WO2018071576A1 - Treatment of tumors by inhibition of cd300f - Google Patents
Treatment of tumors by inhibition of cd300f Download PDFInfo
- Publication number
- WO2018071576A1 WO2018071576A1 PCT/US2017/056192 US2017056192W WO2018071576A1 WO 2018071576 A1 WO2018071576 A1 WO 2018071576A1 US 2017056192 W US2017056192 W US 2017056192W WO 2018071576 A1 WO2018071576 A1 WO 2018071576A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cd300f
- cells
- tumor
- subject
- dendritic cells
- Prior art date
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 228
- 238000011282 treatment Methods 0.000 title description 30
- 230000005764 inhibitory process Effects 0.000 title description 10
- 101000990055 Homo sapiens CMRF35-like molecule 1 Proteins 0.000 claims abstract description 240
- 210000004443 dendritic cell Anatomy 0.000 claims abstract description 222
- 102100029390 CMRF35-like molecule 1 Human genes 0.000 claims abstract description 184
- 238000000034 method Methods 0.000 claims abstract description 150
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 92
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 74
- 239000003112 inhibitor Substances 0.000 claims abstract description 56
- 150000007523 nucleic acids Chemical class 0.000 claims description 114
- 239000000427 antigen Substances 0.000 claims description 89
- 108091007433 antigens Proteins 0.000 claims description 89
- 102000036639 antigens Human genes 0.000 claims description 89
- 102000039446 nucleic acids Human genes 0.000 claims description 79
- 108020004707 nucleic acids Proteins 0.000 claims description 79
- 108091033409 CRISPR Proteins 0.000 claims description 65
- 239000002773 nucleotide Substances 0.000 claims description 53
- 239000013598 vector Substances 0.000 claims description 47
- 125000003729 nucleotide group Chemical group 0.000 claims description 46
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 44
- 108020005004 Guide RNA Proteins 0.000 claims description 37
- 239000000203 mixture Substances 0.000 claims description 35
- 230000001640 apoptogenic effect Effects 0.000 claims description 29
- 210000004881 tumor cell Anatomy 0.000 claims description 29
- 230000002401 inhibitory effect Effects 0.000 claims description 23
- 239000013603 viral vector Substances 0.000 claims description 21
- 102100024125 Embryonal Fyn-associated substrate Human genes 0.000 claims description 18
- 101710156582 Embryonal Fyn-associated substrate Proteins 0.000 claims description 18
- 239000002246 antineoplastic agent Substances 0.000 claims description 17
- 230000028993 immune response Effects 0.000 claims description 17
- 206010009944 Colon cancer Diseases 0.000 claims description 16
- 229940045513 CTLA4 antagonist Drugs 0.000 claims description 14
- 101710163270 Nuclease Proteins 0.000 claims description 14
- 208000024891 symptom Diseases 0.000 claims description 14
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 13
- 230000003247 decreasing effect Effects 0.000 claims description 11
- 206010027476 Metastases Diseases 0.000 claims description 10
- 206010039491 Sarcoma Diseases 0.000 claims description 10
- 229940127089 cytotoxic agent Drugs 0.000 claims description 10
- 108091034117 Oligonucleotide Proteins 0.000 claims description 9
- 201000009030 Carcinoma Diseases 0.000 claims description 8
- 150000003384 small molecules Chemical class 0.000 claims description 8
- 229940124060 PD-1 antagonist Drugs 0.000 claims description 7
- 208000000453 Skin Neoplasms Diseases 0.000 claims description 7
- 108020004459 Small interfering RNA Proteins 0.000 claims description 6
- 239000013604 expression vector Substances 0.000 claims description 6
- 230000001939 inductive effect Effects 0.000 claims description 6
- 108091027967 Small hairpin RNA Proteins 0.000 claims description 5
- 201000000849 skin cancer Diseases 0.000 claims description 5
- 239000004055 small Interfering RNA Substances 0.000 claims description 5
- 230000001131 transforming effect Effects 0.000 claims description 4
- 239000003124 biologic agent Substances 0.000 claims description 3
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 claims 1
- 230000005855 radiation Effects 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 163
- 108090000765 processed proteins & peptides Proteins 0.000 description 85
- 230000014509 gene expression Effects 0.000 description 61
- 102000004196 processed proteins & peptides Human genes 0.000 description 59
- 229920001184 polypeptide Polymers 0.000 description 52
- 235000018102 proteins Nutrition 0.000 description 47
- 102000004169 proteins and genes Human genes 0.000 description 47
- 241000699670 Mus sp. Species 0.000 description 45
- 239000012634 fragment Substances 0.000 description 35
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 34
- 108020004414 DNA Proteins 0.000 description 34
- 210000001616 monocyte Anatomy 0.000 description 34
- 201000011510 cancer Diseases 0.000 description 33
- 235000001014 amino acid Nutrition 0.000 description 32
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 31
- 239000002243 precursor Substances 0.000 description 29
- 150000001413 amino acids Chemical class 0.000 description 28
- 108060003951 Immunoglobulin Proteins 0.000 description 25
- 102000018358 immunoglobulin Human genes 0.000 description 25
- 102000040430 polynucleotide Human genes 0.000 description 24
- 108091033319 polynucleotide Proteins 0.000 description 24
- 150000001875 compounds Chemical class 0.000 description 23
- 230000000670 limiting effect Effects 0.000 description 23
- 239000002157 polynucleotide Substances 0.000 description 23
- -1 DNA oligonucleotides Chemical class 0.000 description 20
- 125000003275 alpha amino acid group Chemical group 0.000 description 20
- 238000003776 cleavage reaction Methods 0.000 description 20
- 102000054256 human CD300LF Human genes 0.000 description 20
- 230000007017 scission Effects 0.000 description 20
- 102100035360 Cerebellar degeneration-related antigen 1 Human genes 0.000 description 19
- 230000000694 effects Effects 0.000 description 19
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 18
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 18
- 238000000338 in vitro Methods 0.000 description 18
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 17
- 230000002950 deficient Effects 0.000 description 16
- 230000006870 function Effects 0.000 description 16
- 108020004999 messenger RNA Proteins 0.000 description 16
- 241000700605 Viruses Species 0.000 description 15
- 239000003814 drug Substances 0.000 description 15
- 239000012636 effector Substances 0.000 description 15
- 239000003550 marker Substances 0.000 description 15
- 108091079001 CRISPR RNA Proteins 0.000 description 14
- 230000007423 decrease Effects 0.000 description 14
- 210000004698 lymphocyte Anatomy 0.000 description 14
- 230000035800 maturation Effects 0.000 description 14
- 230000001225 therapeutic effect Effects 0.000 description 14
- 238000013518 transcription Methods 0.000 description 14
- 230000035897 transcription Effects 0.000 description 14
- 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 13
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 13
- 206010025323 Lymphomas Diseases 0.000 description 13
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 13
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 13
- 230000000692 anti-sense effect Effects 0.000 description 13
- 210000000612 antigen-presenting cell Anatomy 0.000 description 13
- 230000000903 blocking effect Effects 0.000 description 12
- 210000004369 blood Anatomy 0.000 description 12
- 239000008280 blood Substances 0.000 description 12
- 239000003795 chemical substances by application Substances 0.000 description 12
- 239000000126 substance Substances 0.000 description 12
- 238000006467 substitution reaction Methods 0.000 description 12
- 102000004127 Cytokines Human genes 0.000 description 11
- 241000124008 Mammalia Species 0.000 description 11
- 201000010099 disease Diseases 0.000 description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 11
- 230000004614 tumor growth Effects 0.000 description 11
- 102000053642 Catalytic RNA Human genes 0.000 description 10
- 108090000994 Catalytic RNA Proteins 0.000 description 10
- 108090000695 Cytokines Proteins 0.000 description 10
- 108010074328 Interferon-gamma Proteins 0.000 description 10
- 206010035226 Plasma cell myeloma Diseases 0.000 description 10
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 10
- 238000003556 assay Methods 0.000 description 10
- 239000002299 complementary DNA Substances 0.000 description 10
- 238000004519 manufacturing process Methods 0.000 description 10
- 230000004044 response Effects 0.000 description 10
- 108091092562 ribozyme Proteins 0.000 description 10
- 238000012384 transportation and delivery Methods 0.000 description 10
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 9
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 9
- 230000004913 activation Effects 0.000 description 9
- 239000005557 antagonist Substances 0.000 description 9
- 210000003719 b-lymphocyte Anatomy 0.000 description 9
- 201000000050 myeloid neoplasm Diseases 0.000 description 9
- 239000002245 particle Substances 0.000 description 9
- 239000013612 plasmid Substances 0.000 description 9
- 230000008569 process Effects 0.000 description 9
- 230000008685 targeting Effects 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- DGAKHGXRMXWHBX-ONEGZZNKSA-N Azoxymethane Chemical compound C\N=[N+](/C)[O-] DGAKHGXRMXWHBX-ONEGZZNKSA-N 0.000 description 8
- 102100037850 Interferon gamma Human genes 0.000 description 8
- 102000004388 Interleukin-4 Human genes 0.000 description 8
- 108090000978 Interleukin-4 Proteins 0.000 description 8
- 230000006907 apoptotic process Effects 0.000 description 8
- 230000000295 complement effect Effects 0.000 description 8
- 238000000684 flow cytometry Methods 0.000 description 8
- 238000009472 formulation Methods 0.000 description 8
- 210000004408 hybridoma Anatomy 0.000 description 8
- 229940028885 interleukin-4 Drugs 0.000 description 8
- 238000010172 mouse model Methods 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 8
- TVZRAEYQIKYCPH-UHFFFAOYSA-N 3-(trimethylsilyl)propane-1-sulfonic acid Chemical compound C[Si](C)(C)CCCS(O)(=O)=O TVZRAEYQIKYCPH-UHFFFAOYSA-N 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 7
- 208000026310 Breast neoplasm Diseases 0.000 description 7
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 7
- 102000053602 DNA Human genes 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 7
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 7
- 102000015696 Interleukins Human genes 0.000 description 7
- 108010063738 Interleukins Proteins 0.000 description 7
- 102100039019 Nuclear receptor subfamily 0 group B member 1 Human genes 0.000 description 7
- 108091005461 Nucleic proteins Proteins 0.000 description 7
- 206010057249 Phagocytosis Diseases 0.000 description 7
- 230000007812 deficiency Effects 0.000 description 7
- 239000002158 endotoxin Substances 0.000 description 7
- 229940088598 enzyme Drugs 0.000 description 7
- 230000009368 gene silencing by RNA Effects 0.000 description 7
- 230000002068 genetic effect Effects 0.000 description 7
- 239000002502 liposome Substances 0.000 description 7
- 230000009401 metastasis Effects 0.000 description 7
- 230000008782 phagocytosis Effects 0.000 description 7
- 102000005962 receptors Human genes 0.000 description 7
- 108020003175 receptors Proteins 0.000 description 7
- 229940124597 therapeutic agent Drugs 0.000 description 7
- 206010006187 Breast cancer Diseases 0.000 description 6
- 108010042407 Endonucleases Proteins 0.000 description 6
- 102000004533 Endonucleases Human genes 0.000 description 6
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 6
- 230000014102 antigen processing and presentation of exogenous peptide antigen via MHC class I Effects 0.000 description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 208000029742 colonic neoplasm Diseases 0.000 description 6
- 230000001472 cytotoxic effect Effects 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- 230000004927 fusion Effects 0.000 description 6
- 210000002865 immune cell Anatomy 0.000 description 6
- 239000003446 ligand Substances 0.000 description 6
- 210000000066 myeloid cell Anatomy 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- 239000008194 pharmaceutical composition Substances 0.000 description 6
- 230000010076 replication Effects 0.000 description 6
- 241000894007 species Species 0.000 description 6
- 238000002560 therapeutic procedure Methods 0.000 description 6
- 102000003390 tumor necrosis factor Human genes 0.000 description 6
- 102100034540 Adenomatous polyposis coli protein Human genes 0.000 description 5
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 5
- 101000924577 Homo sapiens Adenomatous polyposis coli protein Proteins 0.000 description 5
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 5
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 5
- 101100407308 Mus musculus Pdcd1lg2 gene Proteins 0.000 description 5
- 102000057297 Pepsin A Human genes 0.000 description 5
- 108090000284 Pepsin A Proteins 0.000 description 5
- 108700030875 Programmed Cell Death 1 Ligand 2 Proteins 0.000 description 5
- 102100024213 Programmed cell death 1 ligand 2 Human genes 0.000 description 5
- 102000004389 Ribonucleoproteins Human genes 0.000 description 5
- 108010081734 Ribonucleoproteins Proteins 0.000 description 5
- 208000009956 adenocarcinoma Diseases 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 230000006023 anti-tumor response Effects 0.000 description 5
- 238000013459 approach Methods 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 238000002512 chemotherapy Methods 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- 238000012217 deletion Methods 0.000 description 5
- 230000037430 deletion Effects 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 230000002255 enzymatic effect Effects 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 238000009396 hybridization Methods 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000001990 intravenous administration Methods 0.000 description 5
- 229920006008 lipopolysaccharide Polymers 0.000 description 5
- 239000003094 microcapsule Substances 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 229930014626 natural product Natural products 0.000 description 5
- 230000035755 proliferation Effects 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 230000001177 retroviral effect Effects 0.000 description 5
- 125000002652 ribonucleotide group Chemical group 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 238000010561 standard procedure Methods 0.000 description 5
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- 238000013519 translation Methods 0.000 description 5
- 230000003612 virological effect Effects 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 238000011740 C57BL/6 mouse Methods 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 4
- 241000702421 Dependoparvovirus Species 0.000 description 4
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 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
- 101000998953 Homo sapiens Immunoglobulin heavy variable 1-2 Proteins 0.000 description 4
- 108091008028 Immune checkpoint receptors Proteins 0.000 description 4
- 102000037978 Immune checkpoint receptors Human genes 0.000 description 4
- 102100036887 Immunoglobulin heavy variable 1-2 Human genes 0.000 description 4
- 102000003812 Interleukin-15 Human genes 0.000 description 4
- 108090000172 Interleukin-15 Proteins 0.000 description 4
- 102000000588 Interleukin-2 Human genes 0.000 description 4
- 108010002350 Interleukin-2 Proteins 0.000 description 4
- 206010027406 Mesothelioma Diseases 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 101000990030 Mus musculus CMRF35-like molecule 1 Proteins 0.000 description 4
- 108091081021 Sense strand Proteins 0.000 description 4
- 241000193996 Streptococcus pyogenes Species 0.000 description 4
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 4
- 241000700618 Vaccinia virus Species 0.000 description 4
- 230000002159 abnormal effect Effects 0.000 description 4
- 101150063416 add gene Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 210000001185 bone marrow Anatomy 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 238000013270 controlled release Methods 0.000 description 4
- 231100000433 cytotoxic Toxicity 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- 230000005782 double-strand break Effects 0.000 description 4
- 239000003937 drug carrier Substances 0.000 description 4
- 238000012377 drug delivery Methods 0.000 description 4
- 210000001671 embryonic stem cell Anatomy 0.000 description 4
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 4
- 229960002949 fluorouracil Drugs 0.000 description 4
- 230000006801 homologous recombination Effects 0.000 description 4
- 238000002744 homologous recombination Methods 0.000 description 4
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 238000001802 infusion Methods 0.000 description 4
- 210000000265 leukocyte Anatomy 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 208000020816 lung neoplasm Diseases 0.000 description 4
- 210000002540 macrophage Anatomy 0.000 description 4
- 238000012423 maintenance Methods 0.000 description 4
- 230000003211 malignant effect Effects 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 201000001441 melanoma Diseases 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 239000011859 microparticle Substances 0.000 description 4
- 239000004005 microsphere Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000002105 nanoparticle Substances 0.000 description 4
- 229960003301 nivolumab Drugs 0.000 description 4
- 229940111202 pepsin Drugs 0.000 description 4
- 210000005259 peripheral blood Anatomy 0.000 description 4
- 239000011886 peripheral blood Substances 0.000 description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 4
- 230000003389 potentiating effect Effects 0.000 description 4
- 230000037452 priming Effects 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 230000002285 radioactive effect Effects 0.000 description 4
- 230000006798 recombination Effects 0.000 description 4
- 238000005215 recombination Methods 0.000 description 4
- 229960004641 rituximab Drugs 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 230000009885 systemic effect Effects 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 230000005748 tumor development Effects 0.000 description 4
- 241000701161 unidentified adenovirus Species 0.000 description 4
- 241001529453 unidentified herpesvirus Species 0.000 description 4
- 241001430294 unidentified retrovirus Species 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 239000003981 vehicle Substances 0.000 description 4
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 3
- 102100023635 Alpha-fetoprotein Human genes 0.000 description 3
- 108010077544 Chromatin Proteins 0.000 description 3
- 206010058314 Dysplasia Diseases 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 241000206602 Eukaryota Species 0.000 description 3
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 3
- 108010053070 Glutathione Disulfide Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 3
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 3
- 108010047761 Interferon-alpha Proteins 0.000 description 3
- 102000006992 Interferon-alpha Human genes 0.000 description 3
- 102000004889 Interleukin-6 Human genes 0.000 description 3
- 108090001005 Interleukin-6 Proteins 0.000 description 3
- 108091092195 Intron Proteins 0.000 description 3
- 102000017578 LAG3 Human genes 0.000 description 3
- 241000713666 Lentivirus Species 0.000 description 3
- 108091054437 MHC class I family Proteins 0.000 description 3
- 102000043129 MHC class I family Human genes 0.000 description 3
- 241001529936 Murinae Species 0.000 description 3
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 206010033128 Ovarian cancer Diseases 0.000 description 3
- 206010061535 Ovarian neoplasm Diseases 0.000 description 3
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 description 3
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 208000005718 Stomach Neoplasms Diseases 0.000 description 3
- 230000005867 T cell response Effects 0.000 description 3
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 3
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 3
- 102000002689 Toll-like receptor Human genes 0.000 description 3
- 108020000411 Toll-like receptor Proteins 0.000 description 3
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000011230 binding agent Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 208000025997 central nervous system neoplasm Diseases 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000003638 chemical reducing agent Substances 0.000 description 3
- 210000003483 chromatin Anatomy 0.000 description 3
- 239000000470 constituent Substances 0.000 description 3
- 230000016396 cytokine production Effects 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 238000002784 cytotoxicity assay Methods 0.000 description 3
- 231100000263 cytotoxicity test Toxicity 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 239000003651 drinking water Substances 0.000 description 3
- 235000020188 drinking water Nutrition 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 125000000524 functional group Chemical group 0.000 description 3
- 238000010362 genome editing Methods 0.000 description 3
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 3
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 3
- 210000005260 human cell Anatomy 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 229940100601 interleukin-6 Drugs 0.000 description 3
- 238000007918 intramuscular administration Methods 0.000 description 3
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 208000032839 leukemia Diseases 0.000 description 3
- 230000036210 malignancy Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 239000002088 nanocapsule Substances 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 210000004940 nucleus Anatomy 0.000 description 3
- 238000011275 oncology therapy Methods 0.000 description 3
- 150000002894 organic compounds Chemical class 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 238000010647 peptide synthesis reaction Methods 0.000 description 3
- 210000005134 plasmacytoid dendritic cell Anatomy 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000001742 protein purification Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 210000004988 splenocyte Anatomy 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 125000003396 thiol group Chemical group [H]S* 0.000 description 3
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 2
- RTQWWZBSTRGEAV-PKHIMPSTSA-N 2-[[(2s)-2-[bis(carboxymethyl)amino]-3-[4-(methylcarbamoylamino)phenyl]propyl]-[2-[bis(carboxymethyl)amino]propyl]amino]acetic acid Chemical compound CNC(=O)NC1=CC=C(C[C@@H](CN(CC(C)N(CC(O)=O)CC(O)=O)CC(O)=O)N(CC(O)=O)CC(O)=O)C=C1 RTQWWZBSTRGEAV-PKHIMPSTSA-N 0.000 description 2
- OIVLITBTBDPEFK-UHFFFAOYSA-N 5,6-dihydrouracil Chemical compound O=C1CCNC(=O)N1 OIVLITBTBDPEFK-UHFFFAOYSA-N 0.000 description 2
- LQLQRFGHAALLLE-UHFFFAOYSA-N 5-bromouracil Chemical compound BrC1=CNC(=O)NC1=O LQLQRFGHAALLLE-UHFFFAOYSA-N 0.000 description 2
- KSNXJLQDQOIRIP-UHFFFAOYSA-N 5-iodouracil Chemical compound IC1=CNC(=O)NC1=O KSNXJLQDQOIRIP-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 208000030507 AIDS Diseases 0.000 description 2
- 206010000830 Acute leukaemia Diseases 0.000 description 2
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 208000010839 B-cell chronic lymphocytic leukemia Diseases 0.000 description 2
- 108010074708 B7-H1 Antigen Proteins 0.000 description 2
- 206010004593 Bile duct cancer Diseases 0.000 description 2
- 206010005003 Bladder cancer Diseases 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- COVZYZSDYWQREU-UHFFFAOYSA-N Busulfan Chemical compound CS(=O)(=O)OCCCCOS(C)(=O)=O COVZYZSDYWQREU-UHFFFAOYSA-N 0.000 description 2
- 102100035793 CD83 antigen Human genes 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- GAGWJHPBXLXJQN-UORFTKCHSA-N Capecitabine Chemical compound C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](C)O1 GAGWJHPBXLXJQN-UORFTKCHSA-N 0.000 description 2
- DLGOEMSEDOSKAD-UHFFFAOYSA-N Carmustine Chemical compound ClCCNC(=O)N(N=O)CCCl DLGOEMSEDOSKAD-UHFFFAOYSA-N 0.000 description 2
- 206010007953 Central nervous system lymphoma Diseases 0.000 description 2
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 108010051219 Cre recombinase Proteins 0.000 description 2
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 2
- 102000007260 Deoxyribonuclease I Human genes 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
- 241000283086 Equidae Species 0.000 description 2
- 208000006168 Ewing Sarcoma Diseases 0.000 description 2
- 108010046276 FLP recombinase Proteins 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 229920001917 Ficoll Polymers 0.000 description 2
- 208000000666 Fowlpox Diseases 0.000 description 2
- 241000713813 Gibbon ape leukemia virus Species 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 208000017604 Hodgkin disease Diseases 0.000 description 2
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 2
- 101100384024 Homo sapiens CD300LF gene Proteins 0.000 description 2
- 101000946856 Homo sapiens CD83 antigen Proteins 0.000 description 2
- 101001002552 Homo sapiens Immunoglobulin superfamily member 11 Proteins 0.000 description 2
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 2
- 101000623901 Homo sapiens Mucin-16 Proteins 0.000 description 2
- 101000574648 Homo sapiens Retinoid-inducible serine carboxypeptidase Proteins 0.000 description 2
- 101000600903 Homo sapiens Substance-P receptor Proteins 0.000 description 2
- 101000669447 Homo sapiens Toll-like receptor 4 Proteins 0.000 description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- VSNHCAURESNICA-UHFFFAOYSA-N Hydroxyurea Chemical compound NC(=O)NO VSNHCAURESNICA-UHFFFAOYSA-N 0.000 description 2
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 2
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 2
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 2
- 229930010555 Inosine Natural products 0.000 description 2
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 2
- 102000008070 Interferon-gamma Human genes 0.000 description 2
- 102000013462 Interleukin-12 Human genes 0.000 description 2
- 108010065805 Interleukin-12 Proteins 0.000 description 2
- 102000003816 Interleukin-13 Human genes 0.000 description 2
- 108090000176 Interleukin-13 Proteins 0.000 description 2
- 108010002586 Interleukin-7 Proteins 0.000 description 2
- 102000000704 Interleukin-7 Human genes 0.000 description 2
- 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 2
- 101150030213 Lag3 gene Proteins 0.000 description 2
- GQYIWUVLTXOXAJ-UHFFFAOYSA-N Lomustine Chemical compound ClCCN(N=O)C(=O)NC1CCCCC1 GQYIWUVLTXOXAJ-UHFFFAOYSA-N 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 208000031422 Lymphocytic Chronic B-Cell Leukemia Diseases 0.000 description 2
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 2
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 2
- 102100023123 Mucin-16 Human genes 0.000 description 2
- 241000714177 Murine leukemia virus Species 0.000 description 2
- 101000870135 Mus musculus Cytohesin-1 Proteins 0.000 description 2
- 241001467552 Mycobacterium bovis BCG Species 0.000 description 2
- 201000003793 Myelodysplastic syndrome Diseases 0.000 description 2
- ZDZOTLJHXYCWBA-VCVYQWHSSA-N N-debenzoyl-N-(tert-butoxycarbonyl)-10-deacetyltaxol Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 ZDZOTLJHXYCWBA-VCVYQWHSSA-N 0.000 description 2
- 206010029260 Neuroblastoma Diseases 0.000 description 2
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 2
- 108090000526 Papain Proteins 0.000 description 2
- 206010060862 Prostate cancer Diseases 0.000 description 2
- 201000000582 Retinoblastoma Diseases 0.000 description 2
- 102100025483 Retinoid-inducible serine carboxypeptidase Human genes 0.000 description 2
- 108091028664 Ribonucleotide Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 241000713311 Simian immunodeficiency virus Species 0.000 description 2
- 241000251131 Sphyrna Species 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- 230000006044 T cell activation Effects 0.000 description 2
- 208000031673 T-Cell Cutaneous Lymphoma Diseases 0.000 description 2
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 description 2
- 208000024313 Testicular Neoplasms Diseases 0.000 description 2
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 102100039360 Toll-like receptor 4 Human genes 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 206010046458 Urethral neoplasms Diseases 0.000 description 2
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 2
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 2
- 206010046865 Vaccinia virus infection Diseases 0.000 description 2
- 102000055135 Vasoactive Intestinal Peptide Human genes 0.000 description 2
- 108010003205 Vasoactive Intestinal Peptide Proteins 0.000 description 2
- 208000008383 Wilms tumor Diseases 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
- 229940100198 alkylating agent Drugs 0.000 description 2
- 239000002168 alkylating agent Substances 0.000 description 2
- 230000000340 anti-metabolite Effects 0.000 description 2
- 229940125644 antibody drug Drugs 0.000 description 2
- 230000030741 antigen processing and presentation Effects 0.000 description 2
- 229940100197 antimetabolite Drugs 0.000 description 2
- 239000002256 antimetabolite Substances 0.000 description 2
- 229940034982 antineoplastic agent Drugs 0.000 description 2
- 230000025194 apoptotic cell clearance Effects 0.000 description 2
- 229960000190 bacillus calmette–guérin vaccine Drugs 0.000 description 2
- 238000002869 basic local alignment search tool Methods 0.000 description 2
- 210000002459 blastocyst Anatomy 0.000 description 2
- 210000000481 breast Anatomy 0.000 description 2
- 229960002092 busulfan Drugs 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 210000003068 cdc Anatomy 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 229910052804 chromium Inorganic materials 0.000 description 2
- 239000011651 chromium Substances 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 208000032852 chronic lymphocytic leukemia Diseases 0.000 description 2
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 2
- 238000012411 cloning technique Methods 0.000 description 2
- 201000010276 collecting duct carcinoma Diseases 0.000 description 2
- 210000001072 colon Anatomy 0.000 description 2
- 238000009833 condensation Methods 0.000 description 2
- 230000005494 condensation Effects 0.000 description 2
- 239000000599 controlled substance Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000000139 costimulatory effect Effects 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000009295 crossflow filtration Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 229960000975 daunorubicin Drugs 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 238000000432 density-gradient centrifugation Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- VHJLVAABSRFDPM-ZXZARUISSA-N dithioerythritol Chemical compound SC[C@H](O)[C@H](O)CS VHJLVAABSRFDPM-ZXZARUISSA-N 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 210000000750 endocrine system Anatomy 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 108091006047 fluorescent proteins Proteins 0.000 description 2
- 102000034287 fluorescent proteins Human genes 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 206010017758 gastric cancer Diseases 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 210000003128 head Anatomy 0.000 description 2
- 208000014829 head and neck neoplasm Diseases 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 2
- 229940022353 herceptin Drugs 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229960001001 ibritumomab tiuxetan Drugs 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 229960001438 immunostimulant agent Drugs 0.000 description 2
- 230000003308 immunostimulating effect Effects 0.000 description 2
- 239000007943 implant Substances 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 229960003786 inosine Drugs 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 230000002601 intratumoral effect Effects 0.000 description 2
- 229960005386 ipilimumab Drugs 0.000 description 2
- 238000007834 ligase chain reaction Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 210000001165 lymph node Anatomy 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
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 229960004961 mechlorethamine Drugs 0.000 description 2
- HAWPXGHAZFHHAD-UHFFFAOYSA-N mechlorethamine Chemical compound ClCCN(C)CCCl HAWPXGHAZFHHAD-UHFFFAOYSA-N 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 208000023356 medullary thyroid gland carcinoma Diseases 0.000 description 2
- 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 2
- 229960000485 methotrexate Drugs 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
- 230000000877 morphologic effect Effects 0.000 description 2
- 239000002077 nanosphere Substances 0.000 description 2
- 210000000822 natural killer cell Anatomy 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 238000007899 nucleic acid hybridization Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 210000003463 organelle Anatomy 0.000 description 2
- 230000002611 ovarian Effects 0.000 description 2
- YPZRWBKMTBYPTK-UHFFFAOYSA-N oxidized gamma-L-glutamyl-L-cysteinylglycine Natural products OC(=O)C(N)CCC(=O)NC(C(=O)NCC(O)=O)CSSCC(C(=O)NCC(O)=O)NC(=O)CCC(N)C(O)=O YPZRWBKMTBYPTK-UHFFFAOYSA-N 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 201000002528 pancreatic cancer Diseases 0.000 description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 description 2
- 229960002621 pembrolizumab Drugs 0.000 description 2
- 238000002823 phage display Methods 0.000 description 2
- 229950010773 pidilizumab Drugs 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 229960003171 plicamycin Drugs 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 208000016800 primary central nervous system lymphoma Diseases 0.000 description 2
- CPTBDICYNRMXFX-UHFFFAOYSA-N procarbazine Chemical compound CNNCC1=CC=C(C(=O)NC(C)C)C=C1 CPTBDICYNRMXFX-UHFFFAOYSA-N 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 201000009410 rhabdomyosarcoma Diseases 0.000 description 2
- 239000002336 ribonucleotide Substances 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 230000003007 single stranded DNA break Effects 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 238000005063 solubilization Methods 0.000 description 2
- 230000007928 solubilization Effects 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 201000011549 stomach cancer Diseases 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- WYWHKKSPHMUBEB-UHFFFAOYSA-N tioguanine Chemical compound N1C(N)=NC(=S)C2=C1N=CN2 WYWHKKSPHMUBEB-UHFFFAOYSA-N 0.000 description 2
- UCFGDBYHRUNTLO-QHCPKHFHSA-N topotecan Chemical compound C1=C(O)C(CN(C)C)=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 UCFGDBYHRUNTLO-QHCPKHFHSA-N 0.000 description 2
- 229960005267 tositumomab Drugs 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 241000701447 unidentified baculovirus Species 0.000 description 2
- 229940035893 uracil Drugs 0.000 description 2
- 210000003708 urethra Anatomy 0.000 description 2
- 210000003932 urinary bladder Anatomy 0.000 description 2
- 210000002229 urogenital system Anatomy 0.000 description 2
- 208000007089 vaccinia Diseases 0.000 description 2
- JXLYSJRDGCGARV-CFWMRBGOSA-N vinblastine 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-CFWMRBGOSA-N 0.000 description 2
- 229960004528 vincristine Drugs 0.000 description 2
- 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 2
- 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 2
- 230000003442 weekly effect Effects 0.000 description 2
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 1
- LOGFVTREOLYCPF-KXNHARMFSA-N (2s,3r)-2-[[(2r)-1-[(2s)-2,6-diaminohexanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxybutanoic acid Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-KXNHARMFSA-N 0.000 description 1
- 102100025573 1-alkyl-2-acetylglycerophosphocholine esterase Human genes 0.000 description 1
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 1
- BFPYWIDHMRZLRN-UHFFFAOYSA-N 17alpha-ethynyl estradiol Natural products OC1=CC=C2C3CCC(C)(C(CC4)(O)C#C)C4C3CCC2=C1 BFPYWIDHMRZLRN-UHFFFAOYSA-N 0.000 description 1
- HLYBTPMYFWWNJN-UHFFFAOYSA-N 2-(2,4-dioxo-1h-pyrimidin-5-yl)-2-hydroxyacetic acid Chemical compound OC(=O)C(O)C1=CNC(=O)NC1=O HLYBTPMYFWWNJN-UHFFFAOYSA-N 0.000 description 1
- SGAKLDIYNFXTCK-UHFFFAOYSA-N 2-[(2,4-dioxo-1h-pyrimidin-5-yl)methylamino]acetic acid Chemical compound OC(=O)CNCC1=CNC(=O)NC1=O SGAKLDIYNFXTCK-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
- GJAKJCICANKRFD-UHFFFAOYSA-N 4-acetyl-4-amino-1,3-dihydropyrimidin-2-one Chemical compound CC(=O)C1(N)NC(=O)NC=C1 GJAKJCICANKRFD-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- LMZHZBVAKAMCEG-FJGDRVTGSA-N 4-amino-1-[(2r,3r,4r,5r)-3-amino-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@](O)(N)[C@H](O)[C@@H](CO)O1 LMZHZBVAKAMCEG-FJGDRVTGSA-N 0.000 description 1
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- OVONXEQGWXGFJD-UHFFFAOYSA-N 4-sulfanylidene-1h-pyrimidin-2-one Chemical compound SC=1C=CNC(=O)N=1 OVONXEQGWXGFJD-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
- VKLFQTYNHLDMDP-PNHWDRBUSA-N 5-carboxymethylaminomethyl-2-thiouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=S)NC(=O)C(CNCC(O)=O)=C1 VKLFQTYNHLDMDP-PNHWDRBUSA-N 0.000 description 1
- ZFTBZKVVGZNMJR-UHFFFAOYSA-N 5-chlorouracil Chemical compound ClC1=CNC(=O)NC1=O ZFTBZKVVGZNMJR-UHFFFAOYSA-N 0.000 description 1
- XZIIFPSPUDAGJM-UHFFFAOYSA-N 6-chloro-2-n,2-n-diethylpyrimidine-2,4-diamine Chemical compound CCN(CC)C1=NC(N)=CC(Cl)=N1 XZIIFPSPUDAGJM-UHFFFAOYSA-N 0.000 description 1
- 208000002008 AIDS-Related Lymphoma Diseases 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 1
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 208000009746 Adult T-Cell Leukemia-Lymphoma Diseases 0.000 description 1
- 208000016683 Adult T-cell leukemia/lymphoma Diseases 0.000 description 1
- IPWKGIFRRBGCJO-IMJSIDKUSA-N Ala-Ser Chemical compound C[C@H]([NH3+])C(=O)N[C@@H](CO)C([O-])=O IPWKGIFRRBGCJO-IMJSIDKUSA-N 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 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
- 241000710929 Alphavirus Species 0.000 description 1
- 206010073478 Anaplastic large-cell lymphoma Diseases 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- 101100067974 Arabidopsis thaliana POP2 gene Proteins 0.000 description 1
- JQFZHHSQMKZLRU-IUCAKERBSA-N Arg-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N JQFZHHSQMKZLRU-IUCAKERBSA-N 0.000 description 1
- CKAJHWFHHFSCDT-WHFBIAKZSA-N Asp-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O CKAJHWFHHFSCDT-WHFBIAKZSA-N 0.000 description 1
- 108010024976 Asparaginase Proteins 0.000 description 1
- 206010003571 Astrocytoma Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 102100029822 B- and T-lymphocyte attenuator Human genes 0.000 description 1
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 1
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 1
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- 206010004146 Basal cell carcinoma Diseases 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- 241000701822 Bovine papillomavirus Species 0.000 description 1
- CIUUIPMOFZIWIZ-UHFFFAOYSA-N Bropirimine Chemical compound NC1=NC(O)=C(Br)C(C=2C=CC=CC=2)=N1 CIUUIPMOFZIWIZ-UHFFFAOYSA-N 0.000 description 1
- 241000208199 Buxus sempervirens Species 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 102100024217 CAMPATH-1 antigen Human genes 0.000 description 1
- 102000039864 CD300 family Human genes 0.000 description 1
- 108091068264 CD300 family Proteins 0.000 description 1
- 101150013553 CD40 gene Proteins 0.000 description 1
- 108010065524 CD52 Antigen Proteins 0.000 description 1
- 101710157060 CMRF35-like molecule 1 Proteins 0.000 description 1
- 238000010354 CRISPR gene editing Methods 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
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- GAGWJHPBXLXJQN-UHFFFAOYSA-N Capecitabine Natural products C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1C1C(O)C(O)C(C)O1 GAGWJHPBXLXJQN-UHFFFAOYSA-N 0.000 description 1
- 206010007270 Carcinoid syndrome Diseases 0.000 description 1
- 206010007275 Carcinoid tumour Diseases 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- JWBOIMRXGHLCPP-UHFFFAOYSA-N Chloditan Chemical compound C=1C=CC=C(Cl)C=1C(C(Cl)Cl)C1=CC=C(Cl)C=C1 JWBOIMRXGHLCPP-UHFFFAOYSA-N 0.000 description 1
- 208000005243 Chondrosarcoma Diseases 0.000 description 1
- 208000006332 Choriocarcinoma Diseases 0.000 description 1
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 1
- PTOAARAWEBMLNO-KVQBGUIXSA-N Cladribine Chemical compound C1=NC=2C(N)=NC(Cl)=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 PTOAARAWEBMLNO-KVQBGUIXSA-N 0.000 description 1
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 1
- 241000938605 Crocodylia Species 0.000 description 1
- 239000004971 Cross linker Substances 0.000 description 1
- 102100021906 Cyclin-O Human genes 0.000 description 1
- YXQDRIRSAHTJKM-IMJSIDKUSA-N Cys-Ser Chemical compound SC[C@H](N)C(=O)N[C@@H](CO)C(O)=O YXQDRIRSAHTJKM-IMJSIDKUSA-N 0.000 description 1
- 101710112752 Cytotoxin Proteins 0.000 description 1
- 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 1
- 239000012625 DNA intercalator Substances 0.000 description 1
- 230000007067 DNA methylation Effects 0.000 description 1
- 230000007023 DNA restriction-modification system Effects 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 108010092160 Dactinomycin Proteins 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- 208000001976 Endocrine Gland Neoplasms Diseases 0.000 description 1
- 241000991587 Enterovirus C Species 0.000 description 1
- 206010014967 Ependymoma Diseases 0.000 description 1
- 208000031637 Erythroblastic Acute Leukemia Diseases 0.000 description 1
- 208000036566 Erythroleukaemia Diseases 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- BFPYWIDHMRZLRN-SLHNCBLASA-N Ethinyl estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@](CC4)(O)C#C)[C@@H]4[C@@H]3CCC2=C1 BFPYWIDHMRZLRN-SLHNCBLASA-N 0.000 description 1
- 229910052693 Europium Inorganic materials 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 108091092566 Extrachromosomal DNA Proteins 0.000 description 1
- 201000008808 Fibrosarcoma Diseases 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 description 1
- 208000032612 Glial tumor Diseases 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- FYYSIASRLDJUNP-WHFBIAKZSA-N Glu-Asp Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(O)=O FYYSIASRLDJUNP-WHFBIAKZSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 1
- 102000006354 HLA-DR Antigens Human genes 0.000 description 1
- 108010058597 HLA-DR Antigens Proteins 0.000 description 1
- 208000009889 Herpes Simplex Diseases 0.000 description 1
- WSDOHRLQDGAOGU-BQBZGAKWSA-N His-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CN=CN1 WSDOHRLQDGAOGU-BQBZGAKWSA-N 0.000 description 1
- 102000018713 Histocompatibility Antigens Class II Human genes 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 208000021519 Hodgkin lymphoma Diseases 0.000 description 1
- 101000864344 Homo sapiens B- and T-lymphocyte attenuator Proteins 0.000 description 1
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 1
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 1
- 101000897441 Homo sapiens Cyclin-O Proteins 0.000 description 1
- 101100118549 Homo sapiens EGFR gene Proteins 0.000 description 1
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 1
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 1
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 1
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 1
- 101001137987 Homo sapiens Lymphocyte activation gene 3 protein Proteins 0.000 description 1
- 101001063392 Homo sapiens Lymphocyte function-associated antigen 3 Proteins 0.000 description 1
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 1
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 1
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 1
- 101000831496 Homo sapiens Toll-like receptor 3 Proteins 0.000 description 1
- 101000669402 Homo sapiens Toll-like receptor 7 Proteins 0.000 description 1
- 101000617285 Homo sapiens Tyrosine-protein phosphatase non-receptor type 6 Proteins 0.000 description 1
- 101100321817 Human parvovirus B19 (strain HV) 7.5K gene Proteins 0.000 description 1
- DOMWKUIIPQCAJU-LJHIYBGHSA-N Hydroxyprogesterone caproate Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(C)=O)(OC(=O)CCCCC)[C@@]1(C)CC2 DOMWKUIIPQCAJU-LJHIYBGHSA-N 0.000 description 1
- XDXDZDZNSLXDNA-TZNDIEGXSA-N Idarubicin Chemical compound C1[C@H](N)[C@H](O)[C@H](C)O[C@H]1O[C@@H]1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2C[C@@](O)(C(C)=O)C1 XDXDZDZNSLXDNA-TZNDIEGXSA-N 0.000 description 1
- XDXDZDZNSLXDNA-UHFFFAOYSA-N Idarubicin Natural products C1C(N)C(O)C(C)OC1OC1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2CC(O)(C(C)=O)C1 XDXDZDZNSLXDNA-UHFFFAOYSA-N 0.000 description 1
- 102000037982 Immune checkpoint proteins Human genes 0.000 description 1
- 108091008036 Immune checkpoint proteins Proteins 0.000 description 1
- 108010009817 Immunoglobulin Constant Regions Proteins 0.000 description 1
- 102000009786 Immunoglobulin Constant Regions Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 1
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 1
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 1
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 1
- 102000013463 Immunoglobulin Light Chains Human genes 0.000 description 1
- 108010065825 Immunoglobulin Light Chains Proteins 0.000 description 1
- 102100026720 Interferon beta Human genes 0.000 description 1
- 108090000467 Interferon-beta Proteins 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 102000003777 Interleukin-1 beta Human genes 0.000 description 1
- 108090000193 Interleukin-1 beta Proteins 0.000 description 1
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 1
- 206010061252 Intraocular melanoma Diseases 0.000 description 1
- 208000007766 Kaposi sarcoma Diseases 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- TYYLDKGBCJGJGW-UHFFFAOYSA-N L-tryptophan-L-tyrosine Natural products C=1NC2=CC=CC=C2C=1CC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 TYYLDKGBCJGJGW-UHFFFAOYSA-N 0.000 description 1
- 239000005411 L01XE02 - Gefitinib Substances 0.000 description 1
- 239000005551 L01XE03 - Erlotinib Substances 0.000 description 1
- 239000002136 L01XE07 - Lapatinib Substances 0.000 description 1
- 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 1
- 208000031671 Large B-Cell Diffuse Lymphoma Diseases 0.000 description 1
- 208000032004 Large-Cell Anaplastic Lymphoma Diseases 0.000 description 1
- 208000018142 Leiomyosarcoma Diseases 0.000 description 1
- 206010024305 Leukaemia monocytic Diseases 0.000 description 1
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 1
- 102100030984 Lymphocyte function-associated antigen 3 Human genes 0.000 description 1
- NPBGTPKLVJEOBE-IUCAKERBSA-N Lys-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CCCNC(N)=N NPBGTPKLVJEOBE-IUCAKERBSA-N 0.000 description 1
- 108091054438 MHC class II family Proteins 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 208000005410 Mediastinal Neoplasms Diseases 0.000 description 1
- 208000007054 Medullary Carcinoma Diseases 0.000 description 1
- 208000037196 Medullary thyroid carcinoma Diseases 0.000 description 1
- 208000000172 Medulloblastoma Diseases 0.000 description 1
- 102000003792 Metallothionein Human genes 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 102000029749 Microtubule Human genes 0.000 description 1
- 108091022875 Microtubule Proteins 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 1
- 102100025243 Myeloid cell surface antigen CD33 Human genes 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
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 206010029098 Neoplasm skin Diseases 0.000 description 1
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 1
- 208000009905 Neurofibromatoses Diseases 0.000 description 1
- 201000010133 Oligodendroglioma Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 206010033701 Papillary thyroid cancer Diseases 0.000 description 1
- 206010034016 Paronychia Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 208000002471 Penile Neoplasms Diseases 0.000 description 1
- PYOHODCEOHCZBM-RYUDHWBXSA-N Phe-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 PYOHODCEOHCZBM-RYUDHWBXSA-N 0.000 description 1
- 208000007641 Pinealoma Diseases 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 101100123851 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) HER1 gene Proteins 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 201000010208 Seminoma Diseases 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- LDEBVRIURYMKQS-WISUUJSJSA-N Ser-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](N)CO LDEBVRIURYMKQS-WISUUJSJSA-N 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000710960 Sindbis virus Species 0.000 description 1
- 206010041067 Small cell lung cancer Diseases 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 108091027544 Subgenomic mRNA Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 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 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 230000037453 T cell priming Effects 0.000 description 1
- 206010042971 T-cell lymphoma Diseases 0.000 description 1
- 208000027585 T-cell non-Hodgkin lymphoma Diseases 0.000 description 1
- 229940123237 Taxane Drugs 0.000 description 1
- GKLVYJBZJHMRIY-OUBTZVSYSA-N Technetium-99 Chemical compound [99Tc] GKLVYJBZJHMRIY-OUBTZVSYSA-N 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- GXDLGHLJTHMDII-WISUUJSJSA-N Thr-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(O)=O GXDLGHLJTHMDII-WISUUJSJSA-N 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 108010060818 Toll-Like Receptor 9 Proteins 0.000 description 1
- 102100024324 Toll-like receptor 3 Human genes 0.000 description 1
- 102100039390 Toll-like receptor 7 Human genes 0.000 description 1
- 102100033117 Toll-like receptor 9 Human genes 0.000 description 1
- 108091028113 Trans-activating crRNA Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- TYYLDKGBCJGJGW-WMZOPIPTSA-N Trp-Tyr Chemical compound C([C@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)N)C(O)=O)C1=CC=C(O)C=C1 TYYLDKGBCJGJGW-WMZOPIPTSA-N 0.000 description 1
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 1
- 108091005906 Type I transmembrane proteins Proteins 0.000 description 1
- BMPPMAOOKQJYIP-WMZOPIPTSA-N Tyr-Trp Chemical compound C([C@H]([NH3+])C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C([O-])=O)C1=CC=C(O)C=C1 BMPPMAOOKQJYIP-WMZOPIPTSA-N 0.000 description 1
- 102100021657 Tyrosine-protein phosphatase non-receptor type 6 Human genes 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- 201000005969 Uveal melanoma Diseases 0.000 description 1
- 208000014070 Vestibular schwannoma Diseases 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
- 229940122803 Vinca alkaloid Drugs 0.000 description 1
- 208000033559 Waldenström macroglobulinemia Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- VWQVUPCCIRVNHF-OUBTZVSYSA-N Yttrium-90 Chemical compound [90Y] VWQVUPCCIRVNHF-OUBTZVSYSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- SWPYNTWPIAZGLT-UHFFFAOYSA-N [amino(ethoxy)phosphanyl]oxyethane Chemical compound CCOP(N)OCC SWPYNTWPIAZGLT-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 208000004064 acoustic neuroma Diseases 0.000 description 1
- 208000017733 acquired polycythemia vera Diseases 0.000 description 1
- 125000000641 acridinyl group Chemical group C1(=CC=CC2=NC3=CC=CC=C3C=C12)* 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
- 208000021841 acute erythroid leukemia Diseases 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 230000004721 adaptive immunity Effects 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 210000004404 adrenal cortex Anatomy 0.000 description 1
- 229940009456 adriamycin Drugs 0.000 description 1
- 201000006966 adult T-cell leukemia Diseases 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 229960000548 alemtuzumab Drugs 0.000 description 1
- 229940098174 alkeran Drugs 0.000 description 1
- 229940045714 alkyl sulfonate alkylating agent Drugs 0.000 description 1
- 150000008052 alkyl sulfonates Chemical class 0.000 description 1
- 230000029936 alkylation Effects 0.000 description 1
- 238000005804 alkylation reaction Methods 0.000 description 1
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- ROBVIMPUHSLWNV-UHFFFAOYSA-N aminoglutethimide Chemical compound C=1C=C(N)C=CC=1C1(CC)CCC(=O)NC1=O ROBVIMPUHSLWNV-UHFFFAOYSA-N 0.000 description 1
- 229960003437 aminoglutethimide Drugs 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 239000003098 androgen Substances 0.000 description 1
- 229940030486 androgens Drugs 0.000 description 1
- 239000004037 angiogenesis inhibitor Substances 0.000 description 1
- 229940121369 angiogenesis inhibitor Drugs 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940046836 anti-estrogen Drugs 0.000 description 1
- 230000001833 anti-estrogenic effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 238000011319 anticancer therapy Methods 0.000 description 1
- 102000025171 antigen binding proteins Human genes 0.000 description 1
- 108091000831 antigen binding proteins Proteins 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 229940045687 antimetabolites folic acid analogs Drugs 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 229940045719 antineoplastic alkylating agent nitrosoureas Drugs 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 238000002617 apheresis Methods 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- 108010062796 arginyllysine Proteins 0.000 description 1
- 108010038633 aspartylglutamate Proteins 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 229940120638 avastin Drugs 0.000 description 1
- DVQHYTBCTGYNNN-UHFFFAOYSA-N azane;cyclobutane-1,1-dicarboxylic acid;platinum Chemical compound N.N.[Pt].OC(=O)C1(C(O)=O)CCC1 DVQHYTBCTGYNNN-UHFFFAOYSA-N 0.000 description 1
- 239000003855 balanced salt solution Substances 0.000 description 1
- BLFLLBZGZJTVJG-UHFFFAOYSA-N benzocaine Chemical compound CCOC(=O)C1=CC=C(N)C=C1 BLFLLBZGZJTVJG-UHFFFAOYSA-N 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 229960000397 bevacizumab Drugs 0.000 description 1
- 229940108502 bicnu Drugs 0.000 description 1
- 201000007180 bile duct carcinoma Diseases 0.000 description 1
- 210000003445 biliary tract Anatomy 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 238000001815 biotherapy Methods 0.000 description 1
- 201000001531 bladder carcinoma Diseases 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
- 230000036760 body temperature Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- 208000014581 breast ductal adenocarcinoma Diseases 0.000 description 1
- 201000010983 breast ductal carcinoma Diseases 0.000 description 1
- 201000003714 breast lobular carcinoma Diseases 0.000 description 1
- 208000003362 bronchogenic carcinoma Diseases 0.000 description 1
- 229950009494 bropirimine Drugs 0.000 description 1
- 235000010633 broth Nutrition 0.000 description 1
- GMRQFYUYWCNGIN-NKMMMXOESA-N calcitriol Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](CCCC(C)(C)O)C)=C\C=C1\C[C@@H](O)C[C@H](O)C1=C GMRQFYUYWCNGIN-NKMMMXOESA-N 0.000 description 1
- 229960005084 calcitriol Drugs 0.000 description 1
- 235000020964 calcitriol Nutrition 0.000 description 1
- 239000011612 calcitriol Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- HXCHCVDVKSCDHU-LULTVBGHSA-N calicheamicin Chemical compound C1[C@H](OC)[C@@H](NCC)CO[C@H]1O[C@H]1[C@H](O[C@@H]2C\3=C(NC(=O)OC)C(=O)C[C@](C/3=C/CSSSC)(O)C#C\C=C/C#C2)O[C@H](C)[C@@H](NO[C@@H]2O[C@H](C)[C@@H](SC(=O)C=3C(=C(OC)C(O[C@H]4[C@@H]([C@H](OC)[C@@H](O)[C@H](C)O4)O)=C(I)C=3C)OC)[C@@H](O)C2)[C@@H]1O HXCHCVDVKSCDHU-LULTVBGHSA-N 0.000 description 1
- 229930195731 calicheamicin Natural products 0.000 description 1
- 229940112129 campath Drugs 0.000 description 1
- 229940088954 camptosar Drugs 0.000 description 1
- 238000002619 cancer immunotherapy Methods 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 229960004117 capecitabine Drugs 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 235000013877 carbamide Nutrition 0.000 description 1
- 125000000837 carbohydrate group Chemical group 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 231100000357 carcinogen Toxicity 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 208000002458 carcinoid tumor Diseases 0.000 description 1
- 229960005243 carmustine Drugs 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 208000019065 cervical carcinoma Diseases 0.000 description 1
- 229960005395 cetuximab Drugs 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 230000000973 chemotherapeutic effect Effects 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 229960004630 chlorambucil Drugs 0.000 description 1
- JCKYGMPEJWAADB-UHFFFAOYSA-N chlorambucil Chemical compound OC(=O)CCCC1=CC=C(N(CCCl)CCCl)C=C1 JCKYGMPEJWAADB-UHFFFAOYSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 208000024207 chronic leukemia Diseases 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 229960002436 cladribine Drugs 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 239000008119 colloidal silica Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000009096 combination chemotherapy Methods 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 201000007241 cutaneous T cell lymphoma Diseases 0.000 description 1
- 208000030381 cutaneous melanoma Diseases 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 229960000684 cytarabine Drugs 0.000 description 1
- 108010057085 cytokine receptors Proteins 0.000 description 1
- 230000002380 cytological effect Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 229960003901 dacarbazine Drugs 0.000 description 1
- 229960002806 daclizumab Drugs 0.000 description 1
- 229960000640 dactinomycin Drugs 0.000 description 1
- 230000009615 deamination Effects 0.000 description 1
- 238000006481 deamination reaction Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 239000003398 denaturant Substances 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
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- RGLYKWWBQGJZGM-ISLYRVAYSA-N diethylstilbestrol Chemical compound C=1C=C(O)C=CC=1C(/CC)=C(\CC)C1=CC=C(O)C=C1 RGLYKWWBQGJZGM-ISLYRVAYSA-N 0.000 description 1
- 229960000452 diethylstilbestrol Drugs 0.000 description 1
- 206010012818 diffuse large B-cell lymphoma Diseases 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 150000002019 disulfides Chemical group 0.000 description 1
- 229960003668 docetaxel Drugs 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 201000011523 endocrine gland cancer Diseases 0.000 description 1
- 230000002357 endometrial effect Effects 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 229960001433 erlotinib Drugs 0.000 description 1
- AAKJLRGGTJKAMG-UHFFFAOYSA-N erlotinib Chemical compound C=12C=C(OCCOC)C(OCCOC)=CC2=NC=NC=1NC1=CC=CC(C#C)=C1 AAKJLRGGTJKAMG-UHFFFAOYSA-N 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 239000000328 estrogen antagonist Substances 0.000 description 1
- 229960002568 ethinylestradiol Drugs 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
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 230000012953 feeding on blood of other organism Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229960000390 fludarabine Drugs 0.000 description 1
- GIUYCYHIANZCFB-FJFJXFQQSA-N fludarabine phosphate Chemical compound C1=NC=2C(N)=NC(F)=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O GIUYCYHIANZCFB-FJFJXFQQSA-N 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- YLRFCQOZQXIBAB-RBZZARIASA-N fluoxymesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1CC[C@](C)(O)[C@@]1(C)C[C@@H]2O YLRFCQOZQXIBAB-RBZZARIASA-N 0.000 description 1
- 229960001751 fluoxymesterone Drugs 0.000 description 1
- 150000002224 folic acids Chemical class 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 1
- 229940044627 gamma-interferon Drugs 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 229960002584 gefitinib Drugs 0.000 description 1
- XGALLCVXEZPNRQ-UHFFFAOYSA-N gefitinib Chemical compound C=12C=C(OCCCN3CCOCC3)C(OC)=CC2=NC=NC=1NC1=CC=C(F)C(Cl)=C1 XGALLCVXEZPNRQ-UHFFFAOYSA-N 0.000 description 1
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 description 1
- 229960000578 gemtuzumab Drugs 0.000 description 1
- 229960003297 gemtuzumab ozogamicin Drugs 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 208000003884 gestational trophoblastic disease Diseases 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 201000009277 hairy cell leukemia Diseases 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 208000025750 heavy chain disease Diseases 0.000 description 1
- 201000002222 hemangioblastoma Diseases 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 238000011577 humanized mouse model Methods 0.000 description 1
- 229940088013 hycamtin Drugs 0.000 description 1
- 229940096120 hydrea Drugs 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 229960001330 hydroxycarbamide Drugs 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 229950000801 hydroxyprogesterone caproate Drugs 0.000 description 1
- 210000003026 hypopharynx Anatomy 0.000 description 1
- 229960000908 idarubicin Drugs 0.000 description 1
- 229960001101 ifosfamide Drugs 0.000 description 1
- HOMGKSMUEGBAAB-UHFFFAOYSA-N ifosfamide Chemical compound ClCCNP1(=O)OCCCN1CCCl HOMGKSMUEGBAAB-UHFFFAOYSA-N 0.000 description 1
- 229960002411 imatinib Drugs 0.000 description 1
- KTUFNOKKBVMGRW-UHFFFAOYSA-N imatinib Chemical compound C1CN(C)CCN1CC1=CC=C(C(=O)NC=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)C=C1 KTUFNOKKBVMGRW-UHFFFAOYSA-N 0.000 description 1
- 229940124669 imidazoquinoline Drugs 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 230000005746 immune checkpoint blockade Effects 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 230000008629 immune suppression Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000010324 immunological assay Methods 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 239000003022 immunostimulating agent Substances 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 229960003130 interferon gamma Drugs 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 206010073095 invasive ductal breast carcinoma Diseases 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229960004768 irinotecan Drugs 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 229960004891 lapatinib Drugs 0.000 description 1
- BCFGMOOMADDAQU-UHFFFAOYSA-N lapatinib Chemical compound O1C(CNCCS(=O)(=O)C)=CC=C1C1=CC=C(N=CN=C2NC=3C=C(Cl)C(OCC=4C=C(F)C=CC=4)=CC=3)C2=C1 BCFGMOOMADDAQU-UHFFFAOYSA-N 0.000 description 1
- 210000000867 larynx Anatomy 0.000 description 1
- 125000003473 lipid group Chemical group 0.000 description 1
- 206010024627 liposarcoma Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229960002247 lomustine Drugs 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 101150030901 mab-21 gene Proteins 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 208000026037 malignant tumor of neck Diseases 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 229950008001 matuzumab Drugs 0.000 description 1
- 201000000349 mediastinal cancer Diseases 0.000 description 1
- 210000001370 mediastinum Anatomy 0.000 description 1
- 229960002985 medroxyprogesterone acetate Drugs 0.000 description 1
- PSGAAPLEWMOORI-PEINSRQWSA-N medroxyprogesterone acetate Chemical compound C([C@@]12C)CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2CC[C@]2(C)[C@@](OC(C)=O)(C(C)=O)CC[C@H]21 PSGAAPLEWMOORI-PEINSRQWSA-N 0.000 description 1
- 229960001924 melphalan Drugs 0.000 description 1
- 206010027191 meningioma Diseases 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
- 208000037819 metastatic cancer Diseases 0.000 description 1
- 208000011575 metastatic malignant neoplasm Diseases 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 210000004688 microtubule Anatomy 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- 229960000350 mitotane Drugs 0.000 description 1
- 229960001156 mitoxantrone Drugs 0.000 description 1
- KKZJGLLVHKMTCM-UHFFFAOYSA-N mitoxantrone Chemical compound O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO KKZJGLLVHKMTCM-UHFFFAOYSA-N 0.000 description 1
- 230000007193 modulation by symbiont of host erythrocyte aggregation Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 201000006894 monocytic leukemia Diseases 0.000 description 1
- HDZGCSFEDULWCS-UHFFFAOYSA-N monomethylhydrazine Chemical class CNN HDZGCSFEDULWCS-UHFFFAOYSA-N 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 206010051747 multiple endocrine neoplasia Diseases 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 201000005962 mycosis fungoides Diseases 0.000 description 1
- 208000001611 myxosarcoma Diseases 0.000 description 1
- 210000003928 nasal cavity Anatomy 0.000 description 1
- 210000001989 nasopharynx Anatomy 0.000 description 1
- 210000000581 natural killer T-cell Anatomy 0.000 description 1
- 229940086322 navelbine Drugs 0.000 description 1
- 210000003739 neck Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 230000017066 negative regulation of growth Effects 0.000 description 1
- 208000025189 neoplasm of testis Diseases 0.000 description 1
- 238000010984 neurological examination Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 229950010203 nimotuzumab Drugs 0.000 description 1
- 230000006780 non-homologous end joining Effects 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 210000001331 nose Anatomy 0.000 description 1
- 239000011824 nuclear material Substances 0.000 description 1
- 239000003865 nucleic acid synthesis inhibitor Substances 0.000 description 1
- 201000002575 ocular melanoma Diseases 0.000 description 1
- 210000003300 oropharynx Anatomy 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 210000003101 oviduct Anatomy 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 239000006179 pH buffering agent Substances 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 230000000242 pagocytic effect Effects 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000009996 pancreatic endocrine effect Effects 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 208000004019 papillary adenocarcinoma Diseases 0.000 description 1
- 201000010198 papillary carcinoma Diseases 0.000 description 1
- 208000007312 paraganglioma Diseases 0.000 description 1
- 210000003695 paranasal sinus Anatomy 0.000 description 1
- 230000000849 parathyroid Effects 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 210000003899 penis Anatomy 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
- 229940023041 peptide vaccine Drugs 0.000 description 1
- 229960002087 pertuzumab Drugs 0.000 description 1
- 208000028591 pheochromocytoma Diseases 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 208000024724 pineal body neoplasm Diseases 0.000 description 1
- 201000004123 pineal gland cancer Diseases 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- YIQPUIGJQJDJOS-UHFFFAOYSA-N plerixafor Chemical compound C=1C=C(CN2CCNCCCNCCNCCC2)C=CC=1CN1CCCNCCNCCCNCC1 YIQPUIGJQJDJOS-UHFFFAOYSA-N 0.000 description 1
- 229960002169 plerixafor Drugs 0.000 description 1
- 208000037244 polycythemia vera Diseases 0.000 description 1
- 229940115272 polyinosinic:polycytidylic acid Drugs 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 235000020004 porter Nutrition 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 239000000843 powder Substances 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
- 238000004237 preparative chromatography Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 208000025638 primary cutaneous T-cell non-Hodgkin lymphoma Diseases 0.000 description 1
- 229960000624 procarbazine Drugs 0.000 description 1
- 239000000583 progesterone congener Substances 0.000 description 1
- 230000005522 programmed cell death Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 201000001514 prostate carcinoma Diseases 0.000 description 1
- 229940048914 protamine Drugs 0.000 description 1
- 210000004777 protein coat Anatomy 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 108010043277 recombinant soluble CD4 Proteins 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 230000008521 reorganization Effects 0.000 description 1
- 238000010405 reoxidation reaction Methods 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 230000002245 ribonucleolytic effect Effects 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 201000008407 sebaceous adenocarcinoma Diseases 0.000 description 1
- 238000007560 sedimentation technique Methods 0.000 description 1
- 229960003440 semustine Drugs 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 201000003708 skin melanoma Diseases 0.000 description 1
- 208000000587 small cell lung carcinoma Diseases 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 229940035044 sorbitan monolaurate Drugs 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000008174 sterile solution Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000010907 stover Substances 0.000 description 1
- 229960001052 streptozocin Drugs 0.000 description 1
- ZSJLQEPLLKMAKR-GKHCUFPYSA-N streptozocin Chemical compound O=NN(C)C(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O ZSJLQEPLLKMAKR-GKHCUFPYSA-N 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000003774 sulfhydryl reagent Substances 0.000 description 1
- 229910021653 sulphate ion Inorganic materials 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 201000010965 sweat gland carcinoma Diseases 0.000 description 1
- 206010042863 synovial sarcoma Diseases 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 101150047061 tag-72 gene Proteins 0.000 description 1
- 229960001603 tamoxifen Drugs 0.000 description 1
- 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 1
- 229940063683 taxotere Drugs 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
- 230000002381 testicular Effects 0.000 description 1
- 201000003120 testicular cancer Diseases 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 238000011287 therapeutic dose Methods 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 208000013818 thyroid gland medullary carcinoma Diseases 0.000 description 1
- 208000030045 thyroid gland papillary carcinoma Diseases 0.000 description 1
- 208000013076 thyroid tumor Diseases 0.000 description 1
- 229960003087 tioguanine Drugs 0.000 description 1
- 239000003104 tissue culture media Substances 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 229960000303 topotecan Drugs 0.000 description 1
- 238000006257 total synthesis reaction Methods 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 229960000575 trastuzumab Drugs 0.000 description 1
- 229950007217 tremelimumab 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
- 108010044292 tryptophyltyrosine Proteins 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 231100000402 unacceptable toxicity Toxicity 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 229960001055 uracil mustard Drugs 0.000 description 1
- 150000003672 ureas Chemical class 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 208000010570 urinary bladder carcinoma Diseases 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 229960003048 vinblastine Drugs 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
- CILBMBUYJCWATM-PYGJLNRPSA-N vinorelbine ditartrate Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O.OC(=O)[C@H](O)[C@@H](O)C(O)=O.C1N(CC=2C3=CC=CC=C3NC=22)CC(CC)=C[C@H]1C[C@]2(C(=O)OC)C1=CC([C@]23[C@H]([C@@]([C@H](OC(C)=O)[C@]4(CC)C=CCN([C@H]34)CC2)(O)C(=O)OC)N2C)=C2C=C1OC CILBMBUYJCWATM-PYGJLNRPSA-N 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 230000017613 viral reproduction Effects 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 210000003905 vulva Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 229940053867 xeloda Drugs 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 229940055760 yervoy Drugs 0.000 description 1
- 229950008250 zalutumumab Drugs 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
Definitions
- This relates to the field of oncology, specifically to the use of CD300f inhibitors and/or the use of dendritic cells with modified genes encoding CD300f, for the treatment of solid tumors.
- Cancer immunotherapy aims to enhance the ability of the patients' own immune response to destroy tumors.
- the magnitude of immune response is determined by the balance between immune activating signals and negative inhibitory signals.
- Checkpoint receptors encompass a specific subset of negative regulators that normally deliver inhibitory signals that dampen stimulatory signals and limit immune activation. Blockade of immune checkpoints represents an effective strategy to enhance the immune response against cancer cells.
- Several checkpoint receptors including cytotoxic T lymphocyte associated protein-4 (CTLA-4) and Programmed Death (PD)-l, have been identified on T cells and targeting these with blocking antibodies has been successful in treating different cancer types (e.g., melanoma, bladder, and gastric cancer).
- CTLA-4 cytotoxic T lymphocyte associated protein-4
- PD Programmed Death
- multiple cancers such as pancreatic and prostate cancer, are resistant to T cell checkpoint blocking, underscoring the importance of identifying novel checkpoints on immune cells for successful cancer therapy.
- the PS -recognizing receptor, CD300f expressed on myeloid cells (i.e., dendritic cells and macrophages) and a small sub-population of B cells in mice, regulates phagocytosis of apoptotic cells by macrophages and dendritic cells, thereby controlling inflammatory immune responses (Tian et al., Nat Commun 5:3146, 2014; Tian et al., Cell Death Differ 23: 1086-96, 2016).
- the use of CD300f inhibitors for the treatment of tumors, such as a solid tumor is disclosed herein. Methods are also disclosed for targeting CD300f in dendritic cells, and the use of adoptive transfer of these dendritic cells or T cells activated by these dendritic cells for the treatment of solid tumors.
- methods for treating a subject with a solid tumor.
- the methods include administering to the subject a therapeutically effective amount of a CD300f inhibitor, thereby treating the solid tumor in the subject.
- methods for treating a solid tumor in a subject, wherein the methods include administering to the subject a therapeutically effective amount of dendritic cells comprising an inactivated gene encoding CD300f.
- methods are treating a solid tumor in a subject that include isolating dendritic cells from the subject and transforming the dendritic cells with one or more vectors comprising: a) a Embryonal Fyn- Associated Substrate (EFS) promoter operably linked to a nucleotide sequence encoding a Type ⁇ Cas9 nuclease, b) a U6 promoter operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas system guide RNAs that hybridize with the CD300f gene in the dendritic cell, wherein components (a) and (b) are located on same or different vectors.
- EFS Embryonal Fyn- Associated Substrate
- a ribonucleoprotein (RNP) complex can be utilized, wherein the guide RNA is attached to the protein and delivered to the dendritic cells.
- the one or more guide RNAs target the CD300f gene in the dendritic cell and the Cas9 protein cleaves the CD300f gene such that the sequence of the CD300f gene is modified or inactivated in the dendritic cell, thereby forming modified dendritic cells.
- the subject is administered a therapeutically effective amount of the modified dendritic cells, thereby treating the solid tumor in the subject.
- methods are disclosed treating a solid tumor in a subject that include isolating dendritic cells from the subject, and transforming the dendritic cells with one or more viral vectors comprising: a) a Embryonal Fyn- Associated Substrate (EFS) promoter operably linked to a nucleotide sequence encoding a Type II Cas9 nuclease, b) a U6 promoter operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas system guide RNAs that hybridize with the CD300f gene in the dendritic cell, wherein components (a) and (b) are located on same or different viral vectors.
- EFS Embryonal Fyn- Associated Substrate
- a RNP complex can be utilized, wherein the guide RNA is attached to the protein and delivered to the dendritic cells.
- the one or more guide RNAs target the CD300f gene in the dendritic cell and the Cas9 protein cleaves the CD300f gene such that the sequence of the CD300f gene is modified or inactivated in the dendritic cell, thereby forming modified dendritic cells.
- the modified dendritic cells are contacted with CD8 + T cells in the presence of tumor-associated antigens expressed by the solid tumor to form activated T cells. A therapeutically effective amount of the activated T cells is administered to the subject to treat the solid tumor.
- FIGS. 1A-1C Schematic overview of methods used to examine CD300f deficiency on solid tumor development in mice.
- A. Generation of Cd300f-/- mice on a C57BL/6 genetic background. Exon 2-3 of Cd300f gene was flanked by loxP sites.
- clones bearing the Cd300ffl/fl locus were established after deletion of PGK-neo selection cassette by Flp recombination, and clones with the Cd300f-/- locus were generated after deletion of the LoxP sites flanking regions together with the PGK-neo cassette using Cre recombinase.
- Identified targeted embryonic stem cell clones were microinjected into the blastocysts of C57BL/6 mice.
- B. Use the mouse model of tumor graft to study the effect of CD300f deficiency on solid tumor development.
- Cd300f +/+ or Cd300f-/- mice were subcutaneously inoculated with 10 6 solid tumor cells, for example EL4-TfOVA cells, on dayO. Some of the mice were systemically irradiated at 3.25 Gy on day 7 (IR) and some of the irradiated mice were transferred with 10 6 tumor antigen specific CD8 + T cells, for example OT-I T cells, on day 9 (OT- I). Irradiation was to deplete some existing immune cells and make space to allow for expansion of anti-tumor effectors. Tumor growth was measured on the indicated days; mice were euthanized when the longitudinal tumor diameter reached 15 mm. C. Use the mouse model of
- azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colon cancer to examine CD300f deficiency on solid tumor development.
- Cd300f +/+ or Cd300f-/- mice were injected with AOM intraperitoneally and then fed with DSS-containing water in three 7-day cycles, with intermittent 14-day intervals of regular drinking water. After 10 weeks, mice were euthanized, and colon tumor sizes were measured.
- FIGS. 2A-2D CD300f functions as a checkpoint for tumor immunity.
- mice subcutaneously inoculated with 10 6 EL4-TfOVA cells on day 0.
- 10 6 EL4-TfOVA cells on day 0.
- Some of the mice were systemically irradiated at 3.25 Gy on day 7 (IR) and some of the irradiated mice were transferred with 10 6 OT-I T cells on day 9 (OT-I).
- Irradiation was to deplete some existing immune cells and make space to allow for expansion of anti-tumor effectors. Tumor growth was measured on the indicated days; mice were euthanized when the longitudinal tumor diameter reached 15 mm.
- the graph on the left shows the progression of tumor growth in Cd300f+I+ and Cd300f -I- mice; the images on the right illustrate tumors dissected on day 18 from Cd300f+I+ and Cd300f-I- mice that were both irradiated and OT-I T cell-transferred.
- B Inhibition of growth of AOM/DSS-induced colorectal cancer in C ⁇ i?0O -deficient mice.
- Cd300f+I+ or Cd300f-I- mice were injected with AOM intraperitoneally and then fed with DSS-containing water in three 7-day cycles, with intermittent 14-day intervals of regular drinking water. After 10 weeks, mice were euthanized, and colon tumor sizes were measured.
- Cd300f+I+ and Cd300f- I- mice show tumors in the colons of Cd300f+I+ and Cd300f- I- mice; arrowheads indicate the position of tumors.
- the graph on the right shows quantification of tumor sizes in Cd300f+I+ and Cd300f-I- mice.
- Cd300f+I+ or Cd300f-I- mice were systemically irradiated at 3.25 Gy on day 1 and transferred with OT-I T cells (10 6 ) on day 3.
- FIGS. 3A-3B Inhibition of grafted tumor growth in CD300f-deficient mice.
- Cd300f+I+ or Cd300f-I- mice were subcutaneously inoculated with 10 6 MC38 cells on day 0. Tumor growth was monitored on the indicated days; mice were euthanized when the longitudinal tumor diameter reached 15 mm.
- A. The graph shows the progression of tumor growth in Cd300f+/+ and Cd300f-/- mice.
- B. The images show the tumors dissected on day 16 from Cd300f+/+ and Cd300f-/- mice.
- FIGS. 4A-4B Human CD300f recognizes PS and regulates the phagocytosis of apoptotic cells.
- A. Human CD300f binds PS by surface plasmon resonance analysis. ImM PS-containing liposomes were captured on the LI sensor chip, followed by injection of Fc-fused control protein ⁇ , mouse CD300f and human CD300f at 10 ⁇ g/ml. The sensorgrams show the resonance units over the indicated times.
- B. Human CD300f expression on L929 cells promotes phagocytosis of apoptotic cells.
- L929 cells transduced with empty virus (EV) or human CD300f, were mixed with pHrodo-labeled unirradiated or irradiated mouse thymocytes at a 1:3 ratio for the indicated times. Cells were suspended in the pH 8.8 buffer, and analyzed for the percentage of pHrodo+ cells, representing the cells that engulfed apoptotic cells.
- EV empty virus
- human CD300f human CD300f
- FIGS. 5A-5B Human CD300f is expressed on human myeloid cell populations including monocytes and immature dendritic cells (DCs).
- A Human peripheral blood mononuclear cells (PBMC) were isolated, stained with antibodies against human CD300f, CD3 (T cells), CD19 (B cells), CD56 (NK cells) and CD16 and CD14 (monocytes) and analyzed by flow cytometry. The Rl, R2, R3 and R4 were gated on the dot plots of forward scattered light (FSC) vs. side scattered light (SSC).
- FSC forward scattered light
- SSC side scattered light
- GM- CSF granulocyte macrophage colony stimulating factor
- IL interleukin
- nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
- sequence Listing is submitted as an ASCII text file [Sequence_Listing, October 11, 2017, 17.0KB], which is incorporated by reference herein. In the accompanying sequence listing:
- SEQ ID Nos: 1-8 are the amino acid sequences of human framework regions.
- SEQ ID NO: 9 is the amino acid sequence of a Cas9 from Streptococcus pyogenes.
- SEQ ID NO: 10 is the nucleic acid sequence of an Embryonal Fyn- Associated Substrate (EFS) promoter.
- EFS Embryonal Fyn- Associated Substrate
- SEQ ID NOs: 11-12 are nucleic acid sequences encoding a crRNA.
- SEQ ID NO: 13 is the nucleic acid sequence of a U6 promoter.
- SEQ ID NO: 14 is a nucleic acid sequence encoding a U6 gRNA.
- SEQ ID NO: 15 is a nucleic acid sequence encoding a tracrRNA.
- SEQ ID NOs: 16-19 are RNAi nucleic acid sequences.
- SEQ ID NO: 20 is a nucleic acid sequence of a target in exon 3 of human CD300LF.
- SEQ ID NOs: 21 is a nucleic acid sequence of a target in exon 4 of human CD300LF. DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
- CD300f inhibitors for the treatment of solid tumors.
- Methods are also disclosed for targeting CD300f in dendritic cells, and the use of adoptive transfer of these dendritic cells or T cells activated by these dendritic cells, for the treatment of solid tumors. These methods can be used in combination with other agents for the treatment of solid tumors, such as, but not limited to, sarcomas and carcinomas.
- Administration The introduction of a composition (such as one containing a CD300f inhibitor) into a subject by a chosen route.
- Administration can be local or systemic.
- the chosen route is intravenous
- the composition is administered by introducing the composition into a vein of the subject.
- routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, and intratumoral), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.
- Animal Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
- mammal includes both human and non-human mammals.
- subject includes both human and veterinary subjects.
- Antibody A polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope (e.g., an antigen, such as a CD300f protein or fragment thereof).
- an epitope e.g., an antigen, such as a CD300f protein or fragment thereof.
- a scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.
- the term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3 rd Ed., W.H. Freeman & Co., New York, 1997.
- an immunoglobulin typically has a heavy and light chain.
- Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains").
- the heavy and the light chain variable regions specifically bind the antigen.
- Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called “complementarity-determining regions” or "CDRs". The extent of the framework region and CDRs has been defined (see, Kabat et al., Sequences of Proteins of
- the Kabat database is now maintained online.
- the sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
- the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
- the CDRs are primarily responsible for binding to an epitope of an antigen.
- the CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N- terminus, and are also typically identified by the chain in which the particular CDR is located.
- a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found
- a VL CDRl is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
- VH refers to the variable region of an immunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.
- VL refers to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.
- a “monoclonal antibody” is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected.
- Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.
- a “humanized” immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic)
- the non-human immunoglobulin providing the CDRs is termed a "donor," and the human immunoglobulin providing the framework is termed an "acceptor.”
- all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical.
- all parts of a humanized immunoglobulin, except possibly the CDRs are substantially identical to corresponding parts of natural human immunoglobulin sequences.
- a "humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs.
- the acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework.
- Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.
- Humanized immunoglobulins can be constructed by means of genetic engineering (e.g., see U.S. Patent No. 5,585,089).
- a “neutralizing antibody” is an antibody that interferes with any of the biological activities of its target polypeptide, such as a CD300f polypeptide.
- Apoptotic cells Non-dividing, non-viable cells that can be distinguished from necrotic cells (other dead cells). Apoptosis is a result of programmed cell death. According to characteristic morphological and biochemical features, apoptosis is characterized by shrinkage of the cell, dramatic reorganization of the cell nucleus, cell membrane and cell metabolism, active membrane blebbing, and ultimate fragmentation of the cell into membrane-enclosed vesicles (apoptotic bodies). The nuclear events of apoptosis begin with collapse of the chromatin against the nuclear periphery and into one or a few large clumps within the nucleus.
- Nuclear features include chromatin aggregation followed by DNA fragmentation (a specific marker of apoptotic process) after activation of endonucleases resulting in multiples subunits of DNA of an approximately 180 base pairs.
- the cellular events include cytoplasmic condensation and partition of the cytoplasm and nucleus into membrane bound-vesicles which contain ribosomes, intact mitochondria and nuclear material which are surrounded by an intact cellular membrane (a specific marker of apoptotic process when compared with necrosis, the other non-physiological cell death process).
- CD300 molecule like family member f (CD300f): The human CD300 receptors are type I transmembrane proteins with single IgV-like extracellular domains that are mainly expressed by myeloid cells.
- Mouse CD300f (CLM-1) possesses both activating and inhibitory signaling potentials for regulation of apoptotic cell engulfment upon PS recognition.
- CD300f deficiency predisposes C57BL/6 mice to develop autoimmune disease. While CD300f functions to promote macrophage efferocytosis, its role in dendritic cells serves to inhibit apoptotic cell engulfment.
- the CD300f gene is also known as CD300f, CLM1, IGSF13, IREM1, NKIR and CD300LF.
- CD300f sequences are publicly avaible.
- GenBank® Accession Nos. NP_620587.2, P 001276011.1, P 001276012.1, P 001276013.1, P 001276014.1, P 001276015.1, and NP_001276016.1 provide exemplary human CD300f protein sequences
- GenBank® Accession Nos. NM_139018.4, NM_001289082.1, NM_001289083.1, NM_001289084.1, NM_001289085.1, NM_001289086.1, and NM_001289087.1 provide exemplary human CD300f nucleic acid sequences (all sequences herein incorporated by reference as of October 14, 2016).
- cDNA complementary DNA: A piece of DNA lacking internal, non-coding segments (introns) and regulatory sequences that determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
- Chemotherapeutic agent Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth.
- chemotherapeutic agents can be useful for the treatment of a solid tumor cancer, such as a sarcoma, carcinoma, lymphoma, colorectal or skin cancer.
- chemotherapeutic agents include microtubule binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors.
- a chemotherapeutic agent is a radioactive compound.
- chemotherapeutic agents that can be used are provided in Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2 nd ed., ⁇ 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby- Year Book, 1995; Fischer, D.S., Knobf, M.F., Durivage, H.J. (eds): The Cancer Chemotherapy Handbook, 4th ed. St.
- Combination chemotherapy is the administration of more than one agent to treat cancer.
- Constant amino acid substitutions are those substitutions that do not substantially affect or decrease an activity of a polypeptide, for example a CD300f peptide's ability to mediate negative regulatory signals by recruiting SHP1 or SHIP.
- Specific, non-limiting examples of a conservative substitution include the following examples:
- conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that the polypeptide binds with the same affinity as the unsubstituted (parental) polypeptide.
- Non-conservative substitutions are those that reduce the ability of the polypeptide.
- a polypeptide that consists essentially of a specified amino acid sequence if it does not include any additional amino acid residues.
- the polypeptide can include additional non-peptide components, such as labels (for example, fluorescent, radioactive, or solid particle labels), sugars or lipids.
- additional non-peptide components such as lipids, sugars or labels.
- Cas9 Clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9): An RNA-guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspersed Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes, among other bacteria.
- Cas9 can cleave nearly any sequence complementary to the guide RNA. Includes Cas9 nucleic acid molecules and proteins. Cas9 sequences are publically available, for example from the GENBANK® sequence database (e.g., Accession Nos. NP_269215.1 and AKS40378.1 provide exemplary Cas9 protein sequences, while Accession No.
- NC_002737.2 provides an exemplary Cas9 nucleic acid sequence therein).
- Cas9 variants A polynucleotide encoding a peptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in this disclosure as long as the amino acid sequence of the polypeptide encoded by the nucleotide sequence is unchanged.
- Dendritic Cell An antigen presenting cell that processes antigens and presents them to T cells.
- dendritic cells are present in the skin, the nose, lungs, stomach, intestines, and in the blood. Once activated, they migrate to the lymph nodes where they interact with T cells and B cells to initiate and shape the adaptive immune response. At certain development stages they grow branched projections, called "dendrities.”
- Dendritic cells include conventional dendritic cells
- cDCs that are similar to monocytes, and plasmacytoid dendritic cells (pDCs).
- Monocyte-derived dendritic cells can be generated in vitro from peripheral blood mononuclear cells (PBMCs).
- IL-4 interleukin 4
- GM-CSF granulocyte-macrophage colony stimulating factor
- TNF tumor necrosis factor
- LPS lipopolysaccharide
- Donor polynucleotide A polynucleotide that is capable of specifically inserting into a genomic locus.
- Downstream A relative position on a polynucleotide, wherein the "downstream" position is closer to the 3 ' end of the polynucleotide than the reference point.
- orientation of 5' and 3' ends are based on the sense strand, as opposed to the antisense strand.
- Expression Control Sequences Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence.
- expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct gene reading frame to permit proper translation of mRNA, and stop codons.
- control sequences includes, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
- Expression control sequences can include a promoter.
- a promoter is a minimal sequence sufficient to direct transcription.
- those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue- specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included (see e.g., Bitter et al., 1987, Methods in Enzymology 153, 516-544).
- inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used.
- promoters derived from the genome of mammalian cells such as the metallothionein promoter
- mammalian viruses such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter
- Promoters produced by recombinant DNA or synthetic techniques can also be used to provide for transcription of the nucleic acid sequences.
- Heterologous Originating from separate genetic sources or species.
- a polypeptide that is heterologous is derived from a different cell or tissue type, or a different species from the recipient, and is cloned into a cell that normally does not express that polypeptide.
- mouse (or human) CD300f cloned in a fibroblast cell line that does not express CD300f generates a heterologous CD300f protein.
- an antibody that specifically binds to a protein of interest, such as CD300f will not specifically bind to a heterologous protein.
- Host cells Cells in which a vector can be propagated and its DNA expressed.
- the cell may be prokaryotic or eukaryotic.
- the cell can be mammalian, such as a human cell.
- the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used.
- Immune response A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus.
- the response is specific for a particular antigen (an "antigen-specific response").
- an immune response is a T cell response, such as a CD4 response or a CD8 response.
- the response is a B cell response, and results in the production of specific antibodies.
- Inhibiting or treating a disease refers to inhibiting the full development of a disease. In several examples, inhibiting a disease refers to lessening symptoms of the particular tumor. “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition related to the disease, such as a tumor, such as reducing the size of a tumor, volume of a tumor, number of tumors, metastasis of a tumor, or combinations thereof.
- Isolated An "isolated" biological component (such as a nucleic acid or protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.
- Nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
- Label A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule.
- Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes.
- Lymphocytes A type of white blood cell that is involved in the immune defenses of the body. There are two main types of lymphocytes: B cells and T cells.
- Mammal This term includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects.
- Oligonucleotide A linear polynucleotide sequence of up to about 100 nucleotide bases in length.
- ORF Open reading frame
- a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
- a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence, such as a sequence that encodes a polypeptide.
- operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
- compositions and formulations suitable for pharmaceutically acceptable carriers are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for
- therapeutic agents such as CD300f inhibitors, dendritic cells with an inactivated CD300f, or T cells activated by such dendritic cells
- parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
- pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
- physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
- solid compositions such as powder, pill, tablet, or capsule forms
- conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
- compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
- non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
- a “therapeutically effective amount” is a quantity of a composition or a cell to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to reduce growth of a tumor, number of tumors, and/or reduce or prevent metastasis.
- a dosage When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations that has been shown to achieve an in vitro effect.
- Polynucleotide refers to a polymeric form of nucleotide at least 10 bases in length.
- a recombinant polynucleotide includes a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
- the term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences.
- the nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide.
- the term includes single- and double- stranded forms of DNA.
- Polypeptide Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
- a polypeptide can be between 5 and 25 amino acids in length. In one embodiment, a polypeptide is from about 10 to about 20 amino acids in length. In yet another embodiment, a polypeptide is from about 11 to about 18 amino acids in length. With regard to polypeptides, the word “about” indicates integer amounts. Thus, in one example, a polypeptide "about” 11 amino acids in length is from 10 to 12 amino acids in length. Similarly, a polypeptide "about” 18 amino acids in length is from about 17 to about 19 amino acids in length.
- a polypeptide "about" a specified number of residues can be one amino acid shorter or one amino acid longer than the specified number.
- a fusion polypeptide includes the amino acid sequence of a first polypeptide and a second different polypeptide (for example, a heterologous polypeptide), and can be synthesized as a single amino acid sequence.
- a probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule.
- Primers are short nucleic acids, such as DNA oligonucleotides, of about 15 nucleotides or more in length. Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, for example by polymerase chain reaction (PCR) or other nucleic-acid amplification methods.
- PCR polymerase chain reaction
- a primer comprising 20 consecutive nucleotides will anneal to a target with a higher specificity than a corresponding primer of only 15 nucleotides.
- probes and primers can be selected that comprise about 20, 25, 30, 35, 40, 50 or more consecutive nucleotides.
- Promoter An array of nucleic acid control sequences which direct transcription of a nucleic acid.
- a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
- a promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.
- polypeptides disclosed herein can be purified (and/or synthesized) by any means known in the art (see, e.g., Guide to Protein Purification, ed. Deutscher, Meth. Enzymol.
- substantially purified protein is at least about 60%, 70%, 80%, 90%, 95%, 98% or 99% pure.
- a substantially purified protein is 90% free of other proteins or cellular components.
- purified does not require absolute purity; rather, it is intended as a relative term.
- a purified nucleic acid is one in which the nucleic acid is more enriched than the nucleic acid in its natural environment within a cell.
- a purified population of nucleic acids, proteins, or cells is greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure, or free other nucleic acids, proteins, or cells, respectively.
- a recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of at least two otherwise separated segments of sequence. This artificial combination is can be accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
- a recombinant polypeptide has an amino acid sequence that is not naturally occurring or that is made by two otherwise separated segments of an amino acid sequence.
- “Homologous recombination (HR)” refers to the specialized form of an exchange that takes place, for example, during repair of double-strand breaks in cells. Nucleotide sequence homology is utilized in recombination, for example using a "donor” molecule to template repair of a "target” molecule (i.e., the one that experienced the double-strand break), and is variously known as “non-crossover gene conversion” or “short tract gene conversion,” because it leads to the transfer of genetic information from the donor to the target.
- nucleic acid hybridization reactions the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (for example, GC v. AT content), and nucleic acid type (for example, RNA versus DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.
- washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each of the conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed.
- optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.
- Sequence identity The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.
- NCBI National Center for Biotechnology Information
- blastp blastn
- blastx blastx
- tblastn tblastx
- the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
- the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).
- Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
- homologs and variants When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and can possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
- Subject Human and non-human animals, including all vertebrates, such as mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles.
- the subject is a human, such as a human with a solid tumor.
- Tumor An abnormal growth of cells, which can be benign or malignant. Cancer is a malignant tumor, which is characterized by abnormal or uncontrolled cell growth. Other features often associated with malignancy include metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels and
- Metalstatic disease refers to cancer cells that have left the original tumor site and migrate to other parts of the body for example via the bloodstream or lymph system.
- the amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, or weight of the tumor.
- a tumor that does not metastasize is referred to as "benign.”
- a tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant.”
- hematological tumors include leukemias, including acute leukemias (such as l lq23-positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma,
- acute leukemias such as l lq23-positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia
- chronic leukemias such as chronic
- the lymphoid malignancy can be adult T cell leukemia, cutaneous T cell lymphoma, anaplastic large cell lymphoma, Hodgkin's lymphoma, or a diffuse large B cell lymphoma.
- solid tumors such as sarcomas and carcinomas
- solid tumors include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
- Lymphoma can be solid tumors is some presentations.
- a tumor is colorectal tumor, skin tumor or lymphoma.
- Transgene An exogenous gene.
- Treating, Treatment, and Therapy Any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject's physical state, such as decreasing tumor volume, tumor size, or a symptom of the tumor.
- the treatment may be assessed by objective or subjective parameters; including the results of a physical examination, neurological examination, or psychiatric evaluations.
- Upstream A relative position on a polynucleotide, wherein the "upstream" position is closer to the 5' end of the polynucleotide than the reference point.
- orientation of 5' and 3' ends are based on the sense strand, as opposed to the antisense strand.
- Vector A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
- a vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
- a vector may also include one or more selectable marker gene and other genetic elements known in the art.
- Vectors include plasmid vectors, including plasmids for expression in gram-negative and gram-positive bacterial cell. Exemplary vectors include those for expression in E. coli and Salmonella.
- Vectors also include viral vectors, such as, but are not limited to, retrovirus, orthopox, avipox, fowlpox, capripox, suipox, adenoviral, herpes virus, alpha virus, baculovirus, Sindbis virus, vaccinia virus and poliovirus vectors.
- viral vectors such as, but are not limited to, retrovirus, orthopox, avipox, fowlpox, capripox, suipox, adenoviral, herpes virus, alpha virus, baculovirus, Sindbis virus, vaccinia virus and poliovirus vectors.
- Vectors also include vectors for expression in yeast cells or mammalian cells.
- Virus Microscopic infectious organism that reproduces inside living cells.
- a virus consists essentially of a core of a single nucleic acid surrounded by a protein coat and has the ability to replicate only inside a living cell.
- "Viral replication” is the production of additional virus by the occurrence of at least one viral life cycle.
- Viral vectors are known in the art, and include, for example, adenovirus, adeno-associated virus (AAV), lentivirus and herpes virus.
- CD300f inhibitors are of use for treating a solid tumor.
- the solid tumor is a carcinoma or a sarcoma.
- the solid tumor is a lymphoma, a colorectal cancer or a skin cancer.
- the CD300f inhibitor can be, for example, a soluble protein, an antibody or aptamer that specifically binds CD300f, or an inhibitory nucleic acid molecule (RNAi), such as, but not limited to, a ribozyme, a siRNA or a shRNA.
- RNAi inhibitory nucleic acid molecule
- the CD300f antagonist can result in the induction of an immune response to the tumor.
- the CD300f inhibitor can be an antibody, such as a monoclonal antibody.
- Antibodies that specifically bind CD300f are commercially available. Exemplary nucleic acid sequences encoding human CD300f are provided in GENBANK® Accession No. NM_139018.4 (August 26, 2016), and GENBANK Accession No. NM_001289082.1 (August 26, 2016), which are both incorporated by reference, and an exemplary amino acid sequence of human CD300f is provided in GENBANK® Accession No. AAH28199.1 (June 6, 2006), which is incorporated by reference herein. Other examples are provided herein. Antibodies that specifically bind CD300f are commercially available.
- a mouse IgGi to human CD300f is available from BioLegend, Clone UP-D2; additional monoclonal antibodies that specifically bind CD300f are available from R&D systems, Catalog Number AF2774, Borbyt, Catalog Number orb 160320, and St. John's Laboratory, Catalog Numbers STJ92113 and STJ96840, eBioscience, Clone UP-D1, Catalog Number 50-3008-41/42.
- Antibodies that specifically bind and substantially reduce or inhibit CD300f activity are of use in the methods disclosed herein.
- Antibodies include monoclonal antibodies, human antibodies, humanized antibodies, deimmunized antibodies, and immunoglobulin (Ig) fusion proteins. Fully human and humanized antibodies that bind CD300f can also be produced using methods known to those of skill in the art.
- Polyclonal anti-CD300f antibodies can be prepared, such as by immunizing a suitable subject (such as a veterinary subject) with a CD300f immunogen.
- a suitable subject such as a veterinary subject
- the anti-CD300f antibody titer in the immunized subject can be monitored over time, such as with an enzyme linked
- the antibody molecules that specifically bind CD300f can be isolated from a mammal (such as from serum) and further purified, for example using protein A chromatography to isolate IgG antibodies.
- Antibody-producing cells can be obtained from a subject and used to prepare monoclonal antibodies (see Kohler and Milstein Nature 256:495 49, 1995; Brown et al., J. Immunol. 127:539 46, 1981; Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77 96, 1985; Gefter, M. L. et al. (1977) Somatic Cell Genet. 3:231 36; Kenneth, R. H. in Monoclonal Antibodies: A New Dimension In Biological Analyses. Plenum Publishing Corp., New York, N.Y. (1980); Kozbor et al. Immunol. Today 4:72, 1983; Lerner, E.
- an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with CD300f, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that specifically binds to the polypeptide of interest.
- an immortal cell line (such as a myeloma cell line) is derived from the same mammalian species as the lymphocytes.
- murine hybridomas can be made by fusing lymphocytes from a mouse immunized with a CD300f peptide with an immortalized mouse cell line.
- a mouse myeloma cell line is utilized that is sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
- myeloma cell lines can be used as a fusion partner, including, for example, P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/0-Agl4 myeloma lines, which are available from the American Type Culture Collection (ATCC), Rockville, MD.
- HAT-sensitive mouse myeloma cells can be fused to mouse splenocytes using polyethylene glycol ("PEG").
- PEG polyethylene glycol
- Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused (and unproductively fused) myeloma cells.
- Hybridoma cells producing a monoclonal antibody of interest can be detected, for example, by screening the hybridoma culture supernatants for the production antibodies that bind a CD300f polypeptide, such as by using an immunological assay (such as an enzyme-linked immunosorbant assay (ELISA) or radioimmunoassay (RIA).
- an immunological assay such as an enzyme-linked immunosorbant assay (ELISA) or radioimmunoassay (RIA).
- a monoclonal antibody that specifically binds CD300f can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (such as an antibody phage display library) with CD300f to isolate immunoglobulin library members that specifically bind the polypeptide.
- Library members can be selected that have particular activities, such as binding CD300f, or activation of T cells in an in vitro assay.
- Kits for generating and screening phage display libraries are commercially available (such as, but not limited to, Pharmacia and Stratagene). Examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Pat. No.
- the sequence of the specificity determining regions of each CDR is determined. Residues outside the SDR (specificity determining region, e.g., the non-ligand contacting sites) are substituted. For example, in any of the CDR sequences, at most one, two or three amino acids can be substituted.
- the production of chimeric antibodies, which include a framework region from one antibody and the CDRs from a different antibody, is known in the art.
- humanized antibodies can be produced.
- the antibody or antibody fragment can be a humanized immunoglobulin having CDRs from a donor monoclonal antibody that binds CD300f, and immunoglobulin and heavy and light chain variable region frameworks from human acceptor immunoglobulin heavy and light chain frameworks.
- Humanized monoclonal antibodies can be produced by transferring CDRs from heavy and light variable chains of the donor mouse immunoglobulin (that specifically binds CD300f) into a human variable domain, and then substituting human residues in the framework regions when required to retain affinity.
- the use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of the constant regions of the donor antibody.
- Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321 :522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239: 1534, 1988; Carter et al., Proc. Natl. Acad. Sci.
- the antibody may be of any isotype, but in several embodiments the antibody is an IgG, including but not limited to, IgGi, IgG2, IgG3 and IgG 4 .
- the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework.
- the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 75%, at least about 85%, at least about 99% or at least about 95%, identical to the sequence of the donor immunoglobulin heavy chain variable region framework.
- Human framework regions, and mutations that can be made in humanized antibody framework regions, are known in the art (see, for example, in U.S. Patent No. 5,585,089, incorporated herein by reference).
- Exemplary human antibodies are LEN and 21/28 CL.
- the sequences of the heavy and light chain frameworks are known.
- Exemplary light chain frameworks of human MAb LEN have the following sequences:
- FR3 G VPDRPFGS GS GTDFTLTIS S LQ AED V A V Y YC (SEQ ID NO: 3)
- FR4 FGQGQTKLEIK (SEQ ID NO: 4)
- Exemplary heavy chain frameworks of human MAb 21/28' CL have the following sequences:
- RVTITRDTSASTAYMELSSLRSEDTAVYYCAR SEQ ID NO: 7
- an antibody such as a human or humanized antibody specifically binds to CD300f with an affinity constant of at least 10 7 M "1 , such as at least 10 8 M “1 at least 5 X 10 8 M “1 or at least 10 9 M “1 .
- the antibody specifically binds CD300f with an affinity constant of at least 10 8 M “1 at least 5 X 10 8 M “1 or at least 10 9 M “1 .
- Antibodies such as murine monoclonal antibodies, chimeric antibodies, and humanized antibodies, include full length molecules as well as fragments thereof, such as Fab, F(ab')2, and Fv which include a heavy chain and light chain variable region and are capable of binding specific epitope determinants. These antibody fragments retain some ability to selectively bind with their antigen or receptor. These fragments include:
- Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
- Fab' the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
- Fv a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
- Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
- variable region includes the variable region of the light chain and the variable region of the heavy chain expressed as individual polypeptides.
- Fv antibodies are typically about 25 kDa and contain a complete antigen-binding site with three CDRs per each heavy chain and each light chain.
- the VH and the VL can be expressed from two individual nucleic acid constructs in a host cell. If the VH and the VL are expressed non-contiguously, the chains of the Fv antibody are typically held together by noncovalent interactions.
- the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chain variable region and the light chain variable region are chemically linked by disulfide bonds.
- dsFv disulfide stabilized Fv
- the Fv fragments comprise VH and VL chains connected by a peptide linker.
- These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
- Methods for producing scFvs are known in the art (see Whitlow et al, Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al, Science 242:423, 1988; U.S. Patent No. 4,946,778; Pack et al,
- Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment.
- Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
- antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
- an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Patent No.
- cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody. Any of the antigen binding fragments described herein are of use.
- Conservative variants of the antibodies can be produced. Such conservative variants employed in antibody fragments, such as dsFv fragments or in scFv fragments, will retain critical amino acid residues necessary for correct folding and stabilizing between the VH and the VL regions, and will retain the charge characteristics of the residues in order to preserve the low pi and low toxicity of the molecules. Amino acid substitutions (such as at most one, at most two, at most three, at most four, or at most five amino acid substitutions) can be made in the VH and the VL regions to increase yield. A table of conservative amino acid substitutions is provided above. One of skill in the art can readily review the amino acid sequence of an antibody of interest, locate one or more of the amino acids in the brief table above, identify a conservative substitution, and produce the conservative variant using well-known molecular techniques.
- Effector molecules such as therapeutic, diagnostic, or detection moieties can be linked to an antibody that specifically binds CD300f, using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used.
- the procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector.
- Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH 2 ) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule.
- the antibody is derivatized to expose or attach additional reactive functional groups.
- the derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce
- the linker can be any molecule used to join the antibody to the effector molecule.
- the linker is capable of forming covalent bonds to both the antibody and to the effector molecule.
- Suitable linkers include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers.
- the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.
- Nucleic acid sequences encoding the antibodies can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68: 109-151, 1979; the
- a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.
- Exemplary nucleic acids encoding sequences encoding an antibody that specifically binds CD300f can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook et al., supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc.
- Nucleic acids can also be prepared by amplification methods.
- Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR).
- PCR polymerase chain reaction
- LCR ligase chain reaction
- TAS transcription-based amplification system
- 3SR self-sustained sequence replication system
- an antibody of use is prepared by inserting the cDNA, which encodes a variable region from an antibody that specifically binds CD300f, into a vector which comprises the cDNA encoding an effector molecule (EM). The insertion is made so that the variable region and the EM are read in frame so that one continuous polypeptide is produced.
- the encoded polypeptide contains a functional Fv region and a functional EM region.
- cDNA encoding a detectable marker (such as an enzyme) is ligated to a scFv so that the marker is located at the carboxyl terminus of the scFv.
- a detectable marker is located at the amino terminus of the scFv.
- cDNA encoding a detectable marker is ligated to a heavy chain variable region of an antibody that specifically binds CD300f, so that the marker is located at the carboxyl terminus of the heavy chain variable region.
- the heavy chain- variable region can subsequently be ligated to a light chain variable region of the antibody that specifically binds CD300f using disulfide bonds.
- cDNA encoding a marker is ligated to a light chain variable region of an antibody that binds CD300f, so that the marker is located at the carboxyl terminus of the light chain variable region.
- the light chain- variable region can subsequently be ligated to a heavy chain variable region of the antibody that specifically binds CD300f using disulfide bonds.
- the protein can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells.
- a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells.
- One or more DNA sequences encoding the antibody or functional fragment thereof can be expressed in vitro by DNA transfer into a suitable host cell.
- the cell may be prokaryotic or eukaryotic.
- the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known.
- Polynucleotide sequences encoding the antibody or functional fragment thereof can be operatively linked to expression control sequences.
- An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences.
- the expression control sequences include, but are not limited to appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
- the polynucleotide sequences encoding the antibody or functional fragment thereof can be inserted into an expression vector including, but not limited to a plasmid, virus or other vehicle that can be manipulated to allow insertion or incorporation of sequences and can be expressed in either prokaryotes or eukaryotes.
- Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art.
- Transformation of a host cell with recombinant DNA may be carried out by conventional techniques.
- the host is prokaryotic, such as E. coli
- competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaC method.
- MgC can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.
- Eukaryotic cells can also be cotransformed with polynucleotide sequences encoding the antibody of functional fragment thereof and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
- Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
- a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
- SV40 simian virus 40
- bovine papilloma virus bovine papilloma virus
- Isolation and purification of a recombinantly expressed polypeptide can be carried out by conventional means including preparative chromatography and immunological separations. Once expressed, the recombinant antibodies can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, R. Scopes, Protein Purification, Springer- Verlag, N.Y., 1982). Substantially pure compositions of at least about 90 to 95% homogeneity are disclosed herein, and 98 to 99% or more homogeneity can be used for pharmaceutical purposes. Once purified, partially or to homogeneity as desired, if to be used therapeutically, the polypeptides should be substantially free of endotoxin.
- An exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).
- Reoxidation of the disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena et al, Biochemistry 9: 5015-5021, 1970, incorporated by reference herein, and especially as described by Buchner et al, supra.
- Renaturation is typically accomplished by dilution (for example, 100-fold) of the denatured and reduced protein into refolding buffer.
- An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L- arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.
- the heavy and light chain regions are separately solubilized and reduced and then combined in the refolding solution.
- An exemplary yield is obtained when these two proteins are mixed in a molar ratio such that a 5 fold molar excess of one protein over the other is not exceeded. It is desirable to add excess oxidized glutathione or other oxidizing low molecular weight compounds to the refolding solution after the redox-shuffling is completed.
- the antibodies and functional fragments thereof that are disclosed herein can also be constructed in whole or in part using standard peptide synthesis.
- Solid phase synthesis of the polypeptides of less than about 50 amino acids in length can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., J. Am. Chem. Soc.
- Proteins of greater length may be synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxyl terminal end (such as by the use of the coupling reagent N, N'-dicycylohexylcarbodimide) are known.
- RNA interference such as, but not limited to, small inhibitory RNA (siRNA) or short hairpin RNA, which can be used for interference or inhibition of expression of a target.
- siRNA small inhibitory RNA
- short hairpin RNA short hairpin RNA
- RNAi sequences 5 ' CGTATCAACGATGACAATAA3 ' (SEQ ID NO: 16), and 5 ' CAGTCTCTGGAGGGTGATCTCTGTT3 ' (SEQ ID NO: 17). Additional RNAi sequences of use are:
- CD300f siRNA 1 CGTATCAACGATGACAATAATUU (SEQ ID NO: 18);
- CD300f siRNA 2 CAGTCTCTGGAGGGTGATCTCTGTTUU (SEQ ID NO: 19).
- siRNAs are generated by the cleavage of relatively long double-stranded RNA molecules by Dicer or DCL enzymes (Zamore, Science, 296: 1265-1269, 2002; Bernstein et ah, Nature, 409:363-366, 2001).
- siRNAs are assembled into RISC and guide the sequence specific ribonucleolytic activity of RISC, thereby resulting in the cleavage of mRNAs or other RNA target molecules in the cytoplasm.
- siRNAs In the nucleus, siRNAs also guide heterochromatin- associated histone and DNA methylation, resulting in transcriptional silencing of individual genes or large chromatin domains.
- RNA suitable for interference or inhibition of expression of CD300f which RNA includes double stranded RNA of about 19 to about 40 nucleotides with the sequence that is substantially identical to a portion of an mRNA or transcript of a target gene, such as CD300f, for which interference or inhibition of expression is desired.
- a sequence of the RNA "substantially identical" to a specific portion of the mRNA or transcript of the target gene for which interference or inhibition of expression is desired differs by no more than about 30%, and in some embodiments no more than about 10% or no more than 5% from the specific portion of the mRNA or transcript of the target gene.
- the sequence of the RNA is exactly identical to a specific portion of the mRNA or transcript of the target gene ⁇ e.g., CD300f).
- siRNAs disclosed herein include double- stranded RNA of about 15 to about 40 nucleotides in length and a 3' or 5' overhang having a length of 0 to 5-nucleotides on each strand, wherein the sequence of the double stranded RNA is substantially identical to (see above) a portion of a mRNA or transcript of a nucleic acid encoding CD300f.
- the double stranded RNA contains about 19 to about 25 nucleotides, for instance 20, 21, or 22 nucleotides substantially identical to a nucleic acid encoding CD300f.
- the double stranded RNA contains about 19 to about 25 nucleotides 100% identical to a nucleic acid encoding CD300f. It should be not that in this context "about” refers to integer amounts only. In one example, "about” 20 nucleotides refers to a nucleotide of 19 to 21 nucleotides in length.
- the length of the overhang is independent between the two strands, in that the length of one overhang is not dependent on the length of the overhang on other strand.
- the length of the 3' or 5' overhang is 0-nucleotide on at least one strand, and in some cases it is 0-nucleotide on both strands (thus, a blunt dsRNA).
- the length of the 3' or 5' overhang is 1 -nucleotide to 5- nucleotides on at least one strand.
- the length of the 3 ' or 5 ' overhang is 2-nucleotides on at least one strand, or 2-nucleotides on both strands.
- the dsRNA molecule has 3' overhangs of 2-nucleotides on both strands.
- the double- stranded RNA contains 20, 21, or 22 nucleotides, and the length of the 3' overhang is 2-nucleotides on both strands.
- the double-stranded RNA contains about 40-60% adenine+uracil (AU) and about 60-40% guanine+cytosine (GC). More particularly, in specific examples the double- stranded RNA contains about 50% AU and about 50% GC.
- RNAs that further include at least one modified ribonucleotide, for instance in the sense strand of the double- stranded RNA.
- the modified ribonucleotide is in the 3' overhang of at least one strand, or more particularly in the 3' overhang of the sense strand. It is contemplated that examples of modified ribonucleotides include
- ribonucleotides that include a detectable label (for instance, a fluorophore, such as rhodamine or
- FITC a thiophosphate nucleotide analog
- a deoxynucleotide (considered modified because the base molecule is ribonucleic acid), a 2'-fluorouracil, a 2'-aminouracil, a 2'-aminocytidine, a 4-thiouracil, a 5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, an inosine, or a 2'0-Me-nucleotide analog.
- Antisense and ribozyme molecules for CD300f are also of use in the method disclosed herein.
- Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American 262:40, 1990). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double- stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA, since the cell will not translate an mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target cell producing CD300f. The use of antisense methods to inhibit the in vitro translation of genes is known (see, for example, Marcus-Sakura, Anal. Biochem. 172:289, 1988).
- An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or
- An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions.
- an antisense nucleic acid molecule can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, such as phosphorothioate derivatives and acridine substituted nucleotides can be used.
- modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridin- e, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, amongst others.
- triplex strategy Use of an oligonucleotide to stall transcription is known as the triplex strategy where an oligonucleotide winds around double-helical DNA, forming a three-strand helix. Therefore, these triplex compounds can be designed to recognize a unique site on a chosen gene (Maher, et al. ,
- This type of inhibitory oligonucleotide is also of use in the methods disclosed herein.
- Ribozymes which are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases, are also of use. Through the modification of nucleotide sequences, which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, /. Amer. Med. Assn. 260:3030, 1988). An advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.
- ribozymes There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff, Nature 334:585, 1988) and "hammerhead"-type. Tetrahymena-type, ribozymes recognize sequences which are four bases in length, while “hammerhead”-type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type, ribozymes for inactivating a specific mRNA species and 18-base recognition sequences are preferable to shorter recognition sequences.
- RNA delivery systems are known and can be used to administer the siRNAs and other inhibitory nucleic acid molecules as therapeutics.
- Such systems include, for example,
- CD300f inhibitors include molecules that are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries. Screening methods that detect decreases in CD300f activity are useful for identifying compounds from a variety of sources for activity. The initial screens may be performed using a diverse library of compounds, a variety of other compounds and compound libraries. Thus, molecules that bind CD300f molecules that inhibit the expression of CD300f, and molecules that inhibit the activity of CD300f can be identified. These small molecules can be identified from combinatorial libraries, natural product libraries, or other small molecule libraries. In addition, CD300f antagonist can be identified as compounds from commercial sources, as well as commercially available analogs of identified inhibitors.
- test extracts or compounds The precise source of test extracts or compounds is not critical to the identification of
- CD300f small molecule antagonists can be identified from virtually any number of chemical extracts or compounds.
- examples of such extracts or compounds that can be CD300f inhibitors include, but are not limited to, plant-, fungal-, prokaryotic- or animal- based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds.
- CD300f inhibitors can be identified from synthetic compound libraries that are commercially available from a number of companies including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N. J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). CD300f inhibitors can be identified from a rare chemical library, such as the library that is available from Aldrich (Milwaukee, Wis.).
- CD300f inhibitors can be identified in libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). Natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.
- Useful compounds may be found within numerous chemical classes, though typically they are organic compounds, including small organic compounds. Small organic compounds have a molecular weight of more than 50 yet less than about 2,500 daltons, such as less than about 750 or less than about 350 daltons can be utilized in the methods disclosed herein. Exemplary classes include heterocycles, peptides, saccharides, steroids, and the like. The compounds may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. In several embodiments, compounds of use has a Kd for CD300f of less than InM, less than ⁇ , less than 1 ⁇ , less than 10 ⁇ , or less than lmM.
- compositions Including a CD 300f Inhibitor Methods are provided herein for treating a subject with a solid tumor.
- the solid tumor is a carcinoma or a sarcoma.
- the solid tumor is a lymphoma, skin cancer, or colorectal cancer.
- administration of one or more CD300f inhibitors increases antigen presentation by dendritic cells, and results in the production of activated T cells, such as CD8+ cytotoxic cells, specific for the tumor.
- a therapeutically effective amount of one or more CD300f inhibitors is administered to a subject to treat a tumor, such as to decrease tumor volume or size (such as a decrease of at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100% relative to absence of administration of the inhibitor).
- a therapeutically effective amount of the CD300f inhibitor is administered to a subject to treat a tumor, such as to decrease the number of tumors (such as a decrease of at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100% relative to absence of administration of the inhibitor).
- a therapeutically effective amount of the CD300f inhibitor is administered to a subject to treat a tumor, such as to decrease metastasis, for example the volume or number of metastases (such as a decrease of at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100% relative to absence of administration of the inhibitor).
- a therapeutically effective amount of the CD300f inhibitor is administered to a subject to delay or prevent a symptom of the tumor.
- the formation of tumors, such as metastasis are delayed, prevented or the number of metastases are decreased.
- the size of the primary tumor is decreased.
- a symptom of the tumor is decreased.
- tumor volume is decreased.
- the method involves selecting a subject with a solid tumor, and administering to the subject a therapeutically effective amount of one or more CD300f inhibitors.
- the subject has carcinoma or a sarcoma.
- the subject has a lymphoma, skin cancer, or colorectal cancer.
- the tumor can be any tumor of interest, including, but not limited to, lymphoma, breast cancer, lung cancer and colon cancer.
- the tumor can be benign or malignant.
- Additional examples are breast, brain, cervical carcinomas, testicular carcinomas, head and neck, lung, mediastinum, gastrointestinal tract, genitourinary system, gynaecological system, breast, endocrine system, skin, childhood, unknown primary site or metastatic cancer, a sarcoma of the soft tissue and bone, a mesothelioma, a melanoma, a neoplasm of the central nervous system, a head and neck tumor, comprising tumors of the nasal cavity, paranasal sinuses, nasopharynx, oral cavity, oropharynx, larynx, hypopharynx, salivary glands and paragangliomas, a cancer of the lung, comprising non-small cell lung cancer, small cell lung cancer, a cancer of the mediastinum, a cancer of the gastrointestinal tract, comprising cancer of the oesophagus, stomach, pancreas, liver, biliary tree, small intestine, colon,
- carcinomastosis a Kaposi's sarcoma
- AIDS -associated lymphomas AIDS -associated primary central nervous system lymphoma
- AIDS -associated anogenital cancers The presence of a tumor can be determined by methods known in the art, and typically include cytological and morphological evaluation.
- Treatment of the conditions described herein are generally initiated after the development of a condition described herein, or after the initiation of a precursor condition (such as dysplasia or development of a benign tumor). Treatment can be initiated at the early stages of cancer, for instance, can be initiated before a subject manifests symptoms of a condition, such as during a stage I diagnosis or at the time dysplasia is diagnosed. However, treatment can be initiated during any stage of the disease, such as but not limited to stage I, stage II, stage III and stage IV cancers. In some examples, such as for breast cancer, treatment can be initiated before or during exposure to an agent that damages DNA, such as a result of an exposure to a carcinogen or UV light, oxidative stress, alkylation damage and deamination.
- an agent that damages DNA such as a result of an exposure to a carcinogen or UV light, oxidative stress, alkylation damage and deamination.
- Treatment prior to the development of the condition is referred to herein as treatment of a subject that is "at risk" of developing the condition.
- administration of a composition can be performed during or after the occurrence of the conditions described herein.
- treatment involves increasing the immune response to the tumor.
- Treatment initiated after the development of a condition may result in decreasing the severity of the symptoms of one of the conditions, or completely removing the symptoms, or reducing metastasis, tumor volume or number of tumors.
- there is an increased T cell response to the tumor such as a CD8 + cytotoxic T cell response.
- compositions described herein may be formulated in a variety of ways for
- Administration can be local, such as to the site of a tumor, or systemic.
- systemic methods for administering the composition into mammals include, but are not limited to, intravenous, intratumoral, intraperitoneal, subcutaneous, intradermal, inhalation, transdermal and intramuscular. Any of the CD300f inhibitors disclosed herein can be used in these methods.
- the method can include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a CD300f inhibitor.
- CD300f inhibitor will typically be used to treat human subjects they may also be used to treat similar or identical diseases in other vertebrates, such as other primates, dogs, cats, horses, and cows.
- a suitable administration format may best be determined by a medical practitioner for each subject individually.
- Various pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises, e.g., Remington 's Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A., Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42: 2S, 1988.
- the dosage form of the pharmaceutical composition will be determined by the mode of administration chosen.
- the amount of active compound(s) administered will be dependent on the subject being treated, the severity of the affliction, and the manner of administration, and is best left to the judgment of the prescribing clinician.
- the formulation to be administered will contain a quantity of the active component(s) in amounts effective to achieve the desired effect in the subject being treated. Multiple treatments are envisioned, such as over defined intervals of time, such as daily, bi-weekly, weekly, bi-monthly or monthly, such that chronic administration is achieved. Administration may begin whenever the suppression or prevention of disease is desired, for example, at a certain age of a subject, or prior to an environmental exposure.
- Antibodies and antigen binding fragments thereof can be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The antibody solution is then added to an infusion bag containing 0.9% Sodium Chloride, USP, and typically administered at a dosage of from 0.5 to 15 mg/kg of body weight.
- an infusion bag containing 0.9% Sodium Chloride, USP, and typically administered at a dosage of from 0.5 to 15 mg/kg of body weight.
- Antibody drugs can be administered by slow infusion, rather than in an IV push or bolus.
- a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level. For example, an initial loading dose of 4 mg/kg may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30 minute period if the previous dose was well tolerated.
- a pharmaceutical composition for intravenous administration would include about 0.1 ⁇ g to 10 mg of CD300f inhibitor, such as an antibody, per patient per day. Dosages from 0.1 up to about 100 mg per subject per day can be used, particularly if the agent is administered to a body cavity or into a lumen of an organ. Actual methods for preparing administrable compositions are described in more detail in such publications as
- a pharmaceutical composition includes a nucleic acid encoding one or more of the antagonists, such as siRNA, shRNA or ribozyme, disclosed herein.
- a therapeutically effective amount of the polynucleotide can be administered to a subject, such as a subject with a solid tumor.
- a therapeutically effective amount of the polynucleotide is administered to a subject to treat a tumor, such as to decrease tumor volume.
- a therapeutically effective amount of the polynucleotide is administered to a subject to treat a tumor, such as to decrease metastasis.
- a therapeutically effective amount of the polynucleotide is administered to a subject to delay or prevent a symptom of the tumor.
- nucleic acids are direct immunization with plasmid DNA, such as with a mammalian expression plasmid.
- plasmid DNA such as with a mammalian expression plasmid.
- the nucleotide sequence encoding a polypeptide can be placed under the control of a promoter to increase expression of the molecule.
- the methods include liposomal delivery of the nucleic acids (or of the synthetic peptides themselves).
- a polypeptide in another approach to using nucleic acids for immunization, can also be expressed by attenuated viral hosts or vectors or bacterial vectors.
- Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, or other viral vectors can be used to express the peptide or protein.
- vaccinia vectors and methods of administration are described in U.S. Patent No. 4,722,848.
- BCG Bacillus Calmette Guerin provides another vector for expression of the peptides (see Stover, Nature 351 :456-460, 1991).
- each recombinant virus in the composition in the range of from about 10 5 to about 10 10 plaque forming units/mg mammal, although a lower or higher dose can be administered.
- the composition of recombinant viral vectors can be introduced into a subject with the solid tumor.
- Examples of methods for administering the composition into mammals include, but are not limited to, intravenous, subcutaneous, intradermal or intramuscular administration of the nucleic acid, such as virus or other vector including the nucleic acid encoding the disclosed polypeptides.
- the quantity of recombinant viral vector, carrying the nucleic acid sequence of a polypeptide to be administered is based on the titer of virus particles.
- An exemplary range of the virus to be administered is 10 5 to 10 10 virus particles per mammal, such as a human.
- the composition should provide a sufficient quantity of at least one of the CD300f inhibitor disclosed herein to effectively treat the patient with the solid tumor.
- the dosage can be administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy.
- a dose of the CD300f inhibitor (such as an antibody) is infused for thirty minutes every other day.
- about one to about ten doses can be administered, such as three or six doses can be administered every other day.
- a continuous infusion is administered for about five to about ten days.
- the subject can be treated at regular intervals, such as monthly, until a desired therapeutic result is achieved.
- the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.
- a therapeutically effective amount of the CD300f inhibitor is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.
- Controlled release parenteral formulations of the compositions can be made as implants, oily injections, or as particulate systems.
- Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles.
- Microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central core. In microspheres the therapeutic is dispersed throughout the particle.
- Particles, microspheres, and microcapsules smaller than about 1 ⁇ are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively.
- Capillaries have a diameter of approximately 5 ⁇ so that only nanoparticles are administered intravenously.
- Microparticles are typically around 100 ⁇ in diameter and are administered subcutaneously or intramuscularly. See, e.g., Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342, 1994; and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, 1992, both of which are incorporated herein by reference.
- the therapeutic agent(s) can be provided as an implant, an oily injection, or as a particulate system.
- the particulate system can be a microparticle, a microcapsule, a microsphere, a nanocapsule, or similar particle.
- Polymers can be used for ion-controlled release of the compositions disclosed herein.
- polaxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., /. Parent. Sci. Tech. 44(2):58-65, 1990).
- hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm.112:215- 224, 1994).
- liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA, 1993). Numerous additional systems for controlled delivery of therapeutic proteins are known. See, e.g., U.S. Patent No. 5,055,303; U.S. Patent No. 5,188,837; U.S. Patent No. 4,235,871 ; U.S. Patent No. 4,501,728; U.S.
- the method can include administering additional therapeutic agents, such as, but not limited to, a chemotherapeutic agent, a biologic (such as a monoclonal antibody), a Programmed Death (PD)-l antagonist, or a cytotoxic T lymphocyte associated protein (CTLA)-4 antagonist,
- additional agent is an antibody or antibody fragment that binds to ⁇ 3.
- the agent is an antibody or antibody fragment that binds to LAG3.
- Such agents can be administered before, after, or concurrently with the CD300f inhibitor.
- chemotherapeutic agents are alkylating agents, antimetabolites, natural products, or hormones and their antagonists.
- alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or
- chlorambucil alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).
- antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.
- Examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitocycin C), and enzymes (such as L-asparaginase).
- vinca alkaloids such as vinblastine, vincristine, or vindesine
- epipodophyllotoxins such as etoposide or teniposide
- antibiotics such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitocycin C
- enzymes such as L-asparaginase
- miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum ⁇ also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide).
- platinum coordination complexes such as cis-diamine-dichloroplatinum ⁇ also known as cisplatin
- substituted ureas such as hydroxyurea
- methyl hydrazine derivatives such as procarbazine
- adrenocrotical suppressants such as mitotane and aminoglutethimide
- hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as
- hydroxyprogesterone caproate medroxyprogesterone acetate, and magestrol acetate
- estrogens such as diethylstilbestrol and ethinyl estradiol
- antiestrogens such as tamoxifen
- androgens such as testerone proprionate and fluoxymesterone.
- chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU,
- Non-limiting examples of immunomodulators that can be used include AS- 101 (Wyeth- Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Jersey, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).
- the additional therapeutic agent is a biologic agent (e.g. , mAb) or a small molecule, such as those shown in Table 1.
- a biologic agent e.g. , mAb
- a small molecule such as those shown in Table 1.
- Table 1 Exemplary Tumor-specific antigens and therapeutics
- Tumor-Specific Exemplary Tumors Exemplary Antibody/Small
- HER1 Adenocarcinoma e.g., Cetuximab, panitumamab,
- colorectal cancer head and zalutumumab, nimotuzumab,
- gefitinib erlotinib
- lapatinib a group consisting of erlotinib, and lapatinib
- HER2 breast cancer ovarian cancer
- Trastuzumab Herceptin®
- stomach cancer uterine pertuzumab
- CD20 Non-Hodgkin lymphoma Tositumomab (Bexxar®); Rituximab
- CD33 Acute myelogenous leukemia Gemtuzumab (Mylotarg, for example
- CEA colorectal cancer some CEA-scan (Fab fragment, approved
- Alpha- fetoprotein hepatocellular carcinoma ab75705 available from Abeam
- AFP other commercially available AFP
- TAG72 adenocarcinomas including B72.3 FDA-approved monoclonal
- Vascular Colorectal cancer Bevacizumab (Avastin®)
- the additional agent can be a PD-1 antagonist or a CTLA-4 antagonist.
- an inhibitory nucleic acid e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., a siRNA, shRNA, or ribozyme
- the PD-1 antagonist or CTLA-4 antagonist is an antibody.
- the CTLA-4 antagonist is an antibody or antigen binding fragment thereof that specifically binds CTLA4 (e.g., ipilimumab (also referred to as MDX-010 and MDX- 101 , and marketed as YERVOY®; Bristol-Myers Squibb; Tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675,206).).
- CTLA4 e.g., ipilimumab (also referred to as MDX-010 and MDX- 101 , and marketed as YERVOY®; Bristol-Myers Squibb; Tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675,206).
- the CD300f inhibitor is administered with a PD-1 antagonist.
- PD-1 is an inhibitor ⁇ ' member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA.
- PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75).
- Two ligands for PD-1, PD-LI and PD-L2 have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al. 2000 J Exp Med 192: 1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43).
- PD-LI is abundant in human cancers (Dong et al. 2003 J Mol Med 81 :281-7; Blank et al. 2005 Cancer Immunol.
- the PD-1 antagonist can be an antibody or antigen binding fragment thereof that binds to PD-1 , PD-LI , or PD-L2.
- Antibodies, antibody fragments, and other inhibitors of PD-1, PD-LI and PD-L2 are available in the art and may be used in the methods disclosed herein.
- nivolumab also referred to as BMS-936558 or MDX1106; Bristol-Myers Squibb
- BMS-936558 or MDX1106 is a fully human IgG 4 monoclonal antibody which specifically blocks PD-L Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in U.S. Pat. No. 8,008,449 and PCX Publication No.WO2006/121168.
- Pidilizumab (CT-011; Cure Tech) is a humanized IgGlk monoclonal antibody that binds to PD-1.
- Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in PCT Publication No.
- Lambrolizumab (also referred to as MK03475; Merck) is a humanized IgG 4 monoclonal antibody that binds to PD-1. Lambrolizumab and other humanized anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,354,509 and PCT Publication No. WO2009/ 114335.
- MDPL3280A (Genentech/Roche) is a human Fc optimized IgGi monoclonal antibody that binds to PD-L1.
- MDPL3280A and other human monoclonal antibodies to PD-L1 are disclosed in U.S. Patent No. 7,943,743 and U.S. Publication No. 2012/0039906.
- anti-PD-Ll binding agents include YW243.55.S70 (heavy and light chain variable regions are shown in SEQ ID NOs: 20 and 21 in PCT Publication No. WO2010/077634) and MDX-1 105 (also referred to as BMS-936559, and, e.g., anti-PD-Ll binding agents disclosed in PCT Publication No. WO2007/005874).
- AMP- 224 (B7-DCIg; Amplimmune; e.g., disclosed in PCT Publication No. WO2010/027827 and PCT Publication No. WO2011/066342), is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD-1 and B7-H1.
- anti-PD-1 antibodies include AMP 514 (Amplimmune), among others, e.g., anti-PD-1 antibodies disclosed in U.S. Patent No. 8,609,089, U.S. Publication No. 2010/028330, and/or U.S. Publication No. 2012/0114649. Any of these PD- 1 antagonists are of use in the methods disclosed herein.
- CD300f gene such as in monocytes, monocyte-derived cells (such as monocyte-derived dendritic cells), CD34 +
- CD300f gene is also known as CD300LF, CLM1, IGSF13, IREM1, and NKIR.
- nucleic acid sequences encoding human CD300f are provided in GENBANK®
- NM_001289082.1 August 26, 2016
- an exemplary amino acid sequence of human CD300f is provided in GENBANK® Accession No. AAH28199.1 (June 6, 2006), which is incorporated by reference herein.
- Other examples are provided herein.
- a typical set of CRISPR system is composed of two components, a CRISPR-associated nuclease 9 (Cas9) and one or more guide RNAs (gRNAs), each of which contains a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA).
- Cas9 CRISPR-associated nuclease 9
- gRNAs guide RNAs
- crRNA CRISPR RNA
- tracrRNA trans-activating CRISPR RNA
- Simple gene disruptions can be generated by cleavage of the target site, followed by alteration of nucleic acids, such as a deletion, and repair by the non-homologous-end-joining pathway (NHEJ).
- NHEJ non-homologous-end-joining pathway
- Target recognition by crRNAs occurs through complementary base pairing with target DNA, which directs cleavage of foreign sequences by means of Cas proteins.
- DNA recognition by guide RNA and consequent cleavage by the endonuclease requires complementary base-pairing with a protospacer adjacent motif (PAM) (e.g. 5'-NGG-3') and with a protospacer region in the target.
- PAM protospacer adjacent motif
- the PAM motif recognized by a Cas9 varies for different Cas9 proteins. Any Cas9 protein can be used in the systems and methods disclosed herein.
- One Cas9 of use is from Streptococcus pyogenes as depicted in below (SEQ ID NO: 9) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQ DLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIK
- the systems and methods disclosed herein can be used with the wild type Cas9 protein having double- stranded nuclease activity, Cas9 mutants that act as single stranded nickases, or other mutants with modified nuclease activity.
- the Cas9 includes a catalytically active nuclease domain.
- the Cas9 nuclease includes an HNH-like endonuclease and a RuvC-like endonuclease.
- the HNH-like endonuclease cleaves the DNA strand complementary to the gRNA, and the RuvC-like domain cleaves the non- complementary DNA strand.
- a Cas9 endonuclease can be guided to specific genomic targets using specific gRNA (see below).
- a promoter such as the Embryonal Fyn- Associated Substrate (EFS) promoter is operably linked to the nucleic acid encoding Cas9.
- EFS Embryonal Fyn- Associated Substrate
- This promoter provides for cell specific expression of Cas9. The sequence of this promoter is shown below (SEQ ID NO: 10):
- the promoter can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions in SEQ ID NO: 10, provided the promoter allows for expression in dendritic cells.
- the promoter can be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical SEQ ID NO: 10, provided the promoter allows for expression in dendritic cells.
- promoters of use include, but are not limited to, a cytomegalovirus (CMV) promoter, a synthetic promoter such as CAG promoter, a simian virus (SV)40 promoter, a 35S promoter, and an alcohol dehydrogenase (ADH)l promoter.
- CMV cytomegalovirus
- SV simian virus
- ADH alcohol dehydrogenase
- a nucleic acid molecule encoding a marker also can be operably linked to the EFS promoter.
- Markers include, but are not limited to, enzymes and fluorescent proteins.
- the marker is tdTomato fluorescent protein.
- a nucleic acid molecule encoding a marker is not operably linked the EFS.
- the Cas9 RNA guide system includes a mature crRNA that is base-paired to trans-activating crRNA (tracrRNA), forming a two-RNA structure that directs Cas9 to the locus of a desired double- stranded (ds) break in target DNA, namely the CD300f gene.
- tracrRNA trans-activating crRNA
- base-paired tracrRNA: crRNA combination is engineered as a single RNA chimera to produce a guide sequence (e.g., gRNA) which preserves the ability to direct sequence-specific Cas9 dsDNA cleavage (see Jinek et al., Science. 337:816-821, 2012).
- the Cas9- guide sequence complex results in cleavage of one or both strands at a target sequence within the CD300f gene, such as in exons 1 or 3 of the CD300f gene.
- the Cas9 endonuclease Jinek et al., Science. 337:816-821, 2012; Mali et. al., Nat Methods.
- the cleavage site is at a specific nucleotide, such as, but not limited to the 16, 17, or 18 th nucleotide of a 20 nucleotide target. In one non-limiting example, the cleavage site is at the 17 th nucleotide of a 20-nt target sequence.
- the cleavage can be a double stranded cleavage.
- the gRNA molecule is selected so that the target genomic targets bear a protospacer adjacent motif (PAM).
- DNA recognition by guide RNA and consequent cleavage by the endonuclease requires the presence of a protospacer adjacent motif (PAM) (e.g., 5'-NGG-3') in immediately after the target.
- PAM protospacer adjacent motif
- the PAM is present in the targeted nucleic acid sequence but not in the crRNA that is produced to target it.
- the proto-spacer adjacent motif (PAM) corresponds to 2 to 5 nucleotides starting immediately or in the vicinity of the proto-spacer at the leader distal end.
- the PAM motif also can be NNAGAA, NAG, NGGNG, AWG, CC, CC, CCN, TCN, or TTC.
- cleavage occurs at a site about 3 base-pairs upstream from the PAM.
- the Cas9 nuclease cleaves a double stranded nucleic acid sequence.
- the guide sequence is selected to reduce the degree of secondary structure within the sequence.
- Secondary structure may be determined by any suitable
- polynucleotide folding algorithm Some programs are based on calculating the minimal Gibbs free energy.
- An example of one such algorithm is mFold (Zuker and Stiegler, Nucleic Acids Res. 9 (1981), 133-148).
- Another example folding algorithm is the online webserver RNAfold, which uses the centroid structure prediction algorithm (see e.g., Gruber et al., 2008, Cell 106(1): 23-24; and Can and Church, 2009, Nature Biotechnology 27(12): 1151-62).
- Guide sequences can be designed using the MIT CRISPR design tool found at crispr.mit.edu, Harvard and University of Bergen CHOPCHOP web tool found at chopchop.cbu.uib.no, or the E-CRISP tool found at www.e- crisp.org/E-CRISP. Additional tools for designing tracrRNA and guide sequences are described in Naito et al., Bioinformatics. 2014 Nov 20, and Ma et al. BioMed Research International,
- the crRNA can be 18-48 nucleotides in length.
- the crRNA can be 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
- the crRNA is 20 nucleotides in length.
- the tracrRNA is pre-optimized, and is 83 nucleotides in length, see SEQ ID NO: 7.
- the human CD300f gene is targeted, such as exon 3 or exon 4, and the crRNA is encoded by a nucleic acid sequence set forth as one of SEQ ID NO: 11 or 12.
- the crRNA is encoded by one of the DNA sequences below.
- the PAM, which is recognized by Cas9 is shown next to the target DNA sequence below.
- the DNA encoding the crRNA includes or consists of, one of the nucleic acid sequence sent forth as one of SEQ ID NO: 11 or 12, which target Exon 3 and Exon 4, respectively.
- the PAM which is recognized by Cas9, is shown next to the target DNA sequence.
- the DNA encoding the crRNA can alternatively target another exon, such as, but not limited to, Exon 1 or Exon 2.
- the system disclosed herein introduces double stranded DNA breaks at the CD300f gene, such that the CD300f target is cleaved by Cas9. This results in functional CD300f protein not being produced.
- the system disclosed herein can include a promoter, such as, but not limited to, a U6 or HI promoter operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas RNAs.
- a promoter such as, but not limited to, a U6 or HI promoter operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas RNAs.
- the U6 promoter can include the following nucleic acid sequence:
- the tracer sequence includes seven thymidines for terminating RNA transcription.
- the small "g,” “ga,” and the second "g” border the Saplrev and Sapl sites where the nucleic acid encoding the gRNA is inserted.
- the tracrRNA is encoded by the nucleic acid sequence set forth as:
- more than one DNA break can be introduced by using more than one gRNA.
- two gRNAs can be utilized, such that two breaks are achieved.
- the two or more cleavage events may be made by the same or different Cas9 proteins.
- a single Cas9 nuclease may be used to create both double strand breaks.
- both of the gRNAs corresponding to the DNA sequences set forth as SEQ ID NOs: 11 and 12 can be used.
- the disclosed methods include the use of one or more vectors comprising: a) a EFS promoter operably linked to a nucleotide sequence encoding a Type II Cas9 nuclease, b) a U6 promoter operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas guide RNAs that hybridize with the CD300f gene in a target cell, such as a human cell.
- Components (a) and (b) can be located on same or different vectors, whereby the one or more guide RNAs target the CD300f gene in the target cell and the Cas9 protein cleaves the CD300f gene.
- the one or more vectors are viral vectors such as lentiviral vectors.
- the viral vectors are adenovirus vectors, adeno- associated virus vectors, or retroviral vectors.
- Lentiviral vectors are retroviral vectors that are able to transduce or infect non- dividing cells and typically produce high viral titers. Retroviral vectors are comprised of cis- acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis- acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the desired nucleic acid into the target cell to provide permanent expression.
- lentiviral vector is the lentiCRISPRv2 vector (Adgene Plasmid #52961, see the addgene website, addgene.org/52961/).
- Retroviral vectors of use include murine leukemia virus (MuLV) vectors, gibbon ape leukemia virus (GaLV) vectprs, Simian Immunodeficiency virus (SIV) vectors, human immuno deficiency virus (HIV) vectors, and combinations thereof (see, e.g., Buchscher et ah, (1992) J. Virol. 66:2731-2739; Johanti et al, (1992) J. Virol. 66: 1635-1640; Sommnerfeit et al., (1990) Virol. 176:58-59; Wilson et al, (1998) J. Virol. 63:2374-2378; Miller et al, (1991) J. Virol.
- MiLV murine leukemia virus
- GaLV gibbon ape leukemia virus
- SIV Simian Immunodeficiency virus
- HAV human immuno deficiency virus
- Methods are disclosed herein for altering expression of CD300f in a subject.
- the method included introducing into a dendritic cell, such as a human dendritic cell comprising a gene encoding CD300f an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system comprising one or more viral vectors, such as lentiviral vectors.
- CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
- Cas Clustered Regularly Interspaced Short Palindromic Repeats
- the one or more viral vectors include a) an EFS (or other retina-specific promoter) operably linked to a nucleotide sequence encoding a Cas9 protein, and b) a U6 promoter operably linked to at least one nucleotide sequence encoding a CRISPR-Cas guide RNA that hybridizes with the CD300f gene, wherein components (a) and (b) are located on same or different vectors of the system, whereby the guide RNA targets the CD300f gene and the Cas9 protein cleaves the CD300f gene.
- EFS retina-specific promoter
- U6 promoter operably linked to at least one nucleotide sequence encoding a CRISPR-Cas guide RNA that hybridizes with the CD300f gene
- the Cas9 protein is expressed in a recombinant cell, such as E. coli, and purified.
- the resulting purified Cas9 protein, along with an appropriate guide molecule specific for the target, is then introduced into a cell or organism where one or genomic sequences can be targeted.
- the Cas9 protein and guide nucleic acid molecule i.e., gRNA
- the purified Cas9 protein is complexed with the guide nucleic acid, and this ribonucleoprotein (RNP) complex is introduced into target cells (e.g., using transfection or injection).
- the Cas9 protein and guide molecule are injected into the cell of interest. Once the Cas9 protein and guide nucleic acid molecule are in the cell, one or more genomic sequences can be targeted. Modification of Dendritic Cells and Their Use
- dendritic cells deficient for expression of CD300f can be used to induce an immune response to a solid tumor.
- the dendritic cells deficient for expression of CD300f can be in combination with a CD300f inhibitor (or other chemotherapeutic or anti-neoplastic agent), using any of the methods disclosed above, for the treatment of a tumor in a subject, such as a solid tumor.
- the dendritic cells deficient for expression of CD300f can also be used without the administration of a CD300f inhibitor.
- the dendritic cells are used to treat a leukemia or a lymphoma.
- the dendritic cells are used to treat a lymphoma.
- a therapeutically effective amount of the dendritic cells deficient for expression of CD300f are administered to a subject with the solid tumor.
- the dendritic cells deficient for expression of CD300f are used to induce activated T cells, such as activated cytotoxic CD8 + T cells, specific for the tumor.
- a therapeutically effective amount of the activated T cells can be administered to subject with the tumor.
- the T cells and/or dendritic cells are autologous.
- the subject can be administered an additional therapeutic agent, such as, but not limited to, a CD300f inhibitor, a chemotherapeutic agent, an anti-neoplastic agent, a PD- 1 antagonist or a CTLA-4 antagonist, as discussed above.
- dendritic cells The production of dendritic cells is disclosed, for example, in U.S. Application No.
- dendritic cells can be generated in vivo or ex vivo from immature precursors (e.g., monocytes, CD34 + hematopoietic precursor cells).
- immature precursors e.g., monocytes, CD34 + hematopoietic precursor cells.
- a cell population enriched for dendritic cell precursor cells e.g., peripheral blood mononuclear cells (PBMCs)
- PBMCs peripheral blood mononuclear cells
- monocytic precursors are first enriched or purified from other cell types.
- peripheral blood mononuclear cells PBMCs
- PBMCs peripheral blood mononuclear cells
- the PBMCs will be used to generate monocytic dendritic cell precursors.
- monocytic dendritic cell precursors are isolated by adherence to a monocyte-binding substrate.
- a population of leukocytes e.g., isolated by
- leukapheresis can be contacted with a monocytic dendritic cell precursor adhering substrate.
- a monocytic dendritic cell precursor adhering substrate When the population of leukocytes is contacted with the substrate, the monocytic dendritic cell precursors in the leukocyte population preferentially adhere to the substrate.
- monocytes are isolated through adherence of the monocytic precursors to a plastic (polystyrene) surface, as the monocytes have a greater tendency to stick to plastic than other cells found in, for example, peripheral blood, such as lymphocytes
- dendritic cell precursors and immature dendritic cells can be isolated by phlebotomy, by apheresis or leukapheresis, by collecting heparinized blood, by preparation of buffy coats, rosetting, centrifugation, density gradient centrifugation (e.g., using Ficoll, Percoll (colloidal silica particles of 15-30 mm diameter coated with polyvinylpyrrolidone (PVP)), sucrose, and the like), differential lysis of cells, filtration, and the like.
- dendritic cell precursors can be selected using CD 14 selection of G-CSF mobilized peripheral blood. See U.S. Patent No. 8,728,806, incorporated herein by reference.
- GM-CSF granulocyte macrophage colony stimulating factor
- the subject may be administered at a dose ranging from about 10 ⁇ g/day to about 500 ⁇ g/day, from about 20 ⁇ g/day to about 300 ⁇ g/day, from about 50 ⁇ g/day to about 250 ⁇ g/day, from about 100 ⁇ g/day to about 300 ⁇ g/day, from about 200 ⁇ g/day to about 300 ⁇ g/day, about 200 ⁇ g/day, or about 250 ⁇ g/day.
- GM-CSF may be administered for about 1 day, about 2 days, about 3 days, about 4 day, about 5 days, about 6 days, about 1 week, about 1.5 weeks, about 2 weeks, or longer.
- the effect of GM-CSF may be potentiated by another immunostimulant (such as plerixafor).
- monocytes, monocyte-derived cells including dendritic cells, or monocyte precursors are treated with the CRISPR/Cas9 system disclosed herein to reduce expression of CD300f.
- Variations on this method include different methods of purifying monocytes, including, for example, tangential flow filtration (TFF), or by binding antibodies attached to beads to surface molecules on the monocytes.
- the beads with the bound cells are then concentrated in a column, or on a magnetic surface, such that contaminating cells can be washed away, after which the monocytes are eluted off the beads.
- TNF tangential flow filtration
- cells expressing the stem cell marker CD34 either from blood (see U.S. Patent No. 5,994,126, incorporated herein by reference) or from the bone marrow are purified. These cells are cultured with GM-CSF to differentiate into immature dendritic cells.
- Isolated dendritic cell precursors can be cultured ex vivo for differentiation, maturation and/or expansion.
- monocytic dendritic cells precursors are differentiated to form immature dendritic cells.
- the dendritic cell precursors, such as CD34 + dendritic cell precursors, or immature dendritic cells are then treated using the CRISPR/Cas9 system disclosed above to reduce or eliminate expression of CD300f. These cells are then matured to mature dendritic cells.
- Dendritic cell precursors and/or immature dendritic cells can be cultured and differentiated in suitable culture conditions.
- the tissue culture media can be supplemented with, e.g., plasma, serum, amino acids, vitamins, cytokines (e.g., granulocyte-macrophage colony- stimulating factor (GM-CSF), interleukins such as interleukin 4 (IL-4), interleukin 13 (IL-13), interleukin 15 (IL-15), or combinations thereof), purified proteins (such as serum albumin), divalent cations (e.g., calcium and/or magnesium ions), growth factors, and the like, to promote differentiation of the cells (Sallusto et al., J. Exp.
- a dendritic cell culture medium contains about 200 units/ml to about 1500 units/ml (e.g., about 1000 units/ml, about 500 units/ml, etc.) of GM-CSF and about 200 units/ml to about 1500 units/ml (e.g., about 800 units/ml, about 500 units/ml, etc.) IL-4.
- Immature dendritic cell have a high capacity for taking up and processing antigen, but have a limited ability to initiate immune responses.
- the ability to initiate an immune response is acquired by maturation of the immature dendritic cell. This maturation is also referred to as activating, or activation of, the dendritic cell.
- the maturation process may be initiated and/or induced through contact with maturation-inducing cytokines, tumor- associated antigens or tumor- associated peptide antigens and/or nucleic acids encoding tumor- associated antigens or tumor- associated peptide antigens, and the like.
- mature dendritic cells can be selected by expression of one or more markers.
- the markers include, but are not limited to, CD86, CD80, CD83, CD58, CDla, HLA-DR, CD40, CDllc, IL-2-beta, TLR-4 and combinations thereof.
- the dendritic cells can also be identified as lacking or expressing low levels of markers such as CD14.
- mature dendritic cells are identified as being CD80+, CD83+, CD86+, and CD14-.
- Cell surface markers can be detected in suitable assays, such as flow cytometry, immunohistochemistry, and the like.
- the cells can also be monitored for cytokine production (e.g., by ELISA, FACS, or other immune assay).
- CRISPR/Cas9 system disclosed herein can be cryopreserved for use at a later date.
- Tumor cells such as apoptotic or killed cells, may be used to deliver antigen to either immature or mature dendritic cells, either freshly isolated or obtained from in vitro culture, wherein expression of CD300f is decreased in the dendritic cells.
- tumor cells comprising an antigen e.g., tumor antigen
- immature dendritic cells are co-cultured with immature dendritic cells for a time sufficient to allow the antigen to be internalized by the immature dendritic cells.
- immature dendritic cells are then caused to mature by the addition of a maturation factor to the culture medium.
- the matured dendritic cells expressing processed antigen on their surface are then exposed to T cells for potent cytotoxic T cell induction to the tumor.
- one or more specific tumor antigens can be used. Exemplary tumor antigens that can be used include but are not limited to those shown in Table 1 above.
- peripheral blood mononuclear cells can be isolated from blood by sedimentation techniques.
- Human CD8 + T cell are purified with a commercial kit from Miltenyi Biotec.
- Dendritic cells are prepared, treated using the CRISPR/Cas9 system disclosed herein, and are cultured for 7 days to 10 days in the presence of GM-CSF and IL-4. On about day 7 through 10, apoptotic tumor cells can be co-cultured with the dendritic cells and the dendritic cells caused to mature over the next four days with the addition of monocyte conditioned medium, a signal for maturation.
- a combination of cytokines may be used to induce maturation of the immature dendritic cells.
- cytokines which may be used alone or in combination with each other include, but are not limited to, TNF-a, IL- ⁇ , IL-6, and IFN- ⁇ .
- a therapeutically effective amount of these dendritic cells are administered to a subject with the solid tumor.
- the dendritic cells are contacted with CD8 + T cells, and a therapeutically effective amount of the CD8 + T cells are administered to the subject with the solid tumor.
- dendritic cells are treated with the CRISPR/Cas9 system disclosed herein to reduce expression of CD300f.
- These dendritic cells are then loaded with one or more peptide antigens (e.g., a tumor- associated peptide antigen, such as those listed in Table 1).
- a tumor-associated peptide antigen such as those listed in Table 1.
- 1, 2, 3, 4, 5, 6, 7, 8, 9, or more tumor-associated peptide antigens are used.
- these peptide antigens are expressed by the solid tumor.
- a cell or membrane bound composition e.g., a liposome
- antigen presenting cells e.g., dendritic cells
- APCs antigen presenting cells
- tumor-associated peptide antigens can be incubated with one or more tumor- associated peptide antigens under conditions that are needed to load the major histocompatibility complex (MHC) of the dendritic cells.
- MHC major histocompatibility complex
- Suitable conditions for antigen loading are provided that permit a dendritic cell to contact, process and/or present one or more antigens on its MHC, whether intracellularly or on the cell surface.
- the incubation time may range from about 10 minutes to about 3 days or longer, from about 30 minutes to about 36 hours, from about 1 hour to about 28 hours, from about 2 hours to about 24 hours, from about 4 hours to about 24 hours, from about 4 hours to about 16 hours, from about 16 hours to about 24 hours, from about 20 hours to about 28 hours, from about 2 hours to about 4 hours, from about 1 hour to about 12 hours, from about 2 hours to about 8 hours, from about 3 hours to about 5 hours, for less than about a week, illustratively, for about 1 minute to about 48 hours, about 2 minutes to about 36 hours, about 3 minutes to about 24 hours, about 4 minutes to about 12 hours, about 6 minutes to about 8 hours, about 8 minutes to about 6 hours, about 10 minutes to about 5 hours, about 15 minutes to about 4 hours, about 20 minutes to about 3 hours, about 30 minutes to about 2 hours, about 40 minutes to about 1 hour, about 16 hours, about 20 hours, about 24 hours, about 28 hours, about 1 hour, about 2 hours, or about 4 hours.
- the incubation temperature may range from about 4 °C to about 37 °C, from about 25 °C to about 37 °C, about 4 °C, about 25 °C, or about 37 °C.
- the concentration of the peptide for loading can range from about 1 ⁇ g/ml to about 1 mg/ml, from about 5 ⁇ g/ml to about 800 ⁇ g /ml, from about 10 ⁇ g /ml to about 600 ⁇ g /ml, from about 15 ⁇ g /ml to about 400 ⁇ g/ml, from about 10 ⁇ g/ml to about 200 ⁇ g/ml, from about 10 ⁇ g/ml to about 100 ⁇ g/ml, from about 50 ⁇ g/ml to about 100 ⁇ g/ml, from about 20 ⁇ g/ml to about 100 ⁇ g/ml, etc.
- a number of methods for delivery of antigens to the endogenous processing pathway of antigen-presenting cells may be optionally used. Such methods include, but are not limited to, methods involving pH-sensitive liposomes, coupling of antigens to potent adjuvants, apoptotic cell delivery, pulsing cells onto dendritic cells, delivering recombinant chimeric virus-like particles (VLPs) comprising antigen to the MHC class I processing pathway of a dendritic cell line.
- VLPs chimeric virus-like particles
- Dendritic cells also can be contacted with nucleic acids encoding one or tumor-associated antigens under a condition sufficient for the at least one tumor-associated peptide antigen to be presented by the dendritic cell.
- dendritic cells can be transfected with expression vectors or infected with viral vectors for introducing nucleic acids encoding tumor-associated antigens into the dendritic cells.
- Expression can be optionally effected by targeting the expression construct to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue- specific promoter, or combinations thereof.
- Non-limiting viral vectors include adeno-associated viruses, lentiviruses, retroviruses, herpes viruses, adenoviruses, vaccinia viruses, baculoviruses, Fowl pox, AV-pox, modified vaccinia Ankara (MVA) and other recombinant viruses.
- adeno-associated viruses include adeno-associated viruses, lentiviruses, retroviruses, herpes viruses, adenoviruses, vaccinia viruses, baculoviruses, Fowl pox, AV-pox, modified vaccinia Ankara (MVA) and other recombinant viruses.
- the time and amount of antigens, or nucleic acids encoding the antigens, necessary for the antigen presenting cells to process and present the antigens can be determined, for example, by assaying T cell cytotoxic activity in vitro or using antigen-presenting cells as targets of CTLs.
- the antigen-presenting cells such as dendritic cells that are treated with the CRISPR/Cas9 system disclosed herein such that expression of CD300f is decreased, are loaded with the antigen, and then can be used to stimulate CTL proliferation in vivo or ex vivo.
- the ability of the loaded dendritic cells to stimulate a cytotoxic T lymphocyte (CTL) response can be measured by assaying the ability of the effector cells to lyse target cells.
- CTL cytotoxic T lymphocyte
- the non-radioactive LDH cytotoxicity assay or the europium release assay can be used. Volgmann et al., J. Immunol. Methods 119:45-51, 1989.
- ex vivo or in vitro maturation of dendritic cells can be induced by various maturation factors, including, but not limited to, tumor necrosis factor alpha (TNF-a), interferon alpha (IFN-a), poly (I:C), interferon gamma (IFN- ⁇ ), Interleukin 1 beta (IL- ⁇ ), Interleukin 6 (IL-6), prostaglandin E2 (PGE2), poly- dldC, vasointestinal peptide (VIP), bacterial lipopolysaccharide (LPS), mycobacteria or components of mycobacteria (such as cell wall constituents), or combinations thereof.
- TNF-a tumor necrosis factor alpha
- IFN-a interferon alpha
- poly I:C
- IFN- ⁇ Interleukin 1 beta
- IL-6 Interleukin 6
- PGE2 prostaglandin E2
- poly- dldC vasointestinal peptide
- Additional maturation factors include, for example, an imidazoquinoline compound, e.g., R848 (see PCT Publication No. WO 00/47719, incorporated herein by reference), a synthetic double stranded polyribonucleotide, agonists of a Toll-like receptor (TLR), such as TLR3, TLR4, TLR7 and/or TLR9, a sequence of nucleic acids containing unmethylated CpG motifs known to induce the maturation of dendritic cells, and the like. Further, a combination of any of the above agents can be used in inducing the maturation of immature dendritic cells or dendritic precursor cells.
- TLR Toll-like receptor
- mature dendritic cells or T cells can be expanded in vitro from freshly isolated or frozen cell stocks to generate sufficient numbers of cells for effective adoptive immunotherapy.
- Methods are provided for administration of mature dendritic cells to a subject in need of immunostimulation.
- such methods are performed by obtaining dendritic cell precursors or immature dendritic cells, differentiating and maturing those cells, using the CDRISPR/Cas9 system disclosed herein to produce dendritic cells with reduced expression of CD300f, and then culturing the cells in the presence of a tumor-associated antigen or a tumor- associated peptide antigen, a nucleic acid composition, and/or apoptotic tumor cells, to form a mature dendritic cell population.
- a therapeutically effective amount of any of these dendritic cells can be administered to a subject with a solid tumor.
- the present methods induces an immune response to a tumor in a subject.
- Such methods can include one or more steps of (a) obtaining monocytes (which may act as dendritic cell precursors) from a patient; (b) culturing the monocytes (e.g., with specific cytokines) to induce differentiation into immature dendritic cells; (c) contacting the immature dendritic cells with apoptotic tumor cells to induce engulfment and presentation of tumor-associated antigens; (d) differentiating the immature dendritic cells into mature dendritic cells with maturation factors such as cytokines or TLR ligands ;and (e) administering the mature dendritic cells to the patient.
- monocytes which may act as dendritic cell precursors
- culturing the monocytes e.g., with specific cytokines
- the monocytes, monocyte-derived cells or CD34 + progenitor-derived cells including immature dendritic cells can be treated with the CDRISPR/Cas9 system disclosed herein to produce cells with reduced expression of CD300f.
- Dendritic cells can be administered to the subject once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, or more, within a treatment regime to a subject/patient.
- Dendritic cells can be administered to a subject every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, every 11 days, every 12 days, every 13 days, every 14 days, every 16 days, every 18 days, every 20 days, every 1 month, every 2 months, every 3 months, every 6 months, or at different frequencies.
- the cells are administered intranodally or intradermally.
- the dendritic cells can be administered at a dose ranging from about 1 X 10 3 dendritic cells to about 1 X 10 12 dendritic cells, from about 1 X10 4 dendritic cells to about 1 X 10 10 dendritic cells, from about 1 X 10 5 dendritic cells to about 1 X 10 9 dendritic cells, from about 1 X 10 6 dendritic cells to about 11 X 10 8 dendritic cells, from about 1 X 10 6 dendritic cells to about 1 X 10 7 dendritic cells, from about 1 X 10 7 dendritic cells to about 1 X 10 8 dendritic cells, or from about 1 X 10 8 dendritic cells to about 1 X 10 9 dendritic cells.
- the mature dendritic cells can be contacted with, and thus, activate, lymphocytes.
- the activated, polarized lymphocytes optionally followed by clonal expansion in cell culture, can be administered to a subject with a solid tumor.
- the dendritic cells that engulfed apoptotic tumor cells, or dendritic cells loaded with antigen are contacted with lymphocytes under conditions sufficient to produce tumor-associated antigen- specific lymphocyte capable of eliciting an immune response against a tumor cell, such as cytotoxic CD8 + lymphocytes.
- CD8 + T lymphocytes are contacted with the dendritic cells described above for a period of time, such as for at least about 10 days, for priming and expanding the tumor antigen specific CD8 + T lymphocytes.
- the ability to induce lymphocytes to exhibit an immune response can be determined by any method including, but not limited to, determining T lymphocyte cytotoxic activity in vitro using for example tumor-associated antigen- specific antigen-presenting cells as targets of tumor- associated antigen- specific cytotoxic T lymphocytes (CTL); assaying tumor-associated antigen- specific T lymphocyte proliferation and ELISA methods.
- CD8 + T lymphocytes can be obtained, for example, from peripheral blood and used as purified preparations, which can be obtained by standard techniques including, but not limited to, methods involving immunomagnetic or flow cytometry techniques using antibodies.
- resulting CTL are reinfused autologously to the subject.
- the method includes administering to a subject antigen-presenting cells, T lymphocytes, or both, where the antigen-presenting cells have engulfed apoptotic tumor cells and presented tumor- associated antigens, or wherein the antigen-presenting cells have been loaded with at least one tumor-associated peptide antigen, or where the antigen-presenting cells comprise nucleic acids encoding at least one tumor-associated antigen, under a condition sufficient for at least one tumor- associated peptide antigen to be presented by the antigen-presenting cells.
- the T lymphocytes have been contacted with antigen-presenting cells presenting at least one tumor-associated antigen.
- the dendritic cells and the T cells are autologous.
- the APCs and the responder T cells are from the same individual.
- the APCs and the responder T cells can be syngeneic.
- the APC can be used to present any antigen to a population of autologous T cells.
- antigenic peptides that bind to MHC class I and II molecules can be generated ex vivo (for example instead of being processed from a full-length protein in a cell), and allowed to interact with (such as bind) MHC I and II molecules on a cell surface.
- APCs present antigen in the context of both MHC class I and ⁇ .
- the amount of antigen used to prime T cells can be readily determined using methods known in the art. Generally, if the antigen is used in a purified form, about 1-10 ⁇ g/ ml of peptide is used.
- lymphocytes are primed in vitro by incubating them with soluble antigen or viral lysate for 5-7 days under conditions that permit priming of T cells.
- Viable T cells are recovered, for example by Ficoll-Hypaque centrifugation, thereby generating primed T cells.
- the viable primed T cells can be primed again one or more times, for example by incubation with the antigen for another 5-7 days under the same conditions as those used for the first priming, and viable T cells recovered.
- proliferation of the cells can be stimulated, for example by incubation in the presence of a cytokine, such as interleukin (IL)-2, IL-7, IL-12 and IL-15.
- a cytokine such as interleukin (IL)-2, IL-7, IL-12 and IL-15.
- the amount of cytokine added is sufficient to stimulate production and proliferation of T cells, and can be determined using routine methods.
- the amount of IL-2, IL-7, IL-12, or IL-15 added is about 0.1-100 IU/mL, such as at least 1 IU/mL, at least 10 IU/mL, or at least 20 IU/mL.
- the cells can be counted to determine the cell number. When the desired number of cells is achieved, purity is determined. Purity can be determined, for example, using markers present on the surface of activated T cells concomitant with the assessment of cytokine production upon antigen recognition, such as interferon (IFN)y, tumor necrosis factor (TNF)oc, or interleukin (IL)-2.
- IFN interferon
- TNF tumor necrosis factor
- IL interleukin
- activated CD8 + T cells are positive for the CD3 marker, along with the CD8 marker, and IFN- ⁇ (which is specific for activated T cells).
- FACS fluorescence activated cell sorting
- stimulated T activated cells are permeabilized and incubated in the presence of anti-CD3, anti-CD8 and anti-IFN- ⁇ (each having a different fluorophore attached), for a time sufficient for the antibody to bind to the cells. After removing unbound antibody, cells are analyzed by FACS using routine methods. Antigen- specific T cells are those that are INF- ⁇ positive.
- the method further includes determining the cytotoxicity of the antigen- specific T cells.
- Methods for determining cytotoxicity are known in the art, for example a 51 Cr- release assay (for example see Walker et al. Nature 328:345-8, 1987; Qin et al. Acta Pharmacol. Sin. 23(6):534-8, 2002; all herein incorporated by reference).
- the present disclosure also provides therapeutic compositions that include the enriched (such as purified) activated T cells.
- the resulting enriched population of activated T cells are placed in a therapeutic dose form for administration to a subject with a solid tumor.
- Expanded and selected activated CD8 + T cells can be tested for mycoplasma, sterility, endotoxin and quality controlled for function and purity prior cryopreservation or prior to infusion into the recipient.
- a therapeutically effective amount of activated CD8 + T cells is administered to the subject.
- a therapeutically effective amount of purified activated T cells include purified activated T cells administered at a dose of about 1 X 10 5 cells per kilogram of subject to about 1 X 10 9 cells per kilogram of subject, such as from about 1 X 10 6 cells per kilogram to about 1 X 10 8 cells per kilogram, such as from about 5 X 10 6 cells per kilogram to about 75 X 10 6 cells per kilogram, such as at about 25 X 10 6 cells per kilogram, or at about 50 X 10 6 cells per kilogram.
- Purified activated T cells can be administered in single or multiple doses as determined by a clinician. For example, the cells can be administered at intervals of approximately two weeks depending on the response desired and the response obtained. In some examples, once the desired response is obtained, no further activated T cells are administered. However, if the recipient displays one or more symptoms associated with the presence or growth of a tumor, a therapeutically effective amount of activated T cells can be administered at that time.
- the administration can be local or systemic.
- the purified activated T cells disclosed herein can be administered with a pharmaceutically acceptable carrier, such as saline.
- a pharmaceutically acceptable carrier such as saline.
- Other therapeutic agents can be administered before, during, or after administration of the activated T cells, depending on the desired effect.
- exemplary therapeutic agents include, but are not limited to, anti-microbial agents, immune stimulants such as interferon- alpha, chemotherapeutic agents, biologic agents (such as those listed in Table 1) or peptide vaccines of the same antigen used to stimulate T cells in vitro.
- compositions containing purified activated T cells also include one or more therapeutic agents.
- CD300f function in dendritic cells markedly enhanced their ability to phagocytose and process apoptotic tumor cells, and cross-present tumor cell antigens to cytotoxic CD8 + T cells (CTL), leading to substantial inhibition of tumor growth.
- CTL cytotoxic CD8 + T cells
- Anti-CD300f blocking antibodies can release a checkpoint on human DC in order to enhance tumor cell antigen presentation by DC and induce the expansion and activation of tumor- specific autologous CTL.
- CD300f uniquely functions as a checkpoint receptor to inhibit DC- mediated apoptotic cell phagocytosis and antigen cross-presentation for T cell priming.
- An overview of the method is provided in FIG. 1.
- Mouse with the deficiency of Cd300f gene supplies a very useful tool to examine CD300f function.
- exon 2-3 of Cd300f gene was flanked by loxP sites.
- a PGK-neo cassette flanked by Flp recombinase target sites was used for selection.
- mice Following homologous recombination of the vector in embryonic stem cells, clones bearing the Cd300fil/fl locus were established after deletion of PGK-neo selection cassette by Flp recombination, and clones with the Cd300f-I- locus were generated after deletion of the LoxP sites flanking regions together with the PGK-neo cassette using Cre recombinase. Identified targeted embryonic stem cell clones were microinjected into the blastocysts of C57BL/6 mice. With the Cd300f-I- mice, two tumor models were developed to examine CD300f deficiency on solid tumor development. For tumor graft mouse model, mice were subcutaneously inoculated with 10 6 solid tumor cells on day 0.
- mice were systemically irradiated at 3.25 Gy on day 7 (IR) and some of the irradiated mice were transferred with 10 6 tumor antigen specific CD8 + T cells on day 9 (OT-I). Irradiation was to deplete some existing immune cells and make space to allow for expansion of anti-tumor effectors. Tumor growth was measured on the indicated days; mice were euthanized when the longitudinal tumor diameter reached 15 mm. For the mouse model of
- mice were injected with AOM intraperitoneally and then fed with DSS-containing water in three 7-day cycles, with intermittent 14-day intervals of regular drinking water. After 10 weeks, mice were euthanized, and colon tumor sizes were measured. The results showed that G£?0O -deficient mice have a significantly inhibited growth of solid tumors (e.g., EL4-TfOVA lymphoma grafts, AOM/DSS-induced colorectal cancer; FIGS. 2A, 2B), indicating that CD300f negatively regulates anti-tumor responses against different cancer types.
- solid tumors e.g., EL4-TfOVA lymphoma grafts, AOM/DSS-induced colorectal cancer; FIGS. 2A, 2B
- CD300f checkpoint inhibition leads to inhibition of grafted adenocarcinoma cells (MC38) in a mouse model.
- Cd300f+I+ or Cd300f -I- mice were subcutaneously inoculated with 10 6 MC38 cells on day 0. Tumor growth was monitored on the indicated days; mice were euthanized when the longitudinal tumor diameter reached 15 mm.
- Cd300f-deficient mice had significantly inhibited growth of the grafted adenocarcinoma cells, see FIGS. 3A-3B. Furthermore, the data indicated that the improved tumor clearance in Cd300f- deficient mice is due to enhanced activation of CD8 + T cells (FIG. 2C), indicating that the blockade of CD300f function in DC enhances their cross-presentation of tumor antigens.
- CD300f expression is found on immune cells and immune-associated tissues, but not on non-immune tissues (e.g., heart, muscle, liver, kidneys). It was demonstrated that human CD300f is present on human myeloid cell populations (e.g., monocytes and DCs, see FIGS. 5A-5B). Furthermore, it was found that in human blood CD300f is not expressed on lymphoid cells (T, B, NK or NKT cells), but is highly expressed on myeloid cells (monocytes, neutrophils, DC). Thus, targeting CD300f to release a checkpoint on human is a unique and potent therapeutic method for cancer management, as DC function upstream of T cells.
- CD300f antibodies can block PS recognition and enhance apoptotic cell phagocytosis by mouse DC in vitro. Once a blocking antibody is selected, mouse models and assays are used to demonstrate that antibody-mediated blocking of CD300f functions to inhibit tumor progression (e.g., lymphoma grafts, colorectal cancer).
- tumor progression e.g., lymphoma grafts, colorectal cancer.
- a humanized monoclonal antibody is produced that specifically recognizes human CD300f, with no cross-reactivity with other CD300 family members.
- Such an antibody has blocking potential, i.e., interfere with CD300f ability to recognize its ligand, phosphatidylserine, and thus neutralize CD300f signaling potential (function).
- Selected clones of the antibodies are tested using standard ELISA, flow cytometry and apoptotic cell phagocytosis assays, in order to determine the best clone capable of blocking PS recognition by CD300f and inhibiting CD300f function. Once selected, anti-CD300f blocking antibody (or antibodies) are used in vitro.
- Monocytes were isolated from patient blood samples (using a negative selection kit), and then differentiated into DC in vitro, using standard methods (i.e., culture with GM-CSF and IL-4; Dauer M. et al., J Immunol 2003).
- CD300f -blocked DC were incubated with apoptotic tumor cells (generated from patient's tumor samples), allowing them to engulf and process apoptotic tumor cells.
- Human CD300f like mouse CD300f, recognized phosphatidylserine and regulated the phagocytosis of apoptosis (see FIGS. 4A-4B).
- these DC are incubated with autologous CTL (isolated from patient's blood samples using a negative selection kit), in order to cross-present apoptotic tumor cell-derived antigens and prime CTL.
- autologous CTL isolated from patient's blood samples using a negative selection kit
- Enhanced CTL activation over control treated CTL is verified by monitoring cytokine production (e.g., TNF-oc, IFN- ⁇ ).
- the activated CTL will be tested for their ability to kill patient tumor cells that were the source of the DC-presented antigens, by standard cytotoxicity assays (e.g., chromium release assay).
- cytotoxicity assays e.g., chromium release assay
- mice e.g., NSG mice
- a combinatorial therapeutic approach combining the blockade of checkpoints on DC (CD300f) and CTL, (PD-1, CTLA-4, and/or LAG-3, can be used.
- the engineered humanized antibodies are used in patients to activate DC and stimulate antigen cross-presentation, and thus mediate a potent anti-tumor response.
- the biological readout of physiological efficacy and measureable biomarkers for the therapy is the monitoring of IFN- ⁇ (and/or other cytokines, e.g., IL-2) production by the activated CTL, and the presence of activated CTL themselves in the patients, as already tested in the mouse models.
- An advantage of this approach over the existing methods is the independence from the requirement for a definitive tumor antigen, as DC are fed intact patient tumor cells, and can cross- present a multitude of tumor antigens.
- the method can be used to treat a broad range of cancer types, and be quickly adapted to treat a particular patient's cancer.
- the CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
- CD300LF CD300LF
- the target sequences in the genomic DNA are designed using E-CRISP Designer (v. 4.2; e-crisp.org/E- CRISP/designcrispr_html), or CHOPCHOP web tool for genome editing
- CD300LF 5 different sequences targeting CD300LF gene are generated. Their nucleotide sequences are aligned against those present in the human genomic and transcript database, to verify the specificity of CD300LF targeting.
- the oligomers are synthesized, annealed and cloned into lentiCRISPRv2 (AddGene.org), a one vector system co-expressing a mammalian codon-optimized Cas9 nuclease along with a single guide RNA, according to the protocol found at the Zhang Lab GeCKO website (genome-engineering_org/gecko/).
- the lentiviral expression constructs, verified by DNA sequencing, are transfected into 293T cells with the psPAX2 and pMD2.G helper plasmids (AddGene.org) using PolyJet transfection reagent
- the 293T cell culture medium containing lentivirus particles are used to infect 2xl0 6 human monocytes, monocyte-derived cells (e.g., DCs, macrophages), in the presence of 10 ⁇ g/ml protamine sulphate (Sigma).
- CD300f expression on the cell surface is monitored routinely at protein levels by flow cytometry, to verify the lack of CD300f expression by the transduced cells. All CRISPR constructs are evaluated for their ability to disrupt CD300LF and generate human CD300f-deficient cells.
- GTGGTGGCCGGTCAGAGTTGGGG-3 ' (SEQ ID NO: 21) sequence of CD300LF (exon 3 and 4, respectively) were determined to be the most optimal to mediate gene disruption, and are chosen for generation of CD300f-deficeint human cells.
- Monocytes are isolated from patient' s blood samples using a negative selection kit (Miltenyi Monocyte Isolation Kit II), and then differentiated into DC in vitro, using standard methods (for example, culture with GM-CSF and IL-4, see Dauer M. et al., J Immunol 2003). Those monocyte- derived DC are treated with the anti-CD300f antibody to block CD300f, thereby enhancing DC activity and promoting antigen cross-presentation. In parallel, tumor cells isolated from cancer patients (e.g., biopsy samples) are exposed to UV irradiation to induce apoptosis and generate apoptotic tumor cells.
- a negative selection kit for example, culture with GM-CSF and IL-4, see Dauer M. et al., J Immunol 2003.
- Those monocyte- derived DC are treated with the anti-CD300f antibody to block CD300f, thereby enhancing DC activity and promoting antigen cross-presentation.
- tumor cells isolated from cancer patients e.g
- CD300f -blocked DC is incubated with the apoptotic tumor cells (generated from a patient's tumor sample), allowing them to engulf and process the apoptotic tumor cells.
- Those DC are then matured with 10 ng/ml LPS plus 100 IU/ml IFN- ⁇ and incubated with autologous cytotoxic T lymphocytes (CTLs) (isolated from patient's blood samples using a negative selection kit, such as the Miltenyi CD8 + T Cell Isolation Kit) using the protocol described in Nature Protocols 9, 950-966 (2014), incorporated herein by reference, in order to cross-present apoptotic tumor cell-derived antigens and prime CTL.
- CTLs autologous cytotoxic T lymphocytes
- CTL by DC in co-cultures is verified by monitoring the ability of CTL to produce different cytokines (e.g., TNF-a, IFN- ⁇ ), using ELISA and flow cytometry.
- the activated CTL are subsequently tested for their ability to kill patient tumor cells that were the source of the DC-presented antigens, by standard cytotoxicity assays (e.g., DELFIA or chromium release assay).
- standard cytotoxicity assays e.g., DELFIA or chromium release assay.
- combining the blockade of checkpoints on DC and CTL, using anti-CD300f in combination with anti-PD-1, anti-CTLA-4, and/or anti-LAG-3 antibodies is also tested.
- CD300f knock-out of CD300f in human monocyte-derived DC cells using CRISPR is performed.
- the CD300f deficiency on DCs results in enhanced engulfment of apoptotic tumor cells and cross-presentation of tumor-specific antigens, leading to elevated priming and cytotoxicity of CTL against a variety of tumor cell lines.
- the CD300f-deficient DCs are used to prime and expand the tumor antigen-specific CTL in vitro or are administered directly alone or with CTL to the patient to depress tumor growth.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Genetics & Genomics (AREA)
- Environmental Sciences (AREA)
- Zoology (AREA)
- Public Health (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Biotechnology (AREA)
- General Chemical & Material Sciences (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Methods are disclosed for treating a subject with a solid tumor. The methods can include administering to the subject a therapeutically effective amount of (1) a CD300f inhibitor, (2) dendritic cells comprising an inactivated gene encoding CD300f, (3) T cells activated by the dendritic cells comprising an inactivated gene encoding CD300f, or combinations thereof, thereby treating the solid tumor in the subject.
Description
TREATMENT OF TUMORS BY INHIBITION OF CD300F
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 62/408,596, filed October 14, 2016, which is herein incorporated by reference in its entirety.
FIELD OF THE DISCLOSURE
This relates to the field of oncology, specifically to the use of CD300f inhibitors and/or the use of dendritic cells with modified genes encoding CD300f, for the treatment of solid tumors.
BACKGROUND
Cancer immunotherapy aims to enhance the ability of the patients' own immune response to destroy tumors. The magnitude of immune response is determined by the balance between immune activating signals and negative inhibitory signals. Checkpoint receptors encompass a specific subset of negative regulators that normally deliver inhibitory signals that dampen stimulatory signals and limit immune activation. Blockade of immune checkpoints represents an effective strategy to enhance the immune response against cancer cells. Several checkpoint receptors, including cytotoxic T lymphocyte associated protein-4 (CTLA-4) and Programmed Death (PD)-l, have been identified on T cells and targeting these with blocking antibodies has been successful in treating different cancer types (e.g., melanoma, bladder, and gastric cancer). However, multiple cancers, such as pancreatic and prostate cancer, are resistant to T cell checkpoint blocking, underscoring the importance of identifying novel checkpoints on immune cells for successful cancer therapy.
In addition to uncontrolled cell proliferation and expansion, malignant tumors display a high rate of cell loss due to apoptosis. As apoptotic cells expose phosphatidylserine (PS) on their cell surface, the engagement of PS on the tumor cell surface by PS -recognizing receptors of phagocytic immune cells could be a factor for the anti-tumor response, as it would regulate engulfment of the dying tumor cells and subsequent tumor antigen cross-presentation. A need remains for treatments for malignant tumors, such as treatments that target cell checkpoints.
SUMMARY OF THE DISCLOSURE
The PS -recognizing receptor, CD300f, expressed on myeloid cells (i.e., dendritic cells and macrophages) and a small sub-population of B cells in mice, regulates phagocytosis of apoptotic
cells by macrophages and dendritic cells, thereby controlling inflammatory immune responses (Tian et al., Nat Commun 5:3146, 2014; Tian et al., Cell Death Differ 23: 1086-96, 2016). The use of CD300f inhibitors for the treatment of tumors, such as a solid tumor, is disclosed herein. Methods are also disclosed for targeting CD300f in dendritic cells, and the use of adoptive transfer of these dendritic cells or T cells activated by these dendritic cells for the treatment of solid tumors.
In some embodiments, methods are disclosed for treating a subject with a solid tumor. The methods include administering to the subject a therapeutically effective amount of a CD300f inhibitor, thereby treating the solid tumor in the subject.
In additional embodiments, methods are disclosed for treating a solid tumor in a subject, wherein the methods include administering to the subject a therapeutically effective amount of dendritic cells comprising an inactivated gene encoding CD300f.
In further embodiments, methods are treating a solid tumor in a subject that include isolating dendritic cells from the subject and transforming the dendritic cells with one or more vectors comprising: a) a Embryonal Fyn- Associated Substrate (EFS) promoter operably linked to a nucleotide sequence encoding a Type Π Cas9 nuclease, b) a U6 promoter operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas system guide RNAs that hybridize with the CD300f gene in the dendritic cell, wherein components (a) and (b) are located on same or different vectors. Alternatively, a ribonucleoprotein (RNP) complex can be utilized, wherein the guide RNA is attached to the protein and delivered to the dendritic cells. The one or more guide RNAs target the CD300f gene in the dendritic cell and the Cas9 protein cleaves the CD300f gene such that the sequence of the CD300f gene is modified or inactivated in the dendritic cell, thereby forming modified dendritic cells. The subject is administered a therapeutically effective amount of the modified dendritic cells, thereby treating the solid tumor in the subject.
In yet other embodiments, methods are disclosed treating a solid tumor in a subject that include isolating dendritic cells from the subject, and transforming the dendritic cells with one or more viral vectors comprising: a) a Embryonal Fyn- Associated Substrate (EFS) promoter operably linked to a nucleotide sequence encoding a Type II Cas9 nuclease, b) a U6 promoter operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas system guide RNAs that hybridize with the CD300f gene in the dendritic cell, wherein components (a) and (b) are located on same or different viral vectors. Alternatively, a RNP complex can be utilized, wherein the guide RNA is attached to the protein and delivered to the dendritic cells. The one or more guide RNAs target the CD300f gene in the dendritic cell and the Cas9 protein cleaves the CD300f gene such that the sequence of the CD300f gene is modified or inactivated in the dendritic cell, thereby
forming modified dendritic cells. The modified dendritic cells are contacted with CD8+ T cells in the presence of tumor-associated antigens expressed by the solid tumor to form activated T cells. A therapeutically effective amount of the activated T cells is administered to the subject to treat the solid tumor.
The foregoing and other features of the disclosure will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES FIGS. 1A-1C. Schematic overview of methods used to examine CD300f deficiency on solid tumor development in mice. A. Generation of Cd300f-/- mice on a C57BL/6 genetic background. Exon 2-3 of Cd300f gene was flanked by loxP sites. A PGK-neo cassette flanked by Flp recombinase target sites was used for selection. Following homologous recombination of the vector in embryonic stem cells, clones bearing the Cd300ffl/fl locus were established after deletion of PGK-neo selection cassette by Flp recombination, and clones with the Cd300f-/- locus were generated after deletion of the LoxP sites flanking regions together with the PGK-neo cassette using Cre recombinase. Identified targeted embryonic stem cell clones were microinjected into the blastocysts of C57BL/6 mice. B. Use the mouse model of tumor graft to study the effect of CD300f deficiency on solid tumor development. Cd300f +/+ or Cd300f-/- mice were subcutaneously inoculated with 106 solid tumor cells, for example EL4-TfOVA cells, on dayO. Some of the mice were systemically irradiated at 3.25 Gy on day 7 (IR) and some of the irradiated mice were transferred with 106 tumor antigen specific CD8+ T cells, for example OT-I T cells, on day 9 (OT- I). Irradiation was to deplete some existing immune cells and make space to allow for expansion of anti-tumor effectors. Tumor growth was measured on the indicated days; mice were euthanized when the longitudinal tumor diameter reached 15 mm. C. Use the mouse model of
azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colon cancer to examine CD300f deficiency on solid tumor development. Cd300f +/+ or Cd300f-/- mice were injected with AOM intraperitoneally and then fed with DSS-containing water in three 7-day cycles, with intermittent 14-day intervals of regular drinking water. After 10 weeks, mice were euthanized, and colon tumor sizes were measured.
FIGS. 2A-2D. CD300f functions as a checkpoint for tumor immunity. A. Inhibition of grafted tumor growth in G£?0O -deficient mice. Cd300f+I+ or Cd300f-I- mice were
subcutaneously inoculated with 106 EL4-TfOVA cells on day 0. Some of the mice were
systemically irradiated at 3.25 Gy on day 7 (IR) and some of the irradiated mice were transferred with 106 OT-I T cells on day 9 (OT-I). Irradiation was to deplete some existing immune cells and make space to allow for expansion of anti-tumor effectors. Tumor growth was measured on the indicated days; mice were euthanized when the longitudinal tumor diameter reached 15 mm. The graph on the left shows the progression of tumor growth in Cd300f+I+ and Cd300f -I- mice; the images on the right illustrate tumors dissected on day 18 from Cd300f+I+ and Cd300f-I- mice that were both irradiated and OT-I T cell-transferred. B. Inhibition of growth of AOM/DSS-induced colorectal cancer in C<i?0O -deficient mice. Cd300f+I+ or Cd300f-I- mice were injected with AOM intraperitoneally and then fed with DSS-containing water in three 7-day cycles, with intermittent 14-day intervals of regular drinking water. After 10 weeks, mice were euthanized, and colon tumor sizes were measured. The images on the left show tumors in the colons of Cd300f+I+ and Cd300f- I- mice; arrowheads indicate the position of tumors. The graph on the right shows quantification of tumor sizes in Cd300f+I+ and Cd300f-I- mice. C. Enhanced activation of CD8+ T cells in Cd300f- deficient mice. Cd300f+I+ or Cd300f-I- mice were systemically irradiated at 3.25 Gy on day 1 and transferred with OT-I T cells (106) on day 3. Splenocytes were isolated on day 12, and stimulated with 10 μg/ml OT-1 peptide for 6 hours, and the percentage of OT-I cells positive for IFN-γ was determined by flow cytometry. D. Enhanced infiltration of CD8+ T cells in the EL4-TfOVA tumor tissues in Gi?0O -deficient mice. The images show a representative staining of CD8+ T cells (arrowheads) in paraffin-embedded tumor tissues isolated from Cd300f+I+ and Cd300f-I- mice irradiated at 3.25 Gy.
FIGS. 3A-3B. Inhibition of grafted tumor growth in CD300f-deficient mice. Cd300f+I+ or Cd300f-I- mice were subcutaneously inoculated with 106 MC38 cells on day 0. Tumor growth was monitored on the indicated days; mice were euthanized when the longitudinal tumor diameter reached 15 mm. A. The graph shows the progression of tumor growth in Cd300f+/+ and Cd300f-/- mice. B. The images show the tumors dissected on day 16 from Cd300f+/+ and Cd300f-/- mice.
FIGS. 4A-4B. Human CD300f recognizes PS and regulates the phagocytosis of apoptotic cells. A. Human CD300f binds PS by surface plasmon resonance analysis. ImM PS-containing liposomes were captured on the LI sensor chip, followed by injection of Fc-fused control protein ΝΓΓΡν, mouse CD300f and human CD300f at 10μg/ml. The sensorgrams show the resonance units over the indicated times. B. Human CD300f expression on L929 cells promotes phagocytosis of apoptotic cells. L929 cells, transduced with empty virus (EV) or human CD300f, were mixed with pHrodo-labeled unirradiated or irradiated mouse thymocytes at a 1:3 ratio for the indicated times.
Cells were suspended in the pH 8.8 buffer, and analyzed for the percentage of pHrodo+ cells, representing the cells that engulfed apoptotic cells.
FIGS. 5A-5B. Human CD300f is expressed on human myeloid cell populations including monocytes and immature dendritic cells (DCs). A. Human peripheral blood mononuclear cells (PBMC) were isolated, stained with antibodies against human CD300f, CD3 (T cells), CD19 (B cells), CD56 (NK cells) and CD16 and CD14 (monocytes) and analyzed by flow cytometry. The Rl, R2, R3 and R4 were gated on the dot plots of forward scattered light (FSC) vs. side scattered light (SSC). B. Human peripheral blood monocytes were isolated and induced to differentiate into immature DCs with the combination of granulocyte macrophage colony stimulating factor (GM- CSF) and interleukin (IL)-4. The CD300f expression was tested with EFLUOR™ 660-conjuaged anti-human CD300f antibody and analyzed by flow cytometry.
SEQUENCE LISTING
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file [Sequence_Listing, October 11, 2017, 17.0KB], which is incorporated by reference herein. In the accompanying sequence listing:
SEQ ID NOs: 1-8 are the amino acid sequences of human framework regions.
SEQ ID NO: 9 is the amino acid sequence of a Cas9 from Streptococcus pyogenes.
SEQ ID NO: 10 is the nucleic acid sequence of an Embryonal Fyn- Associated Substrate (EFS) promoter.
SEQ ID NOs: 11-12 are nucleic acid sequences encoding a crRNA.
SEQ ID NO: 13 is the nucleic acid sequence of a U6 promoter.
SEQ ID NO: 14 is a nucleic acid sequence encoding a U6 gRNA.
SEQ ID NO: 15 is a nucleic acid sequence encoding a tracrRNA.
SEQ ID NOs: 16-19 are RNAi nucleic acid sequences.
SEQ ID NO: 20 is a nucleic acid sequence of a target in exon 3 of human CD300LF.
SEQ ID NOs: 21 is a nucleic acid sequence of a target in exon 4 of human CD300LF.
DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
The use of CD300f inhibitors for the treatment of solid tumors is disclosed herein. Methods are also disclosed for targeting CD300f in dendritic cells, and the use of adoptive transfer of these dendritic cells or T cells activated by these dendritic cells, for the treatment of solid tumors. These methods can be used in combination with other agents for the treatment of solid tumors, such as, but not limited to, sarcomas and carcinomas.
Terms
Administration: The introduction of a composition (such as one containing a CD300f inhibitor) into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, and intratumoral), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.
Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects.
Antibody: A polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope (e.g., an antigen, such as a CD300f protein or fragment thereof). This includes intact immunoglobulins and the variants and portions of them well known in the art, such as Fab' fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.
Typically, an immunoglobulin has a heavy and light chain. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as "domains"). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and
heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs". The extent of the framework region and CDRs has been defined (see, Kabat et al., Sequences of Proteins of
Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N- terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDRl is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab. References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.
A "monoclonal antibody" is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected.
Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.
A "humanized" immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic)
immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a "donor," and the human immunoglobulin providing the framework is termed an "acceptor." In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that
provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework.
Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by means of genetic engineering (e.g., see U.S. Patent No. 5,585,089).
A "neutralizing antibody" is an antibody that interferes with any of the biological activities of its target polypeptide, such as a CD300f polypeptide.
Apoptotic cells: Non-dividing, non-viable cells that can be distinguished from necrotic cells (other dead cells). Apoptosis is a result of programmed cell death. According to characteristic morphological and biochemical features, apoptosis is characterized by shrinkage of the cell, dramatic reorganization of the cell nucleus, cell membrane and cell metabolism, active membrane blebbing, and ultimate fragmentation of the cell into membrane-enclosed vesicles (apoptotic bodies). The nuclear events of apoptosis begin with collapse of the chromatin against the nuclear periphery and into one or a few large clumps within the nucleus. Nuclear features include chromatin aggregation followed by DNA fragmentation (a specific marker of apoptotic process) after activation of endonucleases resulting in multiples subunits of DNA of an approximately 180 base pairs. The cellular events include cytoplasmic condensation and partition of the cytoplasm and nucleus into membrane bound-vesicles which contain ribosomes, intact mitochondria and nuclear material which are surrounded by an intact cellular membrane (a specific marker of apoptotic process when compared with necrosis, the other non-physiological cell death process).
CD300 molecule like family member f (CD300f): The human CD300 receptors are type I transmembrane proteins with single IgV-like extracellular domains that are mainly expressed by myeloid cells. Mouse CD300f (CLM-1) possesses both activating and inhibitory signaling potentials for regulation of apoptotic cell engulfment upon PS recognition. CD300f deficiency predisposes C57BL/6 mice to develop autoimmune disease. While CD300f functions to promote macrophage efferocytosis, its role in dendritic cells serves to inhibit apoptotic cell engulfment. Conventional dendritic cells (cDC, CDl lchl B220") and plasmacytoid dendritic cells (pDC, CDllcloB220+PDCA-l+) have similar levels of CD300f expression.
The CD300f gene is also known as CD300f, CLM1, IGSF13, IREM1, NKIR and CD300LF.
CD300f sequences are publicly avaible. For example, GenBank® Accession Nos. NP_620587.2, P 001276011.1, P 001276012.1, P 001276013.1, P 001276014.1, P 001276015.1, and NP_001276016.1, provide exemplary human CD300f protein sequences, and GenBank® Accession
Nos. NM_139018.4, NM_001289082.1, NM_001289083.1, NM_001289084.1, NM_001289085.1, NM_001289086.1, and NM_001289087.1 provide exemplary human CD300f nucleic acid sequences (all sequences herein incorporated by reference as of October 14, 2016).
cDNA (complementary DNA): A piece of DNA lacking internal, non-coding segments (introns) and regulatory sequences that determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
Chemotherapeutic agent: Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. For example, chemotherapeutic agents can be useful for the treatment of a solid tumor cancer, such as a sarcoma, carcinoma, lymphoma, colorectal or skin cancer. Particular examples of chemotherapeutic agents that can be used include microtubule binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. In one embodiment, a chemotherapeutic agent is a radioactive compound. Other chemotherapeutic agents that can be used are provided in Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed., © 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby- Year Book, 1995; Fischer, D.S., Knobf, M.F., Durivage, H.J. (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993; Chabner and Longo, Cancer Chemotherapy and Biotherapy: Principles and Practice (4th ed.). Philadelphia: Lippincott Willians & Wilkins, 2005; Skeel, Handbook of Cancer Chemotherapy (6th ed.). Lippincott Williams & Wilkins, 2003. Combination chemotherapy is the administration of more than one agent to treat cancer.
Conservative variants: "Conservative" amino acid substitutions are those substitutions that do not substantially affect or decrease an activity of a polypeptide, for example a CD300f peptide's ability to mediate negative regulatory signals by recruiting SHP1 or SHIP. Specific, non-limiting examples of a conservative substitution include the following examples:
Original Residue Conservative Substitutions
Ala Ser
Arg Lys
Asn Gin, His
Asp Glu
Cys Ser
Gin Asn
Glu Asp
His Asn; Gin
lie Leu, Val
Leu He; Val
Lys Arg; Gin;
Met Leu; lie
Phe Met; Leu;
Ser Thr
Thr Ser
Trp Tyr
Tyr Trp; Phe
Val lie; Leu
The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that the polypeptide binds with the same affinity as the unsubstituted (parental) polypeptide. Non-conservative substitutions are those that reduce the ability of the polypeptide.
Consists Essentially Of/Consists Of: With regard to a polypeptide, a polypeptide that consists essentially of a specified amino acid sequence if it does not include any additional amino acid residues. However, the polypeptide can include additional non-peptide components, such as labels (for example, fluorescent, radioactive, or solid particle labels), sugars or lipids. With regard to a polypeptide, a polypeptide that consists of a specified amino acid sequence does not include any additional amino acid residues, nor does it include additional non-peptide components, such as lipids, sugars or labels.
Clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9): An RNA-guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspersed Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes, among other bacteria. Cas9 can cleave nearly any sequence complementary to the guide RNA. Includes Cas9 nucleic acid molecules and proteins. Cas9 sequences are publically available, for example from the GENBANK® sequence database (e.g., Accession Nos. NP_269215.1 and AKS40378.1 provide exemplary Cas9 protein sequences, while Accession No. NC_002737.2 provides an exemplary Cas9 nucleic acid sequence therein). One of ordinary skill in the art can identify additional Cas9 nucleic acid and protein sequences, including Cas9 variants.
Degenerate variant: A polynucleotide encoding a peptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in this disclosure as long as the amino acid sequence of the polypeptide encoded by the nucleotide sequence is unchanged.
Dendritic Cell (DC): An antigen presenting cell that processes antigens and presents them to T cells. In vivo, dendritic cells are present in the skin, the nose, lungs, stomach, intestines, and in the blood. Once activated, they migrate to the lymph nodes where they interact with T cells and B cells to initiate and shape the adaptive immune response. At certain development stages they grow branched projections, called "dendrities." Dendritic cells include conventional dendritic cells
(cDCs), that are similar to monocytes, and plasmacytoid dendritic cells (pDCs). Monocyte-derived dendritic cells can be generated in vitro from peripheral blood mononuclear cells (PBMCs).
Treatment of these monocytes with interleukin 4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF) leads to differentiation to immature dendritic cells (iDCs) in about a week. Subsequent treatment with tumor necrosis factor (TNF) or lipopolysaccharide (LPS) further differentiates the iDCs into mature dendritic cells (mDCs).
Donor polynucleotide: A polynucleotide that is capable of specifically inserting into a genomic locus.
Downstream: A relative position on a polynucleotide, wherein the "downstream" position is closer to the 3 ' end of the polynucleotide than the reference point. In the instance of a double- stranded polynucleotide, the orientation of 5' and 3' ends are based on the sense strand, as opposed to the antisense strand.
Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus, expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct gene reading frame to permit proper translation of mRNA, and stop codons. The term "control sequences" includes, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.
A promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue- specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included (see e.g., Bitter et al., 1987, Methods in Enzymology 153, 516-544). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (such as the metallothionein promoter) or from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques can also be used to provide for transcription of the nucleic acid sequences.
Heterologous: Originating from separate genetic sources or species. A polypeptide that is heterologous is derived from a different cell or tissue type, or a different species from the recipient, and is cloned into a cell that normally does not express that polypeptide. In one specific, non- limiting example, mouse (or human) CD300f cloned in a fibroblast cell line that does not express CD300f generates a heterologous CD300f protein. Generally, an antibody that specifically binds to a protein of interest, such as CD300f, will not specifically bind to a heterologous protein.
Host cells: Cells in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The cell can be mammalian, such as a human cell. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used.
Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, an immune response is a T cell response, such as a CD4 response or a CD8 response. In another embodiment, the response is a B cell response, and results in the production of specific antibodies.
Inhibiting or treating a disease: Inhibiting a disease, such as a tumor, refers to inhibiting the full development of a disease. In several examples, inhibiting a disease refers to lessening symptoms of the particular tumor. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition related to the disease, such as a tumor, such as reducing the size of a tumor, volume of a tumor, number of tumors, metastasis of a tumor,
or combinations thereof.
Isolated: An "isolated" biological component (such as a nucleic acid or protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes.
Lymphocytes: A type of white blood cell that is involved in the immune defenses of the body. There are two main types of lymphocytes: B cells and T cells.
Mammal: This term includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects.
Oligonucleotide: A linear polynucleotide sequence of up to about 100 nucleotide bases in length.
Open reading frame (ORF): A series of nucleotide triplets (codons) coding for amino acids without any internal termination codons. These sequences are usually translatable into a protein.
Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence, such as a sequence that encodes a polypeptide. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for
pharmaceutical delivery of the therapeutic agents (such as CD300f inhibitors, dendritic cells with an inactivated CD300f, or T cells activated by such dendritic cells) herein disclosed.
In general, the nature of the carrier will depend on the particular mode of administration
being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
A "therapeutically effective amount" is a quantity of a composition or a cell to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to reduce growth of a tumor, number of tumors, and/or reduce or prevent metastasis. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations that has been shown to achieve an in vitro effect.
Polynucleotide: The term polynucleotide or nucleic acid sequence refers to a polymeric form of nucleotide at least 10 bases in length. A recombinant polynucleotide includes a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single- and double- stranded forms of DNA.
Polypeptide: Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). A polypeptide can be between 5 and 25 amino acids in length. In one embodiment, a polypeptide is from about 10 to about 20 amino acids in length. In yet another embodiment, a polypeptide is from about 11 to about 18 amino acids in length. With regard to polypeptides, the word "about" indicates integer amounts. Thus, in one example, a polypeptide "about" 11 amino acids in length is from 10 to 12 amino acids in length. Similarly, a polypeptide "about" 18 amino acids in length is from about 17 to about 19 amino acids in length. Thus, a polypeptide "about" a specified number of residues can be one amino acid shorter or one amino acid longer than the specified number. A fusion polypeptide includes the amino acid sequence of a first polypeptide and a second different polypeptide (for example, a
heterologous polypeptide), and can be synthesized as a single amino acid sequence.
Probes and primers: A probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule. Primers are short nucleic acids, such as DNA oligonucleotides, of about 15 nucleotides or more in length. Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, for example by polymerase chain reaction (PCR) or other nucleic-acid amplification methods. One of skill in the art will appreciate that the specificity of a particular probe or primer increases with its length. Thus, for example, a primer comprising 20 consecutive nucleotides will anneal to a target with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, in order to obtain greater specificity, probes and primers can be selected that comprise about 20, 25, 30, 35, 40, 50 or more consecutive nucleotides.
Promoter: An array of nucleic acid control sequences which direct transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.
Purified: The polypeptides disclosed herein can be purified (and/or synthesized) by any means known in the art (see, e.g., Guide to Protein Purification, ed. Deutscher, Meth. Enzymol.
185, Academic Press, San Diego, 1990; and Scopes, Protein Purification: Principles and Practice, Springer Verlag, New York, 1982). Substantial purification denotes purification from other proteins or cellular components. A substantially purified protein is at least about 60%, 70%, 80%, 90%, 95%, 98% or 99% pure. Thus, in one specific, non-limiting example, a substantially purified protein is 90% free of other proteins or cellular components. Thus, the term purified does not require absolute purity; rather, it is intended as a relative term.
Similarly, a purified nucleic acid is one in which the nucleic acid is more enriched than the nucleic acid in its natural environment within a cell.
In some examples, a purified population of nucleic acids, proteins, or cells is greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure, or free other nucleic acids, proteins, or cells, respectively.
Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of at least two otherwise
separated segments of sequence. This artificial combination is can be accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. A recombinant polypeptide has an amino acid sequence that is not naturally occurring or that is made by two otherwise separated segments of an amino acid sequence.
Recombination: A process of exchange of genetic information between two
polynucleotides. "Homologous recombination (HR)" refers to the specialized form of an exchange that takes place, for example, during repair of double-strand breaks in cells. Nucleotide sequence homology is utilized in recombination, for example using a "donor" molecule to template repair of a "target" molecule (i.e., the one that experienced the double-strand break), and is variously known as "non-crossover gene conversion" or "short tract gene conversion," because it leads to the transfer of genetic information from the donor to the target.
Selectively hybridize: Hybridization under moderately or highly stringent conditions that excludes non-related nucleotide sequences.
In nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (for example, GC v. AT content), and nucleic acid type (for example, RNA versus DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.
A specific example of progressively higher stringency conditions is as follows: 2 x
SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2 x SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2 x SSC/0.1% SDS at about 42°C (moderate stringency conditions); and 0.1 x SSC at about 68°C (high stringency conditions). One of skill in the art can determine variations on these conditions (e.g., Molecular Cloning: A Laboratory
Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each of the conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.
Sequence identity: The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the
percentage, the more similar the two sequences are. Homologs or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, 1970, J Mol Biol 48, 443-453; Higgins and Sharp, 1988, Gene 73, 237-244; Higgins and Sharp, 1989, CABIOS 5, 151-153; Corpet et al., 1988, Nucleic Acids Research 16, 10881-10890; and Pearson and Lipman, 1988, Proc Natl Acad Sci USA 85, 2444-2448. Altschul et al., 1994, Nature Genet 6, 119-129, presents a detailed consideration of sequence alignment methods and homology calculations.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990, J Mol Biol
215, 403-410) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.
Homologs and variants of a polypeptide are typically characterized by possession of at least
75%, for example at least 80%, sequence identity counted over the full length alignment with the amino acid sequence of a polypeptide using the NCBI Blast 2.0, gapped blastp set to default parameters. For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and can possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
Subject: Human and non-human animals, including all vertebrates, such as mammals and
non-mammals, such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. In many embodiments of the described methods, the subject is a human, such as a human with a solid tumor.
Tumor: An abnormal growth of cells, which can be benign or malignant. Cancer is a malignant tumor, which is characterized by abnormal or uncontrolled cell growth. Other features often associated with malignancy include metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels and
suppression or aggravation of inflammatory or immunological response, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc. "Metastatic disease" refers to cancer cells that have left the original tumor site and migrate to other parts of the body for example via the bloodstream or lymph system.
The amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as "benign." A tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant."
Examples of hematological tumors include leukemias, including acute leukemias (such as l lq23-positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma,
Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia. In specific non-limiting examples, the lymphoid malignancy can be adult T cell leukemia, cutaneous T cell lymphoma, anaplastic large cell lymphoma, Hodgkin's lymphoma, or a diffuse large B cell lymphoma.
Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyrgioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma).
Lymphoma can be solid tumors is some presentations.
In some examples, a tumor is colorectal tumor, skin tumor or lymphoma.
Transgene: An exogenous gene.
Treating, Treatment, and Therapy: Any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject's physical state, such as decreasing tumor volume, tumor size, or a symptom of the tumor. The treatment may be assessed by objective or subjective parameters; including the results of a physical examination, neurological examination, or psychiatric evaluations.
Upstream: A relative position on a polynucleotide, wherein the "upstream" position is closer to the 5' end of the polynucleotide than the reference point. In the instance of a double- stranded polynucleotide, the orientation of 5' and 3' ends are based on the sense strand, as opposed to the antisense strand.
Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker gene and other genetic elements known in the art. Vectors include plasmid vectors, including plasmids for expression in gram-negative and gram-positive bacterial cell. Exemplary vectors include those for expression in E. coli and Salmonella. Vectors also include viral vectors, such as, but are not limited to, retrovirus, orthopox, avipox, fowlpox, capripox, suipox, adenoviral, herpes virus, alpha virus, baculovirus, Sindbis virus, vaccinia virus and poliovirus vectors. Vectors also include vectors for expression in yeast cells or mammalian cells.
Virus: Microscopic infectious organism that reproduces inside living cells. A virus consists essentially of a core of a single nucleic acid surrounded by a protein coat and has the ability to replicate only inside a living cell. "Viral replication" is the production of additional virus by the
occurrence of at least one viral life cycle. Viral vectors are known in the art, and include, for example, adenovirus, adeno-associated virus (AAV), lentivirus and herpes virus.
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
CD300f Inhibitors
It is disclosed herein that CD300f inhibitors are of use for treating a solid tumor. In some embodiments, the solid tumor is a carcinoma or a sarcoma. In particular non-limiting examples, the solid tumor is a lymphoma, a colorectal cancer or a skin cancer.
The CD300f inhibitor can be, for example, a soluble protein, an antibody or aptamer that specifically binds CD300f, or an inhibitory nucleic acid molecule (RNAi), such as, but not limited to, a ribozyme, a siRNA or a shRNA. The CD300f antagonist can result in the induction of an immune response to the tumor.
A. Antibodies and Antigen Binding Fragments Thereof
The CD300f inhibitor can be an antibody, such as a monoclonal antibody. Antibodies that specifically bind CD300f are commercially available. Exemplary nucleic acid sequences encoding human CD300f are provided in GENBANK® Accession No. NM_139018.4 (August 26, 2016), and GENBANK Accession No. NM_001289082.1 (August 26, 2016), which are both incorporated by reference, and an exemplary amino acid sequence of human CD300f is provided in GENBANK® Accession No. AAH28199.1 (June 6, 2006), which is incorporated by reference herein. Other examples are provided herein.
Antibodies that specifically bind CD300f are commercially available. For example, a mouse IgGi to human CD300f is available from BioLegend, Clone UP-D2; additional monoclonal antibodies that specifically bind CD300f are available from R&D systems, Catalog Number AF2774, Borbyt, Catalog Number orb 160320, and St. John's Laboratory, Catalog Numbers STJ92113 and STJ96840, eBioscience, Clone UP-D1, Catalog Number 50-3008-41/42.
Antibodies that specifically bind and substantially reduce or inhibit CD300f activity (such as a reduction of at least 20%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or even 100%) are of use in the methods disclosed herein. Antibodies include monoclonal antibodies, human antibodies, humanized antibodies, deimmunized antibodies, and immunoglobulin (Ig) fusion proteins. Fully human and humanized antibodies that bind CD300f can also be produced using methods known to those of skill in the art.
Polyclonal anti-CD300f antibodies can be prepared, such as by immunizing a suitable subject (such as a veterinary subject) with a CD300f immunogen. The anti-CD300f antibody titer in the immunized subject can be monitored over time, such as with an enzyme linked
immunosorbent assay (ELISA) using immobilized CD300f polypeptide. In one example, the antibody molecules that specifically bind CD300f can be isolated from a mammal (such as from serum) and further purified, for example using protein A chromatography to isolate IgG antibodies.
Antibody-producing cells can be obtained from a subject and used to prepare monoclonal antibodies (see Kohler and Milstein Nature 256:495 49, 1995; Brown et al., J. Immunol. 127:539 46, 1981; Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77 96, 1985; Gefter, M. L. et al. (1977) Somatic Cell Genet. 3:231 36; Kenneth, R. H. in Monoclonal Antibodies: A New Dimension In Biological Analyses. Plenum Publishing Corp., New York, N.Y. (1980); Kozbor et al. Immunol. Today 4:72, 1983; Lerner, E. A. (1981) Yale J. Biol. Med. 54:387 402; Yeh et al., Proc. Natl. Acad. Sci. 76:2927 31, 1976). In one example, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with CD300f, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that specifically binds to the polypeptide of interest.
In one embodiment, to produce a hybridoma, an immortal cell line (such as a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with a CD300f peptide with an immortalized mouse cell line. In one example, a mouse myeloma cell line is utilized that is sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT
medium"). Any of a number of myeloma cell lines can be used as a fusion partner, including, for example, P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/0-Agl4 myeloma lines, which are available from the American Type Culture Collection (ATCC), Rockville, MD. HAT-sensitive mouse myeloma cells can be fused to mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused (and unproductively fused) myeloma cells. Hybridoma cells producing a monoclonal antibody of interest can be detected, for example, by screening the hybridoma culture supernatants for the production antibodies that bind a CD300f polypeptide, such as by using an immunological assay (such as an enzyme-linked immunosorbant assay (ELISA) or radioimmunoassay (RIA).
As an alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody that specifically binds CD300f can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (such as an antibody phage display library) with CD300f to isolate immunoglobulin library members that specifically bind the polypeptide. Library members can be selected that have particular activities, such as binding CD300f, or activation of T cells in an in vitro assay. Kits for generating and screening phage display libraries are commercially available (such as, but not limited to, Pharmacia and Stratagene). Examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO 90/02809; PCT Publication No. WO 91/17271 ; PCT Publication No. WO 92/18619; PCT Publication WO 92/20791 ; PCT
Publication No. WO 92/15679; PCT Publication No. WO 92/01047; PCT Publication WO
93/01288; PCT Publication No. WO 92/09690; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978 7982, 1991; Hoogenboom et al., Nucleic Acids Res. 19:4133 4137, 1991.
In one example the sequence of the specificity determining regions of each CDR is determined. Residues outside the SDR (specificity determining region, e.g., the non-ligand contacting sites) are substituted. For example, in any of the CDR sequences, at most one, two or three amino acids can be substituted. The production of chimeric antibodies, which include a framework region from one antibody and the CDRs from a different antibody, is known in the art. For example, humanized antibodies can be produced. The antibody or antibody fragment can be a humanized immunoglobulin having CDRs from a donor monoclonal antibody that binds CD300f, and immunoglobulin and heavy and light chain variable region frameworks from human acceptor immunoglobulin heavy and light chain frameworks.
Humanized monoclonal antibodies can be produced by transferring CDRs from heavy and light variable chains of the donor mouse immunoglobulin (that specifically binds CD300f) into a
human variable domain, and then substituting human residues in the framework regions when required to retain affinity. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of the constant regions of the donor antibody. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321 :522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239: 1534, 1988; Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285, 1992; Sandhu, Crit. Rev. Biotech.12:437, 1992; and Singer et al., J. Immunol.150:2844, 1993. The antibody may be of any isotype, but in several embodiments the antibody is an IgG, including but not limited to, IgGi, IgG2, IgG3 and IgG4.
In one embodiment, the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Thus, the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 75%, at least about 85%, at least about 99% or at least about 95%, identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Human framework regions, and mutations that can be made in humanized antibody framework regions, are known in the art (see, for example, in U.S. Patent No. 5,585,089, incorporated herein by reference).
Exemplary human antibodies are LEN and 21/28 CL. The sequences of the heavy and light chain frameworks are known. Exemplary light chain frameworks of human MAb LEN have the following sequences:
FR1: DIVMTQS PDSLAVSLGERATINC (SEQ ID NO: 1)
FR2: WYQQKPGQPPLLIY (SEQ ID NO: 2)
FR3: G VPDRPFGS GS GTDFTLTIS S LQ AED V A V Y YC (SEQ ID NO: 3)
FR4: FGQGQTKLEIK (SEQ ID NO: 4)
Exemplary heavy chain frameworks of human MAb 21/28' CL have the following sequences:
FR1: Q VQLVQS G AE VKKPQ AS VKVSCKAS Q YTFT (SEQ ID NO: 5)
FR2: WVRQAPGQRLEWMG (SEQ ID NO: 6)
FR3: RVTITRDTSASTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 7)
FR4: WGQGTLVTVSS (SEQ ID NO: 8).
Generally, an antibody, such as a human or humanized antibody specifically binds to CD300f with an affinity constant of at least 107 M"1, such as at least 108 M"1 at least 5 X 108 M"1 or at least 109 M"1. In several examples, the antibody specifically binds CD300f with an affinity constant of at least 108 M"1 at least 5 X 108 M"1 or at least 109 M"1.
Antibodies, such as murine monoclonal antibodies, chimeric antibodies, and humanized antibodies, include full length molecules as well as fragments thereof, such as Fab, F(ab')2, and Fv which include a heavy chain and light chain variable region and are capable of binding specific epitope determinants. These antibody fragments retain some ability to selectively bind with their antigen or receptor. These fragments include:
(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
(2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
(3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;
(4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and
(5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
Methods of making these fragments are known (see for example, Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). In several examples, the variable region includes the variable region of the light chain and the variable region of the heavy chain expressed as individual polypeptides. Fv antibodies are typically about 25 kDa and contain a complete antigen-binding site with three CDRs per each heavy chain and each light chain. To produce these antibodies, the VH and the VL can be expressed from two individual nucleic acid constructs in a host cell. If the VH and the VL are expressed non-contiguously, the chains of the Fv antibody are typically held together by noncovalent interactions. However, these chains tend to dissociate upon dilution, so methods have been developed to crosslink the chains
through glutaraldehyde, intermolecular disulfides, or a peptide linker. Thus, in one example, the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chain variable region and the light chain variable region are chemically linked by disulfide bonds.
In an additional example, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (scFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are known in the art (see Whitlow et al, Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al, Science 242:423, 1988; U.S. Patent No. 4,946,778; Pack et al,
Bio/Technology 11:1271, 1993; and Sandhu, supra).
Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Patent No.
4,036,945 and U.S. Patent No. 4,331,647, and references contained therein; Nisonhoff et al, Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al, Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).
Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody. Any of the antigen binding fragments described herein are of use.
Conservative variants of the antibodies can be produced. Such conservative variants employed in antibody fragments, such as dsFv fragments or in scFv fragments, will retain critical amino acid residues necessary for correct folding and stabilizing between the VH and the VL regions, and will retain the charge characteristics of the residues in order to preserve the low pi and
low toxicity of the molecules. Amino acid substitutions (such as at most one, at most two, at most three, at most four, or at most five amino acid substitutions) can be made in the VH and the VL regions to increase yield. A table of conservative amino acid substitutions is provided above. One of skill in the art can readily review the amino acid sequence of an antibody of interest, locate one or more of the amino acids in the brief table above, identify a conservative substitution, and produce the conservative variant using well-known molecular techniques.
Effector molecules, such as therapeutic, diagnostic, or detection moieties can be linked to an antibody that specifically binds CD300f, using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used. The procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector.
Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce
Chemical Company, Rockford, IL. The linker can be any molecule used to join the antibody to the effector molecule. The linker is capable of forming covalent bonds to both the antibody and to the effector molecule. Suitable linkers include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the effector molecule are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.
Nucleic acid sequences encoding the antibodies can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68: 109-151, 1979; the
diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22: 1859-1862, 1981; the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetra. Letts. 22(20): 1859- 1862, 1981, for example, using an automated synthesizer as described in, for example, Needham- VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method of U.S. Patent No. 4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill would
recognize that while chemical synthesis of DNA is generally limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.
Exemplary nucleic acids encoding sequences encoding an antibody that specifically binds CD300f can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook et al., supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (San Diego, CA), and Applied Biosystems (Foster City, CA), as well as other commercial sources.
Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are known.
In one example, an antibody of use is prepared by inserting the cDNA, which encodes a variable region from an antibody that specifically binds CD300f, into a vector which comprises the cDNA encoding an effector molecule (EM). The insertion is made so that the variable region and the EM are read in frame so that one continuous polypeptide is produced. Thus, the encoded polypeptide contains a functional Fv region and a functional EM region. In one embodiment, cDNA encoding a detectable marker (such as an enzyme) is ligated to a scFv so that the marker is located at the carboxyl terminus of the scFv. In another example, a detectable marker is located at the amino terminus of the scFv. In a further example, cDNA encoding a detectable marker is ligated to a heavy chain variable region of an antibody that specifically binds CD300f, so that the marker is located at the carboxyl terminus of the heavy chain variable region. The heavy chain- variable region can subsequently be ligated to a light chain variable region of the antibody that specifically binds CD300f using disulfide bonds. In a yet another example, cDNA encoding a marker is ligated to a light chain variable region of an antibody that binds CD300f, so that the marker is located at the carboxyl terminus of the light chain variable region. The light chain- variable region can subsequently be ligated to a heavy chain variable region of the antibody that specifically binds CD300f using disulfide bonds.
Once the nucleic acids encoding the antibody or functional fragment thereof are isolated and cloned, the protein can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells. One or more DNA sequences encoding the antibody or functional fragment thereof can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known.
Polynucleotide sequences encoding the antibody or functional fragment thereof (such as an scFV) can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
The polynucleotide sequences encoding the antibody or functional fragment thereof can be inserted into an expression vector including, but not limited to a plasmid, virus or other vehicle that can be manipulated to allow insertion or incorporation of sequences and can be expressed in either prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art.
Transformation of a host cell with recombinant DNA may be carried out by conventional techniques. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaC method. Alternatively, MgC can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.
When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors, may be used. Eukaryotic cells can also be cotransformed with polynucleotide sequences encoding the antibody of functional fragment thereof
and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). One of skill in the art can readily use expression systems such as plasmids and vectors of use in producing proteins in cells including higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.
Isolation and purification of a recombinantly expressed polypeptide can be carried out by conventional means including preparative chromatography and immunological separations. Once expressed, the recombinant antibodies can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, R. Scopes, Protein Purification, Springer- Verlag, N.Y., 1982). Substantially pure compositions of at least about 90 to 95% homogeneity are disclosed herein, and 98 to 99% or more homogeneity can be used for pharmaceutical purposes. Once purified, partially or to homogeneity as desired, if to be used therapeutically, the polypeptides should be substantially free of endotoxin.
Methods for expression of single chain antibodies and/or refolding to an appropriate active form, including single chain antibodies, from bacteria such as E. coli have been described and are applicable to the antibodies disclosed herein. See, Buchner et al., Anal. Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991; Huse et al, Science 246:1275, 1989 and Ward et al, Nature 341:544, 1989, all incorporated by reference herein.
Often, functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization using strong denaturants, and subsequent refolding. During the solubilization step, a reducing agent must be present to separate disulfide bonds. An exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). Reoxidation of the disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena et al, Biochemistry 9: 5015-5021, 1970, incorporated by reference herein, and especially as described by Buchner et al, supra.
Renaturation is typically accomplished by dilution (for example, 100-fold) of the denatured and reduced protein into refolding buffer. An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L- arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.
As a modification to the two chain antibody purification protocol, the heavy and light chain
regions are separately solubilized and reduced and then combined in the refolding solution. An exemplary yield is obtained when these two proteins are mixed in a molar ratio such that a 5 fold molar excess of one protein over the other is not exceeded. It is desirable to add excess oxidized glutathione or other oxidizing low molecular weight compounds to the refolding solution after the redox-shuffling is completed.
In addition to recombinant methods, the antibodies and functional fragments thereof that are disclosed herein can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of the polypeptides of less than about 50 amino acids in length can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963, and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. , Pierce Chem. Co., Rockford, 111., 1984. Proteins of greater length may be synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxyl terminal end (such as by the use of the coupling reagent N, N'-dicycylohexylcarbodimide) are known.
B. Inhibitory Nucleic Acid Molecules
Inhibitory nucleic acids that decrease the expression and/or activity of CD300f can also be used in the methods disclosed herein. In some examples, such inhibitor nucleic acid molecules decrease CD300f expression or activity by at least 20%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or even 100%. One embodiment is a RNA interference (RNAi), such as, but not limited to, small inhibitory RNA (siRNA) or short hairpin RNA, which can be used for interference or inhibition of expression of a target. RNAis that specifically target CD300f are commercially available, for example from Santa Cruz
Biotechnology, Inc., ThermoFisher Scientific, and Sigma Aldrich. Exemplary commercially available RNAi sequences are: 5 ' CGTATCAACGATGACAATAA3 ' (SEQ ID NO: 16), and 5 ' CAGTCTCTGGAGGGTGATCTCTGTT3 ' (SEQ ID NO: 17). Additional RNAi sequences of use are:
CD300f siRNA 1: CGTATCAACGATGACAATAATUU (SEQ ID NO: 18); and
CD300f siRNA 2: CAGTCTCTGGAGGGTGATCTCTGTTUU (SEQ ID NO: 19).
Generally, siRNAs are generated by the cleavage of relatively long double-stranded RNA
molecules by Dicer or DCL enzymes (Zamore, Science, 296: 1265-1269, 2002; Bernstein et ah, Nature, 409:363-366, 2001). In animals and plants, siRNAs are assembled into RISC and guide the sequence specific ribonucleolytic activity of RISC, thereby resulting in the cleavage of mRNAs or other RNA target molecules in the cytoplasm. In the nucleus, siRNAs also guide heterochromatin- associated histone and DNA methylation, resulting in transcriptional silencing of individual genes or large chromatin domains.
The present disclosure provides RNA suitable for interference or inhibition of expression of CD300f, which RNA includes double stranded RNA of about 19 to about 40 nucleotides with the sequence that is substantially identical to a portion of an mRNA or transcript of a target gene, such as CD300f, for which interference or inhibition of expression is desired. For purposes of this disclosure, a sequence of the RNA "substantially identical" to a specific portion of the mRNA or transcript of the target gene for which interference or inhibition of expression is desired differs by no more than about 30%, and in some embodiments no more than about 10% or no more than 5% from the specific portion of the mRNA or transcript of the target gene. In particular embodiments, the sequence of the RNA is exactly identical to a specific portion of the mRNA or transcript of the target gene {e.g., CD300f).
Thus, siRNAs disclosed herein include double- stranded RNA of about 15 to about 40 nucleotides in length and a 3' or 5' overhang having a length of 0 to 5-nucleotides on each strand, wherein the sequence of the double stranded RNA is substantially identical to (see above) a portion of a mRNA or transcript of a nucleic acid encoding CD300f. In particular examples, the double stranded RNA contains about 19 to about 25 nucleotides, for instance 20, 21, or 22 nucleotides substantially identical to a nucleic acid encoding CD300f. In additional examples, the double stranded RNA contains about 19 to about 25 nucleotides 100% identical to a nucleic acid encoding CD300f. It should be not that in this context "about" refers to integer amounts only. In one example, "about" 20 nucleotides refers to a nucleotide of 19 to 21 nucleotides in length.
Regarding the overhang on the double-stranded RNA, the length of the overhang is independent between the two strands, in that the length of one overhang is not dependent on the length of the overhang on other strand. In specific examples, the length of the 3' or 5' overhang is 0-nucleotide on at least one strand, and in some cases it is 0-nucleotide on both strands (thus, a blunt dsRNA). In other examples, the length of the 3' or 5' overhang is 1 -nucleotide to 5- nucleotides on at least one strand. More particularly, in some examples the length of the 3 ' or 5 ' overhang is 2-nucleotides on at least one strand, or 2-nucleotides on both strands. In particular examples, the dsRNA molecule has 3' overhangs of 2-nucleotides on both strands.
Thus, in one particular provided RNA embodiment, the double- stranded RNA contains 20, 21, or 22 nucleotides, and the length of the 3' overhang is 2-nucleotides on both strands. In embodiments of the RNAs provided herein, the double-stranded RNA contains about 40-60% adenine+uracil (AU) and about 60-40% guanine+cytosine (GC). More particularly, in specific examples the double- stranded RNA contains about 50% AU and about 50% GC.
Also described herein are RNAs that further include at least one modified ribonucleotide, for instance in the sense strand of the double- stranded RNA. In particular examples, the modified ribonucleotide is in the 3' overhang of at least one strand, or more particularly in the 3' overhang of the sense strand. It is contemplated that examples of modified ribonucleotides include
ribonucleotides that include a detectable label (for instance, a fluorophore, such as rhodamine or
FITC), a thiophosphate nucleotide analog, a deoxynucleotide (considered modified because the base molecule is ribonucleic acid), a 2'-fluorouracil, a 2'-aminouracil, a 2'-aminocytidine, a 4-thiouracil, a 5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, an inosine, or a 2'0-Me-nucleotide analog.
Antisense and ribozyme molecules for CD300f are also of use in the method disclosed herein. Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American 262:40, 1990). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double- stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA, since the cell will not translate an mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target cell producing CD300f. The use of antisense methods to inhibit the in vitro translation of genes is known (see, for example, Marcus-Sakura, Anal. Biochem. 172:289, 1988).
An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or
50 nucleotides in length. An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions. For example, an antisense nucleic acid molecule can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, such as phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-
carboxymethylaminomethyl-2-thiouridin- e, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, amongst others.
Use of an oligonucleotide to stall transcription is known as the triplex strategy where an oligonucleotide winds around double-helical DNA, forming a three-strand helix. Therefore, these triplex compounds can be designed to recognize a unique site on a chosen gene (Maher, et al. ,
Antisense Res. and Dev. 1(3):227, 1991 ; Helene, C, Anticancer Drug Design 6(6):569), 1991. This type of inhibitory oligonucleotide is also of use in the methods disclosed herein.
Ribozymes, which are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases, are also of use. Through the modification of nucleotide sequences, which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, /. Amer. Med. Assn. 260:3030, 1988). An advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.
There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff, Nature 334:585, 1988) and "hammerhead"-type. Tetrahymena-type, ribozymes recognize sequences which are four bases in length, while "hammerhead"-type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type, ribozymes for inactivating a specific mRNA species and 18-base recognition sequences are preferable to shorter recognition sequences.
Various delivery systems are known and can be used to administer the siRNAs and other inhibitory nucleic acid molecules as therapeutics. Such systems include, for example,
encapsulation in liposomes, microparticles, microcapsules, nanoparticles, recombinant cells capable of expressing the therapeutic molecule(s) (see, e.g. , Wu et al. , J. Biol. Chem. 262, 4429, 1987), construction of a therapeutic nucleic acid as part of a retroviral or other vector, and the like.
D. Chemical Compounds and Small Molecules
CD300f inhibitors include molecules that are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries. Screening methods that detect decreases in CD300f activity are useful for identifying compounds from a variety of sources for activity. The initial screens may be performed using a diverse library of compounds, a variety of other compounds and compound libraries. Thus, molecules that bind CD300f molecules that inhibit
the expression of CD300f, and molecules that inhibit the activity of CD300f can be identified. These small molecules can be identified from combinatorial libraries, natural product libraries, or other small molecule libraries. In addition, CD300f antagonist can be identified as compounds from commercial sources, as well as commercially available analogs of identified inhibitors.
The precise source of test extracts or compounds is not critical to the identification of
CD300f small molecule antagonists. Accordingly, CD300f inhibitors can be identified from virtually any number of chemical extracts or compounds. Examples of such extracts or compounds that can be CD300f inhibitors include, but are not limited to, plant-, fungal-, prokaryotic- or animal- based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). CD300f inhibitors can be identified from synthetic compound libraries that are commercially available from a number of companies including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N. J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). CD300f inhibitors can be identified from a rare chemical library, such as the library that is available from Aldrich (Milwaukee, Wis.). CD300f inhibitors can be identified in libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). Natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means.
Useful compounds may be found within numerous chemical classes, though typically they are organic compounds, including small organic compounds. Small organic compounds have a molecular weight of more than 50 yet less than about 2,500 daltons, such as less than about 750 or less than about 350 daltons can be utilized in the methods disclosed herein. Exemplary classes include heterocycles, peptides, saccharides, steroids, and the like. The compounds may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. In several embodiments, compounds of use has a Kd for CD300f of less than InM, less than ΙΟηΜ, less than 1 μΜ, less than 10μΜ, or less than lmM.
Methods of Treatment of a Subject with a Solid Tumor
and Pharmaceutical Compositions Including a CD 300f Inhibitor Methods are provided herein for treating a subject with a solid tumor. In some
embodiments the solid tumor is a carcinoma or a sarcoma. In other embodiments, the solid tumor is a lymphoma, skin cancer, or colorectal cancer. Without being bound by theory, administration of one or more CD300f inhibitors increases antigen presentation by dendritic cells, and results in the production of activated T cells, such as CD8+ cytotoxic cells, specific for the tumor.
In one specific, non-limiting example, a therapeutically effective amount of one or more CD300f inhibitors (such as 1, 2, or 3 of such inhibitors) is administered to a subject to treat a tumor, such as to decrease tumor volume or size (such as a decrease of at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100% relative to absence of administration of the inhibitor). In another specific, non-limiting example, a therapeutically effective amount of the CD300f inhibitor is administered to a subject to treat a tumor, such as to decrease the number of tumors (such as a decrease of at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100% relative to absence of administration of the inhibitor). In another specific, non- limiting example, a therapeutically effective amount of the CD300f inhibitor is administered to a subject to treat a tumor, such as to decrease metastasis, for example the volume or number of metastases (such as a decrease of at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100% relative to absence of administration of the inhibitor). In other embodiments, a therapeutically effective amount of the CD300f inhibitor is administered to a subject to delay or prevent a symptom of the tumor. In one aspect of the disclosure, the formation of tumors, such as metastasis, are delayed, prevented or the number of metastases are decreased. In another aspect, the size of the primary tumor is decreased. In a further aspect, a symptom of the tumor is decreased. In yet another aspect, tumor volume is decreased.
Generally, the method involves selecting a subject with a solid tumor, and administering to the subject a therapeutically effective amount of one or more CD300f inhibitors. In some embodiments the subject has carcinoma or a sarcoma. In other embodiments, the subject has a lymphoma, skin cancer, or colorectal cancer. The tumor can be any tumor of interest, including, but not limited to, lymphoma, breast cancer, lung cancer and colon cancer. The tumor can be benign or malignant. Additional examples are breast, brain, cervical carcinomas, testicular carcinomas, head and neck, lung, mediastinum, gastrointestinal tract, genitourinary system, gynaecological system,
breast, endocrine system, skin, childhood, unknown primary site or metastatic cancer, a sarcoma of the soft tissue and bone, a mesothelioma, a melanoma, a neoplasm of the central nervous system, a head and neck tumor, comprising tumors of the nasal cavity, paranasal sinuses, nasopharynx, oral cavity, oropharynx, larynx, hypopharynx, salivary glands and paragangliomas, a cancer of the lung, comprising non-small cell lung cancer, small cell lung cancer, a cancer of the mediastinum, a cancer of the gastrointestinal tract, comprising cancer of the oesophagus, stomach, pancreas, liver, biliary tree, small intestine, colon, rectum and anal region, a cancer of the genitourinary system, comprising cancer of the kidney, urethra, bladder, prostate, urethra, penis and testis, a gynaecologic cancer, comprising cancer of the cervix, vagina, vulva, uterine body, gestational trophoblastic diseases, ovarian, fallopian tube, peritoneal, a cancer of the breast, a cancer of the endocrine system, comprising a tumor of the thyroid, parathyroid, adrenal cortex, pancreatic endocrine tumors, carcinoid tumor and carcinoid syndrome, multiple endocrine neoplasias, a sarcoma of the soft tissue and bone, a mesothelioma, a cancer of the skin, a melanoma, comprising cutaneous melanomas and intraocular melanomas, a neoplasm of the central nervous system, a cancer of the childhood, comprising retinoblastoma, Wilm's tumor, neurofibromatoses, neuroblastoma, Ewing's sarcoma family of tumors, rhabdomyosarcoma, a solid lymphoma, cutaneous T-cell lymphomas, primary central nervous system lymphoma, a cancer of unknown primary origin, a peritoneal
carcinomastosis, a Kaposi's sarcoma, AIDS -associated lymphomas, AIDS -associated primary central nervous system lymphoma and AIDS -associated anogenital cancers. The presence of a tumor can be determined by methods known in the art, and typically include cytological and morphological evaluation.
Treatment of the conditions described herein are generally initiated after the development of a condition described herein, or after the initiation of a precursor condition (such as dysplasia or development of a benign tumor). Treatment can be initiated at the early stages of cancer, for instance, can be initiated before a subject manifests symptoms of a condition, such as during a stage I diagnosis or at the time dysplasia is diagnosed. However, treatment can be initiated during any stage of the disease, such as but not limited to stage I, stage II, stage III and stage IV cancers. In some examples, such as for breast cancer, treatment can be initiated before or during exposure to an agent that damages DNA, such as a result of an exposure to a carcinogen or UV light, oxidative stress, alkylation damage and deamination. Treatment prior to the development of the condition, such as treatment upon detecting dysplasia or an early (benign) precursor condition, is referred to herein as treatment of a subject that is "at risk" of developing the condition. In some embodiments,
administration of a composition can be performed during or after the occurrence of the conditions described herein.
Generally, treatment involves increasing the immune response to the tumor. Treatment initiated after the development of a condition, such as malignant cancer, may result in decreasing the severity of the symptoms of one of the conditions, or completely removing the symptoms, or reducing metastasis, tumor volume or number of tumors. In a specific non-limiting example, there is an increased T cell response to the tumor, such as a CD8+ cytotoxic T cell response.
The compositions described herein may be formulated in a variety of ways for
administration to a subject to induce an immune response to a tumor, or to delay, prevent, reduce the risk of developing, or treat, any tumor of interest. Administration can be local, such as to the site of a tumor, or systemic. Examples of systemic methods for administering the composition into mammals include, but are not limited to, intravenous, intratumoral, intraperitoneal, subcutaneous, intradermal, inhalation, transdermal and intramuscular. Any of the CD300f inhibitors disclosed herein can be used in these methods. Thus, the method can include administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a CD300f inhibitor.
While the CD300f inhibitor will typically be used to treat human subjects they may also be used to treat similar or identical diseases in other vertebrates, such as other primates, dogs, cats, horses, and cows. A suitable administration format may best be determined by a medical practitioner for each subject individually. Various pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises, e.g., Remington 's Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A., Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42: 2S, 1988. The dosage form of the pharmaceutical composition will be determined by the mode of administration chosen.
The amount of active compound(s) administered will be dependent on the subject being treated, the severity of the affliction, and the manner of administration, and is best left to the judgment of the prescribing clinician. Within these bounds, the formulation to be administered will contain a quantity of the active component(s) in amounts effective to achieve the desired effect in the subject being treated. Multiple treatments are envisioned, such as over defined intervals of time, such as daily, bi-weekly, weekly, bi-monthly or monthly, such that chronic administration is achieved. Administration may begin whenever the suppression or prevention of disease is desired, for example, at a certain age of a subject, or prior to an environmental exposure.
Antibodies and antigen binding fragments thereof can be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The antibody solution is then added to an infusion bag containing 0.9% Sodium Chloride, USP, and typically administered at a dosage of from 0.5 to 15 mg/kg of body weight. Considerable experience is available in the art in the administration of antibody drugs, which have been marketed in the U.S. since the approval of Rituxan® in 1997. Antibody drugs can be administered by slow infusion, rather than in an IV push or bolus. In one example, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level. For example, an initial loading dose of 4 mg/kg may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30 minute period if the previous dose was well tolerated.
In one specific, non-limiting example, a pharmaceutical composition for intravenous administration would include about 0.1 μg to 10 mg of CD300f inhibitor, such as an antibody, per patient per day. Dosages from 0.1 up to about 100 mg per subject per day can be used, particularly if the agent is administered to a body cavity or into a lumen of an organ. Actual methods for preparing administrable compositions are described in more detail in such publications as
Remingtons Pharmaceutical Sciences, 19th Ed., Mack Publishing Company, Easton, Pennsylvania, 1995.
In another embodiment, a pharmaceutical composition includes a nucleic acid encoding one or more of the antagonists, such as siRNA, shRNA or ribozyme, disclosed herein. A therapeutically effective amount of the polynucleotide can be administered to a subject, such as a subject with a solid tumor. In one specific, non-limiting example, a therapeutically effective amount of the polynucleotide is administered to a subject to treat a tumor, such as to decrease tumor volume. In another specific, non-limiting example, a therapeutically effective amount of the polynucleotide is administered to a subject to treat a tumor, such as to decrease metastasis. In other embodiments, a therapeutically effective amount of the polynucleotide is administered to a subject to delay or prevent a symptom of the tumor.
One approach to administration of nucleic acids is direct immunization with plasmid DNA, such as with a mammalian expression plasmid. As described above, the nucleotide sequence encoding a polypeptide can be placed under the control of a promoter to increase expression of the molecule.
Administration of nucleic acid constructs is taught, for example, in U.S. Patent No.
5,643,578; U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637; and U.S. Patent No.
5,880,103. The methods include liposomal delivery of the nucleic acids (or of the synthetic peptides themselves).
In another approach to using nucleic acids for immunization, a polypeptide can also be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, or other viral vectors can be used to express the peptide or protein. For example, vaccinia vectors and methods of administration are described in U.S. Patent No. 4,722,848. BCG (Bacillus Calmette Guerin) provides another vector for expression of the peptides (see Stover, Nature 351 :456-460, 1991).
When a viral vector is utilized, it is desirable to provide the recipient with a dosage of each recombinant virus in the composition in the range of from about 105 to about 1010 plaque forming units/mg mammal, although a lower or higher dose can be administered. The composition of recombinant viral vectors can be introduced into a subject with the solid tumor.
Examples of methods for administering the composition into mammals include, but are not limited to, intravenous, subcutaneous, intradermal or intramuscular administration of the nucleic acid, such as virus or other vector including the nucleic acid encoding the disclosed polypeptides. Generally, the quantity of recombinant viral vector, carrying the nucleic acid sequence of a polypeptide to be administered is based on the titer of virus particles. An exemplary range of the virus to be administered is 105 to 1010 virus particles per mammal, such as a human.
For any CD300f inhibitor, single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the subject. In any event, the composition should provide a sufficient quantity of at least one of the CD300f inhibitor disclosed herein to effectively treat the patient with the solid tumor. The dosage can be administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy. In one example, a dose of the CD300f inhibitor (such as an antibody) is infused for thirty minutes every other day. In this example, about one to about ten doses can be administered, such as three or six doses can be administered every other day. In a further example, a continuous infusion is administered for about five to about ten days.
The subject can be treated at regular intervals, such as monthly, until a desired therapeutic result is achieved. Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.
Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. A therapeutically effective amount of the CD300f inhibitor is that
which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.
Controlled release parenteral formulations of the compositions can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, A.J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995) incorporated herein by reference. Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μιη are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μιη so that only nanoparticles are administered intravenously. Microparticles are typically around 100 μιη in diameter and are administered subcutaneously or intramuscularly. See, e.g., Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342, 1994; and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, 1992, both of which are incorporated herein by reference.
To extend the time during which the CD300f inhibitor is available, the therapeutic agent(s) can be provided as an implant, an oily injection, or as a particulate system. The particulate system can be a microparticle, a microcapsule, a microsphere, a nanocapsule, or similar particle.
Polymers can be used for ion-controlled release of the compositions disclosed herein.
Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer, polaxamer 407, exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., /. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm.112:215- 224, 1994). In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA, 1993). Numerous additional systems for controlled delivery of therapeutic proteins are known. See, e.g., U.S. Patent No. 5,055,303; U.S. Patent No. 5,188,837; U.S. Patent No. 4,235,871 ; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; U.S. Patent No.
4,957,735; U.S. Patent No. 5,019,369; U.S. Patent No. 5,055,303; U.S. Patent No. 5,514,670; U.S. Patent No. 5,413,797; U.S. Patent No. 5,268,164; U.S. Patent No. 5,004,697; U.S. Patent No. 4,902,505; U.S. Patent No. 5,506,206; U.S. Patent No. 5,271,961; U.S. Patent No. 5,254,342 and U.S. Patent No. 5,534,496, each of which is incorporated herein by reference.
The method can include administering additional therapeutic agents, such as, but not limited to, a chemotherapeutic agent, a biologic (such as a monoclonal antibody), a Programmed Death (PD)-l antagonist, or a cytotoxic T lymphocyte associated protein (CTLA)-4 antagonist, In an embodiment, the additional agent is an antibody or antibody fragment that binds to ΉΜ3. In an embodiment, the agent is an antibody or antibody fragment that binds to LAG3. Such agents can be administered before, after, or concurrently with the CD300f inhibitor.
Examples of chemotherapeutic agents are alkylating agents, antimetabolites, natural products, or hormones and their antagonists. Examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or
chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine). Examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine. Examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitocycin C), and enzymes (such as L-asparaginase). Examples of miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum Π also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide). Examples of hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as
hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testerone proprionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU,
Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea,
Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP- 16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and
calcitriol. Non-limiting examples of immunomodulators that can be used include AS- 101 (Wyeth- Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).
In one example, the additional therapeutic agent is a biologic agent (e.g. , mAb) or a small molecule, such as those shown in Table 1.
Table 1: Exemplary Tumor- specific antigens and therapeutics
Tumor-Specific Exemplary Tumors Exemplary Antibody/Small
Antigen Molecules
HER1 Adenocarcinoma (e.g., Cetuximab, panitumamab,
colorectal cancer, head and zalutumumab, nimotuzumab,
neck cancer) matuzumab. Small molecule
inhibitors gefitinib, erlotinib, and lapatinib can also be used.
HER2 breast cancer, ovarian cancer, Trastuzumab (Herceptin®),
stomach cancer, uterine pertuzumab
cancer
CD20 Non-Hodgkin lymphoma Tositumomab (Bexxar®); Rituximab
(Rituxan, Mabthera); or Ibritumomab tiuxetan (Zevalin, for example in combination with yttrium-90 or indium-I l l therapy)
CD25 T-cell lymphoma Daclizumab (Zenapax)
CD33 Acute myelogenous leukemia Gemtuzumab (Mylotarg, for example
in combination with calicheamicin therapy)
CD52 chronic lymphocytic leukemia Alemtuzumab (Campath)
CEA colorectal cancer, some CEA-scan (Fab fragment, approved
gastric cancers, biliary cancer by FDA), cololOl
Cancer antigen 125 ovarian cancer, mesothelioma, OC125 monoclonal antibody
(CA125) breast cancer
Alpha- fetoprotein hepatocellular carcinoma ab75705 (available from Abeam) and
(AFP) other commercially available AFP
antibodies
Lewis Y colorectal cancer, biliary B3 (Humanized)
cancer
TAG72 adenocarcinomas including B72.3 (FDA-approved monoclonal
colorectal, pancreatic, gastric, antibody)
ovarian, endometrial,
mammary, and non-small cell
lung cancer
Vascular Colorectal cancer Bevacizumab (Avastin®)
endothelial growth
factor
The additional agent can be a PD-1 antagonist or a CTLA-4 antagonist. Irs embodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., a siRNA, shRNA, or ribozyme, can be used to inhibit expression of PD- 1 or CTLA-4. In other embodiments, the PD-1 antagonist or CTLA-4 antagonist is an antibody.
In some embodiments, the CTLA-4 antagonist is an antibody or antigen binding fragment thereof that specifically binds CTLA4 (e.g., ipilimumab (also referred to as MDX-010 and MDX- 101 , and marketed as YERVOY®; Bristol-Myers Squibb; Tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675,206).).
In some embodiments, the CD300f inhibitor is administered with a PD-1 antagonist. PD-1 is an inhibitor}' member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD-1, PD-LI and PD-L2 have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al. 2000 J Exp Med 192: 1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-LI is abundant in human cancers (Dong et al. 2003 J Mol Med 81 :281-7; Blank et al. 2005 Cancer Immunol.
Immunother 54:307-314; onishi et al. 2004 Clin Cancer Res 10:5094). Immune suppression can be reversed by inhibiting the local interaction of PD-1 with Programmed Death- Ligand (PD-L)l or PD-L2. In some examples, the PD-1 antagonist can be an antibody or antigen binding fragment thereof that binds to PD-1 , PD-LI , or PD-L2. Antibodies, antibody fragments, and other inhibitors of PD-1, PD-LI and PD-L2 are available in the art and may be used in the methods disclosed
herein. For example, nivolumab (also referred to as BMS-936558 or MDX1106; Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody which specifically blocks PD-L Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in U.S. Pat. No. 8,008,449 and PCX Publication No.WO2006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgGlk monoclonal antibody that binds to PD-1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in PCT Publication No.
WO2009/1016H. Lambrolizumab (also referred to as MK03475; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Lambrolizumab and other humanized anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,354,509 and PCT Publication No. WO2009/ 114335. MDPL3280A (Genentech/Roche) is a human Fc optimized IgGi monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies to PD-L1 are disclosed in U.S. Patent No. 7,943,743 and U.S. Publication No. 2012/0039906. Other anti-PD-Ll binding agents include YW243.55.S70 (heavy and light chain variable regions are shown in SEQ ID NOs: 20 and 21 in PCT Publication No. WO2010/077634) and MDX-1 105 (also referred to as BMS-936559, and, e.g., anti-PD-Ll binding agents disclosed in PCT Publication No. WO2007/005874). AMP- 224 (B7-DCIg; Amplimmune; e.g., disclosed in PCT Publication No. WO2010/027827 and PCT Publication No. WO2011/066342), is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD-1 and B7-H1. Other anti-PD-1 antibodies include AMP 514 (Amplimmune), among others, e.g., anti-PD-1 antibodies disclosed in U.S. Patent No. 8,609,089, U.S. Publication No. 2010/028330, and/or U.S. Publication No. 2012/0114649. Any of these PD- 1 antagonists are of use in the methods disclosed herein.
Disruption of the CD300f Gene
Methods and compositions are disclosed herein for altering the CD300f gene, such as in monocytes, monocyte-derived cells (such as monocyte-derived dendritic cells), CD34+
hematopoietic progenitor cells, including dendritic precursor cells). The methods and compositions described herein introduce one or more breaks near the site of the CD300f gene to decrease the production of functional CD300f protein, such as in a dendritic cell. As noted in the section above, the CD300f gene is also known as CD300LF, CLM1, IGSF13, IREM1, and NKIR.
Exemplary nucleic acid sequences encoding human CD300f are provided in GENBANK®
Accession No. NM_139018.4 (August 26, 2016), and GENBANK Accession No.
NM_001289082.1 (August 26, 2016), which are both incorporated by reference, and an exemplary amino acid sequence of human CD300f is provided in GENBANK® Accession No. AAH28199.1
(June 6, 2006), which is incorporated by reference herein. Other examples are provided herein.
A typical set of CRISPR system is composed of two components, a CRISPR-associated nuclease 9 (Cas9) and one or more guide RNAs (gRNAs), each of which contains a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). Simple gene disruptions can be generated by cleavage of the target site, followed by alteration of nucleic acids, such as a deletion, and repair by the non-homologous-end-joining pathway (NHEJ). Target recognition by crRNAs occurs through complementary base pairing with target DNA, which directs cleavage of foreign sequences by means of Cas proteins. In some embodiments, DNA recognition by guide RNA and consequent cleavage by the endonuclease requires complementary base-pairing with a protospacer adjacent motif (PAM) (e.g. 5'-NGG-3') and with a protospacer region in the target. (Jinek et. al., Science. 337:816-821, 2012). The PAM motif recognized by a Cas9 varies for different Cas9 proteins. Any Cas9 protein can be used in the systems and methods disclosed herein.
One Cas9 of use is from Streptococcus pyogenes as depicted in below (SEQ ID NO: 9) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQ DLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEETITPWNFEEWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVK YVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIAN LAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLT RSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVET RQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYK EVKKDLI IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD
In other embodiments, the Streptococcus pyogenes Cas9 peptide can include one or more of the mutations described in the literature, including but not limited to the functional mutations described in: Fonfara et al., Nucleic Acids Res. 2014 Feb;42(4):2577-90; Nishimasu et al. Cell. 2014 Feb 27;156(5):935-49; Jinek M et al. Science. 2012 Aug 17;337(6096):816-21 ; and Jinek et al. Science. 2014 Mar 14;343(6176). Thus in some embodiments the systems and methods disclosed herein can be used with the wild type Cas9 protein having double- stranded nuclease activity, Cas9 mutants that act as single stranded nickases, or other mutants with modified nuclease activity.
The Cas9 includes a catalytically active nuclease domain. In some embodiments, the Cas9 nuclease includes an HNH-like endonuclease and a RuvC-like endonuclease. Thus in some embodiments, to generate a double-stranded DNA break, the HNH-like endonuclease cleaves the DNA strand complementary to the gRNA, and the RuvC-like domain cleaves the non- complementary DNA strand. A Cas9 endonuclease can be guided to specific genomic targets using specific gRNA (see below).
In other embodiments of the the systems and methods disclosed herein, a promoter, such as the Embryonal Fyn- Associated Substrate (EFS) promoter is operably linked to the nucleic acid encoding Cas9. This promoter provides for cell specific expression of Cas9. The sequence of this promoter is shown below (SEQ ID NO: 10):
TGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGA AGTTGGGGGGAGGGGTCGGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAA ACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACC GTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAA CACAGG
The promoter can include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions in SEQ ID NO: 10, provided the promoter allows for expression in dendritic cells. The promoter can be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical SEQ ID NO: 10, provided the promoter allows for expression in dendritic cells.
Other promoters of use, include, but are not limited to, a cytomegalovirus (CMV) promoter, a synthetic promoter such as CAG promoter, a simian virus (SV)40 promoter, a 35S promoter, and an alcohol dehydrogenase (ADH)l promoter. One of skill in the art can readily identify promoters of use.
Optionally, a nucleic acid molecule encoding a marker also can be operably linked to the EFS promoter. Markers include, but are not limited to, enzymes and fluorescent proteins. In one
specific non-limiting example, the marker is tdTomato fluorescent protein. In other embodiments, a nucleic acid molecule encoding a marker is not operably linked the EFS.
As noted above, the Cas9 RNA guide system includes a mature crRNA that is base-paired to trans-activating crRNA (tracrRNA), forming a two-RNA structure that directs Cas9 to the locus of a desired double- stranded (ds) break in target DNA, namely the CD300f gene. In some
embodiments base-paired tracrRNA: crRNA combination is engineered as a single RNA chimera to produce a guide sequence (e.g., gRNA) which preserves the ability to direct sequence-specific Cas9 dsDNA cleavage (see Jinek et al., Science. 337:816-821, 2012). In some embodiments, the Cas9- guide sequence complex results in cleavage of one or both strands at a target sequence within the CD300f gene, such as in exons 1 or 3 of the CD300f gene. Thus, the Cas9 endonuclease (Jinek et al., Science. 337:816-821, 2012; Mali et. al., Nat Methods. 2013 Oct; 10(10): 1028-1034) and the gRNA molecules are used sequence- specific target recognition, cleavage, and genome editing of the CD300f gene. In one embodiment, the cleavage site is at a specific nucleotide, such as, but not limited to the 16, 17, or 18th nucleotide of a 20 nucleotide target. In one non-limiting example, the cleavage site is at the 17th nucleotide of a 20-nt target sequence. The cleavage can be a double stranded cleavage.
In some embodiments, the gRNA molecule is selected so that the target genomic targets bear a protospacer adjacent motif (PAM). In some embodiments, DNA recognition by guide RNA and consequent cleavage by the endonuclease requires the presence of a protospacer adjacent motif (PAM) (e.g., 5'-NGG-3') in immediately after the target. The PAM is present in the targeted nucleic acid sequence but not in the crRNA that is produced to target it. In some embodiments, the proto-spacer adjacent motif (PAM) corresponds to 2 to 5 nucleotides starting immediately or in the vicinity of the proto-spacer at the leader distal end. The PAM motif also can be NNAGAA, NAG, NGGNG, AWG, CC, CC, CCN, TCN, or TTC.
In some embodiments, cleavage occurs at a site about 3 base-pairs upstream from the PAM.
In some embodiments, the Cas9 nuclease cleaves a double stranded nucleic acid sequence.
In some embodiments, the guide sequence is selected to reduce the degree of secondary structure within the sequence. Secondary structure may be determined by any suitable
polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold (Zuker and Stiegler, Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, which uses the centroid structure prediction algorithm (see e.g., Gruber et al., 2008, Cell 106(1): 23-24; and Can and Church, 2009, Nature Biotechnology 27(12): 1151-62). Guide sequences can be
designed using the MIT CRISPR design tool found at crispr.mit.edu, Harvard and University of Bergen CHOPCHOP web tool found at chopchop.cbu.uib.no, or the E-CRISP tool found at www.e- crisp.org/E-CRISP. Additional tools for designing tracrRNA and guide sequences are described in Naito et al., Bioinformatics. 2014 Nov 20, and Ma et al. BioMed Research International,
Volume 2013 (2013), Article ID 270805. The crRNA can be 18-48 nucleotides in length. The crRNA can be 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In one example, the crRNA is 20 nucleotides in length. In additional embodiments, the tracrRNA is pre-optimized, and is 83 nucleotides in length, see SEQ ID NO: 7.
In some embodiments, the human CD300f gene is targeted, such as exon 3 or exon 4, and the crRNA is encoded by a nucleic acid sequence set forth as one of SEQ ID NO: 11 or 12. In specific non-limiting examples, the crRNA is encoded by one of the DNA sequences below. The PAM, which is recognized by Cas9 is shown next to the target DNA sequence below.
Sequence (5'-3') PAM (5'-3')*
GAAAACTGGAAATGACCTTG (SEQ ID NO : 11 ) GGG
GTGGTGGCCGGTCAGAGTTG (SEQ ID NO: 12) GGG
In some embodiments, the DNA encoding the crRNA includes or consists of, one of the nucleic acid sequence sent forth as one of SEQ ID NO: 11 or 12, which target Exon 3 and Exon 4, respectively. The PAM, which is recognized by Cas9, is shown next to the target DNA sequence. The DNA encoding the crRNA can alternatively target another exon, such as, but not limited to, Exon 1 or Exon 2.
The system disclosed herein introduces double stranded DNA breaks at the CD300f gene, such that the CD300f target is cleaved by Cas9. This results in functional CD300f protein not being produced.
The system disclosed herein can include a promoter, such as, but not limited to, a U6 or HI promoter operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas RNAs.
The U6 promoter can include the following nucleic acid sequence:
GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGA GATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGT AGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTA
TCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAA AGGACGAAACACC (SEQ ID NO: 13, see also GENBANK® Accession No. X07425.1, incorporated herein by reference).
Disclosed below is a U6 gRNA sequence, wherein the tracrRNA is underlined. The tracer sequence includes seven thymidines for terminating RNA transcription. The small "g," "ga," and the second "g" border the Saplrev and Sapl sites where the nucleic acid encoding the gRNA is inserted.
GGCGCGCCGGATCCGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATAC AAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTAC
AAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGT
TTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTA
TATATCTTGTGGAAAGGACGAAACACCgGAAGAGCgaGCTCTTCgGnnAGAGCTAGA
AATAGCAAGTTA^
GTGC riTTlTGGTACCGGCGCGCC (SEQ ID NO: 14)
In one example, the tracrRNA is encoded by the nucleic acid sequence set forth as:
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA
GTGGCACCGAGTCGGTGCTTTTTTT (SEP ID NO: 15). In some embodiments, more than one DNA break can be introduced by using more than one gRNA. For example, two gRNAs can be utilized, such that two breaks are achieved. When two or more gRNAs are used to position two or more cleavage events, in a target nucleic acid, it is contemplated that in an embodiment the two or more cleavage events may be made by the same or different Cas9 proteins. For example, when two gRNAs are used to position two double strand breaks, a single Cas9 nuclease may be used to create both double strand breaks. Thus, both of the gRNAs corresponding to the DNA sequences set forth as SEQ ID NOs: 11 and 12 can be used.
In some embodiments, the disclosed methods include the use of one or more vectors comprising: a) a EFS promoter operably linked to a nucleotide sequence encoding a Type II Cas9 nuclease, b) a U6 promoter operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas guide RNAs that hybridize with the CD300f gene in a target cell, such as a human cell. Components (a) and (b) can be located on same or different vectors, whereby the one or more guide RNAs target the CD300f gene in the target cell and the Cas9 protein cleaves the CD300f gene. In specific non-limiting examples, the one or more vectors are viral vectors such as
lentiviral vectors. In other non-limiting examples, the viral vectors are adenovirus vectors, adeno- associated virus vectors, or retroviral vectors.
Lentiviral vectors are retroviral vectors that are able to transduce or infect non- dividing cells and typically produce high viral titers. Retroviral vectors are comprised of cis- acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis- acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the desired nucleic acid into the target cell to provide permanent expression. One non- limiting example of a lentiviral vector is the lentiCRISPRv2 vector (Adgene Plasmid #52961, see the addgene website, addgene.org/52961/).
Retroviral vectors of use include murine leukemia virus (MuLV) vectors, gibbon ape leukemia virus (GaLV) vectprs, Simian Immunodeficiency virus (SIV) vectors, human immuno deficiency virus (HIV) vectors, and combinations thereof (see, e.g., Buchscher et ah, (1992) J. Virol. 66:2731-2739; Johanti et al, (1992) J. Virol. 66: 1635-1640; Sommnerfeit et al., (1990) Virol. 176:58-59; Wilson et al, (1998) J. Virol. 63:2374-2378; Miller et al, (1991) J. Virol.
65:2220-2224; PCT/US94/05700). The use of lentiviral vectors for the delivery of Cas9 and sgRNAs is disclosed in U.S. Published Patent Application No. US20150191744, which is incorporated herein by reference.
Methods are disclosed herein for altering expression of CD300f in a subject. The method included introducing into a dendritic cell, such as a human dendritic cell comprising a gene encoding CD300f an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system comprising one or more viral vectors, such as lentiviral vectors. The one or more viral vectors include a) an EFS (or other retina- specific promoter) operably linked to a nucleotide sequence encoding a Cas9 protein, and b) a U6 promoter operably linked to at least one nucleotide sequence encoding a CRISPR-Cas guide RNA that hybridizes with the CD300f gene, wherein components (a) and (b) are located on same or different vectors of the system, whereby the guide RNA targets the CD300f gene and the Cas9 protein cleaves the CD300f gene.
In some embodiments, the Cas9 protein is expressed in a recombinant cell, such as E. coli, and purified. The resulting purified Cas9 protein, along with an appropriate guide molecule specific for the target, is then introduced into a cell or organism where one or genomic sequences can be targeted. In some examples, the Cas9 protein and guide nucleic acid molecule (i.e., gRNA) are introduced as separate components into the target cell. In other examples, the purified Cas9 protein is complexed with the guide nucleic acid, and this ribonucleoprotein (RNP) complex is
introduced into target cells (e.g., using transfection or injection). In some examples, the Cas9 protein and guide molecule are injected into the cell of interest. Once the Cas9 protein and guide nucleic acid molecule are in the cell, one or more genomic sequences can be targeted. Modification of Dendritic Cells and Their Use
Methods are disclosed herein that utilize dendritic cells deficient for expression of CD300f. These dendritic cells can be used to induce an immune response to a solid tumor. The dendritic cells deficient for expression of CD300f can be in combination with a CD300f inhibitor (or other chemotherapeutic or anti-neoplastic agent), using any of the methods disclosed above, for the treatment of a tumor in a subject, such as a solid tumor. However, the dendritic cells deficient for expression of CD300f can also be used without the administration of a CD300f inhibitor. In some embodiments, the dendritic cells are used to treat a leukemia or a lymphoma. In other
embodiments, the dendritic cells are used to treat a lymphoma.
In some embodiments, a therapeutically effective amount of the dendritic cells deficient for expression of CD300f are administered to a subject with the solid tumor. In another embodiment, the dendritic cells deficient for expression of CD300f are used to induce activated T cells, such as activated cytotoxic CD8+ T cells, specific for the tumor. A therapeutically effective amount of the activated T cells can be administered to subject with the tumor. In particular embodiments, the T cells and/or dendritic cells are autologous. In some non-limiting examples, the subject can be administered an additional therapeutic agent, such as, but not limited to, a CD300f inhibitor, a chemotherapeutic agent, an anti-neoplastic agent, a PD- 1 antagonist or a CTLA-4 antagonist, as discussed above.
The production of dendritic cells is disclosed, for example, in U.S. Application No.
20150335679, incorporated herein by reference. As disclosed in this publication, dendritic cells can be generated in vivo or ex vivo from immature precursors (e.g., monocytes, CD34+ hematopoietic precursor cells). For example, for ex vivo dendritic cell generation, a cell population enriched for dendritic cell precursor cells (e.g., peripheral blood mononuclear cells (PBMCs)) is obtained from a subject, such as with a solid tumor, and then the dendritic cell precursor cells are differentiated ex vivo into mature dendritic cells. Typically, to generate immature dendritic cells, monocytic precursors are first enriched or purified from other cell types. For example, peripheral blood mononuclear cells (PBMCs) are extracted from whole blood (e.g., over Ficoll density gradient centrifugation). Then the PBMCs will be used to generate monocytic dendritic cell precursors.
In certain embodiments, monocytic dendritic cell precursors are isolated by adherence to a monocyte-binding substrate. For example, a population of leukocytes (e.g., isolated by
leukapheresis) can be contacted with a monocytic dendritic cell precursor adhering substrate. When the population of leukocytes is contacted with the substrate, the monocytic dendritic cell precursors in the leukocyte population preferentially adhere to the substrate. In one embodiment, monocytes are isolated through adherence of the monocytic precursors to a plastic (polystyrene) surface, as the monocytes have a greater tendency to stick to plastic than other cells found in, for example, peripheral blood, such as lymphocytes
Additional methods for isolating cell populations enriched for dendritic cell precursors and immature dendritic cells from various sources, including blood and bone marrow, are known in the art. For example, dendritic cell precursors and immature dendritic cells can be isolated by phlebotomy, by apheresis or leukapheresis, by collecting heparinized blood, by preparation of buffy coats, rosetting, centrifugation, density gradient centrifugation (e.g., using Ficoll, Percoll (colloidal silica particles of 15-30 mm diameter coated with polyvinylpyrrolidone (PVP)), sucrose, and the like), differential lysis of cells, filtration, and the like. In one embodiment, dendritic cell precursors can be selected using CD 14 selection of G-CSF mobilized peripheral blood. See U.S. Patent No. 8,728,806, incorporated herein by reference.
Before the subject's blood or bone marrow is obtained to isolate dendritic cell precursors, the subject may be administered granulocyte macrophage colony stimulating factor (GM-CSF) to increase bone marrow production of monocytes and dendritic cell precursors. In certain embodiments, GM-CSF is administered at a dose ranging from about 10 μg/day to about 500 μg/day, from about 20 μg/day to about 300 μg/day, from about 50 μg/day to about 250 μg/day, from about 100 μg/day to about 300 μg/day, from about 200 μg/day to about 300 μg/day, about 200 μg/day, or about 250 μg/day. In certain embodiments, GM-CSF may be administered for about 1 day, about 2 days, about 3 days, about 4 day, about 5 days, about 6 days, about 1 week, about 1.5 weeks, about 2 weeks, or longer. The effect of GM-CSF may be potentiated by another immunostimulant (such as plerixafor). In some embodiments, monocytes, monocyte-derived cells including dendritic cells, or monocyte precursors are treated with the CRISPR/Cas9 system disclosed herein to reduce expression of CD300f.
Variations on this method include different methods of purifying monocytes, including, for example, tangential flow filtration (TFF), or by binding antibodies attached to beads to surface molecules on the monocytes. The beads with the bound cells are then concentrated in a column, or
on a magnetic surface, such that contaminating cells can be washed away, after which the monocytes are eluted off the beads. In yet another method to obtain dendritic cell precursors, cells expressing the stem cell marker CD34, either from blood (see U.S. Patent No. 5,994,126, incorporated herein by reference) or from the bone marrow are purified. These cells are cultured with GM-CSF to differentiate into immature dendritic cells.
Isolated dendritic cell precursors can be cultured ex vivo for differentiation, maturation and/or expansion. In certain embodiments, monocytic dendritic cells precursors are differentiated to form immature dendritic cells. The dendritic cell precursors, such as CD34+ dendritic cell precursors, or immature dendritic cells are then treated using the CRISPR/Cas9 system disclosed above to reduce or eliminate expression of CD300f. These cells are then matured to mature dendritic cells.
Dendritic cell precursors and/or immature dendritic cells can be cultured and differentiated in suitable culture conditions. The tissue culture media can be supplemented with, e.g., plasma, serum, amino acids, vitamins, cytokines (e.g., granulocyte-macrophage colony- stimulating factor (GM-CSF), interleukins such as interleukin 4 (IL-4), interleukin 13 (IL-13), interleukin 15 (IL-15), or combinations thereof), purified proteins (such as serum albumin), divalent cations (e.g., calcium and/or magnesium ions), growth factors, and the like, to promote differentiation of the cells (Sallusto et al., J. Exp. Med., 179:1109-18, 1994, incorporated herein by reference). Such culture conditions can optionally exclude any animal-derived products. In one embodiment, a dendritic cell culture medium contains about 200 units/ml to about 1500 units/ml (e.g., about 1000 units/ml, about 500 units/ml, etc.) of GM-CSF and about 200 units/ml to about 1500 units/ml (e.g., about 800 units/ml, about 500 units/ml, etc.) IL-4.
Immature dendritic cell have a high capacity for taking up and processing antigen, but have a limited ability to initiate immune responses. The ability to initiate an immune response is acquired by maturation of the immature dendritic cell. This maturation is also referred to as activating, or activation of, the dendritic cell. The maturation process may be initiated and/or induced through contact with maturation-inducing cytokines, tumor- associated antigens or tumor- associated peptide antigens and/or nucleic acids encoding tumor- associated antigens or tumor- associated peptide antigens, and the like.
In some embodiments, mature dendritic cells can be selected by expression of one or more markers. The markers include, but are not limited to, CD86, CD80, CD83, CD58, CDla, HLA-DR, CD40, CDllc, IL-2-beta, TLR-4 and combinations thereof. The dendritic cells can also be
identified as lacking or expressing low levels of markers such as CD14. In one embodiment, mature dendritic cells are identified as being CD80+, CD83+, CD86+, and CD14-. Greater MHC expression leads to an increase in antigen density on the dendritic cell surface, while up-regulation of costimulatory molecules CD80 and CD86 strengthens the T cell activation signal through the counterparts of the costimulatory molecules, such as CD28 on the T cells. Any of these cells types can be treated with the CRISPR/Cas9 system disclosed herein such that expression of CD300f is decreased or eliminated.
Cell surface markers can be detected in suitable assays, such as flow cytometry, immunohistochemistry, and the like. The cells can also be monitored for cytokine production (e.g., by ELISA, FACS, or other immune assay). Dendritic cell precursors, immature dendritic cells, and mature dendritic cells, either primed or unprimed with antigens, and/or treated using the
CRISPR/Cas9 system disclosed herein, can be cryopreserved for use at a later date.
Tumor cells, such as apoptotic or killed cells, may be used to deliver antigen to either immature or mature dendritic cells, either freshly isolated or obtained from in vitro culture, wherein expression of CD300f is decreased in the dendritic cells. In one embodiment, tumor cells comprising an antigen (e.g., tumor antigen) are co-cultured with immature dendritic cells for a time sufficient to allow the antigen to be internalized by the immature dendritic cells. These immature dendritic cells are then caused to mature by the addition of a maturation factor to the culture medium. The matured dendritic cells expressing processed antigen on their surface are then exposed to T cells for potent cytotoxic T cell induction to the tumor. Alternatively, one or more specific tumor antigens can be used. Exemplary tumor antigens that can be used include but are not limited to those shown in Table 1 above.
In one embodiment, peripheral blood mononuclear cells (PBMCs) can be isolated from blood by sedimentation techniques. Human CD8+ T cell are purified with a commercial kit from Miltenyi Biotec. Dendritic cells are prepared, treated using the CRISPR/Cas9 system disclosed herein, and are cultured for 7 days to 10 days in the presence of GM-CSF and IL-4. On about day 7 through 10, apoptotic tumor cells can be co-cultured with the dendritic cells and the dendritic cells caused to mature over the next four days with the addition of monocyte conditioned medium, a signal for maturation. Alternatively, a combination of cytokines may be used to induce maturation of the immature dendritic cells. Examples of cytokines which may be used alone or in combination with each other include, but are not limited to, TNF-a, IL-Ιβ, IL-6, and IFN-β. In some embodiments, a therapeutically effective amount of these dendritic cells are administered to a
subject with the solid tumor. In other embodiments, the dendritic cells are contacted with CD8+ T cells, and a therapeutically effective amount of the CD8+ T cells are administered to the subject with the solid tumor.
In further embodiments, dendritic cells (or immature dendritic cells) are treated with the CRISPR/Cas9 system disclosed herein to reduce expression of CD300f. These dendritic cells are then loaded with one or more peptide antigens (e.g., a tumor- associated peptide antigen, such as those listed in Table 1). In certain aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more tumor-associated peptide antigens are used. Generally, these peptide antigens are expressed by the solid tumor. A cell or membrane bound composition (e.g., a liposome) "loaded" (or "pulsed") with a peptide can be used. For example, antigen presenting cells (APCs, e.g., dendritic cells) can be incubated with one or more tumor- associated peptide antigens under conditions that are needed to load the major histocompatibility complex (MHC) of the dendritic cells. Suitable conditions for antigen loading are provided that permit a dendritic cell to contact, process and/or present one or more antigens on its MHC, whether intracellularly or on the cell surface. The incubation time may range from about 10 minutes to about 3 days or longer, from about 30 minutes to about 36 hours, from about 1 hour to about 28 hours, from about 2 hours to about 24 hours, from about 4 hours to about 24 hours, from about 4 hours to about 16 hours, from about 16 hours to about 24 hours, from about 20 hours to about 28 hours, from about 2 hours to about 4 hours, from about 1 hour to about 12 hours, from about 2 hours to about 8 hours, from about 3 hours to about 5 hours, for less than about a week, illustratively, for about 1 minute to about 48 hours, about 2 minutes to about 36 hours, about 3 minutes to about 24 hours, about 4 minutes to about 12 hours, about 6 minutes to about 8 hours, about 8 minutes to about 6 hours, about 10 minutes to about 5 hours, about 15 minutes to about 4 hours, about 20 minutes to about 3 hours, about 30 minutes to about 2 hours, about 40 minutes to about 1 hour, about 16 hours, about 20 hours, about 24 hours, about 28 hours, about 1 hour, about 2 hours, or about 4 hours. The incubation temperature may range from about 4 °C to about 37 °C, from about 25 °C to about 37 °C, about 4 °C, about 25 °C, or about 37 °C. The concentration of the peptide for loading can range from about 1 μg/ml to about 1 mg/ml, from about 5 μg/ml to about 800 μg /ml, from about 10 μg /ml to about 600 μg /ml, from about 15 μg /ml to about 400 ^g/ml, from about 10 μg/ml to about 200 μg/ml, from about 10 μg/ml to about 100 μg/ml, from about 50 μg/ml to about 100 μg/ml, from about 20 μg/ml to about 100 μg/ml, etc.
A number of methods for delivery of antigens to the endogenous processing pathway of antigen-presenting cells may be optionally used. Such methods include, but are not limited to, methods involving pH-sensitive liposomes, coupling of antigens to potent adjuvants, apoptotic cell
delivery, pulsing cells onto dendritic cells, delivering recombinant chimeric virus-like particles (VLPs) comprising antigen to the MHC class I processing pathway of a dendritic cell line.
Dendritic cells also can be contacted with nucleic acids encoding one or tumor-associated antigens under a condition sufficient for the at least one tumor-associated peptide antigen to be presented by the dendritic cell. For example, dendritic cells can be transfected with expression vectors or infected with viral vectors for introducing nucleic acids encoding tumor-associated antigens into the dendritic cells. Expression can be optionally effected by targeting the expression construct to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue- specific promoter, or combinations thereof. Non-limiting viral vectors include adeno-associated viruses, lentiviruses, retroviruses, herpes viruses, adenoviruses, vaccinia viruses, baculoviruses, Fowl pox, AV-pox, modified vaccinia Ankara (MVA) and other recombinant viruses.
The time and amount of antigens, or nucleic acids encoding the antigens, necessary for the antigen presenting cells to process and present the antigens can be determined, for example, by assaying T cell cytotoxic activity in vitro or using antigen-presenting cells as targets of CTLs. The antigen-presenting cells, such as dendritic cells that are treated with the CRISPR/Cas9 system disclosed herein such that expression of CD300f is decreased, are loaded with the antigen, and then can be used to stimulate CTL proliferation in vivo or ex vivo.
The ability of the loaded dendritic cells to stimulate a cytotoxic T lymphocyte (CTL) response can be measured by assaying the ability of the effector cells to lyse target cells. For example, the non-radioactive LDH cytotoxicity assay or the europium release assay can be used. Volgmann et al., J. Immunol. Methods 119:45-51, 1989. As noted above, ex vivo or in vitro maturation of dendritic cells can be induced by various maturation factors, including, but not limited to, tumor necrosis factor alpha (TNF-a), interferon alpha (IFN-a), poly (I:C), interferon gamma (IFN-γ), Interleukin 1 beta (IL-Ιβ), Interleukin 6 (IL-6), prostaglandin E2 (PGE2), poly- dldC, vasointestinal peptide (VIP), bacterial lipopolysaccharide (LPS), mycobacteria or components of mycobacteria (such as cell wall constituents), or combinations thereof. Additional maturation factors include, for example, an imidazoquinoline compound, e.g., R848 (see PCT Publication No. WO 00/47719, incorporated herein by reference), a synthetic double stranded polyribonucleotide, agonists of a Toll-like receptor (TLR), such as TLR3, TLR4, TLR7 and/or TLR9, a sequence of nucleic acids containing unmethylated CpG motifs known to induce the maturation of dendritic cells, and the like. Further, a combination of any of the above agents can be used in inducing the maturation of immature dendritic cells or dendritic precursor cells.
In certain other embodiments, mature dendritic cells or T cells can be expanded in vitro
from freshly isolated or frozen cell stocks to generate sufficient numbers of cells for effective adoptive immunotherapy.
Methods are provided for administration of mature dendritic cells to a subject in need of immunostimulation. In certain embodiments, such methods are performed by obtaining dendritic cell precursors or immature dendritic cells, differentiating and maturing those cells, using the CDRISPR/Cas9 system disclosed herein to produce dendritic cells with reduced expression of CD300f, and then culturing the cells in the presence of a tumor-associated antigen or a tumor- associated peptide antigen, a nucleic acid composition, and/or apoptotic tumor cells, to form a mature dendritic cell population. A therapeutically effective amount of any of these dendritic cells can be administered to a subject with a solid tumor.
In certain embodiments, the present methods induces an immune response to a tumor in a subject. Such methods can include one or more steps of (a) obtaining monocytes (which may act as dendritic cell precursors) from a patient; (b) culturing the monocytes (e.g., with specific cytokines) to induce differentiation into immature dendritic cells; (c) contacting the immature dendritic cells with apoptotic tumor cells to induce engulfment and presentation of tumor-associated antigens; (d) differentiating the immature dendritic cells into mature dendritic cells with maturation factors such as cytokines or TLR ligands ;and (e) administering the mature dendritic cells to the patient. The monocytes, monocyte-derived cells or CD34+ progenitor-derived cells including immature dendritic cells can be treated with the CDRISPR/Cas9 system disclosed herein to produce cells with reduced expression of CD300f. Dendritic cells can be administered to the subject once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, or more, within a treatment regime to a subject/patient. Dendritic cells can be administered to a subject every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, every 11 days, every 12 days, every 13 days, every 14 days, every 16 days, every 18 days, every 20 days, every 1 month, every 2 months, every 3 months, every 6 months, or at different frequencies. In some embodiments, the cells are administered intranodally or intradermally.
The dendritic cells can be administered at a dose ranging from about 1 X 103 dendritic cells to about 1 X 1012 dendritic cells, from about 1 X104 dendritic cells to about 1 X 1010 dendritic cells, from about 1 X 105 dendritic cells to about 1 X 109 dendritic cells, from about 1 X 106 dendritic cells to about 11 X 108 dendritic cells, from about 1 X 106 dendritic cells to about 1 X 107 dendritic cells, from about 1 X 107 dendritic cells to about 1 X 108 dendritic cells, or from about 1 X 108 dendritic cells to about 1 X 109 dendritic cells.
In other embodiments, the mature dendritic cells can be contacted with, and thus, activate, lymphocytes. The activated, polarized lymphocytes, optionally followed by clonal expansion in cell culture, can be administered to a subject with a solid tumor. In these embodiments, the dendritic cells that engulfed apoptotic tumor cells, or dendritic cells loaded with antigen, are contacted with lymphocytes under conditions sufficient to produce tumor-associated antigen- specific lymphocyte capable of eliciting an immune response against a tumor cell, such as cytotoxic CD8+ lymphocytes.
In one embodiment, CD8+ T lymphocytes are contacted with the dendritic cells described above for a period of time, such as for at least about 10 days, for priming and expanding the tumor antigen specific CD8+ T lymphocytes. The ability to induce lymphocytes to exhibit an immune response can be determined by any method including, but not limited to, determining T lymphocyte cytotoxic activity in vitro using for example tumor-associated antigen- specific antigen-presenting cells as targets of tumor- associated antigen- specific cytotoxic T lymphocytes (CTL); assaying tumor-associated antigen- specific T lymphocyte proliferation and ELISA methods.
CD8+ T lymphocytes can be obtained, for example, from peripheral blood and used as purified preparations, which can be obtained by standard techniques including, but not limited to, methods involving immunomagnetic or flow cytometry techniques using antibodies.
In another embodiment, resulting CTL are reinfused autologously to the subject. In one aspect, the method includes administering to a subject antigen-presenting cells, T lymphocytes, or both, where the antigen-presenting cells have engulfed apoptotic tumor cells and presented tumor- associated antigens, or wherein the antigen-presenting cells have been loaded with at least one tumor-associated peptide antigen, or where the antigen-presenting cells comprise nucleic acids encoding at least one tumor-associated antigen, under a condition sufficient for at least one tumor- associated peptide antigen to be presented by the antigen-presenting cells. The T lymphocytes have been contacted with antigen-presenting cells presenting at least one tumor-associated antigen.
Generally, the dendritic cells and the T cells are autologous. In specific, non-limiting examples, the APCs and the responder T cells are from the same individual. However, the APCs and the responder T cells can be syngeneic. The APC can be used to present any antigen to a population of autologous T cells.
One of skill in the art will appreciate that antigenic peptides that bind to MHC class I and II molecules can be generated ex vivo (for example instead of being processed from a full-length protein in a cell), and allowed to interact with (such as bind) MHC I and II molecules on a cell surface. Generally, APCs present antigen in the context of both MHC class I and Π.
The amount of antigen used to prime T cells can be readily determined using methods known in the art. Generally, if the antigen is used in a purified form, about 1-10 μg/ ml of peptide is used.
In a specific example, lymphocytes are primed in vitro by incubating them with soluble antigen or viral lysate for 5-7 days under conditions that permit priming of T cells. Viable T cells are recovered, for example by Ficoll-Hypaque centrifugation, thereby generating primed T cells. If desired, the viable primed T cells can be primed again one or more times, for example by incubation with the antigen for another 5-7 days under the same conditions as those used for the first priming, and viable T cells recovered.
To increase the number of antigen- specific CD8+ T cells, proliferation of the cells can be stimulated, for example by incubation in the presence of a cytokine, such as interleukin (IL)-2, IL-7, IL-12 and IL-15. The amount of cytokine added is sufficient to stimulate production and proliferation of T cells, and can be determined using routine methods. In some examples, the amount of IL-2, IL-7, IL-12, or IL-15 added is about 0.1-100 IU/mL, such as at least 1 IU/mL, at least 10 IU/mL, or at least 20 IU/mL.
In one example, during stimulation of proliferation of activated CD8+ T cells, the cells can be counted to determine the cell number. When the desired number of cells is achieved, purity is determined. Purity can be determined, for example, using markers present on the surface of activated T cells concomitant with the assessment of cytokine production upon antigen recognition, such as interferon (IFN)y, tumor necrosis factor (TNF)oc, or interleukin (IL)-2. Generally, activated CD8+ T cells are positive for the CD3 marker, along with the CD8 marker, and IFN-γ (which is specific for activated T cells). For example, fluorescence activated cell sorting (FACS) can be used to identify (and sort if desired) populations of cells that are positive for CD3, CD8, and IFN-γ by using differently colored anti-CD3, anti-CD8 and anti-IFN-γ. Briefly, stimulated T activated cells are permeabilized and incubated in the presence of anti-CD3, anti-CD8 and anti-IFN-γ (each having a different fluorophore attached), for a time sufficient for the antibody to bind to the cells. After removing unbound antibody, cells are analyzed by FACS using routine methods. Antigen- specific T cells are those that are INF-γ positive.
In another example, the method further includes determining the cytotoxicity of the antigen- specific T cells. Methods for determining cytotoxicity are known in the art, for example a 51Cr- release assay (for example see Walker et al. Nature 328:345-8, 1987; Qin et al. Acta Pharmacol. Sin. 23(6):534-8, 2002; all herein incorporated by reference). The present disclosure also provides therapeutic compositions that include the enriched (such as purified) activated T cells. In particular
examples, the resulting enriched population of activated T cells (specific for the antigen of interest) are placed in a therapeutic dose form for administration to a subject with a solid tumor.
Expanded and selected activated CD8+ T cells can be tested for mycoplasma, sterility, endotoxin and quality controlled for function and purity prior cryopreservation or prior to infusion into the recipient.
A therapeutically effective amount of activated CD8+ T cells is administered to the subject. Specific, non- limiting examples of a therapeutically effective amount of purified activated T cells include purified activated T cells administered at a dose of about 1 X 105 cells per kilogram of subject to about 1 X 109 cells per kilogram of subject, such as from about 1 X 106 cells per kilogram to about 1 X 108 cells per kilogram, such as from about 5 X 106 cells per kilogram to about 75 X 106 cells per kilogram, such as at about 25 X 106 cells per kilogram, or at about 50 X 106 cells per kilogram.
Purified activated T cells can be administered in single or multiple doses as determined by a clinician. For example, the cells can be administered at intervals of approximately two weeks depending on the response desired and the response obtained. In some examples, once the desired response is obtained, no further activated T cells are administered. However, if the recipient displays one or more symptoms associated with the presence or growth of a tumor, a therapeutically effective amount of activated T cells can be administered at that time. The administration can be local or systemic.
The purified activated T cells disclosed herein can be administered with a pharmaceutically acceptable carrier, such as saline. Other therapeutic agents can be administered before, during, or after administration of the activated T cells, depending on the desired effect. Exemplary therapeutic agents include, but are not limited to, anti-microbial agents, immune stimulants such as interferon- alpha, chemotherapeutic agents, biologic agents (such as those listed in Table 1) or peptide vaccines of the same antigen used to stimulate T cells in vitro. In a particular example, compositions containing purified activated T cells also include one or more therapeutic agents.
EXAMPLES
Using mouse cancer models, it was determined that blocking CD300f function in dendritic cells (DCs) markedly enhanced their ability to phagocytose and process apoptotic tumor cells, and cross-present tumor cell antigens to cytotoxic CD8+ T cells (CTL), leading to substantial inhibition of tumor growth. The results show that inhibiting CD300f function on DCs is an anti-cancer therapy. Anti-CD300f blocking antibodies can release a checkpoint on human DC in order to
enhance tumor cell antigen presentation by DC and induce the expansion and activation of tumor- specific autologous CTL.
Example 1
It was demonstrated that CD300f uniquely functions as a checkpoint receptor to inhibit DC- mediated apoptotic cell phagocytosis and antigen cross-presentation for T cell priming. An overview of the method is provided in FIG. 1. Mouse with the deficiency of Cd300f gene supplies a very useful tool to examine CD300f function. To generate the knock out mice, exon 2-3 of Cd300f gene was flanked by loxP sites. A PGK-neo cassette flanked by Flp recombinase target sites was used for selection. Following homologous recombination of the vector in embryonic stem cells, clones bearing the Cd300fil/fl locus were established after deletion of PGK-neo selection cassette by Flp recombination, and clones with the Cd300f-I- locus were generated after deletion of the LoxP sites flanking regions together with the PGK-neo cassette using Cre recombinase. Identified targeted embryonic stem cell clones were microinjected into the blastocysts of C57BL/6 mice. With the Cd300f-I- mice, two tumor models were developed to examine CD300f deficiency on solid tumor development. For tumor graft mouse model, mice were subcutaneously inoculated with 106 solid tumor cells on day 0. Some of the mice were systemically irradiated at 3.25 Gy on day 7 (IR) and some of the irradiated mice were transferred with 106 tumor antigen specific CD8+ T cells on day 9 (OT-I). Irradiation was to deplete some existing immune cells and make space to allow for expansion of anti-tumor effectors. Tumor growth was measured on the indicated days; mice were euthanized when the longitudinal tumor diameter reached 15 mm. For the mouse model of
AOM/DSS-induced colon cancer, mice were injected with AOM intraperitoneally and then fed with DSS-containing water in three 7-day cycles, with intermittent 14-day intervals of regular drinking water. After 10 weeks, mice were euthanized, and colon tumor sizes were measured. The results showed that G£?0O -deficient mice have a significantly inhibited growth of solid tumors (e.g., EL4-TfOVA lymphoma grafts, AOM/DSS-induced colorectal cancer; FIGS. 2A, 2B), indicating that CD300f negatively regulates anti-tumor responses against different cancer types. It was also demonstrated that the release of the CD300f checkpoint inhibition leads to inhibition of grafted adenocarcinoma cells (MC38) in a mouse model. Cd300f+I+ or Cd300f -I- mice were subcutaneously inoculated with 106 MC38 cells on day 0. Tumor growth was monitored on the indicated days; mice were euthanized when the longitudinal tumor diameter reached 15 mm.
Cd300f-deficient mice had significantly inhibited growth of the grafted adenocarcinoma cells, see FIGS. 3A-3B. Furthermore, the data indicated that the improved tumor clearance in Cd300f-
deficient mice is due to enhanced activation of CD8+ T cells (FIG. 2C), indicating that the blockade of CD300f function in DC enhances their cross-presentation of tumor antigens.
In line with this hypothesis, DC from CD300f-deficient mice show increased efferocytosis and enhanced stimulation of CD8+ T cells, and the enhanced anti-tumor response correlates with a significant CD8+ T cell infiltration of the tumor tissues (FIG. 2D). These results demonstrate that releasing a checkpoint on DC has remarkable efficacy, as CTL kill different tumors even when their own checkpoint receptors (e.g., PD-1, CTLA-4, LAG-3) are not blocked.
A database search (Human Protein Atlas, Expression Atlas) indicates that CD300f expression is found on immune cells and immune-associated tissues, but not on non-immune tissues (e.g., heart, muscle, liver, kidneys). It was demonstrated that human CD300f is present on human myeloid cell populations (e.g., monocytes and DCs, see FIGS. 5A-5B). Furthermore, it was found that in human blood CD300f is not expressed on lymphoid cells (T, B, NK or NKT cells), but is highly expressed on myeloid cells (monocytes, neutrophils, DC). Thus, targeting CD300f to release a checkpoint on human is a unique and potent therapeutic method for cancer management, as DC function upstream of T cells.
Releasing a DC checkpoint can be used in patients where blocking T cell checkpoints is ineffective. Moreover, a combined therapy, blocking checkpoints on both DC and T cells may elicit strong anti-tumor responses. Example 2
Commercially available anti-mouse CD300f antibodies (e.g., from R&D systems, Novus, BioLegend, Invitrogen) can block PS recognition and enhance apoptotic cell phagocytosis by mouse DC in vitro. Once a blocking antibody is selected, mouse models and assays are used to demonstrate that antibody-mediated blocking of CD300f functions to inhibit tumor progression (e.g., lymphoma grafts, colorectal cancer).
A humanized monoclonal antibody is produced that specifically recognizes human CD300f, with no cross-reactivity with other CD300 family members. Such an antibody has blocking potential, i.e., interfere with CD300f ability to recognize its ligand, phosphatidylserine, and thus neutralize CD300f signaling potential (function). Selected clones of the antibodies are tested using standard ELISA, flow cytometry and apoptotic cell phagocytosis assays, in order to determine the best clone capable of blocking PS recognition by CD300f and inhibiting CD300f function. Once selected, anti-CD300f blocking antibody (or antibodies) are used in vitro.
Monocytes were isolated from patient blood samples (using a negative selection kit), and then differentiated into DC in vitro, using standard methods (i.e., culture with GM-CSF and IL-4; Dauer M. et al., J Immunol 2003). CD300f -blocked DC were incubated with apoptotic tumor cells (generated from patient's tumor samples), allowing them to engulf and process apoptotic tumor cells. Human CD300f, like mouse CD300f, recognized phosphatidylserine and regulated the phagocytosis of apoptosis (see FIGS. 4A-4B).
Next, these DC are incubated with autologous CTL (isolated from patient's blood samples using a negative selection kit), in order to cross-present apoptotic tumor cell-derived antigens and prime CTL. Enhanced CTL activation over control treated CTL is verified by monitoring cytokine production (e.g., TNF-oc, IFN-γ). The activated CTL will be tested for their ability to kill patient tumor cells that were the source of the DC-presented antigens, by standard cytotoxicity assays (e.g., chromium release assay). With the selected antibodies from the in vitro analysis, it is tested whether they can cross-react with mouse CD300f and block its function. If so, established mouse models are used to test its therapeutic effect, if not, humanized mice (e.g., NSG mice) can be used as the testing vehicle. In addition, a combinatorial therapeutic approach, combining the blockade of checkpoints on DC (CD300f) and CTL, (PD-1, CTLA-4, and/or LAG-3, can be used.
The engineered humanized antibodies are used in patients to activate DC and stimulate antigen cross-presentation, and thus mediate a potent anti-tumor response. The biological readout of physiological efficacy and measureable biomarkers for the therapy is the monitoring of IFN-γ (and/or other cytokines, e.g., IL-2) production by the activated CTL, and the presence of activated CTL themselves in the patients, as already tested in the mouse models.
An advantage of this approach over the existing methods is the independence from the requirement for a definitive tumor antigen, as DC are fed intact patient tumor cells, and can cross- present a multitude of tumor antigens. Thus, the method can be used to treat a broad range of cancer types, and be quickly adapted to treat a particular patient's cancer.
Example 3
The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats )/Cas9 Type II system is used to facilitate a site-specific genome editing to disrupt CD300F (CD300LF) gene. The target sequences in the genomic DNA are designed using E-CRISP Designer (v. 4.2; e-crisp.org/E- CRISP/designcrispr_html), or CHOPCHOP web tool for genome editing
(http://chopchop.cbu.uib.no/). Initially, 5 different sequences targeting CD300LF gene are generated. Their nucleotide sequences are aligned against those present in the human genomic and transcript database, to verify the specificity of CD300LF targeting. The oligomers are synthesized,
annealed and cloned into lentiCRISPRv2 (AddGene.org), a one vector system co-expressing a mammalian codon-optimized Cas9 nuclease along with a single guide RNA, according to the protocol found at the Zhang Lab GeCKO website (genome-engineering_org/gecko/). The lentiviral expression constructs, verified by DNA sequencing, are transfected into 293T cells with the psPAX2 and pMD2.G helper plasmids (AddGene.org) using PolyJet transfection reagent
(Signagen). The 293T cell culture medium containing lentivirus particles are used to infect 2xl06 human monocytes, monocyte-derived cells (e.g., DCs, macrophages), in the presence of 10 μg/ml protamine sulphate (Sigma). CD300f expression on the cell surface is monitored routinely at protein levels by flow cytometry, to verify the lack of CD300f expression by the transduced cells. All CRISPR constructs are evaluated for their ability to disrupt CD300LF and generate human CD300f-deficient cells. The constructs targeting the
5 ' -GAAAACTGGAAATGACCTTGGGG-3 ' (SEQ ID NO: 20), and/or 5'-
GTGGTGGCCGGTCAGAGTTGGGG-3 ' (SEQ ID NO: 21) sequence of CD300LF (exon 3 and 4, respectively) were determined to be the most optimal to mediate gene disruption, and are chosen for generation of CD300f-deficeint human cells.
Example 4
Monocytes are isolated from patient' s blood samples using a negative selection kit (Miltenyi Monocyte Isolation Kit II), and then differentiated into DC in vitro, using standard methods (for example, culture with GM-CSF and IL-4, see Dauer M. et al., J Immunol 2003). Those monocyte- derived DC are treated with the anti-CD300f antibody to block CD300f, thereby enhancing DC activity and promoting antigen cross-presentation. In parallel, tumor cells isolated from cancer patients (e.g., biopsy samples) are exposed to UV irradiation to induce apoptosis and generate apoptotic tumor cells. CD300f -blocked DC is incubated with the apoptotic tumor cells (generated from a patient's tumor sample), allowing them to engulf and process the apoptotic tumor cells. Those DC are then matured with 10 ng/ml LPS plus 100 IU/ml IFN-γ and incubated with autologous cytotoxic T lymphocytes (CTLs) (isolated from patient's blood samples using a negative selection kit, such as the Miltenyi CD8+ T Cell Isolation Kit) using the protocol described in Nature Protocols 9, 950-966 (2014), incorporated herein by reference, in order to cross-present apoptotic tumor cell-derived antigens and prime CTL. The activation of CTL by DC in co-cultures is verified by monitoring the ability of CTL to produce different cytokines (e.g., TNF-a, IFN-γ), using ELISA and flow cytometry. The activated CTL are subsequently tested for their ability to kill patient tumor cells that were the source of the DC-presented antigens, by standard cytotoxicity assays (e.g.,
DELFIA or chromium release assay). In some cases, combining the blockade of checkpoints on DC and CTL, using anti-CD300f in combination with anti-PD-1, anti-CTLA-4, and/or anti-LAG-3 antibodies is also tested.
Additionally, knock-out of CD300f in human monocyte-derived DC cells using CRISPR is performed. The CD300f deficiency on DCs results in enhanced engulfment of apoptotic tumor cells and cross-presentation of tumor-specific antigens, leading to elevated priming and cytotoxicity of CTL against a variety of tumor cell lines. The CD300f-deficient DCs are used to prime and expand the tumor antigen-specific CTL in vitro or are administered directly alone or with CTL to the patient to depress tumor growth.
In view of the many possible embodiments to which the principles the disclosure may be applied, it should be recognized that illustrated embodiments are only examples of the disclosure and should not be considered a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.
Claims
1. A method of treating a subject with a solid tumor, comprising
administering to the subject a therapeutically effective amount of a CD300f inhibitor, thereby treating the solid tumor in the subject.
2. The method of claim 1, wherein the CD300f inhibitor is a monoclonal antibody that specifically binds CD300f, an inhibitory nucleic acid molecule, or a small molecule inhibitor.
3. The method of claim 2, wherein the CD300f inhibitor is the monoclonal antibody, and wherein the monoclonal antibody is a human monoclonal antibody or a humanized monoclonal antibody.
4. The method of claim 1, wherein the CD300f inhibitor is the inhibitory nucleic acid, and wherein the inhibitory nucleic acid is a siRNA or a shRNA.
5. The method of claim 4, wherein the CD300f inhibitor is the inhibitory nucleic acid, and wherein the inhibitory nucleic acid is a morpholino oligonucleotide.
6. A method of treating a solid tumor in a subject, comprising administering to the subject a therapeutically effective amount of dendritic cells comprising an inactivated gene encoding CD300f, thereby treating the solid tumor in the subject.
7. The method of claim 6, wherein the dendritic cells are transformed with one or more expression vectors comprising: a) a Embryonal Fyn-Associated Substrate (EFS) promoter operably linked to a nucleotide sequence encoding a Type II Cas9 nuclease, b) a U6 promoter operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas system guide RNAs that hybridize with the CD300f gene in the dendritic cells, wherein components (a) and (b) are located on same or different expression vectors, whereby the one or more guide RNAs target the CD300f gene in the dendritic cells and the Cas9 protein cleaves the CD300f gene such that the sequence of the CD300f gene is inactivated in the dendritic cells.
8. The method of claim 6 or claim 7, wherein the dendritic cells are autologous.
9. A method of treating a solid tumor in a subject, comprising
isolating dendritic cells from the subject;
transforming the dendritic cells with one or more viral vectors comprising: a) a Embryonal Fyn- Associated Substrate (EFS) promoter operably linked to a nucleotide sequence encoding a Type II Cas9 nuclease, b) a U6 promoter operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas system guide RNAs that hybridize with the CD300f gene in the dendritic cell, wherein components (a) and (b) are located on same or different adeno-associated viral vectors, whereby the one or more guide RNAs target the CD300f gene in the dendritic cell and the Cas9 protein cleaves the CD300f gene such that the sequence of the CD300f gene is modified or inactivated in the dendritic cell, thereby forming modified dendritic cells; and
administering to the subject a therapeutically effective amount of the modified dendritic cells, thereby treating the solid tumor in the subject.
10. A method of treating a solid tumor in a subject, comprising
isolating dendritic cells from the subject;
transforming the dendritic cells with one or more viral vectors comprising: a) a Embryonal Fyn- Associated Substrate (EFS) promoter operably linked to a nucleotide sequence encoding a Type Π Cas9 nuclease, b) a U6 promoter operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas system guide RNAs that hybridize with the CD300f gene in the dendritic cell, wherein components (a) and (b) are located on same or different viral vectors, whereby the one or more guide RNAs target the CD300f gene in the dendritic cell and the Cas9 protein cleaves the CD300f gene such that the sequence of the CD300f gene is modified or inactivated in the dendritic cell, thereby forming modified dendritic cells; and
contacting the modified dendritic cells with T cells in the presence of a tumor antigen expressed by the solid tumor to form activated T cells;
administering to the subject a therapeutically effective amount of the activated T cells, thereby treating the solid tumor in the subject.
11. The method of any of claims 7 to 10, wherein the one or more CRISPR-Cas system guide RNAs comprise:
GAAAACTGGAAATGACCTTG (SEQ ID NO: 11) or
GTGGTGGCCGGTCAGAGTTG (SEQ ID NO: 12).
12. The method any of claims 7 to 11, wherein the one or more expression vector is a lentiviral vector.
13. A method of treating a solid tumor in a subject, comprising
contacting the dendritic cells from the subject with T cells from the subject in the presence of a tumor antigen expressed by the solid tumor and a CD300f inhibitor to form activated T cells; administering to the subject a therapeutically effective amount of the activated T cells, thereby treating the solid tumor in the subject.
14. The method of any one of claims 10 to 13, wherein the dendritic cells and the T cells are autologous.
15. The method of any one of claims 1 to 14, wherein the solid tumor is a carcinoma or a sarcoma.
16. The method of any one of claims 1 to 15, wherein the solid tumor is a colorectal cancer or a skin cancer.
17. The method of any one of claims 1 to 16, further comprising administering an additional anti-tumor agent to the subject.
18. The method of claim 17, wherein the additional anti-tumor agent is a PD- 1 antagonist or a CTLA-4 antagonist.
19. The method of claim 18, wherein the PD-1 antagonist is an anti-PD- 1 antibody or an anti-PD-Ll antibody.
20. The method of claim 18, wherein the CTLA-4 antagonist is an anti-CTLA-4 antibody.
21. The method of claim 17, wherein the additional anti-tumor agent is a chemotherapeutic agent, biologic agent, or radiation.
22. The method of any one of claims 1 to 21, wherein treating the tumor comprises decreasing tumor volume, decreasing the number of size of metastases of the tumor, or lessening a symptom of the tumor.
23. The method of any one of claims 1 to 22, wherein treating the tumor comprises inducing an immune response to the tumor.
24. The method of any one of claims 1-23, wherein the subject is human.
25. The method of any one of claims 10 to 24, wherein contacting the dendritic cells from the subject with T cells from the subject in the presence of the tumor antigen comprises:
contacting dendritic cells from the subject that have engulfed apoptotic tumor cells from the solid tumor with the T cells from the subject.
26. A composition comprising therapeutically effective amount of a CD300f inhibitor for use in treating a solid tumor in the subject.
27. A composition comprising a therapeutically effective amount of dendritic cells comprising a modified or inactivated CD300f gene, for use in treating a solid tumor in a subject.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201662408596P | 2016-10-14 | 2016-10-14 | |
US62/408,596 | 2016-10-14 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2018071576A1 true WO2018071576A1 (en) | 2018-04-19 |
Family
ID=60186394
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2017/056192 WO2018071576A1 (en) | 2016-10-14 | 2017-10-11 | Treatment of tumors by inhibition of cd300f |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2018071576A1 (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021016668A1 (en) * | 2019-07-30 | 2021-02-04 | Dendrocyte Biotech Pty Ltd | Immunoconjugate |
WO2021108868A1 (en) * | 2019-12-05 | 2021-06-10 | Dendrocyte Biotech Pty Ltd | Antigen loading |
JP2021525283A (en) * | 2018-05-31 | 2021-09-24 | セントリックスバイオ インコーポレイテッドCentricsBio, Inc. | A pharmaceutical composition for preventing or treating cancer, which comprises an expression inhibitor or activity inhibitor of CD300c. |
JP2021534791A (en) * | 2018-08-28 | 2021-12-16 | 法▲羅▼斯疫苗株式会社 | Improved wrench viral vector |
JP2021536240A (en) * | 2018-08-28 | 2021-12-27 | 北京永泰瑞科生物科技有限公司 | Improved therapeutic T cells |
WO2022170098A3 (en) * | 2021-02-05 | 2022-09-22 | Sigilon Therapeutics, Inc. | Compositions, devices and methods for treating mps vi disease |
Citations (51)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4036945A (en) | 1976-05-03 | 1977-07-19 | The Massachusetts General Hospital | Composition and method for determining the size and location of myocardial infarcts |
US4235871A (en) | 1978-02-24 | 1980-11-25 | Papahadjopoulos Demetrios P | Method of encapsulating biologically active materials in lipid vesicles |
US4331647A (en) | 1980-03-03 | 1982-05-25 | Goldenberg Milton David | Tumor localization and therapy with labeled antibody fragments specific to tumor-associated markers |
US4458066A (en) | 1980-02-29 | 1984-07-03 | University Patents, Inc. | Process for preparing polynucleotides |
US4501728A (en) | 1983-01-06 | 1985-02-26 | Technology Unlimited, Inc. | Masking of liposomes from RES recognition |
US4722848A (en) | 1982-12-08 | 1988-02-02 | Health Research, Incorporated | Method for immunizing animals with synthetically modified vaccinia virus |
US4837028A (en) | 1986-12-24 | 1989-06-06 | Liposome Technology, Inc. | Liposomes with enhanced circulation time |
US4902505A (en) | 1986-07-30 | 1990-02-20 | Alkermes | Chimeric peptides for neuropeptide delivery through the blood-brain barrier |
WO1990002809A1 (en) | 1988-09-02 | 1990-03-22 | Protein Engineering Corporation | Generation and selection of recombinant varied binding proteins |
US4946778A (en) | 1987-09-21 | 1990-08-07 | Genex Corporation | Single polypeptide chain binding molecules |
US4957735A (en) | 1984-06-12 | 1990-09-18 | The University Of Tennessee Research Corporation | Target-sensitive immunoliposomes- preparation and characterization |
US5004697A (en) | 1987-08-17 | 1991-04-02 | Univ. Of Ca | Cationized antibodies for delivery through the blood-brain barrier |
US5019369A (en) | 1984-10-22 | 1991-05-28 | Vestar, Inc. | Method of targeting tumors in humans |
US5055303A (en) | 1989-01-31 | 1991-10-08 | Kv Pharmaceutical Company | Solid controlled release bioadherent emulsions |
WO1991017271A1 (en) | 1990-05-01 | 1991-11-14 | Affymax Technologies N.V. | Recombinant library screening methods |
WO1992001047A1 (en) | 1990-07-10 | 1992-01-23 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
WO1992009690A2 (en) | 1990-12-03 | 1992-06-11 | Genentech, Inc. | Enrichment method for variant proteins with altered binding properties |
WO1992015679A1 (en) | 1991-03-01 | 1992-09-17 | Protein Engineering Corporation | Improved epitode displaying phage |
WO1992018619A1 (en) | 1991-04-10 | 1992-10-29 | The Scripps Research Institute | Heterodimeric receptor libraries using phagemids |
WO1992020791A1 (en) | 1990-07-10 | 1992-11-26 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
WO1993001288A1 (en) | 1991-07-08 | 1993-01-21 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Phagemide for screening antibodies |
US5188837A (en) | 1989-11-13 | 1993-02-23 | Nova Pharmaceutical Corporation | Lipsopheres for controlled delivery of substances |
US5223409A (en) | 1988-09-02 | 1993-06-29 | Protein Engineering Corp. | Directed evolution of novel binding proteins |
US5254342A (en) | 1991-09-30 | 1993-10-19 | University Of Southern California | Compositions and methods for enhanced transepithelial and transendothelial transport or active agents |
US5268164A (en) | 1990-04-23 | 1993-12-07 | Alkermes, Inc. | Increasing blood-brain barrier permeability with permeabilizer peptides |
US5271961A (en) | 1989-11-06 | 1993-12-21 | Alkermes Controlled Therapeutics, Inc. | Method for producing protein microspheres |
US5413797A (en) | 1992-03-12 | 1995-05-09 | Alkermes Controlled Therapeutics, Inc. | Controlled release ACTH containing microspheres |
US5514670A (en) | 1993-08-13 | 1996-05-07 | Pharmos Corporation | Submicron emulsions for delivery of peptides |
US5534496A (en) | 1992-07-07 | 1996-07-09 | University Of Southern California | Methods and compositions to enhance epithelial drug transport |
US5585089A (en) | 1988-12-28 | 1996-12-17 | Protein Design Labs, Inc. | Humanized immunoglobulins |
US5593972A (en) | 1993-01-26 | 1997-01-14 | The Wistar Institute | Genetic immunization |
US5643578A (en) | 1992-03-23 | 1997-07-01 | University Of Massachusetts Medical Center | Immunization by inoculation of DNA transcription unit |
US5880103A (en) | 1992-08-11 | 1999-03-09 | President And Fellows Of Harvard College | Immunomodulatory peptides |
US5994126A (en) | 1992-04-01 | 1999-11-30 | The Rockefeller University | Method for in vitro proliferation of dendritic cell precursors and their use to produce immunogens |
WO2000047719A2 (en) | 1999-02-11 | 2000-08-17 | 3M Innovative Properties Company | Maturation of dendritic cells with immune response modifying compounds |
WO2006121168A1 (en) | 2005-05-09 | 2006-11-16 | Ono Pharmaceutical Co., Ltd. | Human monoclonal antibodies to programmed death 1(pd-1) and methods for treating cancer using anti-pd-1 antibodies alone or in combination with other immunotherapeutics |
WO2007005874A2 (en) | 2005-07-01 | 2007-01-11 | Medarex, Inc. | Human monoclonal antibodies to programmed death ligand 1 (pd-l1) |
WO2009101611A1 (en) | 2008-02-11 | 2009-08-20 | Curetech Ltd. | Monoclonal antibodies for tumor treatment |
WO2009114335A2 (en) | 2008-03-12 | 2009-09-17 | Merck & Co., Inc. | Pd-1 binding proteins |
US20100028330A1 (en) | 2002-12-23 | 2010-02-04 | Medimmune Limited | Methods of upmodulating adaptive immune response using anti-pd1 antibodies |
WO2010027827A2 (en) | 2008-08-25 | 2010-03-11 | Amplimmune, Inc. | Targeted costimulatory polypeptides and methods of use to treat cancer |
WO2010077634A1 (en) | 2008-12-09 | 2010-07-08 | Genentech, Inc. | Anti-pd-l1 antibodies and their use to enhance t-cell function |
WO2011066342A2 (en) | 2009-11-24 | 2011-06-03 | Amplimmune, Inc. | Simultaneous inhibition of pd-l1/pd-l2 |
US20120039906A1 (en) | 2009-02-09 | 2012-02-16 | INSER (Institut National de la Recherche Medicale) | PD-1 Antibodies and PD-L1 Antibodies and Uses Thereof |
US20120114649A1 (en) | 2008-08-25 | 2012-05-10 | Amplimmune, Inc. Delaware | Compositions of pd-1 antagonists and methods of use |
US8354509B2 (en) | 2007-06-18 | 2013-01-15 | Msd Oss B.V. | Antibodies to human programmed death receptor PD-1 |
US8728806B2 (en) | 2008-12-06 | 2014-05-20 | The Board Of Regents, The University Of Texas System | Methods and compositions related to Th-1 dendritic cells |
US20150191744A1 (en) | 2013-12-17 | 2015-07-09 | University Of Massachusetts | Cas9 effector-mediated regulation of transcription, differentiation and gene editing/labeling |
US20150335679A1 (en) | 2014-05-20 | 2015-11-26 | Kiromic, Llc | Methods and Compositions for Treating Malignancies with Dendritic Cells |
US9405700B2 (en) | 2010-11-04 | 2016-08-02 | Sonics, Inc. | Methods and apparatus for virtualization in an integrated circuit |
WO2016145578A1 (en) * | 2015-03-13 | 2016-09-22 | Syz Cell Therapy Co. | Methods of cancer treatment using activated t cells |
-
2017
- 2017-10-11 WO PCT/US2017/056192 patent/WO2018071576A1/en active Application Filing
Patent Citations (56)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4036945A (en) | 1976-05-03 | 1977-07-19 | The Massachusetts General Hospital | Composition and method for determining the size and location of myocardial infarcts |
US4235871A (en) | 1978-02-24 | 1980-11-25 | Papahadjopoulos Demetrios P | Method of encapsulating biologically active materials in lipid vesicles |
US4458066A (en) | 1980-02-29 | 1984-07-03 | University Patents, Inc. | Process for preparing polynucleotides |
US4331647A (en) | 1980-03-03 | 1982-05-25 | Goldenberg Milton David | Tumor localization and therapy with labeled antibody fragments specific to tumor-associated markers |
US4722848A (en) | 1982-12-08 | 1988-02-02 | Health Research, Incorporated | Method for immunizing animals with synthetically modified vaccinia virus |
US4501728A (en) | 1983-01-06 | 1985-02-26 | Technology Unlimited, Inc. | Masking of liposomes from RES recognition |
US4957735A (en) | 1984-06-12 | 1990-09-18 | The University Of Tennessee Research Corporation | Target-sensitive immunoliposomes- preparation and characterization |
US5019369A (en) | 1984-10-22 | 1991-05-28 | Vestar, Inc. | Method of targeting tumors in humans |
US4902505A (en) | 1986-07-30 | 1990-02-20 | Alkermes | Chimeric peptides for neuropeptide delivery through the blood-brain barrier |
US4837028A (en) | 1986-12-24 | 1989-06-06 | Liposome Technology, Inc. | Liposomes with enhanced circulation time |
US5004697A (en) | 1987-08-17 | 1991-04-02 | Univ. Of Ca | Cationized antibodies for delivery through the blood-brain barrier |
US4946778A (en) | 1987-09-21 | 1990-08-07 | Genex Corporation | Single polypeptide chain binding molecules |
WO1990002809A1 (en) | 1988-09-02 | 1990-03-22 | Protein Engineering Corporation | Generation and selection of recombinant varied binding proteins |
US5223409A (en) | 1988-09-02 | 1993-06-29 | Protein Engineering Corp. | Directed evolution of novel binding proteins |
US5585089A (en) | 1988-12-28 | 1996-12-17 | Protein Design Labs, Inc. | Humanized immunoglobulins |
US5055303A (en) | 1989-01-31 | 1991-10-08 | Kv Pharmaceutical Company | Solid controlled release bioadherent emulsions |
US5271961A (en) | 1989-11-06 | 1993-12-21 | Alkermes Controlled Therapeutics, Inc. | Method for producing protein microspheres |
US5188837A (en) | 1989-11-13 | 1993-02-23 | Nova Pharmaceutical Corporation | Lipsopheres for controlled delivery of substances |
US5506206A (en) | 1990-04-23 | 1996-04-09 | Alkermes, Inc. | Increasing blood-brain barrier permeability with permeabilizer peptides |
US5268164A (en) | 1990-04-23 | 1993-12-07 | Alkermes, Inc. | Increasing blood-brain barrier permeability with permeabilizer peptides |
WO1991017271A1 (en) | 1990-05-01 | 1991-11-14 | Affymax Technologies N.V. | Recombinant library screening methods |
WO1992020791A1 (en) | 1990-07-10 | 1992-11-26 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
WO1992001047A1 (en) | 1990-07-10 | 1992-01-23 | Cambridge Antibody Technology Limited | Methods for producing members of specific binding pairs |
WO1992009690A2 (en) | 1990-12-03 | 1992-06-11 | Genentech, Inc. | Enrichment method for variant proteins with altered binding properties |
WO1992015679A1 (en) | 1991-03-01 | 1992-09-17 | Protein Engineering Corporation | Improved epitode displaying phage |
WO1992018619A1 (en) | 1991-04-10 | 1992-10-29 | The Scripps Research Institute | Heterodimeric receptor libraries using phagemids |
WO1993001288A1 (en) | 1991-07-08 | 1993-01-21 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Phagemide for screening antibodies |
US5254342A (en) | 1991-09-30 | 1993-10-19 | University Of Southern California | Compositions and methods for enhanced transepithelial and transendothelial transport or active agents |
US5413797A (en) | 1992-03-12 | 1995-05-09 | Alkermes Controlled Therapeutics, Inc. | Controlled release ACTH containing microspheres |
US5643578A (en) | 1992-03-23 | 1997-07-01 | University Of Massachusetts Medical Center | Immunization by inoculation of DNA transcription unit |
US5994126A (en) | 1992-04-01 | 1999-11-30 | The Rockefeller University | Method for in vitro proliferation of dendritic cell precursors and their use to produce immunogens |
US5534496A (en) | 1992-07-07 | 1996-07-09 | University Of Southern California | Methods and compositions to enhance epithelial drug transport |
US5880103A (en) | 1992-08-11 | 1999-03-09 | President And Fellows Of Harvard College | Immunomodulatory peptides |
US5817637A (en) | 1993-01-26 | 1998-10-06 | The Trustees Of The University Of Pennsylvania | Genetic immunization |
US5593972A (en) | 1993-01-26 | 1997-01-14 | The Wistar Institute | Genetic immunization |
US5514670A (en) | 1993-08-13 | 1996-05-07 | Pharmos Corporation | Submicron emulsions for delivery of peptides |
WO2000047719A2 (en) | 1999-02-11 | 2000-08-17 | 3M Innovative Properties Company | Maturation of dendritic cells with immune response modifying compounds |
US20100028330A1 (en) | 2002-12-23 | 2010-02-04 | Medimmune Limited | Methods of upmodulating adaptive immune response using anti-pd1 antibodies |
WO2006121168A1 (en) | 2005-05-09 | 2006-11-16 | Ono Pharmaceutical Co., Ltd. | Human monoclonal antibodies to programmed death 1(pd-1) and methods for treating cancer using anti-pd-1 antibodies alone or in combination with other immunotherapeutics |
US8008449B2 (en) | 2005-05-09 | 2011-08-30 | Medarex, Inc. | Human monoclonal antibodies to programmed death 1 (PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics |
US7943743B2 (en) | 2005-07-01 | 2011-05-17 | Medarex, Inc. | Human monoclonal antibodies to programmed death ligand 1 (PD-L1) |
WO2007005874A2 (en) | 2005-07-01 | 2007-01-11 | Medarex, Inc. | Human monoclonal antibodies to programmed death ligand 1 (pd-l1) |
US8354509B2 (en) | 2007-06-18 | 2013-01-15 | Msd Oss B.V. | Antibodies to human programmed death receptor PD-1 |
WO2009101611A1 (en) | 2008-02-11 | 2009-08-20 | Curetech Ltd. | Monoclonal antibodies for tumor treatment |
WO2009114335A2 (en) | 2008-03-12 | 2009-09-17 | Merck & Co., Inc. | Pd-1 binding proteins |
US8609089B2 (en) | 2008-08-25 | 2013-12-17 | Amplimmune, Inc. | Compositions of PD-1 antagonists and methods of use |
US20120114649A1 (en) | 2008-08-25 | 2012-05-10 | Amplimmune, Inc. Delaware | Compositions of pd-1 antagonists and methods of use |
WO2010027827A2 (en) | 2008-08-25 | 2010-03-11 | Amplimmune, Inc. | Targeted costimulatory polypeptides and methods of use to treat cancer |
US8728806B2 (en) | 2008-12-06 | 2014-05-20 | The Board Of Regents, The University Of Texas System | Methods and compositions related to Th-1 dendritic cells |
WO2010077634A1 (en) | 2008-12-09 | 2010-07-08 | Genentech, Inc. | Anti-pd-l1 antibodies and their use to enhance t-cell function |
US20120039906A1 (en) | 2009-02-09 | 2012-02-16 | INSER (Institut National de la Recherche Medicale) | PD-1 Antibodies and PD-L1 Antibodies and Uses Thereof |
WO2011066342A2 (en) | 2009-11-24 | 2011-06-03 | Amplimmune, Inc. | Simultaneous inhibition of pd-l1/pd-l2 |
US9405700B2 (en) | 2010-11-04 | 2016-08-02 | Sonics, Inc. | Methods and apparatus for virtualization in an integrated circuit |
US20150191744A1 (en) | 2013-12-17 | 2015-07-09 | University Of Massachusetts | Cas9 effector-mediated regulation of transcription, differentiation and gene editing/labeling |
US20150335679A1 (en) | 2014-05-20 | 2015-11-26 | Kiromic, Llc | Methods and Compositions for Treating Malignancies with Dendritic Cells |
WO2016145578A1 (en) * | 2015-03-13 | 2016-09-22 | Syz Cell Therapy Co. | Methods of cancer treatment using activated t cells |
Non-Patent Citations (134)
Title |
---|
"Pierce Catalog and Handbook", 1994, PIERCE CHEMICAL CO. |
"Remingtons Pharmaceutical Sciences, 19th Ed.,", 1995, MACK PUBLISHING COMPANY |
ABELOFF: "Clinical Oncology 2nd ed.,", 2000, CHURCHILL LIVINGSTONE, INC, article PERRY ET AL.: "Chemotherapy, Ch. 17" |
AGATA ET AL., INT. IMMUNOL, vol. 8, 1996, pages 765 - 75 |
ALTSCHUL ET AL., J MOL BIOL, vol. 215, 1990, pages 403 - 410 |
ALTSCHUL ET AL., NATURE GENET, vol. 6, 1994, pages 119 - 129 |
AMELIA J TESONE ET AL: "Pathological mobilization and activities of dendritic cells in tumor-bearing hosts: challenges and opportunities for immunotherapy of cancer", FRONTIERS IN IMMUNOLOGY, VOLUME 4, ARTICLE 435, 10 December 2013 (2013-12-10), pages 1 - 11, XP055429519, Retrieved from the Internet <URL:https://www.frontiersin.org/articles/10.3389/fimmu.2013.00435/full> [retrieved on 20171128], DOI: doi: 10.3389/fimmu.2013.00435 * |
BALTZER, L., BERKERY, R.: "Oncology Pocket Guide to Chemotherapy, 2nd ed.", 1995, MOSBY-YEAR BOOK |
BANGA, A.J.: "Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems", 1995, TECHNOMIC PUBLISHING COMPANY, INC. |
BARANY; MERRIFIELD: "The Peptides: Analysis, Synthesis, Biology", SPECIAL METHODS IN PEPTIDE SYNTHESIS, vol. 2, pages 3 - 284 |
BARBAS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 88, 1991, pages 7978 - 7982 |
BEAUCAGE ET AL., TETRA. LETT., vol. 22, 1981, pages 1859 - 1862 |
BEAUCAGE; CARUTHERS, TETRA. LETTS., vol. 22, no. 20, 1981, pages 1859 - 1862 |
BERNSTEIN ET AL., NATURE, vol. 409, 2001, pages 363 - 366 |
BETAGERI ET AL.: "Liposome Drug Delivery Systems", 1993, TECHNOMIC PUBLISHING CO., INC. |
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 |
BITTER ET AL., METHODS IN ENZYMOLOGY, vol. 153, 1987, pages 516 - 544 |
BLANK ET AL., CANCER IMMUNOL. IMMUNOTHER, vol. 54, 2005, pages 307 - 314 |
BROWN ET AL., J. IMMUNOL., vol. 127, 1981, pages 539 - 546 |
BROWN ET AL., METH. ENZYMOL., vol. 68, 1979, pages 109 - 151 |
BUCHNER ET AL., ANAL. BIOCHEM., vol. 205, 1992, pages 263 - 270 |
BUCHSCHER, J. VIROL., vol. 66, 1992, pages 2731 - 2739 |
CAN; CHURCH, NATURE BIOTECHNOLOGY, vol. 27, no. 12, 2009, pages 1151 - 62 |
CARTER ET AL., EUR J IMMUNOL, vol. 32, 2002, pages 634 - 43 |
CARTER ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 89, 1992, pages 4285 |
CECH, J. AMER. MED. ASSN., vol. 260, 1988, pages 3030 |
CHABNER; LONGO: "Cancer Chemotherapy and Biotherapy: Principles and Practice (4th ed.).", 2005, LIPPINCOTT WILLIANS & WILKINS |
COLE ET AL.: "Monoclonal Antibodies and Cancer Therapy", 1985, ALAN R. LISS, INC., pages: 77 96 |
CORPET ET AL., NUCLEIC ACIDS RESEARCH, vol. 16, 1988, pages 10881 - 10890 |
DATABASE GENBANK [O] Database accession no. AAH28199.1 |
DATABASE GenBank [O] Database accession no. NM 001289083.1 |
DATABASE GenBank [O] Database accession no. NM_001289082.1 |
DATABASE GenBank [O] Database accession no. NM_001289084.1 |
DATABASE GenBank [O] Database accession no. NM_001289085.1 |
DATABASE GenBank [O] Database accession no. NM_001289086.1 |
DATABASE GenBank [O] Database accession no. NM_001289087.1 |
DATABASE GenBank [O] Database accession no. NM_139018.4 |
DATABASE GenBank [O] Database accession no. NP 001276011.1 |
DATABASE GenBank [O] Database accession no. NP 001276012.1 |
DATABASE GenBank [O] Database accession no. NP 001276013.1 |
DATABASE GenBank [O] Database accession no. NP 001276014.1 |
DATABASE GenBank [O] Database accession no. NP 001276015.1 |
DATABASE GenBank [O] Database accession no. NP_001276016.1 |
DATABASE GenBank [O] Database accession no. NP_620587.2 |
DAUER M. ET AL., J IMMUNOL, 2003 |
DEUTSCHER,: "Meth. Enzymol.", vol. 185, 1990, ACADEMIC PRESS, article "Guide to Protein Purification" |
DONG ET AL., J MOL MED, vol. 81, 2003, pages 281 - 7 |
E. W. MARTIN: "Remington's Pharmaceutical Sciences", . |
E. W. MARTIN: "Remington's Pharmaceutical Sciences, 15th Edition", 1975, MACK PUBLISHING CO. |
EDELMAN ET AL.: "Methods in Enzymology", vol. 1, 1967, ACADEMIC PRESS, pages: 422 |
FISCHER, D.S., KNOBF, M.F., DURIVAGE, H.J.: "The Cancer Chemotherapy Handbook, 4th ed.", 1993, MOSBY-YEAR BOOK |
FONFARA ET AL., NUCLEIC ACIDS RES., vol. 42, no. 4, February 2014 (2014-02-01), pages 2577 - 90 |
FRANCISCO BORREGO: "The CD300 molecules: an emerging family of regulators of the immune system", BLOOD, vol. 121, no. 11, 13 January 2013 (2013-01-13), pages 1951 - 1960, XP055429724, DOI: 10.1182/blood-2012- 09-435057 * |
FREEMAN ET AL., J EXP MED, vol. 192, 2000, pages 1027 - 34 |
GEFTER, M. L. ET AL.: "Somatic Cell Genet.", vol. 3, 1977, pages: 231 36 |
GLUZMAN: "Eukaryotic Viral Vectors", 1982, COLD SPRING HARBOR LABORATORY |
GRUBER ET AL., CELL, vol. 106, no. 1, 2008, pages 23 - 24 |
HARLOW; LANE: "Antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY |
HASSELHOFF, NATURE, vol. 334, 1988, pages 585 |
HELENE, C., ANTICANCER DRUG DESIGN, vol. 6, no. 6, pages 569 |
HIGGINS; SHARP, CABIOS, vol. 5, 1989, pages 151 - 153 |
HIGGINS; SHARP, GENE, vol. 73, 1988, pages 237 - 244 |
HOOGENBOOM ET AL., NUCLEIC ACIDS RES., vol. 19, 1991, pages 4133 4137 |
HUSE ET AL., SCIENCE, vol. 246, 1989, pages 1275 |
IJNTEMA ET AL., INT. J. PHARM., vol. 112, 1994, pages 215 - 224 |
JAE-KWAN KIM ET AL: "Synthetic Peptides Containing ITIM-Like Domains Block Expression of Inflammatory Mediators and Migration/Invasion of Cancer Cells Through Activation of SHP-1 and PI3K", CANCER INVESTIGATION., vol. 30, no. 5, 23 May 2012 (2012-05-23), US, pages 364 - 371, XP055428924, ISSN: 0735-7907, DOI: 10.3109/07357907.2012.664671 * |
JINEK ET AL., SCIENCE, vol. 337, 2012, pages 816 - 821 |
JINEK ET AL., SCIENCE, vol. 343, 14 March 2014 (2014-03-14), pages 6176 |
JINEK M ET AL., SCIENCE, vol. 337, no. 6096, 17 August 2012 (2012-08-17), pages 816 - 21 |
JINEK, SCIENCE, vol. 337, 2012, pages 816 - 821 |
JOHANTI ET AL., J. VIROL., vol. 66, 1992, pages 1635 - 1640 |
JOHNSTON ET AL., PHARM. RES., vol. 9, 1992, pages 425 - 434 |
JONES ET AL., NATURE, vol. 321, 1986, pages 522 |
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES |
KENNETH, R. H.: "Monoclonal Antibodies: A New Dimension In Biological Analyses", 1980, PLENUM PUBLISHING CORP. |
KOHLER; MILSTEIN, NATURE, vol. 256, 1995, pages 495 49 |
KONISHI ET AL., CLIN CANCER RES, vol. 10, 2004, pages 5094 |
KOZBOR ET AL., IMMUNOL., vol. 4, 1983, pages 72 |
KREUTER, J.: "Colloidal Drug Delivery Systems", 1994, MARCEL DEKKER, INC., pages: 219 - 342 |
KUBY, J.: "Immunology, 3rd ed.,", 1997, W.H. FREEMAN & CO. |
L TIAN ET AL: "Enhanced efferocytosis by dendritic cells underlies memory T-cell expansion and susceptibility to autoimmune disease in CD300f-deficient mice", CELL DEATH AND DIFFERENTIATION., vol. 23, no. 6, 15 January 2016 (2016-01-15), GB, pages 1086 - 1096, XP055428969, ISSN: 1350-9047, DOI: 10.1038/cdd.2015.161 * |
L. SHI ET AL: "DIgR2, dendritic cell-derived immunoglobulin receptor 2, is one representative of a family of IgSF inhibitory receptors and mediates negative regulation of dendritic cell-initiated antigen-specific T-cell responses", BLOOD, vol. 108, no. 8, 15 October 2006 (2006-10-15), US, pages 2678 - 2686, XP055429082, ISSN: 0006-4971, DOI: 10.1182/blood-2006-04-015404 * |
LANGER, ACCOUNTS CHEM. RES., vol. 26, 1993, pages 537 - 542 |
LATCHMAN ET AL., NAT IMMUNOL, vol. 2, 2001, pages 261 - 8 |
LERNER, E. A., YALE J. BIOL. MED., vol. 54, 1981, pages 387 - 402 |
MA ET AL., BIOMED RESEARCH INTERNATIONAL, vol. 2013, 2013 |
MAHER ET AL., ANTISENSE RES. AND DEV., vol. 1, no. 3, 1991, pages 227 |
MALI, NAT METHODS, vol. 10, no. 10, October 2013 (2013-10-01), pages 1028 - 1034 |
MERRIFIELD ET AL., J. AM. CHEM. SOC., vol. 85, 1963, pages 2149 - 2156 |
MILLER ET AL., J. VIROL., vol. 65, 1991, pages 2220 - 2224 |
NAITO ET AL., BIOINFORMATICS, 20 November 2014 (2014-11-20) |
NARANG ET AL., METH. ENZYMOL., vol. 68, 1979, pages 90 - 99 |
NATURE PROTOCOLS, vol. 9, 2014, pages 950 - 966 |
NEEDHAM-VANDEVANTER ET AL., NUCL. ACIDS RES., vol. 12, 1984, pages 6159 - 6168 |
NEEDLEMAN; WUNSCH, J MOL BIOL, vol. 48, 1970, pages 443 - 453 |
NEVILLE E SANJANA ET AL: "Improved vectors and genome-wide libraries for CRISPR screening", NATURE METHODS, vol. 11, no. 8, 30 July 2014 (2014-07-30), pages 783 - 784, XP055294718, ISSN: 1548-7091, DOI: 10.1038/nmeth.3047 * |
NISHIMASU ET AL., CELL, vol. 156, no. 5, 27 February 2014 (2014-02-27), pages 935 - 49 |
NISONHOFF ET AL., BIOCHEM. BIOPHYS., vol. 89, 1960, pages 230 |
PACK ET AL., BIO/TECHNOLOGY, vol. 11, 1993, pages 1271 |
PEARSON; LIPMAN, PROC NATL ACAD SCI, vol. 85, 1988, pages 2444 - 2448 |
PEC ET AL., J. PARENT. SCI. TECH., vol. 44, no. 2, 1990, pages 58 - 65 |
PLUCKTHUN, BIOTECHNOLOGY, vol. 9, 1991, pages 545 |
PORTER, BIOCHEM. J., vol. 73, 1959, pages 119 |
QIN ET AL., ACTA PHARMACOL. SIN., vol. 23, no. 6, 2002, pages 534 - 8 |
R. SCOPES: "Protein Purification", 1982, SPRINGER-VERLAG |
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 |
SALLUSTO ET AL., J. EXP. MED., vol. 179, 1994, pages 1109 - 18 |
SAMBROOK ET AL.,: "Molecular Cloning: A Laboratory Manual, 2nd ed.,", vol. 1-3, 1989, COLD SPRING HARBOR LABORATORY PRESS |
SANDHU, CRIT. REV. BIOTECH., vol. 12, 1992, pages 437 |
SAXENA ET AL., BIOCHEMISTRY, vol. 9, 1970, pages 5015 - 5021 |
SCOPES: "Protein Purification: Principles and Practice", 1982, SPRINGER VERLAG |
SINGER ET AL., J. IMMUNOL., vol. 150, 1993, pages 2844 |
SKEEL: "Handbook of Cancer Chemotherapy (6th ed.).", 2003, LIPPINCOTT WILLIAMS & WILKINS |
SLAPAK; KUFE: "Harrison's Principles of Internal Medicine, 14th edition", article "chapter 86: Principles of Cancer Therapy" |
SMITH; WATERMAN, ADV. APPL. MATH., vol. 2, 1981, pages 482 |
SOMMNERFEIT ET AL., VIROL., vol. 176, 1990, pages 58 - 59 |
STEWART ET AL.: "Solid Phase Peptide Synthesis, 2nd ed.,", 1984, PIERCE CHEM. CO. |
STOVER, NATURE, vol. 351, 1991, pages 456 - 460 |
TIAN ET AL., CELL DEATH DIFFER, vol. 23, 2016, pages 1086 - 96 |
TIAN ET AL., NAT COMMUN, vol. 5, 2014, pages 3146 |
TICE; TABIBI: "Treatise on Controlled Drug Delivery", 1992, MARCEL DEKKER, INC., pages: 315 - 339 |
VERHOEYEN ET AL., SCIENCE, vol. 239, 1988, pages 1534 |
VOLGMANN ET AL., J. IMMUNOL. METHODS, vol. 119, 1989, pages 45 - 51 |
WALKER ET AL., NATURE, vol. 328, 1987, pages 345 - 8 |
WANG, Y. J.; HANSON, M. A., JOURNAL OF PARENTERAL SCIENCE AND TECHNOLOGY, vol. 42, 1988, pages 2S |
WARD ET AL., NATURE, vol. 341, 1989, pages 544 |
WEINTRAUB, SCIENTIFIC AMERICAN, vol. 262, 1990, pages 40 |
WHITLOW ET AL., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, vol. 2, 1991, pages 97 |
WILSON ET AL., J. VIROL., vol. 63, 1998, pages 2374 - 2378 |
WU ET AL., J. BIOL. CHEM., vol. 262, 1987, pages 4429 |
YEH ET AL., PROC. NATL. ACAD. SCI., vol. 76, 1976, pages 2927 31 |
ZAMORE, SCIENCE, vol. 296, 2002, pages 1265 - 1269 |
ZHEN LU ET AL: "DlgR2 knockdown boosts dendritic cell activity and inhibits hepatocellular carcinoma tumor <I>in-situ</I> growth", ONCOTARGET, vol. 8, no. 33, 1 January 2017 (2017-01-01), pages 54993 - 55002, XP055429068, DOI: 10.18632/oncotarget.18990 * |
ZUKER; STIEGLER, NUCLEIC ACIDS RES., vol. 9, 1981, pages 133 - 148 |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2021525283A (en) * | 2018-05-31 | 2021-09-24 | セントリックスバイオ インコーポレイテッドCentricsBio, Inc. | A pharmaceutical composition for preventing or treating cancer, which comprises an expression inhibitor or activity inhibitor of CD300c. |
JP7301264B2 (en) | 2018-05-31 | 2023-07-03 | セントリックスバイオ インコーポレイテッド | Pharmaceutical composition for prevention or treatment of cancer containing a CD300c expression inhibitor or activity inhibitor |
JP2021534791A (en) * | 2018-08-28 | 2021-12-16 | 法▲羅▼斯疫苗株式会社 | Improved wrench viral vector |
JP2021536240A (en) * | 2018-08-28 | 2021-12-27 | 北京永泰瑞科生物科技有限公司 | Improved therapeutic T cells |
JP7386848B2 (en) | 2018-08-28 | 2023-11-27 | 法▲羅▼斯疫苗株式会社 | Improved lentiviral vector |
JP7399157B2 (en) | 2018-08-28 | 2023-12-15 | 北京永泰瑞科生物科技有限公司 | Improved therapeutic T cells |
WO2021016668A1 (en) * | 2019-07-30 | 2021-02-04 | Dendrocyte Biotech Pty Ltd | Immunoconjugate |
WO2021108868A1 (en) * | 2019-12-05 | 2021-06-10 | Dendrocyte Biotech Pty Ltd | Antigen loading |
WO2022170098A3 (en) * | 2021-02-05 | 2022-09-22 | Sigilon Therapeutics, Inc. | Compositions, devices and methods for treating mps vi disease |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11028177B2 (en) | Effective targeting of primary human leukemia using anti-CD123 chimeric antigen receptor engineered T cells | |
WO2018071576A1 (en) | Treatment of tumors by inhibition of cd300f | |
JP2022043043A (en) | Methods for improving efficacy and expansion of immune cells | |
CN114761037A (en) | Chimeric antigen receptor binding to BCMA and CD19 and uses thereof | |
KR20220104217A (en) | CD19 and CD22 chimeric antigen receptors and uses thereof | |
CN111566124A (en) | Method for producing cells expressing chimeric antigen receptor | |
AU2017240667A1 (en) | Chimeric antigen and T cell receptors and methods of use | |
US20200157237A1 (en) | Lymphocyte antigen cd5like (cd5l) monomer, homodimer, and interleukin 12b (p40) heterodimer antagonists and methods of use thereof | |
JP2018516592A (en) | Methods for improving efficacy and proliferation of chimeric antigen receptor expressing cells | |
JP2016514462A (en) | Treatment of cancer using a humanized anti-CD19 chimeric antigen receptor | |
KR20220147109A (en) | Methods for making chimeric antigen receptor-expressing cells | |
CN107074951B (en) | Antagonist anti-OX 40L antibodies and methods of use thereof | |
JP2020523018A (en) | Use of CD39 and CD103 for identification of tumor-reactive human T cells for treatment of cancer | |
CN113980138B (en) | EphA2 chimeric antigen receptor and uses thereof | |
US20210371476A1 (en) | Affinity-enhanced monmeric streptavidin chimeric antigen receptor (car) | |
TW201934575A (en) | An isolated chimeric antigen receptor and modified T cell contacting same and use thereof | |
KR20210136050A (en) | Expansion of tumor-infiltrating lymphocytes from liquid tumors and their therapeutic use | |
AU2022263418A1 (en) | Chimeric costimulatory receptors, chemokine receptors, and the use of same in cellular immunotherapies | |
US20190263911A1 (en) | Anti-bag3 antibodies in combination with inhibitors of immune check-point for therapeutic use | |
US20230087953A1 (en) | Bcma-directed chimeric antigen receptor t cell compositions and methods and uses thereof | |
CN115916833B (en) | Antigen binding polypeptide targeting B7H3 and application thereof | |
CN113631184A (en) | Enhancement of cytolytic T cell activity by inhibition of EBAG9 | |
US20210139601A1 (en) | Lymphocyte antigen cd5-like (cd5l) monomer, homodimer, and interleukin 12b (p40) heterodimer agonists and methods of use thereof | |
CN112996504A (en) | Methods of treating cancer by inhibiting ubiquitin conjugating enzyme E2K (UBE2K) | |
CA3218590A1 (en) | Cxcr5, pd-1, and icos expressing tumor reactive cd4 t cells and their use |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 17791247 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 17791247 Country of ref document: EP Kind code of ref document: A1 |