CAR-T cells in patients (Kershaw et al. 2006)—while third generation CARs have yet to show their utility in a clinical setting.
- To date, the extracellular recognition domains of CARs are almost exclusively scFv's (with the few exceptions instead utilizing the extracellular domains of ligands or receptors), which is due to the fact that a single-chain functional protein (or monomer) is required to facilitate a straightforward fusion design. Accordingly, a tetrameric mAb or even a dimeric Fab would present severe challenges as an extracellular recognition domain.
- Although successes have been demonstrated with various scFv approaches, that construct is an artificial fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an antibody, connected with a short linker peptide of ten to about 25 amino acids. This artificial design leads to scFv-specific problems that are well known in the art. Indeed, scFv's typically display poor biophysical and developability behavior, such as low stability, bad expression yields (Haisma et al. 1998a; Haisma et al. 1998b, Peipp et al. 2004), the potential failure to be efficiently secreted in mammalian cells (Brocks et al. 1997) and, importantly, an oftentimes strong tendency to aggregate (see e.g. Ewert et al. 2003a; Ewert et al. 2003b; Schaefer et al. 2012).
- When an scFv is used for the construction of a CAR, these properties can be deleterious at multiple stages. First, the generation of a well-behaved scFv is difficult, and may require extensive optimization and engineering. Second, in the scFv-containing CAR, poor biophysical behavior can lead to poor and potentially unpredictable cell surface expression, reducing the number of CARs available to bind to a target antigen. Third, the tendency of an scFv to aggregate may lead to an undesired clustering of CARs on the T-cell surface, consequently leading to an undesired spurious activation of the T-cell in the absence of any specific target-bearing cell. The latter two issues can be a serious threat to the efficacy and safety of any given CAR, presenting clear challenges to obtain a predictable and well-behaved CAR-T system.
- The use of a polypeptide that employs a naturally occurring secondary and tertiary structure could provide pronounced benefits to an scFv approach. A mutein of a lipocalin that binds to a desired tumor target is an example of a suitable alternative. A lipocalin mutein, or Anticalin®, is an engineered binding protein based on a lipocalin (preferably of human origin) such as Tear Lipocalin (TLc) or NGAL. Unlike an antibody, an Anticalin is a single-domain protein; and unlike an scFv, an Anticalin is single-domain by nature, therefore per se a well-behaved monomer from a biophysical property standpoint. Apart from exhibiting superior biophysical properties, Anticalins provide tunable affinities from nanomolar to low picomolar range, while having a demonstrated ability to engage targets across a broad spectrum, including immune checkpoints.
- Another application of a CAR-T involves engineering these cells with the capacity to secrete proinflammatory cytokines. In this vein, the engineered CAR-T can lead to accumulation of a proinflammatory cytokine in the tumor micronenvironment where the CAR-T traffics. A proinflammatory cytokine may be desired to facilitate recruiting a second wave of immune cells in a locally restricted fashion to initiate a more complete and potentially target-independent attack of the cells of the tumor. This approach, as recently reviewed (Chmielewski et al. 2014), has been described in particular utilizing an engineered single-chain variant of Interleukin 12, thereafter called scIL-12. Here, secretion of scIL-12 can either be constitutive or be coupled to activation of the T-cell via the CAR. The latter approach has in some instances been termed TRUCK (“T-cells redirected for universal cytokine killing”), which is why in the following the approach and related approaches are termed “TRUCK-like”. A TRUCK-like approach with constitutive scIL-12 expression has already been brought to the clinic in two separate trials, which currently are suspended (NCT01236573) or terminated (NCT01457131), respectively. Potentially, constitutive expression of scIL-12 was not sufficiently localized to lead to the desired effects, or led to intolerable toxicity.
- To improve localized scIL-12 secretion, CAR-T cells have been transduced with the scIL-12 construct expressed under control of the nuclear-factor of the activated T-cell (NFAT)-derived minimal promoter to initiate IL-12 transcription upon signaling of the TCR/CD3 pathway (Chmielewski et al. 2011b; Zhang et al. 2011). Other promoters sensitive to signals of the same pathway, like the IL-2 promoter (Jaalouk et al. 2006) may also be feasible and could allow tuning the produced amount and the selectivity of cytokine production.
- Using inducible scIL-12 expression, improved tumor reduction was observed compared to unmodified CAR-T cells in preclinical models (Chmielewski et al. 2011b; Zhang et al. 2011; Pegram et al. 2012; Pegram et al. 2014). Other cytokines, such as IL15, IL-18, IL-23, IL-27 and IFN-gamma, and other producer cells, such as NK cells, have been proposed for the approach. (Chmielewski et al. 2014).
- The ability to engineer a T-Cell to secrete yet other types of therapeutic proteins upon T-Cell activation would be desirable, further expanding the repertoire to therapeutic modalities available in the tumor microenvironment. Particularly desirable would be specific binding proteins, e.g. a monoclonal antibody, that could bind to an immunomodulatory target, such as an immune checkpoint like CTLA4 or PD1, or to a costimulatory target such as CD137 or OX40. There are at least two drawbacks to using an antibody in this setting, however. First, antibodies are extremely large, tetrameric proteins, limiting the ability of a T-Cell to produce and secrete a functional antibody. Second, antibodies typically have a half-life in man of two to three weeks, which limits their application in an immunomodulatory approach, given potential safety concerns.
- A smaller, more stable monomeric polypeptide having a shorter half-life could overcome these two shortcomings of antibodies. An example for such a molecule would be a mutein of a lipocalin that is specific for an immunomodulatory target and is monomeric, stable and has a half-life tunable from hours to days in man and a molecular weight as low as 18 kilodalton.
- In one aspect, the disclosure provides a CAR that contains a polypeptide defining a naturally occurring monomeric motif (e.g. a lipocalin mutein) specific to a predetermined antigen (e.g., a target differentially expressed on the surface of a tumor cell) that is transduced into a T cell membrane to serve as an antigen receptor, offering benefits over conventionally deployed antibody-derived protein moieties such as a single chain variable fragment (scFv). Benefits include a more stable structure, leading to superior target engagement, for example. As used herein, a “naturally occurring motif” is hereby defined as a motif that can be found endogenously in a reference species, such as human; for example, a lipocalin is a naturally occurring motif (as is a lipocalin mutein that maintains the supersecondary or tertiary structure of a lipocalin), whereas a polypeptide such as a scFv is not a naturally occurring motif. Accordingly, “motif” when used herein in the context of a “naturally occurring motif” is preferably a naturally-occurring structure, preferably a polypeptide. Said structure preferably exists endogenously in a reference species. The skilled worker is able to readily determine whether an engineered polypeptide defines such a naturally occurring motif.
- In a preferred embodiment a polypeptide defining a naturally occurring monomeric motif is not a scFv. However, that being so, a polypeptide defining a naturally occurring monomeric motif may comprise a scFv, however, in addition to a further monomeric polypeptide which is not a scFv. For example, a polypeptide defining a naturally occurring monomeric motif may comprise a scFv and a lipocalin, e.g. fused by a spacer, such as a linker amino acid sequence. Both the scFv and the further monomeric polypeptide which is not a scFV, may be specific to the same predetermined antigen or may be specific for different predetermined targets. This means that the scFv binds to a predetermined target that is different from the predetermined target to which the further monomeric polypeptide which, however, is not a scFv, binds to.
- When referred to herein a “polypeptide that defines a naturally occurring motif” includes that such a polypeptide may be modified (engineered) to be able to bind to a target associated with a tumor. For example, a lipocalin, that is within the context of the present invention a polypeptide that defines a naturally occurring motif, binds to its natural target such as lysozyme in case of tear lipocalin (TLC) or iron in case of LCN2/NGAL. However, such lipocalins can be modified such that they bind to a target other than their natural target. Hence, the term “naturally occurring motif” includes, that a polypeptide defining such a naturally occurring motif that can be found endogenously in a reference species, can preferably be modified or engineered to bind to a target associated with a tumor. Yet, the modification is preferably not a modification resulting in the generation of a scFv.
- The skilled worker will appreciate that the following elements are to be considered when designing a CAR:
-
- (i) The CAR primary sequence, consisting of a fusion of the binding element, linker region, transmembrane domain and intracellular signaling domains
- (ii) The method used to transduce a T-cell
- (iii) Suitable assays such as an in vitro assay, to test the functionality of the CAR
- (iv) In-vivo assays to test the functionality of the CAR
Several designs are possible for such a CAR, with the following to be regarded as a non-limiting example. The tumor-targeting moiety of a CAR-T may comprise a mutein of a polypeptide that, endogenously, defines a monomeric polypeptide, such as a lipocalin. The target to which such mutein (e.g., an Anticalin) may bind includes CAIX, CD19, CD20, CD22, CD30, CD33, CD44v7/8, CEA, EGP-2, EGP-40, FBP, Fetal acethylcholine receptor, GD2, GD3, Her2/neu, IL-13R-a2, KDR, k-light chain, L1 cell adhesion molecule, LeY, MAGE-A1, Mesothelin, MUC1, NKG2D ligands, Oncofetal antigen (h5T4), PSCA, PSMA, TAG-72, VEGF-R2, the α-Folate receptor and others. Other tumor-associated targets that might be considered of interest in this context include EGFR, EPHA2, melanoma 30 associated chondroitin sulfate proteoglycan, IGFR1, fibroblast-activating protein alpha, c-MET, EpCAM, and GPC-3. Further, targets not directly expressed on the tumor cell surface, but associated with the tumor stroma, such as VEGF-A or Ang2, are suitable.
- The linker region and transmembrane domain have the function of stably anchoring the CAR in the cell membrane, while providing for a certain distance between the membrane and the binding element to allow for an effective and activatory formation of the CAR/target complex upon an encounter of the T-cell and the target cell. Typical linker regions described in published examples lead to dimerization of the CAR. When such linker domains are used, the cytoplasmic tail of the dimeric CAR therefore contains two copies of the respective intracellular signaling regions, copying what is found in nature for the T-cell receptor (2 copies of CD3zeta) and the homodimeric receptor CD28. SEQ ID NO: 4 represents the amino acid sequence of one such example, based on a linker region and the transmembrane domain of CD8 alpha (cf. WO2014011988 A2; for all SEQ ID NO's of this application, one of the possible DNA sequences encoding the respective amino acid sequence is provided below as detailed in Table 1).
- As described to date, a CAR may contain up to three intracellular signaling domains in tandem, one of which being a full intracellular signaling domain of CD3zeta (SEQ ID NO: 5) and located at the C-terminus. In second-generation CARs, an additional costimulatory signaling domain is inserted between the transmembrane domain and the CD3zeta intracellular domain; for example, the full intracellular domain of CD28 (SEQ ID NO: 6) or that of CD137 (SEQ ID NO: 7). Alternatively, the intracellular signaling domain of ICOS or of other costimulatory receptors from the TNFR family, including but not limited to Ox-40, CD27 or CD40, may also be employed. In third-generation CARs, three of these costimulatory domains can be combined, with a typical example consisting of the combination CD137, CD28 and CD3zeta. A mutein of a polypeptide that, endogenously, defines a monomeric polypeptide (such as a lipocalin) can be employed in a first, second, third or future generation CAR.
- The tumor-binding moiety may, for example, be an Anticalin specific for a target selected from the group consisting of c-Met and GPC-3. The sequence of such an Anticalin binding c-Met is provided by SEQ ID NO: 1, while the sequence of an Anticalin binding GPC-3 is provided by SEQ ID NO: 2. As the linker and transmembrane region, the CD8 linker and transmembrane region (SEQ ID NO: 4) may be utilized. For intracellular signalling, the intracellular signaling domain of CD3zeta (SEQ ID NO: 5), either in tandem with CD28 (SEQ ID NO: 6) or CD137 (SEQ ID NO: 7), can be employed. The resulting four constructs, accordingly, could be: c-Met-CD28-CD3zeta (SEQ ID NO: 8), c-Met-CD137-CD3zeta (SEQ ID NO: 9), GPC3-CD28-CD3zeta (SEQ ID NO: 10) and GPC3-CD137-CD3zeta (SEQ ID NO: 11).
- A number of methods have been devised to genetically modify lymphocytes ex vivo to overexpress CARs. Retroviral vectors are established and currently widely used, allowing for permanent and heritable CAR expression due to their integration into genomic DNA. For example, retroviral vectors derived from gamma-retrovirus have been utilized for lymphocyte gene transfer in clinical applications since 1990 (Rosenberg et al. 1990). As an alternative, an HIV-based lentiviral vector may provide advantages such as higher and more stable expression of the transgene, and potentially increased safety compared to gamma-retroviral vectors. Other possible methods for gene transfer include electroporation of mRNA constructs, if CAR expression is desired to be transient only (Birkholz et al. 2009); (Zhao et al. 2006), and transposon-based systems such as “piggybac” and “sleeping beauty” (Maiti et al. 2013). A retrovirus-based system based on the vector pBULLET, as described in Willemsen et al. 2000, is utilized in this application to express SEQ ID NOs: 8-11. Alternatively, pSTITCH (Weijtens et al. 1998) also may be employed.
- One or more in vitro assays can be employed to test the functionality of a CAR, using standard methods that serve to demonstrate the activation of T-cells. Investigations include the responses of T-cells to activation, namely proliferation and prolonged survival, the production of cytokines such as IL-2 and IFN-gamma, and the capacity to kill target cells. The latter can be determined, e.g., by direct observation of target cell killing, but also by indirect methods that show for example the release of intracellular components of the target cell or the generation of cytotoxic molecules by the T-cells.
- To investigate the functionality of a given CAR-design in vivo, mouse models may be employed.
- Examples for both in-vitro and in-vivo assays are provided in the literature, cf. for example Hombach et al. 2010; Chmielewski et al. 2011a; Chmielewski et al. 2011b; Kofler et al. 2011; Chmielewski et al. 2012; Maliar et al. 2012; Pegram et al. 2012; Chmielewski et al. 2013a; Chmielewski et al. 2013b; Hombach et al. 2013; Pegram et al. 2014; and Textor et al. 2014.
- The skilled worker also will appreciate that beyond a lipocalin mutein, other monomeric or single domain polypeptides can be employed, including but not limited to a DARPin, a Fynomer, and a Kunitz domain peptide.
- In another aspect, the disclosure provides a CAR that secretes a polypeptide, which may or may not define a naturally occurring monomeric motif (e.g. a lipocalin mutein) specific to a predetermined antigen (e.g. an immunomodulatory target such as an immune checkpoint or costimulatory molecule) that is transduced into a T cell for secretion thereby, bringing an added therapeutic benefit. In this vein, the secreted polypeptide preferably is monomeric; further, the secreted polypeptide preferably is specific for an immunomodulatory target.
- This construct can be based on T-cells with a defined specificity (either without genetic manipulation or by transduction with a CAR or a recombinant TCR) that are equipped with the capacity to secrete therapeutically relevant biologics. In contrast to TRUCK-like approaches, which are based on the secretion of proinflammatory cytokines, the present disclosure provides for a T cell that secretes a specific binding polypeptide that can be antagonistic or agonistic vis-h-vis its target. In the context of oncology indications, tumor-specific T cells that secrete biologics that are antagonistic inhibitors of the interaction of co-inhibitory immune receptors with their ligands (also known as “checkpoint inhibitors”) may be employed. Specific, non-limiting target examples include PD-1 and CTLA-4. Alternatively, constructs that allow the agonistic activation of stimulatory and/or costimulatory immune receptors can be utilized, with examples for the respective targets being CD3 and CD137. Further, antagonists to anti-inflammatory cytokines such as IL-10 or TGF-also can be employed. This approach can be termed T-cell activation-induced localized secretion (TAILS).
- The skilled worker will appreciate that this approach can also be based on other cell types, such as NK cells or B-cells. Further, an analogous approach can be applied to auto-inflammatory conditions; however, in this case antagonists to stimulatory receptors or proinflammatory cytokines or agonists of coinhibitory immune receptors may be applied, and vehicle cells that have no cytotoxic capacity may be employed.
- TAILS can be viewed as an extension of the CAR-T/Recombinant TCR approaches with an improved efficiency. On the other hand, TAILS can also be viewed as an alternative to traditional biologics, where production of the therapeutic occurs in vivo. This firstly provides the advantage of highly localized (ant-)agonism with concomitant minimal unwanted systemic toxicity, in particular if biologics with a low systemic half-life are employed, and secondly facilitates efficient delivery of biologics to any site of action within the human body, including tissues which would otherwise not be reachable by biologics drugs that are systemically applied, such as the brain. Furthermore, local delivery of biologic therapeutics has the potential to not only have a more benign toxicity profile, but also to display an improved efficacy as it is well known that local and systemic effects of agonists or antagonists can be profoundly different.
- The skilled worker will appreciate that the following elements are to be considered when designing the components of a TAILS system according to the foregoing principles:
-
- (i) The biologic used as a secreted antagonist or agonist
- (ii) The target of the CAR or (recombinant) TCR
- (iii) The target of the secreted biologic
- (iv) The cells used as vehicles and producers of the single-chain biologic
- (v) The method used to transduce the vehicle cells
- (vi) In-vitro and in-vivo assays to test the functionality of the TAILS system
- Several designs are possible for the biologic used as a secreted (ant-)agonist, with the following to be regarded as a non-limiting example. In theory, both TRUCK-like or TAILS approaches can be implemented using proteins consisting of multiple subunits (i.e. multiple polypeptide chains); for example, a heterodimeric cytokine such as IL-12 consisting of the IL-12p40 and the IL-12p35 subunits, or an IgG antibody consisting of two heavy chains and two light chains, as such multimeric proteins might be assembled within a vehicle cell that is transduced with all the subunits endcoded on suitable vectors. However, a complex multimeric protein may incorrectly inefficiently assemble, leading either to secretion of an undesired product with concomitant undesired effects (for example an IL-12p40 dimer), or insufficient generation and secretion of the multimer (for example an antibody). Therefore, biologics used for TAILS should preferably define a monomer, with examples including but not limited to Affibody molecules, Affilins, Affimers, Affitins, Anticalins, Avimers, DARPins, Fynomers, Kunitz domain peptides, Monobodies or single-chain variants of Antibodies such as scFv and their derivatives, camelid single-chain Antibodies, nanobodies or domain Antibodies.
- The targets recognized by the T-cell, either by the physiological (T-cell) receptor, a CAR or recombinant TCR, include, but are not limited to the targets described above. Here, it is of note that targets that are tumor-associated but not necessarily tumor cell-associated (such as VEGF-A, Ang2 and others) may be particularly suited to provide local release of a checkpoint inhibitor or an immune receptor agonist without inducing cell killing of specific cells. If a CAR is employed in the approach, the overall design will follow the principles described herein for the Anticalin-based CAR.
- Potential targets of the secreted biologic include coinhibitory receptors or their ligands such as CTLA-4, B7-1, PD-1, PD-L1, LAG3, BTLA, TIM3, TIGIT, CD160 or LAIR1. (Chen et al. 2013) Here, the secreted biologic must be capable of interfering with the ligand/receptor interaction or must otherwise block downstream signaling. Alternatively, constructs that allow the agonistic activation of stimulatory and/or costimulatory immune receptors can be utilized, with examples for receptors being the TCR (e.g. via CD3), CD28, ICOS, CD137, Ox40, GITR, HVEM, CD27, CD30, DR3, SLAM, CD2 or CD226. (Chen et al. 2013) Depending on the target, such agonists can for example be obtained by dimerisation or multimerisation of a single receptor binder.
- For this purpose, mobile cells that have the capacity to strongly proliferate upon activation, and that are capable of secreting sufficient amounts of a given biologic are required. T-, B- and NK cells, and, where applicable, their subtypes are in principle amenable to this task. How this would be done in practice is well known in the art for T-cells (cf. CAR-T approach), and could analogously be implemented for the other cell types.
- Methods for cell transduction of a CAR or TCR can be followed as described herein. Vectors suitable for T-cell activation driven secretion are described in the literature, cf. e.g. Chmielewski et al. 2011b; Zhang et al. 2011; Pegram et al. 2012 and Pegram et al. 2014.
- The assays used to characterize engineered lymphocytes capable of TAILS can be essentially the same as the ones described for CAR- or TCR-transduced T-cells above. Additionally, assays can be used to measure successful secretion of the therapeutic produced by the cells, with ELISA assays being a straightforward possibility.
- A representative example, below, is based on a CAR-T cell generated by transduction of T-cells with the construct GPC3-CD137-CD3zeta (SEQ ID NO: 11) described herein. The T-cell may be co-transduced with a suitable vector encoding the secreted biologic; for this purpose, an Anticalin-based CTLA-4 antagonist (SEQ ID NO: 3), which binds both human and mouse CTLA-4, can be employed.
- Also provided by the present invention is a pharmaceutical composition comprising a T cell as described herein and optionally a pharmaceutically acceptable carrier.
- Furthermore, the present invention provides a T cell as described herein for use in a method of treatment of cancer.
- Similarly, the present invention provides a method of treatment of cancer comprising administering to a subject in need thereof a pharmaceutically efficient amount or numbers of T cells as described herein.
- The treatment of cancer includes the treatment of a tumor. The subject is preferably a mammal, more preferably a human. A pharmaceutically acceptable carrier also includes a pharmaceutically acceptable excipient.
-
FIG. 1 : Inducible expression of CTLA-4 antagonist in primary T cells analyzed by FACS. Human peripheral blood T cells were transduced with pSIN-(NFAT)6-αCTLA-4 and incubated for 48 h at 37° C. under activatory conditions by utilizing plates precoated with anti-CD3 mAb and anti-CD28 mAb. Cells were stained extracellularly using a FITC-labeled anti-CD3 antibody, and intracellulary—after fixation and permeabilization—for SEQ ID NO: 28 utilizing a primary polyclonal anti-NGAL rabbit antibody, followed by secondary staining with a Dylight594-conjugated anti-rabbit IgG antibody. Immunofluorescence was analyzed using a FACS instrument. A detailed protocol of the experiment is provided in Example 1. -
FIG. 2 : Detection of functional T-cell secreted CTLA-4 antagonist by ELISA. The supernatant of human peripheral blood T cells, mock-transduced or transduced with pSIN-(NFAT)6-αCTLA-4 and cultured under activatory conditions (cf. Example 1 andFIG. 1 ) was analyzed for the presence of functionally active CTLA-4 antagonist SEQ ID NO: 28 by ELISA. Recombinant human CTLA-4 was coated on an ELISA plate as described in Example 1, and culture supernatants of transduced and mock-transduced T cells were added. Plate-bound SEQ ID NO: 28 was detected by primary staining with an anti-NGAL polyclonal antibody from the rabbit, secondary staining using goat anti rabbit IgG-HRP and addition of a chromogenic HRP substrate as described in Example 1. -
FIG. 3 : Detection of functional T-cell secreted CTLA-4 antagonist by FACS of CTLA-4-positive CHO cells. The supernatant of Jurkat cells transduced with pSIN-(NFAT)6-αCTLA-4 and cultured under activatory conditions (cf. Example 2) was analyzed for the presence of functionally active CTLA-4 antagonist SEQ ID NO: 28 by incubation of the supernatant with CHO cells stably expressing CTLA-4 on their surface (CHO::CTLA-4). As a control, CHO::CTLA-4 were incubated with isotype control. Cell-bound SEQ ID NO: 28 that had been excreted by the Jurkat cells was then detected using anti-NGAL polyclonal antibody followed by secondary staining with a fluorescently labeled anti-rabbit Antibody as described in Example 2. -
FIG. 4 : Inducible expression of Fc fusion of CTLA-4 antagonist in primary T cells analyzed by FACS. Human peripheral blood T cells were transduced with pSIN-(NFAT)6-αCTLA-4-Fc and incubated for 48 h at 37° C. under activatory conditions by utilizing plates precoated with anti-CD3 mAb and anti-CD28 mAb. Cells were stained extracellularly using a FITC-labeled anti-CD3 antibody, and intracellulary—after fixation and permeabilization—for SEQ ID NO: 29 utilizing a primary polyclonal anti-NGAL antibody, followed by secondary staining with a Dylight594-conjugated anti-rabbit IgG antibody. Immunofluorescence was analyzed using a FACS instrument. Cf. Example 3 for details. -
FIG. 5 : Detection of functional T-cell secreted Fc fusion of a CTLA-4 antagonist by ELISA. The supernatant of human peripheral blood T cells, mock-transduced or transduced with pSIN-(NFAT)6-αCTLA-4-Fc and cultured under activatory conditions (cf. Example 3 andFIG. 4 ) was analyzed for the presence of the functionally active Fc fusion of the CTLA-4 antagonist SEQ ID NO: 29 by ELISA. Recombinant human CTLA-4 was coated on an ELISA Plate as described in Example 3, and culture supernatants of transduced and mock-transduced T cells were added. Plate-bound SEQ ID NO: 29 was detected by primary staining with an anti-NGAL polyclonal rabbit antibody, secondary staining using goat anti rabbit IgG-HRP and addition of a chromogenic HRP substrate as described in Example 3. - A. CAR-Transduced T Cell Containing an Anticalin as CAR Moiety
- Generation of CAR-Transduced T-Cells
- As a first step, the coding DNA corresponding to the SEQ ID NOs: 8-11 is generated by gene synthesis. For that purpose, a set of codons that is compatible with mammalian expression needs to be employed. The DNA stretches encoding the CARs are equipped with suitable restriction sites at the N- and C-termini to allow cloning into the retroviral expression vector pBullet (Willemsen et al. 2000), or, alternatively, pSTITCH (Weijtens et al. 1998). The gene synthesis is straightforwardly performed using a commercial provider, and the resulting DNA is cloned into pBullet using standard molecular biology methods.
- To obtain retroviral particles suitable for peripheral blood T-cell transduction we proceed as described (Weijtens et al. 1998). In short, DNA of the respective retroviral vector (pBullet or pSTITCH) containing the Anticalin-based CAR (6 mg DNA) is co-transfected into 293T cells by calcium phosphate co-precipitation with the retroviral helper plasmid DNA pHIT60 and pCOLT (each 6 mg DNA) encoding the MuLV gag and pol genes (pHIT60) and the GALV envelope gene (pCOLT), respectively, under control of the CMV promotor/enhancer (Weijtens et al. 1998). This procedure results in transient production of high titers of infectious retrovirus. Peripheral blood lymphocytes from healthy donors are isolated by density centrifugation and cultured for 48 h in RPMI-1640 medium supplemented with 10% FCS in the presence of IL-2 (400 U/ml) and OKT3 MAb (100 ng/ml). Cells are harvested, washed, resuspended in medium with IL-2 (400 U/ml), and co-cultured for 48 h with 293T cells that are transiently transfected as described above and therefore produce and excrete the retroviral transducing particles into the medium. In the final step, T cells are harvested and subsequently tested for successful CAR expression and functional assays as described further below.
- Characterisation of CAR-Transduced T-Cells—FACS
- Successful cell-surface expression of the chimeric antigen receptor on transduced T-cells is demonstrated by multicolor immunofluorescence, using the fluorescence-labeled, soluble extracellular domains of the respective targets human c-Met and human GPC-3, or alternatively, an anti-CD8 antibody detected by a suitable fluorescence-labeled secondary antibody. Immunofluorescence is analyzed using a suitable FACS instrument. Transduced cells labeled in this manner show a clear fluorescence signal in FACS compared to a non-transduced T-cell control.
- Characterisation of CAR-Transduced T-Cells—T-Cell Activation on Coated Plates
- To investigate whether signaling via the CAR leads to T-cell activation, we can conduct two experiments with coated protein to cluster the CAR on the transduced T-cells. This is either accomplished by plastic-coating an anti-CD8 antibody on a microtiter plate or by coating the target protein of the CAR (either c-Met or GPC-3 depending on the construct). A non-binding IgG1 Antibody serves as the negative control. The microtiter plates are coated with 1-50 μg/ml of the anti-CD8 antibody, c-Met, GPC-3 or control IgG1. Transduced and non-transduced peripheral blood T cells (1-3 105 cells/well) are incubated for 48 h at 37° C. in the coated microtiter plates. After 48 h, culture supernatants are analyzed for IFN-g by ELISA using a suitable commercial kit.
- The supernatant of CAR-expressing T-cells incubated in plastic well coated with either an anti-CD8-antibody or with the targets c-Met or GPC3, respectively, contains significantly more IFN-gamma than that of the controls. The latter are either T-cells not expressing a CAR, or CAR-transduced T-cells incubated in wells coated with the control IgG1.
- Characterisation of CAR-Transduced T-Cells—T-Cell Activation by Coculture with Target-Expressing Mammalian Cells
- In a second step, which is more akin to the desired situation in-vivo, different amounts of receptor-grafted T cells (1.25 to 10×104 cells/well) are co-cultured for 48 h with GPC-3+ or c-Met+ immortal tumor cell lines. Target-positive cell lines that can be used are HepG2 (GPC-3) and H441 (c-Met), and examples for GPC-3/c-Met-negative control cells are SK-HEP-1 (GPC-3) and A2870 (c-Met). As a further control for T-cell CAR specificity, we co-incubate non-transduced T-cells with GPC-3+ or c-Met+ cells. In all cases, cells are coincubated for 48 h, and supernatants are subsequently analysed for IFN-gamma expression.
- We find that the supernatant of CAR-transduced T-cells cocultured with the target-matched immortal cell lines contains significantly more IFN-gamma than the controls, clearly showing CAR-specific T-cell activation.
- Characterisation of CAR-Transduced T-Cells—Measurement of Cytotoxicity of T-Cells Cocultured with Target-Expressing Mammalian Cells
- Specific cytotoxicity of receptor-grafted T cells to target cells is measured by lactate dehydrogenase (LDH) release. Briefly, receptor-grafted and non-transduced T cells (1.25-10×104 cells/well) are co-cultured for 12 hr with 1-3×104 cells/well of either GPC3+, c-Met+, GPC3− or c-Met-cells, respectively, in round-bottomed microtiter plates. Effector-to-target ratios range from 10 to 1. To determine spontaneous, baseline LDH release, T cells are cultured without target cells. LDH in culture supernatants (100 ml/well) is determined utilizing a suitable commercial kit. The percentage of specific cytotoxicity is calculated by correcting for spontaneous release and normalizing to the maximum LDH release determined after addition of 1% (v/v) Nonidet P-40.
- We find that the specific cytotoxicity of the Anticalin-CAR based T-cells is significantly higher in the presence of the matched, target-positive tumor cell lines than in the presence of the target-negative tumor cell lines. Additionally, non-transduced T-cell preparations show a much reduced specific cytotoxicity compared to the transduced T-cells.
- In-Vivo Characterization of CAR-Transduced T-Cells
- The following experiment is performed in analogous fashion to Chmielewski et al. 2011b. In brief, human T cells are isolated from peripheral blood by magnetic activated cell sorting, using human CD3+ beads. The transduction-competent retroviri encoding the c-Met- and GPC3-specific CARs are generated and used for transduction of human T-cells as described above. For the mouse studies, we use the NIH-Ill mouse (Charles River), which is deficient in NK cells, B cells, and T cells. MC38 cells stably transfected with human GPC-3 or c-Met (9×105 cells/mouse) are s.c. coinjected together with the engineered, specificity matched T cells (2×105 T cells/mouse) into NIH-Ill mice (6-7 mice per group). Alternatively, tumors are induced by s.c. injection of MC38 cells stably transfected with human GPC-3 or c-Met and matched CAR-T cells are applied by i.v. injection at day 6. The respective experiment using CAR-T cells and the mismatched transfected tumor cell line serves as a negative control. Tumor growth is monitored daily by external measurement with a digital caliper.
- In the experiments, we find a significant delay in tumor outgrowth when target-positive cells and target-specific CAR-T cells are injected into the same mouse compared to negative controls.
- B. CAR-Transduced T Cell Containing an Anticalin as TAILS Moiety
- Generation of CAR-Transduced T-Cells Capable of TAILS (CAR/TAILS-T)
- The construct GPC3-CD137-CD3zeta (SEQ ID NO: 11) is generated and cloned into a suitable retroviral vector as described above. Retroviral particles suitable for peripheral blood T-cell transduction are obtained by co-transfection with retroviral helper particles into 293T cells, also as described above.
- A vector suitable to facilitate retroviral expression of the T-cell activation inducible Anticalin-based CTLA4-antagonist (“αCTLA-4”) is obtained by exchanging the mature IL-12 sequence in pSIN-(NFAT)6-IL-12 (Chmielewski et al. 2011b) by the Anticalin sequence (SEQ ID NO: 3) using standard molecular biology methods, to obtain pSIN-(NFAT)6-αCTLA-4. Note that the leader sequence of IL-12 remains in the construct to serve as a secretion signal.
- In a 2-step transduction procedure, T cells are retrovirally transduced with the αCTLA-4 expression cassette and positively selected in the presence of 0.5 mg/mL Geneticin (G418) and IL-2 (500 IU/mL) on plates precoated with anti-CD3 mAb OKT-3 and anti-CD28 mAb 15E8 as previously described (Chmielewski et al. 2011b). Geneticin-resistant clones are transduced with the GPC-3-specific CAR GPC3-CD137-CD3zeta.
- Characterization of CAR/TAILS T-Cells
- Successful cell-surface expression of the chimeric antigen receptor on the doubly transduced CAR/TAILS T-cells is performed as described above: we employ multicolor immunofluorescence, using the fluorescence-labeled, soluble extracellular domain of human GPC-3, or alternatively, an anti-CD8 antibody detected by a suitable fluorescence-labeled secondary antibody. Immunofluorescence is analyzed using a suitable FACS instrument. Transduced cells labeled in this manner show a clear fluorescence signal in FACS compared to a non-transduced T-cell control.
- Characterisation of CAR/TAILS-T-Cells—T-Cell Activation on Coated Plates and CTLA-4 Secretion
- To investigate whether signaling via the CAR leads to T-cell activation in the CAR/TAILS T-cell, we conduct two experiments with coated protein to cluster the CAR on the transduced T-cells, in analogy to the experiment described above. This is either accomplished by plastic-coating an anti-CD8 antibody on a microtiter plate, or, alternatively, by coating GPC-3. A non-binding IgG1 Antibody serves as the negative control. The microtiter plates are coated with 1-50 μg/ml of the anti-CD8 antibody, GPC-3 or control IgG1. Transduced and non-transduced peripheral blood T cells (1-3 105 cells/well) are incubated for 48 h at 37° C. in the coated microtiter plates. After 48 h, culture supernatants are analyzed for IFN-gamma by ELISA using a suitable commercial kit. Further, supernatants are analyzed for the presence of the Anticalin-based CTLA-4 antagonist by a suitable ELISA setup.
- The supernatant of CAR-expressing T-cells incubated in plastic well coated with either an anti-CD8-antibody or with GPC3, respectively, contains significantly more IFN-gamma and Anticalin-based CTLA-4 antagonist than that of the controls. The latter are either T-cells not expressing a CAR, but transduced with αCTLA-4 only (SEQ ID NO: 3), or doubly transduced CAR/TAILS T-cells incubated in wells coated with the control IgG1.
- Characterization of CAR/TAILS-T-Cells—T-Cell Activation by Coculture with Target-Expressing Mammalian Cells
- In a second step, which is more akin to the desired situation in-vivo, different amounts of receptor-grafted T cells (1.25 to 10×104 cells/well) are co-cultured for 48 h with GPC-3+ immortal tumor cell lines. The GPC3-positive cell line used is HepG2, and the GPC-3-negative control cell line is SK-HEP-1. As a further control for T-cell CAR specificity, we co-incubate with GPC-3+ cells with T-cells not expressing a CAR, but transduced with αCTLA-4 (SEQ ID NO: 3) only. In all cases, cells are coincubated for 48 h, and supernatants are subsequently analysed for IFN-γ expression and the expression of the anti-CTLA-4 Anticalin.
- We find that the supernatant of CAR-transduced T-cells co-cultured with the target-matched immortal cell lines contains significantly more IFN-γ and anti-CTLA-4 Anticalin than the controls, clearly showing CAR-specific T-cell activation and T-cell activation induced secretion of the CTLA-4 antagonist.
- Characterisation of CAR/TAILS-T-Cells—Measurement of Cytotoxicity of T-Cells Cocultured with Target-Expressing Mammalian Cells
- Specific cytotoxicity of receptor-grafted T cells to target cells is measured by lactate dehydrogenase (LDH) release as described above. Briefly, doubly transduced CAR/TAILS T-cells, CAR-transduced-cells or non-transduced T cells (1.25-10×104 cells/well) are co-cultured for 12 hr with 1-3×104 cells/well of either GPC3+, or GPC3− cells, respectively, in round-bottomed microtiter plates. Effector-to-target ratios range from 10 to 1. To determine spontaneous, baseline LDH release, T cells are cultured without target cells. LDH in culture supernatants (100 ml/well) is determined utilizing a suitable commercial kit. The percentage of specific cytotoxicity is calculated by correcting for spontaneous release and normalizing to the maximum LDH release determined after addition of 1% (v/v) Nonidet P-40.
- We find that the specific cytotoxicity of the doubly transduced CAR/TAILS T-cells is at least retained compared to the CAR-only based T-cells, and significantly higher in the presence of the matched, target-positive tumor cell lines than in the presence of the target-negative tumor cell lines. Additionally, non-transduced T-cell preparations show a much reduced specific cytotoxicity compared to the transduced T-cells.
- Characterisation of CAR/TAILS-T-Cells—In-Vivo Experiment
- The following experiment is performed in analogy to Chmielewski et al. 2011b and above, but utilizing a different tumor cell line. In brief, human T cells are isolated from peripheral blood by magnetic activated cell sorting, using human CD3+ beads. The GPC-3-specific and αCTLA-4-Anticalin secreting CAR/TAILS-T cells are generated as described herein. For the mouse studies, we use the NIH-Ill mouse (Charles River), which is deficient in NK cells, B cells, and Tcells. AT3 cells stably transfected with human GPC-3 (9×105 cells/mouse) are s.c. coinjected together with the engineered CAR/TAILS-T cells (2×105 T cells/mouse) into NIH-Ill mice (6-7 mice per group). Alternatively, tumors are induced by s.c. injection of AT3 cells stably transfected with human GPC-3 and CAR/TAILS-T cells are applied by i.v. injection at day 6. Negative controls include a vehicle injection control, the respective experiment using CAR/TAILS-T and mock-transfected AT3 cells, and the respective experiment utilizing CAR-T cells that do not secrete α-CTLA-4 Anticalin. Tumor growth is monitored daily by external measurement with a digital caliper.
- In the experiments, we find a significant delay in tumor outgrowth when target-positive cells and target-specific CAR/TAILS-T cells are injected into the same mouse compared to the controls.
- A vector suitable to facilitate retroviral expression of the T-cell activation inducible Anticalin-based CTLA4-antagonist (“αCTLA-4”) was obtained by replacing the IL-12 sequence in pSIN-(NFAT)6-IL-12 (Chmielewski et al. 2011b) by the Anticalin sequence (SEQ ID NO: 3) with a Strep-tag II (SEQ ID NO: 27) using standard molecular biology methods, to obtain pSIN-(NFAT)6-αCTLA-4. The leader sequence of the mouse immunoglobulin kappa light chain (GenBank Acc #CAB46127.1) instead of the IL-12 leader was employed in the construct to serve as a secretion signal. The final amino acid sequence corresponds to SEQ ID NO: 23, encoded by the DNA sequence SEQ ID NO: 24.
- To obtain retroviral particles suitable for peripheral blood T-cell transduction we proceeded as described (Weijtens et al. 1998; Cheadle et al., 2012). In short, DNA of pSIN-(NFAT)6-αCTLA-4 (6 mg DNA) was co-transfected into 293T cells by calcium phosphate co-precipitation with the retroviral helper plasmid DNA pHIT60 and pCOLT (each 6 mg DNA) encoding the MuLV gag and pol genes (pHIT60) and the GALV envelope gene (pCOLT), respectively, under control of the CMV promotor/enhancer (Weijtens et al. 1998). This procedure resulted in the production of high titers of infectious retrovirus.
- In order to obtain T cells transduced with pSIN-(NFAT)6-αCTLA-4, peripheral blood lymphocytes from healthy donors were isolated by density centrifugation in a Ficoll-Paque (GE Healthcare) density gradient and cultured for 48 h in RPM11640 medium supplemented with 10% (v/v) FCS in the presence of IL-2 (400 U/ml) and OKT3 mAb (100 ng/ml). Cells were harvested, washed, resuspended in medium with IL-2 (400 U/ml), and co-cultured for 48 h with 293T cells that were transiently transfected as described above and therefore released the retrovirus particles into the medium. In the final step, T cells were harvested. The same procedure was also carried out using mock-transduction, i.e. transducing the gag, pol and env genes of the retrovirus without the expression cassette.
- To investigate whether activation of the transduced T cells induced the secretion of SEQ ID NO: 28 (the mature processed form of SEQ ID NO:24), 5×106 cells peripheral blood T cells in 10 mL medium—transduced or mock-transduced—were incubated for 48 h at 37° C. on Nunc™ OmniTray™ plates precoated with anti-CD3 mAb OKT-3 and anti-CD28 mAb 15E8 as previously described (Chmielewski et al. 2011b). To determine both successful expression of the anti-CTLA-4 Anticalin (SEQ ID NO: 28) on the transduced TAILS T-cells and the fraction of cells that was successfully transduced, we employed multicolor immunofluorescence using a FITC-labeled anti-CD3 antibody (anti-human CD3, clone OKT3 (ATCC)), and a polyclonal anti-NGAL rabbit antibody, followed by secondary staining with an anti-rabbit antibody labeled with the fluorescent dye Dylight594. The detailed protocol was as follows, performing all incubation steps protected from light: Transduced T cells were stained for CD3 using the FITC-labeled anti-CD3 antibody for 30 minutes at 4° C., followed by fixation and permeabilization by resuspending in 250 μl BD Cytofix/Cytoperm solution and incubating for 20 minutes at 4° C., followed by washing twice in a buffer containing the cell permeabilizing agent saponin (BD Perm/Wash™ buffer, Cat. 554723). Intracellular staining of SEQ ID NO: 28 was then achieved by thoroughly resuspending the fixed and permeabilized cells in 50 μL of a saponin-containing buffer (BD Perm/Wash™ buffer) containing 0.5 μg anti-NGAL mAb per 1 million cells, washing twice with saponin-containing BD Perm/Wash™ buffer, and incubation for 30 minutes at 4° C. with 1 μg of the Dylight594-conjugated anti-rabbit IgG antibody per 1 million cells. Finally, cells were washed twice with PBS buffer and immunofluorescence was analyzed using a BD FACS Canto II instrument. In the FACS analysis, transduced cells labeled in this manner showed a clearly positive anti-NGAL reactivity (
FIG. 1A ) compared to the mock-transduced T-cell control (FIG. 1B ). We found that according to a threshold set based on the mock-transduced T-cell control (FIG. 1B ), 52% of primary T cells had been transduced and were found to be SEQ ID NO: 28-positive. - Supernatants were analyzed for the presence of functionally active Anticalin-based CTLA-4 antagonist by ELISA as described in the following: Recombinant human CTLA-4 at a concentration of 5 μg/mL in PBS was added to each well of a 384 well ELISA plate and incubated over night at 4° C. All following steps were performed with 1 h incubation time and repeated washing with PBS/0.05% (w/v) Tween20 (PBS-T). In the first step, plates were blocked (BSA 2% (w/v) in PBS-T/0.1% (w/v) Tween 20) and 20 μL of culture supernatants were added. Subsequently, rabbit anti-NGAL polyclonal antibody at a concentration of 1 μg/mL in PBS/0.1% (w/v) Tween20/2% (w/v) BSA was added, and bound antibody was detected using goat anti-rabbit IgG-HRP in a dilution of 1:5,000 in PBS/0.1% (w/v) Tween20/2% (w/v) BSA. Chromogenic 3,3′, 5,5;-tetramethylbenzidine (TMB) substrate was used as a detection agent according to the manufacturer's instructions (Life Technologies). Fluorescence signals in RFU (relative fluorescence units) were measured using a plate fluorescence reader.
- We found a clear anti-CTLA-4 reactivity in supernatants of pSIN-(NFAT)6-αCTLA-4-transduced and activated T cells compared to the supernatants of mock-transfected cells (
FIG. 2 ). Note that the mock-transfected cells showed a background fluorescence signal due to nonspecific matrix effects, which was, however, significantly below the positive ELISA signal of the supernatant of pSIN-(NFAT)6-αCTLA-4-transduced cells. - The experiment of this example was carried out in analogy to Example 1, but using the Jurkat T cell line instead of primary T cells. In short, Jurkat T cells were transduced with pSIN-(NFAT)6-αCTLA-4 as described in Example 1, and incubated in medium on plates precoated with anti-CD3 mAb OKT-3 and anti-CD28 mAb 15E8 as previously described (Chmielewski et al. 2011b).
- The culture supernatant containing the anti-CTLA-4 Anticalin (SEQ ID NO: 28) was obtained from the Jurkat cells by centrifugation and added to Chinese Hamster Ovary cells stably transfected with CTLA-4 (CHO::CTLA-4). As a control, CHO::CTLA-4 were incubated with an isotype control antibody. Cell-bound SEQ ID NO: 28 was detected using anti-NGAL rabbit polyclonal antibody followed by staining with anti-rabbit antibody labeled with the fluorescent dye Dylight594. In the FACS fluorescence histogram (
FIG. 3 ), a clear shift in fluorescence intensity for cells incubated with the supernatant from pSIN-(NFAT)6-αCTLA-4-transduced Jurkat cells compared to the isotype control is evident, showing the binding of excreted SEQ ID NO: 28 to CTLA-4 positive cells. - The experiments described here were performed in full analogy to Example 1, but utilizing SEQ ID NO: 25, which is a fusion of the Anticalin-based CTLA-4 Antagonist (SEQ ID NO: 23) to the Fc fragment of a human IgG1 antibody. The DNA sequence encoding SEQ ID NO: 25 is provided by SEQ ID NO: 26. The corresponding inducible expression vector pSIN-(NFAT)6-αCTLA-4-Fc was generated by standard molecular biology methods.
- Generation of retroviral particles suitable for peripheral blood T-cell transduction, as well as T cell preparation and transduction with pSIN-(NFAT)6-αCTLA-4-Fc, were performed as described in Example 1. Mock-transduction was used as a control. To investigate whether activation of the transduced T cells lead to secretion of SEQ ID NO: 29 (the mature processed form of SEQ ID NO:25), we proceeded with multicolor FACS staining of the targets CD3 and SEQ ID NO: 29 as described in Example 1. In the FACS analysis, transduced and fluorescence-labeled cells showed a clearly positive anti-NGAL reactivity (
FIG. 4A ) compared to the mock-transduced T-cell control (FIG. 4B ). We found that according to a threshold set based on the mock-transduced T-cell control (FIG. 4B ), 63% of primaryT cells had been transduced and were found to be SEQ ID NO: 29-positive. - Supernatants were analyzed for the presence of functionally active Anticalin-based CTLA-4 antagonist αCTLA-4-Fc (SEQ ID NO: 29) by ELISA as described in Example 1. In the ELISA, we found a clear anti-CTLA-4 reactivity for T cells transduced with pSIN-(NFAT)6-αCTLA-4-Fc and cultured under activatory conditions, compared to the mock-transfected cells (
FIG. 5 ). Note that the mock-transfected cells showed a background fluorescence signal due to nonspecific matrix effects, which was, however, significantly below the positive ELISA signal of the supernatant of pSIN-(NFAT)6-αCTLA-4-Fc-transduced cells. -
TABLE 1 Amino acid SEQ ID NO's in this application and corresponding SEQ ID NO Amino acid sequence DNA sequence SEQ ID NO: 1 SEQ ID NO: 12 SEQ ID NO: 2: SEQ ID NO: 13 SEQ ID NO: 3 SEQ ID NO: 14 SEQ ID NO: 4: SEQ ID NO: 15 SEQ ID NO: 5 SEQ ID NO: 16 SEQ ID NO: 6 SEQ ID NO: 17 SEQ ID NO: 7 SEQ ID NO: 18 SEQ ID NO: 8 SEQ ID NO: 19 SEQ ID NO: 9 SEQ ID NO: 20 SEQ ID NO: 10 SEQ ID NO: 21 SEQ ID NO: 11 SEQ ID NO: 22 SEQ ID NO: 23 SEQ ID NO: 24 SEQ ID NO: 25 SEQ ID NO: 26 SEQ ID NO: 27 SEQ ID NO: 28 SEQ ID NO: 29 SEQ ID NO: 30 -
- Birkholz, K., et al. (2009). “Transfer of mRNA encoding recombinant immunoreceptors reprograms CD4+ and CD8+ T cells for use in the adoptive immunotherapy of cancer.” Gene Ther 16(5): 596-604.
- Brocks, B., et al. (1997). “A TNF receptor antagonistic scFv, which is not secreted in mammalian cells, is expressed as a soluble mono- and bivalent scFv derivative in insect cells.” Immunotechnology 3(3): 173-84.
- Cheadle, E J., et al. (2012). “Chimeric antigen receptors for T-cell based therapy.” Chapter 36, in: “Antibody engineering: methods and protocols”, 2nd Edition, Ed. P. Chames, Meth. Mol. Biol. 907, 645-666.
- Chen, L., et al. (2013). “Molecular mechanisms of T cell co-stimulation and co-inhibition.” Nat Rev Immunol 13(4): 227-42.
- Chmielewski, M., et al. (2012). “T cells that target carcinoembryonic antigen eradicate orthotopic pancreatic carcinomas without inducing autoimmune colitis in mice.” Gastroenterology 143(4): 1095-107 e2.
- Chmielewski, M., et al. (2011)a. “CD28 signalling does not affect the activation threshold in a chimeric antigen receptor-redirected T-cell attack.” Gene Ther 18(1): 62-72.
- Chmielewski, M., et al. (2013)a. “Antigen-Specific T-Cell Activation Independently of the MHC: Chimeric Antigen Receptor-Redirected T Cells.” Front Immunol 4: 371.
- Chmielewski, M., et al. (2014). “Of CARs and TRUCKs: chimeric antigen receptor (CAR) T cells engineered with an inducible cytokine to modulate the tumor stroma.” Immunol Rev 257(1): 83.90.
- Chmielewski, M., et al. (2011)b. “IL-12 release by engineered T cells expressing chimeric antigen receptors can effectively Muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression.” Cancer Res 71(17): 5697-706.
- Chmielewski, M., et al. (2013)b. “T cells redirected by a CD3zeta chimeric antigen receptor can establish self-antigen-specific tumour protection in the long term.” Gene Ther 20(2): 177-86.
- Ewert, S., et al. (2003)a. “Structure-based improvement of the biophysical properties of immunoglobulin VH domains with a generalizable approach.” Biochemistry 42(6): 1517-28.
- Ewert, S., et al. (2003)b. “Biophysical properties of human antibody variable domains.” J Mol Biol 325(3): 531-53.
- Haisma, H. J., et al. (1998)a. “Construction and characterization of a fusion protein of single-chain anti-carcinoma antibody 323/A3 and human beta-glucuronidase.” Cancer Immunol Immunother 45(5): 266-72.
- Haisma, H. J., et al. (1998)b. “Construction and characterization of a fusion protein of single-chain anti-CD20 antibody and human beta-glucuronidase for antibody-directed enzyme prodrug therapy.” Blood 92(1): 184-90.
- Hombach, A., et al. (2010). “Adoptive immunotherapy with genetically engineered T cells: modification of the IgG1 Fc ‘spacer’ domain in the extracellular moiety of chimeric antigen receptors avoids ‘off-target’ activation and unintended initiation of an innate immune response.” Gene Ther 17(10): 1206-13.
- Hombach, A. A., et al. (2013). “Arming cytokine-induced killer cells with chimeric antigen receptors: CD28 outperforms combined CD28-OX40 “super-stimulation”.” Mol Ther 21(12): 2268-77.
- Jaalouk, D. E., et al. (2006). “A self-inactivating retrovector incorporating the IL-2 promoter for activation-induced transgene expression in genetically engineered T-cells.” Virol J 3: 97.
- Kershaw, M. H., et al. (2006). “A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer.” Clin Cancer Res 12(20 Pt 1): 6106-15.
- Kofler, D. M., et al. (2011). “CD28 costimulation impairs the efficacy of a redirected t-cell antitumor attack in the presence of regulatory t cells which can be overcome by preventing Lck activation.” Mol Ther 19(4): 760-7.
- Maiti, S. N., et al. (2013). “Sleeping beauty system to redirect T-cell specificity for human applications.” J Immunother 36(2): 112-23.
- Maliar, A., et al. (2012). “Redirected T cells that target pancreatic adenocarcinoma antigens eliminate tumors and metastases in mice.” Gastroenterology 143(5): 1375-84 e1-5.
- Maude, S. L., et al. (2014). “Chimeric antigen receptor T cells for sustained remissions in leukemia.” N Engl J Med 371(16): 1507-17.
- Pegram, H. J., et al. (2012). “Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning.” Blood 119(18): 4133-41.
- Pegram, H. J., et al. (2014). “IL-12-secreting CD19-targeted cord blood-derived T cells for the immunotherapy of B-cell acute lymphoblastic leukemia.” Leukemia.
- Peipp, M., et al. (2004). “Efficient eukaryotic expression of fluorescent scFv fusion proteins directed against CD antigens for FACS applications.” J Immunol Methods 285(2): 265-80.
- Rosenberg, S. A., et al. (1990). “Gene transfer into humans—immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction.” N Engl J Med 323(9): 570-8.
- Schaefer, J. V., et al. (2012). “Transfer of engineered biophysical properties between different antibody formats and expression systems.” Protein Eng Des Sel 25(10): 485-506.
- Shi, H., et al. (2014). “Chimeric antigen receptor for adoptive immunotherapy of cancer: latest research and future prospects.” Mol Cancer 13: 219.
- Textor, A., et al. (2014). “Efficacy of CAR T-cell Therapy in Large Tumors Relies upon Stromal Targeting by IFNgamma.” Cancer Res 74(23): 6796-805.
- Weijtens, M. E., et al. (1998). “A retroviral vector system ‘STITCH’ in combination with an optimized single chain antibody chimeric receptor gene structure allows efficient gene transduction and expression in human T lymphocytes.” Gene Ther 5(9): 1195-203.
- Willemsen, R. A., et al. (2000). “Grafting primary human T lymphocytes with cancer-specific chimeric single chain and two chain TCR.” Gene Ther 7(16): 1369.77.
- Zhang, L., et al. (2011). “Improving adoptive T cell therapy by targeting and controlling IL-12 expression to the tumor environment.” Mol Ther 19(4): 751-9.
- Zhao, Y., et al. (2006). “Transduction of an HLA-DP4-restricted NY-ESO-1-specific TCR into primary human CD4+ lymphocytes.” J Immunother 29(4): 398-406.
Claims (17)
1. A modified T cell expressing and secreting a specific binding polypeptide comprising a lipocalin mutein selected from the group consisting of a mutein of human tear lipocalin (hTlc) and a mutein of human neutrophil gelatinase-associated lipocalin (hNGAL), wherein the lipocalin mutein is modified such that it is specific for a target other than the natural target of hTlc and hNGAL, respectively.
2. The modified T cell of claim 1 , wherein the modified T cell secretes the hTlc or the hNGAL mutein upon activation.
3. The modified T cell of claim 1 , wherein the lipocalin mutein is an hNGAL mutein.
4. The modified T cell of claim 3 , wherein the lipocalin mutein is an hNGAL mutein having at least 80% sequence identity to mature, wild-type hNGAL (SEQ ID NO: 30).
5. The modified T cell of claim 4 , wherein the hNGAL mutein retains a supersecondary or tertiary structure of mature, wild-type hNGAL (SEQ ID NO: 30).
6. The modified T cell of claim 4 , wherein the hNGAL mutein comprises an amino acid substitution at each of A40, Q49, L70, R72, K73, D77, W79, R81, L103, K125, S127, and Y132 relative to mature, wild-type hNGAL (SEQ ID NO: 30).
7. The modified T cell of claim 4 , wherein the hNGAL mutein comprises the amino acid sequence of mature, wild-type hNGAL (SEQ ID NO: 30) but for from 13 to 26 amino acid substitutions relative to mature, wild-type hNGAL, wherein at least A40, Q49, L70, R72, K73, D77, W79, R81, C87, L103, K125, S127, and Y132 are replaced with a different amino acid residue.
8. The modified T cell of claim 7 , wherein the hNGAL mutein further comprises at least one additional amino acid replacement at a position selected from E44, K46, D47, K50, N65, F71, P101, G102, T104, V126, Q128, R130, and K134 relative to mature, wild-type hNGAL (SEQ ID NO: 30).
9. The modified T cell of claim 1 , wherein the lipocalin mutein is an hNGAL mutein having at least 80% sequence identity to SEQ ID NO: 3.
10. The modified T cell of claim 9 , wherein the hNGAL mutein comprises an amino acid substitution at each of A40, Q49, L70, R72, K73, D77, W79, R81, L103, K125, S127, and Y132 relative to mature, wild-type hNGAL (SEQ ID NO: 30).
11. The modified T cell of claim 9 , wherein the hNGAL mutein comprises the amino acid sequence of mature, wild-type hNGAL (SEQ ID NO: 30) but for from 13 to 26 amino acid substitutions relative to mature, wild-type hNGAL, wherein at least A40, Q49, L70, R72, K73, D77, W79, R81, C87, L103, K125, S127, and Y132 are replaced with a different amino acid residue.
12. The modified T cell of claim 1 , wherein the modified T cell further comprises a chimeric antigen receptor.
13. The modified T cell of claim 12 , wherein the chimeric antigen receptor comprises: i. an extracellular ligand binding domain; ii. at least one costimulatory domain selected from a CD28 domain, a CD137 domain, an ICOS domain, an OX40 domain, a CD27 domain, and a CD40 domain; and iii. a cytoplasmic domain comprising at least one intracellular signaling domain, wherein the at least one intracellular signaling domain comprises a CD3zeta signaling domain.
14. A modified T cell comprising: (a) single-domain, monomeric polypeptide comprising a mutein of human neutrophil gelatinase-associated lipocalin (hNGAL), wherein the hNGAL mutein has at least 80% sequence identity to mature, wild-type hNGAL (SEQ ID NO: 30); and (b) a chimeric antigen receptor.
15. The modified T cell of claim 14 , wherein the chimeric antigen receptor comprises: i. an extracellular ligand binding domain; ii. at least one costimulatory domain selected from a CD28 domain, a CD137 domain, an ICOS domain, an OX40 domain, a CD27 domain, and a CD40 domain; and iii. a cytoplasmic domain comprising at least one intracellular signaling domain, wherein the at least one intracellular signaling domain comprises a CD3zeta signaling domain.
16. A pharmaceutical composition comprising a modified T cell of claim 1 and optionally a pharmaceutically acceptable carrier or excipient.
17. A pharmaceutical composition comprising a modified T cell of claim 14 and optionally a pharmaceutically acceptable carrier or excipient.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/664,731 US20220296690A1 (en) | 2015-01-12 | 2022-05-24 | Engineered t cells and uses therefor |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP15000047.9 | 2015-01-12 | ||
EP15000047 | 2015-01-12 | ||
PCT/EP2016/050326 WO2016113203A1 (en) | 2015-01-12 | 2016-01-11 | Engineered t cells and uses therefor |
US201715542915A | 2017-07-11 | 2017-07-11 | |
US17/664,731 US20220296690A1 (en) | 2015-01-12 | 2022-05-24 | Engineered t cells and uses therefor |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/542,915 Continuation US11382963B2 (en) | 2015-01-12 | 2016-01-11 | Engineered T cells and uses therefor |
PCT/EP2016/050326 Continuation WO2016113203A1 (en) | 2015-01-12 | 2016-01-11 | Engineered t cells and uses therefor |
Publications (1)
Publication Number | Publication Date |
---|---|
US20220296690A1 true US20220296690A1 (en) | 2022-09-22 |
Family
ID=52434503
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/542,915 Active 2038-08-29 US11382963B2 (en) | 2015-01-12 | 2016-01-11 | Engineered T cells and uses therefor |
US17/664,731 Pending US20220296690A1 (en) | 2015-01-12 | 2022-05-24 | Engineered t cells and uses therefor |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/542,915 Active 2038-08-29 US11382963B2 (en) | 2015-01-12 | 2016-01-11 | Engineered T cells and uses therefor |
Country Status (3)
Country | Link |
---|---|
US (2) | US11382963B2 (en) |
EP (1) | EP3245288A1 (en) |
WO (1) | WO2016113203A1 (en) |
Families Citing this family (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10927154B2 (en) * | 2014-01-13 | 2021-02-23 | Pieris Pharmaceuticals Gmbh | Multi-specific polypeptide useful for localized tumor immunomodulation |
KR20170138574A (en) | 2015-05-04 | 2017-12-15 | 피어이스 파마슈티컬즈 게엠베하 | Anticancer fusion polypeptide |
US11242375B2 (en) | 2015-09-04 | 2022-02-08 | Memorial Sloan Kettering Cancer Center | Immune cell compositions and methods of use |
CA3007980A1 (en) | 2015-12-09 | 2017-06-15 | Memorial Sloan Kettering Cancer Center | Immune cell compositions and methods of using same |
US11549099B2 (en) * | 2016-03-23 | 2023-01-10 | Novartis Ag | Cell secreted minibodies and uses thereof |
US11446398B2 (en) | 2016-04-11 | 2022-09-20 | Obsidian Therapeutics, Inc. | Regulated biocircuit systems |
CA3034691A1 (en) | 2016-08-30 | 2018-03-08 | Memorial Sloan Kettering Cancer Center | Immune cell compositions and methods of use for treating viral and other infections |
US20190209615A1 (en) * | 2016-09-15 | 2019-07-11 | Virex Ab | T-cell immunotherapy |
WO2018148520A1 (en) * | 2017-02-11 | 2018-08-16 | The Regents Of The University Of California | Compositions and multiplexed systems for remote controlled gene expression and cell activation using acoustic mechanogenetics and methods for making and using them |
US11629340B2 (en) | 2017-03-03 | 2023-04-18 | Obsidian Therapeutics, Inc. | DHFR tunable protein regulation |
KR20200010181A (en) * | 2017-03-03 | 2020-01-30 | 옵시디안 테라퓨틱스, 인크. | CD19 Compositions and Methods for Immunotherapy |
WO2018165228A1 (en) * | 2017-03-08 | 2018-09-13 | Memorial Sloan Kettering Cancer Center | Immune cell compositions and methods of use |
EP3502130A1 (en) | 2017-12-20 | 2019-06-26 | St. Anna Kinderkrebsforschung | Ligand regulated protein-protein interaction system |
KR20200101942A (en) | 2017-12-20 | 2020-08-28 | 세인트 안나 킨더크렙스포르슝 | Ligand regulatory protein-protein interaction system |
US10869888B2 (en) | 2018-04-17 | 2020-12-22 | Innovative Cellular Therapeutics CO., LTD. | Modified cell expansion and uses thereof |
KR20210025525A (en) | 2018-06-22 | 2021-03-09 | 카이트 파마 인코포레이티드 | Chimeric transmembrane protein and uses thereof |
US20220348682A1 (en) | 2018-08-30 | 2022-11-03 | Innovative Cellular Therapeutics Holdings, Ltd. | Chimeric antigen receptor cells for treating solid tumor |
CN112680419A (en) * | 2018-11-16 | 2021-04-20 | 上海斯丹赛生物技术有限公司 | Chimeric antigen receptor cell secretion therapeutics |
US10918667B2 (en) | 2018-11-20 | 2021-02-16 | Innovative Cellular Therapeutics CO., LTD. | Modified cell expressing therapeutic agent and uses thereof |
CA3120153A1 (en) * | 2018-11-20 | 2020-05-28 | Innovative Cellular Therapeutics Holdings, Ltd. | Modified cell expressing therapeutic agent and uses thereof |
CA3150224A1 (en) | 2019-09-10 | 2021-03-18 | Obsidian Therapeutics, Inc. | Ca2 compositions and methods for tunable regulation |
US20230227568A1 (en) | 2020-06-05 | 2023-07-20 | Pieris Pharmaceuticals Gmbh | Multimeric immunomodulator targeting 4-1bb |
WO2023118497A1 (en) | 2021-12-22 | 2023-06-29 | Pieris Pharmaceuticals Gmbh | Novel il-18 variants |
WO2023170296A1 (en) * | 2022-03-11 | 2023-09-14 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Nucleic acid system to specifically reprogram b and t cells and uses thereof |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005019255A1 (en) | 2003-08-25 | 2005-03-03 | Pieris Proteolab Ag | Muteins of tear lipocalin |
WO2006056464A2 (en) | 2004-11-26 | 2006-06-01 | Pieris Ag | Compound with affinity for the cytotoxic t lymphocyte-associated antigen (ctla-4) |
WO2006078717A2 (en) * | 2005-01-19 | 2006-07-27 | Beth Israel Deaconess Medical Center | Lipocalin 2 for the treatment, prevention, and management of cancer metastasis, angiogenesis, and fibrosis |
CA2659413C (en) | 2006-08-01 | 2016-06-14 | Pieris Ag | Muteins of tear lipocalin and methods for obtaining the same |
WO2009095447A1 (en) * | 2008-01-30 | 2009-08-06 | Pieris Ag | Muteins of tear lipocalin having affinity to human c-met receptor tyrosine kinase and methods for obtaining the same |
LT2580236T (en) * | 2010-06-08 | 2019-06-25 | Pieris Pharmaceuticals Gmbh | Tear lipocalin muteins binding il-4 r alpha |
DK2640740T3 (en) * | 2010-11-15 | 2017-06-26 | Pieris Pharmaceuticals Gmbh | MUTEINS OF HUMAN LIPOCALIN 2 WITH AFFINITY FOR GLYPICAN-3 (GPC3) |
US9221885B2 (en) * | 2010-12-02 | 2015-12-29 | Pieris Ag | Muteins of human lipocalin 2 with affinity for CTLA-4 |
WO2012136685A1 (en) | 2011-04-04 | 2012-10-11 | Pieris Ag | Methods and compositions for anti-vegf and anti-c-met therapy |
US9522940B2 (en) | 2012-05-23 | 2016-12-20 | Pieris Pharmaceuticals Gmbh | Lipocalin muteins with binding-affinity for glypican-3 (GPC-3) and use of lipocalin muteins for target-specific delivery to cells expressing GPC-3 |
BR112015000638A2 (en) | 2012-07-13 | 2017-08-08 | Univ Pennsylvania | isolated nucleic acid sequence, cell, methods for stimulating a cell-mediated immune response to a target cell or tissue population in a mammal, for providing antitumor immunity in a mammal, for treating a mammal having a disease, disorder or condition associated with high expression of a tumor antigen |
WO2014153114A1 (en) * | 2013-03-14 | 2014-09-25 | Fred Hutchinson Cancer Research Center | Compositions and methods to modify cells for therapeutic objectives |
EP2970426B1 (en) * | 2013-03-15 | 2019-08-28 | Michael C. Milone | Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy |
TWI654206B (en) * | 2013-03-16 | 2019-03-21 | 諾華公司 | Treatment of cancer with a humanized anti-CD19 chimeric antigen receptor |
-
2016
- 2016-01-11 WO PCT/EP2016/050326 patent/WO2016113203A1/en active Application Filing
- 2016-01-11 EP EP16704392.6A patent/EP3245288A1/en active Pending
- 2016-01-11 US US15/542,915 patent/US11382963B2/en active Active
-
2022
- 2022-05-24 US US17/664,731 patent/US20220296690A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
US20180021418A1 (en) | 2018-01-25 |
US11382963B2 (en) | 2022-07-12 |
WO2016113203A1 (en) | 2016-07-21 |
EP3245288A1 (en) | 2017-11-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20220296690A1 (en) | Engineered t cells and uses therefor | |
AU2017301880B2 (en) | Immunomodulatory polypeptides and related compositions and methods | |
AU2015243927B2 (en) | Method and compositions for cellular immunotherapy | |
CN112074279B (en) | Humanized BCMA antibodies and BCMA-CAR-T cells | |
JP2022116230A (en) | Modified cells for immunotherapy | |
JP7438098B2 (en) | Binding molecules that modulate biological activities expressed by cells | |
WO2017219937A1 (en) | Car-t cell for efficiently and stably expressing inhibiting antibody and application thereof | |
JP2018518972A (en) | Masking chimeric antigen receptor T cells for tumor specific activation | |
CN109789164B (en) | Chimeric antigen receptor with GITR intracellular domain as co-stimulatory domain | |
JP7462611B2 (en) | OX40-binding polypeptides and uses thereof | |
CN110678480A (en) | Chimeric polypeptides and methods for altering their localization in cell membranes | |
CN117355600A (en) | Genetically modified NK cells and uses thereof | |
EP3875478A1 (en) | Novel non-immunogenic chimeric antigen receptors and uses thereof | |
CN114555791A (en) | IL-1 superfamily spatio-temporally restricted active cytokine-armed immunoresponsive cells | |
WO2021182929A1 (en) | Bcma-specific antibody and chimeric antigen receptor | |
CN109970859B (en) | Glyphican-3 specific antibody and CAR-T cell specific to the same | |
CN113474012A (en) | EPCAM antibodies and EPCAM-CAR-T cells | |
CN112442126A (en) | Monoclonal antibody of anti-human CS1 antigen and CAR-T cell thereof | |
Hinner | ENGINEERED T CELLS AND USES THEREFOR-Patent Information | |
CN113271965A (en) | CD37 antibody and CD37-CAR-T cells | |
CN116082507B (en) | Humanized BCMA antibodies and BCMA-CAR-T cells | |
Battin et al. | Engineered soluble, trimerized 4-1BBL variants as potent immunomodulatory agents | |
US20230183351A1 (en) | A targeting module comprising pd-l1 and/or pd-l2 for use in a method for stimulating a chimeric antigen receptor mediated immune response in a mammal | |
US20230310601A1 (en) | Chimeric antigen receptor-modified immune cells expressing a secretable bispecific trap protein and uses thereof | |
EP4159760A1 (en) | Antibody specific to cd22, and use thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |