WO2014153114A1 - Compositions and methods to modify cells for therapeutic objectives - Google Patents
Compositions and methods to modify cells for therapeutic objectives Download PDFInfo
- Publication number
- WO2014153114A1 WO2014153114A1 PCT/US2014/029137 US2014029137W WO2014153114A1 WO 2014153114 A1 WO2014153114 A1 WO 2014153114A1 US 2014029137 W US2014029137 W US 2014029137W WO 2014153114 A1 WO2014153114 A1 WO 2014153114A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- antibody
- cell
- cells
- cancer cell
- receptor
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 137
- 239000000203 mixture Substances 0.000 title claims abstract description 84
- 230000001225 therapeutic effect Effects 0.000 title abstract description 30
- 239000003550 marker Substances 0.000 claims abstract description 43
- 238000001727 in vivo Methods 0.000 claims abstract description 23
- 210000004027 cell Anatomy 0.000 claims description 402
- 239000002539 nanocarrier Substances 0.000 claims description 196
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 176
- 239000000427 antigen Substances 0.000 claims description 170
- 102000036639 antigens Human genes 0.000 claims description 169
- 108091007433 antigens Proteins 0.000 claims description 169
- 239000003795 chemical substances by application Substances 0.000 claims description 167
- 210000004698 lymphocyte Anatomy 0.000 claims description 148
- 239000002105 nanoparticle Substances 0.000 claims description 100
- 102000040430 polynucleotide Human genes 0.000 claims description 98
- 108091033319 polynucleotide Proteins 0.000 claims description 98
- 239000002157 polynucleotide Substances 0.000 claims description 98
- 206010028980 Neoplasm Diseases 0.000 claims description 96
- 230000008685 targeting Effects 0.000 claims description 93
- 230000027455 binding Effects 0.000 claims description 77
- 108090000623 proteins and genes Proteins 0.000 claims description 75
- 108020003175 receptors Proteins 0.000 claims description 74
- 102000005962 receptors Human genes 0.000 claims description 73
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims description 65
- 108091008874 T cell receptors Proteins 0.000 claims description 56
- 201000011510 cancer Diseases 0.000 claims description 56
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 claims description 50
- 239000013612 plasmid Substances 0.000 claims description 48
- 241000282414 Homo sapiens Species 0.000 claims description 43
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 43
- 201000010099 disease Diseases 0.000 claims description 42
- 108020004414 DNA Proteins 0.000 claims description 39
- 108010077850 Nuclear Localization Signals Proteins 0.000 claims description 37
- 230000014509 gene expression Effects 0.000 claims description 37
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 claims description 31
- 150000002632 lipids Chemical class 0.000 claims description 31
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 claims description 30
- 101000946843 Homo sapiens T-cell surface glycoprotein CD8 alpha chain Proteins 0.000 claims description 28
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 claims description 28
- 210000002540 macrophage Anatomy 0.000 claims description 26
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 claims description 25
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 claims description 25
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 claims description 25
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 claims description 25
- 210000001616 monocyte Anatomy 0.000 claims description 25
- 102100038080 B-cell receptor CD22 Human genes 0.000 claims description 23
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 claims description 23
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 claims description 23
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 claims description 23
- 210000003719 b-lymphocyte Anatomy 0.000 claims description 23
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 claims description 22
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 claims description 22
- 101001018097 Homo sapiens L-selectin Proteins 0.000 claims description 22
- 101000934341 Homo sapiens T-cell surface glycoprotein CD5 Proteins 0.000 claims description 22
- 102100033467 L-selectin Human genes 0.000 claims description 22
- 102100025244 T-cell surface glycoprotein CD5 Human genes 0.000 claims description 22
- 239000002502 liposome Substances 0.000 claims description 22
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 claims description 21
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 claims description 21
- 102100022748 Wilms tumor protein Human genes 0.000 claims description 21
- 102100036301 C-C chemokine receptor type 7 Human genes 0.000 claims description 20
- 102100032768 Complement receptor type 2 Human genes 0.000 claims description 20
- 101000716065 Homo sapiens C-C chemokine receptor type 7 Proteins 0.000 claims description 20
- 101000941929 Homo sapiens Complement receptor type 2 Proteins 0.000 claims description 20
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 claims description 18
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 claims description 18
- 101001043809 Homo sapiens Interleukin-7 receptor subunit alpha Proteins 0.000 claims description 18
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 claims description 18
- 102100021593 Interleukin-7 receptor subunit alpha Human genes 0.000 claims description 18
- 101150013553 CD40 gene Proteins 0.000 claims description 17
- 102100030886 Complement receptor type 1 Human genes 0.000 claims description 17
- 101000727061 Homo sapiens Complement receptor type 1 Proteins 0.000 claims description 17
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 claims description 17
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 claims description 17
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 claims description 17
- 238000000576 coating method Methods 0.000 claims description 17
- 239000012634 fragment Substances 0.000 claims description 17
- 210000003958 hematopoietic stem cell Anatomy 0.000 claims description 17
- 230000003612 virological effect Effects 0.000 claims description 17
- 102100024217 CAMPATH-1 antigen Human genes 0.000 claims description 16
- 108010065524 CD52 Antigen Proteins 0.000 claims description 16
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 claims description 16
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 claims description 16
- 101000611023 Homo sapiens Tumor necrosis factor receptor superfamily member 6 Proteins 0.000 claims description 16
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 claims description 16
- 108010064548 Lymphocyte Function-Associated Antigen-1 Proteins 0.000 claims description 16
- 102100040403 Tumor necrosis factor receptor superfamily member 6 Human genes 0.000 claims description 16
- 239000011248 coating agent Substances 0.000 claims description 16
- 239000003446 ligand Substances 0.000 claims description 16
- 210000000822 natural killer cell Anatomy 0.000 claims description 16
- 229940045513 CTLA4 antagonist Drugs 0.000 claims description 15
- 210000004881 tumor cell Anatomy 0.000 claims description 15
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 claims description 14
- 102100025390 Integrin beta-2 Human genes 0.000 claims description 14
- 238000003881 globally optimized alternating phase rectangular pulse Methods 0.000 claims description 14
- 101710134031 CCAAT/enhancer-binding protein beta Proteins 0.000 claims description 13
- 108010021064 CTLA-4 Antigen Proteins 0.000 claims description 13
- 101710093674 Cyclic nucleotide-gated cation channel beta-1 Proteins 0.000 claims description 13
- 101710199286 Cytosol aminopeptidase Proteins 0.000 claims description 13
- 101710197780 E3 ubiquitin-protein ligase LAP Proteins 0.000 claims description 13
- 102000001398 Granzyme Human genes 0.000 claims description 13
- 108060005986 Granzyme Proteins 0.000 claims description 13
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 claims description 13
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 claims description 13
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 claims description 13
- 101000801234 Homo sapiens Tumor necrosis factor receptor superfamily member 18 Proteins 0.000 claims description 13
- 101710204480 Lysosomal acid phosphatase Proteins 0.000 claims description 13
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 claims description 13
- 101710089118 Probable cytosol aminopeptidase Proteins 0.000 claims description 13
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 13
- 102100025946 Transforming growth factor beta activator LRRC32 Human genes 0.000 claims description 13
- 101710169732 Transforming growth factor beta activator LRRC32 Proteins 0.000 claims description 13
- 102000008579 Transposases Human genes 0.000 claims description 13
- 108010020764 Transposases Proteins 0.000 claims description 13
- 102100033728 Tumor necrosis factor receptor superfamily member 18 Human genes 0.000 claims description 13
- 101000934372 Homo sapiens Macrosialin Proteins 0.000 claims description 12
- 102100025136 Macrosialin Human genes 0.000 claims description 12
- 102000003735 Mesothelin Human genes 0.000 claims description 12
- 108090000015 Mesothelin Proteins 0.000 claims description 12
- 206010060862 Prostate cancer Diseases 0.000 claims description 12
- 102100027208 T-cell antigen CD7 Human genes 0.000 claims description 12
- 108010033576 Transferrin Receptors Proteins 0.000 claims description 12
- 102100026144 Transferrin receptor protein 1 Human genes 0.000 claims description 12
- 230000001177 retroviral effect Effects 0.000 claims description 12
- 208000032839 leukemia Diseases 0.000 claims description 11
- 239000011148 porous material Substances 0.000 claims description 11
- 230000003834 intracellular effect Effects 0.000 claims description 9
- 108020004999 messenger RNA Proteins 0.000 claims description 9
- 229920001481 poly(stearyl methacrylate) Polymers 0.000 claims description 9
- 206010025323 Lymphomas Diseases 0.000 claims description 8
- 108010079855 Peptide Aptamers Proteins 0.000 claims description 8
- 208000002495 Uterine Neoplasms Diseases 0.000 claims description 8
- 230000002147 killing effect Effects 0.000 claims description 8
- 201000001441 melanoma Diseases 0.000 claims description 8
- 239000002245 particle Substances 0.000 claims description 8
- 201000009030 Carcinoma Diseases 0.000 claims description 7
- 108700018351 Major Histocompatibility Complex Proteins 0.000 claims description 7
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 7
- 206010039491 Sarcoma Diseases 0.000 claims description 7
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 claims description 7
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 claims description 6
- 206010009944 Colon cancer Diseases 0.000 claims description 6
- 101000914324 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 5 Proteins 0.000 claims description 6
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 claims description 6
- 101000623901 Homo sapiens Mucin-16 Proteins 0.000 claims description 6
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 claims description 6
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 claims description 6
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 claims description 6
- 102100023123 Mucin-16 Human genes 0.000 claims description 6
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 claims description 6
- 206010033128 Ovarian cancer Diseases 0.000 claims description 6
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 6
- 229920002873 Polyethylenimine Polymers 0.000 claims description 6
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 claims description 6
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 claims description 6
- 239000000693 micelle Substances 0.000 claims description 6
- 108091008104 nucleic acid aptamers Proteins 0.000 claims description 6
- 206010046766 uterine cancer Diseases 0.000 claims description 6
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 claims description 5
- 206010006187 Breast cancer Diseases 0.000 claims description 5
- 208000026310 Breast neoplasm Diseases 0.000 claims description 5
- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 claims description 5
- 101000777628 Homo sapiens Leukocyte antigen CD37 Proteins 0.000 claims description 5
- 101001024605 Homo sapiens Next to BRCA1 gene 1 protein Proteins 0.000 claims description 5
- 102100031586 Leukocyte antigen CD37 Human genes 0.000 claims description 5
- 239000000232 Lipid Bilayer Substances 0.000 claims description 5
- 206010027406 Mesothelioma Diseases 0.000 claims description 5
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 5
- 101001039269 Rattus norvegicus Glycine N-methyltransferase Proteins 0.000 claims description 5
- 108060008724 Tyrosinase Proteins 0.000 claims description 5
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims description 5
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 5
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 claims description 5
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 claims description 5
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 5
- 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 claims description 5
- 201000002528 pancreatic cancer Diseases 0.000 claims description 5
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 5
- 108090000468 progesterone receptors Proteins 0.000 claims description 5
- 102000006306 Antigen Receptors Human genes 0.000 claims description 4
- 108010083359 Antigen Receptors Proteins 0.000 claims description 4
- 102100035526 B melanoma antigen 1 Human genes 0.000 claims description 4
- 102100021663 Baculoviral IAP repeat-containing protein 5 Human genes 0.000 claims description 4
- 108060000903 Beta-catenin Proteins 0.000 claims description 4
- 102000015735 Beta-catenin Human genes 0.000 claims description 4
- 206010005949 Bone cancer Diseases 0.000 claims description 4
- 208000018084 Bone neoplasm Diseases 0.000 claims description 4
- 208000003174 Brain Neoplasms Diseases 0.000 claims description 4
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 4
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 4
- 108010060385 Cyclin B1 Proteins 0.000 claims description 4
- 206010014759 Endometrial neoplasm Diseases 0.000 claims description 4
- 208000000461 Esophageal Neoplasms Diseases 0.000 claims description 4
- 102100038595 Estrogen receptor Human genes 0.000 claims description 4
- 102000008857 Ferritin Human genes 0.000 claims description 4
- 108050000784 Ferritin Proteins 0.000 claims description 4
- 238000008416 Ferritin Methods 0.000 claims description 4
- 102100032340 G2/mitotic-specific cyclin-B1 Human genes 0.000 claims description 4
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 claims description 4
- 208000017604 Hodgkin disease Diseases 0.000 claims description 4
- 208000010747 Hodgkins lymphoma Diseases 0.000 claims description 4
- 101000874316 Homo sapiens B melanoma antigen 1 Proteins 0.000 claims description 4
- 101000715390 Homo sapiens E3 ubiquitin-protein ligase CBL Proteins 0.000 claims description 4
- 101000833492 Homo sapiens Jouberin Proteins 0.000 claims description 4
- 101000651236 Homo sapiens NCK-interacting protein with SH3 domain Proteins 0.000 claims description 4
- 241000578472 Human endogenous retrovirus H Species 0.000 claims description 4
- 108010061833 Integrases Proteins 0.000 claims description 4
- 208000005016 Intestinal Neoplasms Diseases 0.000 claims description 4
- 208000008839 Kidney Neoplasms Diseases 0.000 claims description 4
- 208000003445 Mouth Neoplasms Diseases 0.000 claims description 4
- 101001016849 Mus musculus Heat shock protein HSP 90-alpha Proteins 0.000 claims description 4
- 101000985444 Mus musculus Heat shock protein HSP 90-beta Proteins 0.000 claims description 4
- 208000001894 Nasopharyngeal Neoplasms Diseases 0.000 claims description 4
- 206010029260 Neuroblastoma Diseases 0.000 claims description 4
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 claims description 4
- 240000007019 Oxalis corniculata Species 0.000 claims description 4
- 108060006580 PRAME Proteins 0.000 claims description 4
- 102000036673 PRAME Human genes 0.000 claims description 4
- 102100034640 PWWP domain-containing DNA repair factor 3A Human genes 0.000 claims description 4
- 108050007154 PWWP domain-containing DNA repair factor 3A Proteins 0.000 claims description 4
- 208000002471 Penile Neoplasms Diseases 0.000 claims description 4
- 108091005804 Peptidases Proteins 0.000 claims description 4
- 208000009565 Pharyngeal Neoplasms Diseases 0.000 claims description 4
- 239000004365 Protease Substances 0.000 claims description 4
- 208000015634 Rectal Neoplasms Diseases 0.000 claims description 4
- 201000010208 Seminoma Diseases 0.000 claims description 4
- 208000000453 Skin Neoplasms Diseases 0.000 claims description 4
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 4
- 101800001271 Surface protein Proteins 0.000 claims description 4
- 108010002687 Survivin Proteins 0.000 claims description 4
- 102100038126 Tenascin Human genes 0.000 claims description 4
- 108010008125 Tenascin Proteins 0.000 claims description 4
- 206010043276 Teratoma Diseases 0.000 claims description 4
- 208000024313 Testicular Neoplasms Diseases 0.000 claims description 4
- 208000024770 Thyroid neoplasm Diseases 0.000 claims description 4
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 4
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 4
- 208000024447 adrenal gland neoplasm Diseases 0.000 claims description 4
- 201000010881 cervical cancer Diseases 0.000 claims description 4
- 208000029742 colonic neoplasm Diseases 0.000 claims description 4
- 108010038795 estrogen receptors Proteins 0.000 claims description 4
- 201000010536 head and neck cancer Diseases 0.000 claims description 4
- 208000014829 head and neck neoplasm Diseases 0.000 claims description 4
- 201000005787 hematologic cancer Diseases 0.000 claims description 4
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 claims description 4
- 206010023841 laryngeal neoplasm Diseases 0.000 claims description 4
- 208000014018 liver neoplasm Diseases 0.000 claims description 4
- 208000020816 lung neoplasm Diseases 0.000 claims description 4
- 201000010453 lymph node cancer Diseases 0.000 claims description 4
- 238000012737 microarray-based gene expression Methods 0.000 claims description 4
- 238000012243 multiplex automated genomic engineering Methods 0.000 claims description 4
- 201000000050 myeloid neoplasm Diseases 0.000 claims description 4
- AEMBWNDIEFEPTH-UHFFFAOYSA-N n-tert-butyl-n-ethylnitrous amide Chemical compound CCN(N=O)C(C)(C)C AEMBWNDIEFEPTH-UHFFFAOYSA-N 0.000 claims description 4
- 210000003800 pharynx Anatomy 0.000 claims description 4
- 208000013139 vaginal neoplasm Diseases 0.000 claims description 4
- 206010055031 vascular neoplasm Diseases 0.000 claims description 4
- 206010005003 Bladder cancer Diseases 0.000 claims description 3
- 206010014733 Endometrial cancer Diseases 0.000 claims description 3
- 206010023825 Laryngeal cancer Diseases 0.000 claims description 3
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 3
- 208000032271 Malignant tumor of penis Diseases 0.000 claims description 3
- 206010030155 Oesophageal carcinoma Diseases 0.000 claims description 3
- 206010034299 Penile cancer Diseases 0.000 claims description 3
- 206010038389 Renal cancer Diseases 0.000 claims description 3
- 108091027076 Spiegelmer Proteins 0.000 claims description 3
- 206010057644 Testis cancer Diseases 0.000 claims description 3
- 201000005188 adrenal gland cancer Diseases 0.000 claims description 3
- 201000004101 esophageal cancer Diseases 0.000 claims description 3
- 206010017758 gastric cancer Diseases 0.000 claims description 3
- 201000002313 intestinal cancer Diseases 0.000 claims description 3
- 201000010982 kidney cancer Diseases 0.000 claims description 3
- 201000004962 larynx cancer Diseases 0.000 claims description 3
- 208000012987 lip and oral cavity carcinoma Diseases 0.000 claims description 3
- 201000007270 liver cancer Diseases 0.000 claims description 3
- 201000005202 lung cancer Diseases 0.000 claims description 3
- 208000025848 malignant tumor of nasopharynx Diseases 0.000 claims description 3
- 239000013528 metallic particle Substances 0.000 claims description 3
- 201000011216 nasopharynx carcinoma Diseases 0.000 claims description 3
- 230000037361 pathway Effects 0.000 claims description 3
- 201000008006 pharynx cancer Diseases 0.000 claims description 3
- 206010038038 rectal cancer Diseases 0.000 claims description 3
- 201000001275 rectum cancer Diseases 0.000 claims description 3
- 201000000849 skin cancer Diseases 0.000 claims description 3
- 201000011549 stomach cancer Diseases 0.000 claims description 3
- 201000003120 testicular cancer Diseases 0.000 claims description 3
- 201000002510 thyroid cancer Diseases 0.000 claims description 3
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 3
- 206010046885 vaginal cancer Diseases 0.000 claims description 3
- 102100023441 Centromere protein J Human genes 0.000 claims 3
- 102100034343 Integrase Human genes 0.000 claims 1
- 102100024407 Jouberin Human genes 0.000 claims 1
- 102100025803 Progesterone receptor Human genes 0.000 claims 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 1
- 102100039094 Tyrosinase Human genes 0.000 claims 1
- 210000000987 immune system Anatomy 0.000 abstract description 39
- -1 CD45RO Proteins 0.000 description 36
- 102000004169 proteins and genes Human genes 0.000 description 36
- 235000001014 amino acid Nutrition 0.000 description 35
- 239000004480 active ingredient Substances 0.000 description 32
- 235000018102 proteins Nutrition 0.000 description 32
- 150000001413 amino acids Chemical class 0.000 description 27
- 241000725303 Human immunodeficiency virus Species 0.000 description 26
- 229940024606 amino acid Drugs 0.000 description 25
- 108090000765 processed proteins & peptides Proteins 0.000 description 25
- 238000011282 treatment Methods 0.000 description 25
- 101150084041 WT1 gene Proteins 0.000 description 19
- 208000023958 prostate neoplasm Diseases 0.000 description 19
- 241000700605 Viruses Species 0.000 description 18
- 108700020467 WT1 Proteins 0.000 description 18
- 238000006467 substitution reaction Methods 0.000 description 18
- 239000007924 injection Substances 0.000 description 17
- 238000002347 injection Methods 0.000 description 17
- 150000007523 nucleic acids Chemical class 0.000 description 17
- 239000000592 Artificial Cell Substances 0.000 description 16
- 241000699670 Mus sp. Species 0.000 description 16
- 230000011664 signaling Effects 0.000 description 16
- 125000003275 alpha amino acid group Chemical group 0.000 description 15
- 239000012636 effector Substances 0.000 description 15
- 230000006378 damage Effects 0.000 description 14
- 230000001086 cytosolic effect Effects 0.000 description 13
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 12
- 102000004196 processed proteins & peptides Human genes 0.000 description 12
- 102100036981 Interferon regulatory factor 1 Human genes 0.000 description 11
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 11
- 210000002865 immune cell Anatomy 0.000 description 11
- 102100034798 CCAAT/enhancer-binding protein beta Human genes 0.000 description 10
- 125000006850 spacer group Chemical group 0.000 description 10
- 238000012546 transfer Methods 0.000 description 10
- 239000013598 vector Substances 0.000 description 10
- 241001465754 Metazoa Species 0.000 description 9
- 108700019146 Transgenes Proteins 0.000 description 9
- 230000008901 benefit Effects 0.000 description 9
- 230000000139 costimulatory effect Effects 0.000 description 9
- 230000006870 function Effects 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- 206010014596 Encephalitis Japanese B Diseases 0.000 description 8
- 201000005807 Japanese encephalitis Diseases 0.000 description 8
- 241000710842 Japanese encephalitis virus Species 0.000 description 8
- 239000011575 calcium Substances 0.000 description 8
- 125000002091 cationic group Chemical group 0.000 description 8
- 230000002538 fungal effect Effects 0.000 description 8
- 208000002672 hepatitis B Diseases 0.000 description 8
- 230000001404 mediated effect Effects 0.000 description 8
- 229920000642 polymer Polymers 0.000 description 8
- 238000011160 research Methods 0.000 description 8
- 102100021671 60S ribosomal protein L29 Human genes 0.000 description 7
- 241000282412 Homo Species 0.000 description 7
- 241001529936 Murinae Species 0.000 description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 description 7
- 230000001363 autoimmune Effects 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 7
- 238000012217 deletion Methods 0.000 description 7
- 230000037430 deletion Effects 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 238000000684 flow cytometry Methods 0.000 description 7
- 230000004068 intracellular signaling Effects 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 210000004940 nucleus Anatomy 0.000 description 7
- 229920001282 polysaccharide Polymers 0.000 description 7
- 239000005017 polysaccharide Substances 0.000 description 7
- 150000004804 polysaccharides Chemical class 0.000 description 7
- 108010025498 ribosomal protein L29 Proteins 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- IUCJMVBFZDHPDX-UHFFFAOYSA-N tretamine Chemical compound C1CN1C1=NC(N2CC2)=NC(N2CC2)=N1 IUCJMVBFZDHPDX-UHFFFAOYSA-N 0.000 description 7
- 101710132601 Capsid protein Proteins 0.000 description 6
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 6
- 101710154606 Hemagglutinin Proteins 0.000 description 6
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 6
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 6
- 101710176177 Protein A56 Proteins 0.000 description 6
- 206010037742 Rabies Diseases 0.000 description 6
- 230000004913 activation Effects 0.000 description 6
- 238000013459 approach Methods 0.000 description 6
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 6
- 239000000185 hemagglutinin Substances 0.000 description 6
- 238000001802 infusion Methods 0.000 description 6
- 238000003780 insertion Methods 0.000 description 6
- 230000037431 insertion Effects 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 230000002093 peripheral effect Effects 0.000 description 6
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 101710158312 DNA-binding protein HU-beta Proteins 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 5
- 101000914321 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 7 Proteins 0.000 description 5
- 108060003951 Immunoglobulin Proteins 0.000 description 5
- 101710128560 Initiator protein NS1 Proteins 0.000 description 5
- 102100034349 Integrase Human genes 0.000 description 5
- 206010027476 Metastases Diseases 0.000 description 5
- 101710144127 Non-structural protein 1 Proteins 0.000 description 5
- 102100022019 Pregnancy-specific beta-1-glycoprotein 2 Human genes 0.000 description 5
- 102100033019 Tyrosine-protein phosphatase non-receptor type 11 Human genes 0.000 description 5
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 5
- 208000010668 atopic eczema Diseases 0.000 description 5
- 230000006470 autoimmune attack Effects 0.000 description 5
- 230000009286 beneficial effect Effects 0.000 description 5
- 229910052791 calcium Inorganic materials 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 238000001415 gene therapy Methods 0.000 description 5
- 102000018358 immunoglobulin Human genes 0.000 description 5
- 239000004615 ingredient Substances 0.000 description 5
- 230000000670 limiting effect Effects 0.000 description 5
- 229920001223 polyethylene glycol Polymers 0.000 description 5
- 210000002307 prostate Anatomy 0.000 description 5
- 239000000377 silicon dioxide Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 210000000130 stem cell Anatomy 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- SNKAWJBJQDLSFF-NVKMUCNASA-N 1,2-dioleoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC SNKAWJBJQDLSFF-NVKMUCNASA-N 0.000 description 4
- 108091023037 Aptamer Proteins 0.000 description 4
- 241000193738 Bacillus anthracis Species 0.000 description 4
- 238000011740 C57BL/6 mouse Methods 0.000 description 4
- 101710098119 Chaperonin GroEL 2 Proteins 0.000 description 4
- 101710198480 Clumping factor A Proteins 0.000 description 4
- 101710139375 Corneodesmosin Proteins 0.000 description 4
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 4
- 241000233866 Fungi Species 0.000 description 4
- 108010010803 Gelatin Proteins 0.000 description 4
- 108091022930 Glutamate decarboxylase Proteins 0.000 description 4
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 4
- 101001030069 Homo sapiens Major vault protein Proteins 0.000 description 4
- 101001103036 Homo sapiens Nuclear receptor ROR-alpha Proteins 0.000 description 4
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 4
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 4
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 4
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 4
- 101710085938 Matrix protein Proteins 0.000 description 4
- 101710127721 Membrane protein Proteins 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 229920002472 Starch Polymers 0.000 description 4
- 102000011923 Thyrotropin Human genes 0.000 description 4
- 108010061174 Thyrotropin Proteins 0.000 description 4
- 101800001690 Transmembrane protein gp41 Proteins 0.000 description 4
- 241000223109 Trypanosoma cruzi Species 0.000 description 4
- 102000003425 Tyrosinase Human genes 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 235000010443 alginic acid Nutrition 0.000 description 4
- 229920000615 alginic acid Polymers 0.000 description 4
- 125000001931 aliphatic group Chemical group 0.000 description 4
- 230000000172 allergic effect Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000029918 bioluminescence Effects 0.000 description 4
- 238000005415 bioluminescence Methods 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 239000002771 cell marker Substances 0.000 description 4
- 229940028617 conventional vaccine Drugs 0.000 description 4
- MWRBNPKJOOWZPW-CLFAGFIQSA-N dioleoyl phosphatidylethanolamine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-CLFAGFIQSA-N 0.000 description 4
- 229920000159 gelatin Polymers 0.000 description 4
- 239000008273 gelatin Substances 0.000 description 4
- 235000019322 gelatine Nutrition 0.000 description 4
- 235000011852 gelatine desserts Nutrition 0.000 description 4
- 239000005090 green fluorescent protein Substances 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 210000002443 helper t lymphocyte Anatomy 0.000 description 4
- 230000002209 hydrophobic effect Effects 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 4
- 230000001506 immunosuppresive effect Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 230000002934 lysing effect Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 201000006417 multiple sclerosis Diseases 0.000 description 4
- 210000005259 peripheral blood Anatomy 0.000 description 4
- 239000011886 peripheral blood Substances 0.000 description 4
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 102000003998 progesterone receptors Human genes 0.000 description 4
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 4
- 210000003289 regulatory T cell Anatomy 0.000 description 4
- 201000005404 rubella Diseases 0.000 description 4
- 235000000346 sugar Nutrition 0.000 description 4
- 241000712461 unidentified influenza virus Species 0.000 description 4
- 229960005486 vaccine Drugs 0.000 description 4
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 3
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 3
- 102100038222 60 kDa heat shock protein, mitochondrial Human genes 0.000 description 3
- 108010058432 Chaperonin 60 Proteins 0.000 description 3
- 241001550206 Colla Species 0.000 description 3
- 229920002261 Corn starch Polymers 0.000 description 3
- 102100031673 Corneodesmosin Human genes 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 102100038132 Endogenous retrovirus group K member 6 Pro protein Human genes 0.000 description 3
- 108090000331 Firefly luciferases Proteins 0.000 description 3
- 102000003886 Glycoproteins Human genes 0.000 description 3
- 108090000288 Glycoproteins Proteins 0.000 description 3
- 241000700721 Hepatitis B virus Species 0.000 description 3
- 208000005176 Hepatitis C Diseases 0.000 description 3
- 208000009889 Herpes Simplex Diseases 0.000 description 3
- 101001103039 Homo sapiens Inactive tyrosine-protein kinase transmembrane receptor ROR1 Proteins 0.000 description 3
- 206010020751 Hypersensitivity Diseases 0.000 description 3
- 102100039615 Inactive tyrosine-protein kinase transmembrane receptor ROR1 Human genes 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 3
- 108060001084 Luciferase Proteins 0.000 description 3
- 239000005089 Luciferase Substances 0.000 description 3
- 241000712079 Measles morbillivirus Species 0.000 description 3
- 102100027673 NCK-interacting protein with SH3 domain Human genes 0.000 description 3
- 102000011931 Nucleoproteins Human genes 0.000 description 3
- 108010061100 Nucleoproteins Proteins 0.000 description 3
- 102100036735 Prostate stem cell antigen Human genes 0.000 description 3
- 101710120463 Prostate stem cell antigen Proteins 0.000 description 3
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 description 3
- 241000269370 Xenopus <genus> Species 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 239000011230 binding agent Substances 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 239000002775 capsule Substances 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 235000012000 cholesterol Nutrition 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 206010012601 diabetes mellitus Diseases 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 238000005538 encapsulation Methods 0.000 description 3
- 230000012202 endocytosis Effects 0.000 description 3
- 230000000799 fusogenic effect Effects 0.000 description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 206010022000 influenza Diseases 0.000 description 3
- 238000001990 intravenous administration Methods 0.000 description 3
- 210000000265 leukocyte Anatomy 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 206010061289 metastatic neoplasm Diseases 0.000 description 3
- 208000010658 metastatic prostate carcinoma Diseases 0.000 description 3
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 238000002823 phage display Methods 0.000 description 3
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 3
- 150000003905 phosphatidylinositols Chemical class 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 3
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 3
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 3
- 238000011321 prophylaxis Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 230000019491 signal transduction Effects 0.000 description 3
- 239000003381 stabilizer Substances 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 150000005846 sugar alcohols Chemical class 0.000 description 3
- 238000013268 sustained release Methods 0.000 description 3
- 239000012730 sustained-release form Substances 0.000 description 3
- 230000009261 transgenic effect Effects 0.000 description 3
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 2
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 description 2
- LVNGJLRDBYCPGB-UHFFFAOYSA-N 1,2-distearoylphosphatidylethanolamine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(COP([O-])(=O)OCC[NH3+])OC(=O)CCCCCCCCCCCCCCCCC LVNGJLRDBYCPGB-UHFFFAOYSA-N 0.000 description 2
- BIABMEZBCHDPBV-MPQUPPDSSA-N 1,2-palmitoyl-sn-glycero-3-phospho-(1'-sn-glycerol) Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@@H](O)CO)OC(=O)CCCCCCCCCCCCCCC BIABMEZBCHDPBV-MPQUPPDSSA-N 0.000 description 2
- OOMDVERDMZLRFX-UHFFFAOYSA-N 2,2-bis(aminomethyl)propane-1,3-diol;cyclobutane-1,1-dicarboxylic acid;platinum Chemical compound [Pt].NCC(CN)(CO)CO.OC(=O)C1(C(O)=O)CCC1 OOMDVERDMZLRFX-UHFFFAOYSA-N 0.000 description 2
- KISWVXRQTGLFGD-UHFFFAOYSA-N 2-[[2-[[6-amino-2-[[2-[[2-[[5-amino-2-[[2-[[1-[2-[[6-amino-2-[(2,5-diamino-5-oxopentanoyl)amino]hexanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-(diaminomethylideneamino)p Chemical compound C1CCN(C(=O)C(CCCN=C(N)N)NC(=O)C(CCCCN)NC(=O)C(N)CCC(N)=O)C1C(=O)NC(CO)C(=O)NC(CCC(N)=O)C(=O)NC(CCCN=C(N)N)C(=O)NC(CO)C(=O)NC(CCCCN)C(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=C(O)C=C1 KISWVXRQTGLFGD-UHFFFAOYSA-N 0.000 description 2
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 2
- 102100021176 ATP-sensitive inward rectifier potassium channel 10 Human genes 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 102400000068 Angiostatin Human genes 0.000 description 2
- 108010079709 Angiostatins Proteins 0.000 description 2
- 101100446590 Arabidopsis thaliana FIM5 gene Proteins 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 241000416162 Astragalus gummifer Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102100022717 Atypical chemokine receptor 1 Human genes 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 2
- 108091008875 B cell receptors Proteins 0.000 description 2
- 102000006942 B-Cell Maturation Antigen Human genes 0.000 description 2
- 108010008014 B-Cell Maturation Antigen Proteins 0.000 description 2
- 102100027203 B-cell antigen receptor complex-associated protein beta chain Human genes 0.000 description 2
- 241000304886 Bacilli Species 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 2
- 102100027207 CD27 antigen Human genes 0.000 description 2
- 210000004366 CD4-positive T-lymphocyte Anatomy 0.000 description 2
- 108091008048 CMVpp65 Proteins 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 229920001661 Chitosan Polymers 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 108010009685 Cholinergic Receptors Proteins 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 102100031162 Collagen alpha-1(XVIII) chain Human genes 0.000 description 2
- 239000004971 Cross linker Substances 0.000 description 2
- 240000005109 Cryptomeria japonica Species 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 201000004624 Dermatitis Diseases 0.000 description 2
- 108700022150 Designed Ankyrin Repeat Proteins Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 108010079505 Endostatins Proteins 0.000 description 2
- 101001095863 Enterobacteria phage T4 RNA ligase 1 Proteins 0.000 description 2
- 101710126487 Envelope glycoprotein B Proteins 0.000 description 2
- 101800001466 Envelope glycoprotein E1 Proteins 0.000 description 2
- 101001065501 Escherichia phage MS2 Lysis protein Proteins 0.000 description 2
- 101150106011 FIM2 gene Proteins 0.000 description 2
- 101150048576 FIM3 gene Proteins 0.000 description 2
- 241000282324 Felis Species 0.000 description 2
- 206010017533 Fungal infection Diseases 0.000 description 2
- 108010001515 Galectin 4 Proteins 0.000 description 2
- 102100039556 Galectin-4 Human genes 0.000 description 2
- 102000008214 Glutamate decarboxylase Human genes 0.000 description 2
- 108010070675 Glutathione transferase Proteins 0.000 description 2
- 102000005720 Glutathione transferase Human genes 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 208000031886 HIV Infections Diseases 0.000 description 2
- 241000606768 Haemophilus influenzae Species 0.000 description 2
- 208000007514 Herpes zoster Diseases 0.000 description 2
- 241000228402 Histoplasma Species 0.000 description 2
- 101000678879 Homo sapiens Atypical chemokine receptor 1 Proteins 0.000 description 2
- 101000914491 Homo sapiens B-cell antigen receptor complex-associated protein beta chain Proteins 0.000 description 2
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 2
- 101100351324 Homo sapiens PDPN gene Proteins 0.000 description 2
- 101000617725 Homo sapiens Pregnancy-specific beta-1-glycoprotein 2 Proteins 0.000 description 2
- 241001135569 Human adenovirus 5 Species 0.000 description 2
- 108010007403 Immediate-Early Proteins Proteins 0.000 description 2
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 2
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- 102000011781 Karyopherins Human genes 0.000 description 2
- 108010062228 Karyopherins Proteins 0.000 description 2
- 108010074781 Kcnj10 (channel) Proteins 0.000 description 2
- 125000000998 L-alanino group Chemical group [H]N([*])[C@](C([H])([H])[H])([H])C(=O)O[H] 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- 102000008201 Lamin Type A Human genes 0.000 description 2
- 108010021099 Lamin Type A Proteins 0.000 description 2
- 101710128836 Large T antigen Proteins 0.000 description 2
- 241000222722 Leishmania <genus> Species 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 241000209082 Lolium Species 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- 201000005505 Measles Diseases 0.000 description 2
- 108010061593 Member 14 Tumor Necrosis Factor Receptors Proteins 0.000 description 2
- 108010057081 Merozoite Surface Protein 1 Proteins 0.000 description 2
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 2
- 208000031888 Mycoses Diseases 0.000 description 2
- 102000047918 Myelin Basic Human genes 0.000 description 2
- 102000055324 Myelin Proteolipid Human genes 0.000 description 2
- 101710107068 Myelin basic protein Proteins 0.000 description 2
- 101710094913 Myelin proteolipid protein Proteins 0.000 description 2
- 208000009869 Neu-Laxova syndrome Diseases 0.000 description 2
- 108010006232 Neuraminidase Proteins 0.000 description 2
- 102000005348 Neuraminidase Human genes 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 229930012538 Paclitaxel Natural products 0.000 description 2
- 108010065129 Patched-1 Receptor Proteins 0.000 description 2
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 2
- 108010081690 Pertussis Toxin Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 101710183389 Pneumolysin Proteins 0.000 description 2
- 102100037265 Podoplanin Human genes 0.000 description 2
- 101710194807 Protective antigen Proteins 0.000 description 2
- 101710192141 Protein Nef Proteins 0.000 description 2
- 102100028680 Protein patched homolog 1 Human genes 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 208000021386 Sjogren Syndrome Diseases 0.000 description 2
- 101001039853 Sonchus yellow net virus Matrix protein Proteins 0.000 description 2
- 101100289792 Squirrel monkey polyomavirus large T gene Proteins 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 101710137302 Surface antigen S Proteins 0.000 description 2
- 230000006044 T cell activation Effects 0.000 description 2
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 2
- 206010043376 Tetanus Diseases 0.000 description 2
- 108010055044 Tetanus Toxin Proteins 0.000 description 2
- 108010034949 Thyroglobulin Proteins 0.000 description 2
- 102000009843 Thyroglobulin Human genes 0.000 description 2
- 241000130764 Tinea Species 0.000 description 2
- 208000002474 Tinea Diseases 0.000 description 2
- 241000223996 Toxoplasma Species 0.000 description 2
- 229920001615 Tragacanth Polymers 0.000 description 2
- 108010030743 Tropomyosin Proteins 0.000 description 2
- 102000005937 Tropomyosin Human genes 0.000 description 2
- 102100028785 Tumor necrosis factor receptor superfamily member 14 Human genes 0.000 description 2
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 2
- 108010059722 Viral Fusion Proteins Proteins 0.000 description 2
- 108020000999 Viral RNA Proteins 0.000 description 2
- 208000008383 Wilms tumor Diseases 0.000 description 2
- 102000013814 Wnt Human genes 0.000 description 2
- 108050003627 Wnt Proteins 0.000 description 2
- 102100036976 X-ray repair cross-complementing protein 6 Human genes 0.000 description 2
- 101710124907 X-ray repair cross-complementing protein 6 Proteins 0.000 description 2
- 102000034337 acetylcholine receptors Human genes 0.000 description 2
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 2
- 108060000200 adenylate cyclase Proteins 0.000 description 2
- 102000030621 adenylate cyclase Human genes 0.000 description 2
- 239000000443 aerosol Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 235000010419 agar Nutrition 0.000 description 2
- 229940072056 alginate Drugs 0.000 description 2
- 239000000783 alginic acid Substances 0.000 description 2
- 229960001126 alginic acid Drugs 0.000 description 2
- 150000004781 alginic acids Chemical class 0.000 description 2
- 208000026935 allergic disease Diseases 0.000 description 2
- 230000007815 allergy Effects 0.000 description 2
- 230000033115 angiogenesis Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 125000000129 anionic group Chemical group 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 210000000612 antigen-presenting cell Anatomy 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- FZCSTZYAHCUGEM-UHFFFAOYSA-N aspergillomarasmine B Natural products OC(=O)CNC(C(O)=O)CNC(C(O)=O)CC(O)=O FZCSTZYAHCUGEM-UHFFFAOYSA-N 0.000 description 2
- 208000006673 asthma Diseases 0.000 description 2
- 208000022362 bacterial infectious disease Diseases 0.000 description 2
- HMFHBZSHGGEWLO-TXICZTDVSA-N beta-D-ribose Chemical group OC[C@H]1O[C@@H](O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-TXICZTDVSA-N 0.000 description 2
- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- 229920000249 biocompatible polymer Polymers 0.000 description 2
- 230000031018 biological processes and functions Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000006172 buffering agent Substances 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000002659 cell therapy Methods 0.000 description 2
- 230000036755 cellular response Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 150000001875 compounds Chemical group 0.000 description 2
- 239000008120 corn starch Substances 0.000 description 2
- 229940099112 cornstarch Drugs 0.000 description 2
- 239000003405 delayed action preparation Substances 0.000 description 2
- 239000000412 dendrimer Substances 0.000 description 2
- 229920000736 dendritic polymer Polymers 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 230000003467 diminishing effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000000428 dust Substances 0.000 description 2
- 210000003162 effector t lymphocyte Anatomy 0.000 description 2
- 210000001163 endosome Anatomy 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- CHPZKNULDCNCBW-UHFFFAOYSA-N gallium nitrate Chemical compound [Ga+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O CHPZKNULDCNCBW-UHFFFAOYSA-N 0.000 description 2
- 210000000973 gametocyte Anatomy 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 229940046528 grass pollen Drugs 0.000 description 2
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- USSYUMHVHQSYNA-SLDJZXPVSA-N indolicidin Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(N)=O)CC1=CNC2=CC=CC=C12 USSYUMHVHQSYNA-SLDJZXPVSA-N 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 229940047124 interferons Drugs 0.000 description 2
- 238000001361 intraarterial administration Methods 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 238000007913 intrathecal administration Methods 0.000 description 2
- 230000002601 intratumoral effect Effects 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 239000007951 isotonicity adjuster Substances 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 230000021633 leukocyte mediated immunity Effects 0.000 description 2
- 229920006008 lipopolysaccharide Polymers 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 229910052752 metalloid Inorganic materials 0.000 description 2
- 150000002738 metalloids Chemical class 0.000 description 2
- 150000002739 metals Chemical class 0.000 description 2
- 230000001394 metastastic effect Effects 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 235000006109 methionine Nutrition 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 2
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 2
- 229960002216 methylparaben Drugs 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- CFCUWKMKBJTWLW-BKHRDMLASA-N mithramycin Chemical compound O([C@@H]1C[C@@H](O[C@H](C)[C@H]1O)OC=1C=C2C=C3C[C@H]([C@@H](C(=O)C3=C(O)C2=C(O)C=1C)O[C@@H]1O[C@H](C)[C@@H](O)[C@H](O[C@@H]2O[C@H](C)[C@H](O)[C@H](O[C@@H]3O[C@H](C)[C@@H](O)[C@@](C)(O)C3)C2)C1)[C@H](OC)C(=O)[C@@H](O)[C@@H](C)O)[C@H]1C[C@@H](O)[C@H](O)[C@@H](C)O1 CFCUWKMKBJTWLW-BKHRDMLASA-N 0.000 description 2
- 210000004296 naive t lymphocyte Anatomy 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 229960001592 paclitaxel Drugs 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- AQIXEPGDORPWBJ-UHFFFAOYSA-N pentan-3-ol Chemical compound CCC(O)CC AQIXEPGDORPWBJ-UHFFFAOYSA-N 0.000 description 2
- 229930192851 perforin Natural products 0.000 description 2
- 108010021711 pertactin Proteins 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 229960003171 plicamycin Drugs 0.000 description 2
- 229920001610 polycaprolactone Polymers 0.000 description 2
- 229920002451 polyvinyl alcohol Polymers 0.000 description 2
- 229920001592 potato starch Polymers 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 2
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 2
- 229960003415 propylparaben Drugs 0.000 description 2
- 239000009342 ragweed pollen Substances 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 description 2
- 108091006024 signal transducing proteins Proteins 0.000 description 2
- 102000034285 signal transducing proteins Human genes 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 235000010356 sorbitol Nutrition 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 210000003046 sporozoite Anatomy 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 239000011593 sulfur Substances 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 230000002459 sustained effect Effects 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 2
- 229940118376 tetanus toxin Drugs 0.000 description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- 229960002175 thyroglobulin Drugs 0.000 description 2
- YFTWHEBLORWGNI-UHFFFAOYSA-N tiamiprine Chemical compound CN1C=NC([N+]([O-])=O)=C1SC1=NC(N)=NC2=C1NC=N2 YFTWHEBLORWGNI-UHFFFAOYSA-N 0.000 description 2
- 229950011457 tiamiprine Drugs 0.000 description 2
- 230000000699 topical effect Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 229910052723 transition metal Inorganic materials 0.000 description 2
- 150000003624 transition metals Chemical class 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 229960004441 tyrosine Drugs 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 241001529453 unidentified herpesvirus Species 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- 238000001086 yeast two-hybrid system Methods 0.000 description 2
- 229950003017 zeniplatin Drugs 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- LNAZSHAWQACDHT-XIYTZBAFSA-N (2r,3r,4s,5r,6s)-4,5-dimethoxy-2-(methoxymethyl)-3-[(2s,3r,4s,5r,6r)-3,4,5-trimethoxy-6-(methoxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6r)-4,5,6-trimethoxy-2-(methoxymethyl)oxan-3-yl]oxyoxane Chemical compound CO[C@@H]1[C@@H](OC)[C@H](OC)[C@@H](COC)O[C@H]1O[C@H]1[C@H](OC)[C@@H](OC)[C@H](O[C@H]2[C@@H]([C@@H](OC)[C@H](OC)O[C@@H]2COC)OC)O[C@@H]1COC LNAZSHAWQACDHT-XIYTZBAFSA-N 0.000 description 1
- XUNKPNYCNUKOAU-VXJRNSOOSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]a Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O XUNKPNYCNUKOAU-VXJRNSOOSA-N 0.000 description 1
- RAVVEEJGALCVIN-AGVBWZICSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-5-amino-2-[[(2s)-2-[[(2s)-2-[[(2s)-6-amino-2-[[(2s)-6-amino-2-[[(2s)-2-[[2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]acetyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]hexanoyl]amino]hexanoyl]amino]-5-(diamino Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCN=C(N)N)NC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 RAVVEEJGALCVIN-AGVBWZICSA-N 0.000 description 1
- ASWBNKHCZGQVJV-UHFFFAOYSA-N (3-hexadecanoyloxy-2-hydroxypropyl) 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(O)COP([O-])(=O)OCC[N+](C)(C)C ASWBNKHCZGQVJV-UHFFFAOYSA-N 0.000 description 1
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 1
- DDMOUSALMHHKOS-UHFFFAOYSA-N 1,2-dichloro-1,1,2,2-tetrafluoroethane Chemical compound FC(F)(Cl)C(F)(F)Cl DDMOUSALMHHKOS-UHFFFAOYSA-N 0.000 description 1
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 1
- UOAFGUOASVSLPK-UHFFFAOYSA-N 1-(2-chloroethyl)-3-(2,2-dimethylpropyl)-1-nitrosourea Chemical compound CC(C)(C)CNC(=O)N(N=O)CCCl UOAFGUOASVSLPK-UHFFFAOYSA-N 0.000 description 1
- FJLUATLTXUNBOT-UHFFFAOYSA-N 1-Hexadecylamine Chemical compound CCCCCCCCCCCCCCCCN FJLUATLTXUNBOT-UHFFFAOYSA-N 0.000 description 1
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- VSNHCAURESNICA-NJFSPNSNSA-N 1-oxidanylurea Chemical compound N[14C](=O)NO VSNHCAURESNICA-NJFSPNSNSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- LDGWQMRUWMSZIU-LQDDAWAPSA-M 2,3-bis[(z)-octadec-9-enoxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)C)OCCCCCCCC\C=C/CCCCCCCC LDGWQMRUWMSZIU-LQDDAWAPSA-M 0.000 description 1
- KSXTUUUQYQYKCR-LQDDAWAPSA-M 2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC KSXTUUUQYQYKCR-LQDDAWAPSA-M 0.000 description 1
- WALUVDCNGPQPOD-UHFFFAOYSA-M 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCOCC(C[N+](C)(C)CCO)OCCCCCCCCCCCCCC WALUVDCNGPQPOD-UHFFFAOYSA-M 0.000 description 1
- UEJJHQNACJXSKW-UHFFFAOYSA-N 2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3(2H)-dione Chemical compound O=C1C2=CC=CC=C2C(=O)N1C1CCC(=O)NC1=O UEJJHQNACJXSKW-UHFFFAOYSA-N 0.000 description 1
- JKNCOURZONDCGV-UHFFFAOYSA-N 2-(dimethylamino)ethyl 2-methylprop-2-enoate Chemical compound CN(C)CCOC(=O)C(C)=C JKNCOURZONDCGV-UHFFFAOYSA-N 0.000 description 1
- BGFTWECWAICPDG-UHFFFAOYSA-N 2-[bis(4-chlorophenyl)methyl]-4-n-[3-[bis(4-chlorophenyl)methyl]-4-(dimethylamino)phenyl]-1-n,1-n-dimethylbenzene-1,4-diamine Chemical compound C1=C(C(C=2C=CC(Cl)=CC=2)C=2C=CC(Cl)=CC=2)C(N(C)C)=CC=C1NC(C=1)=CC=C(N(C)C)C=1C(C=1C=CC(Cl)=CC=1)C1=CC=C(Cl)C=C1 BGFTWECWAICPDG-UHFFFAOYSA-N 0.000 description 1
- CFWRDBDJAOHXSH-SECBINFHSA-N 2-azaniumylethyl [(2r)-2,3-diacetyloxypropyl] phosphate Chemical compound CC(=O)OC[C@@H](OC(C)=O)COP(O)(=O)OCCN CFWRDBDJAOHXSH-SECBINFHSA-N 0.000 description 1
- HZLCGUXUOFWCCN-UHFFFAOYSA-N 2-hydroxynonadecane-1,2,3-tricarboxylic acid Chemical compound CCCCCCCCCCCCCCCCC(C(O)=O)C(O)(C(O)=O)CC(O)=O HZLCGUXUOFWCCN-UHFFFAOYSA-N 0.000 description 1
- NDMPLJNOPCLANR-UHFFFAOYSA-N 3,4-dihydroxy-15-(4-hydroxy-18-methoxycarbonyl-5,18-seco-ibogamin-18-yl)-16-methoxy-1-methyl-6,7-didehydro-aspidospermidine-3-carboxylic acid methyl ester Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 NDMPLJNOPCLANR-UHFFFAOYSA-N 0.000 description 1
- CTSNHMQGVWXIEG-UHFFFAOYSA-N 4-amino-n-(5-chloroquinoxalin-2-yl)benzenesulfonamide Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=CN=C(C(Cl)=CC=C2)C2=N1 CTSNHMQGVWXIEG-UHFFFAOYSA-N 0.000 description 1
- HIQIXEFWDLTDED-UHFFFAOYSA-N 4-hydroxy-1-piperidin-4-ylpyrrolidin-2-one Chemical compound O=C1CC(O)CN1C1CCNCC1 HIQIXEFWDLTDED-UHFFFAOYSA-N 0.000 description 1
- IDPUKCWIGUEADI-UHFFFAOYSA-N 5-[bis(2-chloroethyl)amino]uracil Chemical compound ClCCN(CCCl)C1=CNC(=O)NC1=O IDPUKCWIGUEADI-UHFFFAOYSA-N 0.000 description 1
- LIFHMKCDDVTICL-UHFFFAOYSA-N 6-(chloromethyl)phenanthridine Chemical compound C1=CC=C2C(CCl)=NC3=CC=CC=C3C2=C1 LIFHMKCDDVTICL-UHFFFAOYSA-N 0.000 description 1
- WYWHKKSPHMUBEB-UHFFFAOYSA-N 6-Mercaptoguanine Natural products N1C(N)=NC(=S)C2=C1N=CN2 WYWHKKSPHMUBEB-UHFFFAOYSA-N 0.000 description 1
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 1
- XMCLKYABXSBFLO-UHFFFAOYSA-N 9-[2-[4-[2-[2-[2-[4-[2-[4-[2-[2,5-bis[2-[2,5-didecoxy-4-[2-(9H-(C60-Ih)[5,6]fulleren-1-yl)ethynyl]phenyl]ethynyl]phenyl]ethynyl]-2,5-didecoxyphenyl]ethynyl]-2,5-didecoxyphenyl]ethynyl]-4-[2-[2,5-didecoxy-4-[2-(9H-(C60-Ih)[5,6]fulleren-1-yl)ethynyl]phenyl]ethynyl]phenyl]ethynyl]-2,5-didecoxyphenyl]ethynyl]-1H-(C60-Ih)[5,6]fullerene Chemical compound CCCCCCCCCCOC1=CC(C#CC2=C(OCCCCCCCCCC)C=C(C#CC3=C(C=CC(=C3)C#CC3=CC(OCCCCCCCCCC)=C(C=C3OCCCCCCCCCC)C#CC34C5C6=C7C8=C5C5=C9C%10=C8C8=C%11C%10=C%10C%12=C9C9=C%13C%14=C%12C%12=C%10C%10=C%11C%11=C%15C8=C7C7=C8C6=C6C%16=C8C8=C%17C%18=C(C8=C%157)C%11=C%10C7=C%18C8=C%10C%17=C%16C%11=C%10C(=C%14C8=C%127)C%13=C(C%11=C36)C4=C59)C#CC3=CC(OCCCCCCCCCC)=C(C=C3OCCCCCCCCCC)C#CC34C5C6=C7C8=C5C5=C9C%10=C8C8=C%11C%10=C%10C%12=C9C9=C%13C%14=C%12C%12=C%10C%10=C%11C%11=C%15C8=C7C7=C8C6=C6C%16=C8C8=C%17C%18=C(C8=C%157)C%11=C%10C7=C%18C8=C%10C%17=C%16C%11=C%10C(=C%14C8=C%127)C%13=C(C%11=C36)C4=C59)C(OCCCCCCCCCC)=C2)=C(OCCCCCCCCCC)C=C1C#CC1=CC(=CC=C1C#CC1=CC(OCCCCCCCCCC)=C(C=C1OCCCCCCCCCC)C#CC12C3C4=C5C6=C3C3=C7C8=C6C6=C9C8=C8C%10=C7C7=C%11C%12=C%10C%10=C8C8=C9C9=C%13C6=C5C5=C6C4=C4C%14=C6C6=C%15C%16=C(C6=C%135)C9=C8C5=C%16C6=C8C%15=C%14C9=C8C(=C%12C6=C%105)C%11=C(C9=C14)C2=C37)C#CC1=CC(OCCCCCCCCCC)=C(C=C1OCCCCCCCCCC)C#CC12C3C4=C5C6=C3C3=C7C8=C6C6=C9C8=C8C%10=C7C7=C%11C%12=C%10C%10=C8C8=C9C9=C%13C6=C5C5=C6C4=C4C%14=C6C6=C%15C%16=C(C6=C%135)C9=C8C5=C%16C6=C8C%15=C%14C9=C8C(=C%12C6=C%105)C%11=C(C9=C14)C2=C37 XMCLKYABXSBFLO-UHFFFAOYSA-N 0.000 description 1
- 108010062307 AAVALLPAVLLALLAP Proteins 0.000 description 1
- 241000588626 Acinetobacter baumannii Species 0.000 description 1
- 108010024878 Adenovirus E1A Proteins Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 206010027654 Allergic conditions Diseases 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 102100026882 Alpha-synuclein Human genes 0.000 description 1
- 241000272525 Anas platyrhynchos Species 0.000 description 1
- 235000002198 Annona diversifolia Nutrition 0.000 description 1
- 101000587984 Arabidopsis thaliana Protein SPOROCYTELESS Proteins 0.000 description 1
- 241000712891 Arenavirus Species 0.000 description 1
- 108010014223 Armadillo Domain Proteins Proteins 0.000 description 1
- 102000016904 Armadillo Domain Proteins Human genes 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 102100029822 B- and T-lymphocyte attenuator Human genes 0.000 description 1
- 102100027205 B-cell antigen receptor complex-associated protein alpha chain Human genes 0.000 description 1
- 108010074708 B7-H1 Antigen Proteins 0.000 description 1
- 108700020463 BRCA1 Proteins 0.000 description 1
- 102000036365 BRCA1 Human genes 0.000 description 1
- 101150072950 BRCA1 gene Proteins 0.000 description 1
- 206010004446 Benign prostatic hyperplasia Diseases 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- 241000167854 Bourreria succulenta Species 0.000 description 1
- 208000014644 Brain disease Diseases 0.000 description 1
- 102000003930 C-Type Lectins Human genes 0.000 description 1
- 108090000342 C-Type Lectins Proteins 0.000 description 1
- 102100038078 CD276 antigen Human genes 0.000 description 1
- 108010058905 CD44v6 antigen Proteins 0.000 description 1
- 102100035793 CD83 antigen Human genes 0.000 description 1
- FVLVBPDQNARYJU-XAHDHGMMSA-N C[C@H]1CCC(CC1)NC(=O)N(CCCl)N=O Chemical compound C[C@H]1CCC(CC1)NC(=O)N(CCCl)N=O FVLVBPDQNARYJU-XAHDHGMMSA-N 0.000 description 1
- 101001039256 Caenorhabditis elegans Low-density lipoprotein receptor-related protein Proteins 0.000 description 1
- 101100454807 Caenorhabditis elegans lgg-1 gene Proteins 0.000 description 1
- 101100454808 Caenorhabditis elegans lgg-2 gene Proteins 0.000 description 1
- 101100217502 Caenorhabditis elegans lgg-3 gene Proteins 0.000 description 1
- 101100364669 Caenorhabditis elegans lin-18 gene Proteins 0.000 description 1
- KLWPJMFMVPTNCC-UHFFFAOYSA-N Camptothecin Natural products CCC1(O)C(=O)OCC2=C1C=C3C4Nc5ccccc5C=C4CN3C2=O KLWPJMFMVPTNCC-UHFFFAOYSA-N 0.000 description 1
- 206010058019 Cancer Pain Diseases 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- 101710197658 Capsid protein VP1 Proteins 0.000 description 1
- 101710197665 Capsid protein VP2 Proteins 0.000 description 1
- 102100025466 Carcinoembryonic antigen-related cell adhesion molecule 3 Human genes 0.000 description 1
- 102100025473 Carcinoembryonic antigen-related cell adhesion molecule 6 Human genes 0.000 description 1
- DLGOEMSEDOSKAD-UHFFFAOYSA-N Carmustine Chemical compound ClCCNC(=O)N(N=O)CCCl DLGOEMSEDOSKAD-UHFFFAOYSA-N 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 241000606161 Chlamydia Species 0.000 description 1
- 241000193163 Clostridioides difficile Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 206010009900 Colitis ulcerative Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 1
- 206010010741 Conjunctivitis Diseases 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 241000557626 Corvus corax Species 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- 241001337994 Cryptococcus <scale insect> Species 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- 102000012193 Cystatin A Human genes 0.000 description 1
- 108010061641 Cystatin A Proteins 0.000 description 1
- 108010025905 Cystine-Knot Miniproteins Proteins 0.000 description 1
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 1
- HEBKCHPVOIAQTA-QWWZWVQMSA-N D-arabinitol Chemical compound OC[C@@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-QWWZWVQMSA-N 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-M D-gluconate Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O RGHNJXZEOKUKBD-SQOUGZDYSA-M 0.000 description 1
- XULFJDKZVHTRLG-JDVCJPALSA-N DOSPA trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F.CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)CCNC(=O)C(CCCNCCCN)NCCCN)OCCCCCCCC\C=C/CCCCCCCC XULFJDKZVHTRLG-JDVCJPALSA-N 0.000 description 1
- 108010092160 Dactinomycin Proteins 0.000 description 1
- 101100503636 Danio rerio fyna gene Proteins 0.000 description 1
- 241000289632 Dasypodidae Species 0.000 description 1
- 206010012438 Dermatitis atopic Diseases 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 235000019739 Dicalciumphosphate Nutrition 0.000 description 1
- 239000004338 Dichlorodifluoromethane Substances 0.000 description 1
- 208000003556 Dry Eye Syndromes Diseases 0.000 description 1
- 206010013774 Dry eye Diseases 0.000 description 1
- 102000016942 Elastin Human genes 0.000 description 1
- 108010014258 Elastin Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000032274 Encephalopathy Diseases 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- 241000194031 Enterococcus faecium Species 0.000 description 1
- 241000991587 Enterovirus C Species 0.000 description 1
- 101710091045 Envelope protein Proteins 0.000 description 1
- 101710204837 Envelope small membrane protein Proteins 0.000 description 1
- 108010055196 EphA2 Receptor Proteins 0.000 description 1
- 102100030340 Ephrin type-A receptor 2 Human genes 0.000 description 1
- 239000004386 Erythritol Substances 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 101150018272 FYN gene Proteins 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 102100037362 Fibronectin Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 1
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 1
- 101710177291 Gag polyprotein Proteins 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 244000060234 Gmelina philippensis Species 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 1
- 206010072579 Granulomatosis with polyangiitis Diseases 0.000 description 1
- 102100020948 Growth hormone receptor Human genes 0.000 description 1
- 102100033365 Growth hormone-releasing hormone receptor Human genes 0.000 description 1
- 108010052763 H5WYG peptide Proteins 0.000 description 1
- 208000037357 HIV infectious disease Diseases 0.000 description 1
- 208000030836 Hashimoto thyroiditis Diseases 0.000 description 1
- 241000590002 Helicobacter pylori Species 0.000 description 1
- 102100029360 Hematopoietic cell signal transducer Human genes 0.000 description 1
- 108010014594 Heterogeneous Nuclear Ribonucleoprotein A1 Proteins 0.000 description 1
- 102000017013 Heterogeneous Nuclear Ribonucleoprotein A1 Human genes 0.000 description 1
- 102000006479 Heterogeneous-Nuclear Ribonucleoproteins Human genes 0.000 description 1
- 108010019372 Heterogeneous-Nuclear Ribonucleoproteins Proteins 0.000 description 1
- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 description 1
- 101710103773 Histone H2B Proteins 0.000 description 1
- 102100021639 Histone H2B type 1-K Human genes 0.000 description 1
- 101000694288 Homo sapiens 40S ribosomal protein SA Proteins 0.000 description 1
- 101000834898 Homo sapiens Alpha-synuclein Proteins 0.000 description 1
- 101000864344 Homo sapiens B- and T-lymphocyte attenuator Proteins 0.000 description 1
- 101000914489 Homo sapiens B-cell antigen receptor complex-associated protein alpha chain Proteins 0.000 description 1
- 101000946856 Homo sapiens CD83 antigen Proteins 0.000 description 1
- 101000914337 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 3 Proteins 0.000 description 1
- 101000914326 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 6 Proteins 0.000 description 1
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 1
- 101000882584 Homo sapiens Estrogen receptor Proteins 0.000 description 1
- 101001075287 Homo sapiens Growth hormone receptor Proteins 0.000 description 1
- 101000997535 Homo sapiens Growth hormone-releasing hormone receptor Proteins 0.000 description 1
- 101000990188 Homo sapiens Hematopoietic cell signal transducer Proteins 0.000 description 1
- 101000913074 Homo sapiens High affinity immunoglobulin gamma Fc receptor I Proteins 0.000 description 1
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 description 1
- 101000599048 Homo sapiens Interleukin-6 receptor subunit alpha Proteins 0.000 description 1
- 101001042362 Homo sapiens Leukemia inhibitory factor receptor Proteins 0.000 description 1
- 101001047640 Homo sapiens Linker for activation of T-cells family member 1 Proteins 0.000 description 1
- 101001043594 Homo sapiens Low-density lipoprotein receptor-related protein 5 Proteins 0.000 description 1
- 101001137987 Homo sapiens Lymphocyte activation gene 3 protein Proteins 0.000 description 1
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 1
- 101001133056 Homo sapiens Mucin-1 Proteins 0.000 description 1
- 101001109503 Homo sapiens NKG2-C type II integral membrane protein Proteins 0.000 description 1
- 101001109501 Homo sapiens NKG2-D type II integral membrane protein Proteins 0.000 description 1
- 101000586302 Homo sapiens Oncostatin-M-specific receptor subunit beta Proteins 0.000 description 1
- 101000611936 Homo sapiens Programmed cell death protein 1 Proteins 0.000 description 1
- 101001043564 Homo sapiens Prolow-density lipoprotein receptor-related protein 1 Proteins 0.000 description 1
- 101000702132 Homo sapiens Protein spinster homolog 1 Proteins 0.000 description 1
- 101000738769 Homo sapiens Receptor-type tyrosine-protein phosphatase alpha Proteins 0.000 description 1
- 101000884271 Homo sapiens Signal transducer CD24 Proteins 0.000 description 1
- 101000652359 Homo sapiens Spermatogenesis-associated protein 2 Proteins 0.000 description 1
- 101000946860 Homo sapiens T-cell surface glycoprotein CD3 epsilon chain Proteins 0.000 description 1
- 101000669402 Homo sapiens Toll-like receptor 7 Proteins 0.000 description 1
- 101000679851 Homo sapiens Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 1
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 description 1
- 101001103033 Homo sapiens Tyrosine-protein kinase transmembrane receptor ROR2 Proteins 0.000 description 1
- 101000955999 Homo sapiens V-set domain-containing T-cell activation inhibitor 1 Proteins 0.000 description 1
- 101000851018 Homo sapiens Vascular endothelial growth factor receptor 1 Proteins 0.000 description 1
- 238000012450 HuMAb Mouse Methods 0.000 description 1
- 241000701109 Human adenovirus 2 Species 0.000 description 1
- 241000701085 Human alphaherpesvirus 3 Species 0.000 description 1
- 108700000788 Human immunodeficiency virus 1 tat peptide (47-57) Proteins 0.000 description 1
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 description 1
- 102100037852 Insulin-like growth factor I Human genes 0.000 description 1
- 102100026720 Interferon beta Human genes 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108090000467 Interferon-beta Proteins 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000013462 Interleukin-12 Human genes 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 102000000588 Interleukin-2 Human genes 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 102000004388 Interleukin-4 Human genes 0.000 description 1
- 102100037792 Interleukin-6 receptor subunit alpha Human genes 0.000 description 1
- 102100037795 Interleukin-6 receptor subunit beta Human genes 0.000 description 1
- 101710152369 Interleukin-6 receptor subunit beta Proteins 0.000 description 1
- 108010002586 Interleukin-7 Proteins 0.000 description 1
- 102000000704 Interleukin-7 Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 208000003456 Juvenile Arthritis Diseases 0.000 description 1
- 206010059176 Juvenile idiopathic arthritis Diseases 0.000 description 1
- 208000009319 Keratoconjunctivitis Sicca Diseases 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- 238000012449 Kunming mouse Methods 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- 125000000174 L-prolyl group Chemical group [H]N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(*)=O 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 125000000510 L-tryptophano group Chemical group [H]C1=C([H])C([H])=C2N([H])C([H])=C(C([H])([H])[C@@]([H])(C(O[H])=O)N([H])[*])C2=C1[H] 0.000 description 1
- 102000017578 LAG3 Human genes 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 241000282838 Lama Species 0.000 description 1
- 101150028321 Lck gene Proteins 0.000 description 1
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 108010006444 Leucine-Rich Repeat Proteins Proteins 0.000 description 1
- 102100021747 Leukemia inhibitory factor receptor Human genes 0.000 description 1
- 108010000817 Leuprolide Proteins 0.000 description 1
- 102100024032 Linker for activation of T-cells family member 1 Human genes 0.000 description 1
- 102000019298 Lipocalin Human genes 0.000 description 1
- 108050006654 Lipocalin Proteins 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- GQYIWUVLTXOXAJ-UHFFFAOYSA-N Lomustine Chemical compound ClCCN(N=O)C(=O)NC1CCCCC1 GQYIWUVLTXOXAJ-UHFFFAOYSA-N 0.000 description 1
- 102100021926 Low-density lipoprotein receptor-related protein 5 Human genes 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 108091054437 MHC class I family Proteins 0.000 description 1
- 102000043129 MHC class I family Human genes 0.000 description 1
- 108091054438 MHC class II family Proteins 0.000 description 1
- 102000043131 MHC class II family Human genes 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 235000019759 Maize starch Nutrition 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 238000000585 Mann–Whitney U test Methods 0.000 description 1
- 241000283923 Marmota monax Species 0.000 description 1
- 102000000422 Matrix Metalloproteinase 3 Human genes 0.000 description 1
- 244000246386 Mentha pulegium Species 0.000 description 1
- 235000016257 Mentha pulegium Nutrition 0.000 description 1
- 235000004357 Mentha x piperita Nutrition 0.000 description 1
- 101710081079 Minor spike protein H Proteins 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 1
- 102100034256 Mucin-1 Human genes 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101000930477 Mus musculus Albumin Proteins 0.000 description 1
- 101100226902 Mus musculus Fcrlb gene Proteins 0.000 description 1
- 101100407308 Mus musculus Pdcd1lg2 gene Proteins 0.000 description 1
- 101100364671 Mus musculus Ryk gene Proteins 0.000 description 1
- 101100268066 Mus musculus Zap70 gene Proteins 0.000 description 1
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 1
- HSHXDCVZWHOWCS-UHFFFAOYSA-N N'-hexadecylthiophene-2-carbohydrazide Chemical compound CCCCCCCCCCCCCCCCNNC(=O)c1cccs1 HSHXDCVZWHOWCS-UHFFFAOYSA-N 0.000 description 1
- QJMCKEPOKRERLN-UHFFFAOYSA-N N-3,4-tridhydroxybenzamide Chemical compound ONC(=O)C1=CC=C(O)C(O)=C1 QJMCKEPOKRERLN-UHFFFAOYSA-N 0.000 description 1
- 102100022683 NKG2-C type II integral membrane protein Human genes 0.000 description 1
- 102100022680 NKG2-D type II integral membrane protein Human genes 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 102100023181 Neurogenic locus notch homolog protein 1 Human genes 0.000 description 1
- 108700037638 Neurogenic locus notch homolog protein 1 Proteins 0.000 description 1
- KYRVNWMVYQXFEU-UHFFFAOYSA-N Nocodazole Chemical compound C1=C2NC(NC(=O)OC)=NC2=CC=C1C(=O)C1=CC=CS1 KYRVNWMVYQXFEU-UHFFFAOYSA-N 0.000 description 1
- REYJJPSVUYRZGE-UHFFFAOYSA-N Octadecylamine Chemical compound CCCCCCCCCCCCCCCCCCN REYJJPSVUYRZGE-UHFFFAOYSA-N 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108010062618 Oncogene Proteins v-rel Proteins 0.000 description 1
- 102100030098 Oncostatin-M-specific receptor subunit beta Human genes 0.000 description 1
- 241000713112 Orthobunyavirus Species 0.000 description 1
- 241000150452 Orthohantavirus Species 0.000 description 1
- 241000702244 Orthoreovirus Species 0.000 description 1
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 1
- 108010071083 Patched-2 Receptor Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010067902 Peptide Library Proteins 0.000 description 1
- 241000709664 Picornaviridae Species 0.000 description 1
- 241000224016 Plasmodium Species 0.000 description 1
- 241000223960 Plasmodium falciparum Species 0.000 description 1
- 208000000474 Poliomyelitis Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 108700004821 Polyomavirus major capsid protein VP1 Proteins 0.000 description 1
- 241001505332 Polyomavirus sp. Species 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 102000004257 Potassium Channel Human genes 0.000 description 1
- 108700030875 Programmed Cell Death 1 Ligand 2 Proteins 0.000 description 1
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 description 1
- 102100024213 Programmed cell death 1 ligand 2 Human genes 0.000 description 1
- 102100021923 Prolow-density lipoprotein receptor-related protein 1 Human genes 0.000 description 1
- 208000004403 Prostatic Hyperplasia Diseases 0.000 description 1
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 1
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 description 1
- 101710132593 Protein E2 Proteins 0.000 description 1
- 101710149951 Protein Tat Proteins 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- 229940123924 Protein kinase C inhibitor Drugs 0.000 description 1
- 102100036894 Protein patched homolog 2 Human genes 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 241000125945 Protoparvovirus Species 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 201000004681 Psoriasis Diseases 0.000 description 1
- 201000001263 Psoriatic Arthritis Diseases 0.000 description 1
- 208000036824 Psoriatic arthropathy Diseases 0.000 description 1
- 101710185720 Putative ethidium bromide resistance protein Proteins 0.000 description 1
- 108010081208 RMFPNAPYL Proteins 0.000 description 1
- 108020005067 RNA Splice Sites Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 101710118046 RNA-directed RNA polymerase Proteins 0.000 description 1
- 239000012979 RPMI medium Substances 0.000 description 1
- 241000711798 Rabies lyssavirus Species 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 101001023863 Rattus norvegicus Glucocorticoid receptor Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 101710088839 Replication initiation protein Proteins 0.000 description 1
- 241000725643 Respiratory syncytial virus Species 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 241000702670 Rotavirus Species 0.000 description 1
- 241000710799 Rubella virus Species 0.000 description 1
- 206010039710 Scleroderma Diseases 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 102100038081 Signal transducer CD24 Human genes 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
- 241000700584 Simplexvirus Species 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 101900174659 Staphylococcus aureus Clumping factor A Proteins 0.000 description 1
- 206010042033 Stevens-Johnson syndrome Diseases 0.000 description 1
- 231100000168 Stevens-Johnson syndrome Toxicity 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 230000005867 T cell response Effects 0.000 description 1
- 208000000389 T-cell leukemia Diseases 0.000 description 1
- 208000028530 T-cell lymphoblastic leukemia/lymphoma Diseases 0.000 description 1
- 102100035794 T-cell surface glycoprotein CD3 epsilon chain Human genes 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 102100033456 TGF-beta receptor type-1 Human genes 0.000 description 1
- FOCVUCIESVLUNU-UHFFFAOYSA-N Thiotepa Chemical compound C1CN1P(N1CC1)(=S)N1CC1 FOCVUCIESVLUNU-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 108010045070 Thyroid Hormone Receptors alpha Proteins 0.000 description 1
- 102000008235 Toll-Like Receptor 9 Human genes 0.000 description 1
- 108010060818 Toll-Like Receptor 9 Proteins 0.000 description 1
- 102100039390 Toll-like receptor 7 Human genes 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 108010011702 Transforming Growth Factor-beta Type I Receptor Proteins 0.000 description 1
- 108010082684 Transforming Growth Factor-beta Type II Receptor Proteins 0.000 description 1
- 102000004060 Transforming Growth Factor-beta Type II Receptor Human genes 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- 108010023649 Tripartite Motif Proteins Proteins 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 1
- 102100040247 Tumor necrosis factor Human genes 0.000 description 1
- 102100033732 Tumor necrosis factor receptor superfamily member 1A Human genes 0.000 description 1
- 101710187743 Tumor necrosis factor receptor superfamily member 1A Proteins 0.000 description 1
- 102100033733 Tumor necrosis factor receptor superfamily member 1B Human genes 0.000 description 1
- 101710187830 Tumor necrosis factor receptor superfamily member 1B Proteins 0.000 description 1
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 description 1
- 206010070517 Type 2 lepra reaction Diseases 0.000 description 1
- 102100039616 Tyrosine-protein kinase transmembrane receptor ROR2 Human genes 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- 102100038929 V-set domain-containing T-cell activation inhibitor 1 Human genes 0.000 description 1
- 206010046914 Vaginal infection Diseases 0.000 description 1
- 201000008100 Vaginitis Diseases 0.000 description 1
- 241000700647 Variola virus Species 0.000 description 1
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 description 1
- 108010053100 Vascular Endothelial Growth Factor Receptor-3 Proteins 0.000 description 1
- 102100033178 Vascular endothelial growth factor receptor 1 Human genes 0.000 description 1
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 description 1
- 102100033179 Vascular endothelial growth factor receptor 3 Human genes 0.000 description 1
- OIRDTQYFTABQOQ-UHTZMRCNSA-N Vidarabine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@@H]1O OIRDTQYFTABQOQ-UHTZMRCNSA-N 0.000 description 1
- JXLYSJRDGCGARV-WWYNWVTFSA-N Vinblastine Natural products O=C(O[C@H]1[C@](O)(C(=O)OC)[C@@H]2N(C)c3c(cc(c(OC)c3)[C@]3(C(=O)OC)c4[nH]c5c(c4CCN4C[C@](O)(CC)C[C@H](C3)C4)cccc5)[C@@]32[C@H]2[C@@]1(CC)C=CCN2CC3)C JXLYSJRDGCGARV-WWYNWVTFSA-N 0.000 description 1
- 241000863480 Vinca Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 229930003427 Vitamin E Natural products 0.000 description 1
- 241000710886 West Nile virus Species 0.000 description 1
- 208000026448 Wilms tumor 1 Diseases 0.000 description 1
- 101710127857 Wilms tumor protein Proteins 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 101001038499 Yarrowia lipolytica (strain CLIB 122 / E 150) Lysine acetyltransferase Proteins 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- DSNRWDQKZIEDDB-GCMPNPAFSA-N [(2r)-3-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-2-[(z)-octadec-9-enoyl]oxypropyl] (z)-octadec-9-enoate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C/CCCCCCCC DSNRWDQKZIEDDB-GCMPNPAFSA-N 0.000 description 1
- CWRILEGKIAOYKP-SSDOTTSWSA-M [(2r)-3-acetyloxy-2-hydroxypropyl] 2-aminoethyl phosphate Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCCN CWRILEGKIAOYKP-SSDOTTSWSA-M 0.000 description 1
- KMLCRELJHYKIIL-UHFFFAOYSA-N [1-(azanidylmethyl)cyclohexyl]methylazanide;platinum(2+);sulfuric acid Chemical compound [Pt+2].OS(O)(=O)=O.[NH-]CC1(C[NH-])CCCCC1 KMLCRELJHYKIIL-UHFFFAOYSA-N 0.000 description 1
- XSMVECZRZBFTIZ-UHFFFAOYSA-M [2-(aminomethyl)cyclobutyl]methanamine;2-oxidopropanoate;platinum(4+) Chemical compound [Pt+4].CC([O-])C([O-])=O.NCC1CCC1CN XSMVECZRZBFTIZ-UHFFFAOYSA-M 0.000 description 1
- NAFFDQVVNWTDJD-UHFFFAOYSA-L [4-(azanidylmethyl)oxan-4-yl]methylazanide;cyclobutane-1,1-dicarboxylate;platinum(4+) Chemical compound [Pt+4].[NH-]CC1(C[NH-])CCOCC1.[O-]C(=O)C1(C([O-])=O)CCC1 NAFFDQVVNWTDJD-UHFFFAOYSA-L 0.000 description 1
- 239000003070 absorption delaying agent Substances 0.000 description 1
- 150000001241 acetals Chemical class 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- VUBTYKDZOQNADH-UHFFFAOYSA-N acetyl hexadecanoate Chemical compound CCCCCCCCCCCCCCCC(=O)OC(C)=O VUBTYKDZOQNADH-UHFFFAOYSA-N 0.000 description 1
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 1
- 108091006088 activator proteins Proteins 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 208000036676 acute undifferentiated leukemia Diseases 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 229940009456 adriamycin Drugs 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000009824 affinity maturation Effects 0.000 description 1
- 229940040563 agaric acid Drugs 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 125000002877 alkyl aryl group Chemical group 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 201000009961 allergic asthma Diseases 0.000 description 1
- 208000030961 allergic reaction Diseases 0.000 description 1
- 208000004631 alopecia areata Diseases 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229920000469 amphiphilic block copolymer Polymers 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 230000001512 anti-cytomegaloviral effect Effects 0.000 description 1
- 230000036436 anti-hiv Effects 0.000 description 1
- 230000005809 anti-tumor immunity Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 208000002399 aphthous stomatitis Diseases 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- OPWOOOGFNULJAQ-UHFFFAOYSA-L azane;cyclopentanamine;2-hydroxybutanedioate;platinum(2+) Chemical compound N.[Pt+2].NC1CCCC1.[O-]C(=O)C(O)CC([O-])=O OPWOOOGFNULJAQ-UHFFFAOYSA-L 0.000 description 1
- VSRXQHXAPYXROS-UHFFFAOYSA-N azanide;cyclobutane-1,1-dicarboxylic acid;platinum(2+) Chemical compound [NH2-].[NH2-].[Pt+2].OC(=O)C1(C(O)=O)CCC1 VSRXQHXAPYXROS-UHFFFAOYSA-N 0.000 description 1
- 229960002170 azathioprine Drugs 0.000 description 1
- LMEKQMALGUDUQG-UHFFFAOYSA-N azathioprine Chemical compound CN1C=NC([N+]([O-])=O)=C1SC1=NC=NC2=C1NC=N2 LMEKQMALGUDUQG-UHFFFAOYSA-N 0.000 description 1
- RHISNKCGUDDGEG-UHFFFAOYSA-N bactenecin Chemical compound CCC(C)C1NC(=O)C(C(C)C)NC(=O)C(C(C)C)NC(=O)C(C(C)CC)NC(=O)C(CCCN=C(N)N)NC(=O)C(NC(=O)C(CC(C)C)NC(=O)C(N)CCCN=C(N)N)CSSCC(C(=O)NC(CCCN=C(N)N)C(O)=O)NC(=O)C(C(C)C)NC(=O)C(CCCN=C(N)N)NC1=O RHISNKCGUDDGEG-UHFFFAOYSA-N 0.000 description 1
- 108010016341 bactenecin Proteins 0.000 description 1
- 229960001716 benzalkonium Drugs 0.000 description 1
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 1
- HMFHBZSHGGEWLO-FCAWWPLPSA-N beta-L-ribose Chemical compound OC[C@@H]1O[C@H](O)[C@@H](O)[C@H]1O HMFHBZSHGGEWLO-FCAWWPLPSA-N 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 229940017687 beta-d-ribose Drugs 0.000 description 1
- 108050002883 beta-defensin Proteins 0.000 description 1
- 102000012265 beta-defensin Human genes 0.000 description 1
- 230000008275 binding mechanism Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- HUTDDBSSHVOYJR-UHFFFAOYSA-H bis[(2-oxo-1,3,2$l^{5},4$l^{2}-dioxaphosphaplumbetan-2-yl)oxy]lead Chemical compound [Pb+2].[Pb+2].[Pb+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O HUTDDBSSHVOYJR-UHFFFAOYSA-H 0.000 description 1
- 229960001561 bleomycin Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 229920001400 block copolymer Polymers 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000004067 bulking agent Substances 0.000 description 1
- 230000000981 bystander Effects 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 238000004422 calculation algorithm Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical compound C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 description 1
- 229940127093 camptothecin Drugs 0.000 description 1
- 230000004611 cancer cell death Effects 0.000 description 1
- 230000009702 cancer cell proliferation Effects 0.000 description 1
- 208000020670 canker sore Diseases 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 229960004424 carbon dioxide Drugs 0.000 description 1
- 229960004562 carboplatin Drugs 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 229960005243 carmustine Drugs 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 108010046237 cecropin P1-LI Proteins 0.000 description 1
- PRIVBYDFWSFUFP-RJLJEYQFSA-N cecropin p1 Chemical compound O=C([C@H](CCC(N)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@@H](N)CO)[C@@H](C)O)[C@@H](C)CC)[C@@H](C)CC)NCC(=O)NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PRIVBYDFWSFUFP-RJLJEYQFSA-N 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 238000009172 cell transfer therapy Methods 0.000 description 1
- 239000004568 cement Substances 0.000 description 1
- 229930183167 cerebroside Natural products 0.000 description 1
- 150000001784 cerebrosides Chemical class 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 235000019693 cherries Nutrition 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 208000019069 chronic childhood arthritis Diseases 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 238000010226 confocal imaging Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 208000004921 cutaneous lupus erythematosus Diseases 0.000 description 1
- NZNMSOFKMUBTKW-UHFFFAOYSA-N cyclohexanecarboxylic acid Chemical compound OC(=O)C1CCCCC1 NZNMSOFKMUBTKW-UHFFFAOYSA-N 0.000 description 1
- HPXRVTGHNJAIIH-UHFFFAOYSA-N cyclohexanol Chemical compound OC1CCCCC1 HPXRVTGHNJAIIH-UHFFFAOYSA-N 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 229960000684 cytarabine Drugs 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 229960003901 dacarbazine Drugs 0.000 description 1
- 229960000640 dactinomycin Drugs 0.000 description 1
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 1
- 229960000975 daunorubicin Drugs 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- CFCUWKMKBJTWLW-UHFFFAOYSA-N deoliosyl-3C-alpha-L-digitoxosyl-MTM Natural products CC=1C(O)=C2C(O)=C3C(=O)C(OC4OC(C)C(O)C(OC5OC(C)C(O)C(OC6OC(C)C(O)C(C)(O)C6)C5)C4)C(C(OC)C(=O)C(O)C(C)O)CC3=CC2=CC=1OC(OC(C)C1O)CC1OC1CC(O)C(O)C(C)O1 CFCUWKMKBJTWLW-UHFFFAOYSA-N 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- NEFBYIFKOOEVPA-UHFFFAOYSA-K dicalcium phosphate Chemical compound [Ca+2].[Ca+2].[O-]P([O-])([O-])=O NEFBYIFKOOEVPA-UHFFFAOYSA-K 0.000 description 1
- 229910000390 dicalcium phosphate Inorganic materials 0.000 description 1
- 229940038472 dicalcium phosphate Drugs 0.000 description 1
- RNPXCFINMKSQPQ-UHFFFAOYSA-N dicetyl hydrogen phosphate Chemical compound CCCCCCCCCCCCCCCCOP(O)(=O)OCCCCCCCCCCCCCCCC RNPXCFINMKSQPQ-UHFFFAOYSA-N 0.000 description 1
- 229940093541 dicetylphosphate Drugs 0.000 description 1
- PXBRQCKWGAHEHS-UHFFFAOYSA-N dichlorodifluoromethane Chemical compound FC(F)(Cl)Cl PXBRQCKWGAHEHS-UHFFFAOYSA-N 0.000 description 1
- 235000019404 dichlorodifluoromethane Nutrition 0.000 description 1
- 229940042935 dichlorodifluoromethane Drugs 0.000 description 1
- 229940087091 dichlorotetrafluoroethane Drugs 0.000 description 1
- UMGXUWVIJIQANV-UHFFFAOYSA-M didecyl(dimethyl)azanium;bromide Chemical compound [Br-].CCCCCCCCCC[N+](C)(C)CCCCCCCCCC UMGXUWVIJIQANV-UHFFFAOYSA-M 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- ZGSPNIOCEDOHGS-UHFFFAOYSA-L disodium [3-[2,3-di(octadeca-9,12-dienoyloxy)propoxy-oxidophosphoryl]oxy-2-hydroxypropyl] 2,3-di(octadeca-9,12-dienoyloxy)propyl phosphate Chemical compound [Na+].[Na+].CCCCCC=CCC=CCCCCCCCC(=O)OCC(OC(=O)CCCCCCCC=CCC=CCCCCC)COP([O-])(=O)OCC(O)COP([O-])(=O)OCC(OC(=O)CCCCCCCC=CCC=CCCCCC)COC(=O)CCCCCCCC=CCC=CCCCCC ZGSPNIOCEDOHGS-UHFFFAOYSA-L 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000002612 dispersion medium Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- VSJKWCGYPAHWDS-UHFFFAOYSA-N dl-camptothecin Natural products C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)C5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-UHFFFAOYSA-N 0.000 description 1
- JRBPAEWTRLWTQC-UHFFFAOYSA-N dodecylamine Chemical compound CCCCCCCCCCCCN JRBPAEWTRLWTQC-UHFFFAOYSA-N 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 239000008298 dragée Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002296 dynamic light scattering Methods 0.000 description 1
- 108010031145 eglin proteinase inhibitors Proteins 0.000 description 1
- 229920002549 elastin Polymers 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 229950010625 enloplatin Drugs 0.000 description 1
- 108700004025 env Genes Proteins 0.000 description 1
- 230000001973 epigenetic effect Effects 0.000 description 1
- 230000010502 episomal replication Effects 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 235000019414 erythritol Nutrition 0.000 description 1
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 1
- 229940009714 erythritol Drugs 0.000 description 1
- 229960001842 estramustine Drugs 0.000 description 1
- FRPJXPJMRWBBIH-RBRWEJTLSA-N estramustine Chemical compound ClCCN(CCCl)C(=O)OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 FRPJXPJMRWBBIH-RBRWEJTLSA-N 0.000 description 1
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 1
- 229960005420 etoposide Drugs 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- ODKNJVUHOIMIIZ-RRKCRQDMSA-N floxuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(F)=C1 ODKNJVUHOIMIIZ-RRKCRQDMSA-N 0.000 description 1
- XRECTZIEBJDKEO-UHFFFAOYSA-N flucytosine Chemical compound NC1=NC(=O)NC=C1F XRECTZIEBJDKEO-UHFFFAOYSA-N 0.000 description 1
- 229960004413 flucytosine Drugs 0.000 description 1
- 229960002949 fluorouracil Drugs 0.000 description 1
- 108020005243 folate receptor Proteins 0.000 description 1
- 102000006815 folate receptor Human genes 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 108700004026 gag Genes Proteins 0.000 description 1
- 229940044658 gallium nitrate Drugs 0.000 description 1
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 229940050410 gluconate Drugs 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 239000003979 granulating agent Substances 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 229940037467 helicobacter pylori Drugs 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 208000005252 hepatitis A Diseases 0.000 description 1
- 235000001050 hortel pimenta Nutrition 0.000 description 1
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 1
- 125000001183 hydrocarbyl group Chemical group 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 1
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 1
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 1
- 229960004716 idoxuridine Drugs 0.000 description 1
- 239000012216 imaging agent Substances 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 230000037189 immune system physiology Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 231100000405 induce cancer Toxicity 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 208000037797 influenza A Diseases 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 230000010189 intracellular transport Effects 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 201000002215 juvenile rheumatoid arthritis Diseases 0.000 description 1
- 206010023332 keratitis Diseases 0.000 description 1
- 201000010666 keratoconjunctivitis Diseases 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000001865 kupffer cell Anatomy 0.000 description 1
- 201000010901 lateral sclerosis Diseases 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000004901 leucine-rich repeat Anatomy 0.000 description 1
- GFIJNRVAKGFPGQ-LIJARHBVSA-N leuprolide Chemical compound CCNC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)CC1=CC=C(O)C=C1 GFIJNRVAKGFPGQ-LIJARHBVSA-N 0.000 description 1
- 229960004338 leuprorelin Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229950008991 lobaplatin Drugs 0.000 description 1
- 229960002247 lomustine Drugs 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- RLSSMJSEOOYNOY-UHFFFAOYSA-N m-cresol Chemical compound CC1=CC=CC(O)=C1 RLSSMJSEOOYNOY-UHFFFAOYSA-N 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 239000001095 magnesium carbonate Substances 0.000 description 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 238000002595 magnetic resonance imaging Methods 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- MQXVYODZCMMZEM-ZYUZMQFOSA-N mannomustine Chemical compound ClCCNC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CNCCCl MQXVYODZCMMZEM-ZYUZMQFOSA-N 0.000 description 1
- 229950008612 mannomustine Drugs 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229960004961 mechlorethamine Drugs 0.000 description 1
- HAWPXGHAZFHHAD-UHFFFAOYSA-N mechlorethamine Chemical compound ClCCN(C)CCCl HAWPXGHAZFHHAD-UHFFFAOYSA-N 0.000 description 1
- SGDBTWWWUNNDEQ-LBPRGKRZSA-N melphalan Chemical compound OC(=O)[C@@H](N)CC1=CC=C(N(CCCl)CCCl)C=C1 SGDBTWWWUNNDEQ-LBPRGKRZSA-N 0.000 description 1
- 229960001924 melphalan Drugs 0.000 description 1
- 210000004779 membrane envelope Anatomy 0.000 description 1
- 210000003071 memory t lymphocyte Anatomy 0.000 description 1
- GLVAUDGFNGKCSF-UHFFFAOYSA-N mercaptopurine Chemical compound S=C1NC=NC2=C1NC=N2 GLVAUDGFNGKCSF-UHFFFAOYSA-N 0.000 description 1
- 229960001428 mercaptopurine Drugs 0.000 description 1
- 229940100630 metacresol Drugs 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 229960004452 methionine Drugs 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- OSWPMRLSEDHDFF-UHFFFAOYSA-N methyl salicylate Chemical compound COC(=O)C1=CC=CC=C1O OSWPMRLSEDHDFF-UHFFFAOYSA-N 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- HPNSFSBZBAHARI-UHFFFAOYSA-N micophenolic acid Natural products OC1=C(CC=C(C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-UHFFFAOYSA-N 0.000 description 1
- 210000000274 microglia Anatomy 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 208000005264 motor neuron disease Diseases 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 206010028417 myasthenia gravis Diseases 0.000 description 1
- 108700021654 myb Genes Proteins 0.000 description 1
- 108700024542 myc Genes Proteins 0.000 description 1
- 229960000951 mycophenolic acid Drugs 0.000 description 1
- HPNSFSBZBAHARI-RUDMXATFSA-N mycophenolic acid Chemical compound OC1=C(C\C=C(/C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-RUDMXATFSA-N 0.000 description 1
- 210000004897 n-terminal region Anatomy 0.000 description 1
- 239000006199 nebulizer Substances 0.000 description 1
- 230000002956 necrotizing effect Effects 0.000 description 1
- QZGIWPZCWHMVQL-UIYAJPBUSA-N neocarzinostatin chromophore Chemical compound O1[C@H](C)[C@H](O)[C@H](O)[C@@H](NC)[C@H]1O[C@@H]1C/2=C/C#C[C@H]3O[C@@]3([C@@H]3OC(=O)OC3)C#CC\2=C[C@H]1OC(=O)C1=C(O)C=CC2=C(C)C=C(OC)C=C12 QZGIWPZCWHMVQL-UIYAJPBUSA-N 0.000 description 1
- 230000001613 neoplastic effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229950006344 nocodazole Drugs 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 210000004492 nuclear pore Anatomy 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 102000027450 oncoproteins Human genes 0.000 description 1
- 108091008819 oncoproteins Proteins 0.000 description 1
- 238000000879 optical micrograph Methods 0.000 description 1
- 150000002905 orthoesters Chemical class 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 229960001756 oxaliplatin Drugs 0.000 description 1
- DWAFYCQODLXJNR-BNTLRKBRSA-L oxaliplatin Chemical compound O1C(=O)C(=O)O[Pt]11N[C@@H]2CCCC[C@H]2N1 DWAFYCQODLXJNR-BNTLRKBRSA-L 0.000 description 1
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 108010011903 peptide receptors Proteins 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 230000003836 peripheral circulation Effects 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 229960003742 phenol Drugs 0.000 description 1
- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 210000000557 podocyte Anatomy 0.000 description 1
- 108700004029 pol Genes Proteins 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 229940065514 poly(lactide) Drugs 0.000 description 1
- 229920002246 poly[2-(dimethylamino)ethyl methacrylate] polymer Polymers 0.000 description 1
- 229920000058 polyacrylate Polymers 0.000 description 1
- 229920000447 polyanionic polymer Polymers 0.000 description 1
- 229920005646 polycarboxylate Polymers 0.000 description 1
- 229920002851 polycationic polymer Polymers 0.000 description 1
- 229920001601 polyetherimide Polymers 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 229920006324 polyoxymethylene Polymers 0.000 description 1
- 108020001213 potassium channel Proteins 0.000 description 1
- 229940116317 potato starch Drugs 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- OIGNJSKKLXVSLS-VWUMJDOOSA-N prednisolone Chemical compound O=C1C=C[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OIGNJSKKLXVSLS-VWUMJDOOSA-N 0.000 description 1
- 229960005205 prednisolone Drugs 0.000 description 1
- XOFYZVNMUHMLCC-ZPOLXVRWSA-N prednisone Chemical compound O=C1C=C[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 XOFYZVNMUHMLCC-ZPOLXVRWSA-N 0.000 description 1
- 229960004618 prednisone Drugs 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000003405 preventing effect Effects 0.000 description 1
- 210000004986 primary T-cell Anatomy 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 238000012913 prioritisation Methods 0.000 description 1
- CPTBDICYNRMXFX-UHFFFAOYSA-N procarbazine Chemical compound CNNCC1=CC=C(C(=O)NC(C)C)C=C1 CPTBDICYNRMXFX-UHFFFAOYSA-N 0.000 description 1
- 229960000624 procarbazine Drugs 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000003380 propellant Substances 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 201000005825 prostate adenocarcinoma Diseases 0.000 description 1
- 239000003881 protein kinase C inhibitor Substances 0.000 description 1
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 1
- 239000002510 pyrogen Substances 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 108091008598 receptor tyrosine kinases Proteins 0.000 description 1
- 102000027426 receptor tyrosine kinases Human genes 0.000 description 1
- 230000010837 receptor-mediated endocytosis Effects 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 229940100486 rice starch Drugs 0.000 description 1
- WBHHMMIMDMUBKC-QJWNTBNXSA-M ricinoleate Chemical compound CCCCCC[C@@H](O)C\C=C/CCCCCCCC([O-])=O WBHHMMIMDMUBKC-QJWNTBNXSA-M 0.000 description 1
- 229940066675 ricinoleate Drugs 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229960003440 semustine Drugs 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 239000007909 solid dosage form Substances 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 229940001941 soy protein Drugs 0.000 description 1
- 229940071440 soy protein isolate Drugs 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 229950004330 spiroplatin Drugs 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- SFVFIFLLYFPGHH-UHFFFAOYSA-M stearalkonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 SFVFIFLLYFPGHH-UHFFFAOYSA-M 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 108091007196 stromelysin Proteins 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000008362 succinate buffer Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- WFWLQNSHRPWKFK-ZCFIWIBFSA-N tegafur Chemical compound O=C1NC(=O)C(F)=CN1[C@@H]1OCCC1 WFWLQNSHRPWKFK-ZCFIWIBFSA-N 0.000 description 1
- 229960001674 tegafur Drugs 0.000 description 1
- 239000003277 telomerase inhibitor Substances 0.000 description 1
- NRUKOCRGYNPUPR-QBPJDGROSA-N teniposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@@H](OC[C@H]4O3)C=3SC=CC=3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 NRUKOCRGYNPUPR-QBPJDGROSA-N 0.000 description 1
- 229960001278 teniposide Drugs 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 229960003433 thalidomide Drugs 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 229960001196 thiotepa Drugs 0.000 description 1
- 230000002992 thymic effect Effects 0.000 description 1
- MNRILEROXIRVNJ-UHFFFAOYSA-N tioguanine Chemical compound N1C(N)=NC(=S)C2=NC=N[C]21 MNRILEROXIRVNJ-UHFFFAOYSA-N 0.000 description 1
- 229960003087 tioguanine Drugs 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 210000003956 transport vesicle Anatomy 0.000 description 1
- 108010062760 transportan Proteins 0.000 description 1
- PBKWZFANFUTEPS-CWUSWOHSSA-N transportan Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(N)=O)[C@@H](C)CC)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CN)[C@@H](C)O)C1=CC=C(O)C=C1 PBKWZFANFUTEPS-CWUSWOHSSA-N 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 229950001353 tretamine Drugs 0.000 description 1
- PXSOHRWMIRDKMP-UHFFFAOYSA-N triaziquone Chemical compound O=C1C(N2CC2)=C(N2CC2)C(=O)C=C1N1CC1 PXSOHRWMIRDKMP-UHFFFAOYSA-N 0.000 description 1
- 229960004560 triaziquone Drugs 0.000 description 1
- 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 1
- CYRMSUTZVYGINF-UHFFFAOYSA-N trichlorofluoromethane Chemical compound FC(Cl)(Cl)Cl CYRMSUTZVYGINF-UHFFFAOYSA-N 0.000 description 1
- 229940029284 trichlorofluoromethane Drugs 0.000 description 1
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical class CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 1
- UMKFEPPTGMDVMI-UHFFFAOYSA-N trofosfamide Chemical compound ClCCN(CCCl)P1(=O)OCCCN1CCCl UMKFEPPTGMDVMI-UHFFFAOYSA-N 0.000 description 1
- 229960000875 trofosfamide Drugs 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 229940121358 tyrosine kinase inhibitor Drugs 0.000 description 1
- 239000005483 tyrosine kinase inhibitor Substances 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 229960001055 uracil mustard Drugs 0.000 description 1
- 201000011531 vascular cancer Diseases 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 229960003636 vidarabine Drugs 0.000 description 1
- 229960003048 vinblastine Drugs 0.000 description 1
- JXLYSJRDGCGARV-XQKSVPLYSA-N vincaleukoblastine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-XQKSVPLYSA-N 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
- 229960004355 vindesine Drugs 0.000 description 1
- UGGWPQSBPIFKDZ-KOTLKJBCSA-N vindesine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(N)=O)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1N=C1[C]2C=CC=C1 UGGWPQSBPIFKDZ-KOTLKJBCSA-N 0.000 description 1
- 230000007502 viral entry Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 229940046009 vitamin E Drugs 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
- XLMPPFTZALNBFS-INIZCTEOSA-N vorozole Chemical compound C1([C@@H](C2=CC=C3N=NN(C3=C2)C)N2N=CN=C2)=CC=C(Cl)C=C1 XLMPPFTZALNBFS-INIZCTEOSA-N 0.000 description 1
- 229960001771 vorozole Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 229940100445 wheat starch Drugs 0.000 description 1
- 239000009637 wintergreen oil Substances 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229950009268 zinostatin Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
- A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
-
- 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
- A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464452—Transcription factors, e.g. SOX or c-MYC
- A61K39/464453—Wilms tumor 1 [WT1]
-
- 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
- A61K39/464493—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; Prostatic acid phosphatase [PAP]; Prostate-specific G-protein-coupled receptor [PSGR]
- A61K39/464495—Prostate specific membrane antigen [PSMA]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/464838—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
- A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
- A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
- A61K47/6913—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome the liposome being modified on its surface by an antibody
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
-
- 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
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2815—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD8
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
-
- 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
- A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
- A61K2239/58—Prostate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5123—Organic compounds, e.g. fats, sugars
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—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
- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
- C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
- C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
- C12N2810/859—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from immunoglobulins
Definitions
- compositions and methods that rapidly and selectively modify cells of the immune system to achieve therapeutic objectives.
- the methods can be practiced in vivo and any cell type that expresses or is associated with a known marker can be targeted for a therapeutic objective by the modified cell.
- compositions and methods that rapidly and selectively direct cells of the immune system to achieve therapeutic objectives.
- vaccines are used to prime the immune system to target antigens associated with unwanted cells.
- the biological processes underlying conventional vaccines can render them ineffective against many unwanted cells based on, among other factors, the time it takes to prime the immune system, the amount or degree to which the natural immune system can be primed against certain unwanted cell types and over time, the depletion of immune system resources.
- conventional vaccine approaches can be ineffective against cancer cells and cells affected by certain infectious diseases.
- vaccines can be capable of targeting the immune system to destroy cancer cells in some patients.
- the immune response using this approach requires months to mature and during this time, cancers can significantly progress and become fatal.
- conventional vaccines do not provide an adequate method to target and destroy unwanted cancer cells.
- T-cell transfer therapies are also time and labor-intense and must be personalized for each patient in cell production facilities, which are available only at a few highly specialized cancer centers worldwide. Similar issues are encountered with a number of other unwanted cell types. Thus, additional solutions are needed that allow rapid and selective direction of cells of the immune system to achieve therapeutic objectives
- compositions and methods that can rapidly and selectively direct cells of the immune system to achieve therapeutic objectives.
- the compositions and methods modify cells of the immune system, such as T cells or natural killer (NK) cells, to target and destroy unwanted cell types.
- the compositions and methods modify cells of the immune system, such as monocytes/macrophages, to target and destroy viruses before they infect cells and/or to target bacteria or fungus.
- the compositions and methods modify cells of the immune system, such as B cells, to produce and release antibodies, such as broadly-neutralizing antibodies.
- compositions and methods modify cells of the immune system, such as immunosuppressive regulatory T cells (T RE G) to target and protect, rather than destroy, cell types.
- T RE G immunosuppressive regulatory T cells
- Compositions and methods disclosed herein can also be used to modify stem cells to achieve therapeutic objectives.
- the described methods can be practiced in vivo rather than requiring patient-specific isolation and culturing, as is currently required by many cancer treatments.
- the methods can be practiced in vivo because following administration to a subject, the compositions selectively modify cells of the immune system to achieve selected therapeutic objectives.
- compositions and methods can be used to target any cell type for which a marker is now or later becomes known.
- the compositions and methods achieve this benefit by modifying cells of the immune system to express targeting agents for the marker of interest.
- the cells of the immune system are modified to express targeting agents that bind markers, such as antigens, on unwanted cells. Once bound to an unwanted cell, the immune cells mediate its destruction.
- the cells of the immune system can be modified to express targeting agents that bind markers expressed by wanted cells or cells in the vicinity of wanted cells. Once bound to a wanted cell or in the vicinity of a wanted cell, the immune cells can mediate protection of the wanted cell.
- compositions and methods disclosed herein also provide further advantages over the current state of the art.
- the compositions and methods can selectively destroy unwanted cells leaving healthy tissue undamaged.
- the compositions can be manufactured on a large scale in a stable form with a long shelf life rendering them compatible with wide distribution and inexpensive administration to large patient populations in outpatient settings (i.e., they provide "off-the-shelf directed treatments).
- the compositions can be administered in booster doses to reinforce immune cell targeting.
- the administered composition can be altered over time as a population of unwanted or wanted cell types (collectively "targets" herein) evolves.
- the compositions and methods achieve the described benefits by providing nanocarriers.
- the nanocarriers include a polynucleotide encoding a targeting agent.
- the nanocarrier is taken up by a cell of the immune system, which then expresses the encoded targeting agent.
- the targeting agent selectively binds a marker on a target, directing the cells of the immune system to the site of the therapeutic objective. If the expressed targeting agent is an unwanted cell-targeting agent (such as an antibody or a receptor for a cancer antigen), once bound, the modified immune cell will mediate the destruction of the unwanted cell. If the expressed targeting agent is a wanted cell-targeting agent (such as a receptor for a marker expressed by a cell undergoing autoimmune attack), once bound, the modified immune cell will mediate the protection of the wanted cell.
- an unwanted cell-targeting agent such as an antibody or a receptor for a cancer antigen
- nanocarriers further include lymphocyte-directing agents.
- Lymphocyte-directing agents can achieve selective uptake of the nanocarriers by cells of interest for a particular therapeutic objective.
- the lymphocyte- directing agents can include binding domains extending from the surface of the nanocarriers that facilitate uptake by lymphocytes or particular classes of lymphocytes.
- Nanocarriers can also include lymphocyte-directing agents that achieve selective uptake by more than one cell type.
- Nanocarriers can also further include one or more of: an endosomal release agent to facilitate release of the polynucleotide from endosomal compartments of the lymphocytes and/or a nuclear localization signal (NLS) to direct the polynucleotide into the nucleus of the lymphocyte for expression, particularly when, for example, the polynucleotide comprises plasmid DNA.
- an endosomal release agent to facilitate release of the polynucleotide from endosomal compartments of the lymphocytes
- NLS nuclear localization signal
- the nanocarriers comprise a porous nanoparticle surrounded by a coating.
- the polynucleotide and optionally the NLS
- the optional lymphocyte-directing agent and endosomal release agent can extend from the surface of the coating.
- Fig. 1 Schematic of described strategy to rapidly and selectively modify immune cells for therapeutic objectives using synthetic nanocarriers.
- Nanocarriers are loaded with polynucleotides that encode a targeting agent (e.g. tumor- or virus-specific T-cell receptor).
- a targeting agent e.g. tumor- or virus-specific T-cell receptor.
- Surface-anchored lymphocyte directing agents e.g. anti-CD3 antibody
- the nanocarriers Upon infusion into a patient's bloodstream, the nanocarriers transfer the polynucleotide molecules into lymphocytes, which subsequently express the targeting agent on their surface. Lymphocytes then recognize and lyse cells of interest (e.g. cancerous or virus-infected cells).
- FIG. 2 (A) Schematic representation of the protocell nanoparticle used in studies described herein. (B) A representative TEM image of a protocell nanoparticle.
- FIG. 3 Schematic representation of minicircle DNA construct used in studies described herein. Structure of the P28z minicircle.
- PSMA prostate-specific membrane antigen
- Fig. 4 Redirecting T-cell specificity toward prostate tumor via nanoparticle- mediated gene transfer.
- PSMA Prostate-Specific Membrane Antigen
- B Flow cytometric measurement of surface P28z expression on mouse effector T cells 30 hours after incubation with "empty" (left panel) or P28z minicircle-loaded (right panel) protocell nanoparticles.
- C 51 Cr release assay of T cells 30 h after nanoparticle transfection targeting PSMA-positive TRAMP prostate tumor cells.
- D Light microscope images of nanoparticle-transfected T cells co-cultured on a TRAMP prostate tumor cell monolayer.
- E Flow cytometric measurement of protocell binding to circulating host T cells 6 hours after intravenous injection of 1 x 10 11 fluorescently tagged nanoparticles.
- Fig. 5 Repeated injections of nanocarriers loaded DNA encoding the P28z chimeric antigen receptor brings about T-cell mediated regression of prostate tumor in mice. Luciferase tagged TRAMP-PSMA prostate tumor cells were transplanted into the dorsal lobe of the prostate gland of C57BL/6 mice. Two weeks later (Day 0), mice were treated with five high-dose bolus injections of 1 x10 12 CD3-targeting nanoparticles carrying P28z-encoding transgenes (Day 0, Day 2, Day 4, Day 6, and Day 8). Control mice received no nanoparticles.
- A Sequential bioluminescence imaging of Firefly luciferase-expressing TRAMP-PSMA tumors.
- FIG. 6 Schematic representation of minicircle DNA constructs.
- A Structure of the P28z minicircle.
- the prostate-specific membrane antigen (PSMA)-targeting chimeric antigen receptor P28z is expressed under the control of the T-cell specific CD3-delta promoter.
- B A scaffold/matrix attachment region (S/MAR) is shown upstream of the poly-A signal to allow sustained episomal replication.
- C Alternatively, the gene expression cassette can be flanked by the piggyBac inverted terminal repeats.
- the piggyBac transposon is a mobile genetic element that efficiently transposes between vector and chromosome via a "cut and paste" mechanism.
- compositions and methods that can rapidly and selectively direct cells within the body to achieve therapeutic objectives.
- the compositions and methods modify cells of the immune system, such as T-cells or NK cells, to target and destroy unwanted cell types.
- the compositions and methods modify cells of the immune system, such as monocytes/macrophages to target and destroy viruses before they infect cells and/or bacterial or fungal cells.
- the compositions and methods modify cells of the immune system, such as B cells, to produce and release antibodies, such as broadly-neutralizing antibodies.
- the compositions and methods modify cells of the immune system, such as immunosuppressive T RE G cells to target and protect cell types from, for example, autoimmune attack.
- Compositions and methods disclosed herein can also be used to modify stem cells to achieve therapeutic objectives.
- the described methods can be practiced in vivo rather than requiring patient-specific isolation and culturing, as is currently required by many treatments.
- the methods can be practiced in vivo because following administration to a subject, the compositions selectively modify cells of interest to achieve the therapeutic objective.
- one of the primary goals of clinical health research is to develop compositions and methods to rapidly and selectively direct the immune system to destroy unwanted cells.
- vaccines are used to prime the immune system to target antigens associated with unwanted cells.
- the biological processes underlying conventional vaccines can render them ineffective against many unwanted cells based on, among other factors, the time it takes to prime the immune system, the amount or degree to which the natural immune system can be primed against certain unwanted cell types and over time, the depletion of immune system resources.
- compositions and methods that can rapidly modify cells of the immune system to target and destroy unwanted cell types.
- the methods can be practiced in vivo rather than requiring patient-specific isolation and culturing, as is currently required by many cancer treatments.
- the methods can be practiced in vivo because following administration to a subject, the compositions selectively modify cells of the immune system to target unwanted cell types.
- compositions and methods can be used to target any cell type for which a marker is now or later becomes known.
- the compositions and methods achieve this benefit by modifying cells of the immune system to express targeting agents for the marker expressed by the target or in the vicinity of a target.
- the cells of the immune system are modified to express targeting agents that bind markers, such as antigens, on unwanted cells. Once bound to an unwanted cell, the immune cells mediate its destruction.
- cells of the immune system can be modified to express targeting agents that bind markers on or in the vicinity of wanted cells. Once bound to a wanted cell or in the wanted cell's vicinity, the immune cell can mediate its protection.
- compositions and methods achieve the described benefits by providing nanocarriers that include a polynucleotide encoding a targeting agent.
- Cells that uptake the nanocarrier will begin to express the polynucleotide, thereby expressing the targeting agent.
- the targeting agent directs the modified immune cell to the site of the therapeutic objective.
- a lymphocyte uptakes the nanocarrier and begins to express an unwanted cell targeting agent.
- the lymphocyte then binds and mediates the destruction of the unwanted cell type.
- compositions include lymphocyte-directing agents that selectively deliver the nanocarriers to cells of interest.
- the compositions can further include one or more of: an endosomal release agent to facilitate release of the polynucleotide from endosomal compartments of cells of the immune system and/or a nuclear localization signal (NLS) to direct the polynucleotide into the nucleus of the cell for expression if, for example, the polynucleotide includes plasmid DNA.
- endosomal release agent to facilitate release of the polynucleotide from endosomal compartments of cells of the immune system
- NLS nuclear localization signal
- the nanocarriers comprise a porous nanoparticle surrounded by a coating.
- the polynucleotide and optionally the NLS
- the lymphocyte-directing agent and optionally the endosomal release agent
- Lymphocyte-Directing Agents The lymphocyte-directing agents of the disclosed compositions selectively bind immune cells of interest.
- the cells are lymphocytes.
- lymphocyte-directing agents can direct the compositions to any lymphocyte capable of, without limitation, (i) targeting and killing unwanted cells, (ii) targeting unwanted cells for killing by other cell types, (iii) mediating unwanted cell killing; (iv) targeting viruses for destruction before viral entry into cells, (v) antibody production and/or (vi) targeting and protecting beneficial cells.
- lymphocytes include T-cells, B cells, natural killer (NK) cells, monocytes/macrophages and hematopoietic stem cells.
- lymphocyte-directing agents achieve selective direction to particular lymphocyte populations through receptor-mediated endocytosis.
- TCR T-cell receptor
- the actual T-cell receptor is composed of two separate peptide chains, which are produced from the independent T-cell receptor alpha and beta (TCRa and TCR ) genes and are called a- and ⁇ -TCR chains.
- Lymphocyte directing agents disclosed herein can bind a- and/or ⁇ -TCR chains to achieve selective delivery of a polynucleotide to these T cells.
- ⁇ T-cells represent a small subset of T-cells that possess a distinct T-cell receptor (TCR) on their surface.
- TCR T-cell receptor
- the TCR is made up of one ⁇ -chain and one ⁇ -chain. This group of T-cells is much less common (2% of total T-cells) than the ⁇ T-cells. Nonetheless, lymphocyte-directing agents disclosed herein can bind y- and/or ⁇ TCR chains to achieve selective delivery of a polynucleotide to these T cells.
- CD3 is expressed on all mature T cells. Accordingly, lymphocyte-directing agents disclosed herein can bind CD3 to achieve selective delivery of a polynucleotide to all mature T-cells. Activated T-cells express 4-1 BB (CD137). Accordingly, lymphocyte-directing agents disclosed herein can bind 4-1 BB to achieve selective delivery of a polynucleotide to activated T-cells. CD5 and transferrin receptor are also expressed on T-cells and can be used to achieve selective delivery of a polynucleotide to T-cells.
- T-cells can further be classified into helper cells (CD4+ T-cells) and cytotoxic T-cells (CTLs, CD8+ T-cells), which comprise cytolytic T-cells.
- T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and activation of cytotoxic T-cells and macrophages, among other functions. These cells are also known as CD4+ T-cells because they express the CD4 protein on their surface.
- Helper T-cells become activated when they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. Lymphocyte-directing agents disclosed herein can bind CD4 to achieve selective delivery of a polynucleotide to T helper cells.
- Cytotoxic T-cells destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T-cells because they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body. Lymphocyte-directing agents disclosed herein can bind CD8 to achieve selective delivery of a polynucleotide to CTL.
- Central memory T-cells refers to an antigen experienced CTL that expresses CD62L or CCR7 and CD45RO on the surface thereof, and does not express or has decreased expression of CD45RA as compared to naive cells.
- central memory cells are positive for expression of CD62L, CCR7, CD25, CD127, CD45RO, and CD95, and have decreased expression of CD45RA as compared to naive cells.
- Lymphocyte-directing agents disclosed herein can bind CD62L, CCR7, CD25, CD127, CD45RO and/or CD95 to achieve selective delivery of a polynucleotide to T C M-
- Effective memory T-cell refers to an antigen experienced T-cell that does not express or has decreased expression of CD62L on the surface thereof as compared to central memory cells, and does not express or has decreased expression of CD45RA as compared to a naive cell.
- effector memory cells are negative for expression of CD62L and CCR7, compared to naive cells or central memory cells, and have variable expression of CD28 and CD45RA.
- Effector T-cells are positive for granzyme B and perforin as compared to memory or naive T-cells.
- Lymphocyte-directing agents disclosed herein can bind granzyme B and/or perforin to achieve selective delivery of a polynucleotide to T EM .
- T RE G Regulatory T cells
- T RE G express CD25, CTLA-4, GITR, GARP and LAP.
- Lymphocyte-directing agents disclosed herein can bind CD25, CTLA-4, GITR, GARP and/or LAP to achieve selective delivery of a polynucleotide to na ' fve T REG .
- naive T-cells refers to a non-antigen experienced T cell that expresses CD62L and CD45RA, and does not express CD45RO as compared to central or effector memory cells.
- naive CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naive T-cells including CD62L, CCR7, CD28, CD127, and CD45RA. Lymphocyte-directing agents disclosed herein can bind CD62L, CCR7, CD28, CD127 and/or CD45RA to achieve selective delivery of a polynucleotide to na ' fve T-cells.
- Natural killer cells also known as NK cells, K cells, and killer cells
- NK cells are activated in response to interferons or macrophage-derived cytokines. They serve to contain viral infections while the adaptive immune response is generating antigen- specific cytotoxic T cells that can clear the infection.
- NK cells express CD8, CD16 and CD56 but do not express CDS.
- Lymphocyte-directing agents disclosed herein can bind CD8, CD16 and/or CD56 to achieve selective delivery of a polynucleotide to NK cells.
- Macrophages (and their precursors, monocytes) reside in every tissue of the body (in certain instances as microglia, Kupffer cells and osteoclasts) where they engulf apoptotic cells, pathogens and other non-self components. Because monocytes/macrophages engulf non-self components, a particular macrophage- or monocyte-directing agent is not required on the nanocarriers described herein for selective uptake by these cells.
- lymphocyte-directing agents disclosed herein can bind CD1 1 b, F4/80; CD68; CD1 1 c; IL-4Ra; and/or CD163 to achieve selective delivery of a polynucleotide to monocytes/macrophages.
- B cells can be distinguished from other lymphocytes by the presence of the B cell receptor (BCR).
- BCR B cell receptor
- the principal function of B cells is to make antibodies.
- B cells express CD5, CD19, CD20, CD21 , CD22, CD35, CD40, CD52, and CD80.
- Lymphocyte- directing agents disclosed herein can bind CD5, CD19, CD20, CD21 , CD22, CD35, CD40, CD52, and/or CD80 to achieve selective delivery of a polynucleotide to B-cells.
- Lymphocyte function-associated antigen 1 (LFA-1 ) is expressed by all T- cells, B-cells and monocytes/macrophages. Accordingly, lymphocyte-directing agents disclosed herein can bind LFA-1 to achieve selective delivery of a polynucleotide to T- cells, B-cells and monocytes/macrophages.
- Hematopoietic stem cells can also be targeted for selective delivery of nanocarriers disclosed herein.
- Hematopoietic stem cells express CD34, CD133, Sca-1 and CD1 17.
- Lymphocyte-directing agents disclosed herein can bind CD34, CD133, Sca-1 and/or CD1 17 to achieve selective delivery of a polynucleotide to hematopoietic stem cells.
- Selective delivery means that polynucleotides are delivered and expressed by one or more selected lymphocyte populations. In particular embodiments, selective delivery is exclusive to a selected lymphocyte population. In further embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of administered polynucleotides are delivered and/or expressed by a selected lymphocyte population. In further embodiments, selective delivery ensures that non-lymphocyte cells do not express delivered polynucleotides. For example, when the targeting agent is a T-cell receptor (TCR) gene, selectivity is ensured because only T cells have the zeta chains required for TCR expression.
- TCR T-cell receptor
- Selective delivery can also be based on lack of polynucleotide uptake into unselected cells or based on the presence of a specific promoter within the polynucleotide sequence when the polynucleotide includes plasmid DNA.
- plasmid DNA can include a T-cell-specific CD3-delta promoter.
- Additional promoters that can achieve selective delivery include: the murine stem cell virus promoter or the distal lck promoter for T cells or hematopoietic stem cells; the CD45 promoter, WASP promoter or IFN-beta promoter for hematopoietic stem cells; the B29 promoter for B cells; or the CD14 promoter or the CD1 1 b promoter for monocytes/macrophages.
- lymphocyte-directing agents can include binding domains for motifs found on lymphocyte cells. Lymphocyte-directing agents can also include any selective binding mechanism allowing selective uptake into lymphocytes.
- lymphocyte-directing agents include binding domains for T-cell receptor motifs; T-cell a chains; T-cell ⁇ chains; T-cell ⁇ chains; T-cell ⁇ chains; CCR7; CD3; CD4; CD5; CD7; CD8; CD1 1 b; CD1 1 c; CD16; CD19; CD20; CD21 ; CD22; CD25; CD28; CD34; CD35; CD40; CD45RA; CD45RO; CD52; CD56; CD62L; CD68;CD80; CD95; CD1 17; CD127; CD133; CD137 (4-1 BB); CD163; F4/80; IL-4Ra; Sca-1 ; CTLA-4; GITR; GARP; LAP; granzyme B; LFA-1 ; transferrin receptor;
- binding domains include cell marker ligands, receptor ligands, antibodies, peptides, peptide aptamers, nucleic acids, nucleic acid aptamers, spiegelmers or combinations thereof.
- binding domains include any substance that binds to another substance to form a complex capable of mediating endocytosis.
- Antibodies are one example of binding domains and include whole antibodies or binding fragments of an antibody, e.g., Fv, Fab, Fab', F(ab')2, Fc, and single chain Fv fragments (scFvs) or any biologically effective fragments of an immunoglobulin that bind specifically to a motif expressed by a lymphocyte.
- Antibodies or antigen binding fragments include all or a portion of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, bispecific antibodies, mini bodies, and linear antibodies.
- Antibodies from human origin or humanized antibodies have lowered or no immunogenicity in humans and have a lower number of non-immunogenic epitopes compared to non-human antibodies.
- Antibodies and their fragments will generally be selected to have a reduced level or no antigenicity in human subjects.
- Antibodies that specifically bind a motif expressed by a lymphocyte can be prepared using methods of obtaining monoclonal antibodies, methods of phage display, methods to generate human or humanized antibodies, or methods using a transgenic animal or plant engineered to produce antibodies as is known to those of ordinary skill in the art (see, for example, U.S. Patent Nos. 6,291 ,161 and 6,291 ,158).
- Phage display libraries of partially or fully synthetic antibodies are available and can be screened for an antibody or fragment thereof that can bind to a lymphocyte motif. For example, binding domains may be identified by screening a Fab phage library for Fab fragments that specifically bind to a target of interest (see Hoet et al., Nat. Biotechnol.
- Phage display libraries of human antibodies are also available. Additionally, traditional strategies for hybridoma development using a target of interest as an immunogen in convenient systems ⁇ e.g., mice, HuMAb mouse®, TC mouseTM, KM-mouse ® , llamas, chicken, rats, hamsters, rabbits, etc.) can be used to develop binding domains. In particular embodiments, antibodies specifically bind to motifs expressed by a selected lymphocyte and do not cross react with nonspecific components or unrelated targets. Once identified, the amino acid sequence or polynucleotide sequence coding for the antibody can be isolated and/or determined.
- binding domains of lymphocyte-directing agents include T-cell receptor motif antibodies; T-cell a chain antibodies; T-cell ⁇ chain antibodies; T-cell ⁇ chain antibodies; T-cell ⁇ chain antibodies; CCR7 antibodies; CD3 antibodies; CD4 antibodies; CD5 antibodies; CD7 antibodies; CD8 antibodies; CD1 1 b antibodies; CD1 1 c antibodies; CD16 antibodies; CD19 antibodies; CD20 antibodies; CD21 antibodies; CD22 antibodies; CD25 antibodies; CD28 antibodies; CD34 antibodies; CD35 antibodies; CD40 antibodies; CD45RA antibodies; CD45RO antibodies; CD52 antibodies; CD56 antibodies; CD62L antibodies; CD68 antibodies; CD80 antibodies; CD95 antibodies; CD1 17 antibodies; CD127 antibodies; CD133 antibodies; CD137 (4-1 BB) antibodies; CD163 antibodies; F4/80 antibodies; IL-4Ra antibodies; Sca-1 antibodies; CTLA-4 antibodies; GITR antibodies GARP antibodies; LAP antibodies; granzyme B antibodies; LFA-1 antibodies; or transferrin receptor antibodies.
- binding domains also can consist of scFv fragments of the foregoing antibodies.
- the lymphocyte-directing agent binding domain includes the scFv fragment (SEQ ID NO. 1 ) of the PSMA-specific chimeric antigen receptor (CAR), P28z.
- Peptide aptamers include a peptide loop (which is specific for a target protein) attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to an antibody.
- the variable loop length is typically 8 to 20 amino acids (e.g., 8 to 12 amino acids), and the scaffold may be any protein which is stable, soluble, small, and nontoxic (e.g., thioredoxin-A, stefin A triple mutant, green fluorescent protein, eglin C, and cellular transcription factor Spl).
- Peptide aptamer selection can be made using different systems, such as the yeast two-hybrid system (e.g., Gal4 yeast-two-hybrid system) or the LexA interaction trap system.
- Nucleic acid aptamers are single-stranded nucleic acid (DNA or RNA) ligands that function by folding into a specific globular structure that dictates binding to target proteins or other molecules with high affinity and specificity, as described by Osborne et al., Curr. Opin. Chem. Biol. 1 :5-9, 1997; and Cerchia et al., FEBS Letters 528:12-16, 2002.
- aptamers are small (-15 KD; or between 15-80 nucleotides or between 20-50 nucleotides).
- Aptamers are generally isolated from libraries consisting of 10 14 -10 15 random oligonucleotide sequences by a procedure termed SELEX (systematic evolution of ligands by exponential enrichment; see, for example, Tuerk et al., Science, 249:505-510, 1990; Green et al., Methods Enzymology. 75-86, 1991 ; and Gold et al., Annu. Rev. Biochem., 64: 763-797, 1995). Further methods of generating aptamers are described in, for example, US Patent Nos.
- lymphocytes Other agents that can facilitate internalization by and/or transfection of lymphocytes, such as poly(ethyleneimine)/DNA (PEI/DNA) complexes can also be used.
- PEI/DNA poly(ethyleneimine)/DNA
- PEI/DNA poly(ethyleneimine)/DNA
- polynucleotide includes a nucleic acid molecule that contains a nucleic acid sequence such that upon introduction into a targeted lymphocyte, the nucleic acid molecule can cause transcription and resulting translation of targeting agents encoded by the nucleic acid sequence of the nucleic acid molecule.
- the targeting agent is an unwanted cell targeting agent.
- the targeting agent is a wanted cell targeting agent.
- the term “gene” refers to a nucleic acid sequence that encodes a targeting agent. This definition includes various sequence polymorphisms, mutations, and/or sequence variants wherein such alterations do not affect the function of the encoded targeting agent.
- the term “gene” may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. The term further can include all introns and other DNA sequences spliced from the mRNA transcript, along with variants resulting from alternative splice sites.
- Nucleic acid sequences encoding the targeting agent can be DNA or RNA that directs the expression of the targeting agent.
- nucleic acid sequences may be a DNA strand sequence that is transcribed into RNA or an RNA sequence that is translated into protein.
- the nucleic acid sequences include both the full-length nucleic acid sequences as well as non-full-length sequences derived from the full-length protein.
- the sequences can also include degenerate codons of the native sequence or sequences that may be introduced to provide codon preference in a specific lymphocyte.
- Gene sequences to encode targeting agents disclosed herein are available in publicly available databases and publications, incorporated by reference herein.
- the term "encoding" refers to a property of sequences of nucleotides in a polynucleotide, such as a plasmid, a gene, cDNA, mRNA, to serve as templates for synthesis of targeting agents.
- a polynucleotide can, e.g., encode a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system.
- polynucleotides having a sequence encoding a targeting agent include all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
- the polynucleotides that encode proteins and RNA can also include introns.
- the polynucleotide includes a plasmid, a cDNA, or an mRNA that can include, e.g., a sequence ⁇ e.g., a gene) for expressing a targeting agent.
- Suitable plasmids include standard plasmid vectors and minicircle plasmids that can be used to transfer a gene to a lymphocyte.
- the polynucleotides ⁇ e.g., minicircle plasmids) can further include any additional sequence information to facilitate transfer of the genetic material ⁇ e.g., a sequence encoding a receptor to an antigen) to lymphocytes.
- the polynucleotides can include promoters, such as general promoters, tissue-specific promoters, cell-specific promoters, and/or promoters specific for the nucleus or cytoplasm. Promoters and plasmids ⁇ e.g., minicircle plasmids) are generally well known in the art and can be prepared using conventional techniques.
- the polynucleotides can be used to transfect lymphocytes. Unless otherwise specified, the terms transfect, transfected, or transfecting can be used to indicate the presence of exogenous polynucleotides or the expressed polypeptide therefrom in a lymphocyte.
- a number of vectors are known to be capable of mediating transfer of genes to lymphocytes, as is known in the art.
- the transfected polynucleotides can edit the antigen-specificity of lymphocytes without affecting off-target bystander cells (i.e., provide for selective delivery as defined herein).
- delivered genes can be expressed under the control of a lymphocyte-specific promoter.
- the promoters can be included in minicirde plasmids that are a form of supercoiled DNA molecule for nonviral gene transfer, which have neither bacterial origin of replication nor antibiotic resistance marker. They are thus smaller and potentially safer than the standard plasmids currently used in gene therapy.
- a scaffold/matrix attachment region can also be inserted into the polynucleotides.
- Polynucleotides containing an expression cassette linked to a S/MAR element can autonomously replicate extra-chromosomally in dividing cells.
- PiggyBac or Sleeping Beauty transposase-containing plasmids can also be used to stably integrate nanocarrier-delivered targeting agent genes into the genome of transfected lymphocytes.
- Other options to sustain expression include homo sapiens transposon-derived Busterl transposase-like protein gene; human endogenous retrovirus H protease/integrase-derived ORF1 ; homo sapiens Cas-Br-M (murine) ecotropic retroviral transforming sequence; homo sapiens endogenous retroviral sequence K; homo sapiens endogenous retroviral family W; homo sapiens LINE-1 type transposase domain; or homo sapiens pogo transposable element.
- a delivered polynucleotide is mRNA
- backbone modifications can increase the mRNA's stability making resistant to premature cleavage.
- Targeted Cells & Associated Markers can be unwanted cells or wanted cells.
- Unwanted cells include any cell type that is (i) capable of recognition and destruction by the immune system; and (ii) deemed undesirable by a subject, physician, veterinarian or researcher.
- Unwanted cells include (i) eukaryotic cells that are either cancerous or infected with a pathogen such as a virus and (ii) prokaryotic cells, such as certain bacteria, fungi or yeast.
- Wanted cells include any cell type that is (i) capable of recognition and protection by the immune system; and (ii) deemed desirable by a subject, physician, veterinarian or researcher.
- Wanted cells can include cells undergoing auto-immune attack or bacteria that are beneficial to the health of a microbiome.
- the markers are antigens.
- Antigens refer to substances capable of either binding to an antigen binding region of an immunoglobulin molecule or of eliciting an immune response, e.g., a T cell-mediated immune response by the presentation of the antigen on Major Histocompatibility Antigen (MHC) cellular proteins.
- Antigens include antigenic determinants, haptens, and immunogens, which may be peptides, small molecules, carbohydrates, lipids, nucleic acids or combinations thereof.
- the term "antigen” refers to those portions of the antigen (e.g., a peptide fragment) that is a T cell epitope presented by MHC to the T cell receptor.
- the portion of the antigen that binds to the complementarity determining regions of the variable domains of the antibody (light and heavy) is referenced.
- the bound portion may be a linear or three-dimensional epitope.
- markers are expressed by unwanted cells from cancers.
- Exemplary cancers include adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, leukemias, liver cancers, lymph node cancers, lymphomas, lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non-Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma,
- Particular antigen markers associated with cancers cells include A33; BAGE; Bcl-2; ⁇ -catenin; CA125; CA19-9; CD5; CD19; CD20; CD21 ; CD22; CD33; CD37; CD45; CD123; CEA; c-Met; CS-1 ; cyclin B1 ; DAGE; EBNA; EGFR; ephrinB2; estrogen receptor; FAP; ferritin; folate-binding protein; GAGE; G250; GD-2; GM2; gp75, gp100 (Pmel 17); HER-2/neu; HPV E6; HPV E7; Ki-67; LRP; mesothelin, p53, PRAME; progesterone receptor; PSA; PSMA; MAGE; MART; mesothelin; MUC; MUM-1 -B; myc; NYESO-1 ; ras; RORI; survivin; tenascin; TSTA
- the particular following cancers can be treated by targeting the associated provided antigens: leukemia/lymphoma (CD19, CD20, CD22, ROR1 , CD33); multiple myeloma (B-cell maturation antigen (BCMA)); prostate cancer (PSMA, WT1 , Prostate Stem Cell antigen (PSCA), SV40 T); breast cancer (HER2, ERBB2); stem cell cancer (CD133); ovarian cancer (L1 -CAM, extracellular domain of MUC16 (MUC-CD), folate binding protein (folate receptor), Lewis Y); renal cell carcinoma (carboxy-anhydrase-IX (CAIX); melanoma (GD2); and pancreatic cancer (mesothelin, CEA, CD24).
- B-cell maturation antigen BCMA
- PSMA Prostate Stem Cell antigen
- SV40 T breast cancer
- HER2, ERBB2 Prostate Stem Cell antigen
- CD133 ovarian cancer
- cancer cell antigens include:
- KYHLTVAQVRGGMVFELANSIVLPFDCRDYAWLR KYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEI ASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDP LGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDI ESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA
- modified T cells, NK cells and/or monocytes/macrophages target and destroy cancer cells.
- B cells can also be modified to secrete tumor- specific antibodies.
- Viral Markers In particular embodiments, markers are expressed by unwanted virally-infected cells.
- Exemplary viruses include adenoviruses, arenaviruses, bunyaviruses, coronavirusess, flavirviruses, hantaviruses, hepadnaviruses, herpesviruses, papilomaviruses, paramyxoviruses, parvoviruses, picornaviruses, poxviruses, orthomyxoviruses, retroviruses, reoviruses, rhabdoviruses, rotaviruses, spongiform viruses or togaviruses.
- adenoviruses include adenoviruses, arenaviruses, bunyaviruses, coronavirusess, flavirviruses, hantaviruses, hepadnaviruses, herpesviruses, papilomaviruses, paramyxoviruses, parvoviruses, picornaviruses, poxviruses,
- viral antigen markers include peptides expressed by CMV, cold viruses, Epstein-Barr, flu viruses, hepatitis A, B, and C viruses, herpes simplex, HIV, influenza, Japanese encephalitis, measles, polio, rabies, respiratory syncytial, rubella, smallpox, varicella zoster or West Nile virus.
- cytomegaloviral antigens include envelope glycoprotein B and CMV pp65; Epstein-Barr antigens include EBV EBNAI, EBV P18, and EBV P23; hepatitis antigens include the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, HBCAG DELTA, HBV HBE, hepatitis C viral RNA, HCV NS3 and HCV NS4; herpes simplex viral antigens include immediate early proteins and glycoprotein D; HIV antigens include gene products of the gag, pol, and env genes such as HIV gp32, HIV gp41 , HIV gp120, HIV gp160, HIV P17/24, HIV P24, HIV P55 GAG, HIV P66 POL, HIV TAT, HIV GP36, the Nef protein and reverse transcriptase; influenza antigens include hemagglutinin and neuramini
- Additional particular exemplary viral antigen sequences include:
- modified T cells recognize and destroy virally-infected cells.
- modified monocytes/macrophages can remove viruses from peripheral tissue or the blood stream (extracellular) before cellular infection by a viral particle.
- B cells can also be modified to express broadly neutralizing antibodies.
- B cells can be modified to express broadly neutralizing anti-HIV antibodies.
- the targeting agent targets HIV gag protein, gp120 or the Hepatitis B envelope protein (S domain).
- markers are expressed by cells associated with unwanted bacterial infections.
- Exemplary bacteria include anthrax; gram-negative bacilli, chlamydia, diptheria, haemophilus influenza, Helicobacter pylori, malaria, Mycobacterium tuberculosis, pertussis toxin, pneumococcus, rickettsiae, staphylococcus, streptococcus and tetanus.
- anthrax antigens include anthrax protective antigen; gram-negative bacilli antigens include lipopolysaccharides; haemophilus influenza antigens include capsular polysaccharides; diptheria antigens include diptheria toxin; Mycobacterium tuberculosis antigens include mycolic acid, heat shock protein 65 (HSP65), the 30 kDa major secreted protein and antigen 85A; pertussis toxin antigens include hemagglutinin, pertactin, FIM2, FIM3 and adenylate cyclase; pneumococcal antigens include pneumolysin and pneumococcal capsular polysaccharides; rickettsiae antigens include rompA; streptococcal antigens include M proteins; and tetanus antigens include tetanus toxin.
- HSP65 heat shock protein 65
- bacterial cells can also be wanted cell types.
- Monocytes/macrophages are particularly useful to modify when the therapeutic objective is treatment of a bacterial infection.
- monocytes/macrophages can be modified with a ligand recognizing the surface component lipoteichoic acid of Staphyloccus aureus or the Staphylococcus aureus clumping factor A (ClfA).
- Immunosuppressive T RE G can be useful to modify when a bacteria is a wanted cell type.
- lymphocytes are modified to target multi-drug resistant "superbugs".
- superbugs include Enterococcus faecium, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae (including Escherichia coli, Klebsiella pneumoniae, Enterobacter spp.).
- markers are expressed by cells associated with unwanted fungal infections.
- fungi include Candida, coccidiodes, cryptococcus, histoplasma, leishmania, Plasmodium, protozoa, parasites, schistosomae, tinea, toxoplasma, and trypanosoma cruzi.
- coccidiodes antigens include spherule antigens; cryptococcal antigens include capsular polysaccharides; histoplasma antigens include heat shock protein 60 (HSP60); leishmania antigens include gp63 and lipophosphoglycan; Plasmodium falciparum antigens include merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte/gamete surface antigens, protozoal and other parasitic antigens including the blood-stage antigen pf 155/RESA; schistosomae antigens include glutathione-S- transferase and paramyosin; tinea fungal antigens include trichophytin; toxoplasma antigens include SAG-1 and p30; and trypanosoma cruzi antigens include the 75-77 kDa antigen and the 56 kDa
- Monocytes/macrophages are particularly useful to modify when the therapeutic objective is treatment of a fungal infection.
- markers are expressed by cells associated with unwanted autoimmune or allergic conditions.
- autoimmune conditions include acute necrotizing hemorrhagic encephalopathy, allergic asthma, alopecia areata, anemia, aphthous ulcer, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), asthma, autoimmune thyroiditis, conjunctivitis, Crohn's disease, cutaneous lupus erythematosus, dermatitis (including atopic dermatitis and eczematous dermatitis), diabetes, diabetes mellitus, erythema nodosum leprosum, keratoconjunctivitis, multiple sclerosis, myasthenia gravis, psoriasis, scleroderma, Sjogren's syndrome, including keratoconjunctivitis sicca
- autoimmune antigens include glutamic acid decarboxylase 65 (GAD 65), native DNA, myelin basic protein, myelin proteolipid protein, acetylcholine receptor components, thyroglobulin, and the thyroid stimulating hormone (TSH) receptor.
- GID 65 glutamic acid decarboxylase 65
- native DNA myelin basic protein
- myelin proteolipid protein acetylcholine receptor components
- thyroglobulin thyroglobulin
- TSH thyroid stimulating hormone
- allergic antigens include pollen antigens such as Japanese cedar pollen antigens, ragweed pollen antigens, rye grass pollen antigens, animal derived antigens (such as dust mite antigens and feline antigens), histocompatibility antigens, and penicillin and other therapeutic drugs.
- Immunosuppressive T RE G can be useful to modify to protect wanted cells from autoimmune attack or to reduce immune system activity in an area.
- exemplary wanted cells to protect from autoimmune attack include neurons in multiple sclerosis or amylotrophic lateral sclerosis; connective tissue in rheumatoid arthritis; colon epithelium in Chrohn's disease; and the pancreas in Diabetes mellitus type 1 .
- T RE G are modified to express a chimeric antigen receptor (CAR) against KIR4.1 (a potassium channel) that has been identified as an immune target in multiple sclerosis.
- CAR chimeric antigen receptor
- markers can also include B- cell targets, TNF receptor superfamily members, Hedgehog family members, receptor tyrosine kinases, proteoglycan-related molecules, TGF- ⁇ superfamily members, Wnt-related molecules, T-cell targets, dendritic cell targets, NK cell targets, a monocyte/macrophage cell targets, and angiogenesis targets.
- markers can also include CEACAM6, c-Met, EGFR, ErbB2, ErbB3, ErbB4, EphA2, IGF1 R, GHRHR, GHR, FLT1 , KDR, FLT4, CD44v6, CA125, CEA, BTLA, TGFBR2, TGFBR1 , IL6R, gp130, TNFR1 , TNFR2, PD1 , PD-L1 , PD-L2, HVEM, mesothelin, PSMA, RANK, ROR1 , TNFRSF4, TWEAK-R, HLA, tumor or pathogen derived peptides bound to HLA (such as from hTERT, tyrosinase, or WT-1 ), LT R, LIFR , LRP5, MUC1 , OSMR , TCRa, TCR , B7H4, TLR7, TLR9, PTCH1 , PT
- Targeting agents include any binding domain capable of (i) expression by a lymphocyte; and (ii) binding to a marker associated with a target. Binding of the targeting agent to the marker then mediates destruction or protection of the target.
- Binding domains include any substance that binds to another substance to form a complex.
- binding domains include cell marker ligands, receptor ligands, antibodies, peptides, peptide aptamers, receptors and chimeric antigen receptors (CAR) or combinations thereof.
- targeting agent binding domains can include the same components, options and identification methods as described above in relation to lymphocyte- directing agent binding domains with altered specificity, as appropriate.
- Targeting agent binding domains can particularly include any peptide that specifically binds a marker on a targeted cell.
- Sources of targeting agent binding domains include antibody variable regions from various species (which can be in the form of antibodies, sFvs, scFvs, Fabs, scFv-based grababody, or soluble VH domain or domain antibodies). These antibodies can form antigen-binding regions using only a heavy chain variable region, i.e., these functional antibodies are homodimers of heavy chains only (referred to as "heavy chain antibodies”) (Jespers et al., Nat. Biotechnol. 22:1 161 , 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 72:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008).
- An alternative source of targeting agent binding domains includes sequences that encode random peptide libraries or sequences that encode an engineered diversity of amino acids in loop regions of alternative non-antibody scaffolds, such as scTCR (see, e.g., Lake et al., Int. lmmunol. ⁇ ⁇ ⁇ .745, 1999; Maynard et al., J. Immunol. Methods 306:51 , 2005; U.S. Patent No. 8,361 ,794), fibrinogen domains (see, e.g., Weisel et al., Science 230:1388, 1985), Kunitz domains (see, e.g., US Patent No.
- scTCR see, e.g., Lake et al., Int. lmmunol. ⁇ ⁇ ⁇ .745, 1999; Maynard et al., J. Immunol. Methods 306:51 , 2005; U.S. Patent No. 8,361 ,
- a binding domain is a single chain T cell receptor (scTCR) comprising ⁇ ⁇ ⁇ and C a chains (e.g., V a -C a , V p -C , V a -V p ) or comprising V a -C a , -C , ⁇ ⁇ ⁇ / ⁇ pair specific for a target of interest (e.g., peptide-MHC complex).
- scTCR single chain T cell receptor
- the targeting agent is an unwanted cell targeting agent and the binding domain can be an antibody targeting PSMA.
- a number of antibodies specific for PSMA are known to those of skill in the art and can be readily characterized for sequence, epitope binding, and affinity.
- Unwanted cell targeting agent binding domains can also include anti-Mesothelin ligands (associated with treating ovarian cancer, pancreatic cancer, and mesothelioma); anti-WT-1 (associated with treating leukemia and ovarian cancer); anti-HIV-gag (associated with treating HIV infections); or anti-cytomegalovirus (associated with treating CMV diseases such as herpes virus).
- the unwanted cell targeting agent binding domain can be any ligand that binds to any marker associated with an unwanted cell type as described herein.
- the targeting agent is an unwanted cell targeting agent and the binding domain can be an antibody targeting CD19.
- a binding domain is a single chain Fv fragment (scFv) that comprises VH and VL regions specific for CD19.
- the V H and V L regions are human.
- Exemplary V H and V L regions include the segments of anti-CD19 specific monoclonal antibody FMC63.
- the scFV is a human or humanized ss comprising a variable light chain comprising a CDRL1 sequence of RASQDISKYLN (SEQ ID NO. 14), CDRL2 sequence of SRLHSGV (SEQ ID NO.
- the scFV is a human or humanized ScFv comprising a variable heavy chain comprising CDRHI sequence of DYGVS (SEQ ID NO. 17), CDRH2 sequence of VTWGSETTYYNSALKS (SEQ ID NO. 18), and a CDRH3 sequence of YAMDYWG (SEQ ID NO. 19).
- Other CD19-targeting antibodies such as SJ25C1 and HD37 are known. (SJ25C1 : Bejcek et al. Cancer Res 2005, PMID 7538901 ; HD37: Pezutto et al. Jl 1987, PMID 2437199).
- SEQ ID NO. 20 provides the anti-CD19 scFv (VH-VL) FMC63 DNA sequence
- SEQ ID NO. 21 provides the anti-CD19 scFv (VH-VL) FMC63 amino acid sequence.
- the targeting agent is an unwanted cell targeting agent and the binding domain can be an antibody targeting RORI.
- the scFV is a human or humanized scFv comprising a variable light chain comprising a CDRL1 sequence of ASGFDFSAYYM (SEQ ID NO. 22), CDRL2 sequence of TIYPSSG (SEQ ID NO. 23), and a CDRL3 sequence of ADRATYFCA (SEQ ID NO. 24).
- the scFV is a human or humanized scFv comprising a variable heavy chain comprising CDRH1 sequence of DTIDWY (SEQ ID NO.
- CDRH2 sequence of VQSDGSYTKRPGVPDR SEQ ID NO. 26
- CDRH3 sequence of YIGGYVFG SEQ ID NO. 27
- targeting agent binding domains comprise a sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a TCR V a , V p , C a , or C , wherein each CDR comprises zero changes or at most one, two, or three changes, from a TCR or fragment or derivative thereof that specifically binds to target of interest.
- targeting agent binding domain V a , V p , C a , or C p region of the present disclosure can be derived from or based on a V a , V p , C a , or C of a known TCR ⁇ e.g., a high-affinity TCR) and contains one or more ⁇ e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more ⁇ e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more ⁇ e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions ⁇ e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the V a , V p , C a , or C of a known TCR.
- An insertion, deletion or substitution may be anywhere in a V a , V p , C a , or C region, including at the amino- or carboxy-terminus or both ends of these regions, provided that each CDR comprises zero changes or at most one, two, or three changes and provided a binding domain containing a modified V a , V p , C a , or C region can still specifically bind its target with an affinity similar to wild type.
- a binding domain V H region of the present disclosure can be derived from or based on a V H of a known monoclonal antibody and can contain one or more ⁇ e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more ⁇ e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more ⁇ e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions ⁇ e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the V H of a known monoclonal antibody.
- An insertion, deletion or substitution may be anywhere in the V H region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR comprises zero changes or at most one, two, or three changes and provided a binding domain containing the modified V H region can still specifically bind its target with an affinity similar to the wild type binding domain.
- a V L region in a binding domain of the present disclosure is derived from or based on a V L of a known monoclonal antibody and contains one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the V L of the known monoclonal antibody.
- An insertion, deletion or substitution may be anywhere in the V L region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR comprises zero changes or at most one, two, or three changes and provided a binding domain containing the modified V L region can still specifically bind its target with an affinity similar to the wild type binding domain.
- a binding domain comprises or is a sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a light chain variable region (V L ) or to a heavy chain variable region (V H ), or both, wherein each CDR comprises zero changes or at most one, two, or three changes, from a monoclonal antibody or fragment or derivative thereof that specifically binds to target of interest.
- V L light chain variable region
- V H heavy chain variable region
- cell-targeting agents disclosed herein include chimeric antigen receptors.
- Chimeric antigen receptors or “CARs” refer to synthetically designed receptors comprising at least a binding domain and an effector domain and optionally a spacer domain and/or a transmembrane domain. Binding domains are described elsewhere herein.
- Effector domains are capable of transmitting functional signals to a cell.
- an effector domain will directly or indirectly promote a cellular response by associating with one or more other proteins that directly promote a cellular response.
- Effector domains can provide for activation of at least one function of a transduced lymphocyte expressing the CAR upon binding to the marker expressed on a targeted cell. Activation of the lymphocyte can include one or more of proliferation, differentiation, activation or other effector functions.
- the delivered polynucleotide encodes for the effector domain.
- An effector domain may include one, two, three or more receptor signaling domains, intracellular signaling domains, costimulatory domains, or combinations thereof. Any intracellular effector domain, costimulatory domain or both from any of a variety of signaling molecules ⁇ e.g., signal transduction receptors) may be used in the CARs of this disclosure.
- Exemplary effector domains include those from 4-1 BB, CD3E, CD35, ⁇ 3 ⁇ , CD27, CD28 (e.g., SEQ ID NO.:28), CD79A, CD79B, CARD1 1 , DAP10, FcRa, FcR , FcRy, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH 1 , Wnt, NKG2D, OX40, ROR2, Ryk, SLAMF1 , Slp76, pTa, TCRa, TCR , TRIM, Zap70, PTCH2, or any combination thereof.
- T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation and provide a T cell receptor like signal (primary cytoplasmic signaling sequences) and those that act in an antigen- independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences).
- Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as receptor tyrosine-based activation motifs or iTAMs.
- iTAM containing primary cytoplasmic signaling sequences include those derived from CD3 zeta, FeR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
- an effector domain comprises a cytoplasmic portion that associates with a cytoplasmic signaling protein, wherein the cytoplasmic signaling protein is a lymphocyte receptor or signaling domain thereof, a protein comprising a plurality of ITAMs, a costimulatory factor, or any combination thereof.
- intracellular signaling domains include the cytoplasmic sequences of the CD3 zeta chain, and/or co- receptors that act in concert to initiate signal transduction following CAR engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.
- an intracellular signaling domain of a CAR can be designed to comprise an intracellular signaling domain combined with any other desired cytoplasmic domain(s).
- the intracellular signaling domain of a CAR can comprise an intracellular signaling domain and a costimulatory signaling region.
- the costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule.
- a costimulatory molecule is a cell surface molecule other than the expressed marker ligand that is required for a response of lymphocytes to a marker.
- examples of such molecules include CD27, CD28, 4-1 BB (CD 137), OX40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1 ), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.
- CAR polynucleotides can comprise a sequence encoding for a spacer region.
- the length of the spacer region can be customized for individual markers on targets to optimize target recognition and destruction or protection.
- a spacer length can be selected based upon the location of a marker epitope, affinity of an antibody for the epitope, and/or the ability of the lymphocytes expressing the CAR to proliferate in vitro and/or in vivo in response to marker recognition.
- a spacer region is found between the binding domain and a transmembrane domain of the CAR. Spacer regions can provide for flexibility of the binding domain and allows for high expression levels in the modified cells. In particu l ar embodiments, a spacer region can have at least 10 to 250 amino acids, at least 10 to 200 amino acids, at least 10 to 150 amino acids, at least 10 to 100 amino acids, at least 10 to 50 amino acids or at least 10 to 25 amino acids and including any integer between the endpoints of any of the listed ranges.
- a spacer region has 250 amino acids or less; 200 amino acids or less, 150 amino acids or less; 100 amino acids or less; 50 amino acids or less; 40 amino acids or less; 30 amino acids or less; 20 amino acids or less; or 10 amino acids or less.
- spacer regions can be derived from a hinge region of an immunoglobulin like molecule, for example all or a portion of the hinge region from a human lgG1 , human lgG2, a human lgG3, or a human lgG4. Hinge regions can be modified to avoid undesirable structural interactions such as dimerization.
- all or a portion of a hinge region can be combined with one or more domains of a constant region of an immunoglobulin.
- a portion of a hinge region can be combined with all or a portion of a CH2 or CH3 domain or variant thereof.
- CARs disclosed herein can also include transmembrane domains.
- the CAR polynucleotide encodes the transmembrane domain.
- the transmembrane domain provides for anchoring of the CAR in the lymphocyte membrane.
- the transmembrane domain may be derived either from a natural or a synthetic source. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.
- Transmembrane regions comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3, CD45, CD4, CDS, CD9, CDI6, CD22; CD33, CD37, CD64, CD80, CD86, CDI34, CDI37 and CD154.
- synthetic or variant transmembrane domains comprise predominantly hydrophobic residues such as leucine and valine.
- the CAR comprises a P28z fusion receptor composed of a single-chain antibody (scFv) specific for the extracellular domain of PSMA (J591 ) combined with CD28 and CD3 ⁇ cytoplasmic signaling domains.
- the CAR comprises a P28z CAR of SEQ ID NO. 94.
- SEQ ID NO. 94 includes murine components and was utilized in studies described herein. Amino acid positions 1 -797 include the anti-PSMA scFv (J592) whereas positions 797-1477 include the murine CD8 transmembrane domain, murine CD28 signaling domain and the murine CD3zeta signaling domain. Any P28z domain can be individually replaced with optimized domains.
- the transmembrane domain and signaling domains within positions 797-1477 of SEQ ID NO. 94 can be particularly replaced with domains optimized for use in humans or other animals.
- any whole or portion of a binding domain, any whole or portion of an effector domain, any whole or portion of a spacer domain and/or any whole or portion of a transmembrane domain can be optimized for use in humans or other animals.
- the P28z CAR is optimized for use in humans. When optimized for humans, the P28z CAR can have lowered or no immunogenicity in humans and have a lower number of non-immunogenic epitopes compared to non-human antibodies. [00111] Endosomal Release Agents.
- endosomal release agents include any compound or peptide sequence that facilitates cargo exit from the endosome of a lymphocyte.
- exemplary endosomal release agents include imidazoles, poly or oligoimidazoles, PEIs, peptides, fusogenic peptides, polycarboxylates, polycations, masked oligo or poly cations or anions, acetals, polyacetals, ketals/polyketyals, orthoesters, polymers with masked or unmasked cationic or anionic charges, amphiphilic block copolymers and dendrimers with masked or unmasked cationic or anionic charges.
- H5WYG peptide can be used to induce the lysis of membranes at low pH.
- the histidine-rich peptide H5WYG is a derivative of the N- terminal sequence of the HA-2 subunit of the influenza virus hemagglutinin in which 5 of the amino acids have been replaced with histidine residues.
- H5WYG is able to selectively destabilize membranes at a slightly acidic pH as the histidine residues are protonated.
- the E1 protein from Semliki Forrest virus is also a useful endosomal release agent.
- endosomal release agents include a hydrophobic membrane translocation sequence (MTS).
- MTS hydrophobic membrane translocation sequence
- An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO. 29).
- An RFGF analogue e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO. 30)
- AALLPVLLAAP SEQ ID NO. 30
- Additional exemplary endosomal release agents include:
- NLS Nuclear Localization Signals.
- NLS nuclear localization signals
- NLS are a class of short amino acid sequences from 3 to 100 amino acids in length, from 3 to 50, 4 to 30, or 4 to 20 amino acids in length.
- Exemplary NLS sequences include (i) monopartite NLS exemplified by the SV40 large T antigen NLS (PKKKRKV) (SEQ ID NO: 51 ); (ii) bipartite NLS consisting of two basic domains separated by a variable number of spacer amino acids and exemplified by the Xenopus nucleoplasms NLS (KRXXXXXXXXXKKKL) (SEQ ID NO: 52); and (iii) noncanonical sequences such as M9 of the hnRNP A1 protein, the influenza virus nucleoprotein NLS, and the yeast Gal4 protein NLS (Dingwall and Laskey, Trends Biochem Sci 16:478-481 , 1991 ).
- the NLS can be a highly cationic or basic peptide.
- the NLS comprises two or more Arg or Lys amino acid residues.
- the NLS can bind cytosolic proteins, such as importins and karyopherins, which recognize and transport NLS-containing sequences to the nuclear pore complex.
- polynucleotides in one embodiment nanoparticle-encapsulated plasmids
- SV40 T-Ag-derived NLS peptides include: PKKKRKV (SEQ ID NO. 51 ); PKKKRMV (SEQ ID NO. 53); PKKKRKVEDP (SEQ ID NO. 54); PKKGSKKA (SEQ ID NO. 55); PKTKRKV (SEQ ID NO. 56); CGGPKKKRKVG (SEQ ID NO.
- Additional exemplary NLS sequences include:
- Nanocarriers Compositions disclosed herein include nanocarriers.
- Nanocarriers can include a porous nanoparticle at least substantially covered by a coating.
- polynucleotides and optionally NLSs can be found within the porous nanoparticle whereas optional lymphocyte-directing agents and endosomal release agents can be anchored to the coating.
- Porous Nanoparticles can be constructed from any material capable of forming a porous network.
- Exemplary materials include a variety of material including, without limitation, biocompatible polymers, metals, transition metals and metalloids.
- biocompatible polymers include, but not limited to, agar, agarose, alginate, alginate/calcium phosphate cement (CPC), beta-galactosidase ( ⁇ -GAL), (1 ,2,3,4,6-pentaacetyl a-D-galactose), cellulose, chitin, chitosan, collagen, elastin, gelatin, hyaluronic acid collagen, hydroxyapatite, poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate) (PHBHHx), poly(lactide), poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLG), poly(lactic-co-glycolic acid) (PLGA),poly(vinyl alcohol) (PVA), silk, soy protein, and soy protein isolate, alone or in combination with any other polymer composition, in any concentration and in any ratio.
- CPC alginate/calcium phosphate cement
- Blending different polymer types in different ratios using various grades can result in characteristics that borrow from each of the contributing polymers.
- Various terminal group chemistries can also be adopted.
- Exemplary metals, transition metals and metalloids include lithium, magnesium, zinc, aluminum and silica.
- the porous nanoparticles comprise silica.
- the exceptionally high surface area of mesoporous silica (exceeding 1 ,000 m 2 /g) enables polynucleotide loading at levels exceeding conventional DNA carriers such as liposomes or polymer conjugates.
- pores range in size from 10-20 nm.
- Useful nanocarriers of particular embodiments also include those based on (i) lipid-based delivery systems, including cationic lipids, ionizable cationic lipids, lipid- like molecules and pH-sensitive amphiphiles; and/or (ii) polymeric RNA DNA delivery systems such as polyethyleniminie (PEI)-based polymeric vectors, chitosan-based vectors, dendrimers (highly branched, spherical macromolecules synthesized from poly- amidoamine (PAMAM) and poly-propylene iminie (PPI), and block copolymers such as PAA/BMA/DMAEMA and PDMAEMA.
- PEI polyethyleniminie
- PAMAM poly-amideoamine
- PPI poly-propylene iminie
- block copolymers such as PAA/BMA/DMAEMA and PDMAEMA.
- the porous nanoparticles can be a variety of different shapes, including spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and the like.
- the polynucleotides can be included in the porous nanoparticles in a variety of ways.
- the polynucleotides can be encapsulated in the porous nanoparticles.
- the polynucleotides can be associated (e.g., covalently and/or non-covalently) with the surface or close underlying vicinity of the surface of the porous nanoparticles.
- the polynucleotides can be incorporated in the porous nanoparticles e.g., integrated in the material of the porous nanoparticles.
- the polynucleotides can be incorporated into a polymer matrix of polymer nanoparticles.
- porous nanoparticles include liposomes. Liposomes are microscopic vesicles consisting of at least one concentric lipid bilayer. Vesicle-forming lipids are selected to achieve a specified degree of fluidity or rigidity of the final complex.
- liposomes provide a lipid composition that is an outer layer surrounding a porous nanoparticle.
- PC phosphatidylcholine
- PE phosphatidylethanolamine
- PI phosphatidylinositol
- SM sphingomyelin
- DOPE dioleoylphosphatidylethanolamine
- lipids capable of producing a stable liposome are phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebro sides, distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimido-methyl)cyclohexane-1
- HSPC hydrogenated soy phosphati
- Additional non-phosphorous containing lipids that can become incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, and the cationic lipids mentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Choi).
- Negatively charged lipids include phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylglycerol and (DOPG), dicetylphosphate that are able to form vesicles.
- lipids used to create liposomes disclosed herein include cholesterol, hydrogenated soy phosphatidylcholine (HSPC) and, the derivatized vesicle-forming lipid PEG-DSPE.
- the size of the nanocar ers can vary over a wide range and can be measured in different ways.
- the nanocarhers of the present disclosure can have a minimum dimension of 100 nm.
- the nanocarhers of the present disclosure can also have a minimum dimension of equal to or less than 500 nm, less than 150 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, or less than 10 nm.
- the nanocarriers can have a minimum dimension ranging between 5 nm and 500 nm, between 10 nm and 100 nm, between 20 nm and 90 nm, between 30 nm and 80 nm, between 40 nm and 70 nm, and between 40 nm and 60 nm.
- the dimension is the diameter of nanoparticles or coated nanoparticles.
- a population of nanocarriers of the present disclosure can have a mean minimum dimension of equal to or less than 500 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, or less than 10 nm.
- a population of nanocarriers in a composition of the present disclosure can have a mean diameter ranging between 5 nm and 500 nm, between 10 nm and 100 nm, between 20 nm and 90 nm, between 30 nm and 80 nm, between 40 nm and 70 nm, and between 40 nm and 60 nm.
- Dimensions of the nanocarriers can be determined using, e.g., conventional techniques, such as dynamic lightscattering and/or electron microscopy.
- the compositions include protocells as nanocarriers.
- Protocells can be formed via fusion of liposomes to porous silica nanoparticles.
- the high pore volume and surface area of the spherical mesoporous silica core allow high-capacity encapsulation of a spectrum of cargos, including plasmid DNA.
- the supported lipid bilayer whose composition can be modified for specific biological applications, can serve as a modular, reconfigurable scaffold, allowing the attachment of a variety of molecules, such as lymphocyte-directing agents, to provide cell-specific targeting and controlled intracellular trafficking.
- protocells can efficiently introduce polynucleotides into lymphocytes.
- anti-CD3 antibodies can be coupled onto protocell nanocarriers to selectively target the nanocarriers to T cells for rapid receptor-induced endocytosis.
- Protocells can be formed via fusion of liposomes with porous silica nanoparticles (Fig. 2A, B).
- the high pore volume and surface area of the spherical mesoporous silica core allow high-capacity encapsulation of a spectrum of cargos, including plasmid DNA.
- the membrane serves as a modular scaffold for the attachment of a variety of targeting moieties. In the embodiment depicted in Fig.
- the pH-sensitive fusogenic peptide H5WYG is tethered to the nanocarrier surface to facilitate endosomal escape.
- the plasmid DNA was also modified before encapsulation into nanoparticles with the SV40 large T antigen nuclear localization signal peptide (Fig. 2A).
- Particular nanocarrier embodiments include:
- TCR tumor cells
- T cells 4-1 BB HIV- HIV-gag protein- Cationic- hnRNP
- TREG Anti-CTLA-4 Neurons CAR specific for Cationic SV40 or anti-GARP KIR4.1 for the polymer- antibodies treatment of based
- WT1 antigen
- compositions comprising: The nanoparticles, porous nanoparticles and nanocarriers (all collectively referred to herein as "active ingredients") disclosed herein can be provided as part of compositions that comprise, consist of or consist essentially of the nanoparticles, porous nanoparticles and/or nanocarriers.
- active ingredients can be provided as part of compositions that comprise, consist of or consist essentially of the nanoparticles, porous nanoparticles and/or nanocarriers.
- the compositions can be formulated for administration to subjects.
- the active ingredients are provided as part of a composition that can comprise, for example, at least 0.1 % w/v of active ingredient(s); at least 1 % w/v of active ingredient(s); at least 10% w/v of active ingredient(s); at least 20% w/v of active ingredient(s); at least 30% w/v of active ingredient(s); at least 40% w/v of active ingredient(s); at least 50% w/v of active ingredient(s); at least 60% w/v of active ingredient(s); at least 70% w/v of active ingredient(s); at least 80% w/v of active ingredient(s); at least 90% w/v of active ingredient(s); at least 95% w/v of active ingredient(s); or at least 99% w/v of active ingredient(s).
- the active ingredients are provided as part of a composition that can comprise, for example, at least 0.1 % w/w of active ingredient(s); at least 1 % w/w of active ingredient(s); at least 10% w/w of active ingredient(s); at least 20% w/w of active ingredient(s); at least 30% w/w of active ingredient(s); at least 40% w/w of active ingredient(s); at least 50% w/w of active ingredient(s); at least 60% w/w of active ingredient(s); at least 70% w/w of active ingredient(s); at least 80% w/w of active ingredient(s); at least 90% w/w of active ingredient(s); at least 95% w/w of active ingredient(s); or at least 99% w/w of active ingredient(s).
- compositions disclosed herein can be formulated for administration by, without limitation, injection, inhalation, infusion, perfusion, lavage or ingestion.
- the compositions disclosed herein can further be formulated for, without limitation, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral and/or subcutaneous administration and more particularly by intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral and/or subcutaneous injection.
- compositions can be formulated as aqueous solutions, such as in buffers including Hanks' solution, Ringer's solution, or physiological saline.
- aqueous solutions can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- the formulation can be in lyophilized and/or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
- compositions can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like.
- suitable excipients include binders (gum tragacanth, acacia, cornstarch, gelatin), fillers such as sugars, e.g.
- lactose sucrose, mannitol and sorbitol; dicalcium phosphate, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxy- methylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agents; and binding agents.
- disintegrating agents can be added, such as corn starch, potato starch, alginic acid, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
- solid dosage forms can be sugar-coated or enteric-coated using standard techniques. Flavoring agents, such as peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. can also be used.
- compositions can be formulated as aerosol sprays from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- the dosage unit may
- composition formulation disclosed herein can advantageously include any other pharmaceutically acceptable carriers which include those that do not produce significantly adverse, allergic or other untoward reactions that outweigh the benefit of administration, whether for research, prophylactic and/or therapeutic treatments.
- exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990.
- formulations can be prepared to meet sterility, pyrogenicity, general safety and purity standards as required by United States FDA Office of Biological Standards and/or other relevant foreign regulatory agencies.
- Exemplary generally used pharmaceutically acceptable carriers include any and all bulking agents or fillers, solvents or co-solvents, dispersion media, coatings, surfactants, antioxidants (e.g., ascorbic acid, methionine, vitamin E), preservatives, isotonic agents, absorption delaying agents, salts, stabilizers, buffering agents, chelating agents (e.g., EDTA), gels, binders, disintegration agents, and/or lubricants.
- bulking agents or fillers include any and all bulking agents or fillers, solvents or co-solvents, dispersion media, coatings, surfactants, antioxidants (e.g., ascorbic acid, methionine, vitamin E), preservatives, isotonic agents, absorption delaying agents, salts, stabilizers, buffering agents, chelating agents (e.g., EDTA), gels, binders, disintegration agents, and/or lubricants.
- antioxidants e.g
- Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers and/or trimethylamine salts.
- Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.
- Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol or mannitol.
- Exemplary stabilizers include organic sugars, polyhydric sugar alcohols, polyethylene glycol; sulfur-containing reducing agents, amino acids, low molecular weight polypeptides, proteins, immunoglobulins, hydrophilic polymers or polysaccharides.
- Compositions can also be formulated as depot preparations. Depot preparations can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salts.
- compositions can be formulated as sustained-release systems utilizing semipermeable matrices of solid polymers containing at least one active ingredient.
- sustained-release materials have been established and are well known by those of ordinary skill in the art. Sustained-release systems may, depending on their chemical nature, release active ingredients following administration for a few weeks up to over 100 days.
- compositions can also include plasmid DNA carrying one or more anticancer genes selected from p53, RB, BRCA1 , E1A, bcl-2, MDR-1 , p21 , p16, bax, bcl-xs, E2F, IGF-I VEGF, angiostatin, oncostatin, endostatin, GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN- ⁇ , TNF-a and/or HSV-tk.
- anticancer genes selected from p53, RB, BRCA1 , E1A, bcl-2, MDR-1 , p21 , p16, bax, bcl-xs, E2F, IGF-I VEGF, angiostatin, oncostatin, endostatin, GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN- ⁇ , TNF-a and/or HSV-t
- Compositions can also include or be administered in combination with one or more antineoplastic drugs including adriamycin, angiostatin, azathioprine, bleomycin, busulfane, camptothecin, carboplatin, carmustine, chlorambucile, chlormethamine, chloroquinoxaline sulfonamide, cisplatin, cyclophosphamide, cycloplatam, cytarabine, dacarbazine, dactinomycin, daunorubicin, didox, doxorubicin, endostatin, enloplatin, estramustine, etoposide, extramustinephosphat, flucytosine, fluorodeoxyuridine, fluorouracil, gallium nitrate, hydroxyurea, idoxuridine, interferons, interleukins, leuprolide, lobaplatin, lomustine, mannomustine, mechlorethamine, mechlore
- Methods disclosed herein include treating subjects (humans, veterinary animals, livestock and research animals) with compositions, active ingredients, nanoparticles, porous nanoparticles and/or nanocarriers disclosed herein. Treating subjects includes delivering a therapeutically effective amount.
- An "effective amount" is the amount of a compound necessary to result in a desired physiological change in the subject. Effective amounts are often administered for research purposes. Effective amounts disclosed herein reduce the number of unwanted cell types in a subject.
- a prophylactic treatment includes a treatment administered to a subject who does not display signs or symptoms of a disease or condition associated with or caused by a target or displays only early signs or symptoms of the disease or condition such that treatment is administered for the purpose of diminishing, preventing, or decreasing the risk of developing the disease or condition further.
- a prophylactic treatment functions as a preventative treatment against a disease or disorder associated with or caused by a target.
- a "therapeutic treatment” includes a treatment administered to a subject who displays symptoms or signs of a disease or condition associated with or caused by a target and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the disease or condition.
- Therapeutically effective amounts include those that provide effective amounts, prophylactic treatment and/or therapeutic treatment. Therapeutically effective amounts need not fully prevent or cure the disease or condition but can also provide a partial benefit, such as reduction in the number of unwanted targets; reduction of destruction of wanted targets; and/or a delay of onset or alleviation or improvement of at least one symptom of the disease or condition.
- effective amounts and therapeutically effective amounts can be initially estimated based on results from in vitro assays and/or animal model studies.
- a dose can be formulated in animal models to achieve a circulating concentration range that includes an IC 5 o as determined in cell culture against a particular target. Such information can be used to more accurately determine useful doses in subjects of interest.
- the actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of disease, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.
- a dose can comprise 1 g /kg, 5 g /kg, 10 g /kg, 15 g /kg, 20 pg /kg, 25 pg /kg, 30 pg /kg, 35 pg/kg, 40 pg/kg, 45 pg/kg, 50 pg/kg, 55 pg/kg, 60 pg/kg, 65 pg/kg, 70 pg/kg, 75 pg/kg, 80 pg/kg, 85 pg/kg, 90 pg/kg, 95 pg/kg, 100 pg/kg, 150 Mg/kg, 200 pg/kg, 250 pg/kg, 350 pg/kg, 400 pg/kg, 450 pg/kg, 500 pg/kg, 550 pg/kg, 600 pg
- a dose can comprise 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 150 mg/kg, 200 mg/kg, 250 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 650 mg/kg, 700 mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 mg/kg, 950 mg/kg, 1000 mg/kg or more.
- Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 1 1 months or yearly.
- a treatment regimen e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 1 1 months or yearly.
- Exemplary methods disclosed herein include administering nanocarriers to a subject in need thereof.
- the nanocarriers are directed to chosen lymphocytes in the subject and are designed to be internalized by the lymphocytes. Once internalized, the nanocarriers further deliver a polynucleotide having a sequence that encodes a targeting agent.
- the polynucleotide modifies the lymphocytes to express the targeting agent, which subsequently binds a marker associated with the target.
- the lymphocytes can kill or otherwise trigger the destruction of unwanted targets such as unwanted cells, thereby treating a disease or condition associated with the unwanted cell type.
- the lymphocytes can protect wanted targets such as wanted cells, thereby treating a disease or condition associated with unwanted destruction of the wanted cell type.
- nanocarriers can be loaded with polynucleotides ⁇ e.g., Transgenes) that encode for a defined tumor- or virus-specific TCR.
- polynucleotides ⁇ e.g., Transgenes
- Surface-anchored lymphocyte-directing agents that recognize T-cell-specific proteins enable the nanocarriers to selectively bind T-cells.
- the nanocarriers Upon infusion into a subject's bloodstream, the nanocarriers can deliver TCR genes into T-cells, which can subsequently express this TCR on their surface. Equipped with a therapeutically relevant TCR, the T-cells can recognize and lyse malignant cells or virus-infected cells or other targeted unwanted cell types.
- NK cells are selectively modified to express CARs or high-affinity TCRs.
- hematopoietic stem cells HSCs
- monocytes/macrophages cells are selectively modified to express CARs or high-affinity ligands specific for viruses, bacteria, fungus or yeast antigens.
- B cells are selectively modified to express tumor- or virus- specific antibodies.
- T RE G cells are selectively modified to express CARs or high-affinity ligands specific for autoimmune markers, allergic reaction markers or beneficial bacteria.
- Additional embodiments include methods of delivering pre-designed synthetic nanocarriers to lymphocytes ⁇ e.g., T-cells), in which the nanocarriers can be loaded with polynucleotides ⁇ e.g., plasmids) that encode a receptor for an antigen ⁇ e.g., a prostate tumor-targeting receptor P28z). Internalization of the nanocarriers can render transfected lymphocytes ⁇ e.g., T-cells) capable of lysing cells associated with the antigen ⁇ e.g., a prostate tumor).
- delivery of the nanocarriers including the receptor genes into lymphocytes ⁇ e.g., T-cells can include, e.g., (1 ) specific binding to the lymphocytes ⁇ e.g., T-cells), (2) internalization of the nanocarriers by the lymphocytes, (3) escape from endocytic vesicles into the cytoplasm after internalization, (4) release of the polynucleotide, which (5) can be transported into the nucleus of the lymphocytes and (6) transcribed to deliver genes for expressing a receptor for the antigen.
- the methods are used to target unwanted cancer cells.
- the disclosed methods provide a new paradigm for the treatment of cancer that can involve programming circulating lymphocytes with tumor-recognizing capabilities in vivo.
- This paradigm contrasts with those currently used to generate T cells with defined anti-cancer specificities, which involve isolation of the lymphocytes from the patient and genetically modifying them in the laboratory with tumor antigen- specific receptors using retroviral or lentiviral vectors; the programmed cells are then expanded and infused back into the patient where they can recognize and destroy cancer cells.
- This ex vivo production of modified cells requires the production of a new lymphocyte cohort for each patient, a laborious process that can only be accomplished at elaborate cell-production facilities available at just a few cancer centers worldwide.
- compositions and methods disclosed herein can produce targeting effects within a subject's circulatory system in only days.
- the disclosed methods provide the first implementation of nanocarriers for the genetic engineering of immune cells to selectively target cells associated with markers for various therapeutic objectives. For example, and in relation to cancer cells as an unwanted cell type, previous nanotechnology-based clinical research has focused on particles that selectively accumulate chemotherapeutics, siRNA, or imaging agents at tumor sites while minimizing off-target toxicities.
- the methods described herein are different: instead of introducing therapeutics into tumor tissue, the disclosed methods introduce genes encoding tumor-recognizing receptors into circulating lymphocytes, which in turn bind and destroy tumor cells. This strategy has the advantage that, unlike agent-loaded nanoparticles (which are quickly cleared by phagocytes), the modified lymphocytes can persist and proliferate in the subject for a long-term effect.
- the current disclosure provides a new, more effective therapy.
- the disclosure shifts the focus from broad-impact chemotherapy or radiotherapy (which have many negative side-effects) to tumor-specific immunotherapeutics (which do not harm healthy tissue).
- Nanoparticle gene therapy will provide clinicians with the ability to instantly treat diagnosed patients with an off-the shelf composition that can be widely distributed at low cost, and is amenable to changes in dose and specificity as the treatment evolves.
- therapeutically effective amounts can decrease the number of tumor cells, decrease the number of metastases, decrease tumor volume, increase life expectancy, induce apoptosis of cancer cells, induce cancer cell death, induce chemo- or radiosensitivity in cancer cells, inhibit angiogenesis near cancer cells, inhibit cancer cell proliferation, inhibit tumor growth, prevent metastasis, prolong a subject's life, reduce cancer-associated pain, reduce the number of metastases, and/or reduce relapse or re-occurrence of the cancer following treatment.
- the methods can include obtaining lymphocytes from a subject.
- Lymphocytes can, e.g., be obtained from a subject using any procedure generally known in the art.
- blood can be obtained from a subject and lymphocytes can be isolated.
- the isolated lymphocytes can then be combined with nanocarriers (or a composition comprising nanocarriers) including a polynucleotide having a sequence that encodes a targeting agent.
- the nanocarriers can be internalized by the lymphocytes such that the lymphocytes then incorporate the polynucleotide and express the targeting agent.
- the modified lymphocytes expressing the targeting agent can be administered to the subject such that, after the administering, the lymphocytes bind to the targeted markers on cells associated with the disease, thereby treating the disease.
- the modifying of the lymphocytes can be fully accomplished ex vivo prior to administration, and/or nanocarriers can be internalized and the lymphocytes can be administered to the subject while modifying is being carried out leading to expression of the targeting agents.
- a synthetic nanocarrier comprising (i) a lipid-coated porous nanoparticle (ii) a lymphocyte-directing agent extending from the surface of the lipid-coated porous nanoparticle; and (iii) a polynucleotide encoding a chimeric antigen receptor (CAR) targeting agent within the pores of the lipid-coated porous nanoparticle nanoparticle.
- CAR chimeric antigen receptor
- a synthetic nanocarrier of embodiment 1 further comprising an endosomal release agent extending from the surface of the lipid-coated porous nanoparticle and (ii) a nuclear localization signal (NLS) within the pores of the lipid-coated porous nanoparticle.
- NLS nuclear localization signal
- the synthetic nanocarriers comprise liposomes, polymeric particles, metallic particles, polymeric micelles, polyethyleneimine (PEI)/DNA complexes, or a combination thereof.
- lymphocyte- directing agent comprises a binding domain selected from a lymphocyte receptor ligand, lymphocyte receptor antibody, lymphocyte receptor peptide aptamer, lymphocyte receptor nucleic acid aptamer, lymphocyte receptor aptamer, or a combination thereof.
- a synthetic nanocarrier of embodiment 16 wherein the marker is a cancer antigen is a cancer antigen.
- a synthetic nanocarrier of any one of embodiments 1 -23 comprising a S/MAR element, a PiggyBac transposase-containing plasmid, a Sleeping Beauty transposase-containing plasmid; a homo sapiens transposon-de ved Busterl transposase-like protein gene; a human endogenous retrovirus H protease/integrase-derived ORF1 ; a homo sapiens Cas-Br-M (murine) ecotropic retroviral transforming sequence; a homo sapiens endogenous retroviral sequence K; a homo sapiens endogenous retroviral family W sequence; a homo sapiens LINE-1 type transposase domain; or a homo sapiens pogo transposable element.
- composition comprising a synthetic nanocarrier of any one of embodiments 1 - 24.
- a method of treating a subject having a condition associated with a cell type comprising: administering a therapeutically effective amount of a synthetic nanocarrier of any one of embodiments 1 -24 to the subject thereby treating the subject.
- a method of treating a subject having a condition associated with a cell type comprising: administering a therapeutically effective amount of a composition of embodiment 25 to the subject thereby treating the subject.
- a method of embodiment 28 wherein the unwanted cancer cell is selected from an adrenal cancer cell, a bladder cancer cell, a blood cancer cell, a bone cancer cell, a brain cancer cell, a breast cancer cell, a carcinoma cell, a cervical cancer cell, a colon cancer cell, a colorectal cancer cell, a corpus uterine cancer cell, an ear, nose and throat (ENT) cancer cell, an endometrial cancer cell, an esophageal cancer cell, a gastrointestinal cancer cell, a head and neck cancer cell, a Hodgkin's disease cell, an intestinal cancer cell, a kidney cancer cell, a larynx cancer cell, a leukemia cell, a liver cancer cell, a lymph node cancer cell, a lymphoma cell, a lung cancer cell, a melanoma cell, a mesothelioma cell, a myeloma cell, a nasopharynx cancer cell, a neuroblastoma cell, a non-
- a method for treating a disease associated with an antigen comprising: administering to a subject in need thereof, a composition comprising a therapeutically effective amount of nanocarriers including a polynucleotide having a sequence that encodes a receptor for the antigen, thereby treating the disease.
- a method for treating a disease associated with an antigen comprising:
- lymphocytes from a subject in need thereof obtained by obtaining lymphocytes from a subject in need thereof;
- composition comprising nanocarriers including a polynucleotide having a sequence that encodes a receptor for the antigen, wherein the nanocarriers are selectively incorporated into the lymphocytes such that the lymphocytes express the receptor;
- a method of selectively transfecting lymphocytes in vivo the method comprising: contacting lymphocytes with nanocarriers comprising a polynucleotide having a sequence that encodes a receptor for an antigen associated with a disease, wherein the nanocarriers are selectively incorporated into the lymphocyte to release the polynucleotide such that the lymphocyte expresses the receptor, thereby transfecting the lymphocyte.
- lymphocytes comprise T-cells, NK cells, macrophages, monocytes, B cells, hematopoietic stem cells, or a combination thereof.
- lymphocytes comprise T-cells.
- a method of embodiment 40 wherein the cancer comprises a leukemia, a lymphoma, a carcinoma, a sarcoma, or a melanoma.
- a synthetic nanocarrier comprising (i) a lymphocyte-directing agent; and (ii) a polynucleotide encoding a targeting agent.
- a synthetic nanocarrier of embodiment 1 further comprising a nanoparticle.
- a synthetic nanocarrier of embodiments 1 or 2 further comprising a coating.
- a synthetic nanocarrier of embodiment 3 or 4 wherein the coating is a liposome, a lipid bilayer, or a polymeric micelle.
- the synthetic nanocarrier comprises liposomes, polymeric particles, metallic particles, polymeric micelles, polyethyleneimine (PEI)/DNA complexes, or a combination thereof.
- a synthetic nanocarrier of embodiment 20 wherein the marker is a cancer antigen, a viral antigen, a bacterial antigen, or a fungal antigen.
- anthrax protective antigen lipopolysaccharide
- capsular polysaccharide diptheria toxin
- mycolic acid heat shock protein 65
- HSP65 heat shock protein 65
- antigen 85A hemagglutinin
- pertactin pertactin
- FIM2 FIM
- the marker is a fungal antigen selected from spherule antigens; capsular polysaccharides; heat shock protein 60; gp63; lipophosphoglycan; merozoite surface antigens; sporozoite surface antigens; circumsporozoite antigens;
- a synthetic nanocarrier of embodiment 28 wherein the marker is an autoimmune antigen, an allergic antigen, or a bacterial antigen.
- a synthetic nanocarrier of embodiment 30 wherein the marker is an autoimmune antigen selected from glutamic acid decarboxylase 65 (GAD 65); native DNA; myelin basic protein; myelin proteolipid protein; acetylcholine receptor components; thyroglobulin; and thyroid stimulating hormone (TSH) receptor.
- GID 65 glutamic acid decarboxylase 65
- native DNA myelin basic protein
- myelin proteolipid protein acetylcholine receptor components
- thyroglobulin thyroid stimulating hormone
- a synthetic nanocarrier of embodiment 30 wherein the marker is an allergic antigen selected from Japanese cedar pollen antigens; ragweed pollen antigens; rye grass pollen antigens; dust mite antigens; feline antigens; and canine antigens.
- a synthetic nanocarrier of embodiment 36 wherein the targeting agent is monoclonal antibody FMC63.
- the targeting agent is a human or humanized scFv comprising a variable heavy chain comprising CDRHI sequence of DYGVS (SEQ ID NO. 17), CDRH2 sequence of VTWGSETTYYNSALKS (SEQ ID NO. 18), and a CDRH3 sequence of YAMDYWG (SEQ ID NO. 19).
- a synthetic nanocarrier of embodiment 34 wherein the marker is RORI.
- a synthetic nanocarrier of embodiment 40 wherein the targeting agent is a human or humanized scFv comprising a variable light chain comprising a CDRL1 sequence of ASGFDFSAYYM (SEQ ID NO. 22), CDRL2 sequence of TIYPSSG (SEQ ID NO. 23), and a CDRL3 sequence of ADRATYFCA (SEQ ID NO. 24).
- a synthetic nanocarrier of embodiment 40 wherein the targeting agent is a human or humanized scFv comprising a variable heavy chain comprising CDRH1 sequence of DTIDWY (SEQ ID NO. 25), CDRH2 sequence of VQSDGSYTKRPGVPDR (SEQ ID NO. 26), and a CDRH3 sequence of YIGGYVFG (SEQ ID NO. 27).
- a synthetic nanocarrier of embodiments 43 or 44 wherein the endosomal release agent is selected from any one of SEQ ID NOs. 29-50 or combinations thereof.
- NLS nuclear localization signal
- a synthetic nanocarrier of any one of embodiments 1 -48 comprising a S/MAR element, a PiggyBac transposase-containing plasmid, a Sleeping Beauty transposase-containing plasmid; a homo sapiens transposon-derived Busterl transposase-like protein gene; a human endogenous retrovirus H protease/integrase-derived ORF1 ; a homo sapiens Cas-Br-M (murine) ecotropic retroviral transforming sequence; a homo sapiens endogenous retroviral sequence K; a homo sapiens endogenous retroviral family W sequence; a homo sapiens LINE-1 type transposase domain; or a homo sapiens pogo transposable element.
- composition comprising a synthetic nanocarrier of any one of embodiments 1 - 49.
- a method of treating a subject having a condition associated with a cell type comprising: administering a therapeutically effective amount of a synthetic nanocarrier of any one of embodiments 1 -49 to the subject thereby treating the subject.
- a method of treating a subject having a condition associated with a cell type comprising: administering a therapeutically effective amount of a composition of embodiment 50 to the subject thereby treating the subject.
- a method for treating a disease associated with an antigen comprising: administering to a subject in need thereof, a composition comprising a therapeutically effective amount of nanocarriers including a polynucleotide having a sequence that encodes a receptor for the antigen, thereby treating the disease.
- a method for treating a disease associated with an antigen comprising:
- lymphocytes from a subject in need thereof obtained by obtaining lymphocytes from a subject in need thereof;
- composition comprising nanocarriers including a polynucleotide having a sequence that encodes a receptor for the antigen, wherein the nanocarriers are selectively incorporated into the lymphocytes such that the lymphocytes express the receptor;
- a method of selectively transfecting lymphocytes in vivo the method comprising: contacting lymphocytes with nanocarriers comprising a polynucleotide having a sequence that encodes a receptor for an antigen, wherein the nanocarriers are selectively incorporated into the lymphocyte to release the polynucleotide such that the lymphocyte expresses the receptor, thereby transfecting the lymphocyte.
- the antigen comprises a tumor antigen.
- lymphocytes comprise T-cells, NK cells, macrophages, monocytes, B cells, hematopoietic stem cells, or a combination thereof.
- lymphocytes comprise T-cells.
- a method of embodiment 65 wherein the cancer comprises a leukemia, a lymphoma, a carcinoma, a sarcoma, or a melanoma.
- a method of embodiment 65 wherein the disease is prostate cancer.
- Each of the exemplary embodiments in Set 1 and Set 2 also includes an embodiment wherein the lymphocyte-directing agent can be removed. These embodiments are especially useful when the selected cell types are monocytes/macrophages and broad non-specific uptake of the nanocarriers can be expected.
- Example 1 This example demonstrates that synthetic nanoparticles containing TCR genes can be used to generate functional tumor- or virus-specific T- cells.
- Lipid nanoparticles (Fig. 2A) were loaded with a minicircle gene (Fig. 3) encoding the chimeric antigen receptor P28z.
- P28z is a fusion receptor composed of a single- chain antibody (scFv) specific for the extracellular domain of PSMA (J591 ) combined with CD28 and ⁇ 3 ⁇ cytoplasmic signaling domains (Fig. 4A; SEQ ID NO. 94).
- chimeric antigen receptors are fusion receptors including an antigen- binding domain, a transmembrane domain and an intracellular signaling domain resulting in T-cell activation after antigen binding.
- the P28z CAR directs T-cells toward the prostate-specific membrane antigen (PSMA), which is highly expressed on prostate cancer cells. Therefore, the introduction of the P28z gene into T-cells renders them capable of recognizing and lysing prostate tumor.
- PSMA prostate-specific membrane antigen
- the P28z gene was cloned under the control of the T-cell specific promoter CD3 delta into a minicircle plasmid. Minicircles can include episomal DNA vectors that are produced as circular expression cassettes devoid of any bacterial plasmid DNA backbone. Their smaller molecular size can enable more efficient transfections and offers sustained expression over a period of weeks as compared to standard plasmid vectors that only work for a few days.
- the minicircle plasmid DNA was entrapped into nanocarriers.
- DOPC, DOPE, cholesterol, and 18:1 PEG 2000 PE lipids were first mixed in a 55:5:30:10 mass ratio, dried under a stream of nitrogen, and placed in a vacuum oven overnight to remove residual chloroform.
- the lipid film was then dissolved in tert-butanol and mixed 1 :1 (v/v) with a P28z minicircle plasmid solution (diluted in 10 mM Tris-HCI (pH 7.4) with 0.85% (w/v) NaCI and 0.25 M sucrose) such that the final DOPC:DNA ratio was 10:1 (w/w).
- the mixture was vortexed and passed through a 100 nm filter at least 10 times using a Mini-Extruder set (Avanti Polar Lipids, Inc.; Alabaster, AL, USA).
- anti-mouse CD8 antibodies were coupled to the surface of the lipid envelope.
- Anti-CD8 antibodies (10 mg/ml) were mildly reduced with a 25X molar excess of DTT for 20 min at 25°C in the presence of 10 mM EDTA in PBS to expose free hinge region thiols.
- DTT antibodies were passed through a desalting column.
- the heterobifunctional cross-linker SM(PEG)2 4 was used to anchor antibodies to the surface of DNA-loaded liposomes (Amine groups are present in the head groups of PE lipids, free thiol groups on antibodies were created by DTT, SM(PEG) 24 cross-links between amines and thiol groups).
- Liposomes were first incubated with a tenfold molar excess of SM(PEG)2 4 for 2 h at room temperature and centrifuged to remove unreacted cross-linker. Activated liposomes were then incubated with a fivefold molar excess of reduced anti-CD8 antibody for 2 h at room temperature.
- Nanoparticle-transfected T cells were functional, selectively lysing PSMA-expressing TRAMP prostate tumor cells (Fig. 4C,D).
- Example 2 CD3-targeted protocell nanoparticles selectively bind circulating T cells in mice.
- a goal of the current disclosure is to selectively and quickly edit lymphocyte specificity in vivo to target unwanted cells.
- mice were systemically injected with 1 x 10 11 fluorescently tagged nanoparticles. After 6 hours peripheral blood was collected by retro-orbital puncture and the percentage of fluorescent T cells was quantified by flow cytometry.
- CD3-targeted protocells labeled the majority of T cells in the blood, with relatively low binding to off-target cells (Fig. 4E, left panel). Confocal imaging of sorted T cells showed that nanoparticles are rapidly internalized from the cell surface into the cytoplasm as a result of receptor-induced endocytosis (Fig. 4E, right panel).
- Example 3 Generating an orthotopic bioluminescent mouse model for analyzing treatment of metastatic prostate cancer.
- Male TRAMP transgenic mice spontaneously develop orthotopic prostate tumors following puberty.
- TRAMP tumors do not express significant amounts of PSMA, a target in experiments using the P28z CAR.
- longitudinal studies to measure the prostate cancer volume in TRAMP animals rely on expensive and time- consuming magnetic resonance imaging (MRI) techniques, which preclude analysis of large cohorts of mice.
- MRI magnetic resonance imaging
- tumor cells were also genetically tagged with Firefly luciferase (FLuc).
- FLuc Firefly luciferase
- Example 4 The data shown in Figs. 2 and 4 establish the ability to generate nanoparticles that efficiently program T cells with genes encoding receptors specific for prostate tumor. While this strategy rapidly generates tumor-reactive T cells, expression of transgenes is transient because transferred plasmids are diluted out every time the lymphocyte divides.
- the current example evaluates persistent receptor gene expression in actively dividing T cells caused by inserting into the plasmid either: 1 ) a scaffold/matrix attachment region (S/MAR) sequence (which can undergo episomal self- replication), or 2) a transposable piggyBac element (which integrates the transgene into the genome). Stable and dependable transgene expression in dividing T cells will allow nanoparticle-transfected lymphocytes to serially kill unwanted cell types providing long- lived immunity against such cells.
- S/MAR scaffold/matrix attachment region
- a S/MAR sequence (provided by Dr. Lipps, University Witten/Herdecke) or piggyBac inverted terminal repeats (provided by Dr. Craig, Johns Hopkins University) will be cloned into minicircle plasmids that encode the P28z receptor, as illustrated in Fig. 6.
- Protocell nanoparticles loaded with equivalent amounts of P28z, P28z-S/MAR, or P28z-piggyBac minicircle DNA will be incubated with mouse CD8 + T lymphocytes at a cell:particle ratio of 1 :10. Following nanoparticle transfection, T cells will be expanded with plate-bound anti-CD3/anti-CD28 antibodies. Flow cytometry will be used to assess P28z receptor expression levels and persistence in proliferating T cells every 24 hours during a two week culture period.
- S/MAR-based episomes and piggyBac transposons are two highly efficient tools to modify cells to achieve stable gene expression.
- Incorporating S/MAR sequences or piggyBac transposable elements into nanocarrier-delivered plasmids will also maintain high-level P28z gene expression in T cells over weeks as a result of episomal self-replication or somatic integration, respectively.
- P28z gene expression is independent of chromosomal position effects and therefore not subject to epigenetic silencing and c/s-acting sequences.
- Example 6 determines that systemic injections of DNA nanocarriers can program sufficient quantities of T cells to target and eliminate disseminated prostate cancer. The tests will be conducted using nanoparticles loaded with minicircle DNA encoding the P28z CAR (described above), to generate PSMA- specific lymphocytes. The results of the studies will be positive following testing of the following questions: (1 ) how many peripheral T cells are genetically modified to express P28z following a single intravenous dose of CD3-targeting nanoparticles loaded with genes encoding the receptor?; (2) do the injected nanoparticles selectively edit the antigen-specificity of peripheral T cells without affecting off-target cells? And (3) what nanoparticle dosage is required to bring about T cell-mediated regression of metastatic prostate tumors in mice?
- Example 6(1 ) What percentage of peripheral T cells are modified by nanoparticle gene therapy? The goal of this study is to edit the antigen specificity of at least 10% of peripheral T cells within five days following a single bolus injection of nanocarriers. For comparison, some of the strongest vaccine vectors reported in the literature induce frequencies of self/tumor antigen-specific T cells of 1 -4% following repeated immunizations over weeks. Mice will be systemically injected with 1 x 10 9 , 1 x 10 10 , or 1 x 10 1 1 nanocarriers loaded with minicircle DNA encoding P28z, or with GFP as a control.
- the percentage of P28z + T cells will be quantified by flow cytometry using fluorescent recombinant PSMA protein as the reporter, as performed in previous gene transfer studies (see, e.g., Fig. 4B).
- Example 6(2) Does nanoparticle gene therapy edit the antigen specificity of peripheral T cells without affecting off-target cells? To confirm in vivo studies, showing that CD3-targeting protocells efficiently bind to host T cells after intravenous injection (Fig. 4E), how selectively nanoparticles introduce tumor-targeting receptor genes into circulating T cells will also be determined. To this end, P28z expression by other leukocyte subsets will be evaluated, using the samples obtained in Example 6(1 ).
- the other cell types will be identified using the following reporters: anti-CD8 and anti-CD4 (T-cell markers), anti-B220 (B-cell marker), anti-NK1 .1 (NK-cell marker), anti-CD1 15, anti-F4/80 and anti-CD1 1 b (monocyte markers), anti-Ly6G and anti-CD1 1 b (neutrophil markers), and anti-Gr-1 antibody (granulocyte marker).
- Example 6(3) What nanoparticle dosage is required to bring about T cell- mediated regression of metastatic prostate tumors in mice? To develop a reproducibly effective treatment for metastatic prostate cancer, the therapeutically optimal frequency and dosage of nanocarrier injections must be determined.
- a test system will be created by injecting luciferase-expressing TRAMP-PSMA tumor cells into the prostate of C57BL/6 mice and allowing them to develop for three weeks before performing the tests (see, e.g., Fig. 5).
- mice will be systemically injected with CD3-targeting nanocarriers carrying P28z-encoding transgenes, according to four administration protocols: single high-dose bolus injection (1 x 10 10 nanoparticles, i.v.); high-frequency high-dose injections (1 x 10 10 nanoparticles, i.v. every 3 days for 30 days); single low-dose injection (1 x 10 9 nanoparticles, i.v.); or high-frequency low-dose injections (1 x 10 9 nanoparticles, i.v. every 3 days for 30 days).
- mice To compare the therapeutic efficacy of nanoparticle infusions with conventional adoptive T-cell therapy, one additional group of mice will be treated with a single dose of 10 million T cells transduced ex vivo with P28z-encoding retroviral vectors. Differences in TRAMP-PSMA tumor progression will be measured between treatment and control groups using bioluminescence imaging. To correlate tumor regression with the concentration of nanoparticle-programmed T cells in the peripheral circulation, the percentage of P28z + T cells in whole blood will be quantified by flow cytometry every 6 days.
- circulating T cells can be selectively programmed to target prostate tumors without genetically modifying other cells.
- This specificity can be achieved by coating the nanoparticles with CD3-recognizing antibodies, and by expressing the P28z transgene under the control of the T cell-specific CD3 delta promoter. If flow cytometry shows that more than 20% of P28z-expressing cells in the peripheral blood are not the targeted T cells, the density of anti-CD3 antibodies on the surface of nanocarriers will be increased to improve T cell targeting. If the CD3 delta promoter is too weak to mediate sufficient levels of receptor gene expression in vivo, the murine stem cell leukemia virus (MSCV) promoter can be used to express the P28z CAR in T cells. The MSCV promoter exhibits strong activity in hematopoietic cells and stem cells.
- Example 7 determines that nanocarriers can alternatively modify host T cells with prostate tumor-specific T-cell receptor (TCR) genes that target different antigens.
- TCR tumor-specific T-cell receptor
- WT1 A murine receptor (3D TCR) that has a high affinity for the intracellular oncoprotein Wilms tumor 1 (WT1 ) has been successfully engineered by a team of immunologists led by P. Greenberg at the Fred Hutchinson Cancer Research Center. WT1 was ranked first in a list of 75 cancer antigens in a recent National Cancer Institute prioritization project. It is strongly expressed in high-grade prostate tumor where it promotes the formation of metastases, but is absent in non-neoplastic or benign prostatic hyperplasia tissues. In line with these studies, high WT1 gene expression was detected in the TRAMP prostate tumor cells used herein. WT1 is detected at only very low levels in other normal tissues, particularly hematopoietic stem cells and kidney podocytes.
- T cells have been shown to be capable of selectively recognizing transformed cells expressing high levels without toxicity to normal tissues.
- Example 7 it will be shown that systemic injections of protocells loaded with genes encoding affinity-matured WT1 -specific TCRs can impart specificity for WT1 to host T cells and lead to elimination of prostate cancer.
- mice will be injected with 1 x 10 10 nanoparticles carrying 3D TCR genes. Control nanoparticles will be loaded with GFP-expressing plasmids. Peripheral blood collected by retro-orbital puncture every four days over a 12-day period will be used to quantify WT1 -TCR + T cells and other leukocyte subsets by flow cytometry using a fluorescent conjugate of the WT1 -derived RMFPNAPYL epitope tetramer as the reporter.
- luciferase-expressing TRAMP tumors will be implanted into the prostate of C57BL/6 mice. Three weeks later, animals will be treated with: a single high-dose bolus injection (1 x 10 10 nanoparticles i.v.); high-frequency high- dose injections (1 x 10 10 nanoparticles i.v. every 3 days for 30 days); a single low-dose injection (1 x 10 9 nanoparticles, i.v.); or high-frequency low-dose injections (1 x 10 9 nanoparticles, i.v. every 3 days for 30 days).
- mice will be injected with 10 million T cells, which were ex vivo transduced with 3D TCR genes using retroviral vectors. Differences in TRAMP prostate tumor regression between treatment and control groups will be measured using bioluminescence imaging.
- T cell responses in antitumor immunity can be decisively dependent on the quality of the TCRs involved. Due to thymic selection, the affinities of natural TCRs that target oncogenic self-proteins like WT1 are generally much lower than those of typical virus-targeting TCRs. However, the ability of a naturally occurring TCR to recognize antigens like WT1 can be markedly enhanced by in vitro affinity maturation. Based on these data, if genes for an affinity-optimized, WT1 -specific TCR are introduced into circulating T cells using the disclosed nanoparticle gene therapy approach, T cells will effectively recognize and kill prostate cancer cells.
- 3D TCRs are fully functional in CD4 + and CD8 + T cells, and CD4 + T cells can directly mediate tumor destruction and/or provide cytokine help for CD8 + T cells; however, tumor-specific CD4 + regulatory T cells abrogate CD8 T cell-mediated tumor rejection. If CD3-targeted nanoparticles generate undesirable WT1 -specific CD4 + regulatory T cells, nanoparticles can be targeted to CD8 + T cells only. These studies will demonstrate that nanoparticles can deliver rationally engineered TCR genes into host T-cells and enable them to recognize intracellular tumor-associated antigen.
- Example 8 Modifying host lymphocytes with HIV-specific TCR genes to control HIV infection. HIV-infected humanized NOD/shi-scid/yc null (NOG) mice with nanoparticles carrying HIV-gag protein-specific TCR transgenes, or with control plasmids expressing green fluorescent protein will be studied. Differences in HIV viral titers between treatment groups will be determined and administration of the nanoparticles will show a beneficial result.
- NOG humanized NOD/shi-scid/yc null
- Sequence information provided by public database can be used to identify nucleic acid sequences encoding peptides disclosed herein and vice versa. Variants of the sequences disclosed and referenced herein are also included. [00192] Variants of peptides can include those having one or more conservative amino acid substitutions.
- a "conservative substitution” involves a substitution found in one of the following conservative substitutions groups: Group 1 : Alanine (Ala), Glycine (Gly), Serine (Ser), Threonine (Thr); Group 2: Aspartic acid (Asp), Glutamic acid (Glu); Group 3: Asparagine (Asn), Glutamine (Gin); Group 4: Arginine (Arg), Lysine (Lys), Histidine (His); Group 5: Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Val); and Group 6: Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp).
- amino acids can be grouped into conservative substitution groups by similar function or chemical structure or composition (e.g., acidic, basic, aliphatic, aromatic, sulfur-containing).
- an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Val, Leu, and lie.
- Other groups containing amino acids that are considered conservative substitutions for one another include: sulfur- containing: Met and Cysteine (Cys); acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, lie, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information is found in Creighton (1984) Proteins, W.H. Freeman and Company.
- Variants of the protein and nucleic acid sequences disclosed or referenced herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to he protein and nucleic acid sequences disclosed or referenced herein.
- % sequence identity refers to a relationship between two or more sequences, as determined by comparing the sequences.
- identity also means the degree of sequence relatedness between proteins or nucleic acid sequences as determined by the match between strings of such sequences.
- Identity (often referred to as “similarity") can be readily calculated by known methods, including (but not limited to) those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H.
- each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component.
- the transition term "comprise” or “comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts.
- a material effect would cause a statistically-significant reduction in the ability of a nanocarrier to reduce the number of an unwanted cell type and/or to protect a wanted cell type in vivo.
- the term "about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ⁇ 20% of the stated value; ⁇ 19% of the stated value; ⁇ 18% of the stated value; ⁇ 17% of the stated value; ⁇ 16% of the stated value; ⁇ 15% of the stated value; ⁇ 14% of the stated value; ⁇ 13% of the stated value; ⁇ 12% of the stated value; ⁇ 1 1 % of the stated value; ⁇ 10% of the stated value; ⁇ 9% of the stated value; ⁇ 8% of the stated value; ⁇ 7% of the stated value; ⁇ 6% of the stated value; ⁇ 5% of the stated value; ⁇ 4% of the stated value; ⁇ 3% of the stated value; ⁇ 2% of the stated value; or ⁇ 1 % of the stated value.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Mycology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Dispersion Chemistry (AREA)
- Toxicology (AREA)
- Gynecology & Obstetrics (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Reproductive Health (AREA)
- Pregnancy & Childbirth (AREA)
- Oncology (AREA)
- Endocrinology (AREA)
- Virology (AREA)
- Developmental Biology & Embryology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present disclosure provides compositions and methods that rapidly and selectively modify cells of the immune system to achieve therapeutic objectives. The methods can be practiced in vivo and any cell type that expresses a known marker can be targeted for a therapeutic objective. The present disclosure provides compositions and methods that rapidly and selectively modify cells of the immune system to achieve therapeutic objectives. The methods can be practiced in vivo and any cell type that expresses or is associated with a known marker can be targeted for a therapeutic objective by the modified cell.
Description
COMPOSITIONS AND METHODS
TO MODIFY CELLS FOR THERAPEUTIC OBJECTIVES
CROSS REFERENCE TO RELATED APPLICATIONS
[0001 ] This application claims priority to U.S. Provisional Patent Application No. 61/785,907, filed March 14, 2013, which is incorporated herein by reference in its entirety.
FIELD OF THE DISCLOSURE
[0002] The present disclosure provides compositions and methods that rapidly and selectively modify cells of the immune system to achieve therapeutic objectives. The methods can be practiced in vivo and any cell type that expresses or is associated with a known marker can be targeted for a therapeutic objective by the modified cell.
BACKGROUND OF THE DISCLOSURE
[0003] One of the primary goals of clinical health research is to develop compositions and methods that rapidly and selectively direct cells of the immune system to achieve therapeutic objectives. For example, vaccines are used to prime the immune system to target antigens associated with unwanted cells. The biological processes underlying conventional vaccines, however, can render them ineffective against many unwanted cells based on, among other factors, the time it takes to prime the immune system, the amount or degree to which the natural immune system can be primed against certain unwanted cell types and over time, the depletion of immune system resources. As examples, conventional vaccine approaches can be ineffective against cancer cells and cells affected by certain infectious diseases.
[0004] Using cancer cells as an example of an unwanted cell type, vaccines can be capable of targeting the immune system to destroy cancer cells in some patients. The immune response using this approach, however, requires months to mature and during this time, cancers can significantly progress and become fatal. Thus, conventional vaccines do not provide an adequate method to target and destroy unwanted cancer cells.
[0005] To achieve more rapid and potent cancer cell destruction, infusions of autologous T cells genetically targeted to tumor antigen are currently being tested in the
clinic and represent a promising treatment option. However, T-cell transfer therapies are also time and labor-intense and must be personalized for each patient in cell production facilities, which are available only at a few highly specialized cancer centers worldwide. Similar issues are encountered with a number of other unwanted cell types. Thus, additional solutions are needed that allow rapid and selective direction of cells of the immune system to achieve therapeutic objectives
SUMMARY OF THE DISCLOSURE
[0006] The present disclosure provides compositions and methods that can rapidly and selectively direct cells of the immune system to achieve therapeutic objectives. In particular embodiments, the compositions and methods modify cells of the immune system, such as T cells or natural killer (NK) cells, to target and destroy unwanted cell types. In other embodiments, the compositions and methods modify cells of the immune system, such as monocytes/macrophages, to target and destroy viruses before they infect cells and/or to target bacteria or fungus. In further embodiments, the compositions and methods modify cells of the immune system, such as B cells, to produce and release antibodies, such as broadly-neutralizing antibodies. In additional embodiments, the compositions and methods modify cells of the immune system, such as immunosuppressive regulatory T cells (TREG) to target and protect, rather than destroy, cell types. Compositions and methods disclosed herein can also be used to modify stem cells to achieve therapeutic objectives.
[0007] The described methods can be practiced in vivo rather than requiring patient- specific isolation and culturing, as is currently required by many cancer treatments. The methods can be practiced in vivo because following administration to a subject, the compositions selectively modify cells of the immune system to achieve selected therapeutic objectives.
[0008] The compositions and methods can be used to target any cell type for which a marker is now or later becomes known. The compositions and methods achieve this benefit by modifying cells of the immune system to express targeting agents for the marker of interest.
[0009] In particular examples, the cells of the immune system are modified to express targeting agents that bind markers, such as antigens, on unwanted cells. Once bound to an unwanted cell, the immune cells mediate its destruction. Alternatively, the cells of the immune system can be modified to express targeting agents that bind markers expressed by wanted cells or cells in the vicinity of wanted cells. Once bound to a wanted cell or in the vicinity of a wanted cell, the immune cells can mediate protection of the wanted cell.
[0010] The compositions and methods disclosed herein also provide further advantages over the current state of the art. For example, the compositions and methods can selectively destroy unwanted cells leaving healthy tissue undamaged. The compositions can be manufactured on a large scale in a stable form with a long shelf life rendering them compatible with wide distribution and inexpensive administration to large patient populations in outpatient settings (i.e., they provide "off-the-shelf directed treatments). Further, the compositions can be administered in booster doses to reinforce immune cell targeting. Alternatively, the administered composition can be altered over time as a population of unwanted or wanted cell types (collectively "targets" herein) evolves.
[0011] The compositions and methods achieve the described benefits by providing nanocarriers. In their simplest form, the nanocarriers include a polynucleotide encoding a targeting agent. The nanocarrier is taken up by a cell of the immune system, which then expresses the encoded targeting agent. The targeting agent selectively binds a marker on a target, directing the cells of the immune system to the site of the therapeutic objective. If the expressed targeting agent is an unwanted cell-targeting agent (such as an antibody or a receptor for a cancer antigen), once bound, the modified immune cell will mediate the destruction of the unwanted cell. If the expressed targeting agent is a wanted cell-targeting agent (such as a receptor for a marker expressed by a cell undergoing autoimmune attack), once bound, the modified immune cell will mediate the protection of the wanted cell.
[0012] In some embodiments, nanocarriers further include lymphocyte-directing agents. Lymphocyte-directing agents can achieve selective uptake of the nanocarriers by cells of interest for a particular therapeutic objective. For example, the lymphocyte-
directing agents can include binding domains extending from the surface of the nanocarriers that facilitate uptake by lymphocytes or particular classes of lymphocytes. Nanocarriers can also include lymphocyte-directing agents that achieve selective uptake by more than one cell type.
[0013] Nanocarriers can also further include one or more of: an endosomal release agent to facilitate release of the polynucleotide from endosomal compartments of the lymphocytes and/or a nuclear localization signal (NLS) to direct the polynucleotide into the nucleus of the lymphocyte for expression, particularly when, for example, the polynucleotide comprises plasmid DNA.
[0014] In particular embodiments, the nanocarriers comprise a porous nanoparticle surrounded by a coating. In these embodiments, the polynucleotide (and optionally the NLS) can be within the pores of the nanoparticle and the optional lymphocyte-directing agent and endosomal release agent can extend from the surface of the coating.
BRIEF DESCRIPTION OF THE FIGURES
[0015] Fig. 1 : Schematic of described strategy to rapidly and selectively modify immune cells for therapeutic objectives using synthetic nanocarriers. Nanocarriers are loaded with polynucleotides that encode a targeting agent (e.g. tumor- or virus-specific T-cell receptor). Surface-anchored lymphocyte directing agents (e.g. anti-CD3 antibody) enable these nanocarriers to bind lymphocytes selectively. Upon infusion into a patient's bloodstream, the nanocarriers transfer the polynucleotide molecules into lymphocytes, which subsequently express the targeting agent on their surface. Lymphocytes then recognize and lyse cells of interest (e.g. cancerous or virus-infected cells).
[0016] Fig. 2: (A) Schematic representation of the protocell nanoparticle used in studies described herein. (B) A representative TEM image of a protocell nanoparticle.
[0017] Fig. 3: Schematic representation of minicircle DNA construct used in studies described herein. Structure of the P28z minicircle. The prostate-specific membrane antigen (PSMA)-targeting chimeric antigen receptor P28z is expressed under the control of the T-cell-specific CD3-delta promoter.
[0018] Fig. 4: Redirecting T-cell specificity toward prostate tumor via nanoparticle- mediated gene transfer. (A) Prostate-Specific Membrane Antigen (PSMA)-specific
chimeric antigen receptor P28z (B) Flow cytometric measurement of surface P28z expression on mouse effector T cells 30 hours after incubation with "empty" (left panel) or P28z minicircle-loaded (right panel) protocell nanoparticles. (C) 51 Cr release assay of T cells 30 h after nanoparticle transfection targeting PSMA-positive TRAMP prostate tumor cells. (D) Light microscope images of nanoparticle-transfected T cells co-cultured on a TRAMP prostate tumor cell monolayer. (E) Flow cytometric measurement of protocell binding to circulating host T cells 6 hours after intravenous injection of 1 x 1011 fluorescently tagged nanoparticles.
[0019] Fig. 5: Repeated injections of nanocarriers loaded DNA encoding the P28z chimeric antigen receptor brings about T-cell mediated regression of prostate tumor in mice. Luciferase tagged TRAMP-PSMA prostate tumor cells were transplanted into the dorsal lobe of the prostate gland of C57BL/6 mice. Two weeks later (Day 0), mice were treated with five high-dose bolus injections of 1 x1012 CD3-targeting nanoparticles carrying P28z-encoding transgenes (Day 0, Day 2, Day 4, Day 6, and Day 8). Control mice received no nanoparticles. (A) Sequential bioluminescence imaging of Firefly luciferase-expressing TRAMP-PSMA tumors. (B) Quantified bioluminescent tumor signal. Pairwise differences in bioluminescent photon counts between treatment groups were statistically analyzed with the Wilcoxon rank-sum test. *, ** = Significant P < 0.0001 .
[0020] Fig. 6: Schematic representation of minicircle DNA constructs. (A) Structure of the P28z minicircle. The prostate-specific membrane antigen (PSMA)-targeting chimeric antigen receptor P28z is expressed under the control of the T-cell specific CD3-delta promoter. (B) A scaffold/matrix attachment region (S/MAR) is shown upstream of the poly-A signal to allow sustained episomal replication. (C) Alternatively, the gene expression cassette can be flanked by the piggyBac inverted terminal repeats. The piggyBac transposon is a mobile genetic element that efficiently transposes between vector and chromosome via a "cut and paste" mechanism. This integration event is mediated by piggyBac transposase. Therefore, in piggyBac transposon studies, a plasmid encoding the hyperactive form of piggyBac transposase iPB7 will be co- encapsulated into protocell nanoparticles.
DETAILED DESCRIPTION
[0021] The present disclosure provides compositions and methods that can rapidly and selectively direct cells within the body to achieve therapeutic objectives. In particular embodiments, the compositions and methods modify cells of the immune system, such as T-cells or NK cells, to target and destroy unwanted cell types. In other embodiments, the compositions and methods modify cells of the immune system, such as monocytes/macrophages to target and destroy viruses before they infect cells and/or bacterial or fungal cells. In further embodiments, the compositions and methods modify cells of the immune system, such as B cells, to produce and release antibodies, such as broadly-neutralizing antibodies. In additional embodiments, the compositions and methods modify cells of the immune system, such as immunosuppressive TREG cells to target and protect cell types from, for example, autoimmune attack. Compositions and methods disclosed herein can also be used to modify stem cells to achieve therapeutic objectives.
[0022] The described methods can be practiced in vivo rather than requiring patient- specific isolation and culturing, as is currently required by many treatments. The methods can be practiced in vivo because following administration to a subject, the compositions selectively modify cells of interest to achieve the therapeutic objective.
[0023] As an example, one of the primary goals of clinical health research is to develop compositions and methods to rapidly and selectively direct the immune system to destroy unwanted cells. For example, vaccines are used to prime the immune system to target antigens associated with unwanted cells. The biological processes underlying conventional vaccines, however, can render them ineffective against many unwanted cells based on, among other factors, the time it takes to prime the immune system, the amount or degree to which the natural immune system can be primed against certain unwanted cell types and over time, the depletion of immune system resources.
[0024] The present disclosure provides compositions and methods that can rapidly modify cells of the immune system to target and destroy unwanted cell types. The methods can be practiced in vivo rather than requiring patient-specific isolation and culturing, as is currently required by many cancer treatments. The methods can be
practiced in vivo because following administration to a subject, the compositions selectively modify cells of the immune system to target unwanted cell types.
[0025] The compositions and methods can be used to target any cell type for which a marker is now or later becomes known. The compositions and methods achieve this benefit by modifying cells of the immune system to express targeting agents for the marker expressed by the target or in the vicinity of a target. In particular examples, the cells of the immune system are modified to express targeting agents that bind markers, such as antigens, on unwanted cells. Once bound to an unwanted cell, the immune cells mediate its destruction. Alternatively, cells of the immune system can be modified to express targeting agents that bind markers on or in the vicinity of wanted cells. Once bound to a wanted cell or in the wanted cell's vicinity, the immune cell can mediate its protection.
[0026] The compositions and methods achieve the described benefits by providing nanocarriers that include a polynucleotide encoding a targeting agent. Cells that uptake the nanocarrier will begin to express the polynucleotide, thereby expressing the targeting agent. The targeting agent directs the modified immune cell to the site of the therapeutic objective. In one example, a lymphocyte uptakes the nanocarrier and begins to express an unwanted cell targeting agent. In this embodiment, the lymphocyte then binds and mediates the destruction of the unwanted cell type.
[0027] Additional embodiments of the nanocarriers include lymphocyte-directing agents that selectively deliver the nanocarriers to cells of interest. The compositions can further include one or more of: an endosomal release agent to facilitate release of the polynucleotide from endosomal compartments of cells of the immune system and/or a nuclear localization signal (NLS) to direct the polynucleotide into the nucleus of the cell for expression if, for example, the polynucleotide includes plasmid DNA.
[0028] In particular embodiments, the nanocarriers comprise a porous nanoparticle surrounded by a coating. In these embodiments, the polynucleotide (and optionally the NLS) can be within the pores of the nanoparticle and the lymphocyte-directing agent (and optionally the endosomal release agent) can extend from the surface of the coating. Each of these components is now described in further detail.
[0029] Lymphocyte-Directing Agents. The lymphocyte-directing agents of the disclosed compositions selectively bind immune cells of interest. In particular embodiments, the cells are lymphocytes. In these embodiments, lymphocyte-directing agents can direct the compositions to any lymphocyte capable of, without limitation, (i) targeting and killing unwanted cells, (ii) targeting unwanted cells for killing by other cell types, (iii) mediating unwanted cell killing; (iv) targeting viruses for destruction before viral entry into cells, (v) antibody production and/or (vi) targeting and protecting beneficial cells. As described herein, lymphocytes include T-cells, B cells, natural killer (NK) cells, monocytes/macrophages and hematopoietic stem cells.
[0030] Several different subsets of T-cells have been discovered, each with a distinct function. In particular embodiments, lymphocyte-directing agents achieve selective direction to particular lymphocyte populations through receptor-mediated endocytosis. For example, a majority of T-cells have a T-cell receptor (TCR) existing as a complex of several proteins. The actual T-cell receptor is composed of two separate peptide chains, which are produced from the independent T-cell receptor alpha and beta (TCRa and TCR ) genes and are called a- and β-TCR chains. Lymphocyte directing agents disclosed herein can bind a- and/or β-TCR chains to achieve selective delivery of a polynucleotide to these T cells.
[0031] γδ T-cells represent a small subset of T-cells that possess a distinct T-cell receptor (TCR) on their surface. In γδ T-cells, the TCR is made up of one γ-chain and one δ-chain. This group of T-cells is much less common (2% of total T-cells) than the αβ T-cells. Nonetheless, lymphocyte-directing agents disclosed herein can bind y- and/or δ TCR chains to achieve selective delivery of a polynucleotide to these T cells.
[0032] CD3 is expressed on all mature T cells. Accordingly, lymphocyte-directing agents disclosed herein can bind CD3 to achieve selective delivery of a polynucleotide to all mature T-cells. Activated T-cells express 4-1 BB (CD137). Accordingly, lymphocyte-directing agents disclosed herein can bind 4-1 BB to achieve selective delivery of a polynucleotide to activated T-cells. CD5 and transferrin receptor are also expressed on T-cells and can be used to achieve selective delivery of a polynucleotide to T-cells.
[0033] T-cells can further be classified into helper cells (CD4+ T-cells) and cytotoxic T-cells (CTLs, CD8+ T-cells), which comprise cytolytic T-cells. T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and activation of cytotoxic T-cells and macrophages, among other functions. These cells are also known as CD4+ T-cells because they express the CD4 protein on their surface. Helper T-cells become activated when they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. Lymphocyte-directing agents disclosed herein can bind CD4 to achieve selective delivery of a polynucleotide to T helper cells.
[0034] Cytotoxic T-cells destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T-cells because they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body. Lymphocyte-directing agents disclosed herein can bind CD8 to achieve selective delivery of a polynucleotide to CTL.
[0035] "Central memory" T-cells (or "TCM") as used herein refers to an antigen experienced CTL that expresses CD62L or CCR7 and CD45RO on the surface thereof, and does not express or has decreased expression of CD45RA as compared to naive cells. In particular embodiments, central memory cells are positive for expression of CD62L, CCR7, CD25, CD127, CD45RO, and CD95, and have decreased expression of CD45RA as compared to naive cells. Lymphocyte-directing agents disclosed herein can bind CD62L, CCR7, CD25, CD127, CD45RO and/or CD95 to achieve selective delivery of a polynucleotide to TCM-
[0036] "Effector memory" T-cell (or "TEM") as used herein refers to an antigen experienced T-cell that does not express or has decreased expression of CD62L on the surface thereof as compared to central memory cells, and does not express or has decreased expression of CD45RA as compared to a naive cell. In particular embodiments, effector memory cells are negative for expression of CD62L and CCR7, compared to naive cells or central memory cells, and have variable expression of CD28
and CD45RA. Effector T-cells are positive for granzyme B and perforin as compared to memory or naive T-cells. Lymphocyte-directing agents disclosed herein can bind granzyme B and/or perforin to achieve selective delivery of a polynucleotide to TEM.
[0037] Regulatory T cells ("TREG") are a subpopulation of T cells, which modulate the immune system, maintain tolerance to self-antigens, and abrogate autoimmune disease. TREG express CD25, CTLA-4, GITR, GARP and LAP. Lymphocyte-directing agents disclosed herein can bind CD25, CTLA-4, GITR, GARP and/or LAP to achieve selective delivery of a polynucleotide to na'fve TREG.
[0038] "Naive" T-cells as used herein refers to a non-antigen experienced T cell that expresses CD62L and CD45RA, and does not express CD45RO as compared to central or effector memory cells. In some embodiments, naive CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naive T-cells including CD62L, CCR7, CD28, CD127, and CD45RA. Lymphocyte-directing agents disclosed herein can bind CD62L, CCR7, CD28, CD127 and/or CD45RA to achieve selective delivery of a polynucleotide to na'fve T-cells.
[0039] Natural killer cells (also known as NK cells, K cells, and killer cells) are activated in response to interferons or macrophage-derived cytokines. They serve to contain viral infections while the adaptive immune response is generating antigen- specific cytotoxic T cells that can clear the infection. NK cells express CD8, CD16 and CD56 but do not express CDS. Lymphocyte-directing agents disclosed herein can bind CD8, CD16 and/or CD56 to achieve selective delivery of a polynucleotide to NK cells.
[0040] Macrophages (and their precursors, monocytes) reside in every tissue of the body (in certain instances as microglia, Kupffer cells and osteoclasts) where they engulf apoptotic cells, pathogens and other non-self components. Because monocytes/macrophages engulf non-self components, a particular macrophage- or monocyte-directing agent is not required on the nanocarriers described herein for selective uptake by these cells. Alternatively, lymphocyte-directing agents disclosed herein can bind CD1 1 b, F4/80; CD68; CD1 1 c; IL-4Ra; and/or CD163 to achieve selective delivery of a polynucleotide to monocytes/macrophages.
[0041 ] B cells can be distinguished from other lymphocytes by the presence of the B cell receptor (BCR). The principal function of B cells is to make antibodies. B cells
express CD5, CD19, CD20, CD21 , CD22, CD35, CD40, CD52, and CD80. Lymphocyte- directing agents disclosed herein can bind CD5, CD19, CD20, CD21 , CD22, CD35, CD40, CD52, and/or CD80 to achieve selective delivery of a polynucleotide to B-cells.
[0042] Lymphocyte function-associated antigen 1 (LFA-1 ) is expressed by all T- cells, B-cells and monocytes/macrophages. Accordingly, lymphocyte-directing agents disclosed herein can bind LFA-1 to achieve selective delivery of a polynucleotide to T- cells, B-cells and monocytes/macrophages.
[0043] Hematopoietic stem cells can also be targeted for selective delivery of nanocarriers disclosed herein. Hematopoietic stem cells express CD34, CD133, Sca-1 and CD1 17. Lymphocyte-directing agents disclosed herein can bind CD34, CD133, Sca-1 and/or CD1 17 to achieve selective delivery of a polynucleotide to hematopoietic stem cells.
[0044] "Selective delivery" means that polynucleotides are delivered and expressed by one or more selected lymphocyte populations. In particular embodiments, selective delivery is exclusive to a selected lymphocyte population. In further embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of administered polynucleotides are delivered and/or expressed by a selected lymphocyte population. In further embodiments, selective delivery ensures that non-lymphocyte cells do not express delivered polynucleotides. For example, when the targeting agent is a T-cell receptor (TCR) gene, selectivity is ensured because only T cells have the zeta chains required for TCR expression. Selective delivery can also be based on lack of polynucleotide uptake into unselected cells or based on the presence of a specific promoter within the polynucleotide sequence when the polynucleotide includes plasmid DNA. For example, plasmid DNA can include a T-cell-specific CD3-delta promoter. Additional promoters that can achieve selective delivery include: the murine stem cell virus promoter or the distal lck promoter for T cells or hematopoietic stem cells; the CD45 promoter, WASP promoter or IFN-beta promoter for hematopoietic stem cells; the B29 promoter for B cells; or the CD14 promoter or the CD1 1 b promoter for monocytes/macrophages.
[0045] As indicated, lymphocyte-directing agents can include binding domains for motifs found on lymphocyte cells. Lymphocyte-directing agents can also include any selective binding mechanism allowing selective uptake into lymphocytes. In particular
embodiments, lymphocyte-directing agents include binding domains for T-cell receptor motifs; T-cell a chains; T-cell β chains; T-cell γ chains; T-cell δ chains; CCR7; CD3; CD4; CD5; CD7; CD8; CD1 1 b; CD1 1 c; CD16; CD19; CD20; CD21 ; CD22; CD25; CD28; CD34; CD35; CD40; CD45RA; CD45RO; CD52; CD56; CD62L; CD68;CD80; CD95; CD1 17; CD127; CD133; CD137 (4-1 BB); CD163; F4/80; IL-4Ra; Sca-1 ; CTLA-4; GITR; GARP; LAP; granzyme B; LFA-1 ; transferrin receptor; and combinations thereof.
[0046] In particular embodiments, binding domains include cell marker ligands, receptor ligands, antibodies, peptides, peptide aptamers, nucleic acids, nucleic acid aptamers, spiegelmers or combinations thereof. Within the context of lymphocyte- directing agents, binding domains include any substance that binds to another substance to form a complex capable of mediating endocytosis.
[0047] "Antibodies" are one example of binding domains and include whole antibodies or binding fragments of an antibody, e.g., Fv, Fab, Fab', F(ab')2, Fc, and single chain Fv fragments (scFvs) or any biologically effective fragments of an immunoglobulin that bind specifically to a motif expressed by a lymphocyte. Antibodies or antigen binding fragments include all or a portion of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, bispecific antibodies, mini bodies, and linear antibodies.
[0048] Antibodies from human origin or humanized antibodies have lowered or no immunogenicity in humans and have a lower number of non-immunogenic epitopes compared to non-human antibodies. Antibodies and their fragments will generally be selected to have a reduced level or no antigenicity in human subjects.
[0049] Antibodies that specifically bind a motif expressed by a lymphocyte can be prepared using methods of obtaining monoclonal antibodies, methods of phage display, methods to generate human or humanized antibodies, or methods using a transgenic animal or plant engineered to produce antibodies as is known to those of ordinary skill in the art (see, for example, U.S. Patent Nos. 6,291 ,161 and 6,291 ,158). Phage display libraries of partially or fully synthetic antibodies are available and can be screened for an antibody or fragment thereof that can bind to a lymphocyte motif. For example, binding domains may be identified by screening a Fab phage library for
Fab fragments that specifically bind to a target of interest (see Hoet et al., Nat. Biotechnol. 23:344, 2005). Phage display libraries of human antibodies are also available. Additionally, traditional strategies for hybridoma development using a target of interest as an immunogen in convenient systems {e.g., mice, HuMAb mouse®, TC mouse™, KM-mouse®, llamas, chicken, rats, hamsters, rabbits, etc.) can be used to develop binding domains. In particular embodiments, antibodies specifically bind to motifs expressed by a selected lymphocyte and do not cross react with nonspecific components or unrelated targets. Once identified, the amino acid sequence or polynucleotide sequence coding for the antibody can be isolated and/or determined.
[0050] In particular embodiments, binding domains of lymphocyte-directing agents include T-cell receptor motif antibodies; T-cell a chain antibodies; T-cell β chain antibodies; T-cell γ chain antibodies; T-cell δ chain antibodies; CCR7 antibodies; CD3 antibodies; CD4 antibodies; CD5 antibodies; CD7 antibodies; CD8 antibodies; CD1 1 b antibodies; CD1 1 c antibodies; CD16 antibodies; CD19 antibodies; CD20 antibodies; CD21 antibodies; CD22 antibodies; CD25 antibodies; CD28 antibodies; CD34 antibodies; CD35 antibodies; CD40 antibodies; CD45RA antibodies; CD45RO antibodies; CD52 antibodies; CD56 antibodies; CD62L antibodies; CD68 antibodies; CD80 antibodies; CD95 antibodies; CD1 17 antibodies; CD127 antibodies; CD133 antibodies; CD137 (4-1 BB) antibodies; CD163 antibodies; F4/80 antibodies; IL-4Ra antibodies; Sca-1 antibodies; CTLA-4 antibodies; GITR antibodies GARP antibodies; LAP antibodies; granzyme B antibodies; LFA-1 antibodies; or transferrin receptor antibodies. These binding domains also can consist of scFv fragments of the foregoing antibodies. In one particular embodiment, the lymphocyte-directing agent binding domain includes the scFv fragment (SEQ ID NO. 1 ) of the PSMA-specific chimeric antigen receptor (CAR), P28z.
[0051] Peptide aptamers include a peptide loop (which is specific for a target protein) attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to an antibody. The variable loop length is typically 8 to 20 amino acids (e.g., 8 to 12 amino acids), and the scaffold may be any protein which is stable, soluble, small, and nontoxic (e.g., thioredoxin-A, stefin A triple mutant, green fluorescent protein, eglin C, and
cellular transcription factor Spl). Peptide aptamer selection can be made using different systems, such as the yeast two-hybrid system (e.g., Gal4 yeast-two-hybrid system) or the LexA interaction trap system.
[0052] Nucleic acid aptamers are single-stranded nucleic acid (DNA or RNA) ligands that function by folding into a specific globular structure that dictates binding to target proteins or other molecules with high affinity and specificity, as described by Osborne et al., Curr. Opin. Chem. Biol. 1 :5-9, 1997; and Cerchia et al., FEBS Letters 528:12-16, 2002. In particular embodiments, aptamers are small (-15 KD; or between 15-80 nucleotides or between 20-50 nucleotides). Aptamers are generally isolated from libraries consisting of 1014-1015 random oligonucleotide sequences by a procedure termed SELEX (systematic evolution of ligands by exponential enrichment; see, for example, Tuerk et al., Science, 249:505-510, 1990; Green et al., Methods Enzymology. 75-86, 1991 ; and Gold et al., Annu. Rev. Biochem., 64: 763-797, 1995). Further methods of generating aptamers are described in, for example, US Patent Nos. 6,344,318; 6,331 ,398; 6,1 10,900; 5,817,785; 5,756,291 ; 5,696,249; 5,670,637; 5,637,461 ; 5,595,877; 5,527,894; 5,496,938; 5,475,096; and 5,270,16. Spiegelmers are similar to nucleic acid aptamers except that at least one β-ribose unit is replaced by β- D-deoxyribose or a modified sugar unit selected from, for example, β-D-ribose, a-D- ribose, β-L-ribose.
[0053] Other agents that can facilitate internalization by and/or transfection of lymphocytes, such as poly(ethyleneimine)/DNA (PEI/DNA) complexes can also be used.
[0054] Polynucleotides Encoding Targeting Agents. As used herein, the term "polynucleotide" includes a nucleic acid molecule that contains a nucleic acid sequence such that upon introduction into a targeted lymphocyte, the nucleic acid molecule can cause transcription and resulting translation of targeting agents encoded by the nucleic acid sequence of the nucleic acid molecule. In particular embodiments, the targeting agent is an unwanted cell targeting agent. In further embodiments, the targeting agent is a wanted cell targeting agent.
[0055] As used herein, the term "gene" refers to a nucleic acid sequence that encodes a targeting agent. This definition includes various sequence polymorphisms, mutations, and/or sequence variants wherein such alterations do not affect the function
of the encoded targeting agent. The term "gene" may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. The term further can include all introns and other DNA sequences spliced from the mRNA transcript, along with variants resulting from alternative splice sites. Nucleic acid sequences encoding the targeting agent can be DNA or RNA that directs the expression of the targeting agent. These nucleic acid sequences may be a DNA strand sequence that is transcribed into RNA or an RNA sequence that is translated into protein. The nucleic acid sequences include both the full-length nucleic acid sequences as well as non-full-length sequences derived from the full-length protein. The sequences can also include degenerate codons of the native sequence or sequences that may be introduced to provide codon preference in a specific lymphocyte. Gene sequences to encode targeting agents disclosed herein are available in publicly available databases and publications, incorporated by reference herein.
[0056] As used herein, the term "encoding" refers to a property of sequences of nucleotides in a polynucleotide, such as a plasmid, a gene, cDNA, mRNA, to serve as templates for synthesis of targeting agents. A polynucleotide can, e.g., encode a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Unless otherwise specified, polynucleotides having a sequence encoding a targeting agent include all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The polynucleotides that encode proteins and RNA can also include introns.
[0057] In some embodiments, the polynucleotide includes a plasmid, a cDNA, or an mRNA that can include, e.g., a sequence {e.g., a gene) for expressing a targeting agent. Suitable plasmids include standard plasmid vectors and minicircle plasmids that can be used to transfer a gene to a lymphocyte. The polynucleotides {e.g., minicircle plasmids) can further include any additional sequence information to facilitate transfer of the genetic material {e.g., a sequence encoding a receptor to an antigen) to lymphocytes. For example, the polynucleotides can include promoters, such as general promoters, tissue-specific promoters, cell-specific promoters, and/or promoters specific for the nucleus or cytoplasm. Promoters and plasmids {e.g., minicircle plasmids) are generally well known in the art and can be prepared using conventional techniques. As
described further herein, the polynucleotides can be used to transfect lymphocytes. Unless otherwise specified, the terms transfect, transfected, or transfecting can be used to indicate the presence of exogenous polynucleotides or the expressed polypeptide therefrom in a lymphocyte. A number of vectors are known to be capable of mediating transfer of genes to lymphocytes, as is known in the art.
[0058] In particular embodiments, the transfected polynucleotides can edit the antigen-specificity of lymphocytes without affecting off-target bystander cells (i.e., provide for selective delivery as defined herein). For example, delivered genes can be expressed under the control of a lymphocyte-specific promoter. In particular embodiments, the promoters can be included in minicirde plasmids that are a form of supercoiled DNA molecule for nonviral gene transfer, which have neither bacterial origin of replication nor antibiotic resistance marker. They are thus smaller and potentially safer than the standard plasmids currently used in gene therapy.
[0059] To sustain the expression of transferred targeting agent genes, for example, in rapidly dividing lymphocytes, a scaffold/matrix attachment region can also be inserted into the polynucleotides. Polynucleotides containing an expression cassette linked to a S/MAR element, can autonomously replicate extra-chromosomally in dividing cells. In some embodiments, PiggyBac or Sleeping Beauty transposase-containing plasmids can also be used to stably integrate nanocarrier-delivered targeting agent genes into the genome of transfected lymphocytes. Other options to sustain expression include homo sapiens transposon-derived Busterl transposase-like protein gene; human endogenous retrovirus H protease/integrase-derived ORF1 ; homo sapiens Cas-Br-M (murine) ecotropic retroviral transforming sequence; homo sapiens endogenous retroviral sequence K; homo sapiens endogenous retroviral family W; homo sapiens LINE-1 type transposase domain; or homo sapiens pogo transposable element.
[0060] When a delivered polynucleotide is mRNA, backbone modifications can increase the mRNA's stability making resistant to premature cleavage.
[0061] Targeted Cells & Associated Markers. Targeted cells can be unwanted cells or wanted cells. Unwanted cells include any cell type that is (i) capable of recognition and destruction by the immune system; and (ii) deemed undesirable by a subject, physician, veterinarian or researcher. Unwanted cells include (i) eukaryotic cells that are
either cancerous or infected with a pathogen such as a virus and (ii) prokaryotic cells, such as certain bacteria, fungi or yeast. Wanted cells include any cell type that is (i) capable of recognition and protection by the immune system; and (ii) deemed desirable by a subject, physician, veterinarian or researcher. Wanted cells can include cells undergoing auto-immune attack or bacteria that are beneficial to the health of a microbiome.
[0062] For targeting according to the compositions and methods disclosed herein, unwanted or wanted cells must be associated with a marker that is currently known or later discovered. In particular embodiments, the markers are antigens. Antigens refer to substances capable of either binding to an antigen binding region of an immunoglobulin molecule or of eliciting an immune response, e.g., a T cell-mediated immune response by the presentation of the antigen on Major Histocompatibility Antigen (MHC) cellular proteins. "Antigens" include antigenic determinants, haptens, and immunogens, which may be peptides, small molecules, carbohydrates, lipids, nucleic acids or combinations thereof. When referencing antigens that are processed for presentation to T cells, the term "antigen" refers to those portions of the antigen (e.g., a peptide fragment) that is a T cell epitope presented by MHC to the T cell receptor. When used in the context of a B cell mediated immune response in the form of an antibody that is specific for an "antigen", the portion of the antigen that binds to the complementarity determining regions of the variable domains of the antibody (light and heavy) is referenced. The bound portion may be a linear or three-dimensional epitope.
[0063] Cancer Markers. In particular embodiments, markers are expressed by unwanted cells from cancers. Exemplary cancers include adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, leukemias, liver cancers, lymph node cancers, lymphomas, lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non-Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma,
seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, and metastases thereof.
[0064] Particular antigen markers associated with cancers cells include A33; BAGE; Bcl-2; β-catenin; CA125; CA19-9; CD5; CD19; CD20; CD21 ; CD22; CD33; CD37; CD45; CD123; CEA; c-Met; CS-1 ; cyclin B1 ; DAGE; EBNA; EGFR; ephrinB2; estrogen receptor; FAP; ferritin; folate-binding protein; GAGE; G250; GD-2; GM2; gp75, gp100 (Pmel 17); HER-2/neu; HPV E6; HPV E7; Ki-67; LRP; mesothelin, p53, PRAME; progesterone receptor; PSA; PSMA; MAGE; MART; mesothelin; MUC; MUM-1 -B; myc; NYESO-1 ; ras; RORI; survivin; tenascin; TSTA tyrosinase; VEGF; and WT1 .
[0065] Without limiting the foregoing, the particular following cancers can be treated by targeting the associated provided antigens: leukemia/lymphoma (CD19, CD20, CD22, ROR1 , CD33); multiple myeloma (B-cell maturation antigen (BCMA)); prostate cancer (PSMA, WT1 , Prostate Stem Cell antigen (PSCA), SV40 T); breast cancer (HER2, ERBB2); stem cell cancer (CD133); ovarian cancer (L1 -CAM, extracellular domain of MUC16 (MUC-CD), folate binding protein (folate receptor), Lewis Y); renal cell carcinoma (carboxy-anhydrase-IX (CAIX); melanoma (GD2); and pancreatic cancer (mesothelin, CEA, CD24).
[0066] In further particular examples, cancer cell antigens include:
Cancer Sequence SEQ ID NO.
Antigen
PSMA MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLL 2
GFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKK
FLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSV
ELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEP
PPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYAR
TEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNA
QLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGG
GVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEA
VGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRG
SLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYN
VIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGA
AWHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGL
LGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRV
DCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTK
KSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRA
RYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMF
KYHLTVAQVRGGMVFELANSIVLPFDCRDYAWLR
KYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEI ASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDP LGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDI ESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA
PSCA MKAVLLALLMAGLALQPGTALLCYSCKAQVSNEDC 3
LQVENCTQLGEQCWTARIRAVGLLTVISKGCSLNCV DDSQDYYVGKKNITCCDTDLCNASGAHALQPAAAIL ALLPALGLLLWGPGQL
Mesothelin MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRT 4
LAGETGQEAAPLDGVLANPPNISSLSPRQLLGFPCA
EVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRL
SEPPEDLDALPLDLLLFLNPDAFSGPQACTHFFSRIT
KANVDLLPRGAPERQRLLPAALACWGVRGSLLSEA
DVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPL
DQDQQEAARAALQGGGPPYGPPSTWSVSTMDAL
RGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQP
ERTILRPRFRREVEKTACPSGKKAREIDESLIFYKKW
ELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLD
ELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLE
TLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKD
TLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQD
LDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFL
GGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLT
VAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDD
LDTLGLGLQGGIPNGYLVLDLSVQEALSGTPCLLGP
GPVLTVLALLLASTLA
CD19 MPPPRLLFFLLFLTPMEVRPEEPLWKVEEGDNAVL 5
QCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGL
GIHMRPLASWLFIFNVSQQMGGFYLCQPGPPSEKA
WQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNR
SSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPC
VPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVS
RGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVM
ETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARP
VLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQ
RALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQY
GNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPS
SDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEE
DSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPE
DEDSFSNAESYENEDEELTQPVARTMDFLSPHGSA
WDPSREATSLGSQSYEDMRGILYAAPQLRSIRGQP
GPNHEEDADSYENMDNPDGPDPAWGGGGRMGT
WSTR
CD20 MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRM 6
SSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLL
MIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKN
SRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHF
LKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYC
YSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCS
RPKSNIVLLSAEEKKEQTIEIKEEWGLTETSSQPKN
EEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSS
P
ROR1 MHRPRRRGTRPPLLALLAALLLAARGAAAQETELSV 7
SAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLG
QTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSF
RSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSST
GVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIAC
ARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSH
LSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRD
ECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQ
PESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRG
TVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNG
GHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACD
SKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNN
QKSSSAPVQRQPKHVRGQNVEMSMLNAYKPKSKA
KELPLSAVRFMEELGECAFGKIYKGHLYLPGMDHA
QLVAIKTLKDYNNPQQWTEFQQEASLMAELHHPNI
VCLLGAVTQEQPVCMLFEYINQGDLHEFLIMRSPHS
DVGCSSDEDGTVKSSLDHGDFLHIAIQIAAGMEYLS
SHFFVHKDLAARNILIGEQLHVKISDLGLSREIYSAD
YYRVQSKSLLPIRWMPPEAIMYGKFSSDSDIWSFG
WLWEIFSFGLQPYYGFSNQEVIEMVRKRQLLPCSE
DCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWE
GLSSHTSSTTPSGGNATTQTTSLSASPVSNLSNPR
YPNYMFPSQGITPQGQIAGFIGPPIPQNQRFIPINGY
PIPPGYAAFPAAHYQPTGPPRVIQHCPPPKSRSPSS
ASGSTSTGHVTSLPSSGSNQEANIPLLPHMSIPNHP
GGMGITVFGNKSQKPY
KIDSKQASLLGDANIHGHTESMISAEL
WT1 MGHHHHHHHHHHSSGHIEGRHMRRVPGVAPTLVR 8
SASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRK
HTGEKPYQCDFKDCERRFFRSDQLKRHQRRHTGV
KPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCR
WPSCQKKFARSDELVRHHNMHQRNMTKLQLAL
[0067] In particular embodiments disclosed herein, modified T cells, NK cells and/or monocytes/macrophages target and destroy cancer cells. B cells can also be modified to secrete tumor- specific antibodies.
[0068] Viral Markers. In particular embodiments, markers are expressed by unwanted virally-infected cells. Exemplary viruses include adenoviruses, arenaviruses, bunyaviruses, coronavirusess, flavirviruses, hantaviruses, hepadnaviruses, herpesviruses, papilomaviruses, paramyxoviruses, parvoviruses, picornaviruses, poxviruses, orthomyxoviruses, retroviruses, reoviruses, rhabdoviruses, rotaviruses, spongiform viruses or togaviruses. In additional embodiments, viral antigen markers include peptides expressed by CMV, cold viruses, Epstein-Barr, flu viruses, hepatitis A, B, and C viruses, herpes simplex, HIV, influenza, Japanese encephalitis, measles, polio, rabies, respiratory syncytial, rubella, smallpox, varicella zoster or West Nile virus.
[0069] As further particular examples, cytomegaloviral antigens include envelope glycoprotein B and CMV pp65; Epstein-Barr antigens include EBV EBNAI, EBV P18, and EBV P23; hepatitis antigens include the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, HBCAG DELTA, HBV HBE, hepatitis C viral RNA, HCV NS3 and HCV NS4; herpes simplex viral antigens include immediate early proteins and glycoprotein D; HIV antigens include gene products of the gag, pol, and env genes such as HIV gp32, HIV gp41 , HIV gp120, HIV gp160, HIV P17/24, HIV P24, HIV P55 GAG, HIV P66 POL, HIV TAT, HIV GP36, the Nef protein and reverse transcriptase; influenza antigens include hemagglutinin and neuraminidase; Japanese encephalitis viral antigens include proteins E, M-E, M-E-NS1 , NS1 , NS1 -NS2A and 80% E; measles antigens include the measles virus fusion protein; rabies antigens include rabies glycoprotein and rabies nucleoprotein; respiratory syncytial viral antigens include the RSV fusion protein and the M2 protein; rotaviral antigens include VP7sc; rubella antigens include proteins E1 and E2; and varicella zoster viral antigens include gpl and gpll.
[0070] Additional particular exemplary viral antigen sequences include:
Source Sequence SEQ ID
NO.
Nef (66-97): VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGL 9
Nef (1 16-145) HTQGYFPDWQNYTPGPGVRYPLTFGWLYKL 10
Gag p17 (17-35) EKIRLRPGGKKKYKLKHIV 1 1
Gag p17-p24 NPPIPVGEIYKRWIILGLNKIVRMYSPTSILD 12
(253-284)
Pol 325-355 (RT AIFQSSMTKILEPFRKQNPDIVIYQYMDDLY 13
158-188)
See Fundamental Virology, Second Edition, eds. Fields, B. N. and Knipe, D. M. (Raven Press, New York, 1991 ) for additional examples of viral antigens.
[0071] In particular embodiments disclosed herein, modified T cells recognize and destroy virally-infected cells. Alternatively, or in addition, modified monocytes/macrophages can remove viruses from peripheral tissue or the blood stream (extracellular) before cellular infection by a viral particle. B cells can also be modified to express broadly neutralizing antibodies. In one example, B cells can be modified to express broadly neutralizing anti-HIV antibodies.
[0072] In particular embodiments, the targeting agent targets HIV gag protein, gp120 or the Hepatitis B envelope protein (S domain).
[0073] Bacterial Markers. In particular embodiments, markers are expressed by cells associated with unwanted bacterial infections. Exemplary bacteria include anthrax; gram-negative bacilli, chlamydia, diptheria, haemophilus influenza, Helicobacter pylori, malaria, Mycobacterium tuberculosis, pertussis toxin, pneumococcus, rickettsiae, staphylococcus, streptococcus and tetanus.
[0074] As particular examples of bacterial antigen markers, anthrax antigens include anthrax protective antigen; gram-negative bacilli antigens include lipopolysaccharides; haemophilus influenza antigens include capsular polysaccharides; diptheria antigens include diptheria toxin; Mycobacterium tuberculosis antigens include mycolic acid, heat shock protein 65 (HSP65), the 30 kDa major secreted protein and antigen 85A; pertussis toxin antigens include hemagglutinin, pertactin, FIM2, FIM3 and adenylate cyclase; pneumococcal antigens include pneumolysin and pneumococcal capsular polysaccharides; rickettsiae antigens include rompA; streptococcal antigens include M proteins; and tetanus antigens include tetanus toxin.
[0075] In certain embodiments where the presence of bacteria is beneficial to the health of a microbiome, bacterial cells can also be wanted cell types.
[0076] Monocytes/macrophages are particularly useful to modify when the therapeutic objective is treatment of a bacterial infection. In one particular embodiment, monocytes/macrophages can be modified with a ligand recognizing the surface component lipoteichoic acid of Staphyloccus aureus or the Staphylococcus aureus clumping factor A (ClfA). Immunosuppressive TREG can be useful to modify when a bacteria is a wanted cell type.
[0077] Superbugs. In particular embodiments, lymphocytes are modified to target multi-drug resistant "superbugs". Examples of superbugs include Enterococcus faecium, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae (including Escherichia coli, Klebsiella pneumoniae, Enterobacter spp.).
[0078] Fungal Markers. In particular embodiments, markers are expressed by cells associated with unwanted fungal infections. Exemplary fungi include Candida, coccidiodes, cryptococcus, histoplasma, leishmania, Plasmodium, protozoa, parasites, schistosomae, tinea, toxoplasma, and trypanosoma cruzi.
[0079] As further particular examples of fungal antigens, coccidiodes antigens include spherule antigens; cryptococcal antigens include capsular polysaccharides; histoplasma antigens include heat shock protein 60 (HSP60); leishmania antigens include gp63 and lipophosphoglycan; Plasmodium falciparum antigens include merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte/gamete surface antigens, protozoal and other parasitic antigens including the blood-stage antigen pf 155/RESA; schistosomae antigens include glutathione-S- transferase and paramyosin; tinea fungal antigens include trichophytin; toxoplasma antigens include SAG-1 and p30; and trypanosoma cruzi antigens include the 75-77 kDa antigen and the 56 kDa antigen.
[0080] Monocytes/macrophages are particularly useful to modify when the therapeutic objective is treatment of a fungal infection.
[0081 ] Autoimmune or Allergy Markers. In particular embodiments, markers are expressed by cells associated with unwanted autoimmune or allergic conditions. Exemplary autoimmune conditions include acute necrotizing hemorrhagic encephalopathy, allergic asthma, alopecia areata, anemia, aphthous ulcer, arthritis
(including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), asthma, autoimmune thyroiditis, conjunctivitis, Crohn's disease, cutaneous lupus erythematosus, dermatitis (including atopic dermatitis and eczematous dermatitis), diabetes, diabetes mellitus, erythema nodosum leprosum, keratoconjunctivitis, multiple sclerosis, myasthenia gravis, psoriasis, scleroderma, Sjogren's syndrome, including keratoconjunctivitis sicca secondary to Sjogren's syndrome, Stevens-Johnson syndrome, systemic lupus erythematosis, ulcerative colitis, vaginitis and Wegener's granulomatosis.
[0082] Examples of autoimmune antigens include glutamic acid decarboxylase 65 (GAD 65), native DNA, myelin basic protein, myelin proteolipid protein, acetylcholine receptor components, thyroglobulin, and the thyroid stimulating hormone (TSH) receptor. Examples of allergic antigens include pollen antigens such as Japanese cedar pollen antigens, ragweed pollen antigens, rye grass pollen antigens, animal derived antigens (such as dust mite antigens and feline antigens), histocompatibility antigens, and penicillin and other therapeutic drugs.
[0083] Immunosuppressive TREG can be useful to modify to protect wanted cells from autoimmune attack or to reduce immune system activity in an area. Exemplary wanted cells to protect from autoimmune attack include neurons in multiple sclerosis or amylotrophic lateral sclerosis; connective tissue in rheumatoid arthritis; colon epithelium in Chrohn's disease; and the pancreas in Diabetes mellitus type 1 . In one particular embodiment, TREG are modified to express a chimeric antigen receptor (CAR) against KIR4.1 (a potassium channel) that has been identified as an immune target in multiple sclerosis.
[0084] Without limiting any of the foregoing examples, markers can also include B- cell targets, TNF receptor superfamily members, Hedgehog family members, receptor tyrosine kinases, proteoglycan-related molecules, TGF-β superfamily members, Wnt-related molecules, T-cell targets, dendritic cell targets, NK cell targets, a monocyte/macrophage cell targets, and angiogenesis targets.
[0085] Without limiting any of the foregoing examples, markers can also include CEACAM6, c-Met, EGFR, ErbB2, ErbB3, ErbB4, EphA2, IGF1 R, GHRHR, GHR, FLT1 , KDR, FLT4, CD44v6, CA125, CEA, BTLA, TGFBR2, TGFBR1 , IL6R, gp130, TNFR1 ,
TNFR2, PD1 , PD-L1 , PD-L2, HVEM, mesothelin, PSMA, RANK, ROR1 , TNFRSF4, TWEAK-R, HLA, tumor or pathogen derived peptides bound to HLA (such as from hTERT, tyrosinase, or WT-1 ), LT R, LIFR , LRP5, MUC1 , OSMR , TCRa, TCR , B7H4, TLR7, TLR9, PTCH1 , PTCH1 , Robol , a-fetoprotein (AFP) or Frizzled.
[0086] Targeting Agents. Targeting agents include any binding domain capable of (i) expression by a lymphocyte; and (ii) binding to a marker associated with a target. Binding of the targeting agent to the marker then mediates destruction or protection of the target.
[0087] Binding domains include any substance that binds to another substance to form a complex. Examples of binding domains include cell marker ligands, receptor ligands, antibodies, peptides, peptide aptamers, receptors and chimeric antigen receptors (CAR) or combinations thereof. As will be understood by one of ordinary skill in the art, targeting agent binding domains can include the same components, options and identification methods as described above in relation to lymphocyte- directing agent binding domains with altered specificity, as appropriate.
[0088] Targeting agent binding domains can particularly include any peptide that specifically binds a marker on a targeted cell. Sources of targeting agent binding domains include antibody variable regions from various species (which can be in the form of antibodies, sFvs, scFvs, Fabs, scFv-based grababody, or soluble VH domain or domain antibodies). These antibodies can form antigen-binding regions using only a heavy chain variable region, i.e., these functional antibodies are homodimers of heavy chains only (referred to as "heavy chain antibodies") (Jespers et al., Nat. Biotechnol. 22:1 161 , 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 72:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008).
[0089] An alternative source of targeting agent binding domains includes sequences that encode random peptide libraries or sequences that encode an engineered diversity of amino acids in loop regions of alternative non-antibody scaffolds, such as scTCR (see, e.g., Lake et al., Int. lmmunol.^ ^ ^.745, 1999; Maynard et al., J. Immunol. Methods 306:51 , 2005; U.S. Patent No. 8,361 ,794), fibrinogen domains (see, e.g., Weisel et al., Science 230:1388, 1985), Kunitz domains (see, e.g., US Patent No. 6,423,498), designed ankyrin repeat proteins (DARPins) (Binz et al., J. Mol. Biol. 332:489, 2003 and
Binz et al., Nat. Biotechnol. 22:575, 2004), fibronectin binding domains (adnectins or monobodies) (Richards et al., J. Mol. Biol. 326:1475, 2003; Parker et al., Protein Eng. Des. Selec. 78:435, 2005 and Hackel et al. (2008) J. Mol. Biol. 387:1238-1252), cysteine-knot miniproteins (Vita et al. (1995) Proc. Nat'l. Acad. Sci. (USA) 92:6404- 6408; Martin et al. (2002) Nat. Biotechnol. 27:71 , 2002 and Huang et al. (2005) Structure 73:755, 2005), tetratricopeptide repeat domains (Main et al., Structure 77:497, 2003 and Cortajarena et al., ACS Chem. Biol. 3:161 , 2008), leucine-rich repeat domains (Stumpp et al., J. Mol. Biol. 332:471 , 2003), lipocalin domains (see, e.g., WO 2006/095164, Beste et al., Proc. Nat'l. Acad. Sci. (USA) 96:1898, 1999 and Schonfeld et al., Proc. Nat'l. Acad. Sci. (USA) 706:8198, 2009), V-like domains (see, e.g., US Patent Application Publication No. 2007/0065431 ), C-type lectin domains (Zelensky and Gready, FEBS J. 272:6179, 2005; Beavil et al., Proc. Nat'l. Acad. Sci. (USA) 89:753, 1992 and Sato et al., Proc. Nat'l. Acad. Sci. (USA) 700:7779, 2003), mAb2 or Fcab™ (see, e.g., PCT Patent Application Publication Nos. WO 2007/098934; WO 2006/072620), armadillo repeat proteins (see, e.g., Madhurantakam et al., Protein Sci. 21: 1015, 2012; PCT Patent Application Publication No. WO 2009/040338), affilin (Ebersbach et al., J. Mol. Biol. 372: 172, 2007), affibody, avimers, knottins, fynomers, atrimers, cytotoxic T-lymphocyte associated protein-4 (Weidle et al., Cancer Gen. Proteo. 70:155, 2013) or the like (Nord et al., Protein Eng. 8:601 , 1995; Nord et al., Nat. Biotechnol. 75:772, 1997; Nord et al., Euro. J. Biochem. 268:4269, 2001 ; Binz et al., Nat. Biotechnol. 23:1257, 2005; Boersma and Pluckthun, Curr. Opin. Biotechnol. 22:849, 201 1 ).
[0090] In some embodiments, a binding domain is a single chain T cell receptor (scTCR) comprising ναφ and Ca chains (e.g., Va-Ca, Vp-C , Va-Vp) or comprising Va-Ca, -C , ναΛ/β pair specific for a target of interest (e.g., peptide-MHC complex).
[0091] In another embodiment, the targeting agent is an unwanted cell targeting agent and the binding domain can be an antibody targeting PSMA. A number of antibodies specific for PSMA are known to those of skill in the art and can be readily characterized for sequence, epitope binding, and affinity. Unwanted cell targeting agent binding domains can also include anti-Mesothelin ligands (associated with treating ovarian cancer, pancreatic cancer, and mesothelioma); anti-WT-1 (associated with
treating leukemia and ovarian cancer); anti-HIV-gag (associated with treating HIV infections); or anti-cytomegalovirus (associated with treating CMV diseases such as herpes virus). As will be understood by one of ordinary skill in the art, the unwanted cell targeting agent binding domain can be any ligand that binds to any marker associated with an unwanted cell type as described herein.
[0092] In one embodiment, the targeting agent is an unwanted cell targeting agent and the binding domain can be an antibody targeting CD19. In some embodiments, a binding domain is a single chain Fv fragment (scFv) that comprises VH and VL regions specific for CD19. In certain embodiments, the VH and VL regions are human. Exemplary VH and VL regions include the segments of anti-CD19 specific monoclonal antibody FMC63. In particular embodiments, the scFV is a human or humanized ss comprising a variable light chain comprising a CDRL1 sequence of RASQDISKYLN (SEQ ID NO. 14), CDRL2 sequence of SRLHSGV (SEQ ID NO. 15), and a CDRL3 sequence of GNTLPYTFG (SEQ ID NO. 16). In other embodiments, the scFV is a human or humanized ScFv comprising a variable heavy chain comprising CDRHI sequence of DYGVS (SEQ ID NO. 17), CDRH2 sequence of VTWGSETTYYNSALKS (SEQ ID NO. 18), and a CDRH3 sequence of YAMDYWG (SEQ ID NO. 19). Other CD19-targeting antibodies such as SJ25C1 and HD37 are known. (SJ25C1 : Bejcek et al. Cancer Res 2005, PMID 7538901 ; HD37: Pezutto et al. Jl 1987, PMID 2437199). SEQ ID NO. 20 provides the anti-CD19 scFv (VH-VL) FMC63 DNA sequence and SEQ ID NO. 21 provides the anti-CD19 scFv (VH-VL) FMC63 amino acid sequence.
[0093] In another embodiment, the targeting agent is an unwanted cell targeting agent and the binding domain can be an antibody targeting RORI. In a particular embodiment, the scFV is a human or humanized scFv comprising a variable light chain comprising a CDRL1 sequence of ASGFDFSAYYM (SEQ ID NO. 22), CDRL2 sequence of TIYPSSG (SEQ ID NO. 23), and a CDRL3 sequence of ADRATYFCA (SEQ ID NO. 24). In other embodiments, the scFV is a human or humanized scFv comprising a variable heavy chain comprising CDRH1 sequence of DTIDWY (SEQ ID NO. 25), CDRH2 sequence of VQSDGSYTKRPGVPDR (SEQ ID NO. 26), and a CDRH3 sequence of YIGGYVFG (SEQ ID NO. 27). A number of antibodies specific
for RORI are known to those of skill in the art and can be readily characterized for sequence, epitope binding, and affinity.
[0094] In certain embodiments, targeting agent binding domains comprise a sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a TCR Va, Vp, Ca, or C , wherein each CDR comprises zero changes or at most one, two, or three changes, from a TCR or fragment or derivative thereof that specifically binds to target of interest.
[0095] In certain embodiments, targeting agent binding domain Va, Vp, Ca, or Cp region of the present disclosure can be derived from or based on a Va, Vp, Ca, or C of a known TCR {e.g., a high-affinity TCR) and contains one or more {e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more {e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more {e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions {e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the Va, Vp, Ca, or C of a known TCR. An insertion, deletion or substitution may be anywhere in a Va, Vp, Ca, or C region, including at the amino- or carboxy-terminus or both ends of these regions, provided that each CDR comprises zero changes or at most one, two, or three changes and provided a binding domain containing a modified Va, Vp, Ca, or C region can still specifically bind its target with an affinity similar to wild type.
[0096] In certain embodiments, a binding domain VH region of the present disclosure can be derived from or based on a VH of a known monoclonal antibody and can contain one or more {e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more {e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more {e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions {e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VH of a known monoclonal antibody. An insertion, deletion or substitution may be anywhere in the VH region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR comprises zero changes or at most one, two, or three changes and provided a binding domain containing the modified VH region can still specifically bind its target with an affinity similar to the wild type binding domain.
[0097] In further embodiments, a VL region in a binding domain of the present disclosure is derived from or based on a VL of a known monoclonal antibody and contains one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VL of the known monoclonal antibody. An insertion, deletion or substitution may be anywhere in the VL region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR comprises zero changes or at most one, two, or three changes and provided a binding domain containing the modified VL region can still specifically bind its target with an affinity similar to the wild type binding domain.
[0098] In certain embodiments, a binding domain comprises or is a sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a light chain variable region (VL) or to a heavy chain variable region (VH), or both, wherein each CDR comprises zero changes or at most one, two, or three changes, from a monoclonal antibody or fragment or derivative thereof that specifically binds to target of interest.
[0099] As stated, cell-targeting agents disclosed herein include chimeric antigen receptors. "Chimeric antigen receptors" or "CARs" refer to synthetically designed receptors comprising at least a binding domain and an effector domain and optionally a spacer domain and/or a transmembrane domain. Binding domains are described elsewhere herein.
[00100] Effector domains are capable of transmitting functional signals to a cell. In certain embodiments, an effector domain will directly or indirectly promote a cellular response by associating with one or more other proteins that directly promote a cellular response. Effector domains can provide for activation of at least one function of a transduced lymphocyte expressing the CAR upon binding to the marker expressed on a targeted cell. Activation of the lymphocyte can include one or more of proliferation, differentiation, activation or other effector functions. In particular embodiments, the delivered polynucleotide encodes for the effector domain.
[00101 ] An effector domain may include one, two, three or more receptor signaling domains, intracellular signaling domains, costimulatory domains, or combinations thereof. Any intracellular effector domain, costimulatory domain or both from any of a variety of signaling molecules {e.g., signal transduction receptors) may be used in the CARs of this disclosure.
[00102] Exemplary effector domains include those from 4-1 BB, CD3E, CD35, ΟΌ3ζ, CD27, CD28 (e.g., SEQ ID NO.:28), CD79A, CD79B, CARD1 1 , DAP10, FcRa, FcR , FcRy, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH 1 , Wnt, NKG2D, OX40, ROR2, Ryk, SLAMF1 , Slp76, pTa, TCRa, TCR , TRIM, Zap70, PTCH2, or any combination thereof.
[00103] T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation and provide a T cell receptor like signal (primary cytoplasmic signaling sequences) and those that act in an antigen- independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences). Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as receptor tyrosine-based activation motifs or iTAMs. Examples of iTAM containing primary cytoplasmic signaling sequences include those derived from CD3 zeta, FeR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
[00104] In particular embodiments, an effector domain comprises a cytoplasmic portion that associates with a cytoplasmic signaling protein, wherein the cytoplasmic signaling protein is a lymphocyte receptor or signaling domain thereof, a protein comprising a plurality of ITAMs, a costimulatory factor, or any combination thereof.
[00105] Examples of intracellular signaling domains include the cytoplasmic sequences of the CD3 zeta chain, and/or co- receptors that act in concert to initiate signal transduction following CAR engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability. In particular embodiments, an intracellular signaling domain of a CAR can be designed to comprise an intracellular signaling domain combined with any other desired cytoplasmic domain(s). For example, the intracellular signaling domain of a CAR can comprise an
intracellular signaling domain and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than the expressed marker ligand that is required for a response of lymphocytes to a marker. Examples of such molecules include CD27, CD28, 4-1 BB (CD 137), OX40, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1 ), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.
[00106] In certain embodiments, CAR polynucleotides can comprise a sequence encoding for a spacer region. The length of the spacer region can be customized for individual markers on targets to optimize target recognition and destruction or protection. In particular embodiments, a spacer length can be selected based upon the location of a marker epitope, affinity of an antibody for the epitope, and/or the ability of the lymphocytes expressing the CAR to proliferate in vitro and/or in vivo in response to marker recognition.
[00107] Typically a spacer region is found between the binding domain and a transmembrane domain of the CAR. Spacer regions can provide for flexibility of the binding domain and allows for high expression levels in the modified cells. In particu l ar embodiments, a spacer region can have at least 10 to 250 amino acids, at least 10 to 200 amino acids, at least 10 to 150 amino acids, at least 10 to 100 amino acids, at least 10 to 50 amino acids or at least 10 to 25 amino acids and including any integer between the endpoints of any of the listed ranges. In further embodiments, a spacer region has 250 amino acids or less; 200 amino acids or less, 150 amino acids or less; 100 amino acids or less; 50 amino acids or less; 40 amino acids or less; 30 amino acids or less; 20 amino acids or less; or 10 amino acids or less.
[00108] In particular embodiments, spacer regions can be derived from a hinge region of an immunoglobulin like molecule, for example all or a portion of the hinge region from a human lgG1 , human lgG2, a human lgG3, or a human lgG4. Hinge regions can be modified to avoid undesirable structural interactions such as dimerization. In some embodiments, all or a portion of a hinge region can be combined with one or more domains of a constant region of an immunoglobulin. For
example, a portion of a hinge region can be combined with all or a portion of a CH2 or CH3 domain or variant thereof.
[00109] CARs disclosed herein can also include transmembrane domains. In particular embodiments, the CAR polynucleotide encodes the transmembrane domain. The transmembrane domain provides for anchoring of the CAR in the lymphocyte membrane. The transmembrane domain may be derived either from a natural or a synthetic source. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3, CD45, CD4, CDS, CD9, CDI6, CD22; CD33, CD37, CD64, CD80, CD86, CDI34, CDI37 and CD154. In further particular embodiments, synthetic or variant transmembrane domains comprise predominantly hydrophobic residues such as leucine and valine.
[00110] In a particular embodiment, the CAR comprises a P28z fusion receptor composed of a single-chain antibody (scFv) specific for the extracellular domain of PSMA (J591 ) combined with CD28 and CD3ζ cytoplasmic signaling domains. In another embodiment, the CAR comprises a P28z CAR of SEQ ID NO. 94. SEQ ID NO. 94 includes murine components and was utilized in studies described herein. Amino acid positions 1 -797 include the anti-PSMA scFv (J592) whereas positions 797-1477 include the murine CD8 transmembrane domain, murine CD28 signaling domain and the murine CD3zeta signaling domain. Any P28z domain can be individually replaced with optimized domains. In particularized embodiments, the transmembrane domain and signaling domains within positions 797-1477 of SEQ ID NO. 94 can be particularly replaced with domains optimized for use in humans or other animals. In additional embodiments, any whole or portion of a binding domain, any whole or portion of an effector domain, any whole or portion of a spacer domain and/or any whole or portion of a transmembrane domain can be optimized for use in humans or other animals. In additional embodiments, the P28z CAR is optimized for use in humans. When optimized for humans, the P28z CAR can have lowered or no immunogenicity in humans and have a lower number of non-immunogenic epitopes compared to non-human antibodies.
[00111] Endosomal Release Agents. As used herein, "endosomal release agents" include any compound or peptide sequence that facilitates cargo exit from the endosome of a lymphocyte. Exemplary endosomal release agents include imidazoles, poly or oligoimidazoles, PEIs, peptides, fusogenic peptides, polycarboxylates, polycations, masked oligo or poly cations or anions, acetals, polyacetals, ketals/polyketyals, orthoesters, polymers with masked or unmasked cationic or anionic charges, amphiphilic block copolymers and dendrimers with masked or unmasked cationic or anionic charges.
[00112] Many endosomal release agents are adapted from viral elements that promote escape from the endosome and deliver polynucleotides intact into the nucleus. As one particular example, the H5WYG peptide can be used to induce the lysis of membranes at low pH. The histidine-rich peptide H5WYG is a derivative of the N- terminal sequence of the HA-2 subunit of the influenza virus hemagglutinin in which 5 of the amino acids have been replaced with histidine residues. H5WYG is able to selectively destabilize membranes at a slightly acidic pH as the histidine residues are protonated. The E1 protein from Semliki Forrest virus is also a useful endosomal release agent.
[00113] In particular embodiments, endosomal release agents include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO. 29). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO. 30)) containing a hydrophobic MTS can also be used.
[00114] Additional exemplary endosomal release agents include:
Source Sequence SEQ ID NO.
Influenza virus hemagglutinin HA-2 GLFEAIAGFIENGWEG 31
TAT of HIV YGRKKRRQRRR 32
N-terminal region of the S protein of MSGTFGGILAGLIGLL 33
duck hepatitis B
S protein of woodchuck hepatitis B MSPSSLLGLLAGLQW 34
Synthetic, Duguid et al. 1998 GLFEALLELLESLWELL 35
Synthetic, Gupta & Kothekar, 1997 LKKLLKKLLKKLLKKL 36
Derossi et al., J. Biol. Chem. 269: RQ I Kl WFQN RRM KWKK 37
10444, 1994
Tat fragment (48-60) GRKKRRQRRRPPQC 38
Chaloin et al., Biochem. peptide GALFLGWLGAAGSTMGAWSQ 39
Biophys. Res. Commun., 243: 601 , PKKKRKV
1998
PVEC LLIILRRRIRKQAHAHSK 40
Transportan GWTLNSAGYLLKINLKALAALAK 41
KIL
Amphiphilic model peptide; Oehlke KLALKLALKALKAALKLA 42
et al., Mol. Ther., 2: 339, 2000
Arg9 RRRRRRRRR 43
LL-37 LLGDFFRKSKEKIGKEFKRIVQR 44
IKDFLRNLVPRTES
Cecropin P1 SWLSKTAKKLENSAKKRISEGIA 45
IAIQGGPR
a-defensin ACYC R I PAC I AG E RRYGTC I YQ 46
GRLWAFCC
β-defensin DHYNCVSSGGQCLYSACPIFTK 47
IQGTCYRGKAKCCK
Bactenecin RKCRIWIRVCR 48
PR-3 RRRPRPPYLPRPRPPPFFPPRL 49
PPRIPPGFPPRFPPRFPGKR-
NH2
Indolicidin ILPWKWPWWPWRR-NH2 50
[00115] Nuclear Localization Signals. "Nuclear localization signals" (NLS) refer to sequences that direct associated sequences into the nucleus of a cell. Generally, NLS are a class of short amino acid sequences from 3 to 100 amino acids in length, from 3 to 50, 4 to 30, or 4 to 20 amino acids in length.
[00116] Exemplary NLS sequences include (i) monopartite NLS exemplified by the SV40 large T antigen NLS (PKKKRKV) (SEQ ID NO: 51 ); (ii) bipartite NLS consisting of two basic domains separated by a variable number of spacer amino acids and exemplified by the Xenopus nucleoplasms NLS (KRXXXXXXXXXXKKKL) (SEQ ID NO: 52); and (iii) noncanonical sequences such as M9 of the hnRNP A1 protein, the
influenza virus nucleoprotein NLS, and the yeast Gal4 protein NLS (Dingwall and Laskey, Trends Biochem Sci 16:478-481 , 1991 ). In particular embodiments, the NLS can be a highly cationic or basic peptide. In other embodiments, the NLS comprises two or more Arg or Lys amino acid residues. In further embodiments, the NLS can bind cytosolic proteins, such as importins and karyopherins, which recognize and transport NLS-containing sequences to the nuclear pore complex.
[00117] In particular embodiments, to direct import of delivered polynucleotides, particularly plasmid DNA, into the nucleus, polynucleotides (in one embodiment nanoparticle-encapsulated plasmids) can be conjugated to the SV40 T-Ag-derived NLS peptides. Exemplary SV40 T-Ag-derived NLS peptides include: PKKKRKV (SEQ ID NO. 51 ); PKKKRMV (SEQ ID NO. 53); PKKKRKVEDP (SEQ ID NO. 54); PKKGSKKA (SEQ ID NO. 55); PKTKRKV (SEQ ID NO. 56); CGGPKKKRKVG (SEQ ID NO. 57); PKKKIKV (SEQ ID NO. 58); CYDDEATADSQHSTPPKKKRKVEDPKDFESELLS (SEQ ID NO. 59); and CGYGPKKKRKVGG (SEQ ID NO. 60).
[00118] Additional exemplary NLS sequences include:
Source Sequence SEQ ID
NO.
Polyoma large T protein PKKARED 61
Polyoma large T protein CGYGVSRKRPRPG 62
SV40 VP1 capsid polypeptide APTKRKGS 63
Polyoma virus major capsid protein VP1 APKRKSGVSKC 64
SV40 VP2 capsid protein PNKKKRK 65
Polyoma virus capsid protein VP2 EEDGPQKKKRRL 66
Yeast histone H2B GKKRSKA 67
Adenovirus E1 a KRPRP 68
Adenovirus type 2/5 E1 a CGGLSSKRPRP 69
Xenopus NLS2 LKDKDAKKSKQE 70
v-Rel or p59v"rel GNKAKRQRST 71
Influenza A NS1 protein PFLDRLRRDQK 72
Human lamin A SVTKKRKLE 73
Xenopus lamin A SASKRRRLE 74
Adenovirus 5 DBP PPKKRMRRRIE 75
Rat glucocorticoid receptor YRKCLQAGMNLEARKTKK 76
KIKGIQQATA
Human estrogen receptor RKDRRGGRMLKHKRQRD 77
DGEGRGEVGSAGDMRAM INACIDNLWPSPLMIKRSK
K
Rabbit progesterone receptor RKFKKFNK 78 c-myb gene product PLLKKIKQ 79
N-myc gene product PPQKKIKS 80
p53 PQPKKKP 81
c-erb-A gene product SKRVAKRKL 82
Yeast ribosomal protein L29 MTGSKTRKHRGSGA 83
Yeast ribosomal protein L29 RHRKHP 84
Yeast ribosomal protein L29 KRRKHP 85
Yeast ribosomal protein L29 KYRKHP 86
Yeast ribosomal protein L29 KHRRHP 87
Yeast ribosomal protein L29 KHKKHP 88
Yeast ribosomal protein L29 RHLKHP 89
Hepatitis B core antigen PETTWRRRGRSPRRRTP 90
SPRRRRSPRRRRSQS
Viral jun ASKSRKRKL 91
Human T-cell leukemia virus Tax trans- GGLCSARLHRHALLAT 92
activator protein
Mouse nuclear Mx1 protein DTREKKKFLKRRLLRLDE 93
[00119] Exemplary NLS are also described in Cokol et al., 2000, EMBO Reports, 1 (5):41 1 -415; Boulikas, 1993, Crit. Rev. Eukaryot. Gene Expr., 3:193-227; Collas et al., 1996, Transgenic Research, 5: 451 -458; Collas and Alestrom, 1997, Biochem. Cell Biol. 75: 633-640; Collas and Alestrom, 1998, Transgenic Research, 7: 303-309; Collas and Alestrom, 1996, Mol. Reprod. Devel., 45:431 -438, and US Pat. Nos. 7,531 ,624; 7,498,177; 7,332,586; and 7,550,650.
[00120] Nanocarriers. Compositions disclosed herein include nanocarriers. Nanocarriers can include a porous nanoparticle at least substantially covered by a coating. In particular embodiments, polynucleotides and optionally NLSs can be found within the porous nanoparticle whereas optional lymphocyte-directing agents and endosomal release agents can be anchored to the coating.
[00121] Porous Nanoparticles. Porous nanoparticles of particular compositions can be constructed from any material capable of forming a porous network. Exemplary materials include a variety of material including, without limitation, biocompatible polymers, metals, transition metals and metalloids. Exemplary biocompatible polymers include, but not limited to, agar, agarose, alginate, alginate/calcium phosphate cement (CPC), beta-galactosidase (β-GAL), (1 ,2,3,4,6-pentaacetyl a-D-galactose), cellulose, chitin, chitosan, collagen, elastin, gelatin, hyaluronic acid collagen, hydroxyapatite,
poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate) (PHBHHx), poly(lactide), poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLG), poly(lactic-co-glycolic acid) (PLGA),poly(vinyl alcohol) (PVA), silk, soy protein, and soy protein isolate, alone or in combination with any other polymer composition, in any concentration and in any ratio. Blending different polymer types in different ratios using various grades can result in characteristics that borrow from each of the contributing polymers. Various terminal group chemistries can also be adopted. Exemplary metals, transition metals and metalloids include lithium, magnesium, zinc, aluminum and silica. In one embodiment, the porous nanoparticles comprise silica. The exceptionally high surface area of mesoporous silica (exceeding 1 ,000 m2/g) enables polynucleotide loading at levels exceeding conventional DNA carriers such as liposomes or polymer conjugates. In additional embodiments, pores range in size from 10-20 nm.
[00122] Useful nanocarriers of particular embodiments also include those based on (i) lipid-based delivery systems, including cationic lipids, ionizable cationic lipids, lipid- like molecules and pH-sensitive amphiphiles; and/or (ii) polymeric RNA DNA delivery systems such as polyethyleniminie (PEI)-based polymeric vectors, chitosan-based vectors, dendrimers (highly branched, spherical macromolecules synthesized from poly- amidoamine (PAMAM) and poly-propylene iminie (PPI), and block copolymers such as PAA/BMA/DMAEMA and PDMAEMA.
[00123] The porous nanoparticles can be a variety of different shapes, including spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and the like. The polynucleotides can be included in the porous nanoparticles in a variety of ways. For example, the polynucleotides can be encapsulated in the porous nanoparticles. In other aspects, the polynucleotides can be associated (e.g., covalently and/or non-covalently) with the surface or close underlying vicinity of the surface of the porous nanoparticles. In some embodiments, the polynucleotides can be incorporated in the porous nanoparticles e.g., integrated in the material of the porous nanoparticles. For example, the polynucleotides can be incorporated into a polymer matrix of polymer nanoparticles. One of ordinary skill in the art will appreciate the various ways to carry the polynucleotides so as to allow delivery of the polynucleotide molecules to the lymphocytes.
[00124] In particular embodiments, porous nanoparticles include liposomes. Liposomes are microscopic vesicles consisting of at least one concentric lipid bilayer. Vesicle-forming lipids are selected to achieve a specified degree of fluidity or rigidity of the final complex. In some embodiments, liposomes provide a lipid composition that is an outer layer surrounding a porous nanoparticle.
[00125] Liposomes can be neutral (cholesterol) or bipolar and include phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM) and other type of bipolar lipids including but not limited to dioleoylphosphatidylethanolamine (DOPE), with a hydrocarbon chain length in the range of 14-22, and saturated or with one or more double C=C bonds. Examples of lipids capable of producing a stable liposome, alone, or in combination with other lipid components are phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebro sides, distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimido-methyl)cyclohexane-1 - carboxylate (DOPE-mal). Additional non-phosphorous containing lipids that can become incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, and the cationic lipids mentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Choi). Negatively charged lipids include phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylglycerol and (DOPG), dicetylphosphate that are able to form vesicles. In particular embodiments, lipids used to create liposomes disclosed herein include cholesterol, hydrogenated soy phosphatidylcholine (HSPC) and, the derivatized vesicle-forming lipid PEG-DSPE.
[00126] Methods of forming liposomes are described in, for example, US Patent Nos. 4,229,360; 4,224,179; 4,241 ,046; 4,737,323; 4,078,052; 4,235,871 ; 4,501 ,728; and
4,837,028, as well as in Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980) and Hope et al., Chem. Phys. Lip. 40:89 (1986).
[00127] The size of the nanocar ers can vary over a wide range and can be measured in different ways. For example, the nanocarhers of the present disclosure can have a minimum dimension of 100 nm. The nanocarhers of the present disclosure can also have a minimum dimension of equal to or less than 500 nm, less than 150 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, or less than 10 nm. In certain embodiments, the nanocarriers can have a minimum dimension ranging between 5 nm and 500 nm, between 10 nm and 100 nm, between 20 nm and 90 nm, between 30 nm and 80 nm, between 40 nm and 70 nm, and between 40 nm and 60 nm. In some embodiments, the dimension is the diameter of nanoparticles or coated nanoparticles. In some embodiments, a population of nanocarriers of the present disclosure can have a mean minimum dimension of equal to or less than 500 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, or less than 10 nm. In certain embodiments, a population of nanocarriers in a composition of the present disclosure can have a mean diameter ranging between 5 nm and 500 nm, between 10 nm and 100 nm, between 20 nm and 90 nm, between 30 nm and 80 nm, between 40 nm and 70 nm, and between 40 nm and 60 nm. Dimensions of the nanocarriers can be determined using, e.g., conventional techniques, such as dynamic lightscattering and/or electron microscopy.
[00128] In particular embodiments, the compositions include protocells as nanocarriers. Protocells can be formed via fusion of liposomes to porous silica nanoparticles. The high pore volume and surface area of the spherical mesoporous silica core allow high-capacity encapsulation of a spectrum of cargos, including plasmid DNA. The supported lipid bilayer, whose composition can be modified for specific biological applications, can serve as a modular, reconfigurable scaffold, allowing the attachment of a variety of molecules, such as lymphocyte-directing agents, to provide cell-specific targeting and controlled intracellular trafficking. As provided further herein, protocells can efficiently introduce polynucleotides into lymphocytes.
[00129] In one particular embodiment intended to illustrate the foregoing, anti-CD3 antibodies can be coupled onto protocell nanocarriers to selectively target the nanocarriers to T cells for rapid receptor-induced endocytosis. Protocells can be formed via fusion of liposomes with porous silica nanoparticles (Fig. 2A, B). The high pore volume and surface area of the spherical mesoporous silica core allow high-capacity encapsulation of a spectrum of cargos, including plasmid DNA. The membrane serves as a modular scaffold for the attachment of a variety of targeting moieties. In the embodiment depicted in Fig. 2, the pH-sensitive fusogenic peptide H5WYG is tethered to the nanocarrier surface to facilitate endosomal escape. The plasmid DNA was also modified before encapsulation into nanoparticles with the SV40 large T antigen nuclear localization signal peptide (Fig. 2A).
[00130] Particular nanocarrier embodiments include:
Selected Lymphocyte- Target Targeting Agent Endosomal NLS
Lymphocyte Directing Release
Population Agent Agent
T cells Anti-CD3 Leukemia Anti-CD19 CAR Fusogenic SV40 antibody cells (1928zeta or 194- peptide
1 BBzeta) H5WYG
CD8 T cells Anti-CD8 Ovarian Anti-mesothelin "Proton NLS antibody cancer CAR Sponge" Ku70 cells (with or without effect of
integrated polymeric
costimulatory nanoparticles domains)
T cells Anti-LFA Pancreati Affinity-enhanced TAT peptide None antibody c cancer T cell receptor
cells (TCR) specific for
mesothelin
T cells 4-1 BB HIV- HIV-gag protein- Cationic- hnRNP
(CD137) infected specific T-cell polymer- (M9) targeting cells receptor based aptamers nanocarrier
Monocytes/ Anti-CD14 StaphylClumping factor A Pas nona- SV40 macrophages antibodies ococcus (ClfA) arginine
aureus (PR9)
NK cells Anti-CD56 Prostate Anti-PSMA CAR Cationic None antibodies cancer (P28zeta or P4- lipid-based
cells 1 BB zeta) nanocarrier
TREG Anti-CTLA-4 Neurons CAR specific for Cationic SV40 or anti-GARP KIR4.1 for the polymer-
antibodies treatment of based
multiple sclerosis nanocarrier
Hematopoieti Anti-CD34 Leukemia Affinity-enhanced "Proton NLS c stem cells antibodies cells T cell receptor Sponge" Ku70
(TCR) specific for effect of
Wilms' tumor polymeric
antigen (WT1 ) nanoparticles
[00131] Compositions. The nanoparticles, porous nanoparticles and nanocarriers (all collectively referred to herein as "active ingredients") disclosed herein can be provided as part of compositions that comprise, consist of or consist essentially of the nanoparticles, porous nanoparticles and/or nanocarriers. The compositions can be formulated for administration to subjects.
[00132] In some embodiments, the active ingredients are provided as part of a composition that can comprise, for example, at least 0.1 % w/v of active ingredient(s); at least 1 % w/v of active ingredient(s); at least 10% w/v of active ingredient(s); at least 20% w/v of active ingredient(s); at least 30% w/v of active ingredient(s); at least 40% w/v of active ingredient(s); at least 50% w/v of active ingredient(s); at least 60% w/v of active ingredient(s); at least 70% w/v of active ingredient(s); at least 80% w/v of active ingredient(s); at least 90% w/v of active ingredient(s); at least 95% w/v of active ingredient(s); or at least 99% w/v of active ingredient(s).
[00133] In other embodiments, the active ingredients are provided as part of a composition that can comprise, for example, at least 0.1 % w/w of active ingredient(s); at least 1 % w/w of active ingredient(s); at least 10% w/w of active ingredient(s); at least 20% w/w of active ingredient(s); at least 30% w/w of active ingredient(s); at least 40% w/w of active ingredient(s); at least 50% w/w of active ingredient(s); at least 60% w/w of active ingredient(s); at least 70% w/w of active ingredient(s); at least 80% w/w of active ingredient(s); at least 90% w/w of active ingredient(s); at least 95% w/w of active ingredient(s); or at least 99% w/w of active ingredient(s).
[00134] The compositions disclosed herein can be formulated for administration by, without limitation, injection, inhalation, infusion, perfusion, lavage or ingestion. The compositions disclosed herein can further be formulated for, without limitation, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal,
intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral and/or subcutaneous administration and more particularly by intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral and/or subcutaneous injection.
[00135] For injection, compositions can be formulated as aqueous solutions, such as in buffers including Hanks' solution, Ringer's solution, or physiological saline. The aqueous solutions can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the formulation can be in lyophilized and/or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
[00136] For oral administration, the compositions can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. For oral solid formulations such as, for example, powders, capsules and tablets, suitable excipients include binders (gum tragacanth, acacia, cornstarch, gelatin), fillers such as sugars, e.g. lactose, sucrose, mannitol and sorbitol; dicalcium phosphate, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxy- methylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If desired, disintegrating agents can be added, such as corn starch, potato starch, alginic acid, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, solid dosage forms can be sugar-coated or enteric-coated using standard techniques. Flavoring agents, such as peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. can also be used.
[00137] For administration by inhalation, compositions can be formulated as aerosol sprays from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and
cartridges of gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the therapeutic and a suitable powder base such as lactose or starch.
[00138] Any composition formulation disclosed herein can advantageously include any other pharmaceutically acceptable carriers which include those that do not produce significantly adverse, allergic or other untoward reactions that outweigh the benefit of administration, whether for research, prophylactic and/or therapeutic treatments. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, formulations can be prepared to meet sterility, pyrogenicity, general safety and purity standards as required by United States FDA Office of Biological Standards and/or other relevant foreign regulatory agencies.
[00139] Exemplary generally used pharmaceutically acceptable carriers include any and all bulking agents or fillers, solvents or co-solvents, dispersion media, coatings, surfactants, antioxidants (e.g., ascorbic acid, methionine, vitamin E), preservatives, isotonic agents, absorption delaying agents, salts, stabilizers, buffering agents, chelating agents (e.g., EDTA), gels, binders, disintegration agents, and/or lubricants.
[00140] Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers and/or trimethylamine salts.
[00141] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.
[00142] Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol or mannitol.
[00143] Exemplary stabilizers include organic sugars, polyhydric sugar alcohols, polyethylene glycol; sulfur-containing reducing agents, amino acids, low molecular weight polypeptides, proteins, immunoglobulins, hydrophilic polymers or polysaccharides.
[00144] Compositions can also be formulated as depot preparations. Depot preparations can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salts.
[00145] Additionally, compositions can be formulated as sustained-release systems utilizing semipermeable matrices of solid polymers containing at least one active ingredient. Various sustained-release materials have been established and are well known by those of ordinary skill in the art. Sustained-release systems may, depending on their chemical nature, release active ingredients following administration for a few weeks up to over 100 days.
[00146] When formulated to treat cancer, the disclosed compositions can also include plasmid DNA carrying one or more anticancer genes selected from p53, RB, BRCA1 , E1A, bcl-2, MDR-1 , p21 , p16, bax, bcl-xs, E2F, IGF-I VEGF, angiostatin, oncostatin, endostatin, GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-γ, TNF-a and/or HSV-tk. Compositions can also include or be administered in combination with one or more antineoplastic drugs including adriamycin, angiostatin, azathioprine, bleomycin, busulfane, camptothecin, carboplatin, carmustine, chlorambucile, chlormethamine, chloroquinoxaline sulfonamide, cisplatin, cyclophosphamide, cycloplatam, cytarabine, dacarbazine, dactinomycin, daunorubicin, didox, doxorubicin, endostatin, enloplatin, estramustine, etoposide, extramustinephosphat, flucytosine, fluorodeoxyuridine, fluorouracil, gallium nitrate, hydroxyurea, idoxuridine, interferons, interleukins, leuprolide, lobaplatin, lomustine, mannomustine, mechlorethamine, mechlorethaminoxide, melphalan, mercaptopurine, methotrexate, mithramycin, mitobronitole, mitomycin, mycophenolic acid, nocodazole, oncostatin, oxaliplatin, paclitaxel, pentamustine, platinum-triamine complex, plicamycin, prednisolone, prednisone, procarbazine, protein kinase C inhibitors, puromycine, semustine, signal transduction inhibitors, spiroplatin, streptozotocine, stromelysin inhibitors, taxol, tegafur, telomerase inhibitors, teniposide, thalidomide, thiamiprine, thioguanine, thiotepa, tiamiprine, tretamine, triaziquone, trifosfamide, tyrosine kinase inhibitors, uramustine, vidarabine, vinblastine, vinca alcaloids, vincristine, vindesine, vorozole, zeniplatin, zeniplatin or zinostatin.
[00147] Methods. Methods disclosed herein include treating subjects (humans, veterinary animals, livestock and research animals) with compositions, active ingredients, nanoparticles, porous nanoparticles and/or nanocarriers disclosed herein. Treating subjects includes delivering a therapeutically effective amount. An "effective amount" is the amount of a compound necessary to result in a desired physiological change in the subject. Effective amounts are often administered for research purposes. Effective amounts disclosed herein reduce the number of unwanted cell types in a subject.
[00148] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of a disease or condition associated with or caused by a target or displays only early signs or symptoms of the disease or condition such that treatment is administered for the purpose of diminishing, preventing, or decreasing the risk of developing the disease or condition further. Thus, a prophylactic treatment functions as a preventative treatment against a disease or disorder associated with or caused by a target.
[00149] A "therapeutic treatment" includes a treatment administered to a subject who displays symptoms or signs of a disease or condition associated with or caused by a target and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the disease or condition.
[00150] "Therapeutically effective amounts" include those that provide effective amounts, prophylactic treatment and/or therapeutic treatment. Therapeutically effective amounts need not fully prevent or cure the disease or condition but can also provide a partial benefit, such as reduction in the number of unwanted targets; reduction of destruction of wanted targets; and/or a delay of onset or alleviation or improvement of at least one symptom of the disease or condition.
[00151] For administration, effective amounts and therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and/or animal model studies. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes an IC5o as determined in cell culture against a particular target. Such information can be used to more accurately determine useful doses in subjects of interest.
[00152] The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of disease, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.
[00153] Useful doses often range from 0.1 to 5 pg/kg or from 0.5 to 1 g /kg. In other non-limiting examples, a dose can comprise 1 g /kg, 5 g /kg, 10 g /kg, 15 g /kg, 20 pg /kg, 25 pg /kg, 30 pg /kg, 35 pg/kg, 40 pg/kg, 45 pg/kg, 50 pg/kg, 55 pg/kg, 60 pg/kg, 65 pg/kg, 70 pg/kg, 75 pg/kg, 80 pg/kg, 85 pg/kg, 90 pg/kg, 95 pg/kg, 100 pg/kg, 150 Mg/kg, 200 pg/kg, 250 pg/kg, 350 pg/kg, 400 pg/kg, 450 pg/kg, 500 pg/kg, 550 pg/kg, 600 pg/kg, 650 pg/kg, 700 pg/kg, 750 pg/kg, 800 pg/kg, 850 pg/kg, 900 pg/kg, 950 g/kg, 1000 pg/kg, 0.1 to 5 mg/kg or from 0.5 to 1 mg/kg. In other non-limiting examples, a dose can comprise 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 150 mg/kg, 200 mg/kg, 250 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 650 mg/kg, 700 mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 mg/kg, 950 mg/kg, 1000 mg/kg or more.
[00154] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 1 1 months or yearly.
[00155] Exemplary methods disclosed herein include administering nanocarriers to a subject in need thereof. The nanocarriers are directed to chosen lymphocytes in the subject and are designed to be internalized by the lymphocytes. Once internalized, the nanocarriers further deliver a polynucleotide having a sequence that encodes a targeting agent. The polynucleotide modifies the lymphocytes to express the targeting agent, which subsequently binds a marker associated with the target. Upon binding, the lymphocytes can kill or otherwise trigger the destruction of unwanted targets such as
unwanted cells, thereby treating a disease or condition associated with the unwanted cell type. Alternatively, upon binding, the lymphocytes can protect wanted targets such as wanted cells, thereby treating a disease or condition associated with unwanted destruction of the wanted cell type.
[00156] In another particular embodiment, nanocarriers can be loaded with polynucleotides {e.g., Transgenes) that encode for a defined tumor- or virus-specific TCR. Surface-anchored lymphocyte-directing agents that recognize T-cell-specific proteins enable the nanocarriers to selectively bind T-cells. Upon infusion into a subject's bloodstream, the nanocarriers can deliver TCR genes into T-cells, which can subsequently express this TCR on their surface. Equipped with a therapeutically relevant TCR, the T-cells can recognize and lyse malignant cells or virus-infected cells or other targeted unwanted cell types.
[00157] In additional embodiments, NK cells are selectively modified to express CARs or high-affinity TCRs. In additional embodiments, hematopoietic stem cells (HSCs) are selectively modified to express CARs or high-affinity TCRs. In additional embodiments, monocytes/macrophages cells are selectively modified to express CARs or high-affinity ligands specific for viruses, bacteria, fungus or yeast antigens. In additional embodiments, B cells are selectively modified to express tumor- or virus- specific antibodies. In additional embodiments, TREG cells are selectively modified to express CARs or high-affinity ligands specific for autoimmune markers, allergic reaction markers or beneficial bacteria.
[00158] Additional embodiments include methods of delivering pre-designed synthetic nanocarriers to lymphocytes {e.g., T-cells), in which the nanocarriers can be loaded with polynucleotides {e.g., plasmids) that encode a receptor for an antigen {e.g., a prostate tumor-targeting receptor P28z). Internalization of the nanocarriers can render transfected lymphocytes {e.g., T-cells) capable of lysing cells associated with the antigen {e.g., a prostate tumor). In some embodiments, delivery of the nanocarriers including the receptor genes into lymphocytes {e.g., T-cells) can include, e.g., (1 ) specific binding to the lymphocytes {e.g., T-cells), (2) internalization of the nanocarriers by the lymphocytes, (3) escape from endocytic vesicles into the cytoplasm after internalization, (4) release of the polynucleotide, which (5) can be transported into the
nucleus of the lymphocytes and (6) transcribed to deliver genes for expressing a receptor for the antigen.
[00159] In particular embodiments, the methods are used to target unwanted cancer cells. Thus, the disclosed methods provide a new paradigm for the treatment of cancer that can involve programming circulating lymphocytes with tumor-recognizing capabilities in vivo. This paradigm contrasts with those currently used to generate T cells with defined anti-cancer specificities, which involve isolation of the lymphocytes from the patient and genetically modifying them in the laboratory with tumor antigen- specific receptors using retroviral or lentiviral vectors; the programmed cells are then expanded and infused back into the patient where they can recognize and destroy cancer cells. This ex vivo production of modified cells requires the production of a new lymphocyte cohort for each patient, a laborious process that can only be accomplished at elaborate cell-production facilities available at just a few cancer centers worldwide.
[00160] The disclosed methods provide a more practical and widespread approach, allowing use of an "off-the-shelf solution that can quickly modify lymphocytes to recognize and destroy tumors while they are circulating in a subject, thus avoiding the complications of laboratory modification of extracted cells. In comparison to in vitro methods that modify and expand T cells for each patient, the compositions and methods disclosed herein can produce targeting effects within a subject's circulatory system in only days.
[00161] The disclosed methods provide the first implementation of nanocarriers for the genetic engineering of immune cells to selectively target cells associated with markers for various therapeutic objectives. For example, and in relation to cancer cells as an unwanted cell type, previous nanotechnology-based clinical research has focused on particles that selectively accumulate chemotherapeutics, siRNA, or imaging agents at tumor sites while minimizing off-target toxicities. The methods described herein are different: instead of introducing therapeutics into tumor tissue, the disclosed methods introduce genes encoding tumor-recognizing receptors into circulating lymphocytes, which in turn bind and destroy tumor cells. This strategy has the advantage that, unlike agent-loaded nanoparticles (which are quickly cleared by phagocytes), the modified lymphocytes can persist and proliferate in the subject for a long-term effect. Thus, in
relation to cancer treatments specifically, the current disclosure provides a new, more effective therapy. The disclosure shifts the focus from broad-impact chemotherapy or radiotherapy (which have many negative side-effects) to tumor-specific immunotherapeutics (which do not harm healthy tissue). Nanoparticle gene therapy will provide clinicians with the ability to instantly treat diagnosed patients with an off-the shelf composition that can be widely distributed at low cost, and is amenable to changes in dose and specificity as the treatment evolves.
[00162] In the context of cancers, therapeutically effective amounts can decrease the number of tumor cells, decrease the number of metastases, decrease tumor volume, increase life expectancy, induce apoptosis of cancer cells, induce cancer cell death, induce chemo- or radiosensitivity in cancer cells, inhibit angiogenesis near cancer cells, inhibit cancer cell proliferation, inhibit tumor growth, prevent metastasis, prolong a subject's life, reduce cancer-associated pain, reduce the number of metastases, and/or reduce relapse or re-occurrence of the cancer following treatment.
[00163] While the methods disclosed herein are advantageously practiced in vivo, additional embodiments may also be practiced ex vivo. For example, the methods can include obtaining lymphocytes from a subject. Lymphocytes can, e.g., be obtained from a subject using any procedure generally known in the art. For example, blood can be obtained from a subject and lymphocytes can be isolated. The isolated lymphocytes can then be combined with nanocarriers (or a composition comprising nanocarriers) including a polynucleotide having a sequence that encodes a targeting agent. The nanocarriers can be internalized by the lymphocytes such that the lymphocytes then incorporate the polynucleotide and express the targeting agent. The modified lymphocytes expressing the targeting agent can be administered to the subject such that, after the administering, the lymphocytes bind to the targeted markers on cells associated with the disease, thereby treating the disease. It will be appreciated, for example, that the modifying of the lymphocytes can be fully accomplished ex vivo prior to administration, and/or nanocarriers can be internalized and the lymphocytes can be administered to the subject while modifying is being carried out leading to expression of the targeting agents.
EXEMPLARY EMBODIMENTS - Set 1 .
A synthetic nanocarrier comprising (i) a lipid-coated porous nanoparticle (ii) a lymphocyte-directing agent extending from the surface of the lipid-coated porous nanoparticle; and (iii) a polynucleotide encoding a chimeric antigen receptor (CAR) targeting agent within the pores of the lipid-coated porous nanoparticle nanoparticle.
A synthetic nanocarrier of embodiment 1 further comprising an endosomal release agent extending from the surface of the lipid-coated porous nanoparticle and (ii) a nuclear localization signal (NLS) within the pores of the lipid-coated porous nanoparticle.
A synthetic nanocarrier of embodiments 1 or 2 wherein the CAR is P28z.
A synthetic nanocarrier of any one of embodiments 1 , 2 or 3 wherein the lipid coating is a liposome, a lipid bilayer or a polymeric micelle.
A synthetic nanocarrier of any one of embodiments 1 -4 wherein the synthetic nanocarriers comprise liposomes, polymeric particles, metallic particles, polymeric micelles, polyethyleneimine (PEI)/DNA complexes, or a combination thereof.
A synthetic nanocarrier of any one of embodiments 1 -5 wherein the lipid coating encapsulates the lipid-coated porous nanoparticle.
A synthetic nanocarrier of any one of embodiments 1 -6 wherein the lymphocyte- directing agent selectively binds to lymphocytes in vivo.
A synthetic nanocarrier of any one of embodiments 1 -7 wherein the lymphocyte- directing agent comprises a binding domain selected from a lymphocyte receptor ligand, lymphocyte receptor antibody, lymphocyte receptor peptide aptamer, lymphocyte receptor nucleic acid aptamer, lymphocyte receptor spiegelmer, or a combination thereof.
A synthetic nanocarrier of any one of embodiments 1 -8 wherein the lymphocyte- directing agent selectively binds T cells, NK cells, monocytes, macrophages, B cells, hematopoietic stem cells, or a combination thereof.
A synthetic nanocarrier of any one of embodiments 1 -9 wherein the lymphocyte- directing agent selectively binds T-cell receptor motifs; T-cell a chains; T-cell β chains; T-cell γ chains; T-cell δ chains; CCR7; CD3; CD4; CD5; CD7; CD8;
CD1 1 b; CD1 1 c; CD16; CD19; CD20; CD21 ; CD22; CD25; CD28; CD34; CD35; CD40; CD45RA; CD45RO; CD52; CD56; CD62L; CD68; CD80; CD95; CD1 17; CD127; CD133; CD137 (4-1 BB); CD163; F4/80; IL-4Ra; Sca-1 ; CTLA-4; GITR; GARP; LAP; granzyme B; LFA-1 ; or transferrin receptor.
A synthetic nanocarrier of any one of embodiments 1 -9 wherein the lymphocyte- directing agent selectively binds CCR7; CD3; CD4; CD5; CD8; CD16; CD19; CD20; CD21 ; CD22; CD25; CD28; CD35; CD40; CD45RA; CD45RO; CD52; CD62L; CD80; CD95; CD127; or CD137.
A synthetic nanocarrier of any one of embodiments 1 -9 wherein the lymphocyte- directing agent comprises a binding domain selected from a T-cell a chain antibody; T-cell β chain antibody; T-cell γ chain antibody; T-cell δ chain antibody; CCR7 antibody; CD3 antibody; CD4 antibody; CD5 antibody; CD7 antibody; CD8 antibody; CD1 1 b antibody; CD1 1 c antibody; CD16 antibody; CD19 antibody; CD20 antibody; CD21 antibody; CD22 antibody; CD25 antibody; CD28 antibody; CD34 antibody; CD35 antibody; CD40 antibody; CD45RA antibody; CD45RO antibody; CD52 antibody; CD56 antibody; CD62L antibody; CD68 antibody; CD80 antibody; CD95 antibody; CD1 17 antibody; CD127 antibody; CD133 antibody; CD137 (4-1 BB) antibody; CD163 antibody; F4/80 antibody; IL-4Ra antibody; Sca-1 antibody; CTLA-4 antibody; GITR antibody GARP antibody; LAP antibody; granzyme B antibody; LFA-1 antibody; or transferrin receptor antibody. A synthetic nanocarrier of embodiment 12 wherein the binding domain consists of or consists essentially of an scFv fragment of a T-cell a chain antibody; T-cell β chain antibody; T-cell γ chain antibody; T-cell δ chain antibody; CCR7 antibody; CD3 antibody; CD4 antibody; CD5 antibody; CD7 antibody; CD8 antibody; CD1 1 b antibody; CD1 1 c antibody; CD16 antibody; CD19 antibody; CD20 antibody; CD21 antibody; CD22 antibody; CD25 antibody; CD28 antibody; CD34 antibody; CD35 antibody; CD40 antibody; CD45RA antibody; CD45RO antibody; CD52 antibody; CD56 antibody; CD62L antibody; CD68 antibody; CD80 antibody; CD95 antibody; CD1 17 antibody; CD127 antibody; CD133 antibody; CD137 (4-1 BB) antibody; CD163 antibody; F4/80 antibody; IL-4Ra antibody; Sca-1 antibody; CTLA-4 antibody; GITR antibody GARP antibody; LAP antibody;
granzyme B antibody; LFA-1 antibody; or transferrin receptor antibody.
A synthetic nanocarrier of embodiment 12 wherein the binding domain consists of consists of or consists essentially of the scFv fragment (SEQ ID NO. 1 ) of the PSMA-specific chimeric antigen receptor (CAR), P28z.
A synthetic nanocarrier of any of embodiments 1 -14 wherein the polynucleotide is a plasmid, a minicircle plasmid, or an mRNA molecule.
A synthetic nanocarrier of any of embodiments 1 -15 wherein the CAR targeting agent comprises a binding domain for a marker associated with an unwanted cell type.
A synthetic nanocarrier of embodiment 16 wherein the unwanted cell type is a cancer cell.
A synthetic nanocarrier of embodiment 16 wherein the marker is a cancer antigen.
A synthetic nanocarrier of embodiment 16 wherein the marker is a cancer antigen selected from A33; BAGE; Bcl-2; β-catenin; CA125; CA19-9; CD5; CD19; CD20; CD21 ; CD22; CD33; CD37; CD45; CD123; CEA; c-Met; CS-1 ; cyclin B1 ; DAGE; EBNA; EGFR; ephrinB2; estrogen receptor; FAP; ferritin; folate-binding protein; GAGE; G250; GD-2; GM2; gp75, gp100 (Pmel 17); HER-2/neu; HPV E6; HPV E7; Ki-67; LRP; mesothelin, p53, PRAME; progesterone receptor; PSA; PSMA; MAGE; MART; mesothelin; MUC; MUM-1 -B; myc; NYESO-1 ; ras; RORI; survivin; tenascin; TSTA tyrosinase; VEGF; or WT1 .
A synthetic nanocarrier of embodiment 16 wherein the marker is PSMA.
A synthetic nanocarrier of any of embodiments 1 -20 wherein the CAR targeting agent is a surface antigen receptor or a receptor for an intracellular antigen presented by a Major Histocompatibility Complex antigen-presenting pathway. A synthetic nanocarrier of any one of embodiments 2-21 wherein the endosomal release agent is selected from any one of SEQ ID NOs. 29-50 or combinations thereof.
A synthetic nanocarrier of any one of embodiments 2-22 wherein the NLS is selected from any one of SEQ ID NOs. 51 -93 or combinations thereof.
A synthetic nanocarrier of any one of embodiments 1 -23 comprising a S/MAR
element, a PiggyBac transposase-containing plasmid, a Sleeping Beauty transposase-containing plasmid; a homo sapiens transposon-de ved Busterl transposase-like protein gene; a human endogenous retrovirus H protease/integrase-derived ORF1 ; a homo sapiens Cas-Br-M (murine) ecotropic retroviral transforming sequence; a homo sapiens endogenous retroviral sequence K; a homo sapiens endogenous retroviral family W sequence; a homo sapiens LINE-1 type transposase domain; or a homo sapiens pogo transposable element.
A composition comprising a synthetic nanocarrier of any one of embodiments 1 - 24.
A method of treating a subject having a condition associated with a cell type comprising: administering a therapeutically effective amount of a synthetic nanocarrier of any one of embodiments 1 -24 to the subject thereby treating the subject.
A method of treating a subject having a condition associated with a cell type comprising: administering a therapeutically effective amount of a composition of embodiment 25 to the subject thereby treating the subject.
A method of embodiments 26 or 27 wherein the unwanted cell type is an unwanted cancer cell.
A method of embodiment 28 wherein the unwanted cancer cell is selected from an adrenal cancer cell, a bladder cancer cell, a blood cancer cell, a bone cancer cell, a brain cancer cell, a breast cancer cell, a carcinoma cell, a cervical cancer cell, a colon cancer cell, a colorectal cancer cell, a corpus uterine cancer cell, an ear, nose and throat (ENT) cancer cell, an endometrial cancer cell, an esophageal cancer cell, a gastrointestinal cancer cell, a head and neck cancer cell, a Hodgkin's disease cell, an intestinal cancer cell, a kidney cancer cell, a larynx cancer cell, a leukemia cell, a liver cancer cell, a lymph node cancer cell, a lymphoma cell, a lung cancer cell, a melanoma cell, a mesothelioma cell, a myeloma cell, a nasopharynx cancer cell, a neuroblastoma cell, a non-Hodgkin's lymphoma cell, an oral cancer cell, an ovarian cancer cell, a pancreatic cancer cell, a penile cancer cell, a pharynx cancer cell, a prostate cancer cell, a rectal
cancer cell, a sarcoma cell, a seminoma cell, a skin cancer cell, a stomach cancer cell, a teratoma cell, a testicular cancer cell, a thyroid cancer cell, a uterine cancer cell, a vaginal cancer cell, or a vascular tumor cell.
A method of any one of embodiments 26-29 wherein the administering results in expression of the polynucleotide selectively by lymphocytes within 10 days; within 9 days; within 8 days; within 7 days; within 6 days; within 5 days; within 4 days; or within 3 days of administration.
A method for treating a disease associated with an antigen, the method comprising: administering to a subject in need thereof, a composition comprising a therapeutically effective amount of nanocarriers including a polynucleotide having a sequence that encodes a receptor for the antigen, thereby treating the disease.
A method of embodiment 31 wherein after the administering the nanocarriers are selectively incorporated into lymphocytes in the subject such that the lymphocytes express the receptor and subsequently bind to the antigen on cells associated with the disease thereby killing the cells.
A method for treating a disease associated with an antigen, the method comprising:
obtaining lymphocytes from a subject in need thereof;
combining the lymphocytes with a composition comprising nanocarriers including a polynucleotide having a sequence that encodes a receptor for the antigen, wherein the nanocarriers are selectively incorporated into the lymphocytes such that the lymphocytes express the receptor; and
administering the lymphocytes expressing the receptor to the subject, thereby treating the disease.
A method of embodiment 33 wherein after the administering, the lymphocytes bind to the antigen on cells associated with the disease thereby killing the cells. A method of selectively transfecting lymphocytes in vivo, the method comprising: contacting lymphocytes with nanocarriers comprising a polynucleotide having a sequence that encodes a receptor for an antigen associated with a disease, wherein the nanocarriers are selectively incorporated into the lymphocyte to
release the polynucleotide such that the lymphocyte expresses the receptor, thereby transfecting the lymphocyte.
36. A method of embodiment 35 wherein the antigen comprises a tumor antigen.
37. A method of embodiment 35 wherein the antigen comprises a viral antigen.
38. A method of any one of embodiments 35-37 wherein the lymphocytes comprise T-cells, NK cells, macrophages, monocytes, B cells, hematopoietic stem cells, or a combination thereof.
39. A method of embodiment 38 wherein the lymphocytes comprise T-cells.
40. A method of any one of embodiments 35-39 wherein the disease is a cancer.
41 . A method of embodiment 40 wherein the cancer comprises a leukemia, a lymphoma, a carcinoma, a sarcoma, or a melanoma.
42. A method of embodiment 40 wherein the disease is prostate cancer.
EXEMPLARY EMBODIMENTS - Set 2.
1 . A synthetic nanocarrier comprising (i) a lymphocyte-directing agent; and (ii) a polynucleotide encoding a targeting agent.
2. A synthetic nanocarrier of embodiment 1 further comprising a nanoparticle.
3. A synthetic nanocarrier of embodiments 1 or 2 further comprising a coating.
4. A synthetic nanocarrier of embodiment 2 or 3 wherein the nanoparticle is a porous nanoparticle.
5. A synthetic nanocarrier of embodiment 3 or 4 wherein the coating is a liposome, a lipid bilayer, or a polymeric micelle.
6. A synthetic nanocarrier of any one of embodiments 1 -5 wherein the synthetic nanocarrier comprises liposomes, polymeric particles, metallic particles, polymeric micelles, polyethyleneimine (PEI)/DNA complexes, or a combination thereof.
7. A synthetic nanocarrier of embodiments 3 or 5 wherein the coating encapsulates the nanoparticle.
8. A synthetic nanocarrier of any one of embodiments 1 -7 wherein the polynucleotide is on the surface of the nanocarrier, incorporated in the nanocarrier, encapsulated in the nanocarrier, or a combination thereof.
9. A synthetic nanocarrier of any one of embodiments 3-8 wherein the lymphocyte-
directing agent extends from the outer surface of the coating.
A synthetic nanocarrier of any one of embodiments 4-9 wherein the polynucleotide is within the pores of the porous nanoparticle.
A synthetic nanocarrier of any one of embodiments 1 -10 wherein the lymphocyte- directing agent selectively binds to lymphocytes in vivo.
A synthetic nanocarrier of any one of embodiments 1 -1 1 wherein the lymphocyte- directing agent comprises a binding domain selected from a lymphocyte receptor ligand, lymphocyte receptor antibody, lymphocyte receptor peptide aptamer, lymphocyte receptor nucleic acid aptamer, lymphocyte receptor spiegelmer, or a combination thereof.
A synthetic nanocarrier of any one of embodiments 1 -12 wherein the lymphocyte- directing agent selectively binds T cells, NK cells, monocytes, macrophages, B cells, hematopoietic stem cells, or a combination thereof.
A synthetic nanocarrier of any one of embodiments 1 -13 wherein the lymphocyte- directing agent selectively binds T-cell receptor motifs; T-cell a chains; T-cell β chains; T-cell γ chains; T-cell Δ chains; CCR7; CD3; CD4; CD5; CD7; CD8; CD1 1 b; CD1 1 c; CD16; CD19; CD20; CD21 ; CD22; CD25; CD28; CD34; CD35; CD40; CD45RA; CD45RO; CD52; CD56; CD62L; CD68;CD80; CD95; CD1 17; CD127; CD133; CD137 (4-1 BB); CD163; F4/80; IL-4Ra; Sca-1 ; CTLA-4; GITR; GARP; LAP; granzyme B; LFA-1 ; or transferrin receptor..
A synthetic nanocarrier of any one of embodiments 1 -13 wherein the lymphocyte- directing agent selectively binds CCR7; CD3; CD4; CD5; CD8; CD16; CD19; CD20; CD21 ; CD22; CD25; CD28; CD35; CD40; CD45RA; CD45RO; CD52; CD62L; CD80; CD95; CD127; or CD137.
A synthetic nanocarrier of any one of embodiments 1 -13 wherein the lymphocyte- directing agent comprises a binding domain selected from a T-cell a chain antibody; T-cell β chain antibody; T-cell γ chain antibody; T-cell Δ chain antibody; CCR7 antibody; CD3 antibody; CD4 antibody; CD5 antibody; CD7 antibody; CD8 antibody; CD1 1 b antibody; CD1 1 c antibody; CD16 antibody; CD19 antibody; CD20 antibody; CD21 antibody; CD22 antibody; CD25 antibody; CD28 antibody; CD34 antibody; CD35 antibody; CD40 antibody; CD45RA antibody; CD45RO
antibody; CD52 antibody; CD56 antibody; CD62L antibody; CD68 antibody; CD80 antibody; CD95 antibody; CD1 17 antibody; CD127 antibody; CD133 antibody; CD137 (4-1 BB) antibody; CD163 antibody; F4/80 antibody; IL-4Ra antibody; Sca-1 antibody; CTLA-4 antibody; GITR antibody GARP antibody; LAP antibody; granzyme B antibody; LFA-1 antibody; or transferrin receptor antibody. A synthetic nanocarrier of embodiment 16 wherein the binding domain consists of or consists essentially of an scFv fragment of a T-cell a chain antibody; T-cell β chain antibody; T-cell γ chain antibody; T-cell δ chain antibody; CCR7 antibody; CD3 antibody; CD4 antibody; CD5 antibody; CD7 antibody; CD8 antibody; CD1 1 b antibody; CD1 1 c antibody; CD16 antibody; CD19 antibody; CD20 antibody; CD21 antibody; CD22 antibody; CD25 antibody; CD28 antibody; CD34 antibody; CD35 antibody; CD40 antibody; CD45RA antibody; CD45RO antibody; CD52 antibody; CD56 antibody; CD62L antibody; CD68 antibody; CD80 antibody; CD95 antibody; CD1 17 antibody; CD127 antibody; CD133 antibody; CD137 (4-1 BB) antibody; CD163 antibody; F4/80 antibody; IL-4Ra antibody; Sca-1 antibody; CTLA-4 antibody; GITR antibody GARP antibody; LAP antibody; granzyme B antibody; LFA-1 antibody; or transferrin receptor antibody.
A synthetic nanocarrier of embodiment 17 wherein the binding domain consists of consists of or consists essentially of the scFv fragment (SEQ ID NO. 1 ) of the PSMA-specific chimeric antigen receptor (CAR), P28z.
A synthetic nanocarrier of any of embodiments 1 -18 wherein the polynucleotide is a plasmid, a minicircle plasmid, or an mRNA molecule.
A synthetic nanocarrier of any of embodiments 1 -19 wherein the targeting agent comprises a binding domain for a marker associated with an unwanted cell type. A synthetic nanocarrier of embodiment 20 wherein the unwanted cell type is a cancer cell, a virally infected cell, a bacterial cell, or a fungal cell.
A synthetic nanocarrier of embodiment 20 wherein the marker is a cancer antigen, a viral antigen, a bacterial antigen, or a fungal antigen.
A synthetic nanocarrier of embodiment 20 wherein the marker is a cancer antigen selected from A33; BAGE; Bcl-2; β-catenin; CA125; CA19-9; CD5; CD19; CD20; CD21 ; CD22; CD33; CD37; CD45; CD123; CEA; c-Met; CS-1 ; cyclin B1 ;
DAGE; EBNA; EGFR; ephrinB2; estrogen receptor; FAP; ferritin; folate-binding protein; GAGE; G250; GD-2; GM2; gp75, gp100 (Pmel 17); HER-2/neu; HPV E6; HPV E7; Ki-67; LRP; mesothelin, p53, PRAME; progesterone receptor; PSA; PSMA; MAGE; MART; mesothelin; MUC; MUM-1 -B; myc; NYESO-1 ; ras; RORI; survivin; tenascin; TSTA tyrosinase; VEGF; or WT1 .
A synthetic nanocarrier of embodiment 23 wherein the marker is PSMA.
A synthetic nanocarrier of embodiment 20 wherein the marker is a viral antigen selected from envelope glycoprotein B; CMV pp65; EBV; EBNAI; EBV; P18; EBV P23; S protein of hepatitis B; of M protein of hepatitis B; L proteins of hepatitis B; pre-S antigen of hepatitis B virus; HBCAG DELTA; HBV; HBE; hepatitis C viral RNA; HCV NS3; HCV NS4; herpes simplex immediate early proteins; glycoprotein D; HIV gp32; HIV gp41 ; HIV gp120; HIV gp160; HIV P17/24; HIV P24; HIV P55 GAG; HIV P66 POL; HIV TAT; HIV GP36; Nef protein; hemagglutinin; neuraminidase; Japanese encephalitis protein E; Japanese encephalitis protein M-E; Japanese encephalitis protein M-E-NS1 ; Japanese encephalitis protein NS1 ; Japanese encephalitis protein NS1 -NS2A; Japanese encephalitis protein 80% E; measles virus fusion protein; rabies glycoprotein; rabies nucleoprotein; RSV fusion protein; M2 protein; VP7sc; rubella protein E1 ; rubella protein E2; gpl; gpll; Nef (66-97); Nef (1 16-145); Gag p17 (17-35); Gag p17-p24 (253-284); and Pol 325-355 (RT 158-188).
A synthetic nanocarrier of embodiment 20 wherein the marker is a bacterial antigen selected from anthrax protective antigen; lipopolysaccharide; capsular polysaccharide; diptheria toxin; mycolic acid; heat shock protein 65 (HSP65); the 30 kDa major secreted protein; antigen 85A; hemagglutinin; pertactin; FIM2; FIM3; adenylate cyclase; pneumolysin; pneumococcal capsular polysaccharide; rompA; M proteins; tetanus toxin; lipoteichoic acid; and clumping factor A (ClfA). A synthetic nanocarrier of embodiment 20 wherein the marker is a fungal antigen selected from spherule antigens; capsular polysaccharides; heat shock protein 60; gp63; lipophosphoglycan; merozoite surface antigens; sporozoite surface antigens; circumsporozoite antigens; gametocyte/gamete surface antigens; blood-stage antigen pf 155/RESA; glutathione-S-transferase;
paramyosin; trichophytin; SAG-1 ; p30; trypanosoma cruzi 75-77 kDa antigen; and trypanosoma cruzi 56 kDa antigen.
A synthetic nanocarrier of any of embodiments 1 -19 wherein the targeting agent comprises a binding domain for a marker associated with a wanted cell type. A synthetic nanocarrier of embodiment 28 wherein the wanted cell type is a cell associated with an autoimmune disorder, a cell associated with an allergy, or a bacterial cell.
A synthetic nanocarrier of embodiment 28 wherein the marker is an autoimmune antigen, an allergic antigen, or a bacterial antigen.
A synthetic nanocarrier of embodiment 30 wherein the marker is an autoimmune antigen selected from glutamic acid decarboxylase 65 (GAD 65); native DNA; myelin basic protein; myelin proteolipid protein; acetylcholine receptor components; thyroglobulin; and thyroid stimulating hormone (TSH) receptor.
A synthetic nanocarrier of embodiment 30 wherein the marker is an allergic antigen selected from Japanese cedar pollen antigens; ragweed pollen antigens; rye grass pollen antigens; dust mite antigens; feline antigens; and canine antigens.
A synthetic nanocarrier of any of embodiments 1 -32 wherein the targeting agent is a surface antigen receptor or a receptor for an intracellular antigen presented by a Major Histocompatibility Complex antigen-presenting pathway.
A synthetic nanocarrier of any of embodiments 1 -33 wherein the targeting agent is an antigen receptor or a chimeric antigen receptor.
A synthetic nanocarrier of embodiment 34 wherein the targeting agent is a P28z chimeric antigen receptor.
A synthetic nanocarrier of embodiment 34 wherein the marker is CD19.
A synthetic nanocarrier of embodiment 36 wherein the targeting agent is monoclonal antibody FMC63.
A synthetic nanocarrier of embodiment 36 wherein the targeting agent is a human or humanized scFv comprising a variable light chain comprising a CDRL1 sequence of RASQDISKYLN (SEQ ID NO. 14), CDRL2 sequence of SRLHSGV
(SEQ ID NO. 15), and a CDRL3 sequence of GNTLPYTFG (SEQ ID NO. 16). A synthetic nanocarner of embodiment 36 wherein the targeting agent is a human or humanized scFv comprising a variable heavy chain comprising CDRHI sequence of DYGVS (SEQ ID NO. 17), CDRH2 sequence of VTWGSETTYYNSALKS (SEQ ID NO. 18), and a CDRH3 sequence of YAMDYWG (SEQ ID NO. 19).
A synthetic nanocarrier of embodiment 34 wherein the marker is RORI.
A synthetic nanocarrier of embodiment 40 wherein the targeting agent is a human or humanized scFv comprising a variable light chain comprising a CDRL1 sequence of ASGFDFSAYYM (SEQ ID NO. 22), CDRL2 sequence of TIYPSSG (SEQ ID NO. 23), and a CDRL3 sequence of ADRATYFCA (SEQ ID NO. 24).
A synthetic nanocarrier of embodiment 40 wherein the targeting agent is a human or humanized scFv comprising a variable heavy chain comprising CDRH1 sequence of DTIDWY (SEQ ID NO. 25), CDRH2 sequence of VQSDGSYTKRPGVPDR (SEQ ID NO. 26), and a CDRH3 sequence of YIGGYVFG (SEQ ID NO. 27).
A synthetic nanocarrier of any one of embodiments 1 -42 wherein the synthetic nanocarrier further comprises an endosomal release agent.
A synthetic nanocarrier of any one of embodiments 43 wherein the endosomal release agent extends from the outer surface of the coating.
A synthetic nanocarrier of embodiments 43 or 44 wherein the endosomal release agent is selected from any one of SEQ ID NOs. 29-50 or combinations thereof. A synthetic nanocarrier of any of embodiments 1 -45 wherein the polynucleotide is associated with a nuclear localization signal (NLS).
A synthetic nanocarrier of any one of embodiments 46 wherein the NLS is within a pore of the porous nanoparticle.
A synthetic nanocarrier of embodiments 46 or 47 wherein the NLS is selected from any one of SEQ ID NOs. 51 -93 or combinations thereof.
A synthetic nanocarrier of any one of embodiments 1 -48 comprising a S/MAR element, a PiggyBac transposase-containing plasmid, a Sleeping Beauty transposase-containing plasmid; a homo sapiens transposon-derived Busterl
transposase-like protein gene; a human endogenous retrovirus H protease/integrase-derived ORF1 ; a homo sapiens Cas-Br-M (murine) ecotropic retroviral transforming sequence; a homo sapiens endogenous retroviral sequence K; a homo sapiens endogenous retroviral family W sequence; a homo sapiens LINE-1 type transposase domain; or a homo sapiens pogo transposable element.
A composition comprising a synthetic nanocarrier of any one of embodiments 1 - 49.
A method of treating a subject having a condition associated with a cell type comprising: administering a therapeutically effective amount of a synthetic nanocarrier of any one of embodiments 1 -49 to the subject thereby treating the subject.
A method of treating a subject having a condition associated with a cell type comprising: administering a therapeutically effective amount of a composition of embodiment 50 to the subject thereby treating the subject.
A method of embodiments 51 or 52 wherein the cell type is an unwanted cell type selected from a cancer cell, a virally infected cell, a bacterial cell, or a fungal cell. A method of embodiment 53 wherein the unwanted cell type is a cancer cell selected from an adrenal cancer cell, a bladder cancer cell, a blood cancer cell, a bone cancer cell, a brain cancer cell, a breast cancer cell, a carcinoma cell, a cervical cancer cell, a colon cancer cell, a colorectal cancer cell, a corpus uterine cancer cell, an ear, nose and throat (ENT) cancer cell, an endometrial cancer cell, an esophageal cancer cell, a gastrointestinal cancer cell, a head and neck cancer cell, a Hodgkin's disease cell, an intestinal cancer cell, a kidney cancer cell, a larynx cancer cell, a leukemia cell, a liver cancer cell, a lymph node cancer cell, a lymphoma cell, a lung cancer cell, a melanoma cell, a mesothelioma cell, a myeloma cell, a nasopharynx cancer cell, a neuroblastoma cell, a non-Hodgkin's lymphoma cell, an oral cancer cell, an ovarian cancer cell, a pancreatic cancer cell, a penile cancer cell, a pharynx cancer cell, a prostate cancer cell, a rectal cancer cell, a sarcoma cell, a seminoma cell, a skin cancer cell, a stomach cancer cell, a teratoma cell, a testicular cancer cell, a thyroid cancer cell, a
uterine cancer cell, a vaginal cancer cell, or a vascular tumor cell.
A method of any one of embodiments 51 -54 wherein the administering results in expression of the polynucleotide selectively by lymphocytes within 10 days; within 9 days; within 8 days; within 7 days; within 6 days; within 5 days; within 4 days; or within 3 days of administration.
A method for treating a disease associated with an antigen, the method comprising: administering to a subject in need thereof, a composition comprising a therapeutically effective amount of nanocarriers including a polynucleotide having a sequence that encodes a receptor for the antigen, thereby treating the disease.
A method of embodiment 56 wherein after the administering the nanocarriers are selectively incorporated into lymphocytes in the subject such that the lymphocytes express the receptor and subsequently bind to the antigen on cells associated with the disease thereby killing the cells.
A method for treating a disease associated with an antigen, the method comprising:
obtaining lymphocytes from a subject in need thereof;
combining the lymphocytes with a composition comprising nanocarriers including a polynucleotide having a sequence that encodes a receptor for the antigen, wherein the nanocarriers are selectively incorporated into the lymphocytes such that the lymphocytes express the receptor; and
administering the lymphocytes expressing the receptor to the subject, thereby treating the disease.
A method of embodiment 58 wherein after the administering, the lymphocytes bind to the antigen on cells associated with the disease thereby killing the cells. A method of selectively transfecting lymphocytes in vivo, the method comprising: contacting lymphocytes with nanocarriers comprising a polynucleotide having a sequence that encodes a receptor for an antigen, wherein the nanocarriers are selectively incorporated into the lymphocyte to release the polynucleotide such that the lymphocyte expresses the receptor, thereby transfecting the lymphocyte. A method of any one of embodiments 51 -60 wherein the antigen comprises a
tumor antigen.
62. A method of any one of embodiments 51 -60 wherein the antigen comprises a viral antigen.
63. A method of any one of embodiments 51 -62 wherein the lymphocytes comprise T-cells, NK cells, macrophages, monocytes, B cells, hematopoietic stem cells, or a combination thereof.
64. A method of embodiment 63 wherein the lymphocytes comprise T-cells.
65. A method of any one of embodiment 56 wherein the disease is a cancer.
66. A method of embodiment 65 wherein the cancer comprises a leukemia, a lymphoma, a carcinoma, a sarcoma, or a melanoma.
67. A method of embodiment 65 wherein the disease is prostate cancer.
68. A method of embodiment 62 wherein the antigen is expressed by virus-infected cells associated with the disease.
[00164] Each of the exemplary embodiments in Set 1 and Set 2 also includes an embodiment wherein the lymphocyte-directing agent can be removed. These embodiments are especially useful when the selected cell types are monocytes/macrophages and broad non-specific uptake of the nanocarriers can be expected.
EXAMPLES
[00165] The Examples below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.
[00166] Example 1 . This example demonstrates that synthetic nanoparticles containing TCR genes can be used to generate functional tumor- or virus-specific T- cells. Lipid nanoparticles (Fig. 2A) were loaded with a minicircle gene (Fig. 3) encoding the chimeric antigen receptor P28z. P28z is a fusion receptor composed of a single- chain antibody (scFv) specific for the extracellular domain of PSMA (J591 ) combined with CD28 and ΟΌ3ζ cytoplasmic signaling domains (Fig. 4A; SEQ ID NO. 94). In this Example, chimeric antigen receptors (CARs) are fusion receptors including an antigen-
binding domain, a transmembrane domain and an intracellular signaling domain resulting in T-cell activation after antigen binding. The P28z CAR directs T-cells toward the prostate-specific membrane antigen (PSMA), which is highly expressed on prostate cancer cells. Therefore, the introduction of the P28z gene into T-cells renders them capable of recognizing and lysing prostate tumor. The P28z gene was cloned under the control of the T-cell specific promoter CD3 delta into a minicircle plasmid. Minicircles can include episomal DNA vectors that are produced as circular expression cassettes devoid of any bacterial plasmid DNA backbone. Their smaller molecular size can enable more efficient transfections and offers sustained expression over a period of weeks as compared to standard plasmid vectors that only work for a few days.
[00167] The minicircle plasmid DNA was entrapped into nanocarriers. DOPC, DOPE, cholesterol, and 18:1 PEG 2000 PE lipids were first mixed in a 55:5:30:10 mass ratio, dried under a stream of nitrogen, and placed in a vacuum oven overnight to remove residual chloroform. The lipid film was then dissolved in tert-butanol and mixed 1 :1 (v/v) with a P28z minicircle plasmid solution (diluted in 10 mM Tris-HCI (pH 7.4) with 0.85% (w/v) NaCI and 0.25 M sucrose) such that the final DOPC:DNA ratio was 10:1 (w/w). The mixture was vortexed and passed through a 100 nm filter at least 10 times using a Mini-Extruder set (Avanti Polar Lipids, Inc.; Alabaster, AL, USA).
[00168] To target nanocarriers to T-cells, anti-mouse CD8 antibodies were coupled to the surface of the lipid envelope. Anti-CD8 antibodies (10 mg/ml) were mildly reduced with a 25X molar excess of DTT for 20 min at 25°C in the presence of 10 mM EDTA in PBS to expose free hinge region thiols. To remove DTT, antibodies were passed through a desalting column. The heterobifunctional cross-linker SM(PEG)24 was used to anchor antibodies to the surface of DNA-loaded liposomes (Amine groups are present in the head groups of PE lipids, free thiol groups on antibodies were created by DTT, SM(PEG) 24 cross-links between amines and thiol groups). Liposomes were first incubated with a tenfold molar excess of SM(PEG)24 for 2 h at room temperature and centrifuged to remove unreacted cross-linker. Activated liposomes were then incubated with a fivefold molar excess of reduced anti-CD8 antibody for 2 h at room temperature. Unbound antibody was removed using a centrifugal filtration device (10 kDa MWCO). The final liposome used for subsequent experiments were ~ 100 nm in diameter.
[00169] P28z gene transfer into T-cells using targeted DNA nanocarriers renders them capable of lysing prostate tumor. The transfection efficiency of liposome-mediated gene transfer into primary T-cells was assessed. 60 x 106 mouse effector CD8+ T-cells ml_"1 were resuspended in RPMI medium and an equal volume of lipid nanoparticles (loaded with P28z minicircle DNA) were added with a 100 particles/T-cell ratio. Cells were incubated at 37°C for 30 min with gentle agitation every 10 min and unbound particles were removed by a PBS wash. Two days later, the percentage of T-cells expressing the P28z CAR was determined by flow cytometry. Thirty hours after transfection -23% of the cells expressed the P28z receptor on their surface (Fig. 4B). High P28z expression persisted for three days in vitro before declining toward undetectable expression by eight days (data not shown). Nanoparticle-transfected T cells were functional, selectively lysing PSMA-expressing TRAMP prostate tumor cells (Fig. 4C,D).
[00170] Example 2. CD3-targeted protocell nanoparticles selectively bind circulating T cells in mice. A goal of the current disclosure is to selectively and quickly edit lymphocyte specificity in vivo to target unwanted cells. To examine how selectively protocells bind circulating host T cells, mice were systemically injected with 1 x 1011 fluorescently tagged nanoparticles. After 6 hours peripheral blood was collected by retro-orbital puncture and the percentage of fluorescent T cells was quantified by flow cytometry. CD3-targeted protocells labeled the majority of T cells in the blood, with relatively low binding to off-target cells (Fig. 4E, left panel). Confocal imaging of sorted T cells showed that nanoparticles are rapidly internalized from the cell surface into the cytoplasm as a result of receptor-induced endocytosis (Fig. 4E, right panel).
[00171] Example 3. Generating an orthotopic bioluminescent mouse model for analyzing treatment of metastatic prostate cancer. Male TRAMP transgenic mice spontaneously develop orthotopic prostate tumors following puberty. However, unlike human prostate adenocarcinoma, TRAMP tumors do not express significant amounts of PSMA, a target in experiments using the P28z CAR. Furthermore, longitudinal studies to measure the prostate cancer volume in TRAMP animals rely on expensive and time- consuming magnetic resonance imaging (MRI) techniques, which preclude analysis of large cohorts of mice. To overcome these issues, a cell line from a primary TRAMP
tumor was established and the PSMA gene was introduced through retroviral transduction. To serially monitor tumor burden by bioluminescence imaging, tumor cells were also genetically tagged with Firefly luciferase (FLuc). Following orthotopic transplantation into the dorsal lobe of the prostate gland of C57BL/6 mice, TRAMP- PSMA-FLuc tumor cells reproducibly developed into lesions within three weeks, with all animals displaying progressive metastatic tumor spread to regional (pelvic, paraaortic) lymph nodes (Fig. 5).
[00172] Example 4. The data shown in Figs. 2 and 4 establish the ability to generate nanoparticles that efficiently program T cells with genes encoding receptors specific for prostate tumor. While this strategy rapidly generates tumor-reactive T cells, expression of transgenes is transient because transferred plasmids are diluted out every time the lymphocyte divides. The current example evaluates persistent receptor gene expression in actively dividing T cells caused by inserting into the plasmid either: 1 ) a scaffold/matrix attachment region (S/MAR) sequence (which can undergo episomal self- replication), or 2) a transposable piggyBac element (which integrates the transgene into the genome). Stable and dependable transgene expression in dividing T cells will allow nanoparticle-transfected lymphocytes to serially kill unwanted cell types providing long- lived immunity against such cells.
[00173] In this Example, a S/MAR sequence (provided by Dr. Lipps, University Witten/Herdecke) or piggyBac inverted terminal repeats (provided by Dr. Craig, Johns Hopkins University) will be cloned into minicircle plasmids that encode the P28z receptor, as illustrated in Fig. 6. Protocell nanoparticles loaded with equivalent amounts of P28z, P28z-S/MAR, or P28z-piggyBac minicircle DNA will be incubated with mouse CD8+ T lymphocytes at a cell:particle ratio of 1 :10. Following nanoparticle transfection, T cells will be expanded with plate-bound anti-CD3/anti-CD28 antibodies. Flow cytometry will be used to assess P28z receptor expression levels and persistence in proliferating T cells every 24 hours during a two week culture period.
[00174] To investigate the extent to which S/MAR sequences or piggyBac transposable elements prevent nanoparticle-transferred plasmids from being lost by dilution in dividing T cells, the actual number of P28z gene copies per T cell over time will be quantified. To this end, genomic and low-molecular weight (episomal) DNA will
be isolated from transfected T cells at each time point during the two week period. Vector copy numbers will be measured by multiplex quantitative PCR (qPCR) with a set of primers and probes specific to the P28z minicircle plasmid. A set of primers specific to the gene encoding mouse albumin will be included as an internal two-copy control.
[00175] To discriminate between episomal (extrachromosomal) versus genome- integrated P28z transgenes, Southern blot analysis will be performed by digesting isolated DNA with Not1 . This restriction site is present only once in the P28z minicircle episome; it yields a 2.8-kb band for the extrachromosomal episome but yields fragments of various lengths for plasmids integrated into the genome.
[00176] The described studies will show that S/MAR-based episomes and piggyBac transposons are two highly efficient tools to modify cells to achieve stable gene expression. Incorporating S/MAR sequences or piggyBac transposable elements into nanocarrier-delivered plasmids will also maintain high-level P28z gene expression in T cells over weeks as a result of episomal self-replication or somatic integration, respectively. Because plasmids containing S/MAR elements do not integrate into the host genome, P28z gene expression is independent of chromosomal position effects and therefore not subject to epigenetic silencing and c/s-acting sequences.
[00177] Example 6. This Example determines that systemic injections of DNA nanocarriers can program sufficient quantities of T cells to target and eliminate disseminated prostate cancer. The tests will be conducted using nanoparticles loaded with minicircle DNA encoding the P28z CAR (described above), to generate PSMA- specific lymphocytes. The results of the studies will be positive following testing of the following questions: (1 ) how many peripheral T cells are genetically modified to express P28z following a single intravenous dose of CD3-targeting nanoparticles loaded with genes encoding the receptor?; (2) do the injected nanoparticles selectively edit the antigen-specificity of peripheral T cells without affecting off-target cells? And (3) what nanoparticle dosage is required to bring about T cell-mediated regression of metastatic prostate tumors in mice?
[00178] Example 6(1 ). What percentage of peripheral T cells are modified by nanoparticle gene therapy? The goal of this study is to edit the antigen specificity of at least 10% of peripheral T cells within five days following a single bolus injection of
nanocarriers. For comparison, some of the strongest vaccine vectors reported in the literature induce frequencies of self/tumor antigen-specific T cells of 1 -4% following repeated immunizations over weeks. Mice will be systemically injected with 1 x 109, 1 x 1010, or 1 x 101 1 nanocarriers loaded with minicircle DNA encoding P28z, or with GFP as a control. After collecting peripheral blood by retro-orbital puncture every four days over a 12-day period, the percentage of P28z+ T cells will be quantified by flow cytometry using fluorescent recombinant PSMA protein as the reporter, as performed in previous gene transfer studies (see, e.g., Fig. 4B).
[00179] Example 6(2). Does nanoparticle gene therapy edit the antigen specificity of peripheral T cells without affecting off-target cells? To confirm in vivo studies, showing that CD3-targeting protocells efficiently bind to host T cells after intravenous injection (Fig. 4E), how selectively nanoparticles introduce tumor-targeting receptor genes into circulating T cells will also be determined. To this end, P28z expression by other leukocyte subsets will be evaluated, using the samples obtained in Example 6(1 ). The other cell types will be identified using the following reporters: anti-CD8 and anti-CD4 (T-cell markers), anti-B220 (B-cell marker), anti-NK1 .1 (NK-cell marker), anti-CD1 15, anti-F4/80 and anti-CD1 1 b (monocyte markers), anti-Ly6G and anti-CD1 1 b (neutrophil markers), and anti-Gr-1 antibody (granulocyte marker).
[00180] Example 6(3). What nanoparticle dosage is required to bring about T cell- mediated regression of metastatic prostate tumors in mice? To develop a reproducibly effective treatment for metastatic prostate cancer, the therapeutically optimal frequency and dosage of nanocarrier injections must be determined. A test system will be created by injecting luciferase-expressing TRAMP-PSMA tumor cells into the prostate of C57BL/6 mice and allowing them to develop for three weeks before performing the tests (see, e.g., Fig. 5).
[00181] The mice will be systemically injected with CD3-targeting nanocarriers carrying P28z-encoding transgenes, according to four administration protocols: single high-dose bolus injection (1 x 1010 nanoparticles, i.v.); high-frequency high-dose injections (1 x 1010 nanoparticles, i.v. every 3 days for 30 days); single low-dose injection (1 x 109 nanoparticles, i.v.); or high-frequency low-dose injections (1 x 109 nanoparticles, i.v. every 3 days for 30 days). To compare the therapeutic efficacy of
nanoparticle infusions with conventional adoptive T-cell therapy, one additional group of mice will be treated with a single dose of 10 million T cells transduced ex vivo with P28z-encoding retroviral vectors. Differences in TRAMP-PSMA tumor progression will be measured between treatment and control groups using bioluminescence imaging. To correlate tumor regression with the concentration of nanoparticle-programmed T cells in the peripheral circulation, the percentage of P28z+ T cells in whole blood will be quantified by flow cytometry every 6 days.
[00182] The results will show that circulating T cells can be selectively programmed to target prostate tumors without genetically modifying other cells. This specificity can be achieved by coating the nanoparticles with CD3-recognizing antibodies, and by expressing the P28z transgene under the control of the T cell-specific CD3 delta promoter. If flow cytometry shows that more than 20% of P28z-expressing cells in the peripheral blood are not the targeted T cells, the density of anti-CD3 antibodies on the surface of nanocarriers will be increased to improve T cell targeting. If the CD3 delta promoter is too weak to mediate sufficient levels of receptor gene expression in vivo, the murine stem cell leukemia virus (MSCV) promoter can be used to express the P28z CAR in T cells. The MSCV promoter exhibits strong activity in hematopoietic cells and stem cells.
[00183] Example 7. Example 7 determines that nanocarriers can alternatively modify host T cells with prostate tumor-specific T-cell receptor (TCR) genes that target different antigens.
[00184] Gene transfer of DNA encoding CARs can only target T cells to antigens located on the surface of tumor cells, so the many tumor antigens that are intracellular are inaccessible to these receptors. However, after degradation in the proteasome these intracellular proteins are presented by major histocompatibility complex (MHC) molecules where they can be recognized by specific T cell receptors (TCRs).
[00185] A murine receptor (3D TCR) that has a high affinity for the intracellular oncoprotein Wilms tumor 1 (WT1 ) has been successfully engineered by a team of immunologists led by P. Greenberg at the Fred Hutchinson Cancer Research Center. WT1 was ranked first in a list of 75 cancer antigens in a recent National Cancer Institute prioritization project. It is strongly expressed in high-grade prostate tumor where it
promotes the formation of metastases, but is absent in non-neoplastic or benign prostatic hyperplasia tissues. In line with these studies, high WT1 gene expression was detected in the TRAMP prostate tumor cells used herein. WT1 is detected at only very low levels in other normal tissues, particularly hematopoietic stem cells and kidney podocytes. T cells have been shown to be capable of selectively recognizing transformed cells expressing high levels without toxicity to normal tissues. In Example 7 it will be shown that systemic injections of protocells loaded with genes encoding affinity-matured WT1 -specific TCRs can impart specificity for WT1 to host T cells and lead to elimination of prostate cancer.
[00186] To determine how efficiently nanocarriers transfect T cells with WT1 -TCR genes in vivo, mice will be injected with 1 x 1010 nanoparticles carrying 3D TCR genes. Control nanoparticles will be loaded with GFP-expressing plasmids. Peripheral blood collected by retro-orbital puncture every four days over a 12-day period will be used to quantify WT1 -TCR+ T cells and other leukocyte subsets by flow cytometry using a fluorescent conjugate of the WT1 -derived RMFPNAPYL epitope tetramer as the reporter.
[00187] To investigate whether nanoparticle injections can cause regression of metastatic prostate cancer in mice, luciferase-expressing TRAMP tumors will be implanted into the prostate of C57BL/6 mice. Three weeks later, animals will be treated with: a single high-dose bolus injection (1 x 1010 nanoparticles i.v.); high-frequency high- dose injections (1 x 1010 nanoparticles i.v. every 3 days for 30 days); a single low-dose injection (1 x 109 nanoparticles, i.v.); or high-frequency low-dose injections (1 x 109 nanoparticles, i.v. every 3 days for 30 days). To determine the therapeutic advantage of nanoparticle infusions over conventional adoptive T-cell therapy, one additional group of mice will be injected with 10 million T cells, which were ex vivo transduced with 3D TCR genes using retroviral vectors. Differences in TRAMP prostate tumor regression between treatment and control groups will be measured using bioluminescence imaging.
[00188] The strength of T cell responses in antitumor immunity can be decisively dependent on the quality of the TCRs involved. Due to thymic selection, the affinities of natural TCRs that target oncogenic self-proteins like WT1 are generally much lower
than those of typical virus-targeting TCRs. However, the ability of a naturally occurring TCR to recognize antigens like WT1 can be markedly enhanced by in vitro affinity maturation. Based on these data, if genes for an affinity-optimized, WT1 -specific TCR are introduced into circulating T cells using the disclosed nanoparticle gene therapy approach, T cells will effectively recognize and kill prostate cancer cells. 3D TCRs are fully functional in CD4+ and CD8+ T cells, and CD4+ T cells can directly mediate tumor destruction and/or provide cytokine help for CD8+ T cells; however, tumor-specific CD4+ regulatory T cells abrogate CD8 T cell-mediated tumor rejection. If CD3-targeted nanoparticles generate undesirable WT1 -specific CD4+ regulatory T cells, nanoparticles can be targeted to CD8+ T cells only. These studies will demonstrate that nanoparticles can deliver rationally engineered TCR genes into host T-cells and enable them to recognize intracellular tumor-associated antigen.
[00189] Example 8. Modifying host lymphocytes with HIV-specific TCR genes to control HIV infection. HIV-infected humanized NOD/shi-scid/yc null (NOG) mice with nanoparticles carrying HIV-gag protein-specific TCR transgenes, or with control plasmids expressing green fluorescent protein will be studied. Differences in HIV viral titers between treatment groups will be determined and administration of the nanoparticles will show a beneficial result.
[00190] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2nd Edition (1989); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (1987); the series Methods IN Enzymology (Academic Press, Inc.); M. MacPherson, et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991 ); MacPherson et al., eds. PCR 2: Practical Approach, (1995); Harlow and Lane, eds. Antibodies, A Laboratory Manual, (1988); and R. I. Freshney, ed. Animal Cell Culture (1987).
[00191] Sequence information provided by public database can be used to identify nucleic acid sequences encoding peptides disclosed herein and vice versa. Variants of the sequences disclosed and referenced herein are also included.
[00192] Variants of peptides can include those having one or more conservative amino acid substitutions. As used herein, a "conservative substitution" involves a substitution found in one of the following conservative substitutions groups: Group 1 : Alanine (Ala), Glycine (Gly), Serine (Ser), Threonine (Thr); Group 2: Aspartic acid (Asp), Glutamic acid (Glu); Group 3: Asparagine (Asn), Glutamine (Gin); Group 4: Arginine (Arg), Lysine (Lys), Histidine (His); Group 5: Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Val); and Group 6: Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp).
[00193] Additionally, amino acids can be grouped into conservative substitution groups by similar function or chemical structure or composition (e.g., acidic, basic, aliphatic, aromatic, sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Val, Leu, and lie. Other groups containing amino acids that are considered conservative substitutions for one another include: sulfur- containing: Met and Cysteine (Cys); acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, lie, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information is found in Creighton (1984) Proteins, W.H. Freeman and Company.
[00194] Variants of the protein and nucleic acid sequences disclosed or referenced herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to he protein and nucleic acid sequences disclosed or referenced herein.
[00195] "% sequence identity" refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between proteins or nucleic acid sequences as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including (but not limited to) those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data,
Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151 -153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 1 1 1 -20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.. Within the context of this disclosure it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. As used herein "default values" will mean any set of values or parameters, which originally load with the software when first initialized.
[00196] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. As used herein, the transition term "comprise" or "comprises" means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase "consisting of excludes any element, step, ingredient or component not specified. The transition phrase "consisting essentially of limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. As used herein, a material effect would
cause a statistically-significant reduction in the ability of a nanocarrier to reduce the number of an unwanted cell type and/or to protect a wanted cell type in vivo.
[00197] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term "about" has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±1 1 % of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1 % of the stated value.
[00198] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[00199] The terms "a," "an," "the" and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling
within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[00200] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
[00201] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above- described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
[00202] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference for their particular cited teachings.
[00203] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.
[00204] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and/or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
[00205] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the following examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).
Claims
1 . A synthetic nanocarrier comprising (i) a lipid-coated porous nanoparticle (ii) a lymphocyte-directing agent extending from the surface of the lipid-coated porous nanoparticle; and (iii) a polynucleotide encoding a chimeric antigen receptor (CAR) targeting agent within the pores of the lipid-coated porous nanoparticle.
2. A synthetic nanocarrier of embodiment 1 further comprising an endosomal release agent extending from the surface of the lipid-coated porous nanoparticle and (ii) a nuclear localization signal (NLS) within the pores of the lipid-coated porous nanoparticle.
3. A synthetic nanocarrier of claim 1 wherein the CAR is P28z.
4. A synthetic nanocarrier of claim 1 wherein the lipid coating is a liposome, a lipid bilayer, or a polymeric micelle.
5. A synthetic nanocarrier of claim 1 wherein the synthetic nanocarriers comprise liposomes, polymeric particles, metallic particles, polymeric micelles,
polyethyleneimine (PEI)/DNA complexes, or a combination thereof.
6. A synthetic nanocarrier of claim 1 wherein the lipid coating encapsulates the lipid-coated porous nanoparticle.
7. A synthetic nanocarrier of claim 1 wherein the lymphocyte-directing agent selectively binds to lymphocytes in vivo.
8. A synthetic nanocarrier of claim 1 wherein the lymphocyte-directing agent comprises a binding domain selected from a lymphocyte receptor ligand, lymphocyte receptor antibody, lymphocyte receptor peptide aptamer, lymphocyte receptor nucleic acid aptamer, lymphocyte receptor spiegelmer, or a combination thereof.
9. A synthetic nanocarrier of claim 1 wherein the lymphocyte-directing agent selectively binds T cells, NK cells, monocytes, macrophages, B cells, hematopoietic stem cells, or a combination thereof.
10. A synthetic nanocarrier of claim 1 wherein the lymphocyte-directing agent selectively binds T-cell receptor motifs; T-cell a chains; T-cell β chains; T-cell γ chains; T-cell Δ chains; CCR7; CD3; CD4; CD5; CD7; CD8; CD1 1 b; CD1 1 c; CD16; CD19; CD20; CD21 ; CD22; CD25; CD28; CD34; CD35; CD40; CD45RA;
CD45RO; CD52; CD56; CD62L; CD68; CD80; CD95; CD1 17; CD127; CD133; CD137 (4-1 BB); CD163; F4/80; IL-4Ra; Sca-1 ; CTLA-4; GITR; GARP; LAP; granzyme B; LFA-1 ; or transferrin receptor.
1 1 . A synthetic nanocarrier of claim 1 wherein the lymphocyte-directing agent selectively binds CCR7; CD3; CD4; CD5; CD8; CD16; CD19; CD20; CD21 ; CD22; CD25; CD28; CD35; CD40; CD45RA; CD45RO; CD52; CD62L; CD80; CD95; CD127; or CD137.
12. A synthetic nanocarrier of claim 1 wherein the lymphocyte-directing agent comprises a binding domain selected from a T-cell a chain antibody; T-cell β chain antibody; T-cell γ chain antibody; T-cell δ chain antibody; CCR7 antibody; CD3 antibody; CD4 antibody; CD5 antibody; CD7 antibody; CD8 antibody; CD1 1 b antibody; CD1 1 c antibody; CD16 antibody; CD19 antibody; CD20 antibody; CD21 antibody; CD22 antibody; CD25 antibody; CD28 antibody; CD34 antibody; CD35 antibody; CD40 antibody; CD45RA antibody; CD45RO antibody; CD52 antibody; CD56 antibody; CD62L antibody; CD68 antibody; CD80 antibody; CD95 antibody; CD1 17 antibody; CD127 antibody; CD133 antibody; CD137 (4-1 BB) antibody; CD163 antibody; F4/80 antibody; IL-4Ra antibody; Sca-1 antibody; CTLA-4 antibody; GITR antibody GARP antibody; LAP antibody; granzyme B antibody; LFA-1 antibody; or transferrin receptor antibody.
13. A synthetic nanocarrier of claim 12 wherein the binding domain consists of or consists essentially of an scFv fragment of a T-cell a chain antibody; T-cell β chain antibody; T-cell γ chain antibody; T-cell δ chain antibody; CCR7 antibody; CD3 antibody; CD4 antibody; CD5 antibody; CD7 antibody; CD8 antibody; CD1 1 b antibody; CD1 1 c antibody; CD16 antibody; CD19 antibody; CD20 antibody; CD21 antibody; CD22 antibody; CD25 antibody; CD28 antibody; CD34 antibody; CD35 antibody; CD40 antibody; CD45RA antibody; CD45RO antibody; CD52 antibody; CD56 antibody; CD62L antibody; CD68 antibody; CD80 antibody; CD95 antibody; CD1 17 antibody; CD127 antibody; CD133 antibody; CD137 (4-1 BB) antibody; CD163 antibody; F4/80 antibody; IL-4Ra antibody; Sca-1 antibody; CTLA-4 antibody; GITR antibody GARP antibody; LAP antibody; granzyme B antibody; LFA-1 antibody; or transferrin receptor antibody.
14. A synthetic nanocarner of claim 13 wherein the binding domain consists of consists of or consists essentially of the scFv fragment (SEQ ID NO. 1 ) of the PSMA-specific chimeric antigen receptor (CAR), P28z.
15. A synthetic nanocarrier of claim 1 wherein the polynucleotide is a plasmid, a minicircle plasmid, or an mRNA molecule.
16. A synthetic nanocarrier of claim 1 wherein the CAR targeting agent comprises a binding domain for a marker associated with an unwanted cell type.
17. A synthetic nanocarrier of claim 16 wherein the unwanted cell type is a cancer cell.
18. A synthetic nanocarrier of claim 16 wherein the marker is a cancer antigen.
19. A synthetic nanocarrier of claim 16 wherein the marker is a cancer antigen selected from A33; BAGE; Bcl-2; β-catenin; CA125; CA19-9; CD5; CD19; CD20; CD21 ; CD22; CD33; CD37; CD45; CD123; CEA; c-Met; CS-1 ; cyclin B1 ; DAGE; EBNA; EGFR; ephrinB2; estrogen receptor; FAP; ferritin; folate-binding protein; GAGE; G250; GD-2; GM2; gp75, gp100 (Pmel 17); HER-2/neu; HPV E6; HPV E7; Ki-67; LRP; mesothelin, p53, PRAME; progesterone receptor; PSA; PSMA; MAGE; MART; mesothelin; MUC; MUM-1 -B; myc; NYESO-1 ; ras; RORI; survivin; tenascin; TSTA tyrosinase; VEGF; or WT1 .
20. A synthetic nanocarrier of claim 16 wherein the marker is PSMA.
21 .A synthetic nanocarrier of claim 1 wherein the CAR targeting agent is a surface antigen receptor or a receptor for an intracellular antigen presented by a Major Histocompatibility Complex antigen-presenting pathway.
22. A synthetic nanocarrier of claim 2 wherein the endosomal release agent is selected from any one of SEQ ID NOs. 29-50 or combinations thereof.
23. A synthetic nanocarrier of claim 2 wherein the NLS is selected from any one of SEQ ID NOS. 51 -93 or combinations thereof.
24. A synthetic nanocarrier of claim 1 comprising a S/MAR element, a PiggyBac transposase-containing plasmid, a Sleeping Beauty transposase-containing plasmid; a homo sapiens transposon-derived Busterl transposase-like protein gene; a human endogenous retrovirus H protease/integrase-derived ORF1 ; a homo sapiens Cas-Br-M (murine) ecotropic retroviral transforming sequence; a
homo sapiens endogenous retroviral sequence K; a homo sapiens endogenous retroviral family W sequence; a homo sapiens LINE-1 type transposase domain; or a homo sapiens pogo transposable element.
25. A composition comprising a synthetic nanocarrier of claim 1 .
26. A method of treating a subject having a condition associated with a cell type comprising: administering a therapeutically effective amount of a synthetic nanocarrier of claim 1 to the subject thereby treating the subject.
27. A method of treating a subject having a condition associated with an unwanted cell type comprising: administering a therapeutically effective amount of a composition of claim 25 to the subject thereby treating the subject.
28. A method of claim 27 wherein the unwanted cell type is an unwanted cancer cell.
29. A method of claim 28 wherein the unwanted cancer cell is selected from an adrenal cancer cell, a bladder cancer cell, a blood cancer cell, a bone cancer cell, a brain cancer cell, a breast cancer cell, a carcinoma cell, a cervical cancer cell, a colon cancer cell, a colorectal cancer cell, a corpus uterine cancer cell, an ear, nose and throat (ENT) cancer cell, an endometrial cancer cell, an esophageal cancer cell, a gastrointestinal cancer cell, a head and neck cancer cell, a Hodgkin's disease cell, an intestinal cancer cell, a kidney cancer cell, a larynx cancer cell, a leukemia cell, a liver cancer cell, a lymph node cancer cell, a lymphoma cell, a lung cancer cell, a melanoma cell, a mesothelioma cell, a myeloma cell, a nasopharynx cancer cell, a neuroblastoma cell, a non-Hodgkin's lymphoma cell, an oral cancer cell, an ovarian cancer cell, a pancreatic cancer cell, a penile cancer cell, a pharynx cancer cell, a prostate cancer cell, a rectal cancer cell, a sarcoma cell, a seminoma cell, a skin cancer cell, a stomach cancer cell, a teratoma cell, a testicular cancer cell, a thyroid cancer cell, a uterine cancer cell, a vaginal cancer cell, or a vascular tumor cell.
30. A method of claim 26 wherein the administering results in expression of the polynucleotide selectively by lymphocytes within 10 days; within 9 days; within 8 days; within 7 days; within 6 days; within 5 days; within 4 days; or within 3 days of administration.
31 . A method for treating a disease associated with an antigen, the method
comprising: administering to a subject in need thereof, a composition comprising a therapeutically effective amount of nanocarriers including a polynucleotide having a sequence that encodes a receptor for the antigen, thereby treating the disease.
32. A method of claim 31 wherein after the administering the nanocarriers are selectively incorporated into lymphocytes in the subject such that the lymphocytes express the receptor and subsequently bind to the antigen on cells associated with the disease thereby killing the cells.
33. A method for treating a disease associated with an antigen, the method comprising:
obtaining lymphocytes from a subject in need thereof;
combining the lymphocytes with a composition comprising nanocarriers including a polynucleotide having a sequence that encodes a receptor for the antigen, wherein the nanocarriers are selectively incorporated into the lymphocytes such that the lymphocytes express the receptor; and
administering the lymphocytes expressing the receptor to the subject, thereby treating the disease.
34. A method of claim 33 wherein after the administering, the lymphocytes bind to the antigen on cells associated with the disease thereby killing the cells.
35. A method of selectively transfecting lymphocytes in vivo, the method comprising: contacting lymphocytes with nanocarriers comprising a polynucleotide having a sequence that encodes a receptor for an antigen associated with a disease, wherein the nanocarriers are selectively incorporated into the lymphocyte to release the polynucleotide such that the lymphocyte expresses the receptor, thereby transfecting the lymphocyte.
36. A method of claim 35 wherein the antigen comprises a tumor antigen.
37. A method of claim 35 wherein the antigen comprises a viral antigen.
38. A method of claim 35 wherein the lymphocytes comprise T-cells, NK cells, macrophages, monocytes, B cells, hematopoietic stem cells, or a combination thereof.
39. A method of claim 38 wherein the lymphocytes comprise T-cells.
40. A method of claim 35 wherein the disease is a cancer.
41 . A method of claim 40 wherein the cancer comprises a leukemia, a lymphoma, a carcinoma, a sarcoma, or a melanoma.
42. A method of a claim 40 wherein the disease is prostate cancer.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14/776,661 US20160145348A1 (en) | 2013-03-14 | 2014-03-14 | Compositions and methods to modify cells for therapeutic objectives |
EP14768529.1A EP2970985A1 (en) | 2013-03-14 | 2014-03-14 | Compositions and methods to modify cells for therapeutic objectives |
US15/672,106 US10392446B2 (en) | 2013-03-14 | 2017-08-08 | Compositions and methods to modify cells for therapeutic objectives |
US16/510,646 US20190330373A1 (en) | 2013-03-14 | 2019-07-12 | Compositions and methods to modify cells for therapeutic objectives |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201361785907P | 2013-03-14 | 2013-03-14 | |
US61/785,907 | 2013-03-14 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/776,661 A-371-Of-International US20160145348A1 (en) | 2013-03-14 | 2014-03-14 | Compositions and methods to modify cells for therapeutic objectives |
US15/672,106 Continuation US10392446B2 (en) | 2013-03-14 | 2017-08-08 | Compositions and methods to modify cells for therapeutic objectives |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2014153114A1 true WO2014153114A1 (en) | 2014-09-25 |
Family
ID=51581420
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2014/029137 WO2014153114A1 (en) | 2013-03-14 | 2014-03-14 | Compositions and methods to modify cells for therapeutic objectives |
Country Status (3)
Country | Link |
---|---|
US (3) | US20160145348A1 (en) |
EP (1) | EP2970985A1 (en) |
WO (1) | WO2014153114A1 (en) |
Cited By (34)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016113203A1 (en) * | 2015-01-12 | 2016-07-21 | Pieris Ag | Engineered t cells and uses therefor |
WO2017050884A1 (en) * | 2015-09-22 | 2017-03-30 | Julius-Maximilians-Universität Würzburg | A method for high level and stable gene transfer in lymphocytes |
WO2017181110A1 (en) | 2016-04-14 | 2017-10-19 | Fred Hutchinson Cancer Research Center | Compositions and methods to program therapeutic cells using targeted nucleic acid nanocarriers |
US10188749B2 (en) | 2016-04-14 | 2019-01-29 | Fred Hutchinson Cancer Research Center | Compositions and methods to program therapeutic cells using targeted nucleic acid nanocarriers |
US10392446B2 (en) | 2013-03-14 | 2019-08-27 | Fred Hutchinson Cancer Research Center | Compositions and methods to modify cells for therapeutic objectives |
JP2019527553A (en) * | 2016-07-29 | 2019-10-03 | ジュノー セラピューティクス インコーポレイテッド | Anti-idiotype antibody against anti-CD19 antibody |
CN110526976A (en) * | 2018-05-25 | 2019-12-03 | 深圳宾德生物技术有限公司 | It is a kind of to target the single-chain antibody of PSMA, Chimeric antigen receptor T cell and its preparation method and application |
US10857181B2 (en) | 2015-04-21 | 2020-12-08 | Enlivex Therapeutics Ltd | Therapeutic pooled blood apoptotic cell preparations and uses thereof |
EP3689336A4 (en) * | 2017-09-14 | 2021-01-20 | Shanghai Jiao Tong University | Multicellular targeting liposome |
WO2021014368A1 (en) * | 2019-07-23 | 2021-01-28 | Kabushiki Kaisha Toshiba | Method of producing car-t cells, nucleic acid-introducing carrier and kit |
US11000548B2 (en) | 2015-02-18 | 2021-05-11 | Enlivex Therapeutics Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
US11013764B2 (en) | 2019-04-30 | 2021-05-25 | Myeloid Therapeutics, Inc. | Engineered phagocytic receptor compositions and methods of use thereof |
CN112912727A (en) * | 2018-08-31 | 2021-06-04 | 因维克蒂斯股份有限公司 | Methods of assessing CAR functionality |
US11034749B2 (en) | 2015-07-28 | 2021-06-15 | The Trustees Of The University Of Pennsylvania | Modified monocytes/macrophage expressing chimeric antigen receptors and uses thereof |
US20210253696A1 (en) * | 2020-01-21 | 2021-08-19 | Cero Therapeutics, Inc. | Bivalent chimeric engulfment receptors and uses thereof |
US20220008473A1 (en) * | 2019-06-27 | 2022-01-13 | Crispr Therapeutics Ag | Use of chimeric antigen receptor t cells and nk cell inhibitors for treating cancer |
US11304976B2 (en) | 2015-02-18 | 2022-04-19 | Enlivex Therapeutics Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
US11312939B2 (en) | 2020-06-04 | 2022-04-26 | Carisma Therapeutics Inc. | Constructs for chimeric antigen receptors |
US11318163B2 (en) | 2015-02-18 | 2022-05-03 | Enlivex Therapeutics Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
US20220204935A1 (en) * | 2015-04-29 | 2022-06-30 | Fred Hutchinson Cancer Research Center | Modified hematopoietic stem/progenitor and non-t effector cells, and uses thereof |
US11497773B2 (en) * | 2020-09-23 | 2022-11-15 | Crispr Therapeutics Ag | Genetically engineered t cells with regnase-1 and/or TGFBRII disruption have improved functionality and persistence |
US11497767B2 (en) | 2015-02-18 | 2022-11-15 | Enlivex Therapeutics R&D Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
US11512289B2 (en) | 2015-02-18 | 2022-11-29 | Enlivex Therapeutics Rdo Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
US11517589B2 (en) | 2015-02-19 | 2022-12-06 | Myeloid Therapeutics, Inc. | Chimeric antigen receptor dendritic cell (CAR-DC) for treatment of cancer |
US11530264B2 (en) * | 2016-12-29 | 2022-12-20 | Shenzhen Beike Biotechnology Co., Ltd | Multifunctional protein |
US11566061B2 (en) | 2017-01-05 | 2023-01-31 | Fred Hutchinson Cancer Center | Systems and methods to improve vaccine efficacy |
US11584793B2 (en) | 2015-06-24 | 2023-02-21 | Hoffmann-La Roche Inc. | Anti-transferrin receptor antibodies with tailored affinity |
US11596652B2 (en) | 2015-02-18 | 2023-03-07 | Enlivex Therapeutics R&D Ltd | Early apoptotic cells for use in treating sepsis |
US11603411B2 (en) | 2015-10-02 | 2023-03-14 | Hoffmann-La Roche Inc. | Bispecific anti-human CD20/human transferrin receptor antibodies and methods of use |
US11628218B2 (en) | 2020-11-04 | 2023-04-18 | Myeloid Therapeutics, Inc. | Engineered chimeric fusion protein compositions and methods of use thereof |
US11634497B2 (en) | 2017-02-03 | 2023-04-25 | Novartis Ag | Anti-CCR7 antibody drug conjugates |
US11672874B2 (en) | 2019-09-03 | 2023-06-13 | Myeloid Therapeutics, Inc. | Methods and compositions for genomic integration |
US11730761B2 (en) | 2016-02-18 | 2023-08-22 | Enlivex Therapeutics Rdo Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
RU2806549C2 (en) * | 2018-10-18 | 2023-11-01 | Такеда Фармасьютикал Компани Лимитед | Method of t-cell activation/proliferation |
Families Citing this family (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10434153B1 (en) * | 2015-05-20 | 2019-10-08 | Kim Leslie O'Neill | Use of car and bite technology coupled with an scFv from an antibody against human thymidine kinase 1 to specifically target tumors |
US11352439B2 (en) | 2015-08-13 | 2022-06-07 | Kim Leslie O'Neill | Macrophage CAR (MOTO-CAR) in immunotherapy |
AR106189A1 (en) | 2015-10-02 | 2017-12-20 | Hoffmann La Roche | BIESPECTIFIC ANTIBODIES AGAINST HUMAN A-b AND THE HUMAN TRANSFERRINE RECEIVER AND METHODS OF USE |
US20200055947A1 (en) * | 2016-11-04 | 2020-02-20 | Memorial Sloan Kettering Cancer Center | Bi-specific activators for tumor therapy |
US11590167B2 (en) | 2016-12-03 | 2023-02-28 | Juno Therapeutic, Inc. | Methods and compositions for use of therapeutic T cells in combination with kinase inhibitors |
CN110709065B (en) * | 2017-04-19 | 2023-02-10 | Apa先进技术有限公司 | Fusogenic liposomes, compositions, kits and uses thereof for treating cancer |
IL275567B2 (en) | 2017-12-27 | 2024-03-01 | Takeda Pharmaceuticals Co | Nucleic acid-containing lipid nano-particle and use thereof |
JP2021510548A (en) | 2018-01-17 | 2021-04-30 | アラティンガ・バイオ・ティーエヌピー | Polymer-encapsulated viral vector for gene therapy |
US10982210B2 (en) * | 2018-03-02 | 2021-04-20 | Sixfold Bioscience Ltd. | Compositions for delivery of cargo to cells |
WO2020080475A1 (en) * | 2018-10-18 | 2020-04-23 | 武田薬品工業株式会社 | Method for activation/proliferation of t cells |
WO2020092631A1 (en) * | 2018-10-31 | 2020-05-07 | Fred Hutchinson Cancer Research Center | Compositions and methods for detecting and treating cancers characterized by expression of mesothelin |
EP3972570A4 (en) * | 2019-05-20 | 2022-11-02 | Technion Research & Development Foundation Limited | Metabolite encapsulating nanoparticles to enhance cellular cancer immunotherapy |
AU2020290417A1 (en) * | 2019-06-12 | 2022-01-06 | The Board Of Trustees Of The Leland Stanford Junior University | Methods to treat viral infections |
JP2021016370A (en) * | 2019-07-23 | 2021-02-15 | 株式会社東芝 | Carriers, carrier sets, compositions and methods for introducing nucleic acids |
EP4031185A2 (en) | 2019-09-21 | 2022-07-27 | Ixaka France | Dmso-free synthesis of oligopeptide-modified poly(beta-amino ester)s and their use in nanoparticle delivery systems |
JP2023523345A (en) | 2020-04-27 | 2023-06-02 | シックスフォールド バイオサイエンス リミテッド | Compositions Containing Nucleic Acid Nanoparticles with Modular Functional Groups |
BR112022023410A2 (en) | 2020-05-19 | 2022-12-20 | Ixaka France | PROMOTER SEQUENCES FOR IN VITRO AND IN VIVO EXPRESSION OF GENE THERAPY PRODUCTS IN CD3+ CELLS |
CN111925448B (en) * | 2020-08-03 | 2022-06-21 | 山东大学 | Preparation method of in vivo-generated CAR-macrophage and application of in vivo-generated CAR-macrophage in tumor immunotherapy |
CA3195281A1 (en) * | 2020-10-13 | 2022-04-21 | Hamideh Parhiz | In vivo targeting of cd4+-t cells for mrna therapeutics |
WO2022081694A1 (en) * | 2020-10-13 | 2022-04-21 | The Trustees Of The University Of Pennsylvania | In vivo targeting of fibrosis by anti-cd5-targeted fap-car t mrna-lnp |
CA3195275A1 (en) * | 2020-10-13 | 2022-04-21 | Hamideh Parhiz | In vivo targeting of t cells for mrna therapeutics |
US20240009239A1 (en) * | 2020-11-04 | 2024-01-11 | Fred Hutchinson Cancer Center | Therapeutic targeting of mesothelin in acute myeloid leukemia with chimeric antigen receptor t cell therapy |
WO2022241131A1 (en) * | 2021-05-14 | 2022-11-17 | Medicametrix, Inc. | Compositions and methods of reprogramming t cells to treat disease |
CN113481169B (en) * | 2021-08-11 | 2023-06-16 | 南方医科大学南方医院 | Cell activation dependent secretion system and application |
WO2023147596A1 (en) * | 2022-01-31 | 2023-08-03 | Noureddine Achraf | Triplex nanoparticles |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030072794A1 (en) * | 2000-06-09 | 2003-04-17 | Teni Boulikas | Encapsulation of plasmid DNA (lipogenes™) and therapeutic agents with nuclear localization signal/fusogenic peptide conjugates into targeted liposome complexes |
US20040043401A1 (en) * | 2002-05-28 | 2004-03-04 | Sloan Kettering Institute For Cancer Research | Chimeric T cell receotors |
US20050142114A1 (en) * | 2003-09-17 | 2005-06-30 | Gieseler Robert K. | Targeted lipid-drug formulations for delivery of drugs to myeloid and lymphoid immune cells |
US20080171061A1 (en) * | 2006-07-21 | 2008-07-17 | Douglas Nixon | Human endogenous retrovirus polypeptide compositions and methods of use thereof |
US20110189209A1 (en) * | 2007-08-01 | 2011-08-04 | The Government of the United States of America as represented by the Secretary, Dept. of Health | Fold-back diabody diphtheria toxin immunotoxin and methods of use |
WO2012079000A1 (en) * | 2010-12-09 | 2012-06-14 | The Trustees Of The University Of Pennsylvania | Use of chimeric antigen receptor-modified t cells to treat cancer |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9809658D0 (en) * | 1998-05-06 | 1998-07-01 | Celltech Therapeutics Ltd | Biological products |
CA2395636A1 (en) * | 1999-12-30 | 2001-07-12 | Novartis Ag | Novel colloid synthetic vectors for gene therapy |
ES2387886T3 (en) * | 2001-11-13 | 2012-10-03 | Celator Pharmaceuticals, Inc. | Compositions that transport lipids with better blood stability |
WO2010042555A2 (en) * | 2008-10-06 | 2010-04-15 | The Brigham And Women's Hospital, Inc. | Particles with multiple functionalized surface domains |
US20110229556A1 (en) * | 2010-03-19 | 2011-09-22 | Massachusetts Institute Of Technology | Lipid-coated polymer particles for immune stimulation |
AU2013207687B2 (en) * | 2012-01-13 | 2017-11-30 | Dana-Farber Cancer Institute, Inc. | Controlled delivery of TLR agonists in structural polymeric devices |
WO2014153114A1 (en) | 2013-03-14 | 2014-09-25 | Fred Hutchinson Cancer Research Center | Compositions and methods to modify cells for therapeutic objectives |
-
2014
- 2014-03-14 WO PCT/US2014/029137 patent/WO2014153114A1/en active Application Filing
- 2014-03-14 EP EP14768529.1A patent/EP2970985A1/en not_active Withdrawn
- 2014-03-14 US US14/776,661 patent/US20160145348A1/en not_active Abandoned
-
2017
- 2017-08-08 US US15/672,106 patent/US10392446B2/en active Active
-
2019
- 2019-07-12 US US16/510,646 patent/US20190330373A1/en active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030072794A1 (en) * | 2000-06-09 | 2003-04-17 | Teni Boulikas | Encapsulation of plasmid DNA (lipogenes™) and therapeutic agents with nuclear localization signal/fusogenic peptide conjugates into targeted liposome complexes |
US20040043401A1 (en) * | 2002-05-28 | 2004-03-04 | Sloan Kettering Institute For Cancer Research | Chimeric T cell receotors |
US20050142114A1 (en) * | 2003-09-17 | 2005-06-30 | Gieseler Robert K. | Targeted lipid-drug formulations for delivery of drugs to myeloid and lymphoid immune cells |
US20080171061A1 (en) * | 2006-07-21 | 2008-07-17 | Douglas Nixon | Human endogenous retrovirus polypeptide compositions and methods of use thereof |
US20110189209A1 (en) * | 2007-08-01 | 2011-08-04 | The Government of the United States of America as represented by the Secretary, Dept. of Health | Fold-back diabody diphtheria toxin immunotoxin and methods of use |
WO2012079000A1 (en) * | 2010-12-09 | 2012-06-14 | The Trustees Of The University Of Pennsylvania | Use of chimeric antigen receptor-modified t cells to treat cancer |
Cited By (70)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10392446B2 (en) | 2013-03-14 | 2019-08-27 | Fred Hutchinson Cancer Research Center | Compositions and methods to modify cells for therapeutic objectives |
US11382963B2 (en) | 2015-01-12 | 2022-07-12 | Pieris Pharmaceuticals Gmbh | Engineered T cells and uses therefor |
WO2016113203A1 (en) * | 2015-01-12 | 2016-07-21 | Pieris Ag | Engineered t cells and uses therefor |
US11000548B2 (en) | 2015-02-18 | 2021-05-11 | Enlivex Therapeutics Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
US11318163B2 (en) | 2015-02-18 | 2022-05-03 | Enlivex Therapeutics Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
US11304976B2 (en) | 2015-02-18 | 2022-04-19 | Enlivex Therapeutics Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
US11596652B2 (en) | 2015-02-18 | 2023-03-07 | Enlivex Therapeutics R&D Ltd | Early apoptotic cells for use in treating sepsis |
US11512289B2 (en) | 2015-02-18 | 2022-11-29 | Enlivex Therapeutics Rdo Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
US11717539B2 (en) | 2015-02-18 | 2023-08-08 | Enlivex Therapeutics RDO Ltd. | Combination immune therapy and cytokine control therapy for cancer treatment |
US11497767B2 (en) | 2015-02-18 | 2022-11-15 | Enlivex Therapeutics R&D Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
US11918604B2 (en) | 2015-02-19 | 2024-03-05 | Myeloid Therapeutics, Inc. | Chimeric antigen receptor dendritic cell (CAR-DC) for treatment of cancer |
US11918605B1 (en) | 2015-02-19 | 2024-03-05 | Myeloid Therapeutics, Inc. | Chimeric antigen receptor dendritic cell (CAR-DC) for treatment of cancer |
US11517589B2 (en) | 2015-02-19 | 2022-12-06 | Myeloid Therapeutics, Inc. | Chimeric antigen receptor dendritic cell (CAR-DC) for treatment of cancer |
US11883429B2 (en) | 2015-04-21 | 2024-01-30 | Enlivex Therapeutics Rdo Ltd | Therapeutic pooled blood apoptotic cell preparations and uses thereof |
US10857181B2 (en) | 2015-04-21 | 2020-12-08 | Enlivex Therapeutics Ltd | Therapeutic pooled blood apoptotic cell preparations and uses thereof |
US20220204935A1 (en) * | 2015-04-29 | 2022-06-30 | Fred Hutchinson Cancer Research Center | Modified hematopoietic stem/progenitor and non-t effector cells, and uses thereof |
US11584793B2 (en) | 2015-06-24 | 2023-02-21 | Hoffmann-La Roche Inc. | Anti-transferrin receptor antibodies with tailored affinity |
US11306133B2 (en) | 2015-07-28 | 2022-04-19 | The Trustees Of The University Of Pennsylvania | Modified monocytes/macrophage expressing chimeric antigen receptors and uses thereof |
US11332511B2 (en) | 2015-07-28 | 2022-05-17 | The Trustees Of The University Of Pennsylvania | Modified monocytes/macrophage expressing chimeric antigen receptors and uses thereof |
US11325963B2 (en) | 2015-07-28 | 2022-05-10 | The Trustees Of The University Of Pennsylvania | Modified monocytes/macrophage expressing chimeric antigen receptors and uses thereof |
US11034749B2 (en) | 2015-07-28 | 2021-06-15 | The Trustees Of The University Of Pennsylvania | Modified monocytes/macrophage expressing chimeric antigen receptors and uses thereof |
US11359002B2 (en) | 2015-07-28 | 2022-06-14 | The Trustees Of The University Of Pennsylvania | Modified monocytes/macrophage expressing chimeric antigen receptors and uses thereof |
US11498954B2 (en) | 2015-07-28 | 2022-11-15 | The Trustees Of The University Of Pennsylvania | Modified monocytes/macrophage expressing chimeric antigen receptors and uses thereof |
US11407805B2 (en) | 2015-07-28 | 2022-08-09 | The Trustees Of The University Of Pennsylvania | Modified monocytes/macrophage expressing chimeric antigen receptors and uses thereof |
US11306134B2 (en) | 2015-07-28 | 2022-04-19 | The Trustees Of The University Of Pennsylvania | Modified monocytes/macrophage expressing chimeric antigen receptors and uses thereof |
US11319358B2 (en) | 2015-07-28 | 2022-05-03 | The Trustees Of The University Of Pennsylvania | Modified monocytes/macrophage expressing chimeric antigen receptors and uses thereof |
AU2016325384B2 (en) * | 2015-09-22 | 2021-07-22 | Julius-Maximilians-Universitat Wurzburg | A method for high level and stable gene transfer in lymphocytes |
JP2022097517A (en) * | 2015-09-22 | 2022-06-30 | ユリウス-マクシミリアン-ウニヴェルシテート・ヴュルツブルク | Method for high level and stable gene transfer in lymphocytes |
CN108601849A (en) * | 2015-09-22 | 2018-09-28 | 尤利乌斯·马克西米利安维尔茨堡大学 | A method of high-caliber in lymphocyte and stable gene transfer |
WO2017050884A1 (en) * | 2015-09-22 | 2017-03-30 | Julius-Maximilians-Universität Würzburg | A method for high level and stable gene transfer in lymphocytes |
JP7142571B2 (en) | 2015-09-22 | 2022-09-27 | ユリウス-マクシミリアン-ウニヴェルシテート・ヴュルツブルク | Methods for high-level and stable gene transfer in lymphocytes |
JP2018532426A (en) * | 2015-09-22 | 2018-11-08 | ユリウス−マクシミリアン−ウニヴェルシテート・ヴュルツブルク | Method for high level and stable gene transfer in lymphocytes |
US11603411B2 (en) | 2015-10-02 | 2023-03-14 | Hoffmann-La Roche Inc. | Bispecific anti-human CD20/human transferrin receptor antibodies and methods of use |
US11730761B2 (en) | 2016-02-18 | 2023-08-22 | Enlivex Therapeutics Rdo Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
JP2019511234A (en) * | 2016-04-14 | 2019-04-25 | フレッド ハッチンソン キャンサー リサーチ センター | Compositions and methods for programming therapeutic cells using targeted nucleic acid nanocarriers |
US10188749B2 (en) | 2016-04-14 | 2019-01-29 | Fred Hutchinson Cancer Research Center | Compositions and methods to program therapeutic cells using targeted nucleic acid nanocarriers |
IL262361B2 (en) * | 2016-04-14 | 2023-07-01 | Hutchinson Fred Cancer Res | Compositions and methods to program therapeutic cells using targeted nucleic acid nanocarriers |
EP3442585A4 (en) * | 2016-04-14 | 2019-09-25 | Fred Hutchinson Cancer Research Center | Compositions and methods to program therapeutic cells using targeted nucleic acid nanocarriers |
JP7118340B2 (en) | 2016-04-14 | 2022-08-16 | フレッド ハッチンソン キャンサー センター | Compositions and methods for programming therapeutic cells using targeted nucleic acid nanocarriers |
AU2017250295B2 (en) * | 2016-04-14 | 2022-08-25 | Fred Hutchinson Cancer Center | Compositions and methods to program therapeutic cells using targeted nucleic acid nanocarriers |
KR102542533B1 (en) * | 2016-04-14 | 2023-06-13 | 프레드 허친슨 캔서 센터 | Compositions and methods for programming therapeutic cells using targeting nucleic acid nanotransporters |
WO2017181110A1 (en) | 2016-04-14 | 2017-10-19 | Fred Hutchinson Cancer Research Center | Compositions and methods to program therapeutic cells using targeted nucleic acid nanocarriers |
KR20180133480A (en) * | 2016-04-14 | 2018-12-14 | 프레드 헛친슨 켄서 리서치 센터 | COMPOSITIONS AND METHODS FOR PROGRAMMING TREATMENT CELLS USING TARGETED NUCLEIC ACID NANOCLEANTS |
EP4166161A1 (en) * | 2016-04-14 | 2023-04-19 | Fred Hutchinson Cancer Center | Compositions and methods to program therapeutic cells using targeted nucleic acid nanocarriers |
US11872195B2 (en) | 2016-04-14 | 2024-01-16 | Fred Hutchinson Cancer Center | Compositions and methods to program therapeutic cells using targeted nucleic acid nanocarriers |
IL262361B1 (en) * | 2016-04-14 | 2023-03-01 | Hutchinson Fred Cancer Res | Compositions and methods to program therapeutic cells using targeted nucleic acid nanocarriers |
JP7062640B2 (en) | 2016-07-29 | 2022-05-06 | ジュノー セラピューティクス インコーポレイテッド | Anti-idiotype antibody against anti-CD19 antibody |
JP2019527553A (en) * | 2016-07-29 | 2019-10-03 | ジュノー セラピューティクス インコーポレイテッド | Anti-idiotype antibody against anti-CD19 antibody |
US11530264B2 (en) * | 2016-12-29 | 2022-12-20 | Shenzhen Beike Biotechnology Co., Ltd | Multifunctional protein |
US11566061B2 (en) | 2017-01-05 | 2023-01-31 | Fred Hutchinson Cancer Center | Systems and methods to improve vaccine efficacy |
US11634497B2 (en) | 2017-02-03 | 2023-04-25 | Novartis Ag | Anti-CCR7 antibody drug conjugates |
EP3689336A4 (en) * | 2017-09-14 | 2021-01-20 | Shanghai Jiao Tong University | Multicellular targeting liposome |
CN110526976A (en) * | 2018-05-25 | 2019-12-03 | 深圳宾德生物技术有限公司 | It is a kind of to target the single-chain antibody of PSMA, Chimeric antigen receptor T cell and its preparation method and application |
CN112912727A (en) * | 2018-08-31 | 2021-06-04 | 因维克蒂斯股份有限公司 | Methods of assessing CAR functionality |
RU2806549C2 (en) * | 2018-10-18 | 2023-11-01 | Такеда Фармасьютикал Компани Лимитед | Method of t-cell activation/proliferation |
US11026973B2 (en) | 2019-04-30 | 2021-06-08 | Myeloid Therapeutics, Inc. | Engineered phagocytic receptor compositions and methods of use thereof |
US11013764B2 (en) | 2019-04-30 | 2021-05-25 | Myeloid Therapeutics, Inc. | Engineered phagocytic receptor compositions and methods of use thereof |
US20220008473A1 (en) * | 2019-06-27 | 2022-01-13 | Crispr Therapeutics Ag | Use of chimeric antigen receptor t cells and nk cell inhibitors for treating cancer |
JP2021016371A (en) * | 2019-07-23 | 2021-02-15 | 株式会社東芝 | Methods for producing car-t cells, carriers and kits for introducing nucleic acid |
WO2021014368A1 (en) * | 2019-07-23 | 2021-01-28 | Kabushiki Kaisha Toshiba | Method of producing car-t cells, nucleic acid-introducing carrier and kit |
US11672874B2 (en) | 2019-09-03 | 2023-06-13 | Myeloid Therapeutics, Inc. | Methods and compositions for genomic integration |
US20210253696A1 (en) * | 2020-01-21 | 2021-08-19 | Cero Therapeutics, Inc. | Bivalent chimeric engulfment receptors and uses thereof |
US11312939B2 (en) | 2020-06-04 | 2022-04-26 | Carisma Therapeutics Inc. | Constructs for chimeric antigen receptors |
US11739297B2 (en) | 2020-06-04 | 2023-08-29 | Carisma Therapeutics Inc. | Method of increasing tumor killing activity of macrophages or monocytes comprising chimeric antigen receptor |
US20230263828A1 (en) * | 2020-09-23 | 2023-08-24 | Crispr Therapeutics Ag | Genetically engineered t cells with regnase-1 and/or tgfbrii disruption have improved functionality and persistence |
US11857574B2 (en) | 2020-09-23 | 2024-01-02 | Crispr Therapeutics Ag | Genetically engineered T cells with Regnase-1 and/or TGFBRII disruption have improved functionality and persistence |
US11679130B2 (en) | 2020-09-23 | 2023-06-20 | Crispr Therapeutics Ag | Genetically engineered t cells with Regnase-1 and/or TGFBRII disruption have improved functionality and persistence |
US11679131B2 (en) | 2020-09-23 | 2023-06-20 | Crispr Therapeutics Ag | Genetically engineered T cells with regnase-1 and/or TGFBRII disruption have improved functionality and persistence |
US11497773B2 (en) * | 2020-09-23 | 2022-11-15 | Crispr Therapeutics Ag | Genetically engineered t cells with regnase-1 and/or TGFBRII disruption have improved functionality and persistence |
US11628218B2 (en) | 2020-11-04 | 2023-04-18 | Myeloid Therapeutics, Inc. | Engineered chimeric fusion protein compositions and methods of use thereof |
Also Published As
Publication number | Publication date |
---|---|
EP2970985A1 (en) | 2016-01-20 |
US20160145348A1 (en) | 2016-05-26 |
US20190330373A1 (en) | 2019-10-31 |
US20180030153A1 (en) | 2018-02-01 |
US10392446B2 (en) | 2019-08-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10392446B2 (en) | Compositions and methods to modify cells for therapeutic objectives | |
US11872195B2 (en) | Compositions and methods to program therapeutic cells using targeted nucleic acid nanocarriers | |
US20210128485A1 (en) | Nanoparticles for gene expression and uses thereof | |
US20230181643A1 (en) | Use of trans-signaling approach in chimeric antigen receptors | |
AU2017250295B2 (en) | Compositions and methods to program therapeutic cells using targeted nucleic acid nanocarriers | |
US20230203532A1 (en) | Recombinant polypeptides for programming extracellular vesicles | |
CA3162629A1 (en) | Nanoparticle systems to stimulate and maintain immune system responsiveness at treatment sites | |
CN112876566A (en) | Construction and application of CD3 specific lentivirus | |
NL2028681B1 (en) | Materials and methods for generating antigen-specific t cells and treating diseases | |
WO2024057315A1 (en) | Compositions and methods for virotherapy | |
NZ787266A (en) | Compositions and methods to program therapeutic cells using targeted nucleic acid nanocarriers |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 14768529 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REEP | Request for entry into the european phase |
Ref document number: 2014768529 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2014768529 Country of ref document: EP |