WO2021155305A1 - Methods and compositions for treating cancer or viral infection with a pla2g2d antagonist - Google Patents
Methods and compositions for treating cancer or viral infection with a pla2g2d antagonist Download PDFInfo
- Publication number
- WO2021155305A1 WO2021155305A1 PCT/US2021/015921 US2021015921W WO2021155305A1 WO 2021155305 A1 WO2021155305 A1 WO 2021155305A1 US 2021015921 W US2021015921 W US 2021015921W WO 2021155305 A1 WO2021155305 A1 WO 2021155305A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- pla2g2d
- cancer
- antagonist
- agent
- cells
- Prior art date
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 249
- 239000005557 antagonist Substances 0.000 title claims abstract description 217
- 238000000034 method Methods 0.000 title claims abstract description 211
- 201000011510 cancer Diseases 0.000 title claims abstract description 179
- 230000009385 viral infection Effects 0.000 title claims description 14
- 208000036142 Viral infection Diseases 0.000 title claims description 9
- 239000000203 mixture Substances 0.000 title description 55
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 150
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 124
- 229920001184 polypeptide Polymers 0.000 claims abstract description 121
- 208000015181 infectious disease Diseases 0.000 claims abstract description 86
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 60
- 101000983153 Homo sapiens Group IID secretory phospholipase A2 Proteins 0.000 claims abstract description 58
- 201000010099 disease Diseases 0.000 claims abstract description 55
- 230000019491 signal transduction Effects 0.000 claims abstract description 24
- 102100026828 Group IID secretory phospholipase A2 Human genes 0.000 claims abstract description 21
- 239000003795 chemical substances by application Substances 0.000 claims description 232
- 210000001519 tissue Anatomy 0.000 claims description 203
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 167
- 210000002865 immune cell Anatomy 0.000 claims description 106
- 230000014509 gene expression Effects 0.000 claims description 100
- 230000027455 binding Effects 0.000 claims description 88
- 210000004027 cell Anatomy 0.000 claims description 88
- 238000009739 binding Methods 0.000 claims description 87
- 230000002401 inhibitory effect Effects 0.000 claims description 83
- 108020004459 Small interfering RNA Proteins 0.000 claims description 45
- 230000001965 increasing effect Effects 0.000 claims description 41
- 239000000427 antigen Substances 0.000 claims description 39
- 108091007433 antigens Proteins 0.000 claims description 39
- 102000036639 antigens Human genes 0.000 claims description 39
- 102000043492 human PLA2G2D Human genes 0.000 claims description 38
- 108090000695 Cytokines Proteins 0.000 claims description 34
- 102000004127 Cytokines Human genes 0.000 claims description 34
- 230000008685 targeting Effects 0.000 claims description 33
- 230000003197 catalytic effect Effects 0.000 claims description 32
- 230000007423 decrease Effects 0.000 claims description 27
- -1 TΪM3 Proteins 0.000 claims description 26
- 239000002955 immunomodulating agent Substances 0.000 claims description 25
- 230000002584 immunomodulator Effects 0.000 claims description 25
- 229940121354 immunomodulator Drugs 0.000 claims description 25
- 230000035772 mutation Effects 0.000 claims description 25
- 239000002246 antineoplastic agent Substances 0.000 claims description 22
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 claims description 20
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 claims description 20
- 206010009944 Colon cancer Diseases 0.000 claims description 19
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 claims description 19
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 claims description 19
- 229940127089 cytotoxic agent Drugs 0.000 claims description 18
- 230000002255 enzymatic effect Effects 0.000 claims description 18
- 108020001507 fusion proteins Proteins 0.000 claims description 18
- 102000037865 fusion proteins Human genes 0.000 claims description 18
- 101100407308 Mus musculus Pdcd1lg2 gene Proteins 0.000 claims description 17
- 108700030875 Programmed Cell Death 1 Ligand 2 Proteins 0.000 claims description 17
- 102100024213 Programmed cell death 1 ligand 2 Human genes 0.000 claims description 17
- 239000002253 acid Substances 0.000 claims description 16
- 108010002350 Interleukin-2 Proteins 0.000 claims description 14
- 101710089372 Programmed cell death protein 1 Proteins 0.000 claims description 14
- 230000012010 growth Effects 0.000 claims description 14
- 230000000087 stabilizing effect Effects 0.000 claims description 14
- 108020005544 Antisense RNA Proteins 0.000 claims description 12
- 108010074708 B7-H1 Antigen Proteins 0.000 claims description 12
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 12
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 claims description 12
- 208000024770 Thyroid neoplasm Diseases 0.000 claims description 12
- 239000003184 complementary RNA Substances 0.000 claims description 12
- 238000010362 genome editing Methods 0.000 claims description 12
- 229940127121 immunoconjugate Drugs 0.000 claims description 12
- 201000002510 thyroid cancer Diseases 0.000 claims description 12
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 claims description 11
- 239000004037 angiogenesis inhibitor Substances 0.000 claims description 11
- 239000002679 microRNA Substances 0.000 claims description 11
- 206010006187 Breast cancer Diseases 0.000 claims description 10
- 208000026310 Breast neoplasm Diseases 0.000 claims description 10
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 10
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims description 10
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 10
- 206010014733 Endometrial cancer Diseases 0.000 claims description 10
- 206010014759 Endometrial neoplasm Diseases 0.000 claims description 10
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 claims description 10
- 101000868279 Homo sapiens Leukocyte surface antigen CD47 Proteins 0.000 claims description 10
- 101000955999 Homo sapiens V-set domain-containing T-cell activation inhibitor 1 Proteins 0.000 claims description 10
- 208000008839 Kidney Neoplasms Diseases 0.000 claims description 10
- 102100032913 Leukocyte surface antigen CD47 Human genes 0.000 claims description 10
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 10
- 206010033128 Ovarian cancer Diseases 0.000 claims description 10
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 10
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 10
- 206010038389 Renal cancer Diseases 0.000 claims description 10
- 208000024313 Testicular Neoplasms Diseases 0.000 claims description 10
- 206010057644 Testis cancer Diseases 0.000 claims description 10
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 10
- 102100038929 V-set domain-containing T-cell activation inhibitor 1 Human genes 0.000 claims description 10
- 201000010881 cervical cancer Diseases 0.000 claims description 10
- 201000010536 head and neck cancer Diseases 0.000 claims description 10
- 208000014829 head and neck neoplasm Diseases 0.000 claims description 10
- 201000010982 kidney cancer Diseases 0.000 claims description 10
- 201000007270 liver cancer Diseases 0.000 claims description 10
- 208000014018 liver neoplasm Diseases 0.000 claims description 10
- 201000005202 lung cancer Diseases 0.000 claims description 10
- 208000020816 lung neoplasm Diseases 0.000 claims description 10
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 10
- 201000002528 pancreatic cancer Diseases 0.000 claims description 10
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 10
- 201000003120 testicular cancer Diseases 0.000 claims description 10
- 206010044412 transitional cell carcinoma Diseases 0.000 claims description 10
- 102100027207 CD27 antigen Human genes 0.000 claims description 9
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 claims description 9
- 101000801234 Homo sapiens Tumor necrosis factor receptor superfamily member 18 Proteins 0.000 claims description 9
- 206010060862 Prostate cancer Diseases 0.000 claims description 9
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 9
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 9
- 102100033728 Tumor necrosis factor receptor superfamily member 18 Human genes 0.000 claims description 9
- 150000007513 acids Chemical class 0.000 claims description 9
- 206010017758 gastric cancer Diseases 0.000 claims description 9
- 201000003733 ovarian melanoma Diseases 0.000 claims description 9
- 201000011549 stomach cancer Diseases 0.000 claims description 9
- 102100023990 60S ribosomal protein L17 Human genes 0.000 claims description 8
- 101000831007 Homo sapiens T-cell immunoreceptor with Ig and ITIM domains Proteins 0.000 claims description 8
- 102100024834 T-cell immunoreceptor with Ig and ITIM domains Human genes 0.000 claims description 8
- 229940034982 antineoplastic agent Drugs 0.000 claims description 8
- 239000004471 Glycine Substances 0.000 claims description 7
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 7
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 claims description 7
- 101001137987 Homo sapiens Lymphocyte activation gene 3 protein Proteins 0.000 claims description 3
- 102000017578 LAG3 Human genes 0.000 claims description 3
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 108091070501 miRNA Proteins 0.000 claims 1
- 208000035473 Communicable disease Diseases 0.000 abstract description 31
- 150000007523 nucleic acids Chemical class 0.000 description 90
- 102000039446 nucleic acids Human genes 0.000 description 85
- 108020004707 nucleic acids Proteins 0.000 description 85
- 108090000623 proteins and genes Proteins 0.000 description 83
- 230000000875 corresponding effect Effects 0.000 description 67
- 125000005647 linker group Chemical group 0.000 description 65
- 241000282414 Homo sapiens Species 0.000 description 54
- 238000002560 therapeutic procedure Methods 0.000 description 53
- 235000018102 proteins Nutrition 0.000 description 52
- 102000004169 proteins and genes Human genes 0.000 description 52
- 239000013598 vector Substances 0.000 description 43
- 150000001413 amino acids Chemical class 0.000 description 42
- 125000003729 nucleotide group Chemical group 0.000 description 41
- 239000002773 nucleotide Substances 0.000 description 39
- 230000000692 anti-sense effect Effects 0.000 description 37
- 230000009368 gene silencing by RNA Effects 0.000 description 34
- 108091033319 polynucleotide Proteins 0.000 description 33
- 102000040430 polynucleotide Human genes 0.000 description 33
- 239000002157 polynucleotide Substances 0.000 description 33
- 108091030071 RNAI Proteins 0.000 description 32
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 28
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 28
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 28
- 230000000694 effects Effects 0.000 description 26
- 235000001014 amino acid Nutrition 0.000 description 25
- 238000011282 treatment Methods 0.000 description 25
- 230000028993 immune response Effects 0.000 description 23
- 239000003814 drug Substances 0.000 description 21
- 230000000295 complement effect Effects 0.000 description 20
- 230000001629 suppression Effects 0.000 description 20
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 18
- 229940079593 drug Drugs 0.000 description 18
- 108020004414 DNA Proteins 0.000 description 16
- 230000006052 T cell proliferation Effects 0.000 description 16
- 230000003247 decreasing effect Effects 0.000 description 16
- 201000001441 melanoma Diseases 0.000 description 15
- 108020004999 messenger RNA Proteins 0.000 description 15
- 230000003612 virological effect Effects 0.000 description 15
- 241000725303 Human immunodeficiency virus Species 0.000 description 14
- 210000004408 hybridoma Anatomy 0.000 description 14
- 238000009169 immunotherapy Methods 0.000 description 14
- 230000008595 infiltration Effects 0.000 description 14
- 238000001764 infiltration Methods 0.000 description 14
- 230000035755 proliferation Effects 0.000 description 14
- 208000029742 colonic neoplasm Diseases 0.000 description 13
- 239000012634 fragment Substances 0.000 description 11
- 230000006870 function Effects 0.000 description 11
- 238000000338 in vitro Methods 0.000 description 11
- 230000004614 tumor growth Effects 0.000 description 11
- 108700011259 MicroRNAs Proteins 0.000 description 10
- 241000699670 Mus sp. Species 0.000 description 10
- 108091028043 Nucleic acid sequence Proteins 0.000 description 10
- 241000700605 Viruses Species 0.000 description 10
- 230000003993 interaction Effects 0.000 description 10
- 230000001737 promoting effect Effects 0.000 description 10
- 206010035226 Plasma cell myeloma Diseases 0.000 description 9
- 230000004913 activation Effects 0.000 description 9
- 239000000090 biomarker Substances 0.000 description 9
- 238000009472 formulation Methods 0.000 description 9
- 238000004519 manufacturing process Methods 0.000 description 9
- 201000000050 myeloid neoplasm Diseases 0.000 description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 8
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 description 8
- 241001529936 Murinae Species 0.000 description 8
- 206010042971 T-cell lymphoma Diseases 0.000 description 8
- 208000027585 T-cell non-Hodgkin lymphoma Diseases 0.000 description 8
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 description 8
- 239000000611 antibody drug conjugate Substances 0.000 description 8
- 229940049595 antibody-drug conjugate Drugs 0.000 description 8
- 238000003556 assay Methods 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 8
- 239000000546 pharmaceutical excipient Substances 0.000 description 8
- 238000006467 substitution reaction Methods 0.000 description 8
- 208000024891 symptom Diseases 0.000 description 8
- 239000013603 viral vector Substances 0.000 description 8
- 241000124008 Mammalia Species 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 7
- 108091034117 Oligonucleotide Proteins 0.000 description 7
- 239000011575 calcium Substances 0.000 description 7
- 239000003937 drug carrier Substances 0.000 description 7
- 239000003112 inhibitor Substances 0.000 description 7
- 210000001165 lymph node Anatomy 0.000 description 7
- 230000000770 proinflammatory effect Effects 0.000 description 7
- 238000003127 radioimmunoassay Methods 0.000 description 7
- 230000002829 reductive effect Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 230000000638 stimulation Effects 0.000 description 7
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 6
- 102000037982 Immune checkpoint proteins Human genes 0.000 description 6
- 108091008036 Immune checkpoint proteins Proteins 0.000 description 6
- 108060003951 Immunoglobulin Proteins 0.000 description 6
- 210000004322 M2 macrophage Anatomy 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 229910052791 calcium Inorganic materials 0.000 description 6
- 238000002648 combination therapy Methods 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 6
- 230000029087 digestion Effects 0.000 description 6
- 102000018358 immunoglobulin Human genes 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 230000002452 interceptive effect Effects 0.000 description 6
- 238000011813 knockout mouse model Methods 0.000 description 6
- 210000002751 lymph Anatomy 0.000 description 6
- 230000001404 mediated effect Effects 0.000 description 6
- 229920000642 polymer Polymers 0.000 description 6
- 210000004881 tumor cell Anatomy 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 5
- 238000011740 C57BL/6 mouse Methods 0.000 description 5
- 108091026890 Coding region Proteins 0.000 description 5
- 101100328463 Mus musculus Cmya5 gene Proteins 0.000 description 5
- 101710163270 Nuclease Proteins 0.000 description 5
- 102100026918 Phospholipase A2 Human genes 0.000 description 5
- 101710096328 Phospholipase A2 Proteins 0.000 description 5
- 238000003559 RNA-seq method Methods 0.000 description 5
- 239000004480 active ingredient Substances 0.000 description 5
- 230000009286 beneficial effect Effects 0.000 description 5
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 5
- 239000002254 cytotoxic agent Substances 0.000 description 5
- 231100000599 cytotoxic agent Toxicity 0.000 description 5
- 230000001472 cytotoxic effect Effects 0.000 description 5
- 230000008629 immune suppression Effects 0.000 description 5
- 210000000987 immune system Anatomy 0.000 description 5
- 230000001506 immunosuppresive effect Effects 0.000 description 5
- 238000013507 mapping Methods 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- RPENMORRBUTCPR-UHFFFAOYSA-M sodium;1-hydroxy-2,5-dioxopyrrolidine-3-sulfonate Chemical class [Na+].ON1C(=O)CC(S([O-])(=O)=O)C1=O RPENMORRBUTCPR-UHFFFAOYSA-M 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 238000000176 thermal ionisation mass spectrometry Methods 0.000 description 5
- 238000013055 trapped ion mobility spectrometry Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 108010088751 Albumins Proteins 0.000 description 4
- 102000009027 Albumins Human genes 0.000 description 4
- 201000009030 Carcinoma Diseases 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 4
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000006044 T cell activation Effects 0.000 description 4
- 108090000631 Trypsin Proteins 0.000 description 4
- 102000004142 Trypsin Human genes 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 201000010897 colon adenocarcinoma Diseases 0.000 description 4
- 238000005859 coupling reaction Methods 0.000 description 4
- 231100000433 cytotoxic Toxicity 0.000 description 4
- 230000002950 deficient Effects 0.000 description 4
- 239000003085 diluting agent Substances 0.000 description 4
- 208000035475 disorder Diseases 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 230000002349 favourable effect Effects 0.000 description 4
- 239000000796 flavoring agent Substances 0.000 description 4
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 4
- 125000000524 functional group Chemical group 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 238000003364 immunohistochemistry Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 210000004698 lymphocyte Anatomy 0.000 description 4
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000008194 pharmaceutical composition Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 230000014616 translation Effects 0.000 description 4
- 239000012588 trypsin Substances 0.000 description 4
- 206010003571 Astrocytoma Diseases 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 108091033380 Coding strand Proteins 0.000 description 3
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 108010010803 Gelatin Proteins 0.000 description 3
- 229940123611 Genome editing Drugs 0.000 description 3
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 description 3
- 108091070975 Group 2 family Proteins 0.000 description 3
- 229920002971 Heparan sulfate Polymers 0.000 description 3
- 102100034458 Hepatitis A virus cellular receptor 2 Human genes 0.000 description 3
- 101001068133 Homo sapiens Hepatitis A virus cellular receptor 2 Proteins 0.000 description 3
- 102100029098 Hypoxanthine-guanine phosphoribosyltransferase Human genes 0.000 description 3
- 240000007472 Leucaena leucocephala Species 0.000 description 3
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 3
- 206010025323 Lymphomas Diseases 0.000 description 3
- 101100028420 Mus musculus Pla2g2d gene Proteins 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 108010029485 Protein Isoforms Proteins 0.000 description 3
- 102000001708 Protein Isoforms Human genes 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 102000005473 Secretory Phospholipases A2 Human genes 0.000 description 3
- 108010031873 Secretory Phospholipases A2 Proteins 0.000 description 3
- 108091081021 Sense strand Proteins 0.000 description 3
- 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 3
- 229930006000 Sucrose Natural products 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 208000003721 Triple Negative Breast Neoplasms Diseases 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- 239000000074 antisense oligonucleotide Substances 0.000 description 3
- 238000012230 antisense oligonucleotides Methods 0.000 description 3
- 239000011230 binding agent Substances 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 230000002490 cerebral effect Effects 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 239000007822 coupling agent Substances 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000004132 cross linking Methods 0.000 description 3
- 210000004443 dendritic cell Anatomy 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 239000008273 gelatin Substances 0.000 description 3
- 229920000159 gelatin Polymers 0.000 description 3
- 235000019322 gelatine Nutrition 0.000 description 3
- 235000011852 gelatine desserts Nutrition 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 230000028996 humoral immune response Effects 0.000 description 3
- 230000006028 immune-suppresssive effect Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 230000007108 local immune response Effects 0.000 description 3
- 201000005249 lung adenocarcinoma Diseases 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 239000003068 molecular probe Substances 0.000 description 3
- 239000002539 nanocarrier Substances 0.000 description 3
- 210000000822 natural killer cell Anatomy 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000003755 preservative agent Substances 0.000 description 3
- 238000007639 printing Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000004393 prognosis Methods 0.000 description 3
- 230000002062 proliferating effect Effects 0.000 description 3
- 230000001177 retroviral effect Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 206010041823 squamous cell carcinoma Diseases 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- CCEKAJIANROZEO-UHFFFAOYSA-N sulfluramid Chemical group CCNS(=O)(=O)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)F CCEKAJIANROZEO-UHFFFAOYSA-N 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 239000003826 tablet Substances 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 108700012359 toxins Proteins 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 208000022679 triple-negative breast carcinoma Diseases 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- GKSPIZSKQWTXQG-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-[1-(pyridin-2-yldisulfanyl)ethyl]benzoate Chemical compound C=1C=C(C(=O)ON2C(CCC2=O)=O)C=CC=1C(C)SSC1=CC=CC=N1 GKSPIZSKQWTXQG-UHFFFAOYSA-N 0.000 description 2
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 2
- 206010052747 Adenocarcinoma pancreas Diseases 0.000 description 2
- 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 2
- 241000416162 Astragalus gummifer Species 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 108091033409 CRISPR Proteins 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 108090000994 Catalytic RNA Proteins 0.000 description 2
- 102000053642 Catalytic RNA Human genes 0.000 description 2
- 102000000844 Cell Surface Receptors Human genes 0.000 description 2
- 108010001857 Cell Surface Receptors Proteins 0.000 description 2
- 241000251730 Chondrichthyes Species 0.000 description 2
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 description 2
- 241000711573 Coronaviridae Species 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- 101100421450 Drosophila melanogaster Shark gene Proteins 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000709661 Enterovirus Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 208000032320 Germ cell tumor of testis Diseases 0.000 description 2
- 206010018338 Glioma Diseases 0.000 description 2
- 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 2
- 241000282412 Homo Species 0.000 description 2
- 101000934372 Homo sapiens Macrosialin Proteins 0.000 description 2
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 2
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 2
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 2
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 2
- 101710203526 Integrase Proteins 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- 102100025136 Macrosialin Human genes 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 2
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 2
- 208000000172 Medulloblastoma Diseases 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 108091092724 Noncoding DNA Proteins 0.000 description 2
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 2
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 208000002606 Paramyxoviridae Infections Diseases 0.000 description 2
- 102220552615 Phospholipase A2, membrane associated_H67Q_mutation Human genes 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 2
- 206010039491 Sarcoma Diseases 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 2
- 238000010459 TALEN Methods 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
- 208000033781 Thyroid carcinoma Diseases 0.000 description 2
- 229920001615 Tragacanth Polymers 0.000 description 2
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical group O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 230000009435 amidation Effects 0.000 description 2
- 238000007112 amidation reaction Methods 0.000 description 2
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 2
- 210000000612 antigen-presenting cell Anatomy 0.000 description 2
- 239000002256 antimetabolite Substances 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 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 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 206010005084 bladder transitional cell carcinoma Diseases 0.000 description 2
- 201000001528 bladder urothelial carcinoma Diseases 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000005341 cation exchange Methods 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 210000004671 cell-free system Anatomy 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 235000010980 cellulose Nutrition 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 201000010240 chromophobe renal cell carcinoma Diseases 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 230000004186 co-expression Effects 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 125000000151 cysteine group Chemical class N[C@@H](CS)C(=O)* 0.000 description 2
- 230000001085 cytostatic effect Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 229910052805 deuterium Inorganic materials 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 238000010195 expression analysis Methods 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 238000009459 flexible packaging Methods 0.000 description 2
- 235000013355 food flavoring agent Nutrition 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 201000006585 gastric adenocarcinoma Diseases 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 239000003966 growth inhibitor Substances 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 108010038082 heparin proteoglycan Proteins 0.000 description 2
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 2
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 230000002687 intercalation Effects 0.000 description 2
- 238000009830 intercalation Methods 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 201000005243 lung squamous cell carcinoma Diseases 0.000 description 2
- 235000019359 magnesium stearate Nutrition 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000002483 medication Methods 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 208000037819 metastatic cancer Diseases 0.000 description 2
- 208000011575 metastatic malignant neoplasm Diseases 0.000 description 2
- VPKDCDLSJZCGKE-UHFFFAOYSA-N methanediimine Chemical compound N=C=N VPKDCDLSJZCGKE-UHFFFAOYSA-N 0.000 description 2
- 239000000693 micelle Substances 0.000 description 2
- 239000002480 mineral oil Substances 0.000 description 2
- 235000010446 mineral oil Nutrition 0.000 description 2
- 102000035118 modified proteins Human genes 0.000 description 2
- 108091005573 modified proteins Proteins 0.000 description 2
- 210000000066 myeloid cell Anatomy 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 238000003012 network analysis Methods 0.000 description 2
- 238000011275 oncology therapy Methods 0.000 description 2
- 201000010302 ovarian serous cystadenocarcinoma Diseases 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 201000002094 pancreatic adenocarcinoma Diseases 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 102000042759 phospholipase A2 family Human genes 0.000 description 2
- 108091082051 phospholipase A2 family Proteins 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 230000004481 post-translational protein modification Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 201000005825 prostate adenocarcinoma Diseases 0.000 description 2
- 238000000159 protein binding assay Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- YUOCYTRGANSSRY-UHFFFAOYSA-N pyrrolo[2,3-i][1,2]benzodiazepine Chemical compound C1=CN=NC2=C3C=CN=C3C=CC2=C1 YUOCYTRGANSSRY-UHFFFAOYSA-N 0.000 description 2
- 238000001959 radiotherapy Methods 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 201000001281 rectum adenocarcinoma Diseases 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000000284 resting effect Effects 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 108091092562 ribozyme Proteins 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000000375 suspending agent Substances 0.000 description 2
- 238000007910 systemic administration Methods 0.000 description 2
- 239000000454 talc Substances 0.000 description 2
- 229910052623 talc Inorganic materials 0.000 description 2
- 235000012222 talc Nutrition 0.000 description 2
- 208000002918 testicular germ cell tumor Diseases 0.000 description 2
- 231100001274 therapeutic index Toxicity 0.000 description 2
- 208000013077 thyroid gland carcinoma Diseases 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 235000010487 tragacanth Nutrition 0.000 description 2
- 239000000196 tragacanth Substances 0.000 description 2
- 229940116362 tragacanth Drugs 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- 241001529453 unidentified herpesvirus Species 0.000 description 2
- 241000712461 unidentified influenza virus Species 0.000 description 2
- 201000003701 uterine corpus endometrial carcinoma Diseases 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- LLXVXPPXELIDGQ-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(2,5-dioxopyrrol-1-yl)benzoate Chemical compound C=1C=CC(N2C(C=CC2=O)=O)=CC=1C(=O)ON1C(=O)CCC1=O LLXVXPPXELIDGQ-UHFFFAOYSA-N 0.000 description 1
- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 1
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- KUHSEZKIEJYEHN-BXRBKJIMSA-N (2s)-2-amino-3-hydroxypropanoic acid;(2s)-2-aminopropanoic acid Chemical compound C[C@H](N)C(O)=O.OC[C@H](N)C(O)=O KUHSEZKIEJYEHN-BXRBKJIMSA-N 0.000 description 1
- JPSHPWJJSVEEAX-OWPBQMJCSA-N (2s)-2-amino-4-fluoranylpentanedioic acid Chemical compound OC(=O)[C@@H](N)CC([18F])C(O)=O JPSHPWJJSVEEAX-OWPBQMJCSA-N 0.000 description 1
- FIDLLEYNNRGVFR-CTNGQTDRSA-N (3R)-2-[(11S)-7,8-difluoro-6,11-dihydrobenzo[c][1]benzothiepin-11-yl]-11-hydroxy-5-oxa-1,2,8-triazatricyclo[8.4.0.03,8]tetradeca-10,13-diene-9,12-dione Chemical compound OC1=C2N(C=CC1=O)N([C@@H]1COCCN1C2=O)[C@@H]1C2=C(SCC3=C1C=CC(F)=C3F)C=CC=C2 FIDLLEYNNRGVFR-CTNGQTDRSA-N 0.000 description 1
- VEEGZPWAAPPXRB-BJMVGYQFSA-N (3e)-3-(1h-imidazol-5-ylmethylidene)-1h-indol-2-one Chemical compound O=C1NC2=CC=CC=C2\C1=C/C1=CN=CN1 VEEGZPWAAPPXRB-BJMVGYQFSA-N 0.000 description 1
- SLURUCSFDHKXFR-WWMWMSKMSA-N (7s,9s)-7-[[(1s,3r,4as,9s,9ar,10as)-9-methoxy-1-methyl-3,4,4a,6,7,9,9a,10a-octahydro-1h-pyrano[1,2][1,3]oxazolo[3,4-b][1,4]oxazin-3-yl]oxy]-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7h-tetracene-5,12-dione Chemical compound O=C1C2=CC=CC(OC)=C2C(=O)C(C(O)=C23)=C1C(O)=C3C[C@@](O)(C(=O)CO)C[C@@H]2O[C@H]1C[C@@H]2N3CCO[C@H](OC)[C@H]3O[C@@H]2[C@H](C)O1 SLURUCSFDHKXFR-WWMWMSKMSA-N 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
- ASNTZYQMIUCEBV-UHFFFAOYSA-N 2,5-dioxo-1-[6-[3-(pyridin-2-yldisulfanyl)propanoylamino]hexanoyloxy]pyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCCCCNC(=O)CCSSC1=CC=CC=N1 ASNTZYQMIUCEBV-UHFFFAOYSA-N 0.000 description 1
- GVJXGCIPWAVXJP-UHFFFAOYSA-N 2,5-dioxo-1-oxoniopyrrolidine-3-sulfonate Chemical compound ON1C(=O)CC(S(O)(=O)=O)C1=O GVJXGCIPWAVXJP-UHFFFAOYSA-N 0.000 description 1
- AUVALWUPUHHNQV-UHFFFAOYSA-N 2-hydroxy-3-propylbenzoic acid Chemical class CCCC1=CC=CC(C(O)=O)=C1O AUVALWUPUHHNQV-UHFFFAOYSA-N 0.000 description 1
- 238000005084 2D-nuclear magnetic resonance Methods 0.000 description 1
- CQXXYOLFJXSRMT-UHFFFAOYSA-N 5-diazocyclohexa-1,3-diene Chemical class [N-]=[N+]=C1CC=CC=C1 CQXXYOLFJXSRMT-UHFFFAOYSA-N 0.000 description 1
- 244000215068 Acacia senegal Species 0.000 description 1
- 235000006491 Acacia senegal Nutrition 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
- 241000701386 African swine fever virus Species 0.000 description 1
- 102100021266 Alpha-(1,6)-fucosyltransferase Human genes 0.000 description 1
- 206010061424 Anal cancer Diseases 0.000 description 1
- 244000303258 Annona diversifolia Species 0.000 description 1
- 235000002198 Annona diversifolia Nutrition 0.000 description 1
- 208000007860 Anus Neoplasms Diseases 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 206010060971 Astrocytoma malignant Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 244000063299 Bacillus subtilis Species 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
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 229920002799 BoPET Polymers 0.000 description 1
- 206010005949 Bone cancer Diseases 0.000 description 1
- 208000018084 Bone neoplasm Diseases 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 206010006143 Brain stem glioma Diseases 0.000 description 1
- 238000011357 CAR T-cell therapy Methods 0.000 description 1
- 102100032912 CD44 antigen Human genes 0.000 description 1
- 241001678559 COVID-19 virus Species 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 108010040467 CRISPR-Associated Proteins Proteins 0.000 description 1
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 1
- 229940045513 CTLA4 antagonist Drugs 0.000 description 1
- 101100180402 Caenorhabditis elegans jun-1 gene Proteins 0.000 description 1
- 102000005701 Calcium-Binding Proteins Human genes 0.000 description 1
- 108010045403 Calcium-Binding Proteins Proteins 0.000 description 1
- KLWPJMFMVPTNCC-UHFFFAOYSA-N Camptothecin Natural products CCC1(O)C(=O)OCC2=C1C=C3C4Nc5ccccc5C=C4CN3C2=O KLWPJMFMVPTNCC-UHFFFAOYSA-N 0.000 description 1
- 206010065305 Cancer in remission Diseases 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- 208000017897 Carcinoma of esophagus Diseases 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- 241000282994 Cervidae Species 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 244000089742 Citrus aurantifolia Species 0.000 description 1
- 235000008733 Citrus aurantifolia Nutrition 0.000 description 1
- 208000030808 Clear cell renal carcinoma Diseases 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 241000723655 Cowpea mosaic virus Species 0.000 description 1
- 241000709687 Coxsackievirus Species 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 229920002785 Croscarmellose sodium Polymers 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 241000725619 Dengue virus Species 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- YZCKVEUIGOORGS-OUBTZVSYSA-N Deuterium Chemical group [2H] YZCKVEUIGOORGS-OUBTZVSYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 101150029707 ERBB2 gene Proteins 0.000 description 1
- 241001115402 Ebolavirus Species 0.000 description 1
- 241001466953 Echovirus Species 0.000 description 1
- 101710170658 Endogenous retrovirus group K member 10 Gag polyprotein Proteins 0.000 description 1
- 101710186314 Endogenous retrovirus group K member 21 Gag polyprotein Proteins 0.000 description 1
- 101710162093 Endogenous retrovirus group K member 24 Gag polyprotein Proteins 0.000 description 1
- 101710094596 Endogenous retrovirus group K member 8 Gag polyprotein Proteins 0.000 description 1
- 101710177443 Endogenous retrovirus group K member 9 Gag polyprotein Proteins 0.000 description 1
- 229940123734 Endonuclease inhibitor Drugs 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000991587 Enterovirus C Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 101710177291 Gag polyprotein Proteins 0.000 description 1
- 208000032612 Glial tumor Diseases 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108060003393 Granulin Proteins 0.000 description 1
- 102100035723 Group 3 secretory phospholipase A2 Human genes 0.000 description 1
- 101710118806 Group IID secretory phospholipase A2 Proteins 0.000 description 1
- 102100026825 Group IIE secretory phospholipase A2 Human genes 0.000 description 1
- 101710182177 Group IIE secretory phospholipase A2 Proteins 0.000 description 1
- 102100026833 Group IIF secretory phospholipase A2 Human genes 0.000 description 1
- 101710202202 Group IIF secretory phospholipase A2 Proteins 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 108010002459 HIV Integrase Proteins 0.000 description 1
- 108010010369 HIV Protease Proteins 0.000 description 1
- 108010078851 HIV Reverse Transcriptase Proteins 0.000 description 1
- 108010007712 Hepatitis A Virus Cellular Receptor 1 Proteins 0.000 description 1
- 102100034459 Hepatitis A virus cellular receptor 1 Human genes 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- MDCTVRUPVLZSPG-BQBZGAKWSA-N His-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CNC=N1 MDCTVRUPVLZSPG-BQBZGAKWSA-N 0.000 description 1
- 101000819490 Homo sapiens Alpha-(1,6)-fucosyltransferase Proteins 0.000 description 1
- 101000946926 Homo sapiens C-C chemokine receptor type 5 Proteins 0.000 description 1
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 1
- 101000735510 Homo sapiens Group 3 secretory phospholipase A2 Proteins 0.000 description 1
- 101001103039 Homo sapiens Inactive tyrosine-protein kinase transmembrane receptor ROR1 Proteins 0.000 description 1
- 101001033249 Homo sapiens Interleukin-1 beta Proteins 0.000 description 1
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 1
- 101001103036 Homo sapiens Nuclear receptor ROR-alpha Proteins 0.000 description 1
- 108010071893 Human Immunodeficiency Virus rev Gene Products Proteins 0.000 description 1
- 241000714260 Human T-lymphotropic virus 1 Species 0.000 description 1
- 241000701085 Human alphaherpesvirus 3 Species 0.000 description 1
- 241001502974 Human gammaherpesvirus 8 Species 0.000 description 1
- 101900272909 Human immunodeficiency virus type 1 group M subtype B p6-gag Proteins 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 description 1
- 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
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 102100039615 Inactive tyrosine-protein kinase transmembrane receptor ROR1 Human genes 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 102100034347 Integrase Human genes 0.000 description 1
- 102100039065 Interleukin-1 beta Human genes 0.000 description 1
- 206010061252 Intraocular melanoma Diseases 0.000 description 1
- KJHKTHWMRKYKJE-SUGCFTRWSA-N Kaletra Chemical compound N1([C@@H](C(C)C)C(=O)N[C@H](C[C@H](O)[C@H](CC=2C=CC=CC=2)NC(=O)COC=2C(=CC=CC=2C)C)CC=2C=CC=CC=2)CCCNC1=O KJHKTHWMRKYKJE-SUGCFTRWSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 241001071864 Lethrinus laticaudis Species 0.000 description 1
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 208000006644 Malignant Fibrous Histiocytoma Diseases 0.000 description 1
- 201000005505 Measles Diseases 0.000 description 1
- 208000002030 Merkel cell carcinoma Diseases 0.000 description 1
- 206010027406 Mesothelioma Diseases 0.000 description 1
- 108091007780 MiR-122 Proteins 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 241000127282 Middle East respiratory syndrome-related coronavirus Species 0.000 description 1
- 208000005647 Mumps Diseases 0.000 description 1
- 241000711386 Mumps virus Species 0.000 description 1
- 101100369641 Mus musculus Tigit gene Proteins 0.000 description 1
- 101100425749 Mus musculus Tnfrsf18 gene Proteins 0.000 description 1
- 239000005041 Mylar™ Substances 0.000 description 1
- 230000004988 N-glycosylation Effects 0.000 description 1
- 208000001894 Nasopharyngeal Neoplasms Diseases 0.000 description 1
- 206010061306 Nasopharyngeal cancer Diseases 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 102000005348 Neuraminidase Human genes 0.000 description 1
- 108010006232 Neuraminidase Proteins 0.000 description 1
- 229940123424 Neuraminidase inhibitor Drugs 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 206010029266 Neuroendocrine carcinoma of the skin Diseases 0.000 description 1
- 241000714209 Norwalk virus Species 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 108700022034 Opsonin Proteins Proteins 0.000 description 1
- 241000702259 Orbivirus Species 0.000 description 1
- 240000007019 Oxalis corniculata Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241001631646 Papillomaviridae Species 0.000 description 1
- 206010061332 Paraganglion neoplasm Diseases 0.000 description 1
- 208000000821 Parathyroid Neoplasms Diseases 0.000 description 1
- 206010034016 Paronychia Diseases 0.000 description 1
- 240000002834 Paulownia tomentosa Species 0.000 description 1
- 235000010678 Paulownia tomentosa Nutrition 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 208000007913 Pituitary Neoplasms Diseases 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 241001505332 Polyomavirus sp. Species 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 101710188313 Protein U Proteins 0.000 description 1
- 241000125945 Protoparvovirus Species 0.000 description 1
- 101150085390 RPM1 gene Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 241000711798 Rabies lyssavirus Species 0.000 description 1
- 244000097202 Rathbunia alamosensis Species 0.000 description 1
- 235000009776 Rathbunia alamosensis Nutrition 0.000 description 1
- 208000015634 Rectal Neoplasms Diseases 0.000 description 1
- 206010038111 Recurrent cancer Diseases 0.000 description 1
- 241000702263 Reovirus sp. Species 0.000 description 1
- 201000000582 Retinoblastoma Diseases 0.000 description 1
- 241000724205 Rice stripe tenuivirus Species 0.000 description 1
- NCDNCNXCDXHOMX-UHFFFAOYSA-N Ritonavir Natural products C=1C=CC=CC=1CC(NC(=O)OCC=1SC=NC=1)C(O)CC(CC=1C=CC=CC=1)NC(=O)C(C(C)C)NC(=O)N(C)CC1=CSC(C(C)C)=N1 NCDNCNXCDXHOMX-UHFFFAOYSA-N 0.000 description 1
- 241000702670 Rotavirus Species 0.000 description 1
- 241000315672 SARS coronavirus Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- 206010041067 Small cell lung cancer Diseases 0.000 description 1
- 101800001611 Spacer peptide 1 Proteins 0.000 description 1
- 101800001610 Spacer peptide 2 Proteins 0.000 description 1
- 208000000102 Squamous Cell Carcinoma of Head and Neck Diseases 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 101150052863 THY1 gene Proteins 0.000 description 1
- 229940123237 Taxane Drugs 0.000 description 1
- 241000223892 Tetrahymena Species 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- 235000011941 Tilia x europaea Nutrition 0.000 description 1
- 101710183280 Topoisomerase Proteins 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 102100040396 Transcobalamin-1 Human genes 0.000 description 1
- 101710124861 Transcobalamin-1 Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 101800001690 Transmembrane protein gp41 Proteins 0.000 description 1
- 208000026911 Tuberous sclerosis complex Diseases 0.000 description 1
- 102000004243 Tubulin Human genes 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102100040247 Tumor necrosis factor Human genes 0.000 description 1
- 208000015778 Undifferentiated pleomorphic sarcoma Diseases 0.000 description 1
- 208000023915 Ureteral Neoplasms Diseases 0.000 description 1
- 206010046392 Ureteric cancer Diseases 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 208000002495 Uterine Neoplasms Diseases 0.000 description 1
- 201000005969 Uveal melanoma Diseases 0.000 description 1
- 101150010086 VP24 gene Proteins 0.000 description 1
- 101150026858 VP30 gene Proteins 0.000 description 1
- 101150077651 VP35 gene Proteins 0.000 description 1
- 101150036892 VP40 gene Proteins 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- 229940122803 Vinca alkaloid Drugs 0.000 description 1
- 238000004833 X-ray photoelectron spectroscopy Methods 0.000 description 1
- 241000710772 Yellow fever virus Species 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-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
- 230000002159 abnormal effect Effects 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 125000000641 acridinyl group Chemical group C1(=CC=CC2=NC3=CC=CC=C3C=C12)* 0.000 description 1
- 230000004721 adaptive immunity Effects 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 1
- 238000012867 alanine scanning Methods 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 125000005599 alkyl carboxylate group Chemical group 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 150000001408 amides Chemical group 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 229940121369 angiogenesis inhibitor Drugs 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 201000011165 anus cancer Diseases 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 108010044540 auristatin Proteins 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000003542 behavioural effect Effects 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
- 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
- 238000004166 bioassay Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000000378 calcium silicate Substances 0.000 description 1
- 229910052918 calcium silicate Inorganic materials 0.000 description 1
- 229960003340 calcium silicate Drugs 0.000 description 1
- 235000012241 calcium silicate Nutrition 0.000 description 1
- OYACROKNLOSFPA-UHFFFAOYSA-N calcium;dioxido(oxo)silane Chemical compound [Ca+2].[O-][Si]([O-])=O OYACROKNLOSFPA-UHFFFAOYSA-N 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
- 229940127093 camptothecin Drugs 0.000 description 1
- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical compound C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 description 1
- 239000003560 cancer drug Substances 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 201000007335 cerebellar astrocytoma Diseases 0.000 description 1
- 208000030239 cerebral astrocytoma Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000973 chemotherapeutic effect Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 208000011654 childhood malignant neoplasm Diseases 0.000 description 1
- 208000012191 childhood neoplasm Diseases 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 206010073251 clear cell renal cell carcinoma Diseases 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 229940075614 colloidal silicon dioxide Drugs 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 229960001681 croscarmellose sodium Drugs 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 235000010947 crosslinked sodium carboxy methyl cellulose Nutrition 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 208000030381 cutaneous melanoma Diseases 0.000 description 1
- 208000017763 cutaneous neuroendocrine carcinoma Diseases 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 108010057085 cytokine receptors Proteins 0.000 description 1
- 102000003675 cytokine receptors Human genes 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 239000000412 dendrimer Substances 0.000 description 1
- 229920000736 dendritic polymer Polymers 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 125000005442 diisocyanate group Chemical group 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 239000001177 diphosphate Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000005315 distribution function Methods 0.000 description 1
- VSJKWCGYPAHWDS-UHFFFAOYSA-N dl-camptothecin Natural products C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)C5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-UHFFFAOYSA-N 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 229960005501 duocarmycin Drugs 0.000 description 1
- VQNATVDKACXKTF-XELLLNAOSA-N duocarmycin Chemical compound COC1=C(OC)C(OC)=C2NC(C(=O)N3C4=CC(=O)C5=C([C@@]64C[C@@H]6C3)C=C(N5)C(=O)OC)=CC2=C1 VQNATVDKACXKTF-XELLLNAOSA-N 0.000 description 1
- 229930184221 duocarmycin Natural products 0.000 description 1
- 238000001378 electrochemiluminescence detection Methods 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 239000008393 encapsulating agent Substances 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000006862 enzymatic digestion Effects 0.000 description 1
- 230000006718 epigenetic regulation Effects 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 201000005619 esophageal carcinoma Diseases 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 208000024519 eye neoplasm Diseases 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 108020005243 folate receptor Proteins 0.000 description 1
- 102000006815 folate receptor Human genes 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- 108010027225 gag-pol Fusion Proteins Proteins 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 201000011243 gastrointestinal stromal tumor Diseases 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 229940014259 gelatin Drugs 0.000 description 1
- 108091008053 gene clusters Proteins 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 201000000459 head and neck squamous cell carcinoma Diseases 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 1
- 208000029824 high grade glioma Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002962 histologic effect Effects 0.000 description 1
- 102000048160 human CCR5 Human genes 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- DCPMPXBYPZGNDC-UHFFFAOYSA-N hydron;methanediimine;chloride Chemical compound Cl.N=C=N DCPMPXBYPZGNDC-UHFFFAOYSA-N 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 229940124452 immunizing agent Drugs 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 230000002637 immunotoxin Effects 0.000 description 1
- 229940051026 immunotoxin Drugs 0.000 description 1
- 239000002596 immunotoxin Substances 0.000 description 1
- 231100000608 immunotoxin Toxicity 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000005414 inactive ingredient Substances 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 230000007154 intracellular accumulation Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 201000008893 intraocular retinoblastoma Diseases 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 208000024312 invasive carcinoma Diseases 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 238000003064 k means clustering Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000004571 lime Substances 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 229960004525 lopinavir Drugs 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 208000030883 malignant astrocytoma Diseases 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 201000011614 malignant glioma Diseases 0.000 description 1
- 208000026045 malignant tumor of parathyroid gland Diseases 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 108091051828 miR-122 stem-loop Proteins 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000012434 mixed-mode chromatography Methods 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 208000010805 mumps infectious disease Diseases 0.000 description 1
- 210000004985 myeloid-derived suppressor cell Anatomy 0.000 description 1
- 230000007498 myristoylation Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- CTMCWCONSULRHO-UHQPFXKFSA-N nemorubicin Chemical compound C1CO[C@H](OC)CN1[C@@H]1[C@H](O)[C@H](C)O[C@@H](O[C@@H]2C3=C(O)C=4C(=O)C5=C(OC)C=CC=C5C(=O)C=4C(O)=C3C[C@](O)(C2)C(=O)CO)C1 CTMCWCONSULRHO-UHQPFXKFSA-N 0.000 description 1
- 229950010159 nemorubicin Drugs 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 230000000683 nonmetastatic effect Effects 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 229940127073 nucleoside analogue Drugs 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 201000008106 ocular cancer Diseases 0.000 description 1
- 201000002575 ocular melanoma Diseases 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 235000015205 orange juice Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- VSZGPKBBMSAYNT-RRFJBIMHSA-N oseltamivir Chemical compound CCOC(=O)C1=C[C@@H](OC(CC)CC)[C@H](NC(C)=O)[C@@H](N)C1 VSZGPKBBMSAYNT-RRFJBIMHSA-N 0.000 description 1
- 229960003752 oseltamivir Drugs 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000026792 palmitoylation Effects 0.000 description 1
- 208000007312 paraganglioma Diseases 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 235000010603 pastilles Nutrition 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 230000001175 peptic effect Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- XRQDFNLINLXZLB-CKIKVBCHSA-N peramivir Chemical compound CCC(CC)[C@H](NC(C)=O)[C@@H]1[C@H](O)[C@@H](C(O)=O)C[C@H]1NC(N)=N XRQDFNLINLXZLB-CKIKVBCHSA-N 0.000 description 1
- 229960001084 peramivir Drugs 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 208000028591 pheochromocytoma Diseases 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 208000010916 pituitary tumor Diseases 0.000 description 1
- 210000003720 plasmablast Anatomy 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 108010089520 pol Gene Products Proteins 0.000 description 1
- 230000001884 polyglutamylation Effects 0.000 description 1
- 230000037048 polymerization activity Effects 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 230000032361 posttranscriptional gene silencing Effects 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 108020001580 protein domains Proteins 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 231100000654 protein toxin Toxicity 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 206010038038 rectal cancer Diseases 0.000 description 1
- 201000001275 rectum cancer Diseases 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 201000009410 rhabdomyosarcoma Diseases 0.000 description 1
- NCDNCNXCDXHOMX-XGKFQTDJSA-N ritonavir Chemical compound N([C@@H](C(C)C)C(=O)N[C@H](C[C@H](O)[C@H](CC=1C=CC=CC=1)NC(=O)OCC=1SC=NC=1)CC=1C=CC=CC=1)C(=O)N(C)CC1=CSC(C(C)C)=N1 NCDNCNXCDXHOMX-XGKFQTDJSA-N 0.000 description 1
- 229960000311 ritonavir Drugs 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 201000005404 rubella Diseases 0.000 description 1
- 238000013391 scatchard analysis Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 239000002911 sialidase inhibitor Substances 0.000 description 1
- 230000009450 sialylation Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 201000003708 skin melanoma Diseases 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 208000000587 small cell lung carcinoma Diseases 0.000 description 1
- 201000002314 small intestine cancer Diseases 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007909 solid dosage form Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 208000017572 squamous cell neoplasm Diseases 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- DKPFODGZWDEEBT-QFIAKTPHSA-N taxane Chemical class C([C@]1(C)CCC[C@@H](C)[C@H]1C1)C[C@H]2[C@H](C)CC[C@@H]1C2(C)C DKPFODGZWDEEBT-QFIAKTPHSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000004797 therapeutic response Effects 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 150000007970 thio esters Chemical class 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 208000008732 thymoma Diseases 0.000 description 1
- 239000008181 tonicity modifier Substances 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 230000009752 translational inhibition Effects 0.000 description 1
- LZAJKCZTKKKZNT-PMNGPLLRSA-N trichothecene Chemical compound C12([C@@]3(CC[C@H]2OC2C=C(CCC23C)C)C)CO1 LZAJKCZTKKKZNT-PMNGPLLRSA-N 0.000 description 1
- 229930013292 trichothecene Natural products 0.000 description 1
- 208000009999 tuberous sclerosis Diseases 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 108020005087 unfolded proteins Proteins 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 201000011294 ureter cancer Diseases 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 238000011816 wild-type C57Bl6 mouse Methods 0.000 description 1
- 238000002424 x-ray crystallography Methods 0.000 description 1
- 229940051021 yellow-fever virus Drugs 0.000 description 1
- ARAIBEBZBOPLMB-UFGQHTETSA-N zanamivir Chemical compound CC(=O)N[C@@H]1[C@@H](N=C(N)N)C=C(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO ARAIBEBZBOPLMB-UFGQHTETSA-N 0.000 description 1
- 229960001028 zanamivir Drugs 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- XOOUIPVCVHRTMJ-UHFFFAOYSA-L zinc stearate Chemical compound [Zn+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O XOOUIPVCVHRTMJ-UHFFFAOYSA-L 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
-
- 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
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- 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
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- 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
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
-
- 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
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01004—Phospholipase A2 (3.1.1.4)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to methods and compositions for treating a disease or condition that involve an antagonist targeting PLA2G2D signaling pathway.
- the present application provides methods of treating diseases or conditions (such as cancer or viral infection).
- the present application in one aspect provides methods of treating a cancer or viral infection in an individual comprising administering into the individual an effective amount of an antagonist targeting PLA2G2D signaling pathway.
- the antagonist is an antagonist targeting PLA2G2D.
- the PLA2G2D is a human PLA2G2D.
- the antagonist decreases enzymatic activity level of PLA2G2D,
- the antagonist targeting PLA2G2D signaling pathway blocks a catalytic site on PLA2G2D.
- the antagonist targets the H67 catalytic site on a human PLA2G2D according to SEQ ID NO: 1 or 5.
- the antagonist comprises a siRNA, a miRNA, an antisense RNA, or a gene editing system.
- the antagonist comprises an agent that inhibits PLA2G2D (such as an agent that blocks the binding of PLA2G2D to an immune cell or an agent that inhibits the activity of PLA2G2D).
- the immune cell is aT cell.
- the antagonist comprises an anti-PLA2G2D antibody.
- the anti-PLA2G2D antibody is a monoclonal antibody, in some embodiments, the antagonist is a fusion protein or immunoconjugate comprising an anti-PLA2G2B antibody moiety and a second moiety, in some embodiments, the second moiety comprises a cytokine.
- the antagonist comprises an inhibitory PLA2G2D polypeptide that blocks the binding of PLA2G2D to an immune ceil.
- the inhibitory PLA2G2D polypeptide binds to the immune cell with a greater affinity than for PLA2G2D,
- the immune cells is a T cell.
- the inhibitory polypeptide further comprises a stabilizing domain.
- the stabilizing domain is an Fc domain.
- the inhibitory PLA2G2D polypeptide has a length of about 50 to about 200 amino acids.
- the inhibitory' PLA2G2D polypeptide has a mutation at the position corresponding to histidine at position 67 (H67) according to SEQ ID NO: 1 or 5.
- the inhibitory PLA2G2D polypeptide comprises an ammo acid sequence of SEQ ID NO: 3, 4, 7, or 8,
- the disease or condition is a cancer.
- the cancer is a solid tumor.
- the cancer is an advanced or malignant tumor.
- the cancer has an increased expression level of PLA2G2D.
- the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, renal cancer, prostate cancer, urothelial cancer, testis cancer, ovarian cancer and melanoma.
- the disease or condition is a viral infection.
- the infection site has an increased expression level of PLA2G2D.
- the method further comprises administering a second agent.
- the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomoduiator, an anti-angiogenesis agent, a growth inhibitory' agent, and an antineoplastic agent.
- the second agent is an immunomoduiator.
- the immunomoduiator is an immune checkpoint inhibitor.
- the immune checkpoint inhibitor specifically target PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIMS, LAGS, CD27, 4-1BB, or B7H4.
- the second agent comprises a cell comprising a chimeric antigen receptor that specifically binds to a tumor antigen.
- the antagonist and the second agent is administered simultaneously or concurrently.
- the antagonist and the second agent is administered sequentially.
- the antagonist and/or the second agent is administered parentally.
- the antagonist is administered to the cancer tissue or infection site directly.
- the antagonist is administered at a dose of about 0.001 pg/kg to about 100 mg/kg.
- the individual has an increased number of immune ceils in the cancer tissue or at the infection site after administration of the antagonist.
- the immune ceils are T cells.
- the T cells are activated T cells.
- the number of immune cells in the cancer tissue or at the infection site is increased by at least about 5% (such as at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold) after administration of the antagonist.
- immune ceils in the cancer tissue or at the infection site produce an increased level of a cytokine after administration of the antagonis t.
- the cytokine is IFNy and/or IL-2.
- the level of the cytokine is increased by at least about 5% (such as at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2- fold, 3-fold, 4-fold, or 5 -fold) after administration of the antagonist.
- FIGS. 1 A-1D shows that PLA2G2D is highly differentially expressed in human (FIG. 1 A) lung adenocarcinoma, (FIG. IB) triple negative breast cancer, (FIG. 1C) live hepatocellular carcinoma, and (FIG. ID) stomach adenocarcinoma. Relative expression and significance ofPD-1, CTLA-4 and TIGIT are also indicated.
- FIG. 2A shows that soluble human PLA2G2D-Fc protein dose-dependently suppresses PBMC derived CD4+ and CD8+ T cell proliferation in the presence of anti-CD3 and anti-CD28 stimulation.
- FIG. 2B shows a quantitative graph of the effect of human PLA2G2D-Fe protein on PBMC-derived CD4+ and CD8+ T cell proliferation.
- FIGS. 3A-3C show that soluble PLA2G2D protein dose-dependently suppresses T cell proliferation in different PBMC donors in the presence of anti-CD3 and anti-CD28 stimulation.
- T cell CFSE proliferation as analyzed by flow cytometry is shown on the left, and quantitative representations of percent CD4+ and CD8+ T cell proliferation are shown on the nght.
- FIGS, 4A-4B show that soluble PLA2G2D protein dose-dependently suppresses IFN ⁇ and IL-2 levels in correlation with suppression of T cell proliferation across different donors.
- FIG. 5 shows that immobilized PLA2G2D protein dose-dependently suppresses T cell proliferation in PBMC culture in the presence of anti-CD3 and anti-CD28 stimulation.
- T cell CFSE proliferation as analyzed by flow cytometry is shown on the left, and quantitative representations of percent ⁇ 1)4 and CD8+ T cell proliferation are shown on the right.
- FIG. 6 show's that immobilized PLA2G2D protein dose-dependently suppresses proliferation of isolated T cell cultures in the presence of anti ⁇ CD3 and anti-CD28 stimulation.
- T cell CFSE proliferation as analyzed by flow cytometry is shown on the left, and quantitative representations of percent CD4+ and CD8+ T cell proliferation are shown on the right.
- FIG. 7A shows structural and functional features of in terest of the human PLA2G2D protein (SEQ ID NO: 22) including its signal peptide (the first 20 ammo acids), calcium binding sites, catalytic sites, N-linked glycosylation site, and active site.
- a H67Q catalytic site mutant was generated to create an enzymatic-deficient PLA2G2D protein.
- FIGS. 7B-7C show that aH47Q-PLA2G2D catalytic mutant retains most of the immune suppressive functions on CD4+ (7B) and CD8+ (7C) T cells.
- FIG. 8 shows that a general inhibitor for various PLA2 small molecule Inhibitor
- LY315920 does not rescue immune suppression by PLA2G2D.
- FIGS. 9A-9C show that human PLA2G2D-Fc preferentially hinds activated CD4 + and CD8 + T ceils in different donor T cells compared with controi-Fc protein.
- PLA2G2D binds unstimulated T ceils to a small degree, but binding is dramatically increased upon T cell stimulation.
- FIG. 9C shows a quantitative representation of PLA2G2D ⁇ Fc binding to stimulated T cells.
- FIG. 10A shows a predicted automated 3G structure based upon Swiss-model.
- FIG. 10B shows sequence homology of different PLA2 Group 2 family members to PLA2G2D. SEQ ID NOs from top to bottom are SEQ ID NOs 22-32.
- FIG. IOC shows sequence homology of PLA2G2D from different species vs. human. Human (SEQ ID NO: 33), Mouse (SEQ ID NO: 34), Rat (SEQ ID NO: 35), Rhesus (SEQ ID NO: 36), and Chimp (SEQ ID NO: 37).
- FIG. GIB show's the tumor growth kinetics of individual animals from each group.
- FIG. 11D show's the tumor growth kinetics of individual animals from each group.
- FIG. 1 IF shows the tumor growth kinetics of individual animals from each group.
- FIG. 12A shows that the mean fluorescence intensity (MFI) of PLA2G2D ⁇ Fc binding to activated T cells in PBMC culture can be blocked by anti-PLA2G2D antibodies.
- FIGS. 12B-12C show that PLA2G2D-Fc mediated suppression of IL-2 and IFNy levels in T cell-activated PBMC cultures can he reversed by the addition of function-blocking anti-PLA2G2D antibodies.
- the present application in one aspect pro vides methods of treating a disease or condition (such as cancer or infectious disease) that involves administering an antagonist targeting PLA2G2D signaling pathway.
- the antagonist comprises an agent binding to PLA2G2D (such as an agent comprising an anti-PL, A2G2D antibody moiety).
- the antagonist comprises an inhibitory PLA2G2D polypeptide.
- the antagonist comprises a nucleic acid agent targeting PLA2G2D (such as a siRNA or antisense RNA).
- the antagonist comprises an agent that inhibits PLA2G2D enzymatic activity.
- the present application in another aspect provides non-naturally occurring polypeptides such as the inhibitory PLA2G2D polypeptides that can he used for treatment.
- the present application is at least partly based upon the striking finding that PLA2G2D plays a crucial role in suppressing a key player in immune system, T cells. Specifically, it was found that PLA2G2D was expressed 56 times higher in CD8+ high tumors than CD8+ low' tumors. As shown in Examples in more details, PLA2G2D can both directly and indirectly (e.g., via cross-linking with antigen-presenting cells) inhibit both CB4-t- and CD8+ T cells" proliferation, activation and/or cytokine production, which can lead to a significant level of suppression of immune response m diseases such as cancer, especially in the diseased tissue such as a cancer tissue.
- PLA2G2D enzymatic activity only partially contributes to its role in suppressing immune response, and that PLA2G2D can directly hind to T cells, especially activated T cells. Without being bound to the theory, it is believed that at least part of the suppressive function of PLA2G2D on T cells is exerted by the binding of PLA2G2D to the cells.
- the present application has for the first time promising novel methods of using an antagonist that targets PLA2G2D pathway (such as an agent comprising an anti ⁇ PLA2G2D antibody moiety' or an inhibitory' PLA2G2D polypeptide) to treat a disease or condition in which the immune response is suppressed, including, for example, cancer and infectious disease (such as viral infectious disease).
- wild type is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
- variable should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature.
- nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.
- expression refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins.
- Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
- therapeutic agent refers to a molecule or compound that confers some beneficial effect upon administration to an individual.
- the beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.
- antibody is used in its broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, chimeric antibodies, full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity.
- Antibodies and/or antibody fragments may be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, eamelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camebzed antibody variable domains.
- Percent (%) amino acid sequence identity or “homology” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identify'. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.
- % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5): 1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1): 113, 2004, each of which are incorporated herein by reference in their entirety for all purposes).
- “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or ammo acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous.
- the DNA sequences ATTGCC and TATGGC share 50% homology'. Generally, a comparison is made when two sequences are aligned to give maximum homolog ⁇ '.
- epitope refers to the specific group of atoms or amino acids on an antigen to which an antibody or diabody binds. Two antibodies or antibody moieties may bind the same epitope within an antigen if they exhibit competitive binding for the antigen.
- polypeptide or “peptide” are used herein to encompass ah kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, forrnylation, amidation, polyglutamylation, ADP-ribosyiation, pegylalion, biotinylation, etc.).
- modified proteins e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, forrnylation, amidation, polyglutamylation, ADP-ribosyiation, pegylalion, biotinylation, etc.
- the terms “specifically binds,” “specifically recognizing,” and “is specific for” refer to measurable and reproducible interactions, such as binding between a target and an antibody (such as a diabody).
- specific binding is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules (e.g., cell surface receptors).
- an antibody that specifically recognizes a target is an antibody (such as a diabody) that binds this target with greater affinity, avidity, more readily, and/or with greater duration than its bindings to other molecules.
- the extent of binding of an antibody to an unrelated molecule is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA).
- an antibody that specifically binds a target has a dissociation constant (KD) of ⁇ 10 -5 M, ⁇ 10 '6 M, ⁇ 10 -7 M, ⁇ 10 -8 M, ⁇ 10 -5 M, ⁇ 10 -10 M, ⁇ 10 -11 M, or ⁇ 10 -12 M.
- KD dissociation constant
- an antibody specifically binds an epitope on a protein that is conserved among the protein from different species.
- specific binding can include, but does not require exclusive binding.
- Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA, RIA, ECL, IRMA, ETA, BIACORETM and peptide scans.
- compositions includes and is applicable to compositions of the application.
- the application also provides pharmaceutical compositions comprising the components described herein.
- treatment or “treating” is an approach for obtaining beneficial or desired results including clinical results.
- beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g, preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival.
- the methods of the application contemplate any one or more of these aspects of treatment.
- the benefit to an individual to be treated is either statistically significant or at least perceptible to the patient or to the physician.
- the term “effective amount” used herein refers to an amount of an agent or composition sufficient to treat a specified state, disorder, condition, or disease such as ameliorate, palliate, lessen, and/or delay one or more of its symptoms (e.g., clinical or sub- clinical symptoms).
- beneficial or desired results include, e.g., decreasing one or more symptoms resulting from the disease (biochemical, histologic and/or behavioral), including its complications and intermediate pathological phenotypes presenting during development of the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication, delaying the progression of the disease, and/or prolonging survival of patients, in reference to a cancer, an effective amount comprises an amount sufficient to cause a cancer tissue to shrink and/or to decrease the growth rate of the cancer tissue or to prevent or delay other unwanted cell proliferation in the cancer. In some embodiments, an effective amount is an amount sufficient to delay development of a cancer. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. In the case of cancer, the effective amount of the drug or composition may: (i) reduce the number of tumor cells:
- the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The exact amount required will vary from individual to individual, depending on the species, age, and general condition of the individual, the se verity of die condition being treated, the particular drug or drugs employed, the mode of administration, and the like.
- 'simultaneous administration means that a first therapy and second therapy in a combination therapy are administered with a time separation of no more than about 15 minutes, such as no more than about any of 10, 5, or 1 minutes.
- the first and second therapies may be contained in the same composition (e.g, a composition comprising both a first and second therapy) or in separate compositions (e.g., a first therapy in one composition and a second therapy is contained in another composition).
- the term “sequential administration” means that the first therapy and second therapy in a combination therapy are administered with a time separation of more than about 15 minutes, such as more than about any of 20, 30, 40, 50, 60, or more minutes. Either the first therapy or the second therapy may be administered first.
- the first and second therapies are contained in separate compositions, which may be contained in the same or different packages or kits.
- the term “concurrent administration” means that the administration of the first therapy and that of a second therapy in a combination therapy overlap with each other.
- pharmaceutically acceptable or “pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.
- Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and/or are included on the Inactive ingredient Guide prepared by the U.S. Food and Drug administration or other state/federal government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.
- carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
- Such pharmaceutical carriers can he sterile liquids, such as water and oils, including those of petroleum, animal , vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
- Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
- the earner can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant.
- Suitable pharmaceutical carriers are described in ‘ ‘ Remington’s Pharmaceutical Sciences” by E.W. Martin, incorporated by reference in its entirety for all purposes.
- tumor refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth and includes benign or malignant abnormal growth of tissue.
- tumor includes cancer.
- the terms “individual,” “subject,” and “patient” are used interchangeably herein to refer to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate, in some embodiments, the individual is a human. In a preferred embodiment, the individual is a human.
- Reference to "about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se.
- description referring to "about X” includes description of "X".
- a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range.
- the allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.
- the present application in one aspect provides methods of treating a disease or condition (such as cancer or infectious disease) in an individual, comprising administering to the individual an effective amount of an antagonist that targets PLA2G2D signaling pathway, in some embodiments, the antagonist comprises an agent binding to PLA2G2D (such as an agent comprising an anti ⁇ PLA2G2D antibody moiety). In some embodiments, the antagonist comprises an inhibitory PLA2G2D polypeptide. In some embodiments, the antagonist comprises a nucleic acid agent targeting PLA2G2D (such as a siRNA or antisense RNA). In some embodiments, the antagonist comprises an agent that inhibits PLA2G2D enzymatic activity .
- a method of treating a cancer comprising administering into the individual an effective amount of an antagonist that comprises an agent that inhibits PLA2G2D (such as an agent that blocks the binding of PLA2G2D to an immune cell or an agent that inhibits the activity of PLA2G2D).
- an antagonist that comprises an agent that inhibits PLA2G2D (such as an agent that blocks the binding of PLA2G2D to an immune cell or an agent that inhibits the activity of PLA2G2D).
- the immune cell is a T cell (such as an activated T cell, such as activated CD4+ T cells or CD8+ T cells), in some embodiments, the antagonist comprises an anti-PLA2G2D antibody.
- the anti-PLA2G2D antibody is a monoclonal antibody.
- the antagonist is a fusion protein or immunoconj ugate comprising an anti ⁇ PLA2G2D antibody moiety' and a second moiety', such as a second moiety' comprising a cytokine (such as a pro-inflammatory cytokine).
- the PLA2G2D is a human PLA2G2D.
- the cancer tissue has an increased expression level of PLA2G2D as compared to a reference tissue (such as a corresponding tissue in a healthy individual). In some embodiments, the cancer is an advanced or malignant tumor.
- the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, renal cancer, prostate cancer, urothelial cancer, testis cancer, ovarian cancer and melanoma.
- the method further comprises administering a second agent.
- the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulator, an anti-angiogenesis agent, a growth inhibitory agent, and an antineoplastic agent.
- the second agent is ait immunomodulator.
- the immunomodulator is an immune checkpoint inhibitor.
- the immune checkpoint inhibitor specifically target PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIMS,
- the second agent comprises a ceil comprising a chimeric antigen receptor that specifically binds to a tumor antigen.
- the antagonist and the second agent is administered simultaneously or concurrently.
- the antagonist and the second agent is administered sequentially.
- the antagonist and/or the second agent is administered parentally.
- the antagonist is administered to diseased tissue directly.
- a method of treating an infectious disease comprising administering into the individual an effecti ve amount of an antagonist that comprises an agent that inhibits PLA2G2D (such as an agent that blocks the binding of PLA2G2D to an immune cell or an agent that inhibits the activity' of PLA2G2D).
- the immune cell is a T cell (such as ait activated T cell, such as activated CD4+ T cells or CD8+ T cells).
- the antagonist comprises an anti-PLA2G2D antibody, in some embodiments, the anti-PLA2G2D antibody is a monoclonal antibody.
- the antagonist is a fusion protein or immunoconjugate comprising an anii-PLA2G2D antibody moiety and a second moiety, in some embodiments, the second moiety comprises a cytokine (such as a pro-inflammatory cytokine).
- the PLA2G2D is a human PLA2G2D.
- the infection site has an increased expression level of PLA2G2D as compared to a reference tissue (such as a corresponding tissue in a healthy individual).
- the method further comprises administering a second agent.
- the second agent comprises an immune therapy.
- the antagonist and the second agent is administered simultaneously or concurrently.
- the antagonist and the second agent is administered sequentially.
- the antagonist and/or the second agent is administered parentally.
- the antagonist is administered to diseased tissue directly.
- a method of treating a cancer comprising administering into the individual an effective amount of an antagonist comprises an inhibitory PLA2G2D polypeptide that inhibits PLA2G2D (such as an inhibitory polypeptide that blocks the binding of PLA2G2D to an immune cell).
- an inhibitory PLA2G2D polypeptide that inhibits PLA2G2D (such as an inhibitory polypeptide that blocks the binding of PLA2G2D to an immune cell).
- the inhibitory PLA2G2D polypeptide binds to the immune cell with a greater affinity' than for PLA2G2D (such as a wildtype PLA2G2D).
- the immune cell is a T ceil (such as an activated T cell, such as activated CD4+ T cells or CD 8+ T cells).
- the inhibitory' PLA2G2D polypeptide further comprises a stabilizing domain.
- the stabilizing domain is an Fc domain.
- the inhibitory PLA2G2D polypeptide has a length of about 50 to about 200 amino acids.
- the inhibitory' PLA2G2D polypeptide has a) a mutation at the position corresponding to histidine at position 67 (H67) according to 8EQ ID NO: 1 or 5 or b) a mutation at the position corresponding to glycine at position 80 (G80) according to SEQ ID NO: 5.
- the inhibitory' PLA2G2D polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, and 7-12 or a variant thereof.
- the PLA2G2D is a human PLA2G2B.
- the cancer tissue has an increased expression level of PLA2G2D as compared to a reference tissue (such as a corresponding tissue in a healthy individual).
- the cancer is an advanced or malignant tumor.
- the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, renal cancer, prostate cancer, urothelial cancer, testis cancer, ovarian cancer and melanoma
- the method further comprises administering a second agent, in some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulator, an anti-angiogenesis agent, a growth inhibitory agent, and an antmeoplastic agent.
- the second agent is an immunomodulator.
- the immunomodulator is an immune checkpoint inhibitor.
- the immune checkpoint inhibitor specifically target PD-L1, PD-L2, CTLA4, PD-1,2, PD-1, CD47, ! Kil l GITR, TIMS, LAGS, CD27, 4-IBB, or B7H4.
- the second agent comprises a cell comprising a chimeric antigen receptor that specifically binds to a tumor antigen.
- the antagonist and the second agent is administered simultaneously or concurrently.
- the antagonist and the second agent is administered sequentially.
- the antagonist and''orthe second agent is administered parentally, in some embodiments, the antagonist is administered to diseased tissue directly.
- a method of treating an infections disease comprising administering into the individual an effective amount of an antagonist comprises an inhibitory PLA2G2D polypeptide that blocks the binding of PLA2G2D to an immune cell
- the inhibitory PLA2G2D polypeptide binds to the immune cell with a greater affinity' than for PLA2G2D (such as a wildtype PLA2G2D).
- the immune cell is a T cell (such as an activated T ceil, such as activated CD4+ T cells or CD8+ T cells).
- the inhibitor ⁇ ' PLA2G2D polypeptide further comprises a stabilizing domain, in some embodiments, the stabilizing domain is an Fe domain, in some embodiments, the inhibitory PLA2G2D polypeptide has a length of about 50 to about 200 amino acids. In some embodiments, the inhibitory PLA2G2D polypeptide has a) a mutation at the position corresponding to histidine at position 67 (H67) according to SEQ ID NO: 1 or 5 or b) a mutation at the position corresponding to glycine at position 80 (G80) according to SEQ ID NO: 5.
- the inhibitor ⁇ ' PLA2G2D polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, and 7-12 or a variant thereof.
- the PLA2G2D is a human PLA2G2D.
- the infection site has an increased expression level of PLA2G2D as compared to a reference tissue (such as a corresponding tissue in a healthy individual).
- tire method further comprises administering a second agent, in some embodiments, the second agent comprises an immune therapy.
- the antagonist and the second agent is administered simultaneously or concurrently, in some embodiments, the antagonist and the second agent is administered sequentially, in some embodiments, the antagonist and/or the second agent is administered parentally.
- the antagonist is administered to diseased tissue directly.
- a method of treating a cancer comprising administering into the individual an effective amount of an antagonist that comprises a nucleic acid agent that inhibits the expression of PLA2G2D.
- the nucleic acid agent comprises a siRNA, a miRNA, or an antisense RNA.
- the PLA2G2D is a human PLA2G2D.
- the cancer tissue has an increased expression level of PLA2G2D as compared to a reference tissue (such as a corresponding tissue in a healthy individual).
- the cancer is an advanced or malignant tumor.
- the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, renal cancer, prostate cancer, urothelial cancer, testis cancer, ovarian cancer and melanoma.
- the method further comprises administering a second agent.
- the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodtilator, an anti-angiogenesis agent, a growth inhibitory agent, and an antmeoplastic agent, in some embodiments, the second agent is an immunomodulator.
- the immunomodulator is an immune checkpoint inhibitor.
- the immune checkpoint inhibitor specifically target PD-LL PD-L2, CTLA4, PB-L2, PD-1, CD47, TIGIT, GITR, TIMS, LAGS, CD27, 4-1BB, or B7H4.
- the second agent comprises a cell comprising a chimeric antigen receptor that specifically binds to a tumor antigen.
- the antagonist and the second agent is administered simultaneously or concurrently.
- the antagonist and the second agent is administered sequentially.
- the antagonist and/or the second agent is administered parentally, in some embodiments, the antagonist is administered to diseased tissue directly.
- a method of treating a cancer comprising administering into the individual an effective amount of an antagonist that comprises a nucleic acid agent that inhibits the expression of PLA2G2D, wherein the individual has high T cell infiltration in cancer tissue
- the high T cell infiltration comprises a high number, percentage or density of T cells (e.g., CD3 T cells, CD4 T cells, CD8 T cells, activated T cells, activated CD4 T cells, activated CD8 T cells) in the cancer tissue
- the high T cell infiltration is present when the number of the T cells in the cancer is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% more than the number of the corresponding T cells in a reference tissue, in some embodiments, the high T cell infiltration is present when
- the reference tissue is the corresponding tissue in a healthy individual.
- the number of the corresponding T ceils in a reference tiss ue is the average number of the corresponding T cells in the same tissue in a group of individuals (such as 10, 30, 50, 100 individuals) with same or similar cancer.
- the reference tissue is the corresponding tissue in an individual who also has a cancer but has a less suppressed immune response in the cancer tissue as indicated by a biomarker.
- biomarker indicative of immunosuppressive tumor microenvironment includes: a) a high number, percentage and/or density of M2 macrophages (e.g., CD68+CD163+ cells) in the tissue; b) a high expression level of an immune checkpoint agent (e.g., PD-1 or PD-L1).
- an immune checkpoint agent e.g., PD-1 or PD-L1.
- the nucleic acid agent comprises a siRNA, a miRNA, or an antisense RNA.
- the PLA2G2D is a human PLA2G2D.
- the cancer tissue has an increased expression level of PLA2G2D as compared to a reference tissue (such as a corresponding tissue in a healthy individual), in some embodiments, the cancer is an advanced or malignant tumor.
- the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, renal cancer, prostate cancer, urothelial cancer, testis cancer, ovarian cancer and melanoma.
- the method further comprises administering a second agent, in some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulator, an anti-angiogenesis agent, a growth inhibitory agent, and an antineoplastic agent. In some embodiments, the second agent is an immunomodulator.
- the immunomodulator is an immune checkpoint inhibitor, in some embodiments, the immune checkpoint inhibitor specifically target PD-Ll, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, T ⁇ M3, LAGS. CD27, 4-1BB, or B7H4.
- the second agent comprises a cell comprising a chimeric antigen receptor that specifically binds to a tumor antigen, in some embodiments, the antagonist and the second agent is administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent is administered sequentially. In some embodiments, the antagonist and/or the second agent is administered parentally. In some embodiments, the antagonist is administered to diseased tissue directly.
- a, method of treating a, cancer comprising administering into the individual an effective amount of an antagonist that comprises a nucleic acid agent that inhibits the expression of PLA2G2D, wherein the individual has a, high expression level of PLA2G2D in the cancer tissue.
- the cancer tissue has a high expression level of PLA2G2D when the expression level of PLA2G2D (e.g., assessed by immunohistochemistry) is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% higher than the expression level of PLA2G2D in a reference tissue.
- the expression level of PLA2G2D e.g., assessed by immunohistochemistry
- the cancer tissue has a high expression level of PLA2G2D when the expression level of PLA2G2D (e.g., assessed by immunohistochemistry) is at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7 -fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold higher than the expression level of PLA2G2D in a reference tissue.
- the reference tissue is the corresponding tissue in a healthy individual.
- the expression level of PLA2G2D in a reference tissue is the average expression level of PLA2G2D in the same tissue in a group of individuals (such as 10, 30, 50, 100 individuals) with same or similar cancer.
- the reference tissue is the corresponding tissue in an individual who also has a cancer but has a less suppressed immune response in the cancer tissue as indicated by a biomarker (such as high M2 macrophages, or high expression of an immune checkpoint agent such as PD-1 or PD-Ll).
- the nucleic acid agent comprises a siRNA, a miRNA, or an antisense RNA.
- the PLA2G2D is a human PLA2G2D.
- the cancer tissue has an increased expression level of PLA2G2D as compared to a reference tissue (such as a corresponding tissue in a healthy individual), in some embodiments, the cancer is an advanced or malignant tumor.
- the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, renal cancer, prostate cancer, urothelial cancer, testis cancer, ovarian cancer and melanoma.
- the method further comprises administering a second agent, in some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulator, an anti-angiogenesis agent, a growth inhibitor ⁇ ' agent, and an antineoplastic agent.
- the second agent is an immunomodulator.
- the immunomodulator is an immune checkpoint inhibitor, in some embodiments, the immune checkpoint inhibitor specifically target PD-L1, PDA 2. CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIM3,
- the second agent comprises a cell comprising a chimeric antigen receptor that specifically binds to a tumor antigen.
- the antagonist and the second agent is administered simultaneously or concurrently.
- the antagonist and the second agent is administered sequentially.
- the antagonist and/or the second agent is administered parentally.
- the antagonist is administered to diseased tissue directly.
- a method of treating a cancer comprising administering into the individual an effective amount of an antagonist that comprises a nucleic acid agent that inhibits the expression of PLA2G2D, wherein the individual has a) a high T cell infiltration (e.g., CDS T cells, e.g,, CD4 T cells, e.g., CDS T cells, e.g., activated CDS or CD4 or CDS T cells) in the cancer tissue, and/or b) a high PLA2G2D expression in the cancer tissue.
- a cancer such as a solid tumor, a colon cancer, melanoma, or a T ceil lymphoma
- an antagonist that comprises a nucleic acid agent that inhibits the expression of PLA2G2D
- the individual has a) a high T cell infiltration (e.g., CDS T cells, e.g, CD4 T cells, e.g., CDS T cells, e.g., activated CDS or CD4 or CDS
- the methods described herein further comprise selecting an individual for treatment based upon high T cell infiltration (e.g., high CDS T cells, high CDS T cells, high CD4 T ceils, activated T cells, activated CDS T ceils, or activated CD4 T ceils) in the cancer tissue.
- High T cell infiltration can be determined by a) assessing the number of T cells (e.g., CDS T ceils, CD4 T cells, CDS T cells, activated T cells, activated CD4 T cells, activated CDS T ceils) in tumor, and b) comparing the number to the number of corresponding T cells in a reference tissue.
- the high T cell infiltration is present when the number of the T cells in the cancer is at least about 5%, 10%. 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% more than the number of the corresponding T cells in a reference tissue. In some embodiments, the high T cell infiltration is present when the number of the T cells in the cancer is at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-foid, 6-fold, 7 -fold, 8-fold, 9-fold, or 10-fold more than the number of the corresponding T cells in a reference tissue. In some embodiments, the reference tissue is the corresponding tissue in a healthy individual.
- the number of the corresponding T cells in a reference tissue is the average number of the corresponding T cells in the same tissue in a group of individuals (such as 10, 30, 50, 100 individuals) with same or similar cancer.
- the reference tissue is the corresponding tissue in an individual who also has a cancer but has a less suppressed immune response in the cancer tissue as indicated by a biomarker.
- biomarker indicative of immunosuppressive tumor microenvironment includes: a) a high number, percentage and/or density of M2 macrophages (e.g., CD68+CD163+ cells) in the tissue; b) a high expression level of an immune checkpoint agent (e.g., PD-1 or PD-Ll).
- the methods described above further comprise selecting an individual for treatment based upon a high expression level of PLA2G2D in the cancer tissue.
- the cancer tissue has a high expression level of PLA2G2D when the expression level of PLA2G2D (e.g., assessed by immunohistochemistry) is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% higher than the expression level of PLA2G2D in a reference tissue.
- the cancer tissue has a high expression level of PLA2G2D when the expression level of PLA2G2D (e.g., assessed by immunohistochemistry') is at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6- fo!d, 7 -fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold higher than the expression level of PLA2G2D in a reference tissue.
- the reference tissue is the corresponding tissue in a healthy individual.
- the expression level of PLA2G2D in a reference tissue is the average expression level of PLA2G2D in the same tissue in a group of individuals (such as 10, 30, 50, 100 individuals) with same or similar cancer.
- the reference tissue is the corresponding tissue in an individual who also has a cancer but has a less suppressed immune response in the cancer tissue as indicated by a biomarker (such as high M2 macrophages, or high expression of an immune checkpoint agent such as PD-1 or PD-L1).
- the methods described herein comprise selecting an individual for treatment, wherein the individual has a) a high T cell infiltration (e.g., CD3 T ceils, e.g., CD4 T cells, e.g., CDS T cells, e.g., activated CD3 or CD4 or CDS T cells) m the cancer tissue, and/or b) a high PLA2G2D expression in the cancer tissue.
- a high T cell infiltration e.g., CD3 T ceils, e.g., CD4 T cells, e.g., CDS T cells, e.g., activated CD3 or CD4 or CDS T cells
- a method of treating an infectious disease comprising administering into the individual an effective amount of an antagonist that comprises a nucleic acid agent that inhibits the expression of PLA2G2D.
- the nucleic acid agent comprises a siRNA, a miRNA, or an antisense RNA.
- the PLA2G2D is a human PLA2G2D.
- the infection site has an increased expression level of PLA2G2D as compared to a reference tissue (such as a corresponding tissue in a healthy individual).
- the method further comprises administering a second agent.
- the second agent comprises an immune therapy.
- the antagonist and the second agent is administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent is administered sequentially. In some embodiments, the antagonist and/or the second agent is administered parentally. In some embodiments, the antagonist is administered to diseased tissue directly.
- a method of treating a cancer comprising administering into the individual an effective amount of an antagonist decreasing enzymatic activity level of PLA2G2D.
- the antagonist targeting PLA2G2D signaling pathway blocks a catalytic site on PLA2G2D.
- the PLA2G2D is a human PLA2G2D.
- the antagonist comprises an agent that specifically inhibits the cataly tic His67-Asp68 Dyad of human PLA2G2D as set forth in 8EQ ID NO: 1 or 5.
- the antagonist targets the H67 catalytic site on a human PLA2G2D according to SEQ ID NO: 1 or 5.
- the agent interferes with the binding of calcium to PLA2G2D.
- the agent blocks the binding of calcium to residues at one or more o ⁇ ' 1147. G49, G51, and D68 according to SEQ ID NO: 1 or 5.
- the antagonist comprises an agent that specifically decreases enzymatic activity of the catalytic His67-Asp68 Dyad of human PLA2G2D as set forth in SEQ ID NO: I or 5 by at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%,
- the cancer tissue has an increased expression level of PLA2G2D as compared to a reference tissue (such as a corresponding tissue in a healthy individual).
- the cancer is an advanced or malignant tumor, in some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, renal cancer, prostate cancer, urothelial cancer, testis cancer, ovarian cancer and melanoma.
- the method further comprises administering a second agent.
- the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulator, an anti -angiogenesis agent, a growth inhibitory agent, and an antineoplastic agent.
- the second agent is an immunomodulator.
- the immunomodulator is an immune checkpoint inhibitor.
- the immune checkpoint inhibitor specifically target PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, T1GIT, GITR, TIM3, LAG3, CD27, 4-1BB, or B7H4.
- the second agent comprises a cell comprising a chimeric antigen receptor that specifically hinds to a tumor antigen.
- the antagonist and the second agent is administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent is administered sequentially. In some embodiments, the antagonist and/or the second agent is administered parentally. In some embodiments, the antagonist is administered to diseased tissue directly.
- a method of treating an infectious disease comprising administering into the individual an effective amount of an antagonist decreasing enzymatic activity level of PLA2G2D.
- the PLA2G2D is a human PLA2G2D.
- the antagonist targeting PLA2G2D signaling pathway blocks a catalytic site on PLA2G2D.
- the antagonist comprises an agent that specifically inhibits the catalytic His67-Asp68 Dyad of human PLA2G2D as set forth in SEQ ID NO: 1 or 5.
- the antagonist targets the H67 catalytic site on a human PLA2G2D according to SEQ ID NO: 1 or 5.
- the agent interferes with the binding of calcium to PLA2G2D.
- the agent blocks the binding of calcium to residues at one or more of H47, G49, G51, and D68 according to SEQ ID NO: 1 or 5.
- the antagonist comprises an agent that specifically decreases enzymatic activity of the catalytic His67-Asp68 Dyad of human PLA2G2D as set forth in SEQ ID NO:
- the infection site has ait increased expression level of PLA2G2D as compared to a reference tissue (such as a corresponding tissue in a healthy individual), in some embodiments, the method further comprises administering a second agent.
- the second agent comprises an immune therapy.
- the antagonist and the second agent is administered simultaneously or concurrently, in some embodiments, the antagonist and the second agent is administered sequentially, in some embodiments, the antagonist and/or the second agent is administered parentally. In some embodiments, the antagonist is administered to diseased tissue directly.
- the administration of the antagonists described herein can also be useful for promoting local immune response, promoting proliferation and/or activation of immune cells (such as T cells), and promoting a favorable tumor microenvironment.
- a method of promoting proliferation and/or activation of T cells in an infection site in an individual having an infection comprising administering any of the antagonists described herein.
- the T cells are CD4+ T cells.
- the T cells are CD8+ T cells.
- a method of promoting a favorable tumor microenvironment in a cancer tissue in an individual having a cancer comprising administering any of the antagoni sts described herein.
- a method of promoting a favorable microenvironment in an infection site m an individual having an infection comprising administering any of the antagonists described herein.
- “Promoting favorable tumor microenvironment” generally refers to or comprises conversion of a tumor tissue that is resistant to a cancer therapy (such as an immunotherapy) to a tumor tissue that is less resistant to the cancer therapy.
- Tiie antagonist may be any of an antibody, a polypeptide, a peptide, a polynucleotide, apeptidomimeiic, a natural product, a carbohydrate, an aptamer an avimer, an anticalm, a speigelmer, or a small molecule that targets (i.e., inhibits or downregulates) PLA2G2D signaling pathway.
- the antagonist targets (i.e., inhibits or downregulates) PLA2G2D.
- the antagonist is a fusion protein (such as a fusion protein that comprises a half-life extending domain (e.g., an Fc domain)).
- PLA2G2D (phospholipase A2 group III), sPLA2-IID) is a secreted member of the phospholipase A2 family. Phospholipase A2 family members hydrolyze the sn-2 fatty acid ester bond of giycerophospholipids to produce lysophospholipids and free fatty acid. To date, 10 sPLA2isoforms (IB, II A, IIC, IID, TIE, IIF, III, V, X, and XII) have been identified in mammals.
- isoforms except for group III isoforms, have a highly conserved catalytic site, a Ca 2+ binding loop, and a common molecular weight of 14-19 kDa.
- sPLA2isoforms sPLA2-IIA, sPLA2-lIC, sPI,A2 ⁇ IID, sPLA2-IIE, sPLA2-IIF, and sPLA 2-V have the same chromosomal locus (Ip34-p36), which are often referred to as group II subfamily sPLA2.
- the biological feature of the group II subfamily sPLA2 is that almost all isoforms, except sPLA 2-IIC (a pseudogene in humans), are associated with inflammatory and immune processes.
- PLA2G2D is a basic protein (pl ⁇ 8,7) with 14 cysteines at exactly conserved positions. Likely because of its cationic nature, PLA2G2D binds to heparin in vitro or heparin sulfate on the cell surface when overexpressed in cultured cells.
- the PLA2G2D comprises an ammo acid sequence set forth in SEQ ID NO: I or 2. In some embodiments, the PLA2G2D comprises an amino acid sequence set forth in SEQ ID NO: 5 or 6.
- the antagonist decreases expression level of PLA2G2D. In some embodiments, the antagonist decreases enzymatic activity level of PLA2G2D. In some embodiments, the anti-PLA2G2D antibody does not completely inhibit or block the catalytic activity of PLA2G2D (such as blocking the catalytic activity no more than about 90%), 8Q%, 7Q%, 60%, 50%, 40%), 30 ( 1 ⁇ 2, 20% or 10% of the full catalytic activity). In some embodiments, the anti-PLA2G2D antibody does not inhibit or block the catalytic activity of PLA2G2D.
- the antagonist comprises an agent that inhibits PLA2G2D (such as an agent that blocks the binding of PLA2G2D to an immune cell or an agent that inhibits the activity of PLA2G2D) (such as a T cell, such as an activated T cell, such as an activated CD4+ T cell, such as an activated CD8+- T cell).
- an agent that inhibits PLA2G2D such as an agent that blocks the binding of PLA2G2D to an immune cell or an agent that inhibits the activity of PLA2G2D
- a T cell such as an activated T cell, such as an activated CD4+ T cell, such as an activated CD8+- T cell.
- the antagonist is an agent that recognizes and binds specifically to PLA2G2D.
- the agent comprises an anti-PLA2G2D antibody moiety (such as an anii-PLA2G2D antibody).
- the anti-PL A2G2D antibody moiety blocks or decreases the binding of PLA2G2D to an immune cell. In some embodiments, the anti-PLA2G2D antibody moiety decreases the binding of PLA2G2D to an immune cell by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the binding of the PLA2G2D to an immune cell is independent from binding through heparin sulfate on cell surface.
- the PLA2G2D recognized by the anti-PLA2G2D antibody is a human PLA2G2D.
- the human PLA2G2D comprises or has the amino acid sequence of SEQ ID NO: 1 or a natural variant of human PLA2G2D.
- the natural variant of human PLA2G2D is derived from a tumor tissue, in some embodiments, the natural variant of human PLA2G2D is derived from a virus infection site.
- the anti-PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two, three, four, or five of) Q65, H73, S80, 1196. and R121 according to SEQ ID NO: 1.
- the anti-PLA2G2D antibody moiety 7 binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids from R121 to Cl 45 according to SEQ ID NO: 1. In some embodiments, the anti-PLA2G2D antibody moiety 7 binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids from V32 to A59 according to SEQ ID NO: I.
- the anti-PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids from T60 to T76.
- the an ⁇ i ⁇ PLA2G2D antibody moiety' binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids from Q77 to Y85 according to SEQ ID NO: 1.
- the anti-PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) ammo acids from G21 to Q31 according to SEQ ID NO: 1.
- the anti-PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids from Y86 to W103 according to SEQ ID NO: 1, In some embodiments, the anti-PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) ammo acids from Cl 04 to R121 according to SEQ ID NO: 1. In some embodiments, the epitope is a discontinuous epitope. In some embodiments, the epitope is a continuous epitope.
- Sequence homology ' of human PLA2G2D to different PLA2 group 2 family members is analyzed and shown in FIG. 10B. Sequence homology of human PLA2G2D to PLA2G2D of different species is analyzed and shown m FIG. IOC.
- the anti- PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising one or more residues at positions a) that are different from corresponding residues in other PLA2 group 2 family members and/or b) that are same as PLA2G2D in other species.
- the anti ⁇ PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) ammo acids at the position of 22, 23, 25, 26, 27, or 31 according to SEQ ID NO: 1.
- the anti ⁇ PLA2G2D antibody moiety' binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids at the position of 36, 37, 38, 42, 43, 55, or 59 according to SEQ ID NO: 1.
- the anti-PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids at the position of 62, 65, 66, 72, 73, or 76, according to SEQ ID NO: 1.
- the anti-PL, A2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids at the position of 77, 80, 81, 83, 84, or 85 according to SEQ ID NO: 1.
- the anti-PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids at the position of 87, 89, 90, 92, 93, 94, 96, 98, 99, 100, 101, 102, or 103 according to SEQ ID NO: 1.
- the anti-PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids at the position of 105, 106, 107, 108, 110, 114, 115, 117, 119, or 120 according to SEQ ID NO: 1.
- the anti-PLA2G2D antibody moiety' binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids at the position of 123, 124, 127, 129, 130, 131, 132, 134, 135, 136, 137, 139, 141, 144, or 145 according to SEQ ID NO: 1.
- the anti-PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids ai the position of 22, 26, 31, 36, 42, 43, 72, 73, 76, 77, 80, 81, 83, 85, 87, 89, 90, 92, 94, 96, 100, 101, 102, 103, 106, 110, 114, 115, 117, 120, 134, 135, 136, 141, or 144 according to SEQ ID NO: 1.
- the epitope is a discontinuous epitope. In some embodiments, the epitope is a continuous epitope.
- the agent comprises an anti-PLA2G2D antibody.
- the anti-PLA2G2D antibody is a polyclonal antibody.
- the anti-PL A2G2D antibody is a monoclonal antibody.
- the anti-PLA2G2D antibody is an anti-human PLA2G2D antibody.
- the anti-PLA2G2D antibody is humanized or chimeric.
- the anti-PLA2G2D antibody is a full-length antibody or an immunoglobulin derivative.
- the anli-PLA2G2D antibody is an antigen-bindmg fragment, for example an antigen-binding fragment selected from the group consisting of a single-chain Fv (scFv), a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFvfi, a V H H, a Fv-Fc fusion, a scFv-Fc fusion, a scFv-Fv fusion, a diabody, a tribody, and a letrabody.
- the anti-PLA2G2D antibody is a scFv. In some embodiments, the anti-PLA2G2D antibody is a Fab or Fab’, in some embodiments, the anti-PLA2G2D antibody is chimeric, human, partially humanized, fully humanized, or semi-synthetic. Antibodies and/or antibody fragments may be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, eamelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains. [0100] In some embodiments, the anti-PLA2G2D antibody comprises an Fc fragment (such as any of the Fc fragments described herein).
- the Fc fragment is selected from the group consisting of Fc fragments from IgG, IgA, IgD, IgE, IgM. and combinations and hybrids thereof.
- the Fc fragment is derived from a human IgG.
- the Fc fragment comprises the Fc region of human IgGl, IgG2, IgG3, IgG4, or a combination or hybrid IgG.
- the anti ⁇ PLA2G2D antibody does not completely inhibit or block the catalytic activity of PLA2G2D (such as blocking the catalytic activity no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the full catalytic activity). In some embodiments, the anti-PLA2G2D antibody does not inhibit or block the catalytic activity of PLA2G2D.
- the anti-PLA2G2D antibody blocks the binding of PLA2G2D to a T cell by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.
- the anti-PLA2G2D antibody is capable of restoring T cell activation to at least about 50%, 60%, 70%, 80%, 90%, 95%, or 100%.
- the activation of the T cell can be indicated, for example, by its cytokine secretion level.
- Exemplary cytokines include IL-2 and IFN-v.
- an epitope region for an anti-PLA2G2D antibody may be determined by epitope "foot-printing" using chemical modification of the exposed amines/carboxyls in the PLA2G2D protein.
- a foot-printing technique is the use of HXMS (hydrogen-deuterium exchange detected by mass spectrometry) wherein a hydrogen/deuterium exchange of receptor and ligand protein amide protons, binding, and back exchange occurs, wherein the backbone amide groups participating in protein binding are protected from back exchange and therefore will remain deulerated.
- NMR nuclear magnetic resonance epitope mapping
- the antigen typically is selectively isotopieally labeled with 15N so that only signals corresponding to the antigen and no signals from the antigen binding peptide are seen in the NMR-spectrum.
- Antigen signals originating from amino acids involved in the interaction with the antigen binding peptide typically will shift position in the spectrum of the complex compared to the spectrum of the free antigen, and the amino acids invol v ed in the binding can be identified that way. See, e.g, Ernst Sobering Res Found Workshop. 2004;
- Epitope mapping/characterization also can he performed using mass spectrometry methods. See, e.g., Downard, J Mass Spectrom. 2000 Apr; 35 (4): 493-503 and Kiselar and Downard, Anal Chem. 1999 May 1; 71 (9): 1792-1801, each of which is incorporated herein by reference in their entirety for all purposes.
- Protease digestion techniques also can be useful in the context of epitope mapping and identification. Antigenic determinant-relevant regions/sequences can be determined by protease digestion, e.g.
- trypsin in a ratio of about 1 :50 to PLA2G2D or o/n digestion at and pH 7-8, followed by mass spectrometry (MS) analysis for peptide identification.
- MS mass spectrometry
- the peptides protected from trypsin cleavage by the anti-PL A2G2D binder can subsequently be identified by comparison of samples individuated to trypsin digestion and samples incubated with antibody and then individual ed to digestion by e.g. trypsin (thereby revealing a footprint for the binder).
- Other enzymes like chymotrypsin, pepsin, etc., also or alternatively can he used in similar epitope characterization methods.
- enzy matic digestion can provide a quick method for analyzing whether a potential antigenic determinant sequence is within a region of the PLA2G2D polypeptide (such as a polypeptide set forth in SEQ ID NO: 1 ) that is not surface exposed and, accordingly, most likely not relevant in terms of immunogen! city /antigenicity.
- Site-directed mutagenesis is another technique useful for elucidation of a binding epitope. For example, m "alanine-scanning", each residue within a protein segment is re- placed with an alanine residue, and the consequences for binding affinity' measured, if the mutation leads to a significant reduction in binding affinity', it is most likely involved in binding.
- Monoclonal antibodies specific for structural epitopes i.e., antibodies which do not bind the unfolded protein
- Electron microscopy can also be used for epitope "foot-printing".
- Wang et al, Nature 1992; 355:275-278 used coordinated application of ciyoelectron micros-copy, three-dimensional image reconstruction, and X-ray crystallography to determine the physical footprint of a Fab-fragment on the capsid surface of native cowpea mosaic virus.
- label-free assay for epitope evaluation include surface plasmon resonance (SPR, BIACORE) and ref!ectometric interference spectroscopy (RifS).
- SPR surface plasmon resonance
- RifS ref!ectometric interference spectroscopy
- the agents that bind to PLA2G2D described herein further comprises a second moiety, in some embodiments, the second moiety comprises a therapeutic agent, in some embodiments, the second moiety comprises a label.
- the anti-PLA2G2D antibody moiety and the second moiety is linked via a linker (such as any of the linkers described in the "‘linkers” section).
- the second agent is a cytotoxic agent.
- the cytotoxic agent is a chemotherapeutic agent.
- the cytotoxic agent is a growth inhibitory agent.
- the cytotoxic agent is a toxin (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), in some embodiments, the cytotoxic agent is a radioactive isotype (i.e., a radioconjugage).
- Immunoconjugates allow for the targeted delivery of a drug moiety to a tissues (such as a tumor), and, in some embodiments intracellular accumulation therein, where systemic administration of unconjugated drugs may result in unacceptable levels of toxicity' to normal cells (Polakis P. (2005) Current Opinion in Pharmacology 5:382-387).
- ADC Antibody-drug conjugates
- ADC are targeted chemo therapeutic molecules which combine properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to antigen-expressing tumor cells (Teicher, B. A. (2009) Current Cancer Drug Targets 9:982- 1004), thereby enhancing the therapeutic index by maximizing efficacy and minimizing off- target toxicity (Carter, P. J. and Senter P. B. (2008) The Cancer Jour: 14(3): 154-169; Chan,
- the ADC compounds of the application include those with anticancer activity.
- the ADC compounds include an antibody conjugated, i.e. covalently attached, to the drug moiety, in some embodiments, the antibody is covalently attached to the drug moiety through a linker.
- the second agent is connected to the anti-PLA2G2D antibody moiety via a linker (such as a linker described herein), in some embodiments, the linker is a cleavable. In some embodiments, the linker is non-cleavable.
- the antibody-drug conjugates (ADC) of the application selectively deliver an effective dose of a drug to tumor tissue whereby greater selectivity, i.e. a lower efficacious dose, may be achieved while increasing the therapeutic index (‘'therapeutic window').
- the drug moiety of the antibody-drug conjugates (ADC) may include any compound, moiety or group that has a cytotoxic or cytostatic effect. Drug moieties may impart their cytotoxic and cytostatic effects by mechanisms including but not limited to tubulin binding, DNA binding or intercalation, and inhibition of RNA polymerase, protein synthesis, and/or topoisomerase.
- Exemplary drug moieties include, but are not limited to, a maytansinoid, dolasiatin, auristatin, calicheamicin, pyrrolobenzodiazepine (PBD), nemorubicin and its derivatives, PNU-159682, anthracy dine, duocarmycin, Vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, e!inafkle, and stereoisomers, isos teres, analogs, and derivati ves thereof that have cytotoxic activity.
- PPD pyrrolobenzodiazepine
- nemorubicin and its derivatives PNU-159682, anthracy dine, duocarmycin, Vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, e!inafkle, and stereoisomers, isos teres, analogs, and derivati ves thereof that have
- the agent binds to PLA2G2D comprises a fusion protein that comprises an anti-PLA2G2D antibody moiety and a second moiety .
- the second moiety comprises an Fc fragment (such as any of the Fc fragments described herein).
- the half-life extending moiety- is an albumin binding moiety (e.g., an albumin binding antibody moiety).
- the second moiety comprises a cytokine.
- the cytokine is a proinflammatory cytokine (such as TNF-a, IL-1B, 1L-6, or IL-10).
- the anti-PLA2G2B antibody moiety and the second moiety is linked via a linker (such as any of the linkers described in the “Linkers” section).
- Fc region refers to a C -terminal non-antigen binding region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
- the term includes native Fc regions and variant Fc regions, in some embodiments, a human IgG heavy chain Fc region extends from Cys226 to the carboxyl -terminus of the heavy chain.
- the C -terminal lysine (Lys447) of the Fc region may or may not be present, without affecting the structure or stability of the Fc region.
- numbering of amino acid residues in the IgG or Fc region is according to the EU numbering system for antibodies, also called the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
- the Fc fragment is sel ected from the group consisting of Fc fragments from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc fragment is selected from the group consisting of Fc fragments from IgG I, IgG2, IgG3, IgG4, and combinations and hybrids thereof.
- the Fc fragment has a reduced effector function as compared to corresponding wildtype Fc fragment (such as at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%), or 95%) reduced effector function as measured by the level of antibody-dependent cellular cytotoxicity (ADCC)).
- ADCC antibody-dependent cellular cytotoxicity
- the Fc fragment is an IgGl Fc fragment.
- the IgGl Fc fragment comprises a L234A mutation and/or a L235A mutation.
- the Fc fragment is an IgG2 or IgG4 Fc fragment.
- the Fc fragment is an IgG4 Fc fragment comprising a S228P, F234A, and/or a L235A mutation.
- the Fc fragment comprises a N297A mutation, in some embodiments, the Fc fragment comprises aN297G mutation.
- the an ⁇ i-PLA2G2D immunoconjugates or fusion proteins described herein comprise an anti ⁇ PLA2G2D antibody described herein fused to the second moiety via a linker.
- the length, the degree of flexibility and/or other properties of the linker used in the anti-PLA2G2D immunoconj ugates or fusion proteins may have some influence on properties, including but not limited to the affinity', specificity or avidity of the anti ⁇ PLA2G2D, and/or affinity, specificity or avidity' for one or more particular antigens or epitopes present on PLA2G2D.
- longer linkers may be selected to ensure that two adjacent antibody moieties do not statically interfere with one another.
- a linker (such as peptide linker) comprises flexible residues (such as glycine and serine) so that the adjacent antibody moieties are free to move relative to each other.
- a glycine-serine doublet can be a suitable peptide linker.
- the linker is anon-peptide linker.
- the linker is a peptide linker.
- the linker is a non-cleavable linker.
- the linker is a cleavable linker.
- linker considerations include the effect on physical or pharmacokinetic properties of the resulting an anti-PLA2G2D immunoconj ugate or fusion protein, such as solubility', lipophil icily, hydrophilicity, hydrophobicily, stability (more or less stable as well as planned degradation), rigidity', flexibility ' , immunogemcity, modulation of antibody binding, the ability' to be incorporated into a micelle or liposome, and the like.
- any one or all of the linkers described herein can be peptide linkers.
- the peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence.
- a sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. See, for example, WO 1996/34103, ineorporated by reference in its entirety for all purposes, in some embodiments, the peptide linker comprises the ammo acid sequence of CPPCP, a sequence found in the native IgGl lunge region.
- the peptide linker can be of any suitable length.
- the length of the peptide linker is any of about 1 aa to about 10 aa, about 1 aa to about 20 aa, about 1 aa to about 30 aa, about 5 aa to about 15 aa, about 10 aa to about 25 aa, about 5 aa to about 30 aa, about 10 aa to about 30 aa, about 30 aa to about 50 aa about 50 aa to about 100 aa, or about 1 aa to about 100 aa.
- peptide linker does not comprise any polymerization activity.
- the characteristics of a peptide linker, which comprise the absence of the promotion of secondary structures, are known in the art and described, e.g., in DalFAcquaef al. (Biochem. (1998) 37, 9266-9273), Cheadle el al. (Mol Immunol (1992) 29, 21-30) and Raag and Whitlow (FA8EB (1995) 9(1), 73-80, each incorporated by reference in their entirety for all purposes).
- a particularly preferred amino acid in context of the ‘'peptide linker” is Gly.
- peptide linkers that also do not promote any secondary' structures are preferred.
- the linkage of the molecules to each other can be provided by, e.g., genetic engineering.
- Methods for preparing fused and operatively linked antibody constructs and expressing them in mammalian cells or bacteria are well- known in the art (e.g. WO 99/54440, Ausubel, Current Protocols in Molecular Biology ' , Green Publishing Associates and Wiley Interscience, N. Y. 1989 and 1994 or Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N, Y., 2001, each incorporated by reference in their entirety for all purposes).
- the peptide linker is a stable linker, which is not cleavable by protease, such as by Matrix metalloproteinases (MMPs).
- MMPs Matrix metalloproteinases
- the peptide linker tends not to adopt a rigid three-dimensional structure, but rather provide flexibility' to a polypeptide (e.g., first and/or second components), such as providing flexibility' between the anti-PLA2G2D and the second moiety.
- the peptide linker is a flexible linker.
- Exemplary 7 flexible linkers include glycine polymers (G)n (SEQ ID NO: 13), glycine-serine polymers (including, for example, (GS)n (SEQ ID NO: 14), (GSGGS)n (SEQ ID NO: 15), (GGGGS)n (SEQ ID NO: 16), and (GGGS)n (SEQ ID NO: 17), where n is an integer of at least one), glycine- alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between components.
- an anti ⁇ PLA2G2D can include linkers that are all or partially flexible, such that the linker can include a flexible linker portion as well as one or more portions that confer less flexible structure to provide a desired immunoconjugate or fusion protein structure.
- exemplary linkers also include the amino acid sequence of such as (GGGGS)n(SEQ ID NO: 16), wherein n is an integer between 1 and 8, e.g. (GGGGS)3 ⁇ 4 (SEQ ID NO: 18; hereinafter referred to as (048)3 ' or “G83”), or (GGGGS)e (SEQ ID NO: 19; hereinafter referred to as “(G4S)6” or “GS6”).
- the peptide linker comprises the amino acid sequence of (GSTSGSGKPGSGEGS)n (SEQ ID NO: 20), wherein n is an integer between 1 and 3.
- Natural linkers adopt various conformations in secondary' structure, such as helical, b- strand, coil/bend and turns, to exert their functions.
- Linkers in an a-helix structure might serve as rigid spacers to effectively separate protein domains, thus reducing their unfavorable interactions.
- Non-helical linkers with Pro-rich sequence could increase the linker rigidity and function in reducing inter-domain interference.
- the anti-PLA2G2D antibody moiety' and the second moiety is linked together by an a-helical linker with an amino acid sequence of A(EAAAK)4A (SEQ ID NO: 21).
- any one or all of the linkers described herein can be accomplished by any chemical reaction that will bind the two molecules so long as the components or fragments retain their respective activities, e.g. binding to target PLA2G2D, function of the second moiety (such as binding to a FcR or cytokine receptor).
- This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and compiexation.
- the binding is covalent binding.
- Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules.
- Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as a second moiety to the anti-PLA2G2D antibody of the present invention.
- representative coupling agents can include organic compounds such as thioesters, carbodiimides, succmimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines.
- organic compounds such as thioesters, carbodiimides, succmimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines.
- non-peptide linkers used herein include: (i) EDC (l-etbyl-3-(3-dimethy]amino-propy]) carbodiimide hydrochloride; (ii) SMPT (4- succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio) ⁇ toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succimmidyi-6 [3-(2-pyridyldithio) propionamidojhexanoate (Pierce Chem.
- the linkers described above contain components that have different attributes, thus leading to agents binding to PLA2G2D (such as anti ⁇ PLA2G2D imnmnoconjugates or fusion proteins) with differing physio-chemical properties.
- PLA2G2D such as anti ⁇ PLA2G2D imnmnoconjugates or fusion proteins
- sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates.
- NHS-ester containing linkers are less soluble than sulfo-NHS esters.
- the linker SMPT contains a sterical!y hindered disulfide bond, and can form fusion protein with increased stability.
- Disulfide linkages are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less fusion protein available.
- Sulfo-NHS m particular, can enhance the stability of carbodimide couplings.
- Carhodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.
- Die methods described herein in some embodiments involve use of inhibitory PLA2G2D polypeptides that block the binding between PLA2G2D (e.g., a wildtype PLA2G2D) and immune cells completely or partially (such as blocks the binding between the PLA2G2D and an immune cell by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 95%).
- the present application in one aspect provides novel and non-naturally occurring polypeptide comprising an inhibitory PLA2G2D polypeptide that blocks the binding of PLA2G2D to an immune cell, in some embodiments, the inhibitory PLA2G2D polypeptide is a soluble polypeptide.
- the inhibitory PLA2G2D polypeptide is membrane bound. In some embodiments, the membrane bound inhibitory PLA2G2D polypeptide binds to the immune ceil but does not trigger PLA2G2D signaling pathway in the immune cell. In some embodiments, the membrane bound inhibitory PLA2G2D polypeptide binds to the immune cell and attenuates PLA2G2D signaling pathway in the immune cell. In some embodiments, the membrane bound inhibitory PLA2G2D polypeptide is introduced by a gene editing system or an mRNA delivery vehicle. [0139] In some embodiments, the inhibitory PLA2G2D polypeptide comprises a naturally occurring PLA2G2D polypeptide.
- the naturally occurring PLA2G2D polypeptide is from a human who has an autoimmune or inflammatory disease (such as chronic obstructive pulmonary disease (COPD)).
- COPD chronic obstructive pulmonary disease
- the inhibitory' PLA2G2D polypeptides has a mutation at a position corresponding to a polymorphism described in Takabatake et al (Am I Respir Crii Care Med. 2005 Nov 1 ; 172(9): 1097-104) or Igarashi etal. (Respiration. 2009;78(3): 312-21).
- the inhibitory PLA2G2D polypeptide comprises a mutation at the position corresponding to histidine at position 67 (H67) according to 8EQ ID NO: 1 or 5.
- the inhibitory' PLA2G2D polypeptide comprises an amino acid sequence of SEQ ID NO: 3, 4, 7, or 8 or a variant thereof, in some embodiments, the variant has at least about 80% (such as about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 3, 4, 7, or 8.
- the inhibitory' PLA2G2D polypeptide comprises a mutation at a position corresponding to G80 according to SEQ ID NO: 5.
- the inhibitory' PLA2G2D polypeptide comprises a comprises an amino acid sequence of SEQ ID NO: 9 or 10 or a variant thereof.
- the variant has at least about 80% (such as about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, or 99%) sequence identity' ' to the ammo acid sequence of SEQ ID NO: 9 or 10.
- the inhibitory PLA2G2D poly peptide comprises a) a mutation at the position corresponding to histidine at position 67 (H67) and b) a mutation at a position corresponding to G80 according to SEQ ID NO: 5.
- the inhibitory' PLA2G2D polypeptide comprises a comprises an amino acid sequence of SEQ ID NO: 11 or 12 or a variant thereof.
- the variant has at least about 80% (such as about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 11 or 12.
- the inhibitory PLA2G2D polypeptide further comprises at least one or more (such as about at least 10, 15, 20, 25, 30, 35, 45, 50, or all) of residues at the position of 22, 23, 25, 26, 27, 31, 36, 37, 38, 42, 43, 55, 59, 62, 65, 66, 72, 73, 76, 77, 80, 81, 83, 84, 85, 87, 89, 90, 92, 93, 94, 96, 98, 99, 100, 101, 102, 103, 105, 106, 107, 108, 110, 114, 115, 117, 119, 120, 123, 124, 127, 129, 130, 131, 132, 134, 135, 136, 137, 139, 141,
- the inhibitory PLA2G2B polypeptide further comprises at least one or more (such as about at least 10, 15, 20, 25, 30, or all) of residues at the position of 22, 26, 31, 36, 42, 43, 72, 73, 76, 77, 80, 81, 83, 85, 87, 89, 90, 92, 94, 96, 100, 101, 102, 103, 106, 110, 114, 115, 117, 120, 134, 135, 136, 141, or 144 wherein the ammo acid numbering is based on 8EQ ID NO:l.
- the variant described herein is a natural variant.
- the variant does not comprise anon-conservative substitution.
- the variant only comprises one or more conservative substitution.
- the one or more conservative substitutions comprise or consist of the substitutions shown m Table 1 below under the heading of “Preferred substitutions.”
- the inhibitory PLA2G2D polypeptide binds to the immune ceil with a greater affinity than for a wildtype PLA2G2D. In some embodiments, the inhibitory PLA2G2D polypeptide binds to the immune cell with a KD of at most half, one-fifth, one- tenth, one-twentieth, one-fiftieth, one-hundredth, one-thousandth of that of the binding between the wildtype PLA2G2D and the immune ceil.
- the inhibitory PLA2G2D polypeptide further comprises a stabilizing domain.
- the stabilizing domain can be any domain that stabilizes the inhibitory ' PLA2G2D polypeptide (for example, extending half-life of the inhibitory' PLA2G2D polypeptide in vivo).
- the stabilizing domain comprises an Fc fragment. Exemplary ' Fc fragment include those described under “Fc fragment” section.
- the inhibitory PLA2G2D polypeptide is about 50 to about 1000 ammo acids in length, such as about 50-800, 50-500, 50-400, 50-300 or 50-200 ammo acids m length. In some embodiments, the inhibitory polypeptide is about 50 to about 100 amino acids, about 100 to about 150 amino acids, or about 150 amino acids to about 200 ammo acids in length.
- the antagonist targeting PLA2G2D comprises a nucleic acid agent (such as a siRNA, a shRNA, a miRNA, or an antisense RNA) that targets PLA2G2D (such as a human PLA2G2D).
- a nucleic acid agent such as a siRNA, a shRNA, a miRNA, or an antisense RNA
- the antagonist comprises a siRNA or RNAi. In some embodiments, the antagonist comprises an antisense RNA. In some embodiments, the antagonist comprises a short hairpin ribonucleic acid (shRNA). In some embodiments, the antagonist comprises a microRNA (miRNA).
- shRNA short hairpin ribonucleic acid
- miRNA microRNA
- RNAi interfering RNA
- siRN A specific targeting PLA2G2D.
- the nucleic acid selected sometimes is the RNAi or siRNA or a nucleic acid that encodes such products.
- RNAi refers to double-stranded RNA (dsRNA) which mediates degradation of specific mRNAs, and can also be used to lower or eliminate gene expression.
- short interfering nucleic acid refers to any nucleic acid molecule directed against a gene.
- a siRNA is capable of inhibiting or down regulating gene expression or viral replication, for example by mediating RNA interference “RNAi” or gene silencing in a sequence-specific manner; see for example Zamore et af, 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428- 429; Elbashir et al., 2001, Nature, 411 , 494-498; and Kreutzer et af, International PCT Publication No. WO 00/44895; Zemicka-Goetz et a!., international PCT Publication No. WO 01/36646; Fire, International PCT Publication No.
- siRNA Science, 297, 1818-1819; Voipe et al., 2002, Science, 297, 1833-1837; Jenuwem, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831).
- ' There is no particular limitation in the length of siRNA as long as it does not show toxicity.
- modified RNAi and siRNA examples include STEALTH ' TM forms (Invitrogen Corp., Carlsbad, Calif.), forms described in U.S. Patent Publication No. 2004/0014956 (application Ser. No. 10/357,529) and U.S. patent application Ser. No. 11/049,636, filed Feb. 2, 2005), and other forms described hereafter.
- a siRNA can be a double-stranded polynucleotide molecule comprising self- complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
- the siRNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary' (i.e.
- each strand comprises nucleotide sequence that is complementary' to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 19 base pairs); the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
- the siRNA is assembled from a single oligonucleotide, where the self- eompiementary sense and antisense regions of the siRNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s).
- the siRNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self- complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
- Hie siRNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.
- the siRNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary' to nucleotide sequence m a target nucleic acid molecule or a portion thereof (for example, where such siRNA molecule does not require the presence within the siRNA molecule of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5 f -phosphate (see for example Martinez et al, 2002, Cell, 110, 563-574 and Schwarz et al, 2002, Molecular CeIl, 10, 537-568), or 5 ',3 '-diphosphate.
- a terminal phosphate group such as a 5 f -phosphate (see for example Martinez et al, 2002, Cell, 110, 563-574 and Schwarz et al, 2002, Molecular CeIl, 10, 537-568), or 5 ',3
- the siRNA molecule of the invention comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions, in certain embodiments, the siRNA molecules of the invention comprise nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, tire siRNA molecule of the invention interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.
- the double-stranded RNA portions of siRNAs in which two RNA strands pair are not limited to the completely paired forms, and may contain non-pairing portions due to mismatch (the corresponding nucleotides are not complementary), bulge (lacking in the corresponding complementary nucleotide on one strand), and the like.
- Non-pairing portions can be contained to the extent that they do not interfere with siRNA formation.
- the ' ‘bulge” used herein often comprises 1 to 2 non-pairing nucleotides, and the double-stranded RN A region of siRNAs in which two RNA strands pair up sometimes contains 1 to 7, and at limes 1 to 5 bulges.
- the “mismatch” used herein is contained in the double-stranded RNA region of siRNAs in which two RNA strands pair up, sometimes 1 to 7, and at times 1 to 5, in number.
- one of the nucleotides is guanine, and the other is uracil.
- Such a mismatch is due to a mutation from C to T, G to A, or mixtures thereof in DNA coding for sense RNA, but not particularly limited to them.
- the double-stranded RNA region of siRNAs in which two RNA strands pair up may contain both bulge and mismatched, which sum up to, sometimes Ito 7, and at times Ito 5, in number.
- the terminal structure of siRNA may be either blunt or cohesive (overhanging) as long as siRNA enables to silence the target gene expression due to its RNAi effect.
- siRNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides.
- RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics.
- siRNA molecules of the invention can be used to epigenetical!y silence genes at both the post-transcriptional level and the pre- transcriptional level, in a non-limiting example, epigenetic regulation of gene expression by siRNA molecules of the invention can result from siRNA mediated modification of chromatin structure to alter gene expression (see, for example, Verdel et a!., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Yolpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Flail et al, 2002, Science, 297, 2232-2237).
- RNAi may be designed by those methods known to those of ordinary' skill m the art.
- siRNA may be designed by classifying RNAi sequences, for example 1000 sequences, based on functionality, with afunctional group being classified as having greater than 85% knockdown activity and a non-functional group with less than 85% knockdown activity.
- the distribution of base composition was calculated for entire the entire RNAi target sequence for both the functional group and the non-functional group.
- the ratio of base distribution of functional and non-functional group may then be used to build a score matrix for each position of RNAi sequence. For a given target sequence, the base for each position is scored, and then the log ratio of the multiplication of all the positions is taken as a final score. Using this score system, a very strong correlation may be found of the functional knockdown activity and the log ratio score.
- the target sequence Once the target sequence is selected, it may be filtered through both fast NCBI blast and slow Smith Waterman algorithm search against the Uni gene database to identify the gene-specific RNAi or siRNA. Sequences with at least one mismatch in the last 12 bases may he selected.
- An antisense nucleic acid can he designed, prepared and/or utilized by the artisan to inhibit a nucleic acid encoding PLA2G2D.
- An ‘ ' antisense '' nucleic acid refers to a nucleotide sequence complementary to a ‘sense 55 nucleic acid encoding PLA2G2D or fragment (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence).
- the antisense nucleic acid can be complementary to an entire coding strand, or to a portion thereof or a substantially identical sequence thereof.
- the antisense nucleic acid molecule is antisense to a “noncoding region 55 of the coding strand of a nucleotide sequence.
- An antisense nucleic acid can he complementary ' to the entire coding region of an mRNA encoded by a PLA2G2D nucleotide sequence, and often the antisense nucleic acid is an oligonucleotide antisense to only a portion of a coding or noncoding region of the mRNA.
- the antisense oligonucleotide can be complementary' to the region surrounding the translation start site of the mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest.
- An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.
- An antisense nucleic acid can he constructed using chemical synthesis or enzyme ligation reactions using standard procedures.
- an antisense nucleic acid e.g., an antisense oligonucleotide
- an antisense nucleic acid 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 (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).
- Antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
- antisense nucleic acids When utilized in subjects, antisense nucleic acids typically are administered to a subject (e.g., by direct injection at a tissue site) or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a polypeptide and thereby inhibit expression of the polypeptide, for example, by inhibiting transcription and/or translation.
- antisense nucleic acid molecules can he modified to target selected cells and then are administered systemieaily.
- antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, for example, by linking antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
- Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. Sufficient intracellular concentrations of antisense molecules are achieved by incorporating a strong promoter, such as a pot II or pol III promoter, in the vector construct. Antisense nucleic acid molecules sometimes are alpha-anomeric nucleic acid molecules. An alpha-anomerie nucleic acid molecule forms specific double-stranded hybrids with complementary 7 RNA in which, contrary to the usual beta-units, the strands run parallel to each other (Gaultier et al. Nucleic Acids. Res. 15: 6625-6641 (1987)).
- Antisense nucleic acid molecules also can comprise a 2' ⁇ o-methylnbonucleotide (Inoue et al., Nucleic Acids Res. 15: 6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et al., FEES Lett. 215: 327-330 (1987)).
- Antisense nucleic acids sometimes are composed of DNA or PNA or any other nucleic acid derivatives described previously.
- An antisense nucleic acid is a ribozyme in some embodiments.
- a nbozyme having specificity for an Aid nucleotide sequence can include one or more sequences complementary to such a nucleotide sequence, and a sequence having a known catalytic region responsible for mRNA cleavage (e.g., U.S. Pat. No. 5,093,246 or Haselhoff and Geriach, Nature 334: 585-591 (1988)).
- a derivative of a Tetrahymena L-19 TVS RNA is sometimes utilized in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved m an mRNA (e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742).
- PLA2G2D mRNA sequences also may be utilized to select a catalytic RNA having a specific ribonuciease activity from a pool of RNA molecules (e.g,, Bartel & Szostak, Science 261: 1411-1418 (1993)).
- the nucleic acid agents targeting PLA2G2D are nucleic acids that can form triple helix structures with an Aid nucleic acid.
- PLA2G2D expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of a nucleotide sequence referenced herein or a substantially identical sequence (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of a gene in target cells (see e.g., Helene, Anticancer Drug Des. 6(6): 569-84 (1991); Helene et al., Ann. N.Y. Acad. Sci.
- Switchback molecules are synthesized in an alternating 5'-3',3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of purines or pyrimidines being present on one strand of a duplex.
- the antagonist targeting PLA2G2D comprises a genome- editing system that targets PLA2G2D.
- the genome-editing system comprises a DNA nuclease such as an engineered (e.g., programmable or targetable) DNA nuclease to induce genome editing of a target DNA sequence of PLA2G2D.
- sui table DN A nuclease can be used including, but not limited to, CRISPR-associated protein (Cas) nucleases, zmc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, other endo- or exo-nucleases, variants thereof, fragments thereof, and combinations thereof.
- Cas CRISPR-associated protein
- ZFNs zmc finger nucleases
- TALENs transcription activator-like effector nucleases
- meganucleases other endo- or exo-nucleases, variants thereof, fragments thereof, and combinations thereof.
- the genome editing comprises modifying PLA2G2D so that die modified PLA2G2D no longer suppresses an immune cell (such as a T cell, such as an activated T cell, such as an activated ( 1)4 T cell, such as an activated CD8+ T cell) or suppresses the immune ceil to a less extent than wildtype PLA2G2D.
- the genome editing comprises modifying PLA2G2D so that the modified PLA2G2D no longer binds to an immune ceil (such as a T cell, such as an activated T cell, such as an activated CD4+ T cell, such as an activated CD8+ T cell) or binds to the immune cell to a less extent than wildtype PLA2G2D.
- the modification comprises inserting a transgene comprising a variant of PLA2G2D (such as any of the variants of PLA2G2D described herein).
- the variant PLA2G2D has a mutation at H67 based upon SEQ ID NO: 1.
- the variant PLA2G2D has aH67A mutation based upon SEQ ID NO: 1.
- the antagonist comprises an agent that inhibits PLA2G2D enzymatic activity (i.e., hydrolyzing fatty acids). In some embodiments, the antagonist comprises an agent that specifically inhibits enzymatic activity' of the catalytic His67-Asp68 Dyad of human PLA2G2D as set forth in SEQ ID NO: 1 or 5. In some embodiments, the antagonist comprises an agent that specifically decreases enzymatic activity of the catalytic His67-Asp68 Dyad of human PLA2G2D as set forth in SEQ ID NO: 1 or 5 by at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%.
- the agent interferes with the binding of calcium to PLA2G2D.
- the agent blocks the binding of calcium to residues at one or more of H47, G49, G51, and D68 according to SEQ ID NO: 1 or 5.
- the methods described herein are applicable to diseases and conditions for which there are suppressed immune responses in the body that at least partly contribute to the less effective treating of the disease.
- exemplary diseases include cancer or infectious disease (such as viral infectious disease).
- the disease or condition described herein is a cancer.
- Cancers that may be treated using any of the methods described herein include any types of cancers.
- Types of cancers to be treated with the agent as described in this application include, but are not limited to, carcinoma, blastema, sarcoma, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas.
- sarcomas e.g., sarcomas, carcinomas, and melanomas.
- Adult tumors/cancers and pediatric tumors/cancers are also included.
- the cancer is early stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, cancer in remission, recurrent cancer, cancer in an adjuvant setting, cancer in a neoadjuvant setting, or cancer substantially refractory to a therapy.
- the cancer is a solid tumor.
- the cancer is a liquid tumor.
- the cancer tissue has a high expression level of PLA2G2D when the expression level of PLA2G2D (e.g., assessed by immimohistochemistry) is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% higher than the expression level of PLA2G2D in a reference tissue.
- the expression level of PLA2G2D e.g., assessed by immimohistochemistry
- the cancer tissue has a high expression level of PLA2G2D when the expression level of PLA2G2D (e.g., assessed by immimohistochemistry) is at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7 -fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold higher than the expression level ofPLA2G2D in a reference tissue.
- the reference tissue is the corresponding tissue in a healthy individual.
- the expression level of PLA2G2D in a reference tissue is the average expression level of PLA2G2D in the same tissue in a group of individuals (such as 10, 30, 50, 100 individuals) with same or similar cancer.
- the reference tissue is the corresponding tissue in an individual who also has a cancer but has a less suppressed immune response in the cancer tissue as indicated by a biomarker (such as high M2 macrophages, or high expression of an immune checkpoint agent such as PD-1 or PD-L1).
- a biomarker such as high M2 macrophages, or high expression of an immune checkpoint agent such as PD-1 or PD-L1.
- the cancer tissue has a high T cell infiltration (e.g., high CD3 T cells, high CD8 T cells, high CD4 T cells, activated T cells, activated CDS T cells, or activated CD4 T cells) in the cancer tissue.
- the cancer tissue has a high T cell infiltration when the number of the T cells in the cancer is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% more than the number of the corresponding T ceils in a reference tissue.
- the high T cell infiltration is present when the number of the T ceils m the cancer is at least about 1-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7 -fold, 8-fold, 9-fold, or 10-fold more than the number of the corresponding T ceils in a reference tissue.
- the reference tissue is the corresponding tissue in a healthy individual.
- the number of the corresponding T cells in a reference tissue is the average number of the corresponding T cells in the same tissue in a group of individuals (such as 10, 30, 50, 100 individuals) with same or similar cancer, in some embodiments, the reference tissue is the corresponding tissue in an individual who also has a cancer but has a less suppressed immune response in the cancer tissue as indicated by a biomarker (such as high M2 macrophages, high expression of an immune checkpoint agent such as PD- 1 or PD-L1, high expression level of PLA2G2D).
- a biomarker such as high M2 macrophages, high expression of an immune checkpoint agent such as PD- 1 or PD-L1, high expression level of PLA2G2D.
- the cancer has a decreased number (such as a decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of immune cells (such as activated T cells, activated CD4+ T cells, or activated CD8+ T cells) in the cancer tissue as compared to that of a reference tissue.
- immune cells such as activated T cells, activated CD4+ T cells, or activated CD8+ T cells
- the cancer has a decreased number (such as a decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of activated immune cells (such as activated T cells, activated CD4+ T cells, or activated CD8+ T cells) m the cancer tissue as compared to that of a reference tissue.
- the cancer tissue has a decreased level (such as a decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of a cytokine (such as a pro-inflammatory cytokine, such as IFNy or IL-2) as compared to that of a reference tissue.
- a cytokine such as a pro-inflammatory cytokine, such as IFNy or IL-2
- the reference tissue is the corresponding tissue in a healthy individual. In some embodiments, the reference tissue is the corresponding tissue in an individual who also has a cancer but has a less suppressed immune response in the cancer tissue.
- the suppression of immune response can be assessed by measuring a) the number of immune cells (e.g., CD3+ cells); b) the proliferating/expanding status of immune cells; c) the activation status of immune cells; and/or d) the cytokine level. In some embodiments!, any one or more of the a) -d) is measured in the cancer tissue.
- the immune cells are T cells. In some embodiments, the immune cells are CD8+ T cells (such as activated CD8-f- T cells). In some embodiments, the immune cells are CD4+ T cells (such as activated CD4+ T cells).
- cancers that may be treated by the methods of this application include, but are not limited to, anal cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bladder cancer, bone cancer, (osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., astrocytoma, malignant glioma, medulloblastoma, and glioblastoma), breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer (e.g., uterine cancer), esophageal cancer, eye cancer (e.g, intraocular melanoma and retinoblastoma), gastric (stomach) cancer, gastrointestinal stromal tumor (GIST), head and neck cancer, hepatocellular (liver) cancer (e.g,
- the disease or condition is a colon cancer.
- the disease or condition is melanoma.
- the disease or condition is aT ceil lymphoma.
- the disease or condition is an infectious disease.
- the infectious disease is a viral infectious disease.
- the viral infectious disease is characterized by infection with hepatitis virus, human immunodeficiency virus (HIV), picomavirus, poliovirus, enterovirus, human Coxsackie virus, influenza virus, rhino virus, echovirus, rubella vims, encephalitis virus, rabies virus, herpes virus, papillomavirus, polyoma virus, RSV, adenovirus, yellow fever virus, dengue virus, parainfluenza virus, hemorrhagic virus, pox virus, varicella zoster virus, parainfluenza virus, reo virus, orbivirus, rotavirus, parvovirus, African swine fever virus, measles, coronavirus (such as SARS-CoV, MERS-CoV, 2019-nCoV), Ebola virus, mumps or Norwalk virus.
- HCV human immunodeficiency virus
- picomavirus poliovirus
- enterovirus human Coxsackie virus
- the viral infectious disease is characterized by infection with an oncogenic virus such as CMV, EBV, HBV, KSHV, HPV, MCV, HTLV-1 , HIV- 1, or HCV.
- the one or more genes encoding proteins involved in the viral infectious disease development and/or progression include, but are not limited to, genes encoding RSV nucleocapsid, Pre-gen/Pre-C, Pre-Si, Pre-S2/S,X, HBV conserved sequences, HIV Gag polyprotein (p55), HIV Pol polyprotein, HIV Gag-Pol precursor (pi 60), HIV matrix protein (MA, pl7), HIV capsid protein (CA, p24), HIV spacer peptide 1 (SP1, p2), HTV nucleocapsid protein (NC, p9), HIV spacer peptide 2 (SP2, pi), HIV P6 protein, HIV reverse transcriptase (RT, p50), HIV RNase H (pi 5), HIV integrase (IN,
- the viral infectious disease is characterized by infection with coronavirus. In some embodiments, the viral infectious disease is characterized by infection with influenza virus.
- An infection site refers to a tissue m the body where virus appear in a significant number and/or causes significant damages.
- the infection site comprises has an increased expression level of PLA2G2D as compared to a reference tissue.
- the PLA2G2D expression level in the infection site is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% as compared to that of the reference tissue.
- the PLA2G2D expression level in the infection site is increased by at least about 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold as compared to that of the reference tissue.
- the infection site has a decreased number (such as a decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of immune cells (such as activated T ceils, activated CD4+ T cells, or activated CD8+ T ceils) in the infection site as compared to that of a reference tissue
- the infection site has a decreased number (such as a decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of activated immune cells (such as activated T cells, activated ⁇ 1)4 T ceils, or activated CD8+ T cells) in the infection site as compared to that of a reference tissue.
- the infection site has a decreased level (such as a decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of a cytokine (such as a pro-inflammatory cytokine, such as IFNy or TL-2) as compared to that of a reference tissue.
- a cytokine such as a pro-inflammatory cytokine, such as IFNy or TL-2
- the reference tissue is the corresponding tissue in a healthy individual. In some embodiments, the reference tissue is the corresponding tissue in an individual who also has a virus infection (such as a virus infection of the same type) but has a less suppressed immune response in the infection site.
- the suppression of immune response can be assessed by measuring a) the number of immune cells; b) the proliferating/expanding status of immune cells; c) the activation status of immune cells; and/or d) the cytokine level, in some embodiments, the immune cells in circulation are assessed, in some embodiments, the immune ceils in diseased tissue are assessed, in some embodiments, the immune cells in lymph tissue (such as lymph node) are assessed.
- the immune cells are T cells. In some embodiments, the immune cells are CD8+ T cells (such as activated CD8+ T cells). In some embodiments, the immune cells are CD4+ T ceils (such as activated CD4+ T cells).
- the individual is a mammal (such as a human).
- the indi vidual is selected for treatment based upon a high expression of PLA2G2D in a diseased tissue.
- the tissue is a cancer tissue.
- the tissue is an infection site.
- the PLA2G2D expression level in the infection site is increased by at least about 10%, 20%, 3Q%, 40%, 50%, 6014, 70%, 80%, 90% as compared to that of the reference tissue. In some embodiments, the PLA2G2D expression level in the infection site is increased by at least about 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold as compared to that of the reference tissue.
- the individual is selected for treatment based upon the indication of a suppressed immune response.
- the individual has a suppressed immune response in a diseased tissue.
- the tissue is a cancer tissue.
- the tissue is an infection site.
- the suppression of immune response can be assessed by measuring a) the number of immune cells; b) the proliferating/ expan dmg status of immune cells; c) the activation status of immune cells; and/or d) the cytokine level.
- the immune cells in circulation are assessed.
- the immune cells in diseased tissue are assessed.
- the immune cells in lymph tissue are assessed.
- the immune cells are T cells.
- the immune cells are CD8+ T cells (such as activated CD8+ T cells), in some embodiments, the immune cells are CD4+ T ceils (such as activated CD4+ T cells).
- the individual has a decreased number (such as a decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of immune cells (such as activated T ceils, activated CD4+ T cells, or activated CD8+ T ceils) in the tissue (such as the cancer tissue or infection site) as compared to that of a reference tissue.
- immune cells such as activated T ceils, activated CD4+ T cells, or activated CD8+ T ceils
- the individual has a decreased number (such as a decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of activated immune cells (such as activated T cells, activated CD4+ T cells, or activated CD 8+ T cells) in the tissue (such as the cancer tissue or infection site) as compared to that of a reference tissue.
- activated immune cells such as activated T cells, activated CD4+ T cells, or activated CD 8+ T cells
- the individual has a decreased level (such as a decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of a cytokine (such as a pro-inflammatory cytokine, such as iFNy or IL-2) in the tissue (such as the cancer tissue or infection site) as compared to that of a reference tissue.
- a cytokine such as a pro-inflammatory cytokine, such as iFNy or IL-2
- the reference tissue is the corresponding tissue in a healthy individual. In some embodiments, the reference tissue is the corresponding tissue in an individual who also has a same or similar disease or condition but has a less suppressed immune response in the cancer tissue.
- the individual has a compromised immune system
- the individual is at least about 60, 65, 70, 75, 80, 85, or 90 years old.
- the individual has at least one prior therapy, in some embodiments, the prior therapy comprises a radiation therapy, a chemotherapy and-'or an immunotherapy. In some embodiments, the individual is resistant, refractory, or recurrent to the prior therapy.
- the present application also provides methods administering an effective amount of an antagonist targeting PLA2G2D signaling pathway into an individual for treating a disease or condition (such as cancer or infectious disease), wherein the method further comprises administering a second agent or therapy.
- a disease or condition such as cancer or infectious disease
- the second agent or therapy is a standard or commonly used agent or therapy for treating the disease or condition.
- the antagonist is administered simultaneously with the second agent or therapy. In some embodiments, the antagonist is administered concurrently with the second agent or therapy. In some embodiments, the antagonist is administered sequentially with the second agent or therapy.
- the second agent or therapy comprises a chemotherapeutic agent.
- the second agent or therapy comprises a, surgery.
- the second agent or therapy comprises a radiation therapy.
- the second agent or therapy comprises an immunotherapy.
- the second agent or therapy comprises a ceil therapy (such as a ceil therapy comprising an immune cell (e.g., CAR T cell)).
- the second agent or therapy comprises an angiogenesis inhibitor.
- the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulator, an anti-angiogenesis agent, a growth inhibitor ⁇ ' agent, and an antineoplastic agent.
- the second agent is a chemotherapeutic agent. In some embodiments, the second agent is antimetabolite agent. In some embodiments, the antimetabolite agent is 5-FU.
- the second agent is an immunomodulator.
- the immunomodulatory is an immune checkpoint inhibitor.
- the checkpoint inhibitor specifically targets PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIMS, LAGS, CD27, 4-1BB, or B7H4.
- the second agent is an anti -PD- 1 antibody or fragment thereof.
- the second agent is an anti-PD-Ll antibody or fragment thereof.
- the second agent comprises a cell (such as an immune ceil, such as a T cell) comprising a chimeric antigen receptor that specifically binds to a tumor antigen.
- Exemplary combination therapies for infectious disease (such as viral infectious disease).
- the second agent or therapy comprises a nucleotide analogue.
- the second agent or therapy comprises a nucleoside analogue.
- the second agent or therapy comprises a protease inhibitor. In some embodiments, the second agent or therapy comprises Lopinavir. In some embodiments, the second agent or therapy comprises ritonavir.
- the second agent or therapy comprises a neuraminidase inhibitor. In some embodiments, the second agent or therapy comprises zanamivir. In some embodiments, the second agent or therapy comprises oseltamivir. In some embodiments, the second agent or therapy comprises peramivir.
- the second agent or therapy comprises a Cap-dependent endonuclease inhibitor. In some embodiments, the second agent or therapy comprises baloxavir.
- the second agent or therapy comprises a sialidase.
- the second agent and the antagonist can be administered sequentially, concurrently, or simultaneously. In some embodiments, the second agent is administered prior to the antagonist. In some embodiments, the second agent is administered after the antagonist.
- the dose of the antagonist and, in some embodiments, the second agent as described herein, administered to an individual may vary with the particular composition, the method of administration, and the particular kind and stage of disease or condition being treated.
- the amount should be sufficient to produce a desirable response, such as a therapeutic response against the disease or condition.
- the amount of the antagonist and/or the second agent is a therapeutically effective amount.
- the amount of the antagonist is an amount sufficient to decrease the suppression of the immune response in the indi vidual. Whether there is a decrease in the suppression of the immune response and the extent of the decrease in suppression can be indicated by any of the following.
- the amount of the antagonist is an amount sufficient to increase the number of immune cells (such as T ceils, such as ⁇ 1)4 and/or CD8+ T cells) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% post administration of the antagonist.
- the immune ceils in circulation are assessed, in some embodiments, the immune cells in diseased tissue are assessed.
- the immune cells in lymph tissue are assessed.
- the immune cells comprises myeloid ceils (such as dendritic cells).
- the immune cells comprises NK cells.
- the immune cells comprises T cells, such as CD4 ⁇ and/or CD8+ T cells.
- the number of immune cells is assessed about 1, 2, 3, 4, 5, 6, or 7 days post administration of the antagonist.
- the amount of the antagonist is an amount sufficient to increase the number of activated immune cells (such as activated CD4+ and/or CD8+ T cells) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% post administration of the antagonist.
- the activated immune cells in circulation are assessed.
- the activated immune cells in diseased tissue are assessed, in some embodiments, the activated immune cells in lymph tissue (such as lymph node) are assessed.
- the immune cells comprises myeloid cells (such as dendritic cells).
- the immune cells comprises NK cells.
- the immune cells comprises T cells, such as CD4+ and/or CD8+ T cells.
- the number of activated immune cells is assessed about 1, 2, 3, 4, 5, 6, or 7 days post administration of the antagonist,
- the amount of the antagonist is an amount sufficient to increase the proliferation of immune ceils or activated immune cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% post administration of the antagonist.
- the immune cells or the activated immune cells in circulation are assessed, in some embodiments, the immune cells or the activated immune cells in diseased tissue are assessed, in some embodiments, the immune cells or the activated immune ceils in lymph tissue (such as lymph node) are assessed.
- the immune cells comprises myeloid cells (such as dendritic cells).
- the immune cells comprises NK cells.
- the immune ceils comprises T ceils, such as CD4+ and/or CD8+ T cells.
- the proliferation of immune cells or activated immune cells is assessed about 1, 2, 3, 4, 5, 6, or 7 days post administration of the antagonist.
- the amount of the antagonist is an amount sufficient to increase the cytokine level (such as a pro-inflammatory cytokine, such as IFNv or IL-2) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% post administration of the antagonist.
- the cytokine level in diseased tissue is assessed.
- the level of cytokine is assessed about 1, 2, 3, 4, 5, 6, or 7 days post administration of the antagonist.
- the amount of the antagonist is an amount sufficient to decrease the suppressive immune cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% post administration.
- the suppressive immune cells comprise regulator ⁇ ' T cells.
- the suppressive immune cells comprise myeloid derived suppressor cells.
- the suppressive immune cells in circulation are assessed, in some embodiments, the suppressive immune cells in diseased tissue are assessed.
- the suppressive immune cells in lymph tissue (such as lymph node) are assessed.
- the number of suppressive immune cells is assessed about 1, 2, 3, 4, 5, 6, or 7 days post administration of the antagonist.
- the amount of the antagonist is an amount sufficient to increase humoral immune response in the individual by at least about 10%, 20%, 30%, 40%, 50%, 60%), 70%, 80%, 90%), or 100% post administration of the antagonist.
- the humoral immune response can be assessed by measuring antibodies (such as IgG antibodies) that target a disease-associated antigen or plasmablasts that produce such antibodies in circulation, in some embodiments, the humoral immune response is assessed about 7-28 days (such as about 7-14 days) post administration of the antagonist.
- the amount of the antagonist is an amount sufficient to produce a decrease of the size of a tumor, decrease the number of cancer ceils, or decrease the growth rate of a tumor by at least about any of 10%, 20%, 30%, 40%, 50 ( 1 ⁇ 2, 60%, 70%o, 80%, 90%, 95% or 100% compared to the corresponding tumor size, number of cancer cells, or tumor growth rate in the same individual prior to treatment or compared to the corresponding activity m other individuals not receiving the treatment.
- the antagonist is administered at a dose of about 0.001 pg/kg to about 100 mg/kg of total body weight, for example, about 0.005 pg/kg to about 50 mg/kg, about 0,01 ug kg to about 10 mg/kg, or about 0.01 pg/kg to about 1 mg/kg.
- the antagonist and/or the second agent composition is administered intravenously, intraarterially, mtraperitoneally, intravesicularly, subcutaneously, intrathecally, intrapuimonanly, intramuscularly, intratracheal ly, intraocularly, topically, transdermally, orally, or by inhalation.
- the antagonist and/or the second agent is administered intravenously.
- the antagonist is administered directly to the diseased tissue.
- Provided herein also include methods of diagnosing or prognosing an individual, including, determining the suitability' of an individual for the treatment as described in section 11 or a different therapy comprising an immunotherapy, determining the likelihood of responsiveness of an indi vidual to the methods as described in section 11 or the different therapy.
- a method of determining the suitability of an individual for a treatment comprising measuring levels of PLA2G2D expression in the diseased tissue of an individual.
- the individual has a cancer, and the tissue is a tumor tissue.
- the individual has an infectious disease (such as a viral infectious disease, and the tissue is an infection site.
- a method of prognosis in an individual having cancer comprising measuring levels of PLA2G2D expression in a tumor sample in vitro or in vivo, wherein a higher PLA2G2D expression level as compared to a reference level indicates a higher possibility of not responding or responding poorly to a therapy (such as an immunotherapy).
- the reference level is a level of PLA2G2D expression (such as an average PLA2G2D expression) in a non-tumor sample in the individual or a corresponding tissue in a different individual (or a group of individuals) who does not have cancer.
- a method of prognosis in an individual having an infectious disease comprising measuring levels of PLA2G2D expression in a sample from the infection site in vitro or in vivo, wherein a higher PLA2G2D expression level as compared to a reference level indicates a higher possibility' of not responding or responding poorly to a therapy (such as an immunotherapy).
- the reference level is a level of PLA2G2D expression (such as an average PLA2G2D expression) in a non-infection site sample in the individual or a corresponding tissue in a different individual (or a group of individuals) who does not have the infectious disease.
- the therapy comprises a cell therapy (such as a CAR-T cell therapy).
- a cell therapy such as a CAR-T cell therapy.
- the therapy further comprises assessing suppression of immune response m the individual. Exemplary' methods of assessing immune response suppression are discussed above.
- a method of preparing the antagonist such as an siRNA targeting PLA2G2D, anti-PLA2G2D agents, inhibitory' PLA2G2D polypeptides, agents inhibiting PLA2G2D enzymatic activity as described herein
- composition comprising the agents, nucleic acid construct, vector, host cell, or culture medium that is produced during the preparation of the agents.
- Tiie agents targeting PLA2G2D e.g, polypeptide comprising an anti-PLA2G2D antibody moiety as described in Section P
- inhibitory PLA2G2D polypeptides described herein can be prepared using any known methods in the art, including those described below.
- Polypeptides comprising anii-PLA2G2D antibody moiety
- Monoclonal antibodies targeting PLA2G2D can be obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post- translational modifications (e.g., isomenzations, amidations) that may be present in minor amounts.
- the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
- the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al, Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
- a mouse or other appropriate host animal such as a hamster or a llama
- lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986).
- the immunizing agent will typically include the antigenic protein or a fusion variant thereof.
- PBLs peripheral blood lymphocytes
- spleen cells or lymph node cells are used if non-human mammalian sources are desired.
- the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.
- a suitable fusing agent such as polyethylene glycol
- Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
- the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
- the culture medium for the hybridomas typically will include hypoxanthine, ammopterin, and thymidine (HAT medium), which are substances that prevent the growth of HGPRT-deficient cells.
- HGPRT hypoxanthine guanine phosphoribosyl transferase
- Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
- preferred are murine myeloma lines such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 cells (and derivatives thereof, e.g., X63-Ag8-653) available from the American Type Culture Collection, Manassas, Va, USA.
- Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J.
- Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
- the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
- RIA radioimmunoassay
- ELISA enzyme-linked immunosorbent assay
- the culture medium in winch the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies directed against the desired antigen.
- the binding affinity and specificity of the monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked assay (ELISA).
- RIA radioimmunoassay
- ELISA enzyme-linked assay
- binding affinity may be determined by the Scatchard analysis of Munson el al..
- the clones may be subcloned by limiting dilution procedures and grown by standard methods (Coding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium.
- the hybridoma cells may be grown in vivo as tumors in a mammal.
- the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as. for example, protein A-Sepharose, hydroxylapalite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
- Monoclonal antibodies may also be made by recombinant DNA methods, such as those described in L ! .S. Pat. No. 4,816,567, and as described above.
- DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
- the hybridoma cells serve as a preferred source of such DNA.
- the DNA may be placed into expression vectors, which are then transfected into host cells such as E.
- antibodies can be isolated from antibody phage libraries generated using the techniques described in MeCafferty et ai, Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks eta!., J. Mol. Biol., 222:581-597 (1991), each of which are incorporated by reference in their entirety for all purposes, describe the isolation of murine and human antibodies, respectively, using phage libraries.
- the DN A also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Nad Acad. Sci. USA, 81:6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
- non- immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity ' for a different antigen.
- the monoclonal antibodies described herein may by monovalent, the preparation of which is well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and a modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinkmg.
- cysteine residues may be substituted with another ammo acid residue or are deleted so as to prevent crosslinking.
- In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art.
- Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry', including those involving crosslinking agents.
- immunotoxins may be constructed using a disulfide-exchange reaction or by forming a thioether bond.
- suitable reagents for this purpose include immothiolate and me thy 1 -4-mercaptob uty ri mi date.
- a nucleic acid molecule comprises a polynucleotide that encodes a heavy chain or a light chain of an antibody (e.g., anti-PL A2G2D antibody).
- a nucleic acid molecule comprises a polynucleotide that encodes an inhibitory' PLA2G2D polypeptide.
- a nucleic acid molecule comprises both a polynucleotide that encodes a heavy chain and a polynucleotide that encodes a light chain, of an antibody (e.g., anti-PLA2G2D antibody).
- a first nucleic acid molecule comprises a first polynucleotide that encodes a heavy chain and a second nucleic acid molecule comprises a second polynucleotide that encodes a light chain.
- a nucleic acid molecule encoding a scFv (e.g., anti-PLA2G2D scFv) is provided.
- a nucleic acid molecule comprises a polynucleotide that encodes an inhibitor ⁇ ' PLA2G2D polypeptide.
- the heavy chain and the light chain of an antibody are expressed from one nucleic acid molecule, or from two separate nucleic acid molecules, as two separate polypeptides.
- an antibody e.g., anti-PLA2G2D antibody
- a single polynucleotide encodes a single polypeptide comprising both a heavy chain and a light chain linked together.
- a polynucleotide encoding a heavy chain or light chain of an antibody comprises a nucleotide sequence that encodes a leader sequence, which, when translated, is located at the N terminus of the heavy chain or light chain.
- the leader sequence may be the native heavy or light chain leader sequence, or may be another heterologous leader sequence.
- the polynucleotide is a DNA. In some embodiments, the polynucleotide is an RNA. In some embodiments, the RNA is an mRNA.
- Nucleic acid molecules may be constructed using recombinant DNA techniques conventional in the art.
- a nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell.
- nucleic acid construct comprising any one of the polynucleotides described herein. In some embodiments, there is provided a nucleic acid construct prepared using any method described herein.
- the nucleic acid construct further comprises a promoter operabiy linked to the polynucleotide, in some embodiments, the polynucleotide corresponds to a gene, wherein the promoter is a wild-type promoter for the gene.
- vector means the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to genetically modify the host and promote expression (e.g., transcription and translation) of the introduced sequence.
- Vectors include plasmids, synthesized RNA and DNA molecules, phages, viruses, etc. in certain embodiments, the vector is a viral vector such as, but not limited to, viral vector is an adenoviral, adeno-associated, alphaviral, herpes, lenti viral, retroviral, or vaccinia vector.
- a vector comprising any polynucleotides that encode the heavy chains ami/or light chains of any one of the antibodies (e.g., anti-PLA2G2B antibodies) described herein.
- a vector comprising any polynucleotides that encode polypeptides (e.g, inhibitory PLA2G2D polypeptides) described herein.
- polypeptides e.g, inhibitory PLA2G2D polypeptides
- nucleic acid construct described herein.
- a vector prepared using any method described herein V ectors comprising polynucleotides that encode any of polypeptides (such as anti-PLA2G2D antibodies or inhibitor ⁇ ' PLA2G2D polypeptides) are also provided.
- Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc.
- a vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain.
- the heavy chain and light chain are expressed from the vector as two separate polypeptides.
- a first vector comprises a polynucleotide that encodes a heavy chain of an antibody (e.g., anti-PLA2G2D antibody) and a second vector comprises a polynucleotide that encodes a light chain of an antibody (e.g., anti-PLA2G2D antibody).
- the first vector and second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts).
- a mole- or mass-ratio of between 5:1 and 1:5 of the first vector and the second vector is transfected into host cells.
- a mass ratio of between 1:1 and 1:5 for the vector encoding the heavy chain and the vector encoding the light chain is used. In some embodiments, a mass ratio of 1 :2 for the vector encoding the heavy 7 chain and the vector encoding the light chain is used.
- a vector is selected that is optimized for expression of polypeptides in CHO or CHO-derived cells, or in NSO cells. Exemplary such vectors are described, e.g., in Running Deer eta!.., Biotechnol. Prog. 20:880-889 (2004).
- the vector is a viral vector
- the viral vector can he, but is not limited to, a retroviral vector, an adenoviral vector, an adeno- associated virus vector, an alphaviral vector, a herpes virus vector, and a vaccinia virus vector.
- the viral vector is a ientiviral vector.
- the vector is a non-viral vector.
- the viral vector may be a plasmid or a transposon (such as a Piggy Bac- or a Sleeping Beauty transposon).
- a host ceil comprising any polypeptide, nucleic acid construct and/or vector described herein.
- a host cell prepared using any method described herein.
- the host cell is capable of producing any of polypeptides (such as antibodies or inhibitory polypeptides) described herein under a fermentation condition.
- the polypeptides described herein may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art.
- exemplary' eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E ceils; CHO cells, including CHO-S, DG44.
- Led 3 CHO cells Led 3 CHO cells, and FUT8 CHO cells; PER.C6® cells (Crucell); andNSO cells.
- the polypeptides described herein may be expressed in yeast. See, e.g., U.S. Publication No. US 2006/0270045 Al.
- a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and/or light chains of the desired antibody.
- CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.
- nucleic acids may be transiently or stably transfected in the desired host cells, according to any suitable method.
- the invention also provides host cells comprising any of the polynucleotides or vectors described herein, in some embodiments, the invention provides a host cell comprising an anti-PLA2G2D antibody.
- Any host cells capable of over-expressing heterologous DNAs can be used for the purpose of isolating the genes encoding the antibody, polypeptide or protein of interest.
- Non-limiting examples of mammalian host cells include but not limited to COS, HeLa, and CHO cells. See also PCX Publication No. WO 87/04462.
- Suitable non- mammalian host cells include prokaryotes (such as E. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; or K lactis).
- the polypeptide is produced in a cell -free system.
- a cell -free system Non-limiting exemplary cell-free systems are described, e.g., in Sitaraman etal., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol, 22: 538-45 (2004); Endo etal., Biotechnol. Adv. 21: 695-713 (2003).
- the polypeptides may be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include the ROR1 BCD and ligands that bind antibody constant regions. In some embodiments, a Protein A, Protein G, Protein A/G, or an antibody affinity column may be used to bind the constant region and to purify an antibody comprising an Fc fragment. Hydrophobic interactive chromatography, for example, a butyl or phenyl column, may also suitable for purifying some polypeptides such as antibodies.
- affinity ligands include the ROR1 BCD and ligands that bind antibody constant regions.
- a Protein A, Protein G, Protein A/G, or an antibody affinity column may be used to bind the constant region and to purify an antibody comprising an Fc fragment.
- Hydrophobic interactive chromatography for example, a butyl or phenyl column, may also suitable for purifying some polypeptides such
- Ion exchange chromatography e.g. anion exchange chromatography and/or cation exchange chromatography
- Mixed-mode chromatography e.g. reversed phase/anion exchange, reversed phase/cation exchange, hydrophilic interaction/amon exchange, hydrophilic interaction/cation exchange, etc.
- Many methods of purify ing polypeptides are known in the art.
- compositions, kits, medicines, and unit dosage forms for use in any of the methods described herein.
- any of the antagonists described herein can be present in a composition (such as a formulation) that includes other agents, excipients, or stabilizers.
- the composition further comprises a target agent or a carrier that promotes the delivery of the antagonist to a diseased tissue.
- exemplary carriers include liposomes, micelles, nanodisperse albumin and its modifications, polymer nanoparticles, dendrimers, inorganic nanoparticles of different compositions.
- the antagonist is packaged in a nanocarrier.
- the nanocarrier has an average diameter of about 20 nnt to about 200 nm. In some embodiments, the nanocarrier has an average diameter of about 50 nm.
- the antagonist described herein is coated with a serum protein (such as albumin). In some embodiments, the antagonist is coated with opsonin.
- the antagonist comprises or is coupled with a moiety that facilitate the delivery of the antagonist to the diseased tissue (such as the cancer tissue or infection site as described above).
- the moiety bind to an antigen that is expressed (e.g., overexpressed) or clustered on the diseased tissue (such as cancer tissue) or cells within the diseased tissue.
- the antigen is a tumor associated antigen (such as Her2, folate receptor, CD44). See, for example, Rosenblum et al. , Nat Common. 2018 Apr 12;9(1): 1410.
- the composition is suitable for administration to a human.
- the composition is suitable for administration to a mammal such as, in the veterinary context, domestic pets and agricultural animals.
- suitable formulations of the composition comprising the antagonist There are a wide variety of suitable formulations of the composition comprising the antagonist. The following formulations and methods are merely exemplary' and are in no way limiting.
- Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice, (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules, (c) suspensions in an appropriate liquid, and (d) suitable emulsions.
- liquid solutions such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice
- capsules, sachets or tablets each containing a predetermined amount of the active ingredient, as solids or granules
- suspensions in an appropriate liquid and (d) suitable emulsions.
- Tablet forms can include one or more of lactose, mannitol, com starch, potato starch, microcrysialline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients.
- Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
- a flavor usually sucrose and acacia or tragacanth
- pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
- Suitable carriers, excipients, and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline solution, syrup, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate, arid mineral oil.
- the composition comprising the antagonist with a carrier as discussed herein is present in a dry formulation (such as iyophilized composition).
- the formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.
- Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation compatible with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
- the formulations can be presented in unit- dose or multi-dose sealed containers, such as ampules and viols, and can be stored in a freeze- dried (Iyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
- Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Injectable formulations are preferred.
- the composition is formulated to have a pH range of about 4.5 to about 9.0, including for example pH ranges of about any of 5,0 to about 8.0, about 6.5 to about 7.5, and about 6.5 to about 7.0. in some embodiments, the pH of the composition is formulated to no less than about 6, including for example no less than about any of 6.5, 7, or 8 (such as about 8).
- the composition can also be made to be isotonic with blood by the addition of a suitable tonicity modifier, such as glycerol.
- Kits provided herein include one or more containers comprising the antagonist or a pharmaceutical composition comprising the antagonist described herein and/or other agent(s), and in some embodiments, further comprise instructions for use in accordance with any of the methods described herein.
- the kit may further comprise a description of selection of individual suitable for treatment. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
- the kit comprises a) a composition comprising an antagonist targeting PLA2G2D signaling pathway comprising an agent comprising an anti ⁇ PLA2G2D antibody moiety, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable earner: and optionally b) instructions for administering the agent for treatment of a disease or condition.
- the agent is an anti-PLA2G2D antibody
- the agent is an anti-PLA2G2D fusion protein.
- the agent is an anti-PL A2G2D immunoconjugate.
- the kit comprises a) a composition comprising an antagonist targeting PLA2G2D signaling pathway comprising an inhibitory PLA2G2D polypeptide, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; and optionally b) instructions for administering the agent for treatment of a disease or condition
- the inhibitory PLA2G2D polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, and 7-12.
- the kit comprises a) a composition comprising an antagonist targeting PLA2G2D signaling pathway comprising a nucleic acid agent targeting PLA2G2D (such as siRNA, shRNA, miRNA, or antisense RNA), or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; and optionally b) instructions for administering the agent for treatment of a disease or condition.
- an antagonist targeting PLA2G2D signaling pathway comprising a nucleic acid agent targeting PLA2G2D (such as siRNA, shRNA, miRNA, or antisense RNA), or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; and optionally b) instructions for administering the agent for treatment of a disease or condition.
- the kit comprises a) a composition comprising an antagonist targeting PLA2G2D signaling pathway comprising a genome editing system that targets PLA2G2D, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; and optionally b) instructions for administering the agent for treatment of a disease or condition.
- the kit comprises a) a composition comprising an antagonist targeting PLA2G2D signaling pathway comprising an agent inhibiting PLA2G2D enzymatic activity, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; and optionally b) instructions for administering the agent for treatment of a disease or condition.
- Die kits of the invention are in suitable packaging.
- suitable packaging includes, but is not limited to, viols, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretative information.
- the present application thus also pro vides articles of manufacture, which include vials (such as sealed vials), bottles, jars, flexible packaging, and the like.
- kits comprise one or more components that facilitate delivery of the antagonist, or a composition comprising the agent, and/or additional therapeutic agents to the individual.
- the kit comprises, e.g., syringes and needles suitable for delivery of cells to the individual, and the like, in such embodiments, the antagonist, or a composition comprising the agent may be contained in the kit m a bag, or in one or more vials.
- the kit comprises components that facilitate intravenous or intra-arterial delivery of the antagonist, or a composition comprising the agent to the individual.
- the antagonist, or a composition comprising the agent may be contained, e.g., within a bottle or bag (for example, a blood bag or similar bag able to contain up to about 1.5 L solution comprising the cells), and the kit further comprises tubing and needles suitable for the delivery of the antagonist, or a composition comprising the agent to the individual.
- a bottle or bag for example, a blood bag or similar bag able to contain up to about 1.5 L solution comprising the cells
- the kit further comprises tubing and needles suitable for the delivery of the antagonist, or a composition comprising the agent to the individual.
- the instructions relating to the use of the compositions generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
- Tire containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.
- kits may be provided that contain sufficient dosages of the zinc as disclosed herein to provide effective treatment of an individual for an extended period, such as any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or more.
- Kits may also include multiple unit doses of the pharmaceutical compositions and instructions for use and packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.
- Embodiment 1 A method of treating a cancer or viral infection in an individual, comprising administering into the individual an effective amount of an antagonist targeting PLA2G2D signaling pathway,
- Embodiment 2 The method of embodiment 1, wherein the antagonist is an antagonist targeting PLA2G2D.
- Embodiment 3. The method of embodiment 2, wherein the PLA2G2D is a human PLA2G2D.
- Embodiment 4 The method of embodiment 2 or embodiment 3, wherein the antagonist decreases enzymatic activity level of PLA2G2D.
- Embodiment 5 The method of embodiment 4, wherein the antagonist targeting PLA2G2D signaling pathway blocks a catalytic site on PLA2G2D.
- Embodiment 6 Hie method of embodiment 5, wherein the antagonist targets the H67 catalytic site on a human PLA2G2D according to SEQ ID NO: 1 or 5.
- Embodiment 7 The method of any one of embodiments 1-3, wherein the antagonist comprises a siRNA, an miRNA, an antisense RNA, or a gene editing system.
- Embodiment 8 The method of any one of embodiments 1-3, wherein the antagonist comprises an agent that inhibits PLA2G2D (such as an agent that blocks the binding of PLA2G2D to an immune cell or an agent that inhibits the activity of PLA2G2D).
- an agent that inhibits PLA2G2D such as an agent that blocks the binding of PLA2G2D to an immune cell or an agent that inhibits the activity of PLA2G2D.
- Embodiment 9 The method of embodiment 8, wherein the immune cell is a T cell.
- Embodiment 10 The method of any one of embodiments 1-3, wherein the antagonist comprises an anti ⁇ PLA2G2D antibody.
- Embodiment 11 The method of embodiment 10, wherein the anti-PL A2G2D antibody is a monoclonal antibody.
- Embodiment 12 The method of embodiment 10, wherein the antagonist is a fusion protein further comprising a second moiety.
- Embodiment 13 The method of embodiment 12, wherein the second moiety comprises a cytokine.
- Embodiment 14 The method of any one of embodiments 1-3, wherein the antagonist comprises an inhibitory PLA2G2D polypeptide that blocks the binding of PLA2G2D to an immune cell.
- Embodiment 15 The method of embodiment 14, wherein the inhibitory PLA2G2D polypeptide binds to the immune cell with a greater affinity than for PLA2G2D.
- Embodiment 16 The method of embodiment 15, wherein the immune cells is a T ceil.
- Embodiment 17 The method of any one of embodiments 14-16, wherein the inhibitory PLA2G2D polypeptide further comprises a stabilizing domain.
- Embodiment 18 The method of embodiment 17, wherein the stabilizing domain is an Fe domain.
- Embodiment 19 The method of any one of embodiments 14-18, wherein the inhibitory' PLA2G2D polypeptide has a length of about 50 to about 200 amino acids.
- Embodiment 20 The method of any one of embodiments 14- 19, wherein inhibitory' PLA2G2D polypeptide has a mutation at the position corresponding to histidine at position 67 (H67) according to SEQ ID NO: 1 or 5.
- Embodiment 21 The method of embodiment 20, wherein the inhibitory PLA2G2D polypeptide comprises an amino acid sequence of SEQ ID NO: 3, 4, 7, or 8.
- Embodiment 22 The method of any one of embodiments 1-21, wherein the disease or condition is a cancer.
- Embodiment 23 The method of embodiment 22, wherein the cancer is a solid tumor.
- Embodiment 24 The method of embodiment 22 or embodiment 23, wherein the cancer is an advanced or malignant tumor.
- Embodiment 25 The method of any one of embodiments 22-24, wherein the cancer has an increased expression level of PLA2G2D.
- Embodiment 26 The method of any one of embodiments 22-25, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, renal cancer, prostate cancer, urothelial cancer, testis cancer, ovarian cancer and melanoma.
- the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, renal cancer, prostate cancer, urothelial cancer, testis cancer, ovarian cancer and melanoma.
- Embodiment 27 The method of any one of embodiments 1-21, wherein the disease or condition is a viral infection
- Embodiment 28 The method of embodiment 27, wherein the infection site has an increased expression level of PLA2G2D.
- Embodiment 29 The method of any one of embodiments 1-28, wherein the method further comprises administering a second agent.
- Embodiment 30 The method of embodiment 29, wherein the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulator, an anti- angiogenesis agent, a growth inhibitory' agent, and an antineoplastic agent,
- Embodiment 31 The method of embodiment 30, wherein the second agent is an immunomodulator.
- Embodiment 32 The method of embodiment 31, wherein the immunomodulator is an immune checkpoint inhibitor.
- Embodiment 33 The method of embodiment 28, wherein the immune checkpoint inhibitor specifically target PD-L1, PD-L2, CTLA4, PD-L2, PL)-! .
- Embodiment 34 The method of embodiment 33, wherein the second agent comprises a cell comprising a chimeric antigen receptor that specifically binds to a tumor antigen.
- Embodiment 35 The method of any one of embodiments 29-34, wherein the antagonist and the second agent is administered simultaneously or concurrently.
- Embodiment 36 The method of any one of embodiments 29-34, wherein the antagonist and the second agent is administered sequentially.
- Embodiment 37 The method of any one of embodiments 1-36, wherein the antagonist and/or the second agent is administered parentally.
- Embodiment 38 The method of any one of embodiments 22-37, wherein the antagonist is administered to the cancer tissue or infection site directly.
- Embodiment 39 The method of any one of embodiments 1-38, wherein the antagonist is administered at a dose of about 0.001 pg/kg to about 100 mg/kg.
- Embodiment 40 The method of any one of embodiments 22-39, wherein the individual has an increased number of immune ceils in the cancer tissue or at the infection site after administration of the antagonist.
- Embodiment 41 The method of embodiment 40, wherein the immune cells are T cells.
- Embodiment 42 The method of embodiment 40 or embodiment 41, wherein the T cells are activated T cells.
- Embodiment 43 The method of any one of embodiment 40-42, wherein the number of immune cells in the cancer tissue or at the infection site is increased by at ieast about 5% after administration of the antagonist,
- Embodiment 44 The method of any one of embodiments 22-43, wherein immune ceils in the cancer tissue or at the infection site produce an increased level of a cytokine after administration of the antagonist.
- Embodiment 45 The method of embodiment 44, wherein the cytokine is IFNy and/or
- Embodiment 46 The method of embodiment 39 or embodiment 40, wherein the level of the cytokine is increased by at least about 5% after administration of the antagonist.
- TCGA Cancer Genome Atlas
- HNS C Head and Neck squamous cell carcinoma
- KICH Kidney Chromophobe
- K1RC Kidney renal clear cell carcinoma
- KIRI 5 Kidney renal papillary cell carcinoma
- L1HC Liver hepatocellular carcinoma
- Lung adenocarcinoma Lung adenocarcinoma
- Lung squamous cell carcinoma Lung squamous cell carcinoma
- OV Ovarian serous cystadenocarcinoma
- PAAD Pancreatic adenocarcinoma
- PCPG Paraganglioma
- PRAB Prostate adenocarcinoma
- READ Rectum Adenocarcinoma
- SARC Prostate adenocarcinoma
- SARC Skin Cutaneous Melanoma
- ST AD Testicular Germ Cell Tumors
- Thyroid carcinoma THCA
- Thymoma ⁇ UM
- Triple negative breast cancer TN-BRCA
- Uterine Corpus Endometrial Carcinoma UCEC
- WGCNA Weighted gene co-expression network analysis
- RNA-seq read count matrix for the tumor was then downloaded from the Google Cloud Pilot RNA-Sequencing for CCLE and TCGA project data repository (https :// osf. io/gqrz9/ 3 ⁇ 4. which contains RNA-seq data from TCGA processed using kallisto.
- PLA2G2D is highly differentially expressed in four types of cancers. See FIGS. lA-lD. Specifically and strikingly, PLA2G2D was expressed 56 times higher in CD8+ high tumors as compared to CD8+ low tumors.
- PBMC peripheral blood mononuclear cell
- PBMC peripheral blood mononuclear cell
- 2x1 P labeled PBMC per well of a 96 well round bottom plate were then stimulated with 1 , ug/ml anti-CD3 (OKT3, Invitrogen) and 0.2 ⁇ g/ml anti-CD28 (CD28.2, Invitrogen) in the presence of soluble 0-20 ⁇ g/ml human PLA2G2D-Fc or control human IgGl-Fc protein (Sino Biological) as indicated in a final volume of 200 m ⁇ RPM1 (Coming). PBMC cultures were incubated for 72 hours at 37°C before the supernatants were harvested and measured for IFNy and IL-2 levels using MSB V-plex assays (Meso Scale Discovery).
- T cell proliferation within the PBMC culture was simultaneously assessed by staining cells with fluoroph ore-conjugated anti ⁇ CD3, anti-CD4, and anti-CD8 antibodies (Biolegend) and Live/Dead Fixable dead cell stain (Molecular Probes), and running on a BD LSRFortessaX-20 flow cytometer (Becton Dickinson). FACS data was analyzed using FlowJo software.
- FIGS. 2A-2B show CFSE histograms and quantitation of T cell proliferation for one PBMC donor with increasing concentrations of PLA2G2D-Fc.
- FIGS. 3A-3C show CFSE histograms and quantitation of proliferation for three more independent PBMC donors in which increasing concentrations of soluble PLA2G2D-Fc protein dose dependently suppressed CD4+ and CD8+ T cell proliferation, whereas equivalent concentrations of Control-Fc protein had no significant effect.
- FIG. 4A-4B show' that, consistent with T cell proliferation, the levels of IFNy and IL-2 in the different PBMC cultures were similarly dose-dependently and significantly decreased by increasing concentrations of soluble PLA2G2D-Fc protein, but not control-Fc.
- the same assay was utilized except 100 m ⁇ of 0-10 ⁇ g/ml human PLA2G2D- Fc or control human IgGl -Fc protein was coated overnight in PBS at 4°C on 96-well flat bottom plates the day before PBMC were prepared and added along with anti-CD 3 and anti- CD28 antibodies.
- FIG. 5 show's that immobilized human PLA2G2D-Fc protein coated on the plate surface also suppressed CD4+ and CD8+ T cell proliferation in stimulated PBMC cultures.
- T cells 1x10 5 purified and labeled T cells were added to each well of the coated plate in a final volume of 200 pi RPMI. T cells were allowed to proliferate for 72 hours at 37°C before supernatants were collected for IFNy and IL-2 analysis by MSD and proliferation was analyzed by FACS.
- FIG. 6 shows that immobilized human PLA2G2D-Fc protein dose-dependently suppresses proliferation of isolated T cell cultures in the presence of anti ⁇ CD3 and anti-CD28 stimulation.
- soluble PLA2G2D-Fc protein was added to isolated T cell cultures instead of being coated on plates, it had no suppressive effect on T cells (data not shown).
- Human PLA2G2D is a 145 amino acid secreted protein consisting of an N-terminal 20 residue signal peptide, a highly conserved Ca 2 ⁇ -binding site and a catalytic His-Asp dyad, in addition to these elements, human PLA2G2D features seven disulfide bonds which contribute to a high degree of stability.
- FIG. 7 A show's general structural and functional features of interest of the human PLA2G2D protein.
- an H67Q mutation w3 ⁇ 4s introduced to the highly conserved catalytic His67-Asp68 dyad of human PLA2G2D. Briefly, a human PLA2G2D cDNA fused in frame to a human IgGl-Fc cDNA on the C terminus was synthesized with a CAC ⁇ CAG point mutation corresponding to a His ⁇ Gin substitution at residue 67. The construct was cloned into a high expression mammalian vector and transfected into HEK293 cells.
- FIGS. 7B-7C show that the PLA2G2D-H67Q-Fc catalytic mutant retains most of the immune suppressive function on CD4 + and CD8 + T cells at the dose of 0.5 ⁇ g/ml to 5 ⁇ g/ml, and exhibited a significantly decreased suppression at the dose of 10 ⁇ g/ml.
- FIG. 8 shows that LY315920 does not reverse immune suppression induced by PLA2G2D.
- T cells were isolated from PBMC using a Pan T Ceil Isolation Kit (Miltenyi) and cultured in the presence or absence of beads loaded with anti-human CD2, CD3, and CD28 antibodies (human T Cell Activation/Expansion Kit, Miltenyi) in RPMi for 48 hours at 37°C. Stimulated or unstimulated T ceils were then harvested, washed, and incubated with 0- 10 ⁇ g/ml of human PLA2G2D-Fc protein or human IgGl-Fc protein for 30 minutes at 4°C. Cells were washed 3 times with PBS and then stained with Alexa 488-conjugated goat anti- human IgGl antibodies (Invilrogen) for 30 minutes at 4 o C, washed, then analyzed by FACS.
- FIGS. 9A-9C show' that human PLA2G2D-Fc preferentially hinds activated CD4 + and CD8 + T ceils in two different donor T cells compared with control human IgGl ⁇ Fc protein.
- PLA2G2D binds unstimulated T cells to a small degree, but binding is dramatically increased upon T ceil stimulation.
- FIG. 9C shows a quantitative representation of PLA2G2D-Fc binding to stimulated T cells.
- Addition of heparin sulfate proteoglycan (HSPG) partially reduced PLA2G2D-Fc binding of T cells but did not alter suppression of CD4+ and CD8+ T cell proliferation (data not shown). This suggests that the immune suppression potentially associated with the binding of PLA2G2D to T cells is not dependent on the binding through heparin sulfate on cell surface.
- HSPG heparin sulfate proteoglycan
- PLA2G2D knockout mice were generated by deleting exon 2 of the mouse Pla2g2d gene from C57BL6 mice using CRISPR/Cas9-mediated gene editing. To confirm the absence of a functional Pla2g2d gene in these mice, spleens from wild-type and knockout mice were harvested and total RNA isolated using TRIZol (Invitrogen). Total RNA was subjected to real-time RT-PCRto detect Pla2g2dvdRH A. Hprtl (hypoxanthiiie-guaiune phosphoribosyltransferase) mRNA levels were also measured as a control. The results demonstrated that the knockout mice were deficient i nPla2g2d expression.
- the murine syngeneic tumor cell lines MC38 colon adenocarcinoma
- B16F10 melanoma
- E.G7-OVA T cell lymphoma
- WT age-matched wild-type C57BL6
- Tumor volume was calculated using the formula: tumor volume ::: 0.5 x length x width 2 .
- Body weights were also monitored weekly. Mice were sacrificed after 3-4 weeks, or upon reaching designated endpoints.
- PLA2G2D-binding monoclonal antibodies by immunizing mice and generating hybridomas.
- PLA2G2D preferentially binds to activated T cells, but only minimally to resting T cells, suggesting that PLA2G2D can impart suppressive signaling by directly binding T cells upon their activation.
- T cells within human PBMC cultures were stimulated with anti-CD3 and anii-CD28 antibodies (Invitrogen) for 24 hours at 37°C, PBMC cultures were then harvested, washed, and incubated with 2 ⁇ g/ml of human PLA2G2D-Fc protein or control human IgGl-Fc protein for 30 minutes at 4°C m the presence of 10 pg/ml of PLA2G2D antibodies (developed in house) or mouse IgG2a isotype control antibody (Invitrogen).
- FIG. 12A shows that that two representative PLA2G2D-binding antibodies are able to reduce binding of PLA2G2D to activated T cells, as measured by the mean fluorescence intensity (MFI) of PLA2G2D staining on gated T cells.
- MFI mean fluorescence intensity
- PLA2G2D antibodies could neutralize PLA2G2D-dependent suppression of T cell function, we used an approach similar to the one we took to demonstrate PLA2GD suppressive activity above (FIGS, 4A-4B), Triplicate wells containing 2x1 (P PBMC per well of a 96 well round bottom plate were stimulated with 1 ⁇ g/ml anti- CBS (OKT3, Invitrogen) and 0.2 pg/ ' ml anti-CD28 (CD28.2, Invitrogen) in the presence of 1 pg/ml soluble human PLA2G2D-Fc protein (Sino Biological) and 10 pg/mi PLA2GD antibodies or control mIgG2a isotype control antibody (Invitrogen) as indicated in a final volume of 200 m ⁇ RPMI (Corning). PBMC cultures were incubated for 48 hours at 37°C before the supernatants were harvested and measured for IL-2 and IFNy levels using MSB V- plex as
- F1GS.12B-12C show that two representative function-blocking PLA2G2D antibodies are able to rescue PLA2G2D-mediated suppression of IL-2 and IFNy secretion levels m this assay.
- the murine syngeneic tumor cell lines MC38 colon adenocarcinoma
- B16F10 melanoma
- E.G7- OVA T cell lymphoma
- 1x10 6 MC38 or E.G7-OVA ceils, or 5x10 5 B16F10 cells suspended in 100 ul PBS are subcutaneously injected into C57BL6 mice.
- tumor volumes are in the range of 50-150 mm 5 , mice are randomized into control and treatment groups.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Virology (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Hematology (AREA)
- Developmental Biology & Embryology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present application provides methods of treating a disease (such as cancer or infectious disease) that involves an antagonist that targets PLA2G2D signaling pathway (such as an antagonist that targets PLA2G2D. The present application also provides non-naturally occurring PLA2G2D polypeptides.
Description
METHODS AND COMPOSITIONS FOR TREATING CANCER OR VIRAL INFECTION WITH A PLA2G2D ANTAGONIST
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority' benefit of U.S. Provisional Application 62/968,060 filed January' 30, 2020, the contents of which are incorporated herein by reference in their entirety'.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 196882000140SEQLIST.TXT, date recorded: January' 29, 2021, size: 34 KB),
FIELD OF THE APPLICATION
[0003] The present invention relates to methods and compositions for treating a disease or condition that involve an antagonist targeting PLA2G2D signaling pathway.
BACKGROUND
[0004] The mechanisms by which the immune system responds to an infection or disease depend on a complex interplay between the elements of innate and adaptive immunity. Unwanted suppression of immune response stays as a major hurdle for patients’ own immune system or promising treatments such as immunotherapies to fight against the disease or infection. For example, a fundamental problem in the effort to treat patients with an immunotherapy, is that the tumor-bearing state is associated with immunosuppressive mechanisms derived from both the tumor and the host's disturbed immune system, thereby preventing the therapy to achieve the ideal efficacy,
[0005] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety for all purposes.
BRIEF SUMMARY OF THE APPLICATION [0006] The present application provides methods of treating diseases or conditions (such as cancer or viral infection).
[0007] The present application in one aspect provides methods of treating a cancer or viral infection in an individual comprising administering into the individual an effective amount of an antagonist targeting PLA2G2D signaling pathway. In some embodiments, the
antagonist is an antagonist targeting PLA2G2D. in some embodiments, the PLA2G2D is a human PLA2G2D. In some embodiments, the antagonist decreases enzymatic activity level of PLA2G2D, In some embodiments, the antagonist targeting PLA2G2D signaling pathway blocks a catalytic site on PLA2G2D. In some embodiments, the antagonist targets the H67 catalytic site on a human PLA2G2D according to SEQ ID NO: 1 or 5.
[0008] In some embodiments, the antagonist comprises a siRNA, a miRNA, an antisense RNA, or a gene editing system.
[0009] In some embodiments, the antagonist comprises an agent that inhibits PLA2G2D (such as an agent that blocks the binding of PLA2G2D to an immune cell or an agent that inhibits the activity of PLA2G2D). In some embodiments, the immune cell is aT cell.
[0010] In some embodiments, the antagonist comprises an anti-PLA2G2D antibody. In some embodiments, the anti-PLA2G2D antibody is a monoclonal antibody, in some embodiments, the antagonist is a fusion protein or immunoconjugate comprising an anti-PLA2G2B antibody moiety and a second moiety, in some embodiments, the second moiety comprises a cytokine.
[0011] In some embodiments, the antagonist comprises an inhibitory PLA2G2D polypeptide that blocks the binding of PLA2G2D to an immune ceil.
[0012] In some embodiments, the inhibitory PLA2G2D polypeptide binds to the immune cell with a greater affinity than for PLA2G2D, In some embodiments, the immune cells is a T cell. In some embodiments, the inhibitory polypeptide further comprises a stabilizing domain. In some embodiments, the stabilizing domain is an Fc domain. In some embodiments, the inhibitory PLA2G2D polypeptide has a length of about 50 to about 200 amino acids. In some embodiments, the inhibitory' PLA2G2D polypeptide has a mutation at the position corresponding to histidine at position 67 (H67) according to SEQ ID NO: 1 or 5. In some embodiments, the inhibitory PLA2G2D polypeptide comprises an ammo acid sequence of SEQ ID NO: 3, 4, 7, or 8,
[0013] In some embodiments according to any one of the methods described above, the disease or condition is a cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is an advanced or malignant tumor. In some embodiments, the cancer has an increased expression level of PLA2G2D. In some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer,
pancreatic cancer, renal cancer, prostate cancer, urothelial cancer, testis cancer, ovarian cancer and melanoma. In some embodiments, the disease or condition is a viral infection. In some embodiments, the infection site has an increased expression level of PLA2G2D.
[0014] In some embodiments according to any one of the methods described above, the method further comprises administering a second agent. In some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomoduiator, an anti-angiogenesis agent, a growth inhibitory' agent, and an antineoplastic agent. In some embodiments, the second agent is an immunomoduiator. in some embodiments, the immunomoduiator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor specifically target PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIMS, LAGS, CD27, 4-1BB, or B7H4. In some embodiments, the second agent comprises a cell comprising a chimeric antigen receptor that specifically binds to a tumor antigen. In some embodiments, the antagonist and the second agent is administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent is administered sequentially. In some embodiments, the antagonist and/or the second agent is administered parentally. In some embodiments, the antagonist is administered to the cancer tissue or infection site directly.
[0015] In some embodiments according to any of the methods described above, the antagonist is administered at a dose of about 0.001 pg/kg to about 100 mg/kg.
[0016] In some embodiments according to any of the methods described above, the individual has an increased number of immune ceils in the cancer tissue or at the infection site after administration of the antagonist. In some embodiments, the immune ceils are T cells. In some embodiment, the T cells are activated T cells. In some embodiments, the number of immune cells in the cancer tissue or at the infection site is increased by at least about 5% (such as at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold) after administration of the antagonist.
[0017] In some embodiments according to any of the methods described above, immune ceils in the cancer tissue or at the infection site produce an increased level of a cytokine after administration of the antagonis t. In some embodiments, the cytokine is IFNy and/or IL-2. In some embodiments, the level of the cytokine is increased by at least about 5% (such as at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2- fold, 3-fold, 4-fold, or 5 -fold) after administration of the antagonist.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1 A-1D shows that PLA2G2D is highly differentially expressed in human (FIG. 1 A) lung adenocarcinoma, (FIG. IB) triple negative breast cancer, (FIG. 1C) live hepatocellular carcinoma, and (FIG. ID) stomach adenocarcinoma. Relative expression and significance ofPD-1, CTLA-4 and TIGIT are also indicated.
[0019] FIG. 2A shows that soluble human PLA2G2D-Fc protein dose-dependently suppresses PBMC derived CD4+ and CD8+ T cell proliferation in the presence of anti-CD3 and anti-CD28 stimulation.
[0020] FIG. 2B shows a quantitative graph of the effect of human PLA2G2D-Fe protein on PBMC-derived CD4+ and CD8+ T cell proliferation.
[0021] FIGS. 3A-3C show that soluble PLA2G2D protein dose-dependently suppresses T cell proliferation in different PBMC donors in the presence of anti-CD3 and anti-CD28 stimulation. T cell CFSE proliferation as analyzed by flow cytometry is shown on the left, and quantitative representations of percent CD4+ and CD8+ T cell proliferation are shown on the nght.
[0022] FIGS, 4A-4B show that soluble PLA2G2D protein dose-dependently suppresses IFN □ and IL-2 levels in correlation with suppression of T cell proliferation across different donors.
[0023] FIG. 5 shows that immobilized PLA2G2D protein dose-dependently suppresses T cell proliferation in PBMC culture in the presence of anti-CD3 and anti-CD28 stimulation. T cell CFSE proliferation as analyzed by flow cytometry is shown on the left, and quantitative representations of percent < 1)4 and CD8+ T cell proliferation are shown on the right.
[0024] FIG. 6 show's that immobilized PLA2G2D protein dose-dependently suppresses proliferation of isolated T cell cultures in the presence of anti~CD3 and anti-CD28 stimulation. T cell CFSE proliferation as analyzed by flow cytometry is shown on the left, and quantitative representations of percent CD4+ and CD8+ T cell proliferation are shown on the right.
[0025] FIG. 7A shows structural and functional features of in terest of the human PLA2G2D protein (SEQ ID NO: 22) including its signal peptide (the first 20 ammo acids), calcium binding sites, catalytic sites, N-linked glycosylation site, and active site. A H67Q catalytic site mutant was generated to create an enzymatic-deficient PLA2G2D protein.
[0026] FIGS. 7B-7C show that aH47Q-PLA2G2D catalytic mutant retains most of the immune suppressive functions on CD4+ (7B) and CD8+ (7C) T cells.
[0027] FIG. 8 shows that a general inhibitor for various PLA2 small molecule Inhibitor,
LY315920, does not rescue immune suppression by PLA2G2D.
[0028] FIGS. 9A-9C show that human PLA2G2D-Fc preferentially hinds activated CD4+ and CD8+ T ceils in different donor T cells compared with controi-Fc protein. PLA2G2D binds unstimulated T ceils to a small degree, but binding is dramatically increased upon T cell stimulation. FIG. 9C shows a quantitative representation of PLA2G2D~Fc binding to stimulated T cells.
[0029] FIG. 10A shows a predicted automated 3G) structure based upon Swiss-model.
[0030] FIG. 10B shows sequence homology of different PLA2 Group 2 family members to PLA2G2D. SEQ ID NOs from top to bottom are SEQ ID NOs 22-32.
[0031] FIG. IOC shows sequence homology of PLA2G2D from different species vs. human. Human (SEQ ID NO: 33), Mouse (SEQ ID NO: 34), Rat (SEQ ID NO: 35), Rhesus (SEQ ID NO: 36), and Chimp (SEQ ID NO: 37).
[0032] FIG. 11A shows that the mean tumor volume of syngeneic subcutaneous MC38 colon adenocarcinoma tumors implanted in PLA2G2D knockout (KO) mice (n=16) is significantly reduced compared to wild type (WT) C57BL6 mice in 16). FIG. GIB show's the tumor growth kinetics of individual animals from each group.
[0033] FIG. 11C show's that the mean tumor volume of syngeneic subcutaneous B16F10 melanoma tumors implanted in PLA2G2D knockout (KO) mice (n=16) is significantly reduced compared to wild type (WT) C57BL6 mice (n-16). FIG. 11D show's the tumor growth kinetics of individual animals from each group.
[0034] FIG. 1 IE shows that the mean tumor volume of syngeneic subcutaneous E.G7-OVA T cell lymphoma tumors implanted in PLA2G2D knockout (KO) mice (n=T6) is significantly reduced compared to wild type (WT) C57BL6 mice (n=T6). FIG. 1 IF shows the tumor growth kinetics of individual animals from each group.
[0035] FIG. 12A shows that the mean fluorescence intensity (MFI) of PLA2G2D~Fc binding to activated T cells in PBMC culture can be blocked by anti-PLA2G2D antibodies.
[QQ36] FIGS. 12B-12C show that PLA2G2D-Fc mediated suppression of IL-2 and IFNy levels in T cell-activated PBMC cultures can he reversed by the addition of function-blocking anti-PLA2G2D antibodies.
DETAILED DESCRIPTION OF THE APPLICATION
[0037] The present application in one aspect pro vides methods of treating a disease or condition (such as cancer or infectious disease) that involves administering an antagonist targeting PLA2G2D signaling pathway. In some embodiments, the antagonist comprises an agent binding to PLA2G2D (such as an agent comprising an anti-PL, A2G2D antibody moiety). In some embodiments, the antagonist comprises an inhibitory PLA2G2D polypeptide. In some embodiments, the antagonist comprises a nucleic acid agent targeting PLA2G2D (such as a siRNA or antisense RNA). In some embodiments, the antagonist comprises an agent that inhibits PLA2G2D enzymatic activity. The present application in another aspect provides non-naturally occurring polypeptides such as the inhibitory PLA2G2D polypeptides that can he used for treatment.
[QQ38] The present application is at least partly based upon the striking finding that PLA2G2D plays a crucial role in suppressing a key player in immune system, T cells. Specifically, it was found that PLA2G2D was expressed 56 times higher in CD8+ high tumors than CD8+ low' tumors. As shown in Examples in more details, PLA2G2D can both directly and indirectly (e.g., via cross-linking with antigen-presenting cells) inhibit both CB4-t- and CD8+ T cells" proliferation, activation and/or cytokine production, which can lead to a significant level of suppression of immune response m diseases such as cancer, especially in the diseased tissue such as a cancer tissue. It was also found that PLA2G2D’s enzymatic activity only partially contributes to its role in suppressing immune response, and that PLA2G2D can directly hind to T cells, especially activated T cells. Without being bound to the theory, it is believed that at least part of the suppressive function of PLA2G2D on T cells is exerted by the binding of PLA2G2D to the cells. The present application has for the first time promising novel methods of using an antagonist that targets PLA2G2D pathway (such as an agent comprising an anti~PLA2G2D antibody moiety' or an inhibitory' PLA2G2D polypeptide) to treat a disease or condition in which the immune response is suppressed, including, for example, cancer and infectious disease (such as viral infectious disease).
I. Definitions
[0039] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present application.
For purposes of the present application, the following terms are defined.
[0040] It is understood that embodiments of the application described terms of “comprising” herein include “consisting” and/or “consisting essentially of’ embodiments.
[0041] As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
[0042] As used herein the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature.
[0043] The terms “non-natura!iy occurring” or “engineered” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.
[0044] As used herein, “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
[0045] The terms “therapeutic agent”, “therapeutic capable agent” or “treatment agent” are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to an individual. The beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.
[0046] The term ‘‘antibody” is used in its broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, chimeric antibodies, full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. Antibodies and/or antibody fragments may be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, eamelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camebzed antibody variable domains.
[0047] ‘‘Percent (%) amino acid sequence identity” or “homology” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identify'. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5): 1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1): 113, 2004, each of which are incorporated herein by reference in their entirety for all purposes).
[0048] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or ammo acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology'. Generally, a comparison is made when two sequences are aligned to give maximum homolog}'.
[0049] The term ‘‘epitope” as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody or diabody binds. Two antibodies or antibody moieties may bind the same epitope within an antigen if they exhibit competitive binding for the antigen.
[0050] The terms “polypeptide” or “peptide” are used herein to encompass ah kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, forrnylation, amidation, polyglutamylation, ADP-ribosyiation, pegylalion, biotinylation, etc.).
[0051] As use herein, the terms “specifically binds,” “specifically recognizing,” and “is specific for” refer to measurable and reproducible interactions, such as binding between a target and an antibody (such as a diabody). In certain embodiments, specific binding is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules (e.g., cell surface receptors). For example, an antibody that specifically recognizes a target (which can be art epitope) is an antibody (such as a diabody) that binds this target with greater affinity, avidity, more readily, and/or with greater duration than its bindings to other molecules. In some embodiments, the extent of binding of an antibody to an unrelated molecule is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA). in some embodiments, an antibody that specifically binds a target has a dissociation constant (KD) of ≤ 10-5 M, ≤10'6 M, ≤10-7 M, ≤10-8 M, ≤10 -5 M, ≤10-10 M, ≤10-11 M, or ≤10-12 M. In some embodiments, an antibody specifically binds an epitope on a protein that is conserved among the protein from different species. In some embodiments, specific binding can include, but does not require exclusive binding. Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA, RIA, ECL, IRMA, ETA, BIACORETM and peptide scans.
[0052] As used herein, “the composition” or “compositions” includes and is applicable to compositions of the application. The application also provides pharmaceutical compositions comprising the components described herein.
10053 [ As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g, preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival. The methods of the application contemplate any one or more of these aspects of treatment. The benefit to an individual to be treated is either statistically significant or at least perceptible to the patient or to the physician.
[0054] The term “effective amount” used herein refers to an amount of an agent or composition sufficient to treat a specified state, disorder, condition, or disease such as ameliorate, palliate, lessen, and/or delay one or more of its symptoms (e.g., clinical or sub- clinical symptoms). For therapeutic use, beneficial or desired results include, e.g., decreasing one or more symptoms resulting from the disease (biochemical, histologic and/or behavioral), including its complications and intermediate pathological phenotypes presenting during development of the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication, delaying the progression of the disease, and/or prolonging survival of patients, in reference to a cancer, an effective amount comprises an amount sufficient to cause a cancer tissue to shrink and/or to decrease the growth rate of the cancer tissue or to prevent or delay other unwanted cell proliferation in the cancer. In some embodiments, an effective amount is an amount sufficient to delay development of a cancer. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. In the case of cancer, the effective amount of the drug or composition may: (i) reduce the number of tumor cells:
(ii) reduce the tumor size; (iii) inhibit, retard, slow to some extent and preferably stop a tumor cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms
associated with the cancer. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The exact amount required will vary from individual to individual, depending on the species, age, and general condition of the individual, the se verity of die condition being treated, the particular drug or drugs employed, the mode of administration, and the like.
[0055] 'The term ‘'simultaneous administration,” as used herein, means that a first therapy and second therapy in a combination therapy are administered with a time separation of no more than about 15 minutes, such as no more than about any of 10, 5, or 1 minutes. When the first and second therapies are administered simultaneously, the first and second therapies may be contained in the same composition (e.g, a composition comprising both a first and second therapy) or in separate compositions (e.g., a first therapy in one composition and a second therapy is contained in another composition).
[0056] As used herein, the term “sequential administration” means that the first therapy and second therapy in a combination therapy are administered with a time separation of more than about 15 minutes, such as more than about any of 20, 30, 40, 50, 60, or more minutes. Either the first therapy or the second therapy may be administered first. The first and second therapies are contained in separate compositions, which may be contained in the same or different packages or kits.
[0057] As used herein, the term “concurrent administration” means that the administration of the first therapy and that of a second therapy in a combination therapy overlap with each other.
[0058] As used herein, by “pharmaceutically acceptable” or “pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and/or are included on the Inactive ingredient Guide prepared by the U.S. Food and Drug administration or other state/federal government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.
[0059] The term ‘‘carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can he sterile liquids, such as water and oils, including those of petroleum, animal , vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Alternatively, the earner can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in ‘‘Remington’s Pharmaceutical Sciences” by E.W. Martin, incorporated by reference in its entirety for all purposes.
[0060] The term “tumor” refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth and includes benign or malignant abnormal growth of tissue. The term “tumor” includes cancer.
[0061] 'The terms “individual,” “subject,” and “patient” are used interchangeably herein to refer to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate, in some embodiments, the individual is a human. In a preferred embodiment, the individual is a human.
[0062] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X". in certain embodiments, a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.
[0063] The term “about X-Y” used herein has the same meaning as “about X to about Y,”
[0064] As used herein and in the appended claims, the singular forms "a,” “an,” "or," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading tins disclosure. As is apparent to one skilled in the art, an individual assessed, selected for, and/or receiving treatment is an individual in need of such activities.
[0065] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of statistical analysis, molecular biology (including recombinant techniques), microbiology', cell biology , and biochemistry, which are within the skill of the art. Such tools and techniques are described in detail in e.g., Sambrook et al. (2001)
Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press:
Cold Spring Harbor, New York; Ausubel et al, eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bomfacmo et al. eds. (2005) Current Protocols m Cell Biology. John Wiley and Sons, inc.: Hoboken, NJ; Co!igan et al. eds.
(2005) Current Protocols in immunology , John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ. Additional techniques are explained, e.g., in U.S. Patent No. 7,912,698 and U.S. Patent Appl. Pub. Nos. 2011/0202322 and 2011/0307437, each of which is incorporated by reference in their entirety for all purposes.
[0066] The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of tire technology claimed.
II. Methods of treatment
[0067] The present application in one aspect provides methods of treating a disease or condition (such as cancer or infectious disease) in an individual, comprising administering to the individual an effective amount of an antagonist that targets PLA2G2D signaling pathway, in some embodiments, the antagonist comprises an agent binding to PLA2G2D (such as an agent comprising an anti~PLA2G2D antibody moiety). In some embodiments, the antagonist comprises an inhibitory PLA2G2D polypeptide. In some embodiments, the antagonist comprises a nucleic acid agent targeting PLA2G2D (such as a siRNA or antisense RNA). In some embodiments, the antagonist comprises an agent that inhibits PLA2G2D enzymatic activity .
[0068] In some embodiments, there is provided a method of treating a cancer (such as a solid tumor, a colon cancer, melanoma, or a T cell lymphoma) in an individual, comprising administering into the individual an effective amount of an antagonist that comprises an agent
that inhibits PLA2G2D (such as an agent that blocks the binding of PLA2G2D to an immune cell or an agent that inhibits the activity of PLA2G2D). In some embodiments, the immune cell is a T cell (such as an activated T cell, such as activated CD4+ T cells or CD8+ T cells), in some embodiments, the antagonist comprises an anti-PLA2G2D antibody. In some embodiments, the anti-PLA2G2D antibody is a monoclonal antibody. In some embodiments, the antagonist is a fusion protein or immunoconj ugate comprising an anti~PLA2G2D antibody moiety' and a second moiety', such as a second moiety' comprising a cytokine (such as a pro-inflammatory cytokine). In some embodiment, the PLA2G2D is a human PLA2G2D. In some embodiments, the cancer tissue has an increased expression level of PLA2G2D as compared to a reference tissue (such as a corresponding tissue in a healthy individual). In some embodiments, the cancer is an advanced or malignant tumor. In some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, renal cancer, prostate cancer, urothelial cancer, testis cancer, ovarian cancer and melanoma. In some embodiments, the method further comprises administering a second agent. In some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulator, an anti-angiogenesis agent, a growth inhibitory agent, and an antineoplastic agent. In some embodiments, the second agent is ait immunomodulator. In some embodiments, the immunomodulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor specifically target PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIMS,
LAGS, CB27, 4-1BB, or B7H4. In some embodiments, the second agent comprises a ceil comprising a chimeric antigen receptor that specifically binds to a tumor antigen. In some embodiments, the antagonist and the second agent is administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent is administered sequentially. In some embodiments, the antagonist and/or the second agent is administered parentally. In some embodiments, the antagonist is administered to diseased tissue directly.
[0069] In some embodiments, there is provided a method of treating an infectious disease (such as a viral infectious disease) in an individual, comprising administering into the individual an effecti ve amount of an antagonist that comprises an agent that inhibits PLA2G2D (such as an agent that blocks the binding of PLA2G2D to an immune cell or an agent that inhibits the activity' of PLA2G2D). In some embodiments, the immune cell is a T cell (such as ait activated T cell, such as activated CD4+ T cells or CD8+ T cells). In some
embodiments, the antagonist comprises an anti-PLA2G2D antibody, in some embodiments, the anti-PLA2G2D antibody is a monoclonal antibody. In some embodiments, the antagonist is a fusion protein or immunoconjugate comprising an anii-PLA2G2D antibody moiety and a second moiety, in some embodiments, the second moiety comprises a cytokine (such as a pro-inflammatory cytokine). In some embodiment, the PLA2G2D is a human PLA2G2D. in some embodiments, the infection site has an increased expression level of PLA2G2D as compared to a reference tissue (such as a corresponding tissue in a healthy individual). In some embodiments, the method further comprises administering a second agent. In some embodiments, the second agent comprises an immune therapy. In some embodiments, the antagonist and the second agent is administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent is administered sequentially. In some embodiments, the antagonist and/or the second agent is administered parentally. In some embodiments, the antagonist is administered to diseased tissue directly.
[0070] In some embodiments, there is provided a method of treating a cancer (such as a solid tumor, a colon cancer, melanoma, or a T ceil lymphoma) in an individual, comprising administering into the individual an effective amount of an antagonist comprises an inhibitory PLA2G2D polypeptide that inhibits PLA2G2D (such as an inhibitory polypeptide that blocks the binding of PLA2G2D to an immune cell). In some embodiments, the inhibitory PLA2G2D polypeptide binds to the immune cell with a greater affinity' than for PLA2G2D (such as a wildtype PLA2G2D). In some embodiments, the immune cell is a T ceil (such as an activated T cell, such as activated CD4+ T cells or CD 8+ T cells). In some embodiments, the inhibitory' PLA2G2D polypeptide further comprises a stabilizing domain. In some embodiments, the stabilizing domain is an Fc domain. In some embodiments, the inhibitory PLA2G2D polypeptide has a length of about 50 to about 200 amino acids. In some embodiments, the inhibitory' PLA2G2D polypeptide has a) a mutation at the position corresponding to histidine at position 67 (H67) according to 8EQ ID NO: 1 or 5 or b) a mutation at the position corresponding to glycine at position 80 (G80) according to SEQ ID NO: 5. In some embodiments, the inhibitory' PLA2G2D polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, and 7-12 or a variant thereof. In some embodiment, the PLA2G2D is a human PLA2G2B. In some embodiments, the cancer tissue has an increased expression level of PLA2G2D as compared to a reference tissue (such as a corresponding tissue in a healthy individual). In some embodiments, the cancer is an advanced or malignant tumor. In some embodiments, the cancer is selected from
the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, renal cancer, prostate cancer, urothelial cancer, testis cancer, ovarian cancer and melanoma, in some embodiments, the method further comprises administering a second agent, in some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulator, an anti-angiogenesis agent, a growth inhibitory agent, and an antmeoplastic agent. In some embodiments, the second agent is an immunomodulator. in some embodiments, the immunomodulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor specifically target PD-L1, PD-L2, CTLA4, PD-1,2, PD-1, CD47, ! Kil l GITR, TIMS, LAGS, CD27, 4-IBB, or B7H4. in some embodiments, the second agent comprises a cell comprising a chimeric antigen receptor that specifically binds to a tumor antigen. In some embodiments, the antagonist and the second agent is administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent is administered sequentially. In some embodiments, the antagonist and''orthe second agent is administered parentally, in some embodiments, the antagonist is administered to diseased tissue directly.
[0071] in some embodiments, there is provided a method of treating an infections disease (such as a viral infectious disease) in an individual, comprising administering into the individual an effective amount of an antagonist comprises an inhibitory PLA2G2D polypeptide that blocks the binding of PLA2G2D to an immune cell, in some embodiments, the inhibitory PLA2G2D polypeptide binds to the immune cell with a greater affinity' than for PLA2G2D (such as a wildtype PLA2G2D). In some embodiments, the immune cell is a T cell (such as an activated T ceil, such as activated CD4+ T cells or CD8+ T cells). In some embodiments, the inhibitor}' PLA2G2D polypeptide further comprises a stabilizing domain, in some embodiments, the stabilizing domain is an Fe domain, in some embodiments, the inhibitory PLA2G2D polypeptide has a length of about 50 to about 200 amino acids. In some embodiments, the inhibitory PLA2G2D polypeptide has a) a mutation at the position corresponding to histidine at position 67 (H67) according to SEQ ID NO: 1 or 5 or b) a mutation at the position corresponding to glycine at position 80 (G80) according to SEQ ID NO: 5. In some embodiments, the inhibitor}' PLA2G2D polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, and 7-12 or a variant thereof. In some embodiment, the PLA2G2D is a human PLA2G2D. In some embodiments, the infection site has an increased expression level of PLA2G2D as compared to a reference
tissue (such as a corresponding tissue in a healthy individual). In some embodiments, tire method further comprises administering a second agent, in some embodiments, the second agent comprises an immune therapy. In some embodiments, the antagonist and the second agent is administered simultaneously or concurrently, in some embodiments, the antagonist and the second agent is administered sequentially, in some embodiments, the antagonist and/or the second agent is administered parentally. In some embodiments, the antagonist is administered to diseased tissue directly.
[0072] In some embodiments, there is provided a method of treating a cancer (such as a solid tumor, a colon cancer, melanoma, or a T cell lymphoma) in an individual, comprising administering into the individual an effective amount of an antagonist that comprises a nucleic acid agent that inhibits the expression of PLA2G2D. In some embodiments, the nucleic acid agent comprises a siRNA, a miRNA, or an antisense RNA. in some embodiment, the PLA2G2D is a human PLA2G2D. in some embodiments, the cancer tissue has an increased expression level of PLA2G2D as compared to a reference tissue (such as a corresponding tissue in a healthy individual). In some embodiments, the cancer is an advanced or malignant tumor. In some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, renal cancer, prostate cancer, urothelial cancer, testis cancer, ovarian cancer and melanoma. In some embodiments, the method further comprises administering a second agent. In some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodtilator, an anti-angiogenesis agent, a growth inhibitory agent, and an antmeoplastic agent, in some embodiments, the second agent is an immunomodulator. In some embodiments, the immunomodulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor specifically target PD-LL PD-L2, CTLA4, PB-L2, PD-1, CD47, TIGIT, GITR, TIMS, LAGS, CD27, 4-1BB, or B7H4. in some embodiments, the second agent comprises a cell comprising a chimeric antigen receptor that specifically binds to a tumor antigen. In some embodiments, the antagonist and the second agent is administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent is administered sequentially. In some embodiments, the antagonist and/or the second agent is administered parentally, in some embodiments, the antagonist is administered to diseased tissue directly.
[0073] In some embodiments, there is provided a method of treating a cancer (such as a solid tumor, a colon cancer, melanoma, or a T cell lymphoma) in an individual, comprising administering into the individual an effective amount of an antagonist that comprises a nucleic acid agent that inhibits the expression of PLA2G2D, wherein the individual has high T cell infiltration in cancer tissue, in some embodiments, the high T cell infiltration comprises a high number, percentage or density of T cells (e.g., CD3 T cells, CD4 T cells, CD8 T cells, activated T cells, activated CD4 T cells, activated CD8 T cells) in the cancer tissue, in some embodiments, the high T cell infiltration is present when the number of the T cells in the cancer is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% more than the number of the corresponding T cells in a reference tissue, in some embodiments, the high T cell infiltration is present when the number of the T ceils in the cancer is at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold more than the number of the corresponding T cells in a reference tissue. In some embodiments, the reference tissue is the corresponding tissue in a healthy individual. In some embodiments, the number of the corresponding T ceils in a reference tiss ue is the average number of the corresponding T cells in the same tissue in a group of individuals (such as 10, 30, 50, 100 individuals) with same or similar cancer. In some embodiments, the reference tissue is the corresponding tissue in an individual who also has a cancer but has a less suppressed immune response in the cancer tissue as indicated by a biomarker. Examples of biomarker indicative of immunosuppressive tumor microenvironment (TME) includes: a) a high number, percentage and/or density of M2 macrophages (e.g., CD68+CD163+ cells) in the tissue; b) a high expression level of an immune checkpoint agent (e.g., PD-1 or PD-L1). Methods assessing and evaluating these biomarkers are known. See e.g., Hensler et al. Journal for ImmunoTherapy of Cancer 2020;8:e(X)0979; Chen et ah, J Biomed Sci 26, 78 (2019): Teng et al, Cancer Res. 2015 Jun 1; 75(11): 2139-2145.In some embodiments, the nucleic acid agent comprises a siRNA, a miRNA, or an antisense RNA. in some embodiment, the PLA2G2D is a human PLA2G2D. in some embodiments, the cancer tissue has an increased expression level of PLA2G2D as compared to a reference tissue (such as a corresponding tissue in a healthy individual), in some embodiments, the cancer is an advanced or malignant tumor. In some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, renal cancer, prostate cancer, urothelial cancer, testis cancer, ovarian cancer and melanoma. In some embodiments, the method further comprises administering a second
agent, in some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulator, an anti-angiogenesis agent, a growth inhibitory agent, and an antineoplastic agent. In some embodiments, the second agent is an immunomodulator. In some embodiments, the immunomodulator is an immune checkpoint inhibitor, in some embodiments, the immune checkpoint inhibitor specifically target PD-Ll, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TΊM3, LAGS. CD27, 4-1BB, or B7H4. in some embodiments, the second agent comprises a cell comprising a chimeric antigen receptor that specifically binds to a tumor antigen, in some embodiments, the antagonist and the second agent is administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent is administered sequentially. In some embodiments, the antagonist and/or the second agent is administered parentally. In some embodiments, the antagonist is administered to diseased tissue directly.
[0074] In some embodiments, there is provided a, method of treating a, cancer (such as a solid tumor, a colon cancer, melanoma, or a T cell lymphoma) in an individual, comprising administering into the individual an effective amount of an antagonist that comprises a nucleic acid agent that inhibits the expression of PLA2G2D, wherein the individual has a, high expression level of PLA2G2D in the cancer tissue. In some embodiments, the cancer tissue has a high expression level of PLA2G2D when the expression level of PLA2G2D (e.g., assessed by immunohistochemistry) is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% higher than the expression level of PLA2G2D in a reference tissue. In some embodiments, the cancer tissue has a high expression level of PLA2G2D when the expression level of PLA2G2D (e.g., assessed by immunohistochemistry) is at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7 -fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold higher than the expression level of PLA2G2D in a reference tissue. In some embodiments, the reference tissue is the corresponding tissue in a healthy individual. In some embodiments, the expression level of PLA2G2D in a reference tissue is the average expression level of PLA2G2D in the same tissue in a group of individuals (such as 10, 30, 50, 100 individuals) with same or similar cancer. In some embodiments, the reference tissue is the corresponding tissue in an individual who also has a cancer but has a less suppressed immune response in the cancer tissue as indicated by a biomarker (such as high M2 macrophages, or high expression of an immune checkpoint agent such as PD-1 or PD-Ll). In some embodiments, the nucleic acid agent comprises a siRNA, a miRNA, or an antisense RNA. in some embodiment, the PLA2G2D is a human PLA2G2D.
in some embodiments, the cancer tissue has an increased expression level of PLA2G2D as compared to a reference tissue (such as a corresponding tissue in a healthy individual), in some embodiments, the cancer is an advanced or malignant tumor. In some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, renal cancer, prostate cancer, urothelial cancer, testis cancer, ovarian cancer and melanoma. In some embodiments, the method further comprises administering a second agent, in some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulator, an anti-angiogenesis agent, a growth inhibitor}' agent, and an antineoplastic agent. In some embodiments, the second agent is an immunomodulator. In some embodiments, the immunomodulator is an immune checkpoint inhibitor, in some embodiments, the immune checkpoint inhibitor specifically target PD-L1, PDA 2. CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIM3,
LAGS, CD27, 4-1BB, or B7H4. in some embodiments, the second agent comprises a cell comprising a chimeric antigen receptor that specifically binds to a tumor antigen. In some embodiments, the antagonist and the second agent is administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent is administered sequentially. In some embodiments, the antagonist and/or the second agent is administered parentally. In some embodiments, the antagonist is administered to diseased tissue directly.
[0075] In some embodiments, there is provided a method of treating a cancer (such as a solid tumor, a colon cancer, melanoma, or a T ceil lymphoma) in an individual, comprising administering into the individual an effective amount of an antagonist that comprises a nucleic acid agent that inhibits the expression of PLA2G2D, wherein the individual has a) a high T cell infiltration (e.g., CDS T cells, e.g,, CD4 T cells, e.g., CDS T cells, e.g., activated CDS or CD4 or CDS T cells) in the cancer tissue, and/or b) a high PLA2G2D expression in the cancer tissue.
[0076] In some embodiments, the methods described herein further comprise selecting an individual for treatment based upon high T cell infiltration (e.g., high CDS T cells, high CDS T cells, high CD4 T ceils, activated T cells, activated CDS T ceils, or activated CD4 T ceils) in the cancer tissue. High T cell infiltration can be determined by a) assessing the number of T cells (e.g., CDS T ceils, CD4 T cells, CDS T cells, activated T cells, activated CD4 T cells, activated CDS T ceils) in tumor, and b) comparing the number to the number of corresponding T cells in a reference tissue. In some embodiments, the high T cell infiltration
is present when the number of the T cells in the cancer is at least about 5%, 10%. 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% more than the number of the corresponding T cells in a reference tissue. In some embodiments, the high T cell infiltration is present when the number of the T cells in the cancer is at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-foid, 6-fold, 7 -fold, 8-fold, 9-fold, or 10-fold more than the number of the corresponding T cells in a reference tissue. In some embodiments, the reference tissue is the corresponding tissue in a healthy individual. In some embodiments, the number of the corresponding T cells in a reference tissue is the average number of the corresponding T cells in the same tissue in a group of individuals (such as 10, 30, 50, 100 individuals) with same or similar cancer. In some embodiments, the reference tissue is the corresponding tissue in an individual who also has a cancer but has a less suppressed immune response in the cancer tissue as indicated by a biomarker. Examples of biomarker indicative of immunosuppressive tumor microenvironment (TME) includes: a) a high number, percentage and/or density of M2 macrophages (e.g., CD68+CD163+ cells) in the tissue; b) a high expression level of an immune checkpoint agent (e.g., PD-1 or PD-Ll).
[0077] In some embodiments, the methods described above further comprise selecting an individual for treatment based upon a high expression level of PLA2G2D in the cancer tissue. In some embodiments, the cancer tissue has a high expression level of PLA2G2D when the expression level of PLA2G2D (e.g., assessed by immunohistochemistry) is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% higher than the expression level of PLA2G2D in a reference tissue. In some embodiments, the cancer tissue has a high expression level of PLA2G2D when the expression level of PLA2G2D (e.g., assessed by immunohistochemistry') is at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6- fo!d, 7 -fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold higher than the expression level of PLA2G2D in a reference tissue. In some embodiments, the reference tissue is the corresponding tissue in a healthy individual. In some embodiments, the expression level of PLA2G2D in a reference tissue is the average expression level of PLA2G2D in the same tissue in a group of individuals (such as 10, 30, 50, 100 individuals) with same or similar cancer. In some embodiments, the reference tissue is the corresponding tissue in an individual who also has a cancer but has a less suppressed immune response in the cancer tissue as indicated by a biomarker (such as high M2 macrophages, or high expression of an immune checkpoint agent such as PD-1 or PD-L1).
[0078] In some embodiments, the methods described herein comprise selecting an individual for treatment, wherein the individual has a) a high T cell infiltration (e.g., CD3 T ceils, e.g., CD4 T cells, e.g., CDS T cells, e.g., activated CD3 or CD4 or CDS T cells) m the cancer tissue, and/or b) a high PLA2G2D expression in the cancer tissue.
[0079] In some embodiments, there is pro vided a method of treating an infectious disease (such as a viral infectious disease) in an individual, comprising administering into the individual an effective amount of an antagonist that comprises a nucleic acid agent that inhibits the expression of PLA2G2D. In some embodiments, the nucleic acid agent comprises a siRNA, a miRNA, or an antisense RNA. In some embodiment, the PLA2G2D is a human PLA2G2D. In some embodiments, the infection site has an increased expression level of PLA2G2D as compared to a reference tissue (such as a corresponding tissue in a healthy individual). In some embodiments, the method further comprises administering a second agent. In some embodiments, the second agent comprises an immune therapy. In some embodiments, the antagonist and the second agent is administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent is administered sequentially. In some embodiments, the antagonist and/or the second agent is administered parentally. In some embodiments, the antagonist is administered to diseased tissue directly.
[0080] In some embodiments, there is provided a method of treating a cancer (such as a solid tumor, a colon cancer, melanoma, or a T cell lymphoma) in an individual, comprising administering into the individual an effective amount of an antagonist decreasing enzymatic activity level of PLA2G2D. In some embodiments, the antagonist targeting PLA2G2D signaling pathway blocks a catalytic site on PLA2G2D. In some embodiment, the PLA2G2D is a human PLA2G2D. In some embodiments, the antagonist comprises an agent that specifically inhibits the cataly tic His67-Asp68 Dyad of human PLA2G2D as set forth in 8EQ ID NO: 1 or 5. In some embodiments, the antagonist targets the H67 catalytic site on a human PLA2G2D according to SEQ ID NO: 1 or 5. In some embodiments, the agent interferes with the binding of calcium to PLA2G2D. In some embodiments, the agent blocks the binding of calcium to residues at one or more oί' 1147. G49, G51, and D68 according to SEQ ID NO: 1 or 5. In some embodiments, the antagonist comprises an agent that specifically decreases enzymatic activity of the catalytic His67-Asp68 Dyad of human PLA2G2D as set forth in SEQ ID NO: I or 5 by at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%,
90%, or 95%. In some embodiments, the cancer tissue has an increased expression level of PLA2G2D as compared to a reference tissue (such as a corresponding tissue in a healthy
individual). In some embodiments, the cancer is an advanced or malignant tumor, in some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, renal cancer, prostate cancer, urothelial cancer, testis cancer, ovarian cancer and melanoma. In some embodiments, the method further comprises administering a second agent. In some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulator, an anti -angiogenesis agent, a growth inhibitory agent, and an antineoplastic agent. In some embodiments, the second agent is an immunomodulator. In some embodiments, the immunomodulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor specifically target PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, T1GIT, GITR, TIM3, LAG3, CD27, 4-1BB, or B7H4. In some embodiments, the second agent comprises a cell comprising a chimeric antigen receptor that specifically hinds to a tumor antigen. In some embodiments, the antagonist and the second agent is administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent is administered sequentially. In some embodiments, the antagonist and/or the second agent is administered parentally. In some embodiments, the antagonist is administered to diseased tissue directly.
[0081] In some embodiments, there is pro vided a method of treating an infectious disease (such as a viral infectious disease) in an individual, comprising administering into the individual an effective amount of an antagonist decreasing enzymatic activity level of PLA2G2D. In some embodiments, the PLA2G2D is a human PLA2G2D. In some embodiments, the antagonist targeting PLA2G2D signaling pathway blocks a catalytic site on PLA2G2D. In some embodiments, the antagonist comprises an agent that specifically inhibits the catalytic His67-Asp68 Dyad of human PLA2G2D as set forth in SEQ ID NO: 1 or 5. In some embodiments, the antagonist targets the H67 catalytic site on a human PLA2G2D according to SEQ ID NO: 1 or 5. In some embodiments, the agent interferes with the binding of calcium to PLA2G2D. In some embodiments, the agent blocks the binding of calcium to residues at one or more of H47, G49, G51, and D68 according to SEQ ID NO: 1 or 5. In some embodiments, the antagonist comprises an agent that specifically decreases enzymatic activity of the catalytic His67-Asp68 Dyad of human PLA2G2D as set forth in SEQ ID NO:
1 or 5 by at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the infection site has ait increased expression level of PLA2G2D as
compared to a reference tissue (such as a corresponding tissue in a healthy individual), in some embodiments, the method further comprises administering a second agent. In some embodiments, the second agent comprises an immune therapy. In some embodiments, the antagonist and the second agent is administered simultaneously or concurrently, in some embodiments, the antagonist and the second agent is administered sequentially, in some embodiments, the antagonist and/or the second agent is administered parentally. In some embodiments, the antagonist is administered to diseased tissue directly.
[0082] The administration of the antagonists described herein can also be useful for promoting local immune response, promoting proliferation and/or activation of immune cells (such as T cells), and promoting a favorable tumor microenvironment. In some embodiments, there is provided a method of promoting local immune response m a cancer tissue m an individual having a cancer (such as a solid tumor), comprising administering any of the antagonists described herein. In some embodiments, there is provided a method of promoting local immune response in an infection site in an individual having an infection (such as a virus infection), comprising administering any of the antagonists described herein.
[0083] In some embodiments, there is provided a method of promoting proliferation and/or activation of T cells in a cancer tissue in an individual having a cancer (such as a solid tumor), comprising administering any of the antagonists described herein. In some embodiments, there is provided a method of promoting proliferation and/or activation of T cells in an infection site in an individual having an infection (such as a virus infection), comprising administering any of the antagonists described herein. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells.
[0084] In some embodiments, there is provided a method of promoting a favorable tumor microenvironment in a cancer tissue in an individual having a cancer (such as a solid tumor), comprising administering any of the antagoni sts described herein. In some embodiments, there is provided a method of promoting a favorable microenvironment in an infection site m an individual having an infection (such as a vims infection), comprising administering any of the antagonists described herein. “Promoting favorable tumor microenvironment” generally refers to or comprises conversion of a tumor tissue that is resistant to a cancer therapy (such as an immunotherapy) to a tumor tissue that is less resistant to the cancer therapy.
Antagonist targeting PLA2G2D signaling pathway
[0085] Tiie antagonist may be any of an antibody, a polypeptide, a peptide, a polynucleotide, apeptidomimeiic, a natural product, a carbohydrate, an aptamer an avimer, an anticalm, a speigelmer, or a small molecule that targets (i.e., inhibits or downregulates) PLA2G2D signaling pathway. In some embodiments, the antagonist targets (i.e., inhibits or downregulates) PLA2G2D. Particular examples of what the agent may be are described below. In some embodiments, the antagonist is a fusion protein (such as a fusion protein that comprises a half-life extending domain (e.g., an Fc domain)).
PLA2 G2D
[0086] PLA2G2D (phospholipase A2 group III), sPLA2-IID) is a secreted member of the phospholipase A2 family. Phospholipase A2 family members hydrolyze the sn-2 fatty acid ester bond of giycerophospholipids to produce lysophospholipids and free fatty acid. To date, 10 sPLA2isoforms (IB, II A, IIC, IID, TIE, IIF, III, V, X, and XII) have been identified in mammals. These isoforms, except for group III isoforms, have a highly conserved catalytic site, a Ca 2+ binding loop, and a common molecular weight of 14-19 kDa. Of these sPLA2isoforms, sPLA2-IIA, sPLA2-lIC, sPI,A2~IID, sPLA2-IIE, sPLA2-IIF, and sPLA 2-V have the same chromosomal locus (Ip34-p36), which are often referred to as group II subfamily sPLA2. The biological feature of the group II subfamily sPLA2 is that almost all isoforms, except sPLA 2-IIC (a pseudogene in humans), are associated with inflammatory and immune processes.
[0087] Human PLA2G2D is a basic protein (pl~8,7) with 14 cysteines at exactly conserved positions. Likely because of its cationic nature, PLA2G2D binds to heparin in vitro or heparin sulfate on the cell surface when overexpressed in cultured cells.
[0088] In some embodiments, the PLA2G2D comprises an ammo acid sequence set forth in SEQ ID NO: I or 2. In some embodiments, the PLA2G2D comprises an amino acid sequence set forth in SEQ ID NO: 5 or 6.
Antagonist targeting PLA2G2D
[0089] In some embodiments, the antagonist decreases expression level of PLA2G2D. In some embodiments, the antagonist decreases enzymatic activity level of PLA2G2D. In some embodiments, the anti-PLA2G2D antibody does not completely inhibit or block the catalytic activity of PLA2G2D (such as blocking the catalytic activity no more than about 90%), 8Q%, 7Q%, 60%, 50%, 40%), 30(½, 20% or 10% of the full catalytic activity). In some
embodiments, the anti-PLA2G2D antibody does not inhibit or block the catalytic activity of PLA2G2D.
[0090] In some embodiments, the antagonist comprises an agent that inhibits PLA2G2D (such as an agent that blocks the binding of PLA2G2D to an immune cell or an agent that inhibits the activity of PLA2G2D) (such as a T cell, such as an activated T cell, such as an activated CD4+ T cell, such as an activated CD8+- T cell).
A, Agents binding to PLA2G2D
[0091] In some embodiments, the antagonist is an agent that recognizes and binds specifically to PLA2G2D. in some embodiments, the agent comprises an anti-PLA2G2D antibody moiety (such as an anii-PLA2G2D antibody).
[0092] In some embodiments, the anti-PL A2G2D antibody moiety blocks or decreases the binding of PLA2G2D to an immune cell. In some embodiments, the anti-PLA2G2D antibody moiety decreases the binding of PLA2G2D to an immune cell by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the binding of the PLA2G2D to an immune cell is independent from binding through heparin sulfate on cell surface.
[0093] in some embodiments, the PLA2G2D recognized by the anti-PLA2G2D antibody is a human PLA2G2D. In some embodiments, the human PLA2G2D comprises or has the amino acid sequence of SEQ ID NO: 1 or a natural variant of human PLA2G2D. In some embodiments, the natural variant of human PLA2G2D is derived from a tumor tissue, in some embodiments, the natural variant of human PLA2G2D is derived from a virus infection site.
[0094] 3D structure of the PLA2G2D predicted based upon Swiss-model is shown in FIG. 10A. In some embodiments, the anti-PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two, three, four, or five of) Q65, H73, S80, 1196. and R121 according to SEQ ID NO: 1. (Q65, H73, S80, H96 and R121 are sites that vary natural variants.) in some embodiments, the anti-PLA2G2D antibody moiety7 binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids from R121 to Cl 45 according to SEQ ID NO: 1. In some embodiments, the anti-PLA2G2D antibody moiety7 binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids from V32 to A59 according to SEQ ID NO: I. In some embodiments, the anti-PLA2G2D
antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids from T60 to T76. In some embodiments, the an†i~PLA2G2D antibody moiety' binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids from Q77 to Y85 according to SEQ ID NO: 1. In some embodiments, the anti-PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) ammo acids from G21 to Q31 according to SEQ ID NO: 1. In some embodiments, the anti-PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids from Y86 to W103 according to SEQ ID NO: 1, In some embodiments, the anti-PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) ammo acids from Cl 04 to R121 according to SEQ ID NO: 1. In some embodiments, the epitope is a discontinuous epitope. In some embodiments, the epitope is a continuous epitope.
[0095] Sequence homology' of human PLA2G2D to different PLA2 group 2 family members is analyzed and shown in FIG. 10B. Sequence homology of human PLA2G2D to PLA2G2D of different species is analyzed and shown m FIG. IOC. In some embodiments, the anti- PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising one or more residues at positions a) that are different from corresponding residues in other PLA2 group 2 family members and/or b) that are same as PLA2G2D in other species. In some embodiments, the anti~PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) ammo acids at the position of 22, 23, 25, 26, 27, or 31 according to SEQ ID NO: 1. In some embodiments, the anti~PLA2G2D antibody moiety' binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids at the position of 36, 37, 38, 42, 43, 55, or 59 according to SEQ ID NO: 1. In some embodiments, the anti-PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids at the position of 62, 65, 66, 72, 73, or 76, according to SEQ ID NO: 1. In some embodiments, the anti-PL, A2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids at the position of 77, 80, 81, 83, 84, or 85 according to SEQ ID NO: 1. In some embodiments, the anti-PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or
more of) amino acids at the position of 87, 89, 90, 92, 93, 94, 96, 98, 99, 100, 101, 102, or 103 according to SEQ ID NO: 1. In some embodiments, the anti-PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids at the position of 105, 106, 107, 108, 110, 114, 115, 117, 119, or 120 according to SEQ ID NO: 1. in some embodiments, the anti-PLA2G2D antibody moiety' binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids at the position of 123, 124, 127, 129, 130, 131, 132, 134, 135, 136, 137, 139, 141, 144, or 145 according to SEQ ID NO: 1. In some embodiments, the anti-PLA2G2D antibody moiety binds to an epitope on PLA2G2D comprising any one or more of (such as one, two , three, four, five or more of) amino acids ai the position of 22, 26, 31, 36, 42, 43, 72, 73, 76, 77, 80, 81, 83, 85, 87, 89, 90, 92, 94, 96, 100, 101, 102, 103, 106, 110, 114, 115, 117, 120, 134, 135, 136, 141, or 144 according to SEQ ID NO: 1. In some embodiments, the epitope is a discontinuous epitope. In some embodiments, the epitope is a continuous epitope.
[0096] In some embodiments, the agent comprises an anti-PLA2G2D antibody. In some embodiments, the anti-PLA2G2D antibody is a polyclonal antibody. In some embodiments, the anti-PL A2G2D antibody is a monoclonal antibody.
[0097] In some embodiments, the anti-PLA2G2D antibody is an anti-human PLA2G2D antibody.
[0098] In some embodiments, the anti-PLA2G2D antibody is humanized or chimeric.
[QQ99] In some embodiments, the anti-PLA2G2D antibody is a full-length antibody or an immunoglobulin derivative. In some embodiments, the anli-PLA2G2D antibody is an antigen-bindmg fragment, for example an antigen-binding fragment selected from the group consisting of a single-chain Fv (scFv), a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFvfi, a VHH, a Fv-Fc fusion, a scFv-Fc fusion, a scFv-Fv fusion, a diabody, a tribody, and a letrabody. In some embodiments, the anti-PLA2G2D antibody is a scFv. In some embodiments, the anti-PLA2G2D antibody is a Fab or Fab’, in some embodiments, the anti-PLA2G2D antibody is chimeric, human, partially humanized, fully humanized, or semi-synthetic. Antibodies and/or antibody fragments may be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, eamelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.
[0100] In some embodiments, the anti-PLA2G2D antibody comprises an Fc fragment (such as any of the Fc fragments described herein). In some embodiments, the Fc fragment is selected from the group consisting of Fc fragments from IgG, IgA, IgD, IgE, IgM. and combinations and hybrids thereof. In some embodiments, the Fc fragment is derived from a human IgG. In some embodiments, the Fc fragment comprises the Fc region of human IgGl, IgG2, IgG3, IgG4, or a combination or hybrid IgG.
[0101] In some embodiments, the anti~PLA2G2D antibody does not completely inhibit or block the catalytic activity of PLA2G2D (such as blocking the catalytic activity no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the full catalytic activity). In some embodiments, the anti-PLA2G2D antibody does not inhibit or block the catalytic activity of PLA2G2D.
[0102] In some embodiments, the anti-PLA2G2D antibody blocks the binding of PLA2G2D to a T cell by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.
[0103] In some embodiments, the anti-PLA2G2D antibody is capable of restoring T cell activation to at least about 50%, 60%, 70%, 80%, 90%, 95%, or 100%. The activation of the T cell can be indicated, for example, by its cytokine secretion level. Exemplary cytokines include IL-2 and IFN-v.
Epitope mapping
[0104] Determination of whether an antibody moiety' binds within an epitope region can be carried out in ways known to the person skilled in the art. As one example of such mapping/characterization methods, an epitope region for an anti-PLA2G2D antibody may be determined by epitope "foot-printing" using chemical modification of the exposed amines/carboxyls in the PLA2G2D protein. One specific example of such a foot-printing technique is the use of HXMS (hydrogen-deuterium exchange detected by mass spectrometry) wherein a hydrogen/deuterium exchange of receptor and ligand protein amide protons, binding, and back exchange occurs, wherein the backbone amide groups participating in protein binding are protected from back exchange and therefore will remain deulerated. Relevant regions can be identified at this point by peptic proteolysis, fast microbore high-performance liquid chromatography separation, and/or electrospray ionization mass spectrometry' . See, e.g., Ehring H, Analytical Biochemistry, Vol. 267 (2) pp. 252-259 (1999); Engen, J. R. and Smith, D. L. (2001) Anal. Chem. 73, 256A-265A, each of which is incorporated herein by reference in their entirety for all purposes. Another example
of a suitable epitope identification technique is nuclear magnetic resonance epitope mapping (NMR), where typically the position of the signals in two- dimensional NMR spectra of the free antigen and the antigen complexed with the antigen binding peptide, such as an antibody, are compared. The antigen typically is selectively isotopieally labeled with 15N so that only signals corresponding to the antigen and no signals from the antigen binding peptide are seen in the NMR-spectrum. Antigen signals originating from amino acids involved in the interaction with the antigen binding peptide typically will shift position in the spectrum of the complex compared to the spectrum of the free antigen, and the amino acids invol v ed in the binding can be identified that way. See, e.g, Ernst Sobering Res Found Workshop. 2004;
(44): 149-67; Huang et al., Journal of Molecular Biolog}', Vol. 281 (1) pp. 61 -67 (1998); and Saito and Patterson, Methods. 1996 Jim; 9 (3): 516-24, each of which is incorporated herein by reference in their entirety- for all purposes.
[0105] Epitope mapping/characterization also can he performed using mass spectrometry methods. See, e.g., Downard, J Mass Spectrom. 2000 Apr; 35 (4): 493-503 and Kiselar and Downard, Anal Chem. 1999 May 1; 71 (9): 1792-1801, each of which is incorporated herein by reference in their entirety for all purposes. Protease digestion techniques also can be useful in the context of epitope mapping and identification. Antigenic determinant-relevant regions/sequences can be determined by protease digestion, e.g. by using trypsin in a ratio of about 1 :50 to PLA2G2D or o/n digestion at and pH 7-8, followed by mass spectrometry (MS) analysis for peptide identification. The peptides protected from trypsin cleavage by the anti-PL A2G2D binder can subsequently be identified by comparison of samples individuated to trypsin digestion and samples incubated with antibody and then individual ed to digestion by e.g. trypsin (thereby revealing a footprint for the binder). Other enzymes like chymotrypsin, pepsin, etc., also or alternatively can he used in similar epitope characterization methods. Moreover, enzy matic digestion can provide a quick method for analyzing whether a potential antigenic determinant sequence is within a region of the PLA2G2D polypeptide (such as a polypeptide set forth in SEQ ID NO: 1 ) that is not surface exposed and, accordingly, most likely not relevant in terms of immunogen! city /antigenicity.
[0106] Site-directed mutagenesis is another technique useful for elucidation of a binding epitope. For example, m "alanine-scanning", each residue within a protein segment is re- placed with an alanine residue, and the consequences for binding affinity' measured, if the mutation leads to a significant reduction in binding affinity', it is most likely involved in binding. Monoclonal antibodies specific for structural epitopes (i.e., antibodies which do not
bind the unfolded protein) can be used to verify that the alanine-replacement does not influence over-all fold of the protein. See, e.g., Clackson and Wells, Science 1995; 267:383- 386; and Wells, Proc Natl Acad Sci USA 1996; 93:1-6.
[0107] Electron microscopy can also be used for epitope "foot-printing". For example, Wang et al, Nature 1992; 355:275-278 used coordinated application of ciyoelectron micros-copy, three-dimensional image reconstruction, and X-ray crystallography to determine the physical footprint of a Fab-fragment on the capsid surface of native cowpea mosaic virus.
[0108] Other forms of "label-free" assay for epitope evaluation include surface plasmon resonance (SPR, BIACORE) and ref!ectometric interference spectroscopy (RifS). See, e.g., Fagerstam et al., Journal Of Molecular Recognition 1990;3:208-14; Nice etal. , J. Chrorna- togr. 1993; 646:159-168; Lei peri a at.. Angew. Chem. Int. Ed. 1998; 37:3308- 3311; Kroger et al. Biosensors and Bioelectronics 2002; 17:937-944.
Immunoconju gates
[0109] In some embodiments, the agents that bind to PLA2G2D described herein further comprises a second moiety, in some embodiments, the second moiety comprises a therapeutic agent, in some embodiments, the second moiety comprises a label. In some embodiments, the anti-PLA2G2D antibody moiety and the second moiety is linked via a linker (such as any of the linkers described in the "‘linkers” section).
[0110] In some embodiments, the second agent is a cytotoxic agent. In some embodiments, the cytotoxic agent is a chemotherapeutic agent. In some embodiments, the cytotoxic agent is a growth inhibitory agent. In some embodiments, the cytotoxic agent is a toxin (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), in some embodiments, the cytotoxic agent is a radioactive isotype (i.e., a radioconjugage).
[0111] Immunoconjugates allow for the targeted delivery of a drug moiety to a tissues (such as a tumor), and, in some embodiments intracellular accumulation therein, where systemic administration of unconjugated drugs may result in unacceptable levels of toxicity' to normal cells (Polakis P. (2005) Current Opinion in Pharmacology 5:382-387).
[0112] Antibody-drug conjugates (ADC) are targeted chemo therapeutic molecules which combine properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to antigen-expressing tumor cells (Teicher, B. A. (2009) Current Cancer Drug Targets 9:982- 1004), thereby enhancing the therapeutic index by maximizing efficacy and minimizing off-
target toxicity (Carter, P. J. and Senter P. B. (2008) The Cancer Jour: 14(3): 154-169; Chan,
R. V. (2008) ACC. Chen. Res. 41.98-107.
[0113] In the context of treating cancer, the ADC compounds of the application include those with anticancer activity. In some embodiments, the ADC compounds include an antibody conjugated, i.e. covalently attached, to the drug moiety, in some embodiments, the antibody is covalently attached to the drug moiety through a linker. In some embodiments, the second agent is connected to the anti-PLA2G2D antibody moiety via a linker (such as a linker described herein), in some embodiments, the linker is a cleavable. In some embodiments, the linker is non-cleavable.
[0114] The antibody-drug conjugates (ADC) of the application selectively deliver an effective dose of a drug to tumor tissue whereby greater selectivity, i.e. a lower efficacious dose, may be achieved while increasing the therapeutic index (‘'therapeutic window'). The drug moiety of the antibody-drug conjugates (ADC) may include any compound, moiety or group that has a cytotoxic or cytostatic effect. Drug moieties may impart their cytotoxic and cytostatic effects by mechanisms including but not limited to tubulin binding, DNA binding or intercalation, and inhibition of RNA polymerase, protein synthesis, and/or topoisomerase. Exemplary drug moieties include, but are not limited to, a maytansinoid, dolasiatin, auristatin, calicheamicin, pyrrolobenzodiazepine (PBD), nemorubicin and its derivatives, PNU-159682, anthracy dine, duocarmycin, Vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, e!inafkle, and stereoisomers, isos teres, analogs, and derivati ves thereof that have cytotoxic activity.
[0115] Production of immunoconjugates described herein can be found in, for example, US 9,562,099 and 1187,541,034, which are hereby incorporated by references in their entirety.
Fusion proteins
[0116] In some embodiments, the agent binds to PLA2G2D comprises a fusion protein that comprises an anti-PLA2G2D antibody moiety and a second moiety .
[0117] In some embodiments, the second moiety comprises an Fc fragment (such as any of the Fc fragments described herein). In some embodiments, the half-life extending moiety- is an albumin binding moiety (e.g., an albumin binding antibody moiety).
[0118] In some embodiments, the second moiety comprises a cytokine. In some embodiments, the cytokine is a proinflammatory cytokine (such as TNF-a, IL-1B, 1L-6, or IL-10).
[0119] In some embodiments, the anti-PLA2G2B antibody moiety and the second moiety is linked via a linker (such as any of the linkers described in the “Linkers” section).
1. Fc fragment
[0120] The term “Fc region,” “Fc domain” or “Fc” refers to a C -terminal non-antigen binding region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native Fc regions and variant Fc regions, in some embodiments, a human IgG heavy chain Fc region extends from Cys226 to the carboxyl -terminus of the heavy chain. However, the C -terminal lysine (Lys447) of the Fc region may or may not be present, without affecting the structure or stability of the Fc region. Unless otherwise specified herein, numbering of amino acid residues in the IgG or Fc region is according to the EU numbering system for antibodies, also called the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
[0121] In some embodiments, the Fc fragment is sel ected from the group consisting of Fc fragments from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc fragment is selected from the group consisting of Fc fragments from IgG I, IgG2, IgG3, IgG4, and combinations and hybrids thereof.
[0122] In some embodiments, the Fc fragment has a reduced effector function as compared to corresponding wildtype Fc fragment (such as at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%), or 95%) reduced effector function as measured by the level of antibody-dependent cellular cytotoxicity (ADCC)).
[0123] In some embodiments, the Fc fragment is an IgGl Fc fragment. In some embodiments, the IgGl Fc fragment comprises a L234A mutation and/or a L235A mutation. In some embodiments, the Fc fragment is an IgG2 or IgG4 Fc fragment. In some embodiments, the Fc fragment is an IgG4 Fc fragment comprising a S228P, F234A, and/or a L235A mutation. In some embodiments, the Fc fragment comprises a N297A mutation, in some embodiments, the Fc fragment comprises aN297G mutation.
2. Linkers
[0124] In some embodiments, the an†i-PLA2G2D immunoconjugates or fusion proteins described herein comprise an anti~PLA2G2D antibody described herein fused to the second moiety via a linker.
[012S] The length, the degree of flexibility and/or other properties of the linker used in the anti-PLA2G2D immunoconj ugates or fusion proteins may have some influence on properties, including but not limited to the affinity', specificity or avidity of the anti~PLA2G2D, and/or affinity, specificity or avidity' for one or more particular antigens or epitopes present on PLA2G2D. For example, longer linkers may be selected to ensure that two adjacent antibody moieties do not statically interfere with one another. In some embodiments, a linker (such as peptide linker) comprises flexible residues (such as glycine and serine) so that the adjacent antibody moieties are free to move relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker. In some embodiments, the linker is anon-peptide linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a cleavable linker.
[0126] Other linker considerations include the effect on physical or pharmacokinetic properties of the resulting an anti-PLA2G2D immunoconj ugate or fusion protein, such as solubility', lipophil icily, hydrophilicity, hydrophobicily, stability (more or less stable as well as planned degradation), rigidity', flexibility', immunogemcity, modulation of antibody binding, the ability' to be incorporated into a micelle or liposome, and the like.
Peptide linkers
[0127] Any one or all of the linkers described herein can be peptide linkers. The peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. See, for example, WO 1996/34103, ineorporated by reference in its entirety for all purposes, in some embodiments, the peptide linker comprises the ammo acid sequence of CPPCP, a sequence found in the native IgGl lunge region.
[0128] The peptide linker can be of any suitable length. In some embodiments, the length of the peptide linker is any of about 1 aa to about 10 aa, about 1 aa to about 20 aa, about 1 aa to about 30 aa, about 5 aa to about 15 aa, about 10 aa to about 25 aa, about 5 aa to about 30 aa, about 10 aa to about 30 aa, about 30 aa to about 50 aa about 50 aa to about 100 aa, or about 1 aa to about 100 aa.
[0129] An essential technical feature of such peptide linker is that said peptide linker does not comprise any polymerization activity. The characteristics of a peptide linker, which comprise the absence of the promotion of secondary structures, are known in the art and described, e.g., in DalFAcquaef al. (Biochem. (1998) 37, 9266-9273), Cheadle el al. (Mol
Immunol (1992) 29, 21-30) and Raag and Whitlow (FA8EB (1995) 9(1), 73-80, each incorporated by reference in their entirety for all purposes). A particularly preferred amino acid in context of the ‘'peptide linker” is Gly. Furthermore, peptide linkers that also do not promote any secondary' structures are preferred. The linkage of the molecules to each other can be provided by, e.g., genetic engineering. Methods for preparing fused and operatively linked antibody constructs and expressing them in mammalian cells or bacteria are well- known in the art (e.g. WO 99/54440, Ausubel, Current Protocols in Molecular Biology', Green Publishing Associates and Wiley Interscience, N. Y. 1989 and 1994 or Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N, Y., 2001, each incorporated by reference in their entirety for all purposes).
[0130] In some embodiments, the peptide linker is a stable linker, which is not cleavable by protease, such as by Matrix metalloproteinases (MMPs).
[0131] In some embodiments, the peptide linker tends not to adopt a rigid three-dimensional structure, but rather provide flexibility' to a polypeptide (e.g., first and/or second components), such as providing flexibility' between the anti-PLA2G2D and the second moiety. In some embodiments, the peptide linker is a flexible linker. Exemplary7 flexible linkers include glycine polymers (G)n (SEQ ID NO: 13), glycine-serine polymers (including, for example, (GS)n (SEQ ID NO: 14), (GSGGS)n (SEQ ID NO: 15), (GGGGS)n (SEQ ID NO: 16), and (GGGS)n (SEQ ID NO: 17), where n is an integer of at least one), glycine- alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between components. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). The ordinarily skilled artisan will recognize that design of an anti~PLA2G2D can include linkers that are all or partially flexible, such that the linker can include a flexible linker portion as well as one or more portions that confer less flexible structure to provide a desired immunoconjugate or fusion protein structure.
[0132] Furthermore, exemplary linkers also include the amino acid sequence of such as (GGGGS)n(SEQ ID NO: 16), wherein n is an integer between 1 and 8, e.g. (GGGGS)¾ (SEQ ID NO: 18; hereinafter referred to as (048)3 ' or “G83”), or (GGGGS)e (SEQ ID NO: 19; hereinafter referred to as “(G4S)6” or “GS6”). In some embodiments, the peptide linker
comprises the amino acid sequence of (GSTSGSGKPGSGEGS)n (SEQ ID NO: 20), wherein n is an integer between 1 and 3.
[0133] Natural linkers adopt various conformations in secondary' structure, such as helical, b- strand, coil/bend and turns, to exert their functions. Linkers in an a-helix structure might serve as rigid spacers to effectively separate protein domains, thus reducing their unfavorable interactions. Non-helical linkers with Pro-rich sequence could increase the linker rigidity and function in reducing inter-domain interference. In some embodiments, the anti-PLA2G2D antibody moiety' and the second moiety is linked together by an a-helical linker with an amino acid sequence of A(EAAAK)4A (SEQ ID NO: 21).
Non-peptide linkers
[0134] Any one or all of the linkers described herein can be accomplished by any chemical reaction that will bind the two molecules so long as the components or fragments retain their respective activities, e.g. binding to target PLA2G2D, function of the second moiety (such as binding to a FcR or cytokine receptor). This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and compiexation. In some embodiments, the binding is covalent binding. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as a second moiety to the anti-PLA2G2D antibody of the present invention. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succmimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents ( see Killen and Lindstrom, Jour. Irnmun. 133:1335- 2549 (1984); Jansen et al, Immunological Reviews 62:185-216 (1982); and Vitetta etal., Science 238:1098 (1987), each incorporated by reference in their entirety for all purposes).
[013S] Linkers that can be applied in the present application are described in the literature (see, for example, Ramakrishnan, S. el al. Cancer Res. 44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester), incorporated by reference in its entirety for all purposes). In some embodiments, non-peptide linkers used herein include: (i) EDC (l-etbyl-3-(3-dimethy]amino-propy]) carbodiimide hydrochloride; (ii) SMPT (4- succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)~toluene (Pierce Chem. Co.,
Cat. (21558G); (iii) SPDP (succimmidyi-6 [3-(2-pyridyldithio) propionamidojhexanoate (Pierce Chem. Co., Cat #21651G); (iv) Sulfo-LC-SPDP (sulfosuccinirmdyl 6 [3~(2~ pyridyldithio)~propianarmde] hexanoate (Pierce Chem. Co. Cat. #2165~G); and (v) sulfo- NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC.
[0136] The linkers described above contain components that have different attributes, thus leading to agents binding to PLA2G2D (such as anti~PLA2G2D imnmnoconjugates or fusion proteins) with differing physio-chemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester containing linkers are less soluble than sulfo-NHS esters. Further, the linker SMPT contains a sterical!y hindered disulfide bond, and can form fusion protein with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less fusion protein available. Sulfo-NHS, m particular, can enhance the stability of carbodimide couplings. Carhodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.
B. inhibitory PLA2G2D polypeptides or variants thereof
[0137] Die methods described herein in some embodiments involve use of inhibitory PLA2G2D polypeptides that block the binding between PLA2G2D (e.g., a wildtype PLA2G2D) and immune cells completely or partially (such as blocks the binding between the PLA2G2D and an immune cell by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 95%). The present application in one aspect provides novel and non-naturally occurring polypeptide comprising an inhibitory PLA2G2D polypeptide that blocks the binding of PLA2G2D to an immune cell, in some embodiments, the inhibitory PLA2G2D polypeptide is a soluble polypeptide.
[0138] In some embodiments, the inhibitory PLA2G2D polypeptide is membrane bound. In some embodiments, the membrane bound inhibitory PLA2G2D polypeptide binds to the immune ceil but does not trigger PLA2G2D signaling pathway in the immune cell. In some embodiments, the membrane bound inhibitory PLA2G2D polypeptide binds to the immune cell and attenuates PLA2G2D signaling pathway in the immune cell. In some embodiments, the membrane bound inhibitory PLA2G2D polypeptide is introduced by a gene editing system or an mRNA delivery vehicle.
[0139] In some embodiments, the inhibitory PLA2G2D polypeptide comprises a naturally occurring PLA2G2D polypeptide. In some embodiments, the naturally occurring PLA2G2D polypeptide is from a human who has an autoimmune or inflammatory disease (such as chronic obstructive pulmonary disease (COPD)). In some embodiments, the inhibitory' PLA2G2D polypeptides has a mutation at a position corresponding to a polymorphism described in Takabatake et al (Am I Respir Crii Care Med. 2005 Nov 1 ; 172(9): 1097-104) or Igarashi etal. (Respiration. 2009;78(3): 312-21).
[0140] In some embodiments, the inhibitory PLA2G2D polypeptide comprises a mutation at the position corresponding to histidine at position 67 (H67) according to 8EQ ID NO: 1 or 5. In some embodiments, the inhibitory' PLA2G2D polypeptide comprises an amino acid sequence of SEQ ID NO: 3, 4, 7, or 8 or a variant thereof, in some embodiments, the variant has at least about 80% (such as about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 3, 4, 7, or 8.
[0141] In some embodiments, the inhibitory' PLA2G2D polypeptide comprises a mutation at a position corresponding to G80 according to SEQ ID NO: 5. In some embodiments, the inhibitory' PLA2G2D polypeptide comprises a comprises an amino acid sequence of SEQ ID NO: 9 or 10 or a variant thereof. In some embodiments, the variant has at least about 80% (such as about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, or 99%) sequence identity'' to the ammo acid sequence of SEQ ID NO: 9 or 10.
[0142] In some embodiments, the inhibitory PLA2G2D poly peptide comprises a) a mutation at the position corresponding to histidine at position 67 (H67) and b) a mutation at a position corresponding to G80 according to SEQ ID NO: 5. In some embodiments, the inhibitory' PLA2G2D polypeptide comprises a comprises an amino acid sequence of SEQ ID NO: 11 or 12 or a variant thereof. In some embodiments, the variant has at least about 80% (such as about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 11 or 12.
[0143] In some embodiments, the inhibitory PLA2G2D polypeptide further comprises at least one or more (such as about at least 10, 15, 20, 25, 30, 35, 45, 50, or all) of residues at the position of 22, 23, 25, 26, 27, 31, 36, 37, 38, 42, 43, 55, 59, 62, 65, 66, 72, 73, 76, 77, 80, 81, 83, 84, 85, 87, 89, 90, 92, 93, 94, 96, 98, 99, 100, 101, 102, 103, 105, 106, 107, 108, 110, 114, 115, 117, 119, 120, 123, 124, 127, 129, 130, 131, 132, 134, 135, 136, 137, 139, 141,
144, and 145 wherein the amino acid numbering is based on SEQ ID NO: 1.
[0144] in some embodiments, , the inhibitory PLA2G2B polypeptide further comprises at least one or more (such as about at least 10, 15, 20, 25, 30, or all) of residues at the position of 22, 26, 31, 36, 42, 43, 72, 73, 76, 77, 80, 81, 83, 85, 87, 89, 90, 92, 94, 96, 100, 101, 102, 103, 106, 110, 114, 115, 117, 120, 134, 135, 136, 141, or 144 wherein the ammo acid numbering is based on 8EQ ID NO:l.
[0145] In some embodiments, the variant described herein is a natural variant. In some embodiments, the variant does not comprise anon-conservative substitution. In some embodiments, the variant only comprises one or more conservative substitution. In some embodiments, the one or more conservative substitutions comprise or consist of the substitutions shown m Table 1 below under the heading of “Preferred substitutions.”
[0146] In some embodiments, the inhibitory PLA2G2D polypeptide binds to the immune ceil with a greater affinity than for a wildtype PLA2G2D. In some embodiments, the inhibitory PLA2G2D polypeptide binds to the immune cell with a KD of at most half, one-fifth, one-
tenth, one-twentieth, one-fiftieth, one-hundredth, one-thousandth of that of the binding between the wildtype PLA2G2D and the immune ceil.
[0147] In some embodiments, the inhibitory PLA2G2D polypeptide further comprises a stabilizing domain. The stabilizing domain can be any domain that stabilizes the inhibitory' PLA2G2D polypeptide (for example, extending half-life of the inhibitory' PLA2G2D polypeptide in vivo). In some embodiments, the stabilizing domain comprises an Fc fragment. Exemplary' Fc fragment include those described under “Fc fragment” section.
[0148] In some embodiments, the inhibitory PLA2G2D polypeptide is about 50 to about 1000 ammo acids in length, such as about 50-800, 50-500, 50-400, 50-300 or 50-200 ammo acids m length. In some embodiments, the inhibitory polypeptide is about 50 to about 100 amino acids, about 100 to about 150 amino acids, or about 150 amino acids to about 200 ammo acids in length.
C. Nucleic acid agents targeting PLA2G2D
[0149] In some embodiments, the antagonist targeting PLA2G2D comprises a nucleic acid agent (such as a siRNA, a shRNA, a miRNA, or an antisense RNA) that targets PLA2G2D (such as a human PLA2G2D).
[0150] In some embodiments, the antagonist comprises a siRNA or RNAi. In some embodiments, the antagonist comprises an antisense RNA. In some embodiments, the antagonist comprises a short hairpin ribonucleic acid (shRNA). In some embodiments, the antagonist comprises a microRNA (miRNA).
[0151] One skilled in the art may select an interfering RNA (RNAi) or siRN A specific targeting PLA2G2D. The nucleic acid selected sometimes is the RNAi or siRNA or a nucleic acid that encodes such products. The term “RNAi” as used herein refers to double-stranded RNA (dsRNA) which mediates degradation of specific mRNAs, and can also be used to lower or eliminate gene expression. The term “short interfering nucleic acid”, “siNA”, “short interfering RNA”, “siRNA”, “short interfering nucleic acid molecule”, “short interfering oligonucleotide molecule”, or “chemically -modified short interfering nucleic acid molecule” as used herein refers to any nucleic acid molecule directed against a gene. For example, a siRNA is capable of inhibiting or down regulating gene expression or viral replication, for example by mediating RNA interference “RNAi” or gene silencing in a sequence-specific manner; see for example Zamore et af, 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428- 429; Elbashir et al., 2001, Nature, 411 , 494-498; and Kreutzer et af, International PCT
Publication No. WO 00/44895; Zemicka-Goetz et a!., international PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al, international PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaiilette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914; Allshire,
2002, Science, 297, 1818-1819; Voipe et al., 2002, Science, 297, 1833-1837; Jenuwem, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831). 'There is no particular limitation in the length of siRNA as long as it does not show toxicity. Examples of modified RNAi and siRNA include STEALTH'™ forms (Invitrogen Corp., Carlsbad, Calif.), forms described in U.S. Patent Publication No. 2004/0014956 (application Ser. No. 10/357,529) and U.S. patent application Ser. No. 11/049,636, filed Feb. 2, 2005), and other forms described hereafter.
[0152] A siRNA can be a double-stranded polynucleotide molecule comprising self- complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siRNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary' (i.e. each strand comprises nucleotide sequence that is complementary' to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 19 base pairs); the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, the siRNA is assembled from a single oligonucleotide, where the self- eompiementary sense and antisense regions of the siRNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s). The siRNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self- complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic
acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Hie siRNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi. The siRNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary' to nucleotide sequence m a target nucleic acid molecule or a portion thereof (for example, where such siRNA molecule does not require the presence within the siRNA molecule of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5f-phosphate (see for example Martinez et al, 2002, Cell, 110, 563-574 and Schwarz et al, 2002, Molecular CeIl, 10, 537-568), or 5 ',3 '-diphosphate. In certain embodiments, the siRNA molecule of the invention comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions, in certain embodiments, the siRNA molecules of the invention comprise nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, tire siRNA molecule of the invention interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.
[0153] The double-stranded RNA portions of siRNAs in which two RNA strands pair are not limited to the completely paired forms, and may contain non-pairing portions due to mismatch (the corresponding nucleotides are not complementary), bulge (lacking in the corresponding complementary nucleotide on one strand), and the like. Non-pairing portions can be contained to the extent that they do not interfere with siRNA formation. The '‘bulge” used herein often comprises 1 to 2 non-pairing nucleotides, and the double-stranded RN A region of siRNAs in which two RNA strands pair up sometimes contains 1 to 7, and at limes 1 to 5 bulges. In addition, the “mismatch” used herein is contained in the double-stranded RNA region of siRNAs in which two RNA strands pair up, sometimes 1 to 7, and at times 1
to 5, in number. In an often utilized mismatch, one of the nucleotides is guanine, and the other is uracil. Such a mismatch is due to a mutation from C to T, G to A, or mixtures thereof in DNA coding for sense RNA, but not particularly limited to them. Furthermore, in the present invention, the double-stranded RNA region of siRNAs in which two RNA strands pair up may contain both bulge and mismatched, which sum up to, sometimes Ito 7, and at times Ito 5, in number. The terminal structure of siRNA may be either blunt or cohesive (overhanging) as long as siRNA enables to silence the target gene expression due to its RNAi effect.
[0154] As used herein, siRNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, siRNA molecules of the invention can be used to epigenetical!y silence genes at both the post-transcriptional level and the pre- transcriptional level, in a non-limiting example, epigenetic regulation of gene expression by siRNA molecules of the invention can result from siRNA mediated modification of chromatin structure to alter gene expression (see, for example, Verdel et a!., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Yolpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Flail et al, 2002, Science, 297, 2232-2237).
[0155] RNAi may be designed by those methods known to those of ordinary' skill m the art.
In one example, siRNA may be designed by classifying RNAi sequences, for example 1000 sequences, based on functionality, with afunctional group being classified as having greater than 85% knockdown activity and a non-functional group with less than 85% knockdown activity. The distribution of base composition was calculated for entire the entire RNAi target sequence for both the functional group and the non-functional group. The ratio of base distribution of functional and non-functional group may then be used to build a score matrix for each position of RNAi sequence. For a given target sequence, the base for each position is scored, and then the log ratio of the multiplication of all the positions is taken as a final score. Using this score system, a very strong correlation may be found of the functional knockdown activity and the log ratio score. Once the target sequence is selected, it may be filtered through both fast NCBI blast and slow Smith Waterman algorithm search against the
Uni gene database to identify the gene-specific RNAi or siRNA. Sequences with at least one mismatch in the last 12 bases may he selected.
[0156] An antisense nucleic acid can he designed, prepared and/or utilized by the artisan to inhibit a nucleic acid encoding PLA2G2D. An ‘'antisense'' nucleic acid refers to a nucleotide sequence complementary to a ‘‘sense55 nucleic acid encoding PLA2G2D or fragment (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). The antisense nucleic acid can be complementary to an entire coding strand, or to a portion thereof or a substantially identical sequence thereof. In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region55 of the coding strand of a nucleotide sequence.
[0157] An antisense nucleic acid can he complementary' to the entire coding region of an mRNA encoded by a PLA2G2D nucleotide sequence, and often the antisense nucleic acid is an oligonucleotide antisense to only a portion of a coding or noncoding region of the mRNA. For example, the antisense oligonucleotide can be complementary' to the region surrounding the translation start site of the mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length. An antisense nucleic acid can he constructed using chemical synthesis or enzyme ligation reactions using standard procedures. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) 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 (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used). Antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
[Q158] When utilized in subjects, antisense nucleic acids typically are administered to a subject (e.g., by direct injection at a tissue site) or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a polypeptide and thereby inhibit expression of the polypeptide, for example, by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can he modified to target selected cells and then are administered systemieaily. For systemic administration, antisense
molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, for example, by linking antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. Sufficient intracellular concentrations of antisense molecules are achieved by incorporating a strong promoter, such as a pot II or pol III promoter, in the vector construct. Antisense nucleic acid molecules sometimes are alpha-anomeric nucleic acid molecules. An alpha-anomerie nucleic acid molecule forms specific double-stranded hybrids with complementary7 RNA in which, contrary to the usual beta-units, the strands run parallel to each other (Gaultier et al. Nucleic Acids. Res. 15: 6625-6641 (1987)). Antisense nucleic acid molecules also can comprise a 2'~ o-methylnbonucleotide (Inoue et al., Nucleic Acids Res. 15: 6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et al., FEES Lett. 215: 327-330 (1987)). Antisense nucleic acids sometimes are composed of DNA or PNA or any other nucleic acid derivatives described previously.
[0159] An antisense nucleic acid is a ribozyme in some embodiments. A nbozyme having specificity for an Aid nucleotide sequence can include one or more sequences complementary to such a nucleotide sequence, and a sequence having a known catalytic region responsible for mRNA cleavage (e.g., U.S. Pat. No. 5,093,246 or Haselhoff and Geriach, Nature 334: 585-591 (1988)). For example, a derivative of a Tetrahymena L-19 TVS RNA is sometimes utilized in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved m an mRNA (e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742). PLA2G2D mRNA sequences also may be utilized to select a catalytic RNA having a specific ribonuciease activity from a pool of RNA molecules (e.g,, Bartel & Szostak, Science 261: 1411-1418 (1993)).
[0160] In some embodiments, the nucleic acid agents targeting PLA2G2D are nucleic acids that can form triple helix structures with an Aid nucleic acid. PLA2G2D expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of a nucleotide sequence referenced herein or a substantially identical sequence (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of a gene in target cells (see e.g., Helene, Anticancer Drug Des. 6(6): 569-84 (1991); Helene et al., Ann. N.Y. Acad. Sci. 660: 27-36 (1992); and Maher, Bioassays 14(12): 807-15 (1992). Triple helix formation can be enhanced by generating a “switchback” nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3',3'-5' manner, such that they base pair with
first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of purines or pyrimidines being present on one strand of a duplex.
D . Genome-editing systems that target PLA2G2D
[0161] In some embodiments, the antagonist targeting PLA2G2D comprises a genome- editing system that targets PLA2G2D. in some embodiments, the genome-editing system comprises a DNA nuclease such as an engineered (e.g., programmable or targetable) DNA nuclease to induce genome editing of a target DNA sequence of PLA2G2D. Any sui table DN A nuclease can be used including, but not limited to, CRISPR-associated protein (Cas) nucleases, zmc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, other endo- or exo-nucleases, variants thereof, fragments thereof, and combinations thereof. In some embodiments, the genome editing comprises modifying PLA2G2D so that die modified PLA2G2D no longer suppresses an immune cell (such as a T cell, such as an activated T cell, such as an activated ( 1)4 T cell, such as an activated CD8+ T cell) or suppresses the immune ceil to a less extent than wildtype PLA2G2D. In some embodiments, the genome editing comprises modifying PLA2G2D so that the modified PLA2G2D no longer binds to an immune ceil (such as a T cell, such as an activated T cell, such as an activated CD4+ T cell, such as an activated CD8+ T cell) or binds to the immune cell to a less extent than wildtype PLA2G2D. In some embodiments, the modification comprises inserting a transgene comprising a variant of PLA2G2D (such as any of the variants of PLA2G2D described herein). In some embodiments, the variant PLA2G2D has a mutation at H67 based upon SEQ ID NO: 1. In some embodiments, the variant PLA2G2D has aH67A mutation based upon SEQ ID NO: 1.
E. Agents inhibiting PLA2G2D enzymatic activity
[0162] In some embodiments, the antagonist comprises an agent that inhibits PLA2G2D enzymatic activity (i.e., hydrolyzing fatty acids). In some embodiments, the antagonist comprises an agent that specifically inhibits enzymatic activity' of the catalytic His67-Asp68 Dyad of human PLA2G2D as set forth in SEQ ID NO: 1 or 5. In some embodiments, the antagonist comprises an agent that specifically decreases enzymatic activity of the catalytic His67-Asp68 Dyad of human PLA2G2D as set forth in SEQ ID NO: 1 or 5 by at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%.
[0163] in some embodiments, the agent interferes with the binding of calcium to PLA2G2D. In some embodiments, the agent blocks the binding of calcium to residues at one or more of H47, G49, G51, and D68 according to SEQ ID NO: 1 or 5.
Disease or condition
[0164] The methods described herein are applicable to diseases and conditions for which there are suppressed immune responses in the body that at least partly contribute to the less effective treating of the disease. Exemplary diseases include cancer or infectious disease (such as viral infectious disease).
Cancer
[0165] In some embodiments, the disease or condition described herein is a cancer. Cancers that may be treated using any of the methods described herein include any types of cancers. Types of cancers to be treated with the agent as described in this application include, but are not limited to, carcinoma, blastema, sarcoma, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.
]0166] In various embodiments, the cancer is early stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, cancer in remission, recurrent cancer, cancer in an adjuvant setting, cancer in a neoadjuvant setting, or cancer substantially refractory to a therapy.
[0167] In some embodiments, the cancer is a solid tumor.
[0168] In some embodiments, the cancer is a liquid tumor.
[0169] In some embodiments, the cancer tissue has a high expression level of PLA2G2D when the expression level of PLA2G2D (e.g., assessed by immimohistochemistry) is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% higher than the expression level of PLA2G2D in a reference tissue. In some embodiments, the cancer tissue has a high expression level of PLA2G2D when the expression level of PLA2G2D (e.g., assessed by immimohistochemistry) is at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7 -fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold higher than the expression level ofPLA2G2D in a reference tissue. In some embodiments, the reference tissue is the corresponding tissue in a healthy individual. In some embodiments, the expression level of PLA2G2D in a reference tissue is the average expression level of
PLA2G2D in the same tissue in a group of individuals (such as 10, 30, 50, 100 individuals) with same or similar cancer. In some embodiments, the reference tissue is the corresponding tissue in an individual who also has a cancer but has a less suppressed immune response in the cancer tissue as indicated by a biomarker (such as high M2 macrophages, or high expression of an immune checkpoint agent such as PD-1 or PD-L1).
[0170] In some embodiments, the cancer tissue has a high T cell infiltration (e.g., high CD3 T cells, high CD8 T cells, high CD4 T cells, activated T cells, activated CDS T cells, or activated CD4 T cells) in the cancer tissue. In some embodiments, the cancer tissue has a high T cell infiltration when the number of the T cells in the cancer is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% more than the number of the corresponding T ceils in a reference tissue. In some embodiments, the high T cell infiltration is present when the number of the T ceils m the cancer is at least about 1-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7 -fold, 8-fold, 9-fold, or 10-fold more than the number of the corresponding T ceils in a reference tissue. In some embodiments, the reference tissue is the corresponding tissue in a healthy individual. In some embodiments, the number of the corresponding T cells in a reference tissue is the average number of the corresponding T cells in the same tissue in a group of individuals (such as 10, 30, 50, 100 individuals) with same or similar cancer, in some embodiments, the reference tissue is the corresponding tissue in an individual who also has a cancer but has a less suppressed immune response in the cancer tissue as indicated by a biomarker (such as high M2 macrophages, high expression of an immune checkpoint agent such as PD- 1 or PD-L1, high expression level of PLA2G2D).
[0171] In some embodiments, the cancer has a decreased number (such as a decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of immune cells (such as activated T cells, activated CD4+ T cells, or activated CD8+ T cells) in the cancer tissue as compared to that of a reference tissue. In some embodiments, the cancer has a decreased number (such as a decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of activated immune cells (such as activated T cells, activated CD4+ T cells, or activated CD8+ T cells) m the cancer tissue as compared to that of a reference tissue. In some embodiments, the cancer tissue has a decreased level (such as a decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of a cytokine (such as a pro-inflammatory cytokine, such as IFNy or IL-2) as compared to that of a reference tissue.
[0172] In some embodiments, the reference tissue is the corresponding tissue in a healthy individual. In some embodiments, the reference tissue is the corresponding tissue in an individual who also has a cancer but has a less suppressed immune response in the cancer tissue. The suppression of immune response can be assessed by measuring a) the number of immune cells (e.g., CD3+ cells); b) the proliferating/expanding status of immune cells; c) the activation status of immune cells; and/or d) the cytokine level. In some embodiments!, any one or more of the a) -d) is measured in the cancer tissue. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are CD8+ T cells (such as activated CD8-f- T cells). In some embodiments, the immune cells are CD4+ T cells (such as activated CD4+ T cells).
[0173] Examples of cancers that may be treated by the methods of this application include, but are not limited to, anal cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bladder cancer, bone cancer, (osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., astrocytoma, malignant glioma, medulloblastoma, and glioblastoma), breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer (e.g., uterine cancer), esophageal cancer, eye cancer (e.g, intraocular melanoma and retinoblastoma), gastric (stomach) cancer, gastrointestinal stromal tumor (GIST), head and neck cancer, hepatocellular (liver) cancer (e.g, hepatic carcinoma and heptoma), liver cancer, lung cancer (e.g, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), medulloblastoma, melanoma, mesothelioma, myelodyspiastic syndromes, nasopharyngeal cancer, neuroblastoma, ovarian cancer, pancreatic cancer, parathyroid cancer, cancer of the peritoneal, pituitary tumor, rectal cancer, renal cancer, renal pel v is and ureter cancer (transitional cell cancer), rhabdomyosarcoma, skin cancer (e.g,, non-melanoma (e.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma), small intestine cancer, squamous cell cancer, testicular cancer, thyroid cancer, and tuberous sclerosis. Additional examples of cancers can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, § on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); The Merck Manual of Diagnosis and Therapy, 20th Edition, § on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2018 (ISBN 978-0- 911-91042-1) (2018 digital online editi on at internet website of Merck Manuals); and SEER Program Coding and Staging Manual 2016, each of which are incorporated by reference in their entirety for ail purposes.
[0174] In some embodiments, the disease or condition is a colon cancer.
[0175] In some embodiments, the disease or condition is melanoma.
[0176] In some embodiments, the disease or condition is aT ceil lymphoma.
Infectious disease
[0177] In some embodiments, the disease or condition is an infectious disease. In some embodiments, the infectious disease is a viral infectious disease.
[0178] In some embodiments, the viral infectious disease is characterized by infection with hepatitis virus, human immunodeficiency virus (HIV), picomavirus, poliovirus, enterovirus, human Coxsackie virus, influenza virus, rhino virus, echovirus, rubella vims, encephalitis virus, rabies virus, herpes virus, papillomavirus, polyoma virus, RSV, adenovirus, yellow fever virus, dengue virus, parainfluenza virus, hemorrhagic virus, pox virus, varicella zoster virus, parainfluenza virus, reo virus, orbivirus, rotavirus, parvovirus, African swine fever virus, measles, coronavirus (such as SARS-CoV, MERS-CoV, 2019-nCoV), Ebola virus, mumps or Norwalk virus. In some embodiments, the viral infectious disease is characterized by infection with an oncogenic virus such as CMV, EBV, HBV, KSHV, HPV, MCV, HTLV-1 , HIV- 1, or HCV. In some embodiments, the one or more genes encoding proteins involved in the viral infectious disease development and/or progression include, but are not limited to, genes encoding RSV nucleocapsid, Pre-gen/Pre-C, Pre-Si, Pre-S2/S,X, HBV conserved sequences, HIV Gag polyprotein (p55), HIV Pol polyprotein, HIV Gag-Pol precursor (pi 60), HIV matrix protein (MA, pl7), HIV capsid protein (CA, p24), HIV spacer peptide 1 (SP1, p2), HTV nucleocapsid protein (NC, p9), HIV spacer peptide 2 (SP2, pi), HIV P6 protein, HIV reverse transcriptase (RT, p50), HIV RNase H (pi 5), HIV integrase (IN, p31), HIV protease (PR, plO), HIV Env (gpl60), gpl20, gp41, HIV transactivator (Tat), HIV regulator of expression of virion proteins (Rev), HIV ientivirus protein R (Vpr), HIV Vif, HIV negative factor (Nef), HIV virus protein U (Vpu), human CCR5, miR-122, EBOV polymerase L, VP24, VP40, GP/sGP, VP30, VP35, NPCi, and TIM-1, including mutants thereof
[0179] In some embodiments, the viral infectious disease is characterized by infection with coronavirus. In some embodiments, the viral infectious disease is characterized by infection with influenza virus.
[0180] An infection site refers to a tissue m the body where virus appear in a significant number and/or causes significant damages. In some embodiments, the infection site comprises has an increased expression level of PLA2G2D as compared to a reference tissue.
in some embodiments, the PLA2G2D expression level in the infection site is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% as compared to that of the reference tissue. In some embodiments, the PLA2G2D expression level in the infection site is increased by at least about 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold as compared to that of the reference tissue.
[0181] In some embodiments, the infection site has a decreased number (such as a decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of immune cells (such as activated T ceils, activated CD4+ T cells, or activated CD8+ T ceils) in the infection site as compared to that of a reference tissue, in some embodiments, the infection site has a decreased number (such as a decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of activated immune cells (such as activated T cells, activated < 1)4 T ceils, or activated CD8+ T cells) in the infection site as compared to that of a reference tissue. In some embodiments, the infection site has a decreased level (such as a decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of a cytokine (such as a pro-inflammatory cytokine, such as IFNy or TL-2) as compared to that of a reference tissue.
[0182] In some embodiments, the reference tissue is the corresponding tissue in a healthy individual. In some embodiments, the reference tissue is the corresponding tissue in an individual who also has a virus infection (such as a virus infection of the same type) but has a less suppressed immune response in the infection site. The suppression of immune response can be assessed by measuring a) the number of immune cells; b) the proliferating/expanding status of immune cells; c) the activation status of immune cells; and/or d) the cytokine level, in some embodiments, the immune cells in circulation are assessed, in some embodiments, the immune ceils in diseased tissue are assessed, in some embodiments, the immune cells in lymph tissue (such as lymph node) are assessed. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are CD8+ T cells (such as activated CD8+ T cells). In some embodiments, the immune cells are CD4+ T ceils (such as activated CD4+ T cells).
Individ eal
[0183] In some embodiments, the individual is a mammal (such as a human).
[0184] In some embodiments, the indi vidual is selected for treatment based upon a high expression of PLA2G2D in a diseased tissue. In some embodiments, the tissue is a cancer tissue. In some embodiments, the tissue is an infection site.
[0185] In some embodiments, the PLA2G2D expression level in the infection site is increased by at least about 10%, 20%, 3Q%, 40%, 50%, 6014, 70%, 80%, 90% as compared to that of the reference tissue. In some embodiments, the PLA2G2D expression level in the infection site is increased by at least about 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold as compared to that of the reference tissue.
[0186] In some embodiments, the individual is selected for treatment based upon the indication of a suppressed immune response. In some embodiments, the individual has a suppressed immune response in a diseased tissue. In some embodiments, the tissue is a cancer tissue. In some embodiments, the tissue is an infection site.
[0187] As described above, the suppression of immune response can be assessed by measuring a) the number of immune cells; b) the proliferating/ expan dmg status of immune cells; c) the activation status of immune cells; and/or d) the cytokine level. In some embodiments, the immune cells in circulation are assessed. In some embodiments, the immune cells in diseased tissue are assessed. In some embodiments, the immune cells in lymph tissue (such as lymph node) are assessed. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are CD8+ T cells (such as activated CD8+ T cells), in some embodiments, the immune cells are CD4+ T ceils (such as activated CD4+ T cells).
[0188] In some embodiments, the individual has a decreased number (such as a decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of immune cells (such as activated T ceils, activated CD4+ T cells, or activated CD8+ T ceils) in the tissue (such as the cancer tissue or infection site) as compared to that of a reference tissue. In some embodiments, the individual has a decreased number (such as a decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of activated immune cells (such as activated T cells, activated CD4+ T cells, or activated CD 8+ T cells) in the tissue (such as the cancer tissue or infection site) as compared to that of a reference tissue. In some embodiments, the individual has a decreased level (such as a decrease by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or
95%) of a cytokine (such as a pro-inflammatory cytokine, such as iFNy or IL-2) in the tissue (such as the cancer tissue or infection site) as compared to that of a reference tissue.
[0189] In some embodiments, the reference tissue is the corresponding tissue in a healthy individual. In some embodiments, the reference tissue is the corresponding tissue in an individual who also has a same or similar disease or condition but has a less suppressed immune response in the cancer tissue.
[0190] In some embodiments, the individual has a compromised immune system,
[0191] In some embodiments, the individual is at least about 60, 65, 70, 75, 80, 85, or 90 years old.
[0192] in some embodiments, the individual has at least one prior therapy, in some embodiments, the prior therapy comprises a radiation therapy, a chemotherapy and-'or an immunotherapy. In some embodiments, the individual is resistant, refractory, or recurrent to the prior therapy.
Combination therapy
[0193] The present application also provides methods administering an effective amount of an antagonist targeting PLA2G2D signaling pathway into an individual for treating a disease or condition (such as cancer or infectious disease), wherein the method further comprises administering a second agent or therapy. In some embodiments, the second agent or therapy is a standard or commonly used agent or therapy for treating the disease or condition.
[0194] In some embodiments, the antagonist is administered simultaneously with the second agent or therapy. In some embodiments, the antagonist is administered concurrently with the second agent or therapy. In some embodiments, the antagonist is administered sequentially with the second agent or therapy.
Exemplary combination therapies for cancer
[019S] in some embodiments, the second agent or therapy comprises a chemotherapeutic agent. In some embodiments, the second agent or therapy comprises a, surgery. In some embodiments, the second agent or therapy comprises a radiation therapy. In some embodiments, the second agent or therapy comprises an immunotherapy. In some embodiments, the second agent or therapy comprises a ceil therapy (such as a ceil therapy comprising an immune cell (e.g., CAR T cell)). In some embodiments, the second agent or therapy comprises an angiogenesis inhibitor.
[0196] In some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulator, an anti-angiogenesis agent, a growth inhibitor}' agent, and an antineoplastic agent.
[0197] In some embodiments, the second agent is a chemotherapeutic agent. In some embodiments, the second agent is antimetabolite agent. In some embodiments, the antimetabolite agent is 5-FU.
[0198] In some embodiments, the second agent is an immunomodulator. In some embodiments, the immunomodulatory is an immune checkpoint inhibitor. In some embodiments, the checkpoint inhibitor specifically targets PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIMS, LAGS, CD27, 4-1BB, or B7H4. In some embodiments, the second agent is an anti -PD- 1 antibody or fragment thereof. In some embodiments, the second agent is an anti-PD-Ll antibody or fragment thereof.
[0199] In some embodiments, the second agent comprises a cell (such as an immune ceil, such as a T cell) comprising a chimeric antigen receptor that specifically binds to a tumor antigen.
Exemplary combination therapies for infectious disease (such as viral infectious disease).
[0200] In some embodiments, the second agent or therapy comprises a nucleotide analogue.
[0201] In some embodiments, the second agent or therapy comprises a nucleoside analogue.
[0202] In some embodiments, the second agent or therapy comprises a protease inhibitor. In some embodiments, the second agent or therapy comprises Lopinavir. In some embodiments, the second agent or therapy comprises ritonavir.
[0203] In some embodiments, the second agent or therapy comprises a neuraminidase inhibitor. In some embodiments, the second agent or therapy comprises zanamivir. In some embodiments, the second agent or therapy comprises oseltamivir. In some embodiments, the second agent or therapy comprises peramivir.
[0204] In some embodiments, the second agent or therapy comprises a Cap-dependent endonuclease inhibitor. In some embodiments, the second agent or therapy comprises baloxavir.
[0205] In some embodiments, the second agent or therapy comprises a sialidase.
[0206] The second agent and the antagonist can be administered sequentially, concurrently, or simultaneously. In some embodiments, the second agent is administered prior to the antagonist. In some embodiments, the second agent is administered after the antagonist.
Dosing regimen and routes of administration
[0207] The dose of the antagonist and, in some embodiments, the second agent as described herein, administered to an individual (such as a human) may vary with the particular composition, the method of administration, and the particular kind and stage of disease or condition being treated. The amount should be sufficient to produce a desirable response, such as a therapeutic response against the disease or condition. In some embodiments, the amount of the antagonist and/or the second agent is a therapeutically effective amount.
[0208] In some embodiments, the amount of the antagonist is an amount sufficient to decrease the suppression of the immune response in the indi vidual. Whether there is a decrease in the suppression of the immune response and the extent of the decrease in suppression can be indicated by any of the following.
[0209] In some embodiments, the amount of the antagonist is an amount sufficient to increase the number of immune cells (such as T ceils, such as < 1)4 and/or CD8+ T cells) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% post administration of the antagonist. In some embodiments, the immune ceils in circulation are assessed, in some embodiments, the immune cells in diseased tissue are assessed. In some embodiments, the immune cells in lymph tissue (such as lymph node) are assessed. In some embodiments, the immune cells comprises myeloid ceils (such as dendritic cells). In some embodiments, the immune cells comprises NK cells. In some embodiments, the immune cells comprises T cells, such as CD4÷ and/or CD8+ T cells. In some embodiments, the number of immune cells is assessed about 1, 2, 3, 4, 5, 6, or 7 days post administration of the antagonist.
[Q210] In some embodiments, the amount of the antagonist is an amount sufficient to increase the number of activated immune cells (such as activated CD4+ and/or CD8+ T cells) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% post administration of the antagonist. In some embodiments, the activated immune cells in circulation are assessed. In some embodiments, the activated immune cells in diseased tissue are assessed, in some embodiments, the activated immune cells in lymph tissue (such as lymph node) are assessed. In some embodiments, the immune cells comprises myeloid cells (such as dendritic cells). In some embodiments, the immune cells comprises NK cells. In
some embodiments, the immune cells comprises T cells, such as CD4+ and/or CD8+ T cells. In some embodiments, the number of activated immune cells is assessed about 1, 2, 3, 4, 5, 6, or 7 days post administration of the antagonist,
[0211] In some embodiments, the amount of the antagonist is an amount sufficient to increase the proliferation of immune ceils or activated immune cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% post administration of the antagonist. In some embodiments, the immune cells or the activated immune cells in circulation are assessed, in some embodiments, the immune cells or the activated immune cells in diseased tissue are assessed, in some embodiments, the immune cells or the activated immune ceils in lymph tissue (such as lymph node) are assessed. In some embodiments, the immune cells comprises myeloid cells (such as dendritic cells). In some embodiments, the immune cells comprises NK cells. In some embodiments, the immune ceils comprises T ceils, such as CD4+ and/or CD8+ T cells. In some embodiments, the proliferation of immune cells or activated immune cells is assessed about 1, 2, 3, 4, 5, 6, or 7 days post administration of the antagonist.
[0212] in some embodiments, the amount of the antagonist is an amount sufficient to increase the cytokine level (such as a pro-inflammatory cytokine, such as IFNv or IL-2) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% post administration of the antagonist. In some embodiments, the cytokine level in diseased tissue is assessed. In some embodiments, the level of cytokine is assessed about 1, 2, 3, 4, 5, 6, or 7 days post administration of the antagonist.
[0213] In some embodiments, the amount of the antagonist is an amount sufficient to decrease the suppressive immune cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% post administration. In some embodiments, the suppressive immune cells comprise regulator}' T cells. In some embodiments, the suppressive immune cells comprise myeloid derived suppressor cells. In some embodiments, the suppressive immune cells in circulation are assessed, in some embodiments, the suppressive immune cells in diseased tissue are assessed. In some embodiments, the suppressive immune cells in lymph tissue (such as lymph node) are assessed. In some embodiments, the number of suppressive immune cells is assessed about 1, 2, 3, 4, 5, 6, or 7 days post administration of the antagonist.
[0214] in some embodiments, the amount of the antagonist is an amount sufficient to increase humoral immune response in the individual by at least about 10%, 20%, 30%, 40%,
50%, 60%), 70%, 80%, 90%), or 100% post administration of the antagonist. The humoral immune response can be assessed by measuring antibodies (such as IgG antibodies) that target a disease-associated antigen or plasmablasts that produce such antibodies in circulation, in some embodiments, the humoral immune response is assessed about 7-28 days (such as about 7-14 days) post administration of the antagonist.
[0215] In some embodiments, the amount of the antagonist is an amount sufficient to produce a decrease of the size of a tumor, decrease the number of cancer ceils, or decrease the growth rate of a tumor by at least about any of 10%, 20%, 30%, 40%, 50(½, 60%, 70%o, 80%, 90%, 95% or 100% compared to the corresponding tumor size, number of cancer cells, or tumor growth rate in the same individual prior to treatment or compared to the corresponding activity m other individuals not receiving the treatment.
[0216] in some embodiments, the antagonist is administered at a dose of about 0.001 pg/kg to about 100 mg/kg of total body weight, for example, about 0.005 pg/kg to about 50 mg/kg, about 0,01 ug kg to about 10 mg/kg, or about 0.01 pg/kg to about 1 mg/kg.
[0217] In some embodiments according to any one of the methods described herein, the antagonist and/or the second agent composition is administered intravenously, intraarterially, mtraperitoneally, intravesicularly, subcutaneously, intrathecally, intrapuimonanly, intramuscularly, intratracheal ly, intraocularly, topically, transdermally, orally, or by inhalation. In some embodiments, the antagonist and/or the second agent is administered intravenously.
[0218] In some embodiments, the antagonist is administered directly to the diseased tissue. ML Methods of diagnosis and prognosis
[0219] Provided herein also include methods of diagnosing or prognosing an individual, including, determining the suitability' of an individual for the treatment as described in section 11 or a different therapy comprising an immunotherapy, determining the likelihood of responsiveness of an indi vidual to the methods as described in section 11 or the different therapy.
[0220] In some embodiments, there is provided a method of determining the suitability of an individual for a treatment, comprising measuring levels of PLA2G2D expression in the diseased tissue of an individual. In some embodiments, the individual has a cancer, and the
tissue is a tumor tissue. In some embodiments, the individual has an infectious disease (such as a viral infectious disease, and the tissue is an infection site.
[0221] In some embodiments, there is provided a method of prognosis in an individual having cancer (such as a solid tumor), comprising measuring levels of PLA2G2D expression in a tumor sample in vitro or in vivo, wherein a higher PLA2G2D expression level as compared to a reference level indicates a higher possibility of not responding or responding poorly to a therapy (such as an immunotherapy). In some embodiments, the reference level is a level of PLA2G2D expression (such as an average PLA2G2D expression) in a non-tumor sample in the individual or a corresponding tissue in a different individual (or a group of individuals) who does not have cancer.
[0222] In some embodiments, there is provided a method of prognosis in an individual having an infectious disease (such as a virus infectious disease), comprising measuring levels of PLA2G2D expression in a sample from the infection site in vitro or in vivo, wherein a higher PLA2G2D expression level as compared to a reference level indicates a higher possibility' of not responding or responding poorly to a therapy (such as an immunotherapy). In some embodiments, the reference level is a level of PLA2G2D expression (such as an average PLA2G2D expression) in a non-infection site sample in the individual or a corresponding tissue in a different individual (or a group of individuals) who does not have the infectious disease.
[0223] in some embodiments, the therapy comprises a cell therapy (such as a CAR-T cell therapy).
[0224] In some embodiments, the therapy further comprises assessing suppression of immune response m the individual. Exemplary' methods of assessing immune response suppression are discussed above.
IV. Methods of Preparation, Nucleic Adds, Vectors, Host cells, and Culture medium
[0225] In some embodiments, there is provided a method of preparing the antagonist (such as an siRNA targeting PLA2G2D, anti-PLA2G2D agents, inhibitory' PLA2G2D polypeptides, agents inhibiting PLA2G2D enzymatic activity as described herein) and composition comprising the agents, nucleic acid construct, vector, host cell, or culture medium that is produced during the preparation of the agents.
Polypeptide Expression and Production
[0226] Tiie agents targeting PLA2G2D (e.g, polypeptide comprising an anti-PLA2G2D antibody moiety as described in Section P) and inhibitory PLA2G2D polypeptides described herein can be prepared using any known methods in the art, including those described below.
Polypeptides comprising anii-PLA2G2D antibody moiety
[0227] Monoclonal antibodies targeting PLA2G2D can be obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post- translational modifications (e.g., isomenzations, amidations) that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. For example, the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al, Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567). In the hybridoma method, a mouse or other appropriate host animal, such as a hamster or a llama, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986).
[0228] The immunizing agent will typically include the antigenic protein or a fusion variant thereof. Generally, either peripheral blood lymphocytes (“PBLs”) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103, incorporated by reference in its entirety' for all purposes.
[0229] Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture
medium for the hybridomas typically will include hypoxanthine, ammopterin, and thymidine (HAT medium), which are substances that prevent the growth of HGPRT-deficient cells.
[0230] Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 cells (and derivatives thereof, e.g., X63-Ag8-653) available from the American Type Culture Collection, Manassas, Va, USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol, 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), each of which are incorporated by reference in their entirety for all purposes).
[0231] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
[Q232] The culture medium in winch the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies directed against the desired antigen. Preferably, the binding affinity and specificity of the monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked assay (ELISA). Such techniques and assays are known in the in art. For example, binding affinity may be determined by the Scatchard analysis of Munson el al..
Anal. Biochem., 107:220 (1980).
[0233] After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Coding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as tumors in a mammal.
[0234] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification
procedures such as. for example, protein A-Sepharose, hydroxylapalite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
[0235] Monoclonal antibodies may also be made by recombinant DNA methods, such as those described in L!.S. Pat. No. 4,816,567, and as described above. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, m order to synthesize monoclonal antibodies in such recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol, 5:256-262 (1993) and Pluckthun, Immunol. Revs. 130:151-188 (1992).
[0236] In a further embodiment, antibodies can be isolated from antibody phage libraries generated using the techniques described in MeCafferty et ai, Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks eta!., J. Mol. Biol., 222:581-597 (1991), each of which are incorporated by reference in their entirety for all purposes, describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity' (nM range) human antibodies by chain shuffling (Marks et ai., Bio/Technoiogy, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy' for constructing very' large phage libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.
[0237] The DN A also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Nad Acad. Sci. USA, 81:6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Typically, such non- immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity' for a different antigen.
[0238] The monoclonal antibodies described herein may by monovalent, the preparation of which is well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and a modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinkmg.
Alternatively, the relevant cysteine residues may be substituted with another ammo acid residue or are deleted so as to prevent crosslinking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art.
[0239] Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry', including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide-exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include immothiolate and me thy 1 -4-mercaptob uty ri mi date.
Nucleic Acid Molecules Encoding Polypeptides
[0240] In some embodiments, there is provided a polynucleotide encoding any one of the antibodies (such as anti-PLA2G2D antibodies) or polypeptides (such as inhibitory PLA2G2D polypeptides) described herein. In some embodiments, there is provided a polynucleotide prepared using any one of the methods as described herein. In some embodiments, a nucleic acid molecule comprises a polynucleotide that encodes a heavy chain or a light chain of an antibody (e.g., anti-PL A2G2D antibody). In some embodiments, a nucleic acid molecule comprises a polynucleotide that encodes an inhibitory' PLA2G2D polypeptide. In some embodiments, a nucleic acid molecule comprises both a polynucleotide that encodes a heavy chain and a polynucleotide that encodes a light chain, of an antibody (e.g., anti-PLA2G2D antibody). In some embodiments, a first nucleic acid molecule comprises a first polynucleotide that encodes a heavy chain and a second nucleic acid molecule comprises a second polynucleotide that encodes a light chain. In some embodiments, a nucleic acid molecule encoding a scFv (e.g., anti-PLA2G2D scFv) is provided. In some embodiments, a nucleic acid molecule comprises a polynucleotide that encodes an inhibitor}' PLA2G2D polypeptide.
[0241] In some such embodiments, the heavy chain and the light chain of an antibody (e.g., anti-PLA2G2D antibody) are expressed from one nucleic acid molecule, or from two separate nucleic acid molecules, as two separate polypeptides. In some embodiments, such as when
an antibody is a scFv, a single polynucleotide encodes a single polypeptide comprising both a heavy chain and a light chain linked together.
[0242] In some embodiments, a polynucleotide encoding a heavy chain or light chain of an antibody (e.g, anti-PL, A2G2D antibody) comprises a nucleotide sequence that encodes a leader sequence, which, when translated, is located at the N terminus of the heavy chain or light chain. As discussed above, the leader sequence may be the native heavy or light chain leader sequence, or may be another heterologous leader sequence.
[0243] In some embodiments, the polynucleotide is a DNA. In some embodiments, the polynucleotide is an RNA. in some embodiments, the RNA is an mRNA.
[0244] Nucleic acid molecules may be constructed using recombinant DNA techniques conventional in the art. In some embodiments, a nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell.
Nucleic acid construct
[0245] In some embodiments, there is provided a nucleic acid construct comprising any one of the polynucleotides described herein. In some embodiments, there is provided a nucleic acid construct prepared using any method described herein.
[0246] in some embodiments, the nucleic acid construct further comprises a promoter operabiy linked to the polynucleotide, in some embodiments, the polynucleotide corresponds to a gene, wherein the promoter is a wild-type promoter for the gene.
Vectors
[0247] The terms ‘"vector”, “cloning vector” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to genetically modify the host and promote expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, synthesized RNA and DNA molecules, phages, viruses, etc. in certain embodiments, the vector is a viral vector such as, but not limited to, viral vector is an adenoviral, adeno-associated, alphaviral, herpes, lenti viral, retroviral, or vaccinia vector.
[0248] In some embodiments, there is provided a vector comprising any polynucleotides that encode the heavy chains ami/or light chains of any one of the antibodies (e.g., anti-PLA2G2B antibodies) described herein. In some embodiments, there is provided a vector comprising any polynucleotides that encode polypeptides (e.g, inhibitory PLA2G2D polypeptides)
described herein. In some embodiments, there is provided a vector comprising any nucleic acid construct described herein. In some embodiments, there is provided a vector prepared using any method described herein, V ectors comprising polynucleotides that encode any of polypeptides (such as anti-PLA2G2D antibodies or inhibitor}' PLA2G2D polypeptides) are also provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, a vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy chain and light chain are expressed from the vector as two separate polypeptides.
[0249] In some embodiments, a first vector comprises a polynucleotide that encodes a heavy chain of an antibody (e.g., anti-PLA2G2D antibody) and a second vector comprises a polynucleotide that encodes a light chain of an antibody (e.g., anti-PLA2G2D antibody). In some embodiments, the first vector and second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts). In some embodiments, a mole- or mass-ratio of between 5:1 and 1:5 of the first vector and the second vector is transfected into host cells. In some embodiments, a mass ratio of between 1:1 and 1:5 for the vector encoding the heavy chain and the vector encoding the light chain is used. In some embodiments, a mass ratio of 1 :2 for the vector encoding the heavy7 chain and the vector encoding the light chain is used.
[0250] In some embodiments, a vector is selected that is optimized for expression of polypeptides in CHO or CHO-derived cells, or in NSO cells. Exemplary such vectors are described, e.g., in Running Deer eta!.., Biotechnol. Prog. 20:880-889 (2004).
[0251] In certain embodiments, the vector is a viral vector, in certain embodiments, the viral vector can he, but is not limited to, a retroviral vector, an adenoviral vector, an adeno- associated virus vector, an alphaviral vector, a herpes virus vector, and a vaccinia virus vector. In some embodiments, the viral vector is a ientiviral vector.
[0252] In some embodiments, the vector is a non-viral vector. The viral vector may be a plasmid or a transposon (such as a Piggy Bac- or a Sleeping Beauty transposon).
Host Ceils
[0253] In some embodiments, there is provided a host ceil comprising any polypeptide, nucleic acid construct and/or vector described herein. In some embodiments, there is provided a host cell prepared using any method described herein. In some embodiments, the
host cell is capable of producing any of polypeptides (such as antibodies or inhibitory polypeptides) described herein under a fermentation condition.
[0254] In some embodiments, the polypeptides described herein (e.g., anti-PLA2G2D antibodies or inhibitory' PLA2G2D polypeptides) may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art. Exemplary' eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E ceils; CHO cells, including CHO-S, DG44. Led 3 CHO cells, and FUT8 CHO cells; PER.C6® cells (Crucell); andNSO cells. In some embodiments, the polypeptides described herein (e.g., anti-PLA2G2D antibodies or inhibitory PLA2G2D polypeptides) may be expressed in yeast. See, e.g., U.S. Publication No. US 2006/0270045 Al. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and/or light chains of the desired antibody. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.
[0255] Introduction of one or more nucleic acids into a desired host cell may be accomplished by any method, including but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory' Press (2001), incorporated by reference in its entirety7 for all purposes. Nucleic acids may be transiently or stably transfected in the desired host cells, according to any suitable method.
[0256] The invention also provides host cells comprising any of the polynucleotides or vectors described herein, in some embodiments, the invention provides a host cell comprising an anti-PLA2G2D antibody. Any host cells capable of over-expressing heterologous DNAs can be used for the purpose of isolating the genes encoding the antibody, polypeptide or protein of interest. Non-limiting examples of mammalian host cells include but not limited to COS, HeLa, and CHO cells. See also PCX Publication No. WO 87/04462. Suitable non- mammalian host cells include prokaryotes (such as E. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; or K lactis).
[0257] In some embodiments, the polypeptide is produced in a cell -free system. Non-limiting exemplary cell-free systems are described, e.g., in Sitaraman etal., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol, 22: 538-45 (2004); Endo etal., Biotechnol. Adv. 21: 695-713 (2003).
Purification of polypeptides
[0258] The polypeptides (e.g., anti-PLA2G2D antibodies, e.g., inhibitory PLA2G2D polypeptides) may be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include the ROR1 BCD and ligands that bind antibody constant regions. In some embodiments, a Protein A, Protein G, Protein A/G, or an antibody affinity column may be used to bind the constant region and to purify an antibody comprising an Fc fragment. Hydrophobic interactive chromatography, for example, a butyl or phenyl column, may also suitable for purifying some polypeptides such as antibodies. Ion exchange chromatography (e.g. anion exchange chromatography and/or cation exchange chromatography) may also suitable for purifying some polypeptides such as antibodies. Mixed-mode chromatography (e.g. reversed phase/anion exchange, reversed phase/cation exchange, hydrophilic interaction/amon exchange, hydrophilic interaction/cation exchange, etc.) may also suitable for purifying some polypeptides such as antibodies. Many methods of purify ing polypeptides are known in the art.
V. Compositions, Kits, and articles of manufacture
[0259] The present application also provides compositions, kits, medicines, and unit dosage forms for use in any of the methods described herein.
Compositions
[0260] Any of the antagonists described herein can be present in a composition (such as a formulation) that includes other agents, excipients, or stabilizers.
[0261] In some embodiments, the composition further comprises a target agent or a carrier that promotes the delivery of the antagonist to a diseased tissue. Exemplary carriers include liposomes, micelles, nanodisperse albumin and its modifications, polymer nanoparticles, dendrimers, inorganic nanoparticles of different compositions.
[0262] In some embodiments, the antagonist is packaged in a nanocarrier. In some embodiments, the nanocarrier has an average diameter of about 20 nnt to about 200 nm. In some embodiments, the nanocarrier has an average diameter of about 50 nm.
[0263] In some embodiments, the antagonist described herein is coated with a serum protein (such as albumin). In some embodiments, the antagonist is coated with opsonin.
[0264] In some embodiments, the antagonist comprises or is coupled with a moiety that facilitate the delivery of the antagonist to the diseased tissue (such as the cancer tissue or infection site as described above). In some embodiments, the moiety bind to an antigen that is expressed (e.g., overexpressed) or clustered on the diseased tissue (such as cancer tissue) or cells within the diseased tissue. In some embodiments, the antigen is a tumor associated antigen (such as Her2, folate receptor, CD44). See, for example, Rosenblum et al. , Nat Common. 2018 Apr 12;9(1): 1410.
[0265] In some embodiments, the composition is suitable for administration to a human. In some embodiments, the composition is suitable for administration to a mammal such as, in the veterinary context, domestic pets and agricultural animals. There are a wide variety of suitable formulations of the composition comprising the antagonist. The following formulations and methods are merely exemplary' and are in no way limiting. Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice, (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules, (c) suspensions in an appropriate liquid, and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, com starch, potato starch, microcrysialline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients.
Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
[0266] Examples of suitable carriers, excipients, and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose,
polyvinylpyrrolidone, cellulose, water, saline solution, syrup, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate, arid mineral oil. In some embodiments, the composition comprising the antagonist with a carrier as discussed herein is present in a dry formulation (such as iyophilized composition). The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.
[0267] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation compatible with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit- dose or multi-dose sealed containers, such as ampules and viols, and can be stored in a freeze- dried (Iyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Injectable formulations are preferred.
[0268] In some embodiments, the composition is formulated to have a pH range of about 4.5 to about 9.0, including for example pH ranges of about any of 5,0 to about 8.0, about 6.5 to about 7.5, and about 6.5 to about 7.0. in some embodiments, the pH of the composition is formulated to no less than about 6, including for example no less than about any of 6.5, 7, or 8 (such as about 8). The composition can also be made to be isotonic with blood by the addition of a suitable tonicity modifier, such as glycerol.
Kits
[Q269] Kits provided herein include one or more containers comprising the antagonist or a pharmaceutical composition comprising the antagonist described herein and/or other agent(s), and in some embodiments, further comprise instructions for use in accordance with any of the methods described herein. The kit may further comprise a description of selection of individual suitable for treatment. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
[0270] In some embodiments, the kit comprises a) a composition comprising an antagonist targeting PLA2G2D signaling pathway comprising an agent comprising an anti~PLA2G2D antibody moiety, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable earner: and optionally b) instructions for administering the agent for treatment of a disease or condition. In some embodiments, the agent is an anti-PLA2G2D antibody, in some embodiments, the agent is an anti-PLA2G2D fusion protein. In some embodiments, the agent is an anti-PL A2G2D immunoconjugate.
[0271] In some embodiments, the kit comprises a) a composition comprising an antagonist targeting PLA2G2D signaling pathway comprising an inhibitory PLA2G2D polypeptide, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; and optionally b) instructions for administering the agent for treatment of a disease or condition, in some embodiments, the inhibitory PLA2G2D polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, and 7-12.
[0272] In some embodiments, the kit comprises a) a composition comprising an antagonist targeting PLA2G2D signaling pathway comprising a nucleic acid agent targeting PLA2G2D (such as siRNA, shRNA, miRNA, or antisense RNA), or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; and optionally b) instructions for administering the agent for treatment of a disease or condition.
[Q273] In some embodiments, the kit comprises a) a composition comprising an antagonist targeting PLA2G2D signaling pathway comprising a genome editing system that targets PLA2G2D, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; and optionally b) instructions for administering the agent for treatment of a disease or condition.
[0274] In some embodiments, the kit comprises a) a composition comprising an antagonist targeting PLA2G2D signaling pathway comprising an agent inhibiting PLA2G2D enzymatic activity, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; and optionally b) instructions for administering the agent for treatment of a disease or condition.
[0275] Die kits of the invention are in suitable packaging. Suitable packaging includes, but is not limited to, viols, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and
interpretative information. The present application thus also pro vides articles of manufacture, which include vials (such as sealed vials), bottles, jars, flexible packaging, and the like.
[0276] In some embodiments, the kits comprise one or more components that facilitate delivery of the antagonist, or a composition comprising the agent, and/or additional therapeutic agents to the individual. In some embodiments, the kit comprises, e.g., syringes and needles suitable for delivery of cells to the individual, and the like, in such embodiments, the antagonist, or a composition comprising the agent may be contained in the kit m a bag, or in one or more vials. In some embodiments, the kit comprises components that facilitate intravenous or intra-arterial delivery of the antagonist, or a composition comprising the agent to the individual. In some embodiments, the antagonist, or a composition comprising the agent may be contained, e.g., within a bottle or bag (for example, a blood bag or similar bag able to contain up to about 1.5 L solution comprising the cells), and the kit further comprises tubing and needles suitable for the delivery of the antagonist, or a composition comprising the agent to the individual.
[0277] The instructions relating to the use of the compositions generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. Tire containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.
For example, kits may be provided that contain sufficient dosages of the zinc as disclosed herein to provide effective treatment of an individual for an extended period, such as any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or more. Kits may also include multiple unit doses of the pharmaceutical compositions and instructions for use and packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.
EXEMPLARY EMBODIMENTS
[0278] Embodiment 1. A method of treating a cancer or viral infection in an individual, comprising administering into the individual an effective amount of an antagonist targeting PLA2G2D signaling pathway,
[0279] Embodiment 2. The method of embodiment 1, wherein the antagonist is an antagonist targeting PLA2G2D.
[0280] Embodiment 3. The method of embodiment 2, wherein the PLA2G2D is a human PLA2G2D.
[0281] Embodiment 4. The method of embodiment 2 or embodiment 3, wherein the antagonist decreases enzymatic activity level of PLA2G2D.
[0282] Embodiment 5. The method of embodiment 4, wherein the antagonist targeting PLA2G2D signaling pathway blocks a catalytic site on PLA2G2D.
[0283] Embodiment 6. Hie method of embodiment 5, wherein the antagonist targets the H67 catalytic site on a human PLA2G2D according to SEQ ID NO: 1 or 5.
[0284] Embodiment 7. The method of any one of embodiments 1-3, wherein the antagonist comprises a siRNA, an miRNA, an antisense RNA, or a gene editing system.
[0285] Embodiment 8. The method of any one of embodiments 1-3, wherein the antagonist comprises an agent that inhibits PLA2G2D (such as an agent that blocks the binding of PLA2G2D to an immune cell or an agent that inhibits the activity of PLA2G2D).
[0286] Embodiment 9. The method of embodiment 8, wherein the immune cell is a T cell.
[0287] Embodiment 10. The method of any one of embodiments 1-3, wherein the antagonist comprises an anti~PLA2G2D antibody.
[0288] Embodiment 11, The method of embodiment 10, wherein the anti-PL A2G2D antibody is a monoclonal antibody.
[0289] Embodiment 12. The method of embodiment 10, wherein the antagonist is a fusion protein further comprising a second moiety.
[0290] Embodiment 13. The method of embodiment 12, wherein the second moiety comprises a cytokine.
[0291] Embodiment 14. The method of any one of embodiments 1-3, wherein the antagonist comprises an inhibitory PLA2G2D polypeptide that blocks the binding of PLA2G2D to an immune cell.
[0292] Embodiment 15. The method of embodiment 14, wherein the inhibitory PLA2G2D polypeptide binds to the immune cell with a greater affinity than for PLA2G2D.
[0293] Embodiment 16. The method of embodiment 15, wherein the immune cells is a T ceil.
[0294] Embodiment 17. The method of any one of embodiments 14-16, wherein the inhibitory PLA2G2D polypeptide further comprises a stabilizing domain.
[0295] Embodiment 18. The method of embodiment 17, wherein the stabilizing domain is an Fe domain.
[0296] Embodiment 19. The method of any one of embodiments 14-18, wherein the inhibitory' PLA2G2D polypeptide has a length of about 50 to about 200 amino acids.
[0297] Embodiment 20. The method of any one of embodiments 14- 19, wherein inhibitory' PLA2G2D polypeptide has a mutation at the position corresponding to histidine at position 67 (H67) according to SEQ ID NO: 1 or 5.
[0298] Embodiment 21. The method of embodiment 20, wherein the inhibitory PLA2G2D polypeptide comprises an amino acid sequence of SEQ ID NO: 3, 4, 7, or 8.
[0299] Embodiment 22, The method of any one of embodiments 1-21, wherein the disease or condition is a cancer.
[0300] Embodiment 23. The method of embodiment 22, wherein the cancer is a solid tumor.
[0301] Embodiment 24. The method of embodiment 22 or embodiment 23, wherein the cancer is an advanced or malignant tumor.
[0302] Embodiment 25. The method of any one of embodiments 22-24, wherein the cancer has an increased expression level of PLA2G2D.
[0303] Embodiment 26. The method of any one of embodiments 22-25, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, renal cancer, prostate cancer, urothelial cancer, testis cancer, ovarian cancer and melanoma.
[0304] Embodiment 27. The method of any one of embodiments 1-21, wherein the disease or condition is a viral infection,
[0305] Embodiment 28. The method of embodiment 27, wherein the infection site has an increased expression level of PLA2G2D.
[0306] Embodiment 29. The method of any one of embodiments 1-28, wherein the method further comprises administering a second agent.
[0307] Embodiment 30. The method of embodiment 29, wherein the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulator, an anti- angiogenesis agent, a growth inhibitory' agent, and an antineoplastic agent,
[0308] Embodiment 31. The method of embodiment 30, wherein the second agent is an immunomodulator.
[0309] Embodiment 32. The method of embodiment 31, wherein the immunomodulator is an immune checkpoint inhibitor.
[0310] Embodiment 33, The method of embodiment 28, wherein the immune checkpoint inhibitor specifically target PD-L1, PD-L2, CTLA4, PD-L2, PL)-! . CD47, TiGIT, GiTR, TIM3, LAG3, CD27, 4-1BB, or B7H4.
[0311] Embodiment 34. The method of embodiment 33, wherein the second agent comprises a cell comprising a chimeric antigen receptor that specifically binds to a tumor antigen.
[0312] Embodiment 35. The method of any one of embodiments 29-34, wherein the antagonist and the second agent is administered simultaneously or concurrently.
[0313] Embodiment 36. The method of any one of embodiments 29-34, wherein the antagonist and the second agent is administered sequentially.
[0314] Embodiment 37. The method of any one of embodiments 1-36, wherein the antagonist and/or the second agent is administered parentally.
[0315] Embodiment 38. The method of any one of embodiments 22-37, wherein the antagonist is administered to the cancer tissue or infection site directly.
[0316] Embodiment 39. The method of any one of embodiments 1-38, wherein the antagonist is administered at a dose of about 0.001 pg/kg to about 100 mg/kg.
[0317] Embodiment 40. The method of any one of embodiments 22-39, wherein the individual has an increased number of immune ceils in the cancer tissue or at the infection site after administration of the antagonist.
[0318] Embodiment 41. The method of embodiment 40, wherein the immune cells are T cells.
[0319] Embodiment 42. The method of embodiment 40 or embodiment 41, wherein the T cells are activated T cells.
[0320] Embodiment 43. The method of any one of embodiment 40-42, wherein the number of immune cells in the cancer tissue or at the infection site is increased by at ieast about 5% after administration of the antagonist,
[0321] Embodiment 44. The method of any one of embodiments 22-43, wherein immune ceils in the cancer tissue or at the infection site produce an increased level of a cytokine after administration of the antagonist.
[0322] Embodiment 45, The method of embodiment 44, wherein the cytokine is IFNy and/or
IL-2.
[0323] Embodiment 46. The method of embodiment 39 or embodiment 40, wherein the level of the cytokine is increased by at least about 5% after administration of the antagonist.
EXAMPLES
[0324] The examples below are intended to be purely exemplary of the application and should therefore not he considered to limit the invention m any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.
Example 1. identification of PLA2G2D signaling pathway
[0325] To identify new- signaling pathways that are involved in cancer, gene expression profiles were studied.
[0326] Specifically, the Cancer Genome Atlas (TCGA) batch-corrected RNA-seq dataset was downloaded from the National Cancer institute’s Genomic Data Commons PanCanAtlas website (https://gdc.cancer.gov/about-data/publications/pancanatlas). The pan-cancer dataset was separated into the following tumor types: Bladder urothelial carcinoma (BLCA), Breast invasive carcinoma (BRCA), COAD (Colon adenocarcinoma), ESCA (Esophageal carcinoma). Head and Neck squamous cell carcinoma (HNS C), Kidney Chromophobe (KICH), K1RC (Kidney renal clear cell carcinoma), KIRI5 (Kidney renal papillary cell carcinoma), L1HC (Liver hepatocellular carcinoma). Lung adenocarcinoma (LOAD), Lung squamous cell carcinoma (LUSC), Ovarian serous cystadenocarcinoma (OV), Pancreatic adenocarcinoma (PAAD), Pheochromocytoma and Paraganglioma (PCPG), Prostate adenocarcinoma (PRAB), Rectum Adenocarcinoma (READ), Prostate adenocarcinoma (SARC), Skin Cutaneous Melanoma (SKCM), Stomach adenocarcinoma (ST AD), Testicular Germ Cell Tumors (TGCT), Thyroid carcinoma (THCA), Thymoma (ΊΉUM), Triple negative breast cancer (TN-BRCA), and Uterine Corpus Endometrial Carcinoma (UCEC).
For each tumor type, the following analysis was performed: Genes with consistently low expression (e.g. genes with no counts in more than 80% of samples) were removed and the data was log-transformed using log2(x+l). The Weighted gene co-expression network analysis (WGCNA) R package was then used to build a gene co-expression network and identify clusters of highly correlated genes. See Langfefder, P. & Horvath, S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 9, (2008).
[0327] 'The cluster containing the T signature genes was extracted and gene ontology analysis was performed using the R package clusterProfiier to ensure that the cluster was enriched for T-cell related pathways. See Yu G, Wang L, Han Y, He Q (2012). “clusterProfiier: an R package for comparing biological themes among gene clusters.” OMICS: A Journal of integrative Biology, 16(5), 284-287. doi: 10.1089/omi.2011.0118. The expression dataset was then subset to genes within this T signature genes cluster and the samples were clustered using the R package ConsensusCiusterPlus using k-means clustering for the clustering algorithm, Euclidean for the distance measure, and maximum cluster number k = 20. See Wilkerson, D. M, Hayes, Neil D (2010). “ConsensusCiusterPlus: a class discovery' tool with confidence assessments and item tracking.” Bioinformatics, 26(12), 1572-1573. The cumulative distribution function (CDF) plot and the delta area plot, which shows the relative change in area under the CDF curve comparing k and k - l, were used to identify the optimal number of dusters. The expression values of the genes within each cluster were then summed to generate a single summary value to describe each cluster. Tumors in the highest gene expression cluster were labeled liof and tumors in the lowest gene expression cluster were labeled ‘cold'. The RNA-seq read count matrix for the tumor was then downloaded from the Google Cloud Pilot RNA-Sequencing for CCLE and TCGA project data repository (https :// osf. io/gqrz9/ ¾. which contains RNA-seq data from TCGA processed using kallisto.
See Tatlow, P. & Piccolo, S. R. A cloud-based workflow to quantify transcript-expression levels m public cancer compendia. Scientific Reports 6, (2016). The transcript-level data was summed to the gene-level and subset to protein-coding genes. Differential expression analysis was then performed using the R package !immato compare the ‘hot’ samples to the ‘cold’ samples. See Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK (2015). “limma powers differential expression analyses for RNA-sequencing and microarray studies.” Nucleic Acids Research, 43(7), e47. The differential expression results were visualized using the R package Enhanced Volcano. See Blighe K, Rana S, Lewis M (2019).
Enhanced Volcano: Publication-ready volcano plots with enhanced colouring and labeling. R package version 1.4.0, https:// github.com/kevmbiighe/EnhancedVolcano.
[0328] Results suggests that PLA2G2D is highly differentially expressed in four types of cancers. See FIGS. lA-lD. Specifically and strikingly, PLA2G2D was expressed 56 times higher in CD8+ high tumors as compared to CD8+ low tumors.
Example 2. Role of PLA2G2D in Suppression of T Cell Activation
[0329] The effect of human PLA2G2D-Fc on T cell activation was assessed in peripheral blood mononuclear cell (PBMC) or isolated T cell cultures. PBMC were isolated from ieukoreduetion system (LRS) chambers from healthy human donors by centrifugation over Ficoli-Paque Plus (GE Life Sciences) and labeled in a 5 mM CFSE solution (Molecular Probes) for 12 min at 37°C and washed. 2x1 (P labeled PBMC per well of a 96 well round bottom plate were then stimulated with 1 ,ug/ml anti-CD3 (OKT3, Invitrogen) and 0.2 μg/ml anti-CD28 (CD28.2, Invitrogen) in the presence of soluble 0-20 μg/ml human PLA2G2D-Fc or control human IgGl-Fc protein (Sino Biological) as indicated in a final volume of 200 mΐ RPM1 (Coming). PBMC cultures were incubated for 72 hours at 37°C before the supernatants were harvested and measured for IFNy and IL-2 levels using MSB V-plex assays (Meso Scale Discovery). T cell proliferation within the PBMC culture was simultaneously assessed by staining cells with fluoroph ore-conjugated anti~CD3, anti-CD4, and anti-CD8 antibodies (Biolegend) and Live/Dead Fixable dead cell stain (Molecular Probes), and running on a BD LSRFortessaX-20 flow cytometer (Becton Dickinson). FACS data was analyzed using FlowJo software.
[0330] The addition of soluble PLA2G2D-Fc dose dependently suppressed CD4+ and CD 8+ T cell proliferation in stimulated PBMC cultures, as measured by CFSE generational tracing. FIGS. 2A-2B show CFSE histograms and quantitation of T cell proliferation for one PBMC donor with increasing concentrations of PLA2G2D-Fc. FIGS. 3A-3C show CFSE histograms and quantitation of proliferation for three more independent PBMC donors in which increasing concentrations of soluble PLA2G2D-Fc protein dose dependently suppressed CD4+ and CD8+ T cell proliferation, whereas equivalent concentrations of Control-Fc protein had no significant effect. FIGS. 4A-4B show' that, consistent with T cell proliferation, the levels of IFNy and IL-2 in the different PBMC cultures were similarly dose-dependently and significantly decreased by increasing concentrations of soluble PLA2G2D-Fc protein, but not control-Fc.
[ 0331 ] To assess the effect of immobilized PLA2G2D protein on T cell proliferation in PBMC cultures, the same assay was utilized except 100 mΐ of 0-10 μg/ml human PLA2G2D- Fc or control human IgGl -Fc protein was coated overnight in PBS at 4°C on 96-well flat bottom plates the day before PBMC were prepared and added along with anti-CD 3 and anti- CD28 antibodies. FIG. 5 show's that immobilized human PLA2G2D-Fc protein coated on the plate surface also suppressed CD4+ and CD8+ T cell proliferation in stimulated PBMC cultures.
[0332] To evaluate the effect of PLA2G2D protein on isolated T cells, 96-well fiat bottom plates were coated overnight at 4°C with 0-10 pg/mi of human PLA2G2D-Fc or control human IgGl-Fc protein as well as with 1 μg/ml anti-CD3 (OKT3) and 0,2 μg/ml anti-CD28 (CD28.2) in 100 pi of PBS. The following day, plates were washed twice with PBS before addition of T ceils. T cells were isolated from PBMC using a Pan T Cell isolation Kit (Miltenyi) and labeled with CFSE as described above. 1x105 purified and labeled T cells were added to each well of the coated plate in a final volume of 200 pi RPMI. T cells were allowed to proliferate for 72 hours at 37°C before supernatants were collected for IFNy and IL-2 analysis by MSD and proliferation was analyzed by FACS.
[0333] FIG. 6 shows that immobilized human PLA2G2D-Fc protein dose-dependently suppresses proliferation of isolated T cell cultures in the presence of anti~CD3 and anti-CD28 stimulation. However, when soluble PLA2G2D-Fc protein was added to isolated T cell cultures instead of being coated on plates, it had no suppressive effect on T cells (data not shown). Collectively these results suggest that in order for PLA2G2D to elicit functional suppression of T cells, it requires cross-linking by antigen presenting cells or by immobilization on a plate surface.
Example 3, T Cell Suppression by PLA2G2D
[0334] Human PLA2G2D is a 145 amino acid secreted protein consisting of an N-terminal 20 residue signal peptide, a highly conserved Ca2 ^-binding site and a catalytic His-Asp dyad, in addition to these elements, human PLA2G2D features seven disulfide bonds which contribute to a high degree of stability. FIG. 7 A show's general structural and functional features of interest of the human PLA2G2D protein.
[Q335] To evaluate whether PLA2G2D enzyme activity' is required for its immune suppressive function, an H67Q mutation w¾s introduced to the highly conserved catalytic His67-Asp68 dyad of human PLA2G2D. Briefly, a human PLA2G2D cDNA fused in frame
to a human IgGl-Fc cDNA on the C terminus was synthesized with a CAC→CAG point mutation corresponding to a His→Gin substitution at residue 67. The construct was cloned into a high expression mammalian vector and transfected into HEK293 cells. Secreted human PLA2G2D-H67Q-Fc protein contained in the supernatant was purified by Protein A affinity chromatography. Purified PLA2G2D-H67Q-Fc protein was used in PBMC cultures as described above and compared against wild type PLA2G2D-Fc and control human IgGI-Fc for T cell suppressive activity.
[0336] FIGS. 7B-7C show that the PLA2G2D-H67Q-Fc catalytic mutant retains most of the immune suppressive function on CD4+ and CD8+ T cells at the dose of 0.5 μg/ml to 5 μg/ml, and exhibited a significantly decreased suppression at the dose of 10 μg/ml.
[0337] Alternatively, PBMC culture T ceil proliferation in the presence of wild type PLA2G2D-FC protein, as described above, was assessed with the addition of 0-25 mM of the sPLA2 inhibitor LY315920, or DMSO control. FIG. 8 shows that LY315920 does not reverse immune suppression induced by PLA2G2D.
Example 4. Binding of PLA2G2D to Activated T Cells
[0338] To determine whether PLA2G2D elicits immune suppression by directly binding to T cells in vitro, the level of PLA2G2D binding was assessed on resting and activated primary human T cells. T cells were isolated from PBMC using a Pan T Ceil Isolation Kit (Miltenyi) and cultured in the presence or absence of beads loaded with anti-human CD2, CD3, and CD28 antibodies (human T Cell Activation/Expansion Kit, Miltenyi) in RPMi for 48 hours at 37°C. Stimulated or unstimulated T ceils were then harvested, washed, and incubated with 0- 10 μg/ml of human PLA2G2D-Fc protein or human IgGl-Fc protein for 30 minutes at 4°C. Cells were washed 3 times with PBS and then stained with Alexa 488-conjugated goat anti- human IgGl antibodies (Invilrogen) for 30 minutes at 4ºC, washed, then analyzed by FACS.
[0339] FIGS. 9A-9C show' that human PLA2G2D-Fc preferentially hinds activated CD4+ and CD8+ T ceils in two different donor T cells compared with control human IgGl ~Fc protein. PLA2G2D binds unstimulated T cells to a small degree, but binding is dramatically increased upon T ceil stimulation. FIG. 9C shows a quantitative representation of PLA2G2D-Fc binding to stimulated T cells. Addition of heparin sulfate proteoglycan (HSPG) partially reduced PLA2G2D-Fc binding of T cells but did not alter suppression of CD4+ and CD8+ T cell proliferation (data not shown). This suggests that the immune suppression potentially
associated with the binding of PLA2G2D to T cells is not dependent on the binding through heparin sulfate on cell surface.
Example 5. Syngeneic Tumor Growth in PLA2G20 Deficient Mice
[0340] PLA2G2D knockout mice were generated by deleting exon 2 of the mouse Pla2g2d gene from C57BL6 mice using CRISPR/Cas9-mediated gene editing. To confirm the absence of a functional Pla2g2d gene in these mice, spleens from wild-type and knockout mice were harvested and total RNA isolated using TRIZol (Invitrogen). Total RNA was subjected to real-time RT-PCRto detect Pla2g2dvdRH A. Hprtl (hypoxanthiiie-guaiune phosphoribosyltransferase) mRNA levels were also measured as a control. The results demonstrated that the knockout mice were deficient i nPla2g2d expression.
[0341] To evaluate the effect of Pla2g2d deficiency on tumor growth, the murine syngeneic tumor cell lines MC38 (colon adenocarcinoma), B16F10 (melanoma), and E.G7-OVA (T cell lymphoma) were implanted into age-matched wild-type C57BL6 (WT) or PLA2G2D knockout mice. 1x106 MC38 or E.G7-QVA cells, or 5x105 B16F10 cells suspended m 100 ul PBS were subcutaneously injected into WT (n=16) or PLA2G2D knockout mice (n=16), and tumor growth was monitored every 2 or 3 days. Tumor volume was calculated using the formula: tumor volume ::: 0.5 x length x width2. Body weights were also monitored weekly. Mice were sacrificed after 3-4 weeks, or upon reaching designated endpoints.
[0342] As shown in Figures 11 A-F, tumor growth of all three syngeneic tumor cell lines was significantly reduced in PLA2G2D knockout mice compared to wild type mice, indicating a role for PLA2G2D in tumor progression and supporting targeted inhibition of PLA2G2D as a potential immunotherapy .
Example 6, Perturbation of PLA2G2D Immunosuppressive Function by Anti-
PLA2G2D Monodonai Antibodies
[0343] To demonstrate whether PLA2G2D immunosuppression can be neutralized and reversed by anti-PLA2G2D antibodies, we developed PLA2G2D-binding monoclonal antibodies by immunizing mice and generating hybridomas.
Blocking of PLA2G2D Binding to Activated T Cells
[0344] As shown in the above example (FIGS. 9A-9C), we found that PLA2G2D preferentially binds to activated T cells, but only minimally to resting T cells, suggesting that PLA2G2D can impart suppressive signaling by directly binding T cells upon their activation. We therefore devised an assay to determine whether anti-PL A2G2D antibodies could hinder
PLA2G2D binding to activated T cells and potentially block immune suppression. T cells within human PBMC cultures were stimulated with anti-CD3 and anii-CD28 antibodies (Invitrogen) for 24 hours at 37°C, PBMC cultures were then harvested, washed, and incubated with 2 μg/ml of human PLA2G2D-Fc protein or control human IgGl-Fc protein for 30 minutes at 4°C m the presence of 10 pg/ml of PLA2G2D antibodies (developed in house) or mouse IgG2a isotype control antibody (Invitrogen). Cells were washed 3 times with PBS and then stained with PE-conjugated goat anti-human IgGl antibodies (invitrogen) alongside fluorophore-conjugated anti-CD3, anti-CD4, and anti-CD8 antibodies (Biolegend) and Live/Dead Fixable dead cell stain (Molecular Probes )for 30 minutes at 4°C, washed, then analyzed by FACS.
[Q345] FIG. 12A shows that that two representative PLA2G2D-binding antibodies are able to reduce binding of PLA2G2D to activated T cells, as measured by the mean fluorescence intensity (MFI) of PLA2G2D staining on gated T cells. In contrast, a control mouse IgG2a isotype control had no effect on PLA2G2D binding.
Reversal of PLA2G2D-Dependent T Ceil Suppression in PBMC Culture
[0346] To determine whether PLA2G2D antibodies could neutralize PLA2G2D-dependent suppression of T cell function, we used an approach similar to the one we took to demonstrate PLA2GD suppressive activity above (FIGS, 4A-4B), Triplicate wells containing 2x1 (P PBMC per well of a 96 well round bottom plate were stimulated with 1 μg/ml anti- CBS (OKT3, Invitrogen) and 0.2 pg/'ml anti-CD28 (CD28.2, Invitrogen) in the presence of 1 pg/ml soluble human PLA2G2D-Fc protein (Sino Biological) and 10 pg/mi PLA2GD antibodies or control mIgG2a isotype control antibody (Invitrogen) as indicated in a final volume of 200 mΐ RPMI (Corning). PBMC cultures were incubated for 48 hours at 37°C before the supernatants were harvested and measured for IL-2 and IFNy levels using MSB V- plex assays (Meso Scale Discover}').
[0347] F1GS.12B-12C show that two representative function-blocking PLA2G2D antibodies are able to rescue PLA2G2D-mediated suppression of IL-2 and IFNy secretion levels m this assay.
Example 7. Use of PLA2G2D antibody to treat tumor
[0348] To evaluate the effect of PLA2G2B amtibodies on tumor growth, the murine syngeneic tumor cell lines MC38 (colon adenocarcinoma), B16F10 (melanoma), and E.G7- OVA (T cell lymphoma) are implanted into age-matched wild-type C57BL6 mice. 1x106
MC38 or E.G7-OVA ceils, or 5x105 B16F10 cells suspended in 100 ul PBS are subcutaneously injected into C57BL6 mice. When tumor volumes are in the range of 50-150 mm5, mice are randomized into control and treatment groups. PLA2G2D antibodies are administered on days 1, 4, 7, and 11 post-randomization at a dose of lOmg/kg by IP injection. Tumor growth is monitored every 2 or 3 days. Tumor volume is calculated using the formula: tumor volume = 0.5 x length x width2. Body weights are also monitored weekly. Mice are sacrificed after 3-4 weeks, or upon reaching designated endpoints.
Claims
1. A method of treating a cancer or viral infection in an individual, comprising administering into the individual an effective amount of an antagonist targeting PLA2G2D signaling pathway.
2. The method of claim 1 , wherein the antagonist is an antagonist inhibits or downregtiiates PLA2G2D.
3. The method of claim 2, wherein the PLA2G2D is a human PLA2G2D.
4. The method of claim 2 or claim 3, wherein the antagonist decreases enzymatic activity level of PLA2G2D.
5. The method of claim 4, wherein the antagonist targeting PLA2G2D signaling pathway blocks a catalytic site on PLA2G2D.
6. The method of claim 5, wherein the antagonist targets the H67 catalytic site on a human PLA2G2D according to 8EQ ID NO: 1 or 5.
7. The method of any one of claims 1-3, wherein the antagonist comprises a siRNA, an miRNA, an antisense RNA, or a gene editing system.
8. The method of any one of claims 1-3, wherein the antagonist blocks the binding of PLA2G2D to an immune cell.
9. The method of claim 8, wherein the immune cell is a T cell.
10. The method of any one of claims 1 -3, wherein the antagonist comprises an anti-
PLA2G2D antibody.
11. The method of claim 10, wherein the anti-PL A2G2D antibody is a monoclonal antibody.
12. The method of claim 10, wherein the antagonist is a fusion protein or immunoconjugate further comprising a second moiety.
13. The method of claim 12, wherein the second moiety comprises a cytokine.
14. The method of any one of claims 1-3, wherein the antagonist comprises an inhibitory PLA2G2D polypeptide that blocks the binding of PLA2G2D to an immune cell.
15. Hie method of claim 14, wherein the inhibitory PLA2G2D polypeptide binds to the immune cell with a greater affinity' than a wi!dtype PLA2G2D.
16. Hie method of claim 15, wherein the immune cells is a T cell.
17. Hie method of any one of claims 14-16, wherein inhibitory PLA2G2D polypeptide further comprises a stabilizing domain.
18. Tire method of claim 17, wherein the stabilizing domain is an Fc domain.
19. Tlie method of any one of claims 14-18, wherein the inhibitory PLA2G2B polypeptide has a length of about 50 to about 200 ammo acids.
20. The method of any one of claims 14-19, wherein the inhibitory PLA2G2D polypeptide has a) a mutation at a position corresponding to histidine at position 67 (H67) according to SEQ ID NO: 1 or 5 or b) a mutation at the position corresponding to glycine at position 80 (G80) according to SEQ ID NO: 5.
21. The method of claim 20, wherein the inhibitory PLA2G2D polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, and 7-12 or a variant thereof.
22. Tlie method of any one of claims 1-21, wherein the disease or condition is a cancer.
23. Tlie method of claim 22, wherein the cancer is a solid tumor.
24. Tlie method of claim 22 or claim 23, wherein the cancer is an advanced or malignant tumor.
25. The method of any one of claims 22-24, wherein the cancer has an increased expression level of PLA2G2D,
26. The method of any one of claims 22-25, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic
cancer, renal cancer, prostate cancer, urothelial cancer, testis cancer, ovarian cancer and melanoma.
27. Hie method of any one of claims 1-21, wherein the disease or condition is a viral infection,
28. The method of claim 27, wherein the expression level of PLA2G2D at an infected site is higher than that of an uninfected site.
29. Tire method of any one of claims 1-28, wherein the method further comprises administering a second agent.
30. The method of claim 29, wherein the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulator, an anti -angiogenesis agent, a growth inhibitory agent, and an antineoplastic agent.
31. The method of claim 30, wherein the second agent is an immunomodulator.
32. The method of claim 31, wherein the immunomodulator is an immune checkpoint inhibitor.
33. The method of claim 28, wherein the immune checkpoint inhibitor specifically target PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TΪM3, LAG3, CD27, 4-lBB, or B7H4.
34. Tire method of claim 33, wherein the second agent comprises a cell comprising a chimeric antigen receptor that specifically binds to a tumor antigen.
35. The method of any one of claims 29-34, wherein the antagonist and the second agent are administered simultaneously or concurrently.
36. The method of any one of claims 29-34, wherein the antagonist and the second agent are administered sequentially.
37. The method of any one of claims 1-36, wherein the antagonist and/or the second agent is administered parentally.
38. The method of any one of claims 22-37, wherein the antagonist is administered to the cancer tissue or infection site directly.
39. The method of any one of claims 1-38, wherein the antagonist is administered at a dose of about 0.001 pg/kg to about 100 rng/kg.
40. The method of any one of claims 22-39, wherein the individual has an increased number of immune cells in the cancer tissue or at the infection site after administration of the antagonist.
41. The method of claim 40, wherein the immune cells are T cells.
42. The method of claim 40 or claim 41, wherein the T cells are acti vated T ceils.
43. The method of any one of claim 40-42, wherein the number of immune ceils in the cancer tissue or at the infection site is increased by at least about 5% after administration of the antagonist.
44. The method of any one of claims 22-43, wherein immune cells in the cancer tissue or at the infection site produce an increased level of a cytokine after administration of the antagonist.
45. The method of claim 44, wherein the cytokine is IFNy and/or IL-2.
46. The method of claim 39 or claim 40, wherein the level of the cytokine is increased by at least about 5% after administration of the antagonist.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202180026634.2A CN115916837A (en) | 2020-01-30 | 2021-01-29 | Methods and compositions for treating cancer or viral infection with PLA2G2D antagonists |
JP2022546697A JP2023513477A (en) | 2020-01-30 | 2021-01-29 | Methods and compositions for treating cancer or viral infections with PLA2G2D antagonists |
EP21748349.4A EP4097142A4 (en) | 2020-01-30 | 2021-01-29 | Methods and compositions for treating cancer or viral infection with a pla2g2d antagonist |
US17/796,148 US20230075779A1 (en) | 2020-01-30 | 2021-01-29 | Methods and compositions for treating cancer or viral infection with a pla2g2d antagonist |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202062968060P | 2020-01-30 | 2020-01-30 | |
US62/968,060 | 2020-01-30 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2021155305A1 true WO2021155305A1 (en) | 2021-08-05 |
Family
ID=77079662
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2021/015921 WO2021155305A1 (en) | 2020-01-30 | 2021-01-29 | Methods and compositions for treating cancer or viral infection with a pla2g2d antagonist |
Country Status (6)
Country | Link |
---|---|
US (1) | US20230075779A1 (en) |
EP (1) | EP4097142A4 (en) |
JP (1) | JP2023513477A (en) |
CN (1) | CN115916837A (en) |
TW (1) | TW202142241A (en) |
WO (1) | WO2021155305A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023015225A1 (en) * | 2021-08-04 | 2023-02-09 | Apeximmune Therapeutics Inc. | Anti-pla2g2d constructs and uses thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000024911A2 (en) * | 1998-10-27 | 2000-05-04 | Incyte Pharmaceuticals, Inc. | Human phospholipases |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050123925A1 (en) * | 2002-11-15 | 2005-06-09 | Genentech, Inc. | Compositions and methods for the diagnosis and treatment of tumor |
-
2021
- 2021-01-29 CN CN202180026634.2A patent/CN115916837A/en active Pending
- 2021-01-29 US US17/796,148 patent/US20230075779A1/en active Pending
- 2021-01-29 WO PCT/US2021/015921 patent/WO2021155305A1/en unknown
- 2021-01-29 JP JP2022546697A patent/JP2023513477A/en active Pending
- 2021-01-29 EP EP21748349.4A patent/EP4097142A4/en active Pending
- 2021-02-01 TW TW110103715A patent/TW202142241A/en unknown
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000024911A2 (en) * | 1998-10-27 | 2000-05-04 | Incyte Pharmaceuticals, Inc. | Human phospholipases |
Non-Patent Citations (4)
Title |
---|
See also references of EP4097142A4 * |
VIJAY R. ET AL.: "Critical role of phospholipase A2 group 11D in age-related susceptibility to sever acute respiratory syndrome-CoV infection", THE JOURNAL OF EXPERIMENTAL MEDICINE, vol. 212, no. 11, 2015, pages 1851 - 1868, XP055774423, DOI: 10.1084/jem.20150632 * |
VON ALLMEN C.E. ET AL.: "Secretory phospholipase A2-IID is an effector molecule of CD 4+ CD 25+ regulatory T cells", PNAS, vol. 106, no. 28, 2009, pages 11673 - 11678, XP055072720, DOI: 10.1073/pnas.0812569106 * |
YOSHIMI MIKI, YUH KIDOGUCHI , MARIKO SATO , YOSHITAKA TAKETOMI , CHOJI TAYA , KAZUAKI MURAMATSU , MICHAEL H GELB , KEI YAMAMOTO ,: "Dual Roles of Group IID Phospholipase A2 in inflammation and Cancer", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 291, no. 30, 2016, pages 15588 - 15601, XP055845274 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023015225A1 (en) * | 2021-08-04 | 2023-02-09 | Apeximmune Therapeutics Inc. | Anti-pla2g2d constructs and uses thereof |
Also Published As
Publication number | Publication date |
---|---|
EP4097142A4 (en) | 2024-05-22 |
TW202142241A (en) | 2021-11-16 |
US20230075779A1 (en) | 2023-03-09 |
EP4097142A1 (en) | 2022-12-07 |
CN115916837A (en) | 2023-04-04 |
JP2023513477A (en) | 2023-03-31 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
TWI781120B (en) | Antibodies to pd-1 and uses thereof | |
JP7334177B2 (en) | Anti-CD73 antibody and use thereof | |
RU2742241C2 (en) | Antibodies to icos | |
US20220281978A1 (en) | Compositions and Methods for the Treatment of Cancer (Anti-ICOS Antibody Dosing) | |
US10968277B2 (en) | Gene signatures for determining ICOS expression | |
JP2022512132A (en) | Anti-claudin antibodies and their use | |
KR20220042056A (en) | Anti-SIGLEC-9 compositions and methods for modulating myeloid cell inflammatory phenotype, and uses thereof | |
JP2022547850A (en) | Anti-TIGIT immune inhibitor and application | |
KR20230017778A (en) | Anti-VSIG4 compositions and methods for modulating myeloid cell inflammatory phenotype, and uses thereof | |
US20230075779A1 (en) | Methods and compositions for treating cancer or viral infection with a pla2g2d antagonist | |
US20220041733A1 (en) | Methods of treating tumor | |
WO2021216417A1 (en) | Compositions and methods for vaccination and the treatment of infectious diseases | |
CN118139894A (en) | Anti-PLA 2G2D constructs and uses thereof | |
KR20240058149A (en) | Treatment and prevention of cancer using VISTA antigen-binding molecules | |
WO2023015225A1 (en) | Anti-pla2g2d constructs and uses thereof | |
WO2024167830A2 (en) | ANTI-SIRPα ANTIBODIES AND USES THEREOF |
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: 21748349 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2022546697 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2021748349 Country of ref document: EP Effective date: 20220830 |