US20230201305A9 - Immune checkpoint inhibitor combinations - Google Patents
Immune checkpoint inhibitor combinations Download PDFInfo
- Publication number
- US20230201305A9 US20230201305A9 US17/366,463 US202117366463A US2023201305A9 US 20230201305 A9 US20230201305 A9 US 20230201305A9 US 202117366463 A US202117366463 A US 202117366463A US 2023201305 A9 US2023201305 A9 US 2023201305A9
- Authority
- US
- United States
- Prior art keywords
- ltx
- cells
- amino
- tumour
- group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 title claims abstract description 21
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 title claims abstract description 21
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 title claims abstract description 16
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 title claims abstract description 16
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 224
- 150000001875 compounds Chemical class 0.000 claims abstract description 113
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 67
- 150000001413 amino acids Chemical class 0.000 claims abstract description 55
- 238000000034 method Methods 0.000 claims abstract description 48
- -1 cationic amino acids Chemical group 0.000 claims abstract description 29
- 125000002091 cationic group Chemical group 0.000 claims abstract description 8
- 229960002621 pembrolizumab Drugs 0.000 claims description 8
- 150000003839 salts Chemical class 0.000 claims description 8
- 150000001408 amides Chemical class 0.000 claims description 5
- 150000002148 esters Chemical class 0.000 claims description 4
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 4
- 229960003301 nivolumab Drugs 0.000 claims description 4
- SAUDSWFPPKSVMK-LBPRGKRZSA-N (2s)-2-(n-phenylanilino)propanoic acid Chemical compound C=1C=CC=CC=1N([C@@H](C)C(O)=O)C1=CC=CC=C1 SAUDSWFPPKSVMK-LBPRGKRZSA-N 0.000 claims description 3
- VVJSFKRZWZKXLP-UHFFFAOYSA-N 2-amino-3-[4-(4-phenylphenyl)phenyl]propanoic acid Chemical compound C1=CC(CC(N)C(O)=O)=CC=C1C1=CC=C(C=2C=CC=CC=2)C=C1 VVJSFKRZWZKXLP-UHFFFAOYSA-N 0.000 claims description 3
- MRVJUNXMEDRMRO-UHFFFAOYSA-N 2-amino-3-anthracen-9-ylpropanoic acid Chemical compound C1=CC=C2C(CC(N)C(O)=O)=C(C=CC=C3)C3=CC2=C1 MRVJUNXMEDRMRO-UHFFFAOYSA-N 0.000 claims description 3
- JCZLABDVDPYLRZ-AWEZNQCLSA-N biphenylalanine Chemical compound C1=CC(C[C@H](N)C(O)=O)=CC=C1C1=CC=CC=C1 JCZLABDVDPYLRZ-AWEZNQCLSA-N 0.000 claims description 3
- 229950010773 pidilizumab Drugs 0.000 claims description 3
- IQMAPACPGQFJNG-UHFFFAOYSA-N 2-amino-3-(2,5,7-tritert-butyl-1h-indol-3-yl)propanoic acid Chemical compound C1=C(C(C)(C)C)C=C2C(CC(N)C(O)=O)=C(C(C)(C)C)NC2=C1C(C)(C)C IQMAPACPGQFJNG-UHFFFAOYSA-N 0.000 claims description 2
- OWUAFTDENICQEO-UHFFFAOYSA-N 2-amino-3-(3,5-diphenylphenyl)propanoic acid Chemical compound C=1C(CC(N)C(O)=O)=CC(C=2C=CC=CC=2)=CC=1C1=CC=CC=C1 OWUAFTDENICQEO-UHFFFAOYSA-N 0.000 claims description 2
- ATLYCIDRXNGNDM-UHFFFAOYSA-N 2-amino-3-(4-naphthalen-2-ylphenyl)propanoic acid Chemical compound C1=CC(CC(N)C(O)=O)=CC=C1C1=CC=C(C=CC=C2)C2=C1 ATLYCIDRXNGNDM-UHFFFAOYSA-N 0.000 claims description 2
- SQKISBBTEJIIRT-UHFFFAOYSA-N 2-amino-3-[4-(2,2-diphenylethyl)phenyl]propanoic acid Chemical compound C1=CC(CC(N)C(O)=O)=CC=C1CC(C=1C=CC=CC=1)C1=CC=CC=C1 SQKISBBTEJIIRT-UHFFFAOYSA-N 0.000 claims description 2
- RHTGQPNFDRVYEY-UHFFFAOYSA-N 2-amino-3-[4-(2-phenylphenyl)phenyl]propanoic acid Chemical compound C1=CC(CC(N)C(O)=O)=CC=C1C1=CC=CC=C1C1=CC=CC=C1 RHTGQPNFDRVYEY-UHFFFAOYSA-N 0.000 claims description 2
- DVGAAJDATPNKDI-UHFFFAOYSA-N 2-amino-3-[4-(3-phenylphenyl)phenyl]propanoic acid Chemical compound C1=CC(CC(N)C(O)=O)=CC=C1C1=CC=CC(C=2C=CC=CC=2)=C1 DVGAAJDATPNKDI-UHFFFAOYSA-N 0.000 claims description 2
- XIEHNNDCBCRVBQ-UHFFFAOYSA-N 2-amino-3-[4-(4-butylphenyl)phenyl]propanoic acid Chemical compound C1=CC(CCCC)=CC=C1C1=CC=C(CC(N)C(O)=O)C=C1 XIEHNNDCBCRVBQ-UHFFFAOYSA-N 0.000 claims description 2
- JCZLABDVDPYLRZ-UHFFFAOYSA-N 2-azaniumyl-3-(4-phenylphenyl)propanoate Chemical compound C1=CC(CC(N)C(O)=O)=CC=C1C1=CC=CC=C1 JCZLABDVDPYLRZ-UHFFFAOYSA-N 0.000 claims description 2
- OFYAYGJCPXRNBL-UHFFFAOYSA-N 2-azaniumyl-3-naphthalen-1-ylpropanoate Chemical compound C1=CC=C2C(CC(N)C(O)=O)=CC=CC2=C1 OFYAYGJCPXRNBL-UHFFFAOYSA-N 0.000 claims description 2
- JPZXHKDZASGCLU-UHFFFAOYSA-N 2-azaniumyl-3-naphthalen-2-ylpropanoate Chemical compound C1=CC=CC2=CC(CC(N)C(O)=O)=CC=C21 JPZXHKDZASGCLU-UHFFFAOYSA-N 0.000 claims description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 2
- 125000004122 cyclic group Chemical group 0.000 claims description 2
- PECGVEGMRUZOML-UHFFFAOYSA-N diphenylalanine Chemical compound C=1C=CC=CC=1C(C(N)C(O)=O)C1=CC=CC=C1 PECGVEGMRUZOML-UHFFFAOYSA-N 0.000 claims description 2
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 claims description 2
- 238000011282 treatment Methods 0.000 abstract description 106
- 239000002955 immunomodulating agent Substances 0.000 abstract description 19
- 210000001700 mitochondrial membrane Anatomy 0.000 abstract description 8
- 239000000203 mixture Substances 0.000 abstract description 8
- 238000002360 preparation method Methods 0.000 abstract description 6
- 239000013543 active substance Substances 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 272
- 108010029554 LTX-315 Proteins 0.000 description 189
- GGAKLYWEFZCVIT-TVEKFXMRSA-N (2S)-2,6-diamino-N-[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1,6-diamino-1-oxohexan-2-yl]amino]-1-oxo-3,3-diphenylpropan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1-oxohexan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1-oxohexan-2-yl]hexanamide Chemical compound C=1C=CC=CC=1C([C@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](CCCCN)C(N)=O)C1=CC=CC=C1 GGAKLYWEFZCVIT-TVEKFXMRSA-N 0.000 description 172
- 241000699670 Mus sp. Species 0.000 description 88
- 241001465754 Metazoa Species 0.000 description 68
- 210000004881 tumor cell Anatomy 0.000 description 40
- 230000000694 effects Effects 0.000 description 34
- 239000007924 injection Substances 0.000 description 30
- 238000002347 injection Methods 0.000 description 30
- 201000011510 cancer Diseases 0.000 description 29
- 239000006228 supernatant Substances 0.000 description 28
- 238000012360 testing method Methods 0.000 description 28
- 241001529936 Murinae Species 0.000 description 26
- 210000003470 mitochondria Anatomy 0.000 description 25
- 102000004196 processed proteins & peptides Human genes 0.000 description 23
- 230000004044 response Effects 0.000 description 23
- 241000699666 Mus <mouse, genus> Species 0.000 description 22
- 239000001963 growth medium Substances 0.000 description 21
- 230000028993 immune response Effects 0.000 description 20
- 201000001441 melanoma Diseases 0.000 description 20
- 206010027476 Metastases Diseases 0.000 description 19
- 230000015572 biosynthetic process Effects 0.000 description 19
- 238000011534 incubation Methods 0.000 description 19
- 230000009401 metastasis Effects 0.000 description 19
- 239000000816 peptidomimetic Substances 0.000 description 19
- 229940045513 CTLA4 antagonist Drugs 0.000 description 18
- 230000004614 tumor growth Effects 0.000 description 18
- 102100037907 High mobility group protein B1 Human genes 0.000 description 17
- 101001025337 Homo sapiens High mobility group protein B1 Proteins 0.000 description 17
- 239000012980 RPMI-1640 medium Substances 0.000 description 17
- 238000002474 experimental method Methods 0.000 description 17
- 102000037982 Immune checkpoint proteins Human genes 0.000 description 16
- 108091008036 Immune checkpoint proteins Proteins 0.000 description 16
- 206010025323 Lymphomas Diseases 0.000 description 16
- 239000003642 reactive oxygen metabolite Substances 0.000 description 16
- 239000000126 substance Substances 0.000 description 16
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 15
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 15
- 239000000427 antigen Substances 0.000 description 15
- 108091007433 antigens Proteins 0.000 description 15
- 102000036639 antigens Human genes 0.000 description 15
- 208000003950 B-cell lymphoma Diseases 0.000 description 14
- 210000004379 membrane Anatomy 0.000 description 14
- 239000012528 membrane Substances 0.000 description 14
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 13
- 239000003795 chemical substances by application Substances 0.000 description 13
- 230000001472 cytotoxic effect Effects 0.000 description 13
- 230000002354 daily effect Effects 0.000 description 13
- 239000000463 material Substances 0.000 description 13
- 239000002953 phosphate buffered saline Substances 0.000 description 13
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 12
- 230000004721 adaptive immunity Effects 0.000 description 12
- 208000027418 Wounds and injury Diseases 0.000 description 11
- 210000003743 erythrocyte Anatomy 0.000 description 11
- 210000000987 immune system Anatomy 0.000 description 11
- 238000000338 in vitro Methods 0.000 description 11
- 210000004988 splenocyte Anatomy 0.000 description 11
- 230000000259 anti-tumor effect Effects 0.000 description 10
- 210000000170 cell membrane Anatomy 0.000 description 10
- 238000011161 development Methods 0.000 description 10
- 230000002489 hematologic effect Effects 0.000 description 10
- 239000013642 negative control Substances 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 229910001868 water Inorganic materials 0.000 description 10
- 201000009030 Carcinoma Diseases 0.000 description 9
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 9
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 9
- 210000001744 T-lymphocyte Anatomy 0.000 description 9
- 239000013504 Triton X-100 Substances 0.000 description 9
- 229920004890 Triton X-100 Polymers 0.000 description 9
- 230000037396 body weight Effects 0.000 description 9
- 239000003112 inhibitor Substances 0.000 description 9
- 239000013641 positive control Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 210000004369 blood Anatomy 0.000 description 8
- 239000008280 blood Substances 0.000 description 8
- 210000000481 breast Anatomy 0.000 description 8
- 230000030833 cell death Effects 0.000 description 8
- 239000013592 cell lysate Substances 0.000 description 8
- 238000010790 dilution Methods 0.000 description 8
- 239000012895 dilution Substances 0.000 description 8
- 230000012010 growth Effects 0.000 description 8
- 230000001939 inductive effect Effects 0.000 description 8
- 230000002401 inhibitory effect Effects 0.000 description 8
- 230000005764 inhibitory process Effects 0.000 description 8
- 238000011081 inoculation Methods 0.000 description 8
- 239000003446 ligand Substances 0.000 description 8
- 230000002438 mitochondrial effect Effects 0.000 description 8
- 230000017074 necrotic cell death Effects 0.000 description 8
- 230000003389 potentiating effect Effects 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 229960005486 vaccine Drugs 0.000 description 8
- 206010009944 Colon cancer Diseases 0.000 description 7
- 241000204031 Mycoplasma Species 0.000 description 7
- 206010039491 Sarcoma Diseases 0.000 description 7
- 208000009956 adenocarcinoma Diseases 0.000 description 7
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 7
- 229960004316 cisplatin Drugs 0.000 description 7
- 238000002648 combination therapy Methods 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 230000001965 increasing effect Effects 0.000 description 7
- VYNDHICBIRRPFP-UHFFFAOYSA-N pacific blue Chemical compound FC1=C(O)C(F)=C2OC(=O)C(C(=O)O)=CC2=C1 VYNDHICBIRRPFP-UHFFFAOYSA-N 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 239000007787 solid Substances 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 238000005303 weighing Methods 0.000 description 7
- RXGJTUSBYWCRBK-UHFFFAOYSA-M 5-methylphenazinium methyl sulfate Chemical compound COS([O-])(=O)=O.C1=CC=C2[N+](C)=C(C=CC=C3)C3=NC2=C1 RXGJTUSBYWCRBK-UHFFFAOYSA-M 0.000 description 6
- 206010006187 Breast cancer Diseases 0.000 description 6
- 208000026310 Breast neoplasm Diseases 0.000 description 6
- 108090000695 Cytokines Proteins 0.000 description 6
- 102000004127 Cytokines Human genes 0.000 description 6
- 231100000070 MTS assay Toxicity 0.000 description 6
- 238000000719 MTS assay Methods 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 239000010432 diamond Substances 0.000 description 6
- 229960004679 doxorubicin Drugs 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 239000012091 fetal bovine serum Substances 0.000 description 6
- 238000002513 implantation Methods 0.000 description 6
- 230000002101 lytic effect Effects 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 210000003463 organelle Anatomy 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 102000005962 receptors Human genes 0.000 description 6
- 108020003175 receptors Proteins 0.000 description 6
- 239000004017 serum-free culture medium Substances 0.000 description 6
- 239000003981 vehicle Substances 0.000 description 6
- 206010018910 Haemolysis Diseases 0.000 description 5
- 206010033128 Ovarian cancer Diseases 0.000 description 5
- 230000001464 adherent effect Effects 0.000 description 5
- 230000002411 adverse Effects 0.000 description 5
- 238000013459 approach Methods 0.000 description 5
- 210000003719 b-lymphocyte Anatomy 0.000 description 5
- 201000008275 breast carcinoma Diseases 0.000 description 5
- 210000004899 c-terminal region Anatomy 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 230000009089 cytolysis Effects 0.000 description 5
- 231100000433 cytotoxic Toxicity 0.000 description 5
- 230000008588 hemolysis Effects 0.000 description 5
- 229940126546 immune checkpoint molecule Drugs 0.000 description 5
- 201000007270 liver cancer Diseases 0.000 description 5
- 208000014018 liver neoplasm Diseases 0.000 description 5
- 230000007774 longterm Effects 0.000 description 5
- 239000006166 lysate Substances 0.000 description 5
- 230000001338 necrotic effect Effects 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 238000002255 vaccination Methods 0.000 description 5
- 208000036762 Acute promyelocytic leukaemia Diseases 0.000 description 4
- 206010003445 Ascites Diseases 0.000 description 4
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 4
- 208000011691 Burkitt lymphomas Diseases 0.000 description 4
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 4
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 4
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 4
- 229930182816 L-glutamine Natural products 0.000 description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 4
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 4
- 208000002151 Pleural effusion Diseases 0.000 description 4
- 208000033826 Promyelocytic Acute Leukemia Diseases 0.000 description 4
- 108020004459 Small interfering RNA Proteins 0.000 description 4
- 210000001015 abdomen Anatomy 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 238000000540 analysis of variance Methods 0.000 description 4
- 239000012298 atmosphere Substances 0.000 description 4
- 210000000805 cytoplasm Anatomy 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 230000002601 intratumoral effect Effects 0.000 description 4
- 206010073095 invasive ductal breast carcinoma Diseases 0.000 description 4
- 201000010985 invasive ductal carcinoma Diseases 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 201000005202 lung cancer Diseases 0.000 description 4
- 208000020816 lung neoplasm Diseases 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 210000004940 nucleus Anatomy 0.000 description 4
- 230000036961 partial effect Effects 0.000 description 4
- 230000002093 peripheral effect Effects 0.000 description 4
- 239000004055 small Interfering RNA Substances 0.000 description 4
- 238000007619 statistical method Methods 0.000 description 4
- 230000004936 stimulating effect Effects 0.000 description 4
- 230000005945 translocation Effects 0.000 description 4
- 238000004627 transmission electron microscopy Methods 0.000 description 4
- 230000035899 viability Effects 0.000 description 4
- VQVUBYASAICPFU-UHFFFAOYSA-N (6'-acetyloxy-2',7'-dichloro-3-oxospiro[2-benzofuran-1,9'-xanthene]-3'-yl) acetate Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(Cl)=C(OC(C)=O)C=C1OC1=C2C=C(Cl)C(OC(=O)C)=C1 VQVUBYASAICPFU-UHFFFAOYSA-N 0.000 description 3
- PECYZEOJVXMISF-UHFFFAOYSA-N 3-aminoalanine Chemical compound [NH3+]CC(N)C([O-])=O PECYZEOJVXMISF-UHFFFAOYSA-N 0.000 description 3
- 206010052360 Colorectal adenocarcinoma Diseases 0.000 description 3
- 108010052832 Cytochromes Proteins 0.000 description 3
- 102000018832 Cytochromes Human genes 0.000 description 3
- 238000012286 ELISA Assay Methods 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- 206010029260 Neuroblastoma Diseases 0.000 description 3
- 206010061535 Ovarian neoplasm Diseases 0.000 description 3
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 3
- 230000003187 abdominal effect Effects 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000033289 adaptive immune response Effects 0.000 description 3
- 230000001093 anti-cancer Effects 0.000 description 3
- 230000000692 anti-sense effect Effects 0.000 description 3
- 238000003782 apoptosis assay Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000009566 cancer vaccine Methods 0.000 description 3
- 229940022399 cancer vaccine Drugs 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 210000001072 colon Anatomy 0.000 description 3
- 230000003750 conditioning effect Effects 0.000 description 3
- 208000035250 cutaneous malignant susceptibility to 1 melanoma Diseases 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 231100000673 dose–response relationship Toxicity 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 230000007717 exclusion Effects 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 210000005260 human cell Anatomy 0.000 description 3
- 238000011221 initial treatment Methods 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 229960005386 ipilimumab Drugs 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 206010061289 metastatic neoplasm Diseases 0.000 description 3
- 238000007069 methylation reaction Methods 0.000 description 3
- 210000005259 peripheral blood Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000000770 proinflammatory effect Effects 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 230000000069 prophylactic effect Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 210000003491 skin Anatomy 0.000 description 3
- 238000000528 statistical test Methods 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 239000007929 subcutaneous injection Substances 0.000 description 3
- 238000010254 subcutaneous injection Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 3
- 230000003442 weekly effect Effects 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- APRZHQXAAWPYHS-UHFFFAOYSA-N 4-[5-[3-(carboxymethoxy)phenyl]-3-(4,5-dimethyl-1,3-thiazol-2-yl)tetrazol-3-ium-2-yl]benzenesulfonate Chemical compound S1C(C)=C(C)N=C1[N+]1=NC(C=2C=C(OCC(O)=O)C=CC=2)=NN1C1=CC=C(S([O-])(=O)=O)C=C1 APRZHQXAAWPYHS-UHFFFAOYSA-N 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 2
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 2
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 2
- 206010073478 Anaplastic large-cell lymphoma Diseases 0.000 description 2
- 102000014133 Antimicrobial Cationic Peptides Human genes 0.000 description 2
- 108010050820 Antimicrobial Cationic Peptides Proteins 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- 108010074708 B7-H1 Antigen Proteins 0.000 description 2
- 102000008096 B7-H1 Antigen Human genes 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 208000003174 Brain Neoplasms Diseases 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 108091006146 Channels Proteins 0.000 description 2
- 102100030497 Cytochrome c Human genes 0.000 description 2
- 108010075031 Cytochromes c Proteins 0.000 description 2
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 description 2
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 2
- 239000005517 L01XE01 - Imatinib Substances 0.000 description 2
- 208000032004 Large-Cell Anaplastic Lymphoma Diseases 0.000 description 2
- 108060001084 Luciferase Proteins 0.000 description 2
- 239000005089 Luciferase Substances 0.000 description 2
- 231100000002 MTT assay Toxicity 0.000 description 2
- 238000000134 MTT assay Methods 0.000 description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 2
- 241001024304 Mino Species 0.000 description 2
- 102000007999 Nuclear Proteins Human genes 0.000 description 2
- 108010089610 Nuclear Proteins Proteins 0.000 description 2
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 2
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 2
- 108010043958 Peptoids Proteins 0.000 description 2
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 2
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 description 2
- 206010038389 Renal cancer Diseases 0.000 description 2
- 108091008874 T cell receptors Proteins 0.000 description 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 238000003917 TEM image Methods 0.000 description 2
- 102000002689 Toll-like receptor Human genes 0.000 description 2
- 108020000411 Toll-like receptor Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 150000001335 aliphatic alkanes Chemical class 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000005911 anti-cytotoxic effect Effects 0.000 description 2
- 210000000612 antigen-presenting cell Anatomy 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- UORVGPXVDQYIDP-UHFFFAOYSA-N borane Chemical compound B UORVGPXVDQYIDP-UHFFFAOYSA-N 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 238000010226 confocal imaging Methods 0.000 description 2
- 238000004624 confocal microscopy Methods 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 230000001461 cytolytic effect Effects 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 238000009510 drug design Methods 0.000 description 2
- 238000009509 drug development Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000013401 experimental design Methods 0.000 description 2
- 238000007421 fluorometric assay Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 229940080856 gleevec Drugs 0.000 description 2
- 208000005017 glioblastoma Diseases 0.000 description 2
- 244000144993 groups of animals Species 0.000 description 2
- 238000005534 hematocrit Methods 0.000 description 2
- 230000002949 hemolytic effect Effects 0.000 description 2
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 2
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 2
- 125000005842 heteroatom Chemical group 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- YLMAHDNUQAMNNX-UHFFFAOYSA-N imatinib methanesulfonate Chemical compound CS(O)(=O)=O.C1CN(C)CCN1CC1=CC=C(C(=O)NC=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)C=C1 YLMAHDNUQAMNNX-UHFFFAOYSA-N 0.000 description 2
- 230000000899 immune system response Effects 0.000 description 2
- 230000037449 immunogenic cell death Effects 0.000 description 2
- 230000001506 immunosuppresive effect Effects 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000028709 inflammatory response Effects 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 201000010982 kidney cancer Diseases 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000010859 live-cell imaging Methods 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 210000003712 lysosome Anatomy 0.000 description 2
- 230000001868 lysosomic effect Effects 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 229910052749 magnesium Inorganic materials 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 230000001394 metastastic effect Effects 0.000 description 2
- 238000005497 microtitration Methods 0.000 description 2
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 229960003104 ornithine Drugs 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 108010089193 pattern recognition receptors Proteins 0.000 description 2
- 102000007863 pattern recognition receptors Human genes 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 210000002307 prostate Anatomy 0.000 description 2
- 239000013558 reference substance Substances 0.000 description 2
- 208000016691 refractory malignant neoplasm Diseases 0.000 description 2
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- GMKMEZVLHJARHF-UHFFFAOYSA-N (2R,6R)-form-2.6-Diaminoheptanedioic acid Natural products OC(=O)C(N)CCCC(N)C(O)=O GMKMEZVLHJARHF-UHFFFAOYSA-N 0.000 description 1
- OGUSQFGGNLZTKG-BCLLBKCYSA-N (2s)-2,5-diamino-5-oxopentanoic acid;(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal Chemical compound OC(=O)[C@@H](N)CCC(N)=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O OGUSQFGGNLZTKG-BCLLBKCYSA-N 0.000 description 1
- NMDDZEVVQDPECF-LURJTMIESA-N (2s)-2,7-diaminoheptanoic acid Chemical compound NCCCCC[C@H](N)C(O)=O NMDDZEVVQDPECF-LURJTMIESA-N 0.000 description 1
- IYKLZBIWFXPUCS-VIFPVBQESA-N (2s)-2-(naphthalen-1-ylamino)propanoic acid Chemical compound C1=CC=C2C(N[C@@H](C)C(O)=O)=CC=CC2=C1 IYKLZBIWFXPUCS-VIFPVBQESA-N 0.000 description 1
- RWLSBXBFZHDHHX-VIFPVBQESA-N (2s)-2-(naphthalen-2-ylamino)propanoic acid Chemical compound C1=CC=CC2=CC(N[C@@H](C)C(O)=O)=CC=C21 RWLSBXBFZHDHHX-VIFPVBQESA-N 0.000 description 1
- FYMNTAQFDTZISY-QMMMGPOBSA-N (2s)-2-amino-3-[4-(diaminomethylideneamino)phenyl]propanoic acid Chemical compound OC(=O)[C@@H](N)CC1=CC=C(N=C(N)N)C=C1 FYMNTAQFDTZISY-QMMMGPOBSA-N 0.000 description 1
- VHDOAFBNVURINJ-QMMMGPOBSA-N (2s)-5-amino-2-(dimethylamino)-2-methylpentanoic acid Chemical compound CN(C)[C@](C)(C(O)=O)CCCN VHDOAFBNVURINJ-QMMMGPOBSA-N 0.000 description 1
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 description 1
- BLCJBICVQSYOIF-UHFFFAOYSA-N 2,2-diaminobutanoic acid Chemical compound CCC(N)(N)C(O)=O BLCJBICVQSYOIF-UHFFFAOYSA-N 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- AWLMDHXPKGLEQT-UHFFFAOYSA-N 4-amino-1-carbamimidoylpiperidine-4-carboxylic acid Chemical compound NC(=N)N1CCC(N)(C(O)=O)CC1 AWLMDHXPKGLEQT-UHFFFAOYSA-N 0.000 description 1
- KHABBYNLBYZCKP-UHFFFAOYSA-N 4-aminopiperidin-1-ium-4-carboxylate Chemical compound OC(=O)C1(N)CCNCC1 KHABBYNLBYZCKP-UHFFFAOYSA-N 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical class OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical group CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 1
- 101800002011 Amphipathic peptide Proteins 0.000 description 1
- 102000011021 Apoptotic Protease-Activating Factor 1 Human genes 0.000 description 1
- 108010062544 Apoptotic Protease-Activating Factor 1 Proteins 0.000 description 1
- 206010003571 Astrocytoma Diseases 0.000 description 1
- 102100029822 B- and T-lymphocyte attenuator Human genes 0.000 description 1
- 208000028564 B-cell non-Hodgkin lymphoma Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102100027207 CD27 antigen Human genes 0.000 description 1
- 101100463133 Caenorhabditis elegans pdl-1 gene Proteins 0.000 description 1
- 102100029968 Calreticulin Human genes 0.000 description 1
- 108090000549 Calreticulin Proteins 0.000 description 1
- 102000005600 Cathepsins Human genes 0.000 description 1
- 108010084457 Cathepsins Proteins 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- 108010002156 Depsipeptides Proteins 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 239000001263 FEMA 3042 Substances 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102100034458 Hepatitis A virus cellular receptor 2 Human genes 0.000 description 1
- 101710083479 Hepatitis A virus cellular receptor 2 homolog Proteins 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 208000017604 Hodgkin disease Diseases 0.000 description 1
- 208000021519 Hodgkin lymphoma Diseases 0.000 description 1
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 1
- 101000864344 Homo sapiens B- and T-lymphocyte attenuator Proteins 0.000 description 1
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 1
- 101001027128 Homo sapiens Fibronectin Proteins 0.000 description 1
- 101001098175 Homo sapiens P2X purinoceptor 7 Proteins 0.000 description 1
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 1
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 1
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 description 1
- 101000666896 Homo sapiens V-type immunoglobulin domain-containing suppressor of T-cell activation Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000002698 KIR Receptors Human genes 0.000 description 1
- 108010043610 KIR Receptors Proteins 0.000 description 1
- OGNSCSPNOLGXSM-UHFFFAOYSA-N L-2,4-diaminobutyric acid group Chemical group NC(C(=O)O)CCN OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 1
- QUOGESRFPZDMMT-UHFFFAOYSA-N L-Homoarginine Natural products OC(=O)C(N)CCCCNC(N)=N QUOGESRFPZDMMT-UHFFFAOYSA-N 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- QUOGESRFPZDMMT-YFKPBYRVSA-N L-homoarginine Chemical compound OC(=O)[C@@H](N)CCCCNC(N)=N QUOGESRFPZDMMT-YFKPBYRVSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 102000017578 LAG3 Human genes 0.000 description 1
- 101800004361 Lactoferricin-B Proteins 0.000 description 1
- 101150030213 Lag3 gene Proteins 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
- 208000025205 Mantle-Cell Lymphoma Diseases 0.000 description 1
- 206010027480 Metastatic malignant melanoma Diseases 0.000 description 1
- 108020005196 Mitochondrial DNA Proteins 0.000 description 1
- 102000006404 Mitochondrial Proteins Human genes 0.000 description 1
- 108010058682 Mitochondrial Proteins Proteins 0.000 description 1
- 238000007126 N-alkylation reaction Methods 0.000 description 1
- 108010049175 N-substituted Glycines Proteins 0.000 description 1
- 108010057466 NF-kappa B Proteins 0.000 description 1
- 102000003945 NF-kappa B Human genes 0.000 description 1
- 101100281510 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) met-6 gene Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102100037602 P2X purinoceptor 7 Human genes 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 description 1
- PCNDJXKNXGMECE-UHFFFAOYSA-N Phenazine Natural products C1=CC=CC2=NC3=CC=CC=C3N=C21 PCNDJXKNXGMECE-UHFFFAOYSA-N 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 231100000991 Reactive Oxygen Species (ROS) Photosafety Assay Toxicity 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 229940126547 T-cell immunoglobulin mucin-3 Drugs 0.000 description 1
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 1
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 1
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 description 1
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 1
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 description 1
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 1
- 102100038282 V-type immunoglobulin domain-containing suppressor of T-cell activation Human genes 0.000 description 1
- 101100514842 Xenopus laevis mtus1 gene Proteins 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 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
- 210000005006 adaptive immune system Anatomy 0.000 description 1
- 230000002730 additional effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 230000001270 agonistic effect Effects 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 125000003368 amide group Chemical class 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000005809 anti-tumor immunity Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- HOQPTLCRWVZIQZ-UHFFFAOYSA-H bis[[2-(5-hydroxy-4,7-dioxo-1,3,2$l^{2}-dioxaplumbepan-5-yl)acetyl]oxy]lead Chemical compound [Pb+2].[Pb+2].[Pb+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HOQPTLCRWVZIQZ-UHFFFAOYSA-H 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 229910000085 borane Inorganic materials 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 238000002619 cancer immunotherapy Methods 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000003570 cell viability assay Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 230000014564 chemokine production Effects 0.000 description 1
- 230000035605 chemotaxis Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 230000010428 chromatin condensation Effects 0.000 description 1
- 238000011260 co-administration Methods 0.000 description 1
- 201000010989 colorectal carcinoma Diseases 0.000 description 1
- 238000011284 combination treatment Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- MCBBFDNTCVNDCE-UHFFFAOYSA-N cyclopenta-1,3-diene cyclopenta-2,4-diene-1-carbonyl cyanide iron(2+) Chemical compound [Fe++].c1cc[cH-]c1.O=C(C#N)[c-]1cccc1 MCBBFDNTCVNDCE-UHFFFAOYSA-N 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 239000002254 cytotoxic agent Substances 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 230000004076 epigenetic alteration Effects 0.000 description 1
- AEOCXXJPGCBFJA-UHFFFAOYSA-N ethionamide Chemical compound CCC1=CC(C(N)=S)=CC=N1 AEOCXXJPGCBFJA-UHFFFAOYSA-N 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 210000001723 extracellular space Anatomy 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- IJJVMEJXYNJXOJ-UHFFFAOYSA-N fluquinconazole Chemical compound C=1C=C(Cl)C=C(Cl)C=1N1C(=O)C2=CC(F)=CC=C2N=C1N1C=NC=N1 IJJVMEJXYNJXOJ-UHFFFAOYSA-N 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000004077 genetic alteration Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000009931 harmful effect Effects 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 230000003862 health status Effects 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 150000004678 hydrides Chemical class 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000002390 hyperplastic effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000008629 immune suppression Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000002434 immunopotentiative effect Effects 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000001524 infective effect Effects 0.000 description 1
- 108091008042 inhibitory receptors Proteins 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 210000005007 innate immune system Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000005061 intracellular organelle Anatomy 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- CFFMZOZGXDAXHP-HOKBLYKWSA-N lactoferricin Chemical compound C([C@H](NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CSSC[C@@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N2CCC[C@H]2C(=O)N[C@@H](CO)C(=O)N[C@H](C(N[C@H](C(=O)N1)[C@@H](C)O)=O)[C@@H](C)CC)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CFFMZOZGXDAXHP-HOKBLYKWSA-N 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000012280 lithium aluminium hydride Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- GMKMEZVLHJARHF-SYDPRGILSA-N meso-2,6-diaminopimelic acid Chemical compound [O-]C(=O)[C@@H]([NH3+])CCC[C@@H]([NH3+])C([O-])=O GMKMEZVLHJARHF-SYDPRGILSA-N 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 208000021039 metastatic melanoma Diseases 0.000 description 1
- WDWDWGRYHDPSDS-UHFFFAOYSA-N methanimine Chemical compound N=C WDWDWGRYHDPSDS-UHFFFAOYSA-N 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 210000000110 microvilli Anatomy 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000001565 modulated differential scanning calorimetry Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000004264 monolayer culture Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 230000008450 motivation Effects 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 210000004985 myeloid-derived suppressor cell Anatomy 0.000 description 1
- 239000007922 nasal spray Substances 0.000 description 1
- 230000035407 negative regulation of cell proliferation Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 229910052762 osmium Inorganic materials 0.000 description 1
- SYQBFIAQOQZEGI-UHFFFAOYSA-N osmium atom Chemical compound [Os] SYQBFIAQOQZEGI-UHFFFAOYSA-N 0.000 description 1
- 201000003733 ovarian melanoma Diseases 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 229940124531 pharmaceutical excipient Drugs 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- UEZVMMHDMIWARA-UHFFFAOYSA-M phosphonate Chemical compound [O-]P(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-M 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920000768 polyamine Polymers 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920000232 polyglycine polymer Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000006950 reactive oxygen species formation Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 208000015347 renal cell adenocarcinoma Diseases 0.000 description 1
- 230000008521 reorganization Effects 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 125000005420 sulfonamido group Chemical group S(=O)(=O)(N*)* 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 235000015523 tannic acid Nutrition 0.000 description 1
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 1
- 229940033123 tannic acid Drugs 0.000 description 1
- 229920002258 tannic acid Polymers 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 150000007970 thio esters Chemical class 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 229950007217 tremelimumab Drugs 0.000 description 1
- 230000002476 tumorcidal effect Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 208000010576 undifferentiated carcinoma Diseases 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 231100000747 viability assay Toxicity 0.000 description 1
- 238000003026 viability measurement method Methods 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- A61K38/1729—Cationic antimicrobial peptides, e.g. defensins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
-
- 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
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to peptides or peptide like molecules and particularly to combined preparations of such peptides with a further agent, and their uses in therapy, in particular as anti-tumour agents.
- tumours are cancer cells circulating in the blood or lymph systems. Elimination of a tumour or a reduction in its size or reducing the number of cancer cells circulating in the blood or lymph systems may be beneficial in a variety of ways; reducing pain or discomfort, preventing metastasis, facilitating operative intervention, prolonging life.
- tumour-specific antigens typically combined with an adjuvant (a substance which is known to cause or enhance an immune response) to the subject.
- adjuvant a substance which is known to cause or enhance an immune response
- Cancer Vaccine is a term used to describe therapeutic agents which are designed to stimulate the patient's immune system against tumour antigens and lead to an attack on tumour cells and improved patient outcome.
- cancer vaccines are generally intended to generate or enhance an immune response against an existing cancer, rather than to prevent disease.
- a cancer or tumour vaccine may not require administration of a tumour antigen, the administered product may utilise tumour antigens already present in the body as a result of tumour development and serve to modify the immune response to the existing tumour associated antigens (TAAs).
- TAAs tumour associated antigens
- T cells have a key role in the immune response, which is initiated through antigen recognition by the T cell receptor (TCR), and they coordinate a balance between co-stimulatory and inhibitory signals known as immune checkpoints (Pardoll, Nature 2012, vol. 12, 252-264). Inhibitory signals suppress the immune system which is important for maintenance of self-tolerance and to protect tissues from damage when the immune system is responding to pathogenic infection. However, immune suppression reduces what could otherwise be a helpful response by the body to the development of tumours.
- cytokines other stimulatory molecules such as CpG (stimulating dendritic cells), Toll-like receptor ligands and other molecular adjuvants enhance the immune response.
- Co-stimulatory interactions involving T cells directly can be enhanced using agonistic antibodies to receptors including OX40, CD28, CD27 and CD137. These are all push-type approaches to cancer immunotherapy.
- Complementary ‘pull’ therapies may block or deplete inhibitory cells or molecules and include the use of antagonistic antibodies against what are known as immune checkpoints.
- Immune checkpoints include cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) and antibodies against these are known in the art; ipilimumab was the first FDA-approved anti-immune checkpoint antibody licensed for the treatment of metastatic melanoma and this blocks cytotoxic T-lymphocyte antigen 4 (CTLA-4) (Naidoo et al. British Journal of Cancer (2014) 111, 2214-2219).
- Cytotoxic T-lymphocyte-associated protein 4 also known as CD152, is a member of the immunoglobulin superfamily, which is expressed on the surface of helper T cells and transmits an inhibitory signal to T cells. Its genomic sequence is known, NCBI Reference Sequence: NG_011502.1, as is its protein sequence NCBI Reference Sequence: NP_005205.2.
- the present inventors have established that some peptides known to lyse tumour cells through disturbing and permeabilizing the cell membrane, are also highly effective at attacking organelles such as mitochondria and lysosomes and can cause lysis thereof. This may be achieved at low concentrations which do not cause direct lysis of the cell membranes, although loss of cell membrane integrity is seen eventually even on administration of low doses. At higher doses, these molecules can cause lysis of the cell membrane and then of the membranes of organelles.
- the peptides of interest are a sub-set of the group of peptides commonly known as Cationic antimicrobial peptides (CAPs). These are positively charged amphipathic peptides and peptides of this type are found in many species and form part of the innate immune system.
- the CAP Lactoferricin (LfcinB) is a 25 amino acid peptide which has been shown to have an effect on mitochondria (Eliasen et al. Int. J. Cancer (2006) 119, 493-450). It has now surprisingly been found that the much smaller peptide LTX-315, a 9 amino acid peptide (of the type described in WO 2010/060497), also targets the mitochondria.
- DAMPs Denssion-associated molecular pattern molecules
- ATP ATP
- Cytochrome C mitochondrial CpG DNA sequences
- mitochondrial formyl peptides mitochondrial formyl peptides
- cathepsins from lysosomes
- HMGB1 from the nucleus
- Lysis of organelles can also result in release of additional tumour-specific antigens (TAAs).
- TAAs tumour-specific antigens
- the present invention provides:
- a compound preferably a peptide, having the following characteristics:
- the combination therapy proposed herein may, in certain advantageous embodiments, provide a synergistic effect.
- Such surprising synergistic effects have been seen, for example, when using an anti-CTLA-4 antibody (Example 11).
- amino acid containing molecules defined above are conveniently referred to herein as the “peptidic compound of the invention”, which expression includes all of the peptides and peptidomimetics disclosed herein.
- the cationic amino acids which may be the same or different, are preferably lysine or arginine but may be histidine or any non-genetically coded amino acid carrying a positive charge at pH 7.0.
- Suitable non-genetically coded cationic amino acids include analogues of lysine, arginine and histidine such as homolysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid and homoarginine as well as trimethylysine and trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4-amino -1-carbamimidoylpiperidine-4-carboxylic acid and 4-guanidinophenylalanine.
- Non-genetically coded amino acids include modified derivatives of genetically coded amino acids and naturally occurring amino acids other than the 20 standard amino acids of the genetic code.
- a D amino acid while not strictly genetically coded, is not considered to be a “non-genetically coded amino acid”, which should be structurally, not just stereospecifically, different from the 20 genetically coded L amino acids.
- the molecules of the invention may have some or all of the amino acids present in the D form, preferably however all amino acids are in the L form.
- the lipophilic amino acids i.e. amino acids with a lipophilic R group
- the lipophilic amino acids which may be the same or different, all possess an R group with at least 7, preferably at least 8 or 9, more preferably at least 10 non-hydrogen atoms.
- An amino acid with a lipophilic R group is referred to herein as a lipophilic amino acid.
- the lipophilic R group has at least one, preferably two cyclic groups, which may be fused or linked.
- the lipophilic R group may contain hetero atoms such as O, N or S but typically there is no more than one heteroatom, preferably it is nitrogen.
- This R group will preferably have no more than 2 polar groups, more preferably none or one, most preferably none.
- Tryptophan is a preferred lipophilic amino acid and the molecules preferably comprise 1 to 3, more preferably 2 or 3, most preferably 3 tryptophan residues. Further genetically coded lipophilic amino acids which may be incorporated are phenylalanine and tyrosine.
- one of the lipophilic amino acids is a non-genetically coded amino acid.
- the molecule consists of 3 genetically coded lipophilic amino acids, 5 genetically coded cationic amino acids and 1 non-genetically coded lipophilic amino acid.
- the R group of that amino acid preferably contains no more than 35 non-hydrogen atoms, more preferably no more than 30, most preferably no more than 25 non-hydrogen atoms.
- Preferred non-genetically coded amino acids include: 2-amino-3-(biphenyl-4-yl)propanoic acid (biphenylalanine), 2-amino-3,3-diphenylpropanoic acid (diphenylalanine), 2-amino-3-(anthracen-9-yl)propanoic acid, 2-amino-3-(naphthalen-2-yl)propanoic acid, 2-amino-3-(naphthalen-1-yl)propanoic acid, 2-amino-3-[1,1′:4′,1′′-terphenyl -4-yl]-propionic acid, 2-amino-3-(2,5,7-tri-tert-butyl-1H-indol-3-yl)propanoic acid, 2-amino-3-[1,1′:3′,1′′-terphenyl-4-yl]-propionic acid, 2-amino-3-[1,1′:2′,1′′-
- the peptidic compounds of the invention have one of formulae I to V listed below, in which C represents a cationic amino acid as defined above and L represents a lipophilic amino acid as defined above.
- the amino acids being covalently linked, preferably by peptide bonds resulting in a true peptide or by other linkages resulting in a peptidomimetic, peptides being preferred.
- the free amino or carboxy terminals of these molecules may be modified, the carboxy terminus is preferably modified to remove the negative charge, most preferably the carboxy terminus is amidated, this amide group may be substituted.
- a peptidomimetic is typically characterised by retaining the polarity, three dimensional size and functionality (bioactivity) of its peptide equivalent but wherein the peptide bonds have been replaced, often by more stable linkages.
- stable is meant more resistant to enzymatic degradation by hydrolytic enzymes.
- the bond which replaces the amide bond conserves many of the properties of the amide bond, e.g. conformation, steric bulk, electrostatic character, possibility for hydrogen bonding etc. Chapter 14 of “Drug Design and Development”, Krogsgaard, Larsen, Liljefors and Madsen (Eds) 1996, Horwood Acad.
- Suitable amide bond surrogates include the following groups: N-alkylation (Schmidt, R. et al., Int. J. Peptide Protein Res., 1995, 46,47), retro-inverse amide (Chorev, M and Goodman, M., Acc. Chem.
- the peptidomimetic compounds may have 9 identifiable sub-units which are approximately equivalent in size and function to the 9 cationic and lipophilic amino acids.
- amino acid may thus conveniently be used herein to refer to the equivalent sub-units of a peptidomimetic compound.
- peptidomimetics may have groups equivalent to the R groups of amino acids and discussion herein of suitable R groups and of N and C terminal modifying groups applies, mutatis mutandis , to peptidomimetic compounds.
- peptidomimetics may involve the replacement of larger structural moieties with di- or tripeptidomimetic structures and in this case, mimetic moieties involving the peptide bond, such as azole-derived mimetics may be used as dipeptide replacements.
- mimetic moieties involving the peptide bond such as azole-derived mimetics may be used as dipeptide replacements.
- Peptidomimetics and thus peptidomimetic backbones wherein just the amide bonds have been replaced as discussed above are, however, preferred.
- Suitable peptidomimetics include reduced peptides where the amide bond has been reduced to a methylene amine by treatment with a reducing agent e.g. borane or a hydride reagent such as lithium aluminium-hydride. Such a reduction has the added advantage of increasing the overall cationicity of the molecule.
- a reducing agent e.g. borane or a hydride reagent such as lithium aluminium-hydride.
- peptidomimetics include peptoids formed, for example, by the stepwise synthesis of amide-functionalised polyglycines.
- Some peptidomimetic backbones will be readily available from their peptide precursors, such as peptides which have been permethylated, suitable methods are described by Ostresh, J. M. et al. in Proc. Natl. Acad. Sci. USA (1994) 91, 11138-11142. Strongly basic conditions will favour N-methylation over 0-methylation and result in methylation of some or all of the nitrogen atoms in the peptide bonds and the N-terminal nitrogen.
- Preferred peptidomimetic backbones include polyesters, polyamines and derivatives thereof as well as substituted alkanes and alkenes.
- the peptidomimetics will preferably have N and C termini which may be modified as discussed herein.
- amino acids as are N-substituted glycines.
- the peptidic compounds of the invention include beta peptides and depsipeptides.
- the peptidic compounds of the invention incorporate at least one, and preferably one, non-genetically coded amino acid.
- L′ preferred compounds are represented by the following formulae:
- Particularly preferred are compounds (preferably peptides) of formula I and II, and of these, compounds (preferably peptides) of formula I′′ are especially preferred.
- Compound LTX-315 is most preferred.
- the molecules are preferably peptides and preferably have a modified, particularly an amidated, C-terminus.
- Amidated peptides may themselves be in salt form and acetate forms are preferred.
- Suitable physiologically acceptable salts are well known in the art and include salts of inorganic or organic acids, and include trifluoracetate as well as acetate and salts formed with HCl.
- the peptidic compounds described herein are amphipathic in nature, their 2° structure, which may or may not tend towards the formation of an ⁇ -helix, provides an amphipathic molecule in physiological conditions.
- the combination therapies defined herein are for the treatment of tumours, in particular solid tumours and thus for the treatment of cancer.
- the peptidic compounds of the invention destabilise and/or permeabilise the membranes of tumour cell organelles, e.g. mitochondria, the nucleus or lysomome, in particular the mitochondria.
- tumour cell organelles e.g. mitochondria, the nucleus or lysomome, in particular the mitochondria.
- stabilising is meant a perturbation of the normal lipid bi-layer configuration including but not limited to membrane thinning, increased membrane permeability to water, ions or metabolites etc.
- Immune checkpoints are known in the art (Naidoo et al. and Pardoll et al supra) and the term is well understood in the context of cancer therapy. The most well-known are CTLA-4, PD-1 and its ligand PDL-1. Others include TIM-3, KIR, LAG-3, VISTA, BTLA. Inhibitors of immune checkpoints inhibit their normal immunosuppressive function, for example by down regulation of expression of the checkpoint molecules or by binding thereto and blocking normal receptor/ligand interactions. As the immune checkpoints put brakes on the immune system response to an antigen, so an inhibitor thereof reduces this immunosuppressive effect and enhances the immune response.
- anti-immune checkpoint antibodies such as anti-CTLA-4 antibodies (e.g. ipilimumab and tremelimumab), anti-PD-1 antibodies (e.g. nivolumab, pembrolizumab, formerly known as lambrolizumab, pidilizumab and RG7446 (Roche)) and anti-PDL-1 antibodies (e.g. BMS-936559 (Bristol-Myers Squibb), MPDL3280A (Genentech), MSB0010718C (EMD-Serono) and MED14736 (AstraZeneca)).
- anti-CTLA-4 antibodies e.g. ipilimumab and tremelimumab
- anti-PD-1 antibodies e.g. nivolumab, pembrolizumab, formerly known as lambrolizumab, pidilizumab and RG7446 (Roche)
- anti-PDL-1 antibodies e.g
- ICIs are antibodies that bind to a specific immune checkpoint molecule, whether that immune checkpoint molecule is itself a receptor or a ligand therefor.
- Preferred immune checkpoint molecules are listed above.
- Receptors which form part of an immune checkpoint are typically found on the surface of T cells.
- Inhibitors may be selected from proteins, peptides, peptidomimetics, peptoids, antibodies, antibody fragments, small inorganic molecules, small non-nucleic acid organic molecules or nucleic acids such as anti-sense nucleic acids, small interfering RNA (siRNA) molecules or oligonucleotides.
- the inhibitor may act to down regulate expression of an immune checkpoint molecule.
- the inhibitor may for example be a modified version of the natural ligand (e.g. for CTLA-4, CD80 (B7-1) and CD86 (B7-2)), such as a truncated version of one of the ligands. They may be naturally occurring, recombinant or synthetic.
- the inhibitor is either an antibody, a modified ligand or an antisense nucleic acid molecule such as siRNA designed to inhibit a particular immune checkpoint molecule.
- siRNA is capable of preventing or prevents the translation of the immune checkpoint, thus preventing the expression of the protein. Given that the genomic sequence of many immune checkpoints are known, the skilled person would be able to use routine methods to design suitable inhibitory antisense nucleic acid molecules.
- Inhibitors of CTLA-4, PD-1 and PD-L1 are preferred and antibodies thereto are particularly preferred. Such antibodies are known as immune checkpoint-blocking antibodies and can invigorate the antitumour immune response.
- the invention provides methods of treating a tumour and a method of treating tumour cells.
- the combination therapy should be effective to kill all or a proportion of the target tumour cells or to prevent or reduce their rate of multiplication, or to inhibit metastasis or otherwise to lessen the harmful effect of the tumour on the patient.
- the clinician or patient should observe improvement in one or more of the parameters or symptoms associated with the tumour. Administration may also be prophylactic and this is encompassed by the term “treatment”.
- the patient will typically be a human patient but non-human animals, such as domestic or livestock animals may also be treated.
- Cancer targets include melanomas, sarcomas, lymphomas, leukemias, neuroblastomas and glioblastomas (e.g. from the brain), carcinomas and adenocarcinomas. Cancers of the breast, colon, bladder, kidney, liver (e.g. hepatocellular carcinoma), lung, ovary, pancreas, prostate and skin) are preferred targets. Head and neck cancers are also preferred targets. Melanomas, sarcomas and lymphomas are preferred targets. Tumours for treatment are typically solid tumours and may be metastatic lesions that are accessible for transdermal injection.
- the peptides may be synthesised in any convenient way. Generally the reactive groups present (for example amino, thiol and/or carboxyl) will be protected during overall synthesis. The final step in the synthesis will thus be the deprotection of a protected derivative of the invention. In building up the peptide, one can in principle start either at the C-terminal or the N-terminal although the C-terminal starting procedure is preferred. Methods of peptide synthesis are well known in the art but for the present invention it may be particularly convenient to carry out the synthesis on a solid phase support, such supports being well known in the art. A wide choice of protecting groups for amino acids which are used in the synthesis of peptides are known.
- peptidic compounds including salts, esters or amides thereof
- pharmaceutical formulations i.e. incorporating one or more pharmaceutically acceptable diluents, carriers or excipients.
- the active agents according to the invention may be presented, for example, in a form suitable for oral, topical, nasal, parenteral, intravenal, intratumoral, rectal or regional (e.g. isolated limb perfusion) administration.
- administration is typically by a parenteral route, preferably by injection subcutaneously, intramuscularly, intracapsularly, intraspinally. intraperitoneally. intratumourally. transdermally or intravenously.
- administration is preferably intratumoural.
- Particularly preferred are intratumoural injections of the peptidic compound of the invention once a day for several consecutive days, e.g. 2, 3, 4, 5, 6 or 7 days, preferably on 2-4 consecutive days or at 2, 3, 4, 5, 6 or 7 daily intervals, e.g. 2-4 times at 5, 6, 7, 8 or 9 daily intervals.
- administration is preferably intravenous or intralesional.
- the peptidic compound may be administered with or after the immune checkpoint inhibitor, as has been shown to be effective with respect to anti-PD1 and anti-CTLA4 antibodies.
- the peptidic compound may be administered with or before the immune checkpoint inhibitor, as has been shown to be effective with respect to anti-CTLA4 and anti-PD-L1 antibodies.
- the immune system is preferably stimulated over time.
- administrations of the peptidic compound and/or the checkpoint inhibitor may be carried out over the course of eight months, preferably four months, more preferably two months; in such regimens administration is preferably weekly.
- the active compounds defined herein may be presented in the conventional pharmacological forms of administration, such as tablets, coated tablets, nasal sprays, solutions, emulsions, liposomes, powders, capsules or sustained release forms.
- Conventional pharmaceutical excipients as well as the usual methods of production may be employed for the preparation of these forms. Simple solutions are preferred.
- Organ specific carrier systems may also be used.
- Injection solutions may, for example, be produced in the conventional manner, such as by the addition of preservation agents, such as p-hydroxybenzoates, or stabilizers, such as EDTA. The solutions are then filled into injection vials or ampoules.
- preservation agents such as p-hydroxybenzoates, or stabilizers, such as EDTA.
- stabilizers such as EDTA.
- Preferred formulations are those in which the molecules are in saline. Such formulations being suitable for use in preferred methods of administration, especially local administration, i.e. intratumoural, e.g. by injection.
- dosage units containing the peptidic molecules preferably contain 0.1-10 mg, for example 1-5 mg.
- the formulation may additionally comprise further active ingredients, including other cytotoxic agents such as other anti-tumour peptides.
- Other active ingredients may include different types of cytokines e.g. IFN- ⁇ , TNF, CSF and growth factors, immunomodulators, chemotherapeutics e.g. cisplatin or antibodies or cancer vaccines.
- Also provided according to the present invention is the use of a peptidic compound as defined above in the manufacture of a medicament for the treatment of a tumour, wherein said peptidic compound is co-administered with a checkpoint inhibitor as defined above.
- the medicament is for the treatment of multidrug resistant (MDR) tumours.
- MDR multidrug resistant
- a pharmaceutical pack or composition comprising:
- the components can be for administration separately.
- the pharmaceutical pack can of course also comprise instructions for administration.
- the pack and composition are for use in the treatment of a tumour.
- Also provided according to the present invention is a method of treatment of a tumour, comprising the step of administering a peptidic compound as defined herein and a checkpoint inhibitor as described herein, together in pharmaceutically effective amounts, to a patient in need of same.
- the peptidic compounds of the invention are able to destabilise mitochondrial membranes and cause release of DAMPs and antigenic material. This can have a powerful positive effect on the immune system's response to cancer cells.
- the immune response is of primary importance, e.g. to treat unidentified secondary tumours, to prevent formation of metastatic tumours, when surgery or other direct intervention is not possible.
- Different cancers are more or less immunogenic and therefore in some scenarios boosting the immune response to cancer is vital.
- the present invention provides a peptidic compound as defined herein for use in the destabilisation of a mitochondrial membrane, wherein said use is in the treatment of a tumour.
- a peptidic compound as defined herein for use in the destabilisation of a mitochondrial membrane, wherein said use is in the treatment of a tumour.
- This can be regarded as an immunotherapeutic use or treatment and the tumour will typically be cancerous.
- Preferred features and embodiments discussed elsewhere in relation to the combination therapies apply, mutatis mutandis , to this aspect.
- the invention provides a composition comprising or consisting of a peptidic compound of the invention and an immunotherapeutic agent.
- the invention provides a method of treating tumours in a patient, said method comprising administration of an effective amount of a peptidic compound of the invention and simultaneous or sequential administration of an effective amount of an immunotherapeutic agent.
- peptidic compound of the invention and an immunotherapeutic agent for use in the treatment of tumours.
- immunotherapeutic agent an agent which modulates the immune response.
- the immunotherapeutic agent enhances the immune response against one or more tumor antigens, for example by suppressing (preferably selectively) Treg cells and or MDSCs and/or by blocking cytotoxic T lymphocyte antigen-4 (CTLA-4), an inhibitory receptor expressed on T cells.
- CTLA-4 cytotoxic T lymphocyte antigen-4
- the immunotherapeutic agent is preferably an anti-CTLA-4 agent.
- the immunotherapeutic agent e.g. the immune checkpoint inhibitor such as an anti-CTLA-4 agent
- it is administered prior to the peptidic compound of the invention, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to the peptidic compound of the invention.
- the first peptidic administration is 1 or 2 days after the last ICI administration.
- the anti-CTLA-4 agent ipilimumab is typically administered once every three weeks for a total of four doses.
- the anti-PD1 agent nivolumab is typically administered every second week, and the anti-PD1 agent pembrolizumab, formerly known as lambrolizumab, is typically administered every third week.
- Such dosage regimens are preferable.
- Anti-PD-L1 is preferably administered at the same time or after the peptidic compound.
- the peptidic compound is administered on the same day as the first dose of the immunotherapeutic agent, followed by weekly administrations for four to eight weeks, e.g. six weeks, as part of an induction phase.
- the peptidic compound may then be administered every second week thereafter as part of a maintenance phase.
- Co-administration may be simultaneous or sequential and by the same or different routes of administration, e.g. oral and/or parenteral, or i.p. for the ICI and intra-tumoural for the peptidic compound.
- the inventors have also surprisingly found that the treatment of a tumour with a peptidic compound of the invention in conjunction with an immunotherapeutic agent can induce an adaptive immunity against further tumours.
- the above methods, uses and products (compositions) may therefore optionally extend to the induction of an adaptive immunity against further tumours.
- the invention also provides a peptidic compound of the invention and an immunotherapeutic agent for use in the treatment of tumours in a patient and inducing adaptive immunity against tumour growth, development or establishment in said patient.
- a method of inducing adaptive immunity against tumour growth, development or establishment in a patient comprising administration of an effective amount of a peptidic compound of the invention and simultaneous or sequential administration of an effective amount of an immunotherapeutic agent.
- the invention provides a peptidic compound of the invention and an immunotherapeutic agent for use in inducing an adaptive immunity against tumour growth, development or establishment.
- a peptidic compound of the invention and an immunotherapeutic agent in the manufacture of a medicament for use as a vaccine against tumour growth, development or establishment.
- a product containing a peptidic compound of the invention and an immunotherapeutic agent as a combined preparation for separate, simultaneous or sequential use in inducing an adaptive immunity against tumour growth, development or establishment.
- the invention also provides a method of vaccinating a subject against tumour growth, development or establishment through administration of an effective amount of a peptidic compound of the invention and an immunotherapeutic agent to said patient.
- FIG. 1 is a graph showing the percentage of red blood cell death in a series of experiments to test peptide LTX-315 at varying concentrations.
- X-axis shows peptide concentration ( ⁇ g/ml).
- Y-axis shows % cell death;
- FIG. 2 shows tumour growth in mice re-inoculated with murine A20 B cell lymphoma cells compared with growth in the control animals from the initial study.
- Diamonds indicate controls from primary studies.
- Solid squares indicate re-inoculated mice;
- FIG. 3 shows tumour growth in individual mice re-inoculated with murine A20 B cell lymphoma cells having been initially treated with LTX-315.
- Squares indicate Mouse 1.
- Triangles (base at bottom) indicate Mouse 2.
- Triangles (base at top) indicate Mouse 3.
- Diamonds indicate Mouse 4;
- FIG. 4 shows tumour growth in mice re-inoculated with murine CT26WT colon carcinoma cells compared with growth in the control animals.
- Diamonds indicate controls from primary studies.
- Solid squares indicate re-inoculated mice;
- FIG. 5 shows tumour growth in individual mice re-inoculated with murine CT26WT colon carcinoma cells having been initially treated with LTX-315.
- Small squares indicate Mouse 1. Small triangles (base at bottom) indicate Mouse 2. Small triangles (base at top) indicate Mouse 3. Small diamonds indicate Mouse 4; Circles indicate Mouse 5. Large squares indicate Mouse 6. Large triangles (base at bottom) indicate Mouse 7. Large triangles (base at top) indicate Mouse 8. Large diamonds indicate Mouse 9;
- FIG. 6 shows growth of A20 B-cell lymphomas in irradiated mice that received splenocytes from donor mice showing complete tumour regression following treatment with LTX-315 (Group 1) or control mice (Group 2) that received splenocytes from na ⁇ ve donor mice.
- Squares indicate Group 1 (mice that received splenocytes from donors showing complete regression).
- Diamonds indicate Group 2 (mice that received splenocytes from naive donors);
- FIG. 7 shows anti-cancer effect of two different treatment regimes on solid murine A20 tumours (Groups 1 and 2) as compared to non-treated controls (Group 3).
- Inverted solid triangles (base at top) indicate Group 1 (treatment).
- Open squares indicate Group 2 (treatment +adjuvant).
- Open triangles (base at bottom) indicate Group 3 (control).
- Order of tumour size (mm 2 ) at Day 21 is (largest to smallest): Group 3, Group 1, Group 2.
- FIGS. 8 a - 8 b LTX-315 causes rapid cell death in human melanoma cells.
- FIG. 8 a shows IC 50 values at different incubation times.
- FIG. 8 b shows percentage cell viability at different concentrations of L TX-315.
- FIG. 9 LTX-315 internalizes and accumulates close to the mitochondria.
- A375 cells treated 30 minutes with 1.5 ⁇ M fluorescence-labeled LTX-315, and with labeled mitochondria and nucleus. The peptide was internalized and detected in close proximity to the mitochondria.
- FIG. 10 Internalization occurs only in lytic 9-mer compounds such as LTX-315 and not in the non-lytic mock peptide LTX-328.
- FIG. 11 LTX-315 treatment causes ultrastructural changes.
- A&D untreated control cells
- B&E cells treated with 3,5 ⁇ M
- C &F cells treated with 17 ⁇ M.
- FIG. 12 ROS generation in LTX-315 induced cell death.
- A375 cells were treated with LTX-315 at different concentrations for 15 minutes.
- carboxy-H2DCFDA was added to the samples and fluorescence was analyzed with a fluorescence plate reader. The experiment was conducted in duplicate, with bars representing mean fluorescence + ⁇ S. D.
- FIG. 13 Human melanoma cells treated with LTX-315 release cytochrome-C in the supernatant. Cytochrome-C release in the supernatant after LTX-315 treatment of A375 after designated time points (5, 15, 45 min) were determined by ELISA assay.
- FIG. 14 HMGB1 is released in the supernatant after LTX-315 treatment.
- A375 human melanoma cells were treated with 35 ⁇ M LTX-315 (top) or LTX-328 (bottom), and cell lysate (L) and supernatant (S) were analyzed with Western blot, and the LTX-315-treated cells showed a gradual translocation from the cell lysate to the cell supernatant.
- Control cells were treated with media alone, and showed no translocation after 60 minutes.
- FIG. 15 Extracellular ATP levels following LTX-315 treatment: A375 cells were treated with LTX-315 for 5 minutes at different concentrations or maintained under controlled conditions, and the supernatant was analyzed for the quantification of ATP secretion by luciferase bioluminescence. Quantitative data (mean + ⁇ S. D.) for one representative experiment are reported.
- FIG. 16 LTX-315 disintegrates the mitochondria membrane.
- TEM images of FIG. 16 a human A547 melanoma cells treated with LTX-315 (10 ⁇ g/ml) for 60 minutes compared to FIG. 16 b : control cells.
- FIGS. 17 a - 17 e Experimental setting in a MCA205 sarcoma model starting with LTX-315 first and boosting with either anti-programmed cell death protein 1 (PD-1) antibodies or anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibodies (a). Tumour regression is seen with LTX-315 combined with CTLA-4 Ab and with LTX-315 combined with PD-1 Ab ((b) and (d)).
- PD-1 anti-programmed cell death protein 1
- CTLA-4 anti-cytotoxic T-lymphocyte-associated protein 4
- FIGS. 18 a - 18 e Experimental setting in a MCA205 sarcoma model starting with either anti-PD-1 antibodies or anti-CTLA-4 antibodies and boosting with weekly LTX-315 (a). Tumour regression is seen with LTX-315 combined with CTLA-4 Ab and LTX-315 combined with PD-1 Ab ((b) and (d)).
- FIGS. 19 a - 19 e Experimental setting in a MCA205 sarcoma model starting with either anti-PD-1 antibodies or anti-CTLA-4 antibodies followed by iterative LTX-315 local inoculations (a). Tumour regression is seen with LTX-315 combined with CTLA-4 Ab and LTX-315 combined with PD-1 Ab ((b) and (d)).
- FIGS. 20 a - 20 k Adaptive immunity effects of the combination of anti-CTLA-4 antibodies with LTX-315 in a MCA205 sarcoma model.
- Tumour rejection took place in 3 out of 5 (60%) of the treated (right flank) tumours and 3 out of 5 (60%) of the untreated (left flank) tumours following administration of anti-CTLA-4 antibody in combination with LTX-315. Tumour rejection took place in 1 out of 5 (20%) of the untreated (left flank) tumours following administration of anti-CTLA-4 antibody alone.
- FIG. 21 Combined direct antitumour and adaptive immunity effects of the combination of anti-PD-L1 antibodies with LTX-315 in a EMT-6 murine mammary carcinoma model.
- Example 1 that LTX-315 is the most potent of the 5 tested compounds in an in vitro cytotoxic activity study against a panel of 37 human cancer cell lines.
- Example 2 that LTX-315 is the most potent of the 5 tested compounds in an in vitro cytotoxic activity study against a panel of 10 lymphoma cell lines.
- Example 3 that LTX 315 has a mean EC 50 value greater than 1200 ⁇ g/ml (833 pM) against human red blood cells.
- Example 4 that the anti-tumour activity of LTX-315 resulted in a complete tumour response in 3 of 7 treated mice for the Group receiving the optimal dose (Group 1) in an investigation into the effect of LTX-315 at different dose levels on a murine A20 B-cell lymphoma in mice.
- Example 5 that four different LTX-315 treatment regimes demonstrated a strong anti tumour effect against murine CT26WT (multidrug resistant) tumours.
- Example 6 that LTX-315 has a broad spectrum of activity against various multidrug resistant cancer cell lines and, significantly, a much weaker cytotoxic effect on normal human cells.
- Example 7 that complete tumour regression following initial treatment of solid murine tumours with LTX-315 resulted in a form of endogenous long-term protection against growth of the same tumours following re-inoculation.
- Example 8 that treatment with LTX-315 may confer long term protection against specific tumours by eliciting an immune response.
- Example 9 that an anti A20 cell immune response have been induced by the injection of the cocktail of LTX-315 and lysed A20 cells.
- Example 10 that treatment with LTX-315 induces hallmarks of immunogenic cell death by mitochondria distortion in human melanoma cells.
- Example 11 that treatment with LTX-315 in combination with an anti-CTLA-4 antibody caused a complete and long-lasting tumor regression in a high proportion of test subjects and induced an adaptive immune response.
- Anti-PD-1 antibody also showed an ability to act in combination with LTX-315 to inhibit tumour growth.
- Example 12 that treatment with LTX-315 in combination with an anti-PD-L1 antibody caused tumour regression in a high proportion of test subjects and induced an adaptive immune response.
- Test substances LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329 (see Table 1) provided in powder form.
- Triton X-100 was used as positive control, supplied by Oncodesign (Dijon, France) from Sigma (Saint Quentin Fallavier, France).
- Tumor cells were grown as adherent monolayers or as suspensions at 37° C. in a humidified atmosphere (5% CO 2 , 95% air).
- the culture medium was RPMI 1640 containing 2 mM L-glutamine (Lonza, Belgium) and supplemented with 10% fetal bovine serum (FBS, Lonza).
- FBS fetal bovine serum
- the adherent cells were detached from the culture flask by a 5-minute treatment with trypsin-versene (Lonza), diluted in Hanks' medium without calcium or magnesium (Lonza) and neutralized by addition of complete culture medium. Cells were counted in a hemocytometer and their viability was assessed by 0.25% trypan blue exclusion.
- Mycoplasma detection was performed using the MycoAlert (RTM) Mycoplasma Detection Kit (Lonza) in accordance with the manufacturers instructions. All tested cells were found to be negative for mycoplasma contamination.
- Tumor cells were plated in 96-well flat-bottom microtitration plates (Nunc, Dutscher, Brumath, France) and incubated at 37° C. for 24 hours before treatment in 190 ⁇ l of drug-free culture medium supplemented or not with 10% FBS for adherent or suspension growing cell lines, respectively.
- the adherent cell lines were washed once with 200 ⁇ l FBS-free culture medium before treatment.
- Tumor cells were incubated for 4 hours with 10 concentrations of compounds in 1 ⁇ 4 dilution step with a top dose of 400 ⁇ M (range 4 ⁇ 10 ⁇ 4 to 4 ⁇ 10 ⁇ 10 M), with 1% (final concentration) Triton X-100 as positive control and FBS-free culture medium as negative control.
- the cells (190 ⁇ l) were incubated in a 200 ⁇ l final volume of FBS-free culture medium containing test substances at 37° C. under 5% CO 2 .
- Dilutions of tested compound as well as distribution to plates containing cells were performed using a Sciclone ALH 3000 liquid handling system (Caliper Life Sciences S. A.). According to automate use, a single range of concentrations was tested whatever the cell lines to be tested. The range was not adapted for each cell line.
- MTS novel tetrazolium compound
- PMS phenazine methosulfate
- the dose response inhibition of proliferation was expressed as follows:
- I ⁇ C O ⁇ D d ⁇ rug - exposed ⁇ ⁇ wells O ⁇ D d ⁇ rug - free ⁇ ⁇ wells ⁇ 100
- the OD values are the mean of 4 experimental measurements.
- IC 50 drug concentration to obtain a 50% inhibition of cell proliferation.
- the dose-response curves were plotted using XLFit 3 (IDBS, United Kingdom).
- the IC 50 determination values were calculated using the XLFit 3 software from semi-log curves. Individual IC 50 determination values as well as mean and SD values were generated.
- Resistance index was calculated for each compound for each couple of sensitive and resistant cell lines. Individual resistance index was calculated when IC 50 values of both sensitive and corresponding resistant cell lines were determined within same experiment. In addition, resistance index was also calculated ratio of mean IC 50 values obtained during three independent experiments.
- All thirty seven human tumor cell lines tested were sensitive to LTX-302 compound with IC 50 values ranging from 4.83 ⁇ 0.96 ⁇ M to 20.09 ⁇ 4.07 ⁇ M for T-47D and Hep G2 cell lines, respectively.
- Mean IC 50 value for LTX-302 compound obtained on the 37 tumor cell lines was 12.05 ⁇ 4.27 ⁇ M with a median value of 11.70 ⁇ M.
- Mean IC 50 value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.
- LTX-302 compound Activity of LTX-302 compound seemed to be slightly decreased by acquired resistance towards doxorubicin as exhibited by the RI values of both HL-60/ADR and MCF-7/mdr cell lines (1.31 and 1.23 for HL-60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, activity of LTX-302 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a RI value of 0.33 for IGROV-1/CDDP cell line.
- Mean IC 50 value for LTX-313 compound obtained on the 37 tumor cell lines was 9.60 ⁇ 3.73 ⁇ M with a median value of 8.83 ⁇ M.
- Mean IC 50 value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.
- Activity of LTX-313 compound seemed not to be modified by acquired resistance towards doxorubicin as exhibited by the RI values of CCRF-CEM/VLB, HL-60/ADR and MCF-7/mdr cell lines (0.76, 1.16 and 1.24 for CCRF-CEM/VLB, HL-60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, activity of LTX-313 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a RI value of 0.49 for IGROV-1/CDDP cell line.
- All thirty seve human tumor cell lines tested were sensitive to LTX-315 compound with IC 50 values ranging from 1.18 ⁇ 0.25 ⁇ M to 7.16 ⁇ 0.99 ⁇ M for T-47D and SK-OV-3 cell lines, respectively.
- Mean IC 50 value for LTX-315 compound obtained on the 37 tumor cell lines was 3.63 ⁇ 1.45 ⁇ M with a median value of 3.27 ⁇ M.
- Mean IC 50 value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.
- LTX-315 compound Activity of LTX-315 compound seemed to be slightly decreased by acquired resistance towards doxorubicin as exhibited by the RI values of HL-60/ADR and MCF-7/mdr cell lines (1.45 and 1.12 for HL-60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, activity of LTX-315 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a RI value of 0.50 for IGROV-1/CDDP cell line.
- All thirty seven human tumor cell lines tested were sensitive to LTX-320 compound with IC 50 values ranging from 3.46 ⁇ 0.22 ⁇ M to 16.64 ⁇ 3.15 ⁇ M for T-47D and Hep G2 cell lines, respectively.
- Mean IC 50 value for LTX-320 compound obtained on the 37 tumor cell lines was 7.58 ⁇ 2.79 ⁇ M with a median value of 6.92 ⁇ M.
- Mean IC 50 value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.
- LTX-320 compound Activity of LTX-320 compound seemed not to be modified by acquired resistance towards doxorubicin as exhibited by the RI values of HL-60/ADR and MCF-7/mdr cell lines (0.90 and 1.19 for HL-60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, activity of LTX-320 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a RI value of 0.49 for IGROV-1/CDDP cell line.
- All thirty seven human tumor cell lines tested were sensitive to LTX-329 compound with IC 50 values ranging from 2.43 ⁇ 0.34 ⁇ M to 16.90 ⁇ 1.18 ⁇ M for T-47D and U-87 MG cell lines, respectively.
- Mean IC 50 value for LTX-329 compound obtained on the 37 tumor cell lines was 8.17 ⁇ 3.20 ⁇ M with a median value of 7.89 ⁇ M.
- Mean IC 50 value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.
- Activity of LTX-329 compound seemed not to be modified by acquired resistance towards doxorubicin as exhibited by the RI values of CCRF-CEM/VLB, HL-60/ADR and MCF-7/mdr cell lines (0.76, 0.80 and 1.07 for CCRF-CEM/VLB, HL-60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, activity of LTX-329 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a RI value of 0.46 for IGROV-1/CDDP cell line.
- T-47D breast cancer cell line is the most sensitive cell line whatever the LTX compound tested.
- Hematological cancer cell lines are the most sensitive histological type for all five compounds tested, liver and ovarian cancer cell lines being within the most resistant cell lines.
- LTX-315 compound is the most potent compound from the five compounds tested.
- LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329) exhibited cytolytic activity against 37 human cancer cell lines tested with IC 50 values in micromolar to ten micromolar range.
- LTX-315 compound is the most potent compound tested with IC 50 values between 1 and 5 micromolar on all 37 human cancer cell lines tested.
- Test substances LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329 (see Table 1) provided in powder form.
- Triton X-100 was used as positive control and supplied by Oncodesign (Dijon, France) from Sigma (Saint Quentin Fallavier, France).
- Tumor cells were grown as suspensions at 37° C. in a humidified atmosphere (5% CO 2 , 95% air). The culture medium for each cell line is described in Table 4 below. For experimental use, cells were counted in a hemocytometer and their viability was assessed by 0.25% trypan blue exclusion.
- Mycoplasma detection was performed using the MycoAlert (RTM) Mycoplasma Detection Kit (Lonza) in accordance with the manufacturers instructions. All tested cells were found to be negative for mycoplasma contamination.
- Tumor cells were plated in 96-well flat-bottom microtitration plates (Nunc, Dutscher, Brumath, France) and incubated at 37° C. for 24 hours before treatment in 190 ⁇ l of drug-free and FBS-free culture medium.
- Tumor cells were incubated for 4 hours with 10 concentrations of compounds in 1 ⁇ 4 dilution step with a top dose of 400 ⁇ M (range 4 ⁇ 10 ⁇ 4 to 4 ⁇ 10 ⁇ 10 M), with 1% (final concentration) Triton X-100 as positive control and FBS-free culture medium as negative control.
- the cells (190 ⁇ l) were incubated in a 200 ⁇ l final volume of FBS-free culture medium containing test substances at 37° C. under 5% CO 2 .
- Dilutions of tested compound as well as distribution to plates containing cells were performed using a Sciclone ALH 3000 liquid handling system (Caliper Life Sciences S. A.). According to automate use, a single range of concentrations was tested whatever the cell lines to be tested. The range was not adapted for each cell line.
- MTS novel tetrazolium compound
- PMS phenazine methosulfate
- Mean IC 50 value for LTX-302 compound obtained on 10 sensitive cell lines was 8.11 ⁇ 2.44 ⁇ M with a median value of 7.53 ⁇ M.
- Mean IC 50 value for LTX-313 compound obtained on 10 sensitive cell lines was 7.05 ⁇ 3.91 ⁇ M with a median value of 5.89 ⁇ M.
- Mean IC 50 value for LTX-315 compound obtained on 10 sensitive cell lines was 3.01 ⁇ 1.36 ⁇ M with a median value of 2.93 ⁇ M.
- Mean IC 50 value for LTX-320 compound obtained on 10 sensitive cell lines was 5.03 ⁇ 2.82 ⁇ M with a median value of 4.84 ⁇ M.
- Mean IC 50 value for LTX-329 compound obtained on 10 sensitive cell lines was 5.76 ⁇ 2.27 ⁇ M with a median value of 5.72 ⁇ M.
- KARPAS-299 and Raji cell lines are the most resistant cell lines whatever the LTX compound tested.
- Hs 445, Ramos and U-937 cell lines are the most sensitive cell lines whatever the LTX compound tested.
- LTX-315 compound is the most potent compound from the five compounds tested.
- LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329) exhibited cytolytic activity against the 10 human lymphoma cell lines tested with IC 50 values in micromolar range.
- LTX-315 compound is the most potent compound tested with IC 50 values between 1 and 5 micromolar on all 10 human lymphoma cell lines tested.
- the haemolytic activity of the peptide LTX-315 against human red blood cells was measured.
- Freshly collected human blood was centrifuged at 1500 rpm for 10 minutes in order to isolate the red blood cells.
- the red blood cells (RBC) were washed three times with PBS [35 mM phosphate buffer with 150 mM NaCl, pH 7.4] by centrifugation at 1500 rpm for 10 minutes, and adjusted to 10% haematocrit with PBS.
- LTX-315 solutions were added to give a final concentration range of the peptide from 1200 ⁇ g/ml to 1 ⁇ g/ml and an RBC concentration of 1%.
- the resulting suspension was incubated with agitation for one hour at 37° C.
- the suspension was centrifuged at 4000 rpm for 5 minutes, and the released haemoglobin were monitored by measuring the absorbance of the supernatant at 405 nm.
- PBS was used as negative control and assumed to cause no haemolysis.
- 0.1% Triton was used as positive control and assumed to cause complete haemolysis.
- LTX-315 concentration corresponding to 50% haemolysis (EC 50 ) was determined from a dose-response curve.
- FIG. 1 shows that LTX-315 has a mean value of EC 50 higher than 1200 ⁇ g/ml (833 ⁇ M).
- the aim of the study was to investigate the effect of LTX-315 at different dose levels on a murine A20 B-cell lymphoma in mice.
- the administration took place by intratumoural injection of LTX-315 dissolved in sterile saline.
- mice Female mice were inoculated subcutaneously in the abdomen with 5 million murine A20 cells (ATCC, LGC Promochem AB, Middlesex, England) in a volume of 50 ⁇ l. The mice were divided into four groups (see Table 8 below for details). The intratumoural treatment was initiated when the tumours had reached the desired size of approximately 5 mm in diameter (minimum of 20 mm 2 ). Three dose levels of LTX-315, 1 mg (Group 1), 0.5 mg (Group 2) and 0.25 mg (Group 3) per injection, were investigated. The volume was 50 ⁇ l for all injections. LTX-315 was dissolved in sterile 0.9% NaCl water solution. This vehicle was used as control (Group 4). All four groups received three injections.
- mice were monitored during the study by measuring the tumours and weighing the animals regularly. The mice were followed until the maximum tumour burden of 125 mm 2 was reached, or until serious adverse events occurred (i.e. wound formation upon repeated treatments during the follow up period), then the mice were sacrificed. A calliper was used for tumour size measurements and weighing and physical examination were used as health control.
- the degree of tumour response in the different treatment groups is summarised in Table 10 below.
- the administration takes place by intra-tumoural injection of LTX-315 dissolved in sterile saline (0.9% NaCl in sterile water).
- mice Each of a total of 40 female mice was inoculated with five million murine CT26WT cells (ATCC, LGC Promochem AB, Boras, Sweden) subcutaneously on the abdomen surface in a volume of 50 ⁇ l.
- the mice were divided into five groups, 8 mice in each group.
- the tumours reached the desired size of 20 mm 2 the treatment by intra tumoural injection was initiated.
- Group one was treated solely on day 1, Group two on day 1 and 2, Group three on day 1 and 3 and Group four on day 1, 2 and 3. All daily treatments were one single injection of 1.0 mg LTX-315 dissolved in 50 ⁇ l (20 mg/ml).
- Group five was treated with the 50 ⁇ l of vehicle for LTX-315 (Group 5).
- mice were monitored during the study by measuring the tumours (digital calliper) and weighing the animals regularly. The mice were followed until the maximum tumour burden of 125 mm 2 was reached, or until serious adverse events occurred (i.e. wound formation due to repeated injections), then the mice were sacrificed. Weighing and physical examination were used as health controls.
- the treatment was started when the tumours had reached the desired size of a minimum of 20 mm 2 and animals were sacrificed when the tumours reached the maximum tumour burden of 125 mm 2 .
- necrosis at the tumour site was seen in all treatment groups. Generally, animals in Group 2, 3 and 4 showed more necrosis, wound and crust formation than the animals in Group 1 that were given only one injection of LTX-315. Group 4 animals, which were given three injections, showed the most necrosis, wound and crust formation. The difference in necrosis between Group 1 and Group 4 was quite large but the animals given the highest number of treatments seemed to cope well. No toxic or other adverse effects besides local necrotic tissue and wound formation were observed in either of the treated groups of animals.
- the amount of necrosis, wound and crust formation was proportional to the number of LTX-315 treatments given.
- Naive receiver mice were irradiated and divided into 2 groups. Group 1 received isolated splenocytes from cured mice, whereas group 2 received isolated splenocytes from naive mice. Freshly prepared cells were injected (20 ⁇ 106 per 100 ⁇ l) via the tail vein. Twenty four hours later receiver mice were inoculated with 5 million murine A20 B-cell lymphoma cells on the abdominal surface as described above. Tumour size and body weight were monitored until the maximum tumour burden of ⁇ 125mm 2 was reached, or a serious adverse events occurred (i.e. wound formation due to tumour tissue necrosis) at which point mice were sacrificed.
- the data provides evidence for an adaptive immune response in the animals that received splenocytes from animals that previously showed complete regression of A20-B lymphoma tumours following treatment with LTX-315.
- This data suggests that treatment with LTX-315 may confer long term protection against specific tumours by eliciting an immune response.
- the objective of the study was to investigate the anti-cancer effect of prophylactic vaccination with A20 lymphoma cells lysed by 10 mg/ml LTX-315:
- LTX-315 LTX-315 dissolved in growth media containing A20 lymphoma cells.
- the cell-LTX-315 “cocktail” was left for 30 min prior to injection in order to assure complete lysis of the cancer cells.
- Group 1 (“vaccine”) mice were injected subcutaneously on the abdomen surface with 50 ⁇ l of a “cocktail” of ten million murine A20 cells (ATCC, LGC Promochem AB, Boras, Sweden) and 10 mg/ml LTX-315 (“A20 lysate”).
- Group 2 (“vaccine +adjuvant”) mice were treated as per Group 1, but in addition were given 25 ⁇ l of 20 mg/ml LTX-315 subcutaneously at the site of vaccination 5 minutes prior to the A20 lysate injection.
- Group 3 (“control”) mice received no treatment.
- mice Six weeks after the treatment, all mice were inoculated with 5 million viable A20 B-cell lymphoma cells subcutaneously on the abdomen surface in a volume of 50 ⁇ l.
- mice were monitored during the study by measuring the tumour size and weighing the animals regularly. The mice were followed until the maximum tumour burden of ⁇ 130 mm 2 was reached, at which point the mice were sacrificed.
- Test substance Murine A20 cells lysed by LTX-315 (Lot 1013687), and LTX-315 (Lot 1013687) alone
- Test substance preparation 10 ⁇ 10 6 A20 cells were added to a 50 ⁇ l 10mg/ml LTX-315/vehicle (“A20 lysate”). The test substance was ready for use 30 minutes after mixing. LTX-315 alone was dissolved in 0.9% NaCl in sterile H 2 O
- the anti cancer effect of the various treatments is presented as mean tumour size in Table 16 below and a graphical presentation of the data is provided in FIG. 7 .
- Day 1 was the day of inoculation of viable A20 cells six weeks post-vaccination.
- the results show that the tumours developed more slowly in both LTX-315/A20-lysate treatment Groups as compared to the control Group.
- the median survival of Group 1 was 28 days, 33 days for Group 2, and 25 days for the control group (Group 3). Increase in median survival was 12% for Group 1 and 35% for Group 2 as compared to the control group (Group 3).
- the data indicate a prolonged survival of the treated groups compared to the untreated control group.
- 50% of the animals in Group 2 were still alive while 37.5% of the animals in Group 1 were still alive.
- End of study was defined as day 60.
- a total of 3 of the 16 treated animals had a complete regression of an initially developing tumour and were tumour free.
- 25% of animals from Group 1, and 12.5% of animals from Group 2 were observed to be tumour free.
- LTX-315 may have a dual use by lysing the tumour cells and inducing release of danger signals from normal cells at the injection site.
- LTX-315 on human melanoma cells.
- the peptide internalized and was shown in association with mitochondria, ultimately leading to a lytic cell death.
- the LTX-315 peptide was designed to treat solid tumors with intratumoral injections through a two-stage mode of action: the first is the collapse of the tumor itself, while the second is the released damage-associated molecular pattern molecules (DAMPs) from the dying tumor cell, which can induce a subsequent immune protection against recurrences and metastastis.
- DAMPs damage-associated molecular pattern molecules
- LTX-315 and LTX-328 were made on request by Bachem AG (Bubendorf, Switzerland) and Innovagen (Lund, Sweden), respectively.
- LTX-315 Pacific Blue and LTX-328 Pacific Blue were purchased on request from Innovagen (Lund, Sweden) Norud (Tromso, Norway), respectively.
- the A375 cell line A375 (ECACC, 88113005) is a human malignant melanoma derived from patient material, and was purchased from Public Health England (PHE Culture Collections, Porton Down, Salisbury,UK). Cells were maintained as monolayer cultures in high glucose 4.5% DMEM supplemented with 10% FBS and 1% L-glutamine, but not as antibiotics (complete media). The cell line was grown in a humidified 5% CO 2 atmosphere at 37° C., and was regularly tested for the presence of mycoplasma with MycoAlert (Lonza).
- the cytotoxic effect of LTX-315 was investigated using the colorimetric MTT viability assay as described in Eliassen et al. (2002), 22(5): pp2703-10.
- the A375 cells were seeded at a concentration of 1 ⁇ 10 5 cells/ml in a volume of 0.1 ml in 96-well plates, and allowed to adhere in a complete growth media overnight. The media was then removed and the cells were washed twice in serum-free, RPMI-1650 media, before adding LTX-315 dissolved in serum-free RPMI at concentrations ranging from 2.5-300 ⁇ g/ml, and incubated for 5-180 minutes.
- Live cell imaging with unlabeled cells A375 cells were seeded at 10,000 cells/well in a complete media in Nunc Lab-Tec 8-wells chambered covered glass (Sigma) precoated with 25 ⁇ g/ml human fibronectin (Sigma) that were allowed to adhere overnight. Cells were washed twice with a serum-free RPMI, treated with peptide dissolved in RPMI and investigated using Bright on a Leica TCS SP5 confocal microscope, with a 63X/1.2W objective. The microscope was equipped with an incubation chamber with CO 2 and temperature control.
- LTX-315 PB exhibited a similar cytotoxic profile as the unlabeled LTX-315 as determined by MTT assay.
- Control cells were treated with unlabeled LTX-315 and also with serum-free RPMI only. After incubation, cells were fixed with 4% paraformaldehyde in PBS, and the wells were covered with Prolong Gold antifade (Invitrogen). Cells were further analyzed by use of a Leica TCS SP5 confocal microscope, with a 693, 1.2 W objective.
- UV Pacific Blue, GFP and Ds Red were excited using UV, with 488 and 561 lasers, and fluorescence channels were sequentially detected using the following band passes: UV: 420-480nm (with attenuation), 488: 501-550nm and 561: 576-676nm.
- A375 cells were seeded at 1 ⁇ 10 5 cells per well in 6-well plates and allowed to grow for three days to optimize membrane structures in the culture, and the media was changed on the second day.
- Cells were washed twice in serum-free RPMI before being treated with LTX-315 dissolved in serum-free RPMI at 5, 10 and 25 ⁇ g/ml, with serum-free RPMI as a negative control.
- Cells were then washed with PBS twice before fixation for 24 hours in 4° C. with 4% formaldehyde and 1% gluteralaldehyde in a Hepes buffer at pH 7.8.
- Dehydration and post-fixation protocols included incubation in a 5% buffered tannic acid and incubation in a 1% osmium-reduced ferrocyanide. Ultrathin sections were prepared, and uranyl acetate (5%) and Reynolds's lead citrate were used for staining and contrasting. Samples were examined on a JEOL JEM-1010 transmission electron microscope, and images were taken with an Olympus Morada side-mounted TEM CCD camera (Olympus soft imaging solutions, GmbH, Germany).
- ROS Reactive Oxygen Species
- a DCFDA cellular reactive oxygen species detection assay kit was purchased from abcam®, and A375 cells seeded in a 96-well Costar black clear bottom plate with 20,000 cells per well incubated in 37° C. 16 hours prior to DCFDA assay. Cells were washed with a 100 ⁇ L/well of pre-warmed PBS one time, and incubated with 20 ⁇ M of DCFDA in a buffer solution supplied with the kit at 37° C. in a cell culture incubator for 45 min, and then washed again with a buffer solution of 100 ⁇ L/well.
- the cells were then stimulated with a 100 ⁇ L/well LTX-315 peptide dissolved in a buffer solution at concentrations of 17 ⁇ M for 30 min, and cells not treated were used as a negative control.
- the fluorescence intensity was determined at an excitation wavelength of 485nm and an emission wavelength of 530 nm on a FLUOstar Galaxy plate reader.
- A375 cells were seeded with 3 ⁇ 10 5 cells/well in 6-well plates in a complete media, and allowed to adhere overnight.
- Cells were treated with LTX-315 or LTX-328 at 35 ⁇ M, and incubated at 37° C. and 5% CO 2 for different time points (5, 10, 15, 30, 60 min), and negative controls were serum-free RPMI-1650.
- Supernatants (S) were collected and centrifuged at 1,400g for five minutes, and cell lysates (L) were harvested after washing with PBS twice and then subsequently lysed using a 4X Sample buffer (Invitrogen, number), 0.1 M DTT (Sigma number) and water.
- HMGB1 antibody rabbit, polyclonal, abcam ab 18256
- TBST incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody
- abcam ab6721 horseradish peroxidase-conjugated secondary antibody
- A375 cells were seeded as with HMGB1 studies, and treated with 35 ⁇ M for different time points (5, 15, 45). Supernatants were collected and concentrated as with HMGB1 studies, and samples from the supernatants were analyzed using a 4.5 hour solid form Cytochrome C- Elisa kit (R&D Systems, USA, #DCTCO) following the manufacturer's description. Shortly thereafter, a 50% diluted sample was analyzed and the optical density was determined using a microplate reader set at 450 nm, and this reading was then subtracted from the reading at 540 nm. A standard curve was generated for each set of samples assayed. Samples were run in four parallels, and the cytochrome-c released into the supernatant was expressed as a fold over the level of cytochrome-c in the supernatant of untreated cells.
- LTX-315-treated A375 cells was analyzed using an Enliten ATP luciferase assay kit (Promega, USA). Cells were then seeded as with an ROS assay, and treated with LTX-315 in different incubation times, from 1 to 15 minutes with two parallels, which was then conducted three times. Negative controls were untreated A375 cells exposed to serum-free media alone. Samples were diluted at 1:50 and 1:100, and analyzed with a Luminoscan RT luminometer according to the manufacturer's protocol.
- IC-50 values for the peptide were 30 ⁇ M after only five minutes of incubation, and progressed to 14 ⁇ M after 90 minutes. Further incubation up to 180 minutes did not offer any additional effect ( FIG. 8 ).
- LTX-315 was labeled with Pacific Blue and incubated with cells at concentrations of 3 ⁇ M and 1.5 ⁇ M, respectively.
- the labeled LTX-315 rapidly penetrated the plasma membrane and at 1.5 ⁇ M, the peptide showed an accumulation around the mitochondria after 30 minutes of incubation but was not detected in the cell nucleus ( FIG. 9 ).
- the labeled non-lytic mock-sequence peptide LTX-328 did not demonstrate any internalization at any concentration or incubation time tested ( FIG. 10 ).
- FIG. 11 B Another common finding in these samples were peripherally placed vacuoles, which were lined with a single membrane layer containing a homogenous material.
- FIG. 11 C &E Another common finding in these samples were peripherally placed vacuoles, which were lined with a single membrane layer containing a homogenous material.
- FIG. 11 C &E Another common finding in these samples were peripherally placed vacuoles, which were lined with a single membrane layer containing a homogenous material.
- FIG. 11 C &E Another common finding in these samples were peripherally placed vacuoles, which were lined with a single membrane layer containing a homogenous material.
- LTX-315 kills the tumor cells with a lytic mode of action, while lower concentrations cause the cells to undergo ultrastructure changes, such as vacuolization and an altered mitochondrial morphology. Moreover, no significant morphological changes suggestive of apoptotic cell death were observed.
- DAMPs are molecules that are released from intracellular sources during cellular damage. DAMPs can initiate and perpetuate an immune response through binding to Pattern Recognition Receptors (PRRs) on Antigen Presenting Cells (APCs).
- PRRs Pattern Recognition Receptors
- APCs Antigen Presenting Cells
- DAMPs are ATP, HMGB1, Calreticulin, Cytochrome C, mitochondrial DNA and Reactive oxygen species (ROS).
- ROS Reactive oxygen species
- LTX-315-treated cells released cytochrome-C into the medium
- A375 cells were treated with LTX-315 at 35 ⁇ M at different time points (5, 15, 45 min). The supernatant was subsequently analyzed using an ELISA assay. Cells treated with 35 ⁇ M value had three times more cytochrome-C in the supernatant compared to untreated control cells. The increase in cytochrome-C was detected after only five minutes of treatment, and there was also an increase after 15 and 45 minutes of peptide treatment, respectively ( FIG. 13 ).
- HMGB1 is a non-histone, chromatin-binding nuclear protein. Once passively released from necrotic cells, HMGB1 is able to trigger the functional maturation of dendritic cells, cytokine stimulation and chemotaxis among several immunopotentiating effects.
- HMGB1 is normally found in the cell nucleus and would be expected in a cell lysate of healthy cells, though not in the culture media (supernatant).
- HMGB1 is normally found in the cell nucleus and would be expected in a cell lysate of healthy cells, though not in the culture media (supernatant).
- Both cell lysate and the cell supernatant of LTX-315- and LTX-328-treated A375 melanoma cells were analyzed using a Western blot.
- Cells were treated with 35 ⁇ M of either LTX-315 or LTX-328, with a gradual translocation from the cell lysate to the supernatant detected in the LTX-315-treated melanoma cells, but not in the cells treated with the mock sequence peptide LTX-328 or a serum-free medium only ( FIG. 14 ).
- ROS generation following LTX-315 treatment was measured by CH2DCFDA fluorometric assay. Significant amounts of ROS were generated after 15 minutes of incubation with LTX-315, and the ROS levels were concentration-dependent ( FIG. 12 ).
- LTX-315 labeled with the fluorescent molecule Pacific Blue was internalized within minutes after incubation with A375 melanoma cells, and was distributed in the cytoplasm ( FIG. 9 ). At low concentrations, accumulation of the peptide around the mitochondria was evident, whereas at higher concentrations the peptide was more spread within the cytoplasm and accumulated in circular structures closer to the cell membrane ( FIG. 10 ). If the peptide attacks the mitochondrial membrane, a decrease or even a total collapse of the mitochondrial membrane potential would be expected.
- a confocal imaging of cells with the membrane potential-dependent mitochondrial stain Mitotracker CMXh2ROS showed a loss of mitochondrial signal a short time after peptide treatment (data not shown).
- Cytochrome-C is a mitochondrial protein released from the intermembrane space and into the cytosol when the outer mitochondrial membrane is perturbed, and by binding to the apoptotic protease activating factor-1 (Apaf-1) it is also a part of the apoptotic cascade that eventually leads to cell death by apoptosis.
- cytochrome-C is found in the extracellular space, it has been reported to act as a pro-inflammatory mediator, thus activating NF-kB and inducing cytokine and chemokine production.
- the transition of HMGB1 from the cellular compartment to the extracellular compartment was detected using a western blot ( FIG. 14 ).
- the nuclear protein HMBG1 When the nuclear protein HMBG1 is released into the extracellular fluid, it functions as a DAMP, and can bind to both the PRR TLRs and to the RAGE receptors; the activation of these may lead to a number of inflammatory responses such as the transcription of pro-inflammatory cytokines.
- LTX-315 induces lytic cell death in cancer cells, not only by direct attack on the plasma membrane, but also as a result of an injury to vital intracellular organelles after the internalization of the peptide at concentrations too low to cause an immediate loss of plasma membrane integrity.
- DAMPs may affect the cellular integrity of the damaged cells in several ways, but are also associated with so-called immunogenic cell death.
- DAMPs potent immune stimulatory molecules
- LTX-315 The combination of LTX-315 with either an anti-programmed cell death protein 1 (PD-1) antibody or an anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody was assayed in a mouse MCA205 sarcoma model. Seven animals were studied per group. For each study, two independent experiments were performed yielding identical results. Anova statistical analyses were performed.
- PD-1 anti-programmed cell death protein 1
- CTLA-4 anti-cytotoxic T-lymphocyte-associated protein 4
- the anti-PD-1 antibody used in this study was a murine IgG isotype.
- the anti-CTLA-4 antibody was a rat IgG isotype. Both antibodies were purchased from eBioscience.
- FIGS. 17 a , 18 a , 19 a and 20 a show the timings and administration routes of LTX-315 and the antibodies in the various studies.
- mice were inoculated with MCA205 cancer cells and palpable tumours were allowed to form.
- LTX-315 was then administered intratumorally on the days shown in the Figures and the anti-PD-1 or anti- CTLA-4 antibodies were administered i.p. on the days shown.
- LTX-315 with an anti-programmed cell death protein 1 ligand (PD-L1) antibody was assayed in a mouse EMT-6 mammary carcinoma model.
- PD-L1 anti-programmed cell death protein 1 ligand
- the anti-PD-L1 antibody had the following characteristics: ref: BE0101, Bioxcell; clone 10F.9G2; reactivity: mouse; isotype: Rat IgG2b.
- LTX-315 was prepared at a dose of 0.5 mg/50 ⁇ L in 0.9% sodium chloride solution.
- Anti-PD-L1 antibody was prepared at a concentration in phosphate-buffered saline and was administered at a dose of 10 mg/kg.
- LTX-315 was injected into the tumour grafted on the right flank of the mice, anti-PD-L1 antibody was injected into the peritoneal cavity of the mice.
- the EMT-6 cell line was established from a transplantable murine mammary carcinoma that arose in a BALB/cCRGL mouse after implantation of a hyperplastic mammary alveolar nodule (VOLENEC FJ., et al., J Surg Oncol. 13(1):39-44, 1980).
- EMT-6 tumor cells were grown as a monolayer at 37° C. in a humidified atmosphere (5% CO 2 , 95% air).
- the culture medium was RPMI 1640 containing 2 mM L-glutamine (ref: BE12-702F, Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (ref: 3302, Lonza).
- EMT-6 tumor cells are adherent to plastic flasks.
- tumor cells were detached from the culture flask by a 5-minute treatment with trypsin-versene (ref: BE17-161E, Lonza), in Hanks' medium without calcium or magnesium (ref: BE10-543F, Lonza) and neutralized by addition of complete culture medium. The cells were counted in a hemocytometer and their viability were assessed by 0.25% trypan blue exclusion assay.
- mice Healthy female Balb/C mice, 6-8 weeks old at reception, were obtained from CHARLES RIVER (L'Arbresles, France) and from Janvier (France). Mice were maintained in SPF health status according to the FELASA guidelines. Mouse housing and experimental procedures were realized according to the French and European Regulations (Principe d'éthique de I'plastation animale, Directive n°2010/63 CEE du 22 groove 2010, Décret n°2013-118 du 01 complicat 2013) and the NRC Guide for the Care and Use of Laboratory Animals (NRC Guide for the Care and Use of Laboratory Animals). The animal facility was authorized by the French authorities (Agreement N° B 21 231 011 EA). All procedures using mice were submitted to the Animal Care and Use Committee of Oncodesign (Oncomet) agreed by French authorities (CNREEA agreement N° 91). Mice were individually identified with a RFID transponder and each cage was labeled with a specific code.
- Mice enclosures provided sterile and adequate space with bedding material, food and water, environmental and social enrichment (group housing) as described: Top filter polycarbonate Eurostandard Type III or IV cages, Corn cob bedding (ref: LAB COB 12, SERLAB, France), 25 kGy Irradiated diet (Ssniff® Soest, Germany), Complete food for immunecompetent rodents—R/M-H Extrudate, Sterile, filtrated at 0.2 ⁇ m water, and Environmental enrichment (SIZZLE-dri kraft—D20004 SERLAB, France).
- a first tumor was induced by subcutaneous injection of 1 ⁇ 10 6 of EMT-6 cells in 200 ⁇ L of RPMI 1640 into the right flank of Balb/C female mice. The day of tumor cell injection in the right flank was considered as D0.
- a second tumor was induced by subcutaneous injection of 1 ⁇ 10 5 of EMT-6 cells in 200 ⁇ L of RPMI 1640 into the left flank of Balb/C female mice. The day of tumor cell injection in the left flank was considered as D3.
- mice were randomized according to their body weight on D3 into four groups each of 5 mice (group 1) or 10 mice (groups 3, 5 and 7) using Vivo manager® software (Biosystemes, Couternon, France).
- a statistical test analysis of variance was performed to test for homogeneity between groups.
- a statistical test analysis of variance was performed to test for homogeneity between groups.
- the treatment schedule was as follows: the mice from group 1 were not treated; the mice from group 3 received a total of 3 intratumoral injections of LTX135; the mice from group 5 received a total of 6 IP injections of anti-PD-L1; and the mice from group 7 received a total of 3 intratumoral injections of LTX315 and a total of 6 IP injections of anti-PD-L1.
- tumour volume (width 2 ⁇ length)/2 (SIMPSON-HERREN L. et al. Cancer Chemotherapy Rep., 54: 143, 1970).
- mice Details regarding the percentage of mice that show total tumour regression in the right and/or left flank are shown in Table 18 below. Mice were tested at day 52 after inoculation or when sacrificed.
- the table shows that the combination of LTX-315 and anti-PD-L1 antibody is particularly effective in inducing adaptive immunity, as 40% of the mice treated with the combination (G7) showed total regression of the tumour in the left flank, compared to 10% of mice treated with LTX-315 alone (G3), 20% of mice treated with anti-PD-L1 antibody alone (G5) and none of the untreated mice (G1).
Abstract
The present invention provides a compound, preferably a peptide, having the following characteristics:
-
- a) consisting of 9 amino acids in a linear arrangement;
- b) of those 9 amino acids, 5 are cationic and 4 have a lipophilic R group;
- c) at least one of said 9 amino acids is a non-genetically coded amino acid (e.g. a modified derivative of a genetically coded amino acid); and optionally
- d) the lipophilic and cationic residues are arranged such that there are no more than two of either type of residue adjacent to one another; and further optionally
- e) the molecule comprises two pairs of adjacent cationic amino acids and one or two pairs of adjacent lipophilic residues;
for use in the treatment of a tumour by combined, sequential or separate administration with an immune checkpoint inhibitor. The present invention further provides pharmaceutical packs or compositions comprising these active agents and methods of treating a tumour comprising administration of these active agents. Also provided are the peptidic compounds as defined above for use in the destabilisation of a mitochondrial membrane, and a peptidic compound as defined above and an immunotherapeutic agent as a combined preparation for separate, simultaneous or sequential use in treating tumours.
Description
- This Application is a Continuation of application Ser. No. 15/534,800 filed on Jun. 9, 2017, which is a 371 National Stage Application of International Application PCT/EP2015/075722 filed on Nov. 4, 2015, which claims priority to Japanese Application 2015-118495 filed on Jun. 11, 2015, and also claims priority to United Kingdom Application 1506127.8 filed on Apr. 10, 2015, and also claims priority to United Kingdom Application 1422084.2 filed on Dec. 11, 2014. The entire contents of these applications are incorporated herein by reference in their entireties.
- The present invention relates to peptides or peptide like molecules and particularly to combined preparations of such peptides with a further agent, and their uses in therapy, in particular as anti-tumour agents.
- The prevalence of cancer in human and animal populations and its role in mortality means there is a continuing need for new drugs which are effective against tumours. Elimination of a tumour or a reduction in its size or reducing the number of cancer cells circulating in the blood or lymph systems may be beneficial in a variety of ways; reducing pain or discomfort, preventing metastasis, facilitating operative intervention, prolonging life.
- Genetic and epigenetic alterations that are characteristic of cancers result in antigens that the immune system can recognise and use to differentiate between tumour cells and their healthy equivalents. In principle, this means that the immune system could be a powerful weapon in controlling tumours. However, the reality is that the immune system usually does not provide a strong response to tumour cells. It is of great therapeutic interest to manipulate and therefore harness the immune system in the fight against cancer (Mellman et al. Nature 2011, vol. 480, 480-489).
- Various attempts have been made to help the immune system to fight tumours. One early approach involved a general stimulation of the immune system, e.g. through the administration of bacteria (live or killed) to elicit a general immune response which would also be directed against the tumour. This is also called nonspecific immunity.
- Recent approaches aimed at helping the immune system specifically to recognise tumour-specific antigens involve administration of tumour-specific antigens, typically combined with an adjuvant (a substance which is known to cause or enhance an immune response) to the subject. This approach requires the in vitro isolation and/or synthesis of antigens, which is costly and time consuming. Often not all the tumour-specific antigens have been identified, e.g. in breast cancer the known antigens are found in 20-30% of the total tumours. The use of tumour-specific vaccines have therefore met with limited success.
- There remains a strong need for alternative methods for treating tumours and for alternative methods for inhibiting the growth or formation of secondary tumours.
- ‘Cancer Vaccine’ is a term used to describe therapeutic agents which are designed to stimulate the patient's immune system against tumour antigens and lead to an attack on tumour cells and improved patient outcome. Despite the name, cancer vaccines are generally intended to generate or enhance an immune response against an existing cancer, rather than to prevent disease. Again, unlike traditional vaccines against infective agents, a cancer or tumour vaccine may not require administration of a tumour antigen, the administered product may utilise tumour antigens already present in the body as a result of tumour development and serve to modify the immune response to the existing tumour associated antigens (TAAs).
- It is recognised that the usual lack of a powerful immune response to TAA is due to a combination of factors. T cells have a key role in the immune response, which is initiated through antigen recognition by the T cell receptor (TCR), and they coordinate a balance between co-stimulatory and inhibitory signals known as immune checkpoints (Pardoll, Nature 2012, vol. 12, 252-264). Inhibitory signals suppress the immune system which is important for maintenance of self-tolerance and to protect tissues from damage when the immune system is responding to pathogenic infection. However, immune suppression reduces what could otherwise be a helpful response by the body to the development of tumours.
- This T cell mediated balance of immune stimulation and suppression has, in recent years, led to the adoption of a principle of tumour immunotherapy known as a ‘push-pull’ approach in which combination therapies could be used to simultaneously enhance the stimulatory factors (push) and reduce the inhibitory factors (pull). A helpful analogy is of a combination therapy which both presses on the accelerator (push) and reduces the brakes (pull). (Berzofsky et al. Semin Oncol. 2012 Jun; 39(3) 348-57).
- For example, cytokines, other stimulatory molecules such as CpG (stimulating dendritic cells), Toll-like receptor ligands and other molecular adjuvants enhance the immune response. Co-stimulatory interactions involving T cells directly can be enhanced using agonistic antibodies to receptors including OX40, CD28, CD27 and CD137. These are all push-type approaches to cancer immunotherapy.
- Complementary ‘pull’ therapies may block or deplete inhibitory cells or molecules and include the use of antagonistic antibodies against what are known as immune checkpoints.
- Immune checkpoints include cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) and antibodies against these are known in the art; ipilimumab was the first FDA-approved anti-immune checkpoint antibody licensed for the treatment of metastatic melanoma and this blocks cytotoxic T-lymphocyte antigen 4 (CTLA-4) (Naidoo et al. British Journal of Cancer (2014) 111, 2214-2219). Cytotoxic T-lymphocyte-associated
protein 4, also known as CD152, is a member of the immunoglobulin superfamily, which is expressed on the surface of helper T cells and transmits an inhibitory signal to T cells. Its genomic sequence is known, NCBI Reference Sequence: NG_011502.1, as is its protein sequence NCBI Reference Sequence: NP_005205.2. - The present inventors have established that some peptides known to lyse tumour cells through disturbing and permeabilizing the cell membrane, are also highly effective at attacking organelles such as mitochondria and lysosomes and can cause lysis thereof. This may be achieved at low concentrations which do not cause direct lysis of the cell membranes, although loss of cell membrane integrity is seen eventually even on administration of low doses. At higher doses, these molecules can cause lysis of the cell membrane and then of the membranes of organelles.
- The peptides of interest are a sub-set of the group of peptides commonly known as Cationic antimicrobial peptides (CAPs). These are positively charged amphipathic peptides and peptides of this type are found in many species and form part of the innate immune system. The CAP Lactoferricin (LfcinB) is a 25 amino acid peptide which has been shown to have an effect on mitochondria (Eliasen et al. Int. J. Cancer (2006) 119, 493-450). It has now surprisingly been found that the much smaller peptide LTX-315, a 9 amino acid peptide (of the type described in WO 2010/060497), also targets the mitochondria. This was unexpected because this small peptide is much more fast acting (causing cell death after 30 minutes of exposure) compared to LfcinB (which is most effective after 24 hours of exposure) and the small peptide acts against a broader spectrum of cell types, which suggests a direct effect on the plasma membrane.
- This disruption of the organelle membrane results in the release of agents therefrom which have a potent immunostimulatory function, such agents are generally known as DAMPs (Damage-associated molecular pattern molecules) and include ATP, Cytochrome C, mitochondrial CpG DNA sequences, mitochondrial formyl peptides, cathepsins (from lysosomes) and HMGB1 (from the nucleus). Lysis of organelles can also result in release of additional tumour-specific antigens (TAAs).
- This ability to stimulate the immune response to tumours through disrupting mitochondrial and other organelle membranes makes these peptides highly suitable as “push” agents in combination “push-pull” immunotherapies designed to treat and protect against tumour development.
- Thus, in a first aspect, the present invention provides:
- A compound, preferably a peptide, having the following characteristics:
-
- a) consisting of 9 amino acids in a linear arrangement;
- b) of those 9 amino acids, 5 are cationic and 4 have a lipophilic R group;
- c) at least one of said 9 amino acids is a non-genetically coded amino acid (e.g. a modified derivative of a genetically coded amino acid); and optionally
- d) the lipophilic and cationic residues are arranged such that there are no more than two of either type of residue adjacent to one another; and further optionally
- e) the molecule comprises two pairs of adjacent cationic amino acids and one or two pairs of adjacent lipophilic residues;
for use in the treatment of a tumour by combined, sequential or separate administration with an immune checkpoint inhibitor (ICI).
- The combination therapy proposed herein may, in certain advantageous embodiments, provide a synergistic effect. Such surprising synergistic effects have been seen, for example, when using an anti-CTLA-4 antibody (Example 11).
- The amino acid containing molecules defined above are conveniently referred to herein as the “peptidic compound of the invention”, which expression includes all of the peptides and peptidomimetics disclosed herein.
- The cationic amino acids, which may be the same or different, are preferably lysine or arginine but may be histidine or any non-genetically coded amino acid carrying a positive charge at pH 7.0. Suitable non-genetically coded cationic amino acids include analogues of lysine, arginine and histidine such as homolysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid and homoarginine as well as trimethylysine and trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4-amino -1-carbamimidoylpiperidine-4-carboxylic acid and 4-guanidinophenylalanine.
- Non-genetically coded amino acids include modified derivatives of genetically coded amino acids and naturally occurring amino acids other than the 20 standard amino acids of the genetic code. In this context, a D amino acid, while not strictly genetically coded, is not considered to be a “non-genetically coded amino acid”, which should be structurally, not just stereospecifically, different from the 20 genetically coded L amino acids. The molecules of the invention may have some or all of the amino acids present in the D form, preferably however all amino acids are in the L form.
- The lipophilic amino acids (i.e. amino acids with a lipophilic R group), which may be the same or different, all possess an R group with at least 7, preferably at least 8 or 9, more preferably at least 10 non-hydrogen atoms. An amino acid with a lipophilic R group is referred to herein as a lipophilic amino acid. Typically the lipophilic R group has at least one, preferably two cyclic groups, which may be fused or linked.
- The lipophilic R group may contain hetero atoms such as O, N or S but typically there is no more than one heteroatom, preferably it is nitrogen. This R group will preferably have no more than 2 polar groups, more preferably none or one, most preferably none.
- Tryptophan is a preferred lipophilic amino acid and the molecules preferably comprise 1 to 3, more preferably 2 or 3, most preferably 3 tryptophan residues. Further genetically coded lipophilic amino acids which may be incorporated are phenylalanine and tyrosine.
- Preferably one of the lipophilic amino acids is a non-genetically coded amino acid. Most preferably the molecule consists of 3 genetically coded lipophilic amino acids, 5 genetically coded cationic amino acids and 1 non-genetically coded lipophilic amino acid.
- When the molecules include a non-genetically coded lipophilic amino acid (e.g. amino acid derivative), the R group of that amino acid preferably contains no more than 35 non-hydrogen atoms, more preferably no more than 30, most preferably no more than 25 non-hydrogen atoms.
- Preferred non-genetically coded amino acids include: 2-amino-3-(biphenyl-4-yl)propanoic acid (biphenylalanine), 2-amino-3,3-diphenylpropanoic acid (diphenylalanine), 2-amino-3-(anthracen-9-yl)propanoic acid, 2-amino-3-(naphthalen-2-yl)propanoic acid, 2-amino-3-(naphthalen-1-yl)propanoic acid, 2-amino-3-[1,1′:4′,1″-terphenyl -4-yl]-propionic acid, 2-amino-3-(2,5,7-tri-tert-butyl-1H-indol-3-yl)propanoic acid, 2-amino-3-[1,1′:3′,1″-terphenyl-4-yl]-propionic acid, 2-amino-3-[1,1′:2′,1″-terphenyl -4-yl]-propionic acid, 2-amino-3-(4-naphthalen-2-yl-phenyl)-propionic acid, 2-amino -3-(4′-butylbiphenyl-4-yl)propanoic acid, 2-amino-3-[1,1′:3′,1″-terphenyl-5′-yl]-propionic acid and 2-amino-3-(4-(2,2-diphenylethyl)phenyl)propanoic acid.
- In a preferred embodiment the peptidic compounds of the invention have one of formulae I to V listed below, in which C represents a cationic amino acid as defined above and L represents a lipophilic amino acid as defined above. The amino acids being covalently linked, preferably by peptide bonds resulting in a true peptide or by other linkages resulting in a peptidomimetic, peptides being preferred. The free amino or carboxy terminals of these molecules may be modified, the carboxy terminus is preferably modified to remove the negative charge, most preferably the carboxy terminus is amidated, this amide group may be substituted.
-
(SEQ ID NO: 1) (I) CCLLCCLLC (SEQ ID NO: 2) (II) LCCLLCCLC (SEQ ID NO: 3) (III) CLLCCLLCC (SEQ ID NO: 4) (IV) CCLLCLLCC (SEQ ID NO: 5) (V) CLCCLLCCL - A peptidomimetic is typically characterised by retaining the polarity, three dimensional size and functionality (bioactivity) of its peptide equivalent but wherein the peptide bonds have been replaced, often by more stable linkages. By ‘stable’ is meant more resistant to enzymatic degradation by hydrolytic enzymes. Generally, the bond which replaces the amide bond (amide bond surrogate) conserves many of the properties of the amide bond, e.g. conformation, steric bulk, electrostatic character, possibility for hydrogen bonding etc.
Chapter 14 of “Drug Design and Development”, Krogsgaard, Larsen, Liljefors and Madsen (Eds) 1996, Horwood Acad. Pub provides a general discussion of techniques for the design and synthesis of peptidomimetics. In the present case, where the molecule is reacting with a membrane rather than the specific active site of an enzyme, some of the problems described of exactly mimicking affinity and efficacy or substrate function are not relevant and a peptidomimetic can be readily prepared based on a given peptide structure or a motif of required functional groups. Suitable amide bond surrogates include the following groups: N-alkylation (Schmidt, R. et al., Int. J. Peptide Protein Res., 1995, 46,47), retro-inverse amide (Chorev, M and Goodman, M., Acc. Chem. Res, 1993, 26, 266), thioamide (Sherman D.B. and Spatola, A.F. J. Am. Chem. Soc., 1990, 112, 433), thioester, phosphonate, ketomethylene (Hoffman, R.V. and Kim, H. O. J. Org. Chem., 1995, 60, 5107), hydroxymethylene, fluorovinyl (Allmendinger, T. et al., Tetrahydron Lett., 1990, 31, 7297), vinyl, methyleneamino (Sasaki, Y and Abe, J. Chem. Pharm. Bull. 1997 45, 13), methylenethio (Spatola, A. F., Methods Neurosci, 1993, 13, 19), alkane (Lavielle, S. et. al., Int. J. Peptide Protein Res., 1993, 42, 270) and sulfonamido (Luisi, G. et al. Tetrahedron Lett. 1993, 34, 2391). - The peptidomimetic compounds may have 9 identifiable sub-units which are approximately equivalent in size and function to the 9 cationic and lipophilic amino acids. The term ‘amino acid’ may thus conveniently be used herein to refer to the equivalent sub-units of a peptidomimetic compound. Moreover, peptidomimetics may have groups equivalent to the R groups of amino acids and discussion herein of suitable R groups and of N and C terminal modifying groups applies, mutatis mutandis, to peptidomimetic compounds.
- As is discussed in “Drug Design and Development”, Krogsgaard et al., 1996, as well as replacement of amide bonds, peptidomimetics may involve the replacement of larger structural moieties with di- or tripeptidomimetic structures and in this case, mimetic moieties involving the peptide bond, such as azole-derived mimetics may be used as dipeptide replacements. Peptidomimetics and thus peptidomimetic backbones wherein just the amide bonds have been replaced as discussed above are, however, preferred.
- Suitable peptidomimetics include reduced peptides where the amide bond has been reduced to a methylene amine by treatment with a reducing agent e.g. borane or a hydride reagent such as lithium aluminium-hydride. Such a reduction has the added advantage of increasing the overall cationicity of the molecule.
- Other peptidomimetics include peptoids formed, for example, by the stepwise synthesis of amide-functionalised polyglycines. Some peptidomimetic backbones will be readily available from their peptide precursors, such as peptides which have been permethylated, suitable methods are described by Ostresh, J. M. et al. in Proc. Natl. Acad. Sci. USA (1994) 91, 11138-11142. Strongly basic conditions will favour N-methylation over 0-methylation and result in methylation of some or all of the nitrogen atoms in the peptide bonds and the N-terminal nitrogen.
- Preferred peptidomimetic backbones include polyesters, polyamines and derivatives thereof as well as substituted alkanes and alkenes. The peptidomimetics will preferably have N and C termini which may be modified as discussed herein.
- β and γ amino acids as well as cc amino acids are included within the term ‘amino acids’, as are N-substituted glycines. The peptidic compounds of the invention include beta peptides and depsipeptides.
- As discussed above, the peptidic compounds of the invention incorporate at least one, and preferably one, non-genetically coded amino acid. When this residue is denoted L′, preferred compounds are represented by the following formulae:
-
(SEQ ID NO: 6) (I′) CCL′LCCLLC (SEQ ID NO: 7) (I′′) CCLLCCLL′C (SEQ ID NO: 8) (I′′′) CCLL′CCLLC (SEQ ID NO: 9) (II′) LCCLL′CCLC - Particularly preferred are compounds (preferably peptides) of formula I and II, and of these, compounds (preferably peptides) of formula I″ are especially preferred.
- The following peptides as presented in Table 1 are most preferred.
-
TABLE 1 Name SEQ ID NO Sequence LTX-301 10 Dip-K-K-W-W-K-K-W-K-NH2 LTX-302 11 W-K-K-W-Dip-K-K-W-K-NH2 LTX-303 12 W-K-K-W-W-K-K-Dip-K-NH2 LTX-304 13 Bip-K-K-W-W-K-K-W-K-NHs LTX-305 14 W-K-K-Bip-W-K-K-W-K-NH2 LTX-306 15 w-k-k-w-dip-k-k-w-k-NH2 LTX-307 16 K-K-W-Dip-K-K-W-W-K-NH2 LTX-308 17 k-k-W-Dip-k-k-W-W-k-NH2 LTX-309 18 K-K-W-Dip-K-K-W-Dip-K-NH2 LTX-310 19 K-K-W-Bip-K-K-W-W-K-NH2 LTX-312 20 K-Bip-K-K-W-W-K-K-W-NH2 LTX-313 21 K-K-Bip-W-K-K-W-W-K-NH2 LTX-314 22 K-K-W-W-K-K-Dip-W-K-NH2 LTX-315 23 K-K-W-W-K-K-W-Dip-K-NH2 LTX-316 24 K-W-Dip-K-K-W-W-K-K-NH2 LTX-317 25 K-K-W-W-K-W-Dip-K-K-NH2 LTX-318 26 Orn-Orn-W-Dip-Orn-Orn-W-W-Orn- NH2 LTX-319 27 Dap-Dap-W-Dip-Dap-Dap-W-W-Dap- NH2 LTX-320 28 R-R-W-Dip-R-R-W-W-R-NH2 LTX-321 29 K-W-W-K-K-Dip-W-K-K-NH2 LTX-323 30 K-Dip-K-K-W-W-K-K-W-NH2 LTX-324 31 K-K-Dip-W-K-K-W-W-K-NH2 LTX-325 32 k-w-w-k-k-dip-w-k-k-NH2 LTX-326 33 R-R-Bip-W-R-R-W-W-R-NH2 LTX-327 34 R-R-Dip-W-R-R-W-W-R-NH2 LTX-329 35 k-k-bip-w-k-k-w-w-k-NH2 LTX-331 36 k-k-Bip-w-k-k-w-w-k-NH2 LTX-332 37 K-K-bip-W-K-K-W-W-K-NH2 LTX-333 38 Dab-Dab-W-Dip-Dab-Dab-W-W-Dab- NH2 LTX-334 39 K-K-W-1-Nal-K-K-W-W-K-NH2 LTX-335 40 K-K-W-2-Nal-K-K-W-W-K-NH2 LTX-336 41 K-K-W-Ath-K-K-W-W-K-NH2 LTX-338 42 K-K-W-Phe(4-4′Bip)-K-K-W-W-K- NH2 - In which:
-
- the standard single letter code is used for the genetically coded amino acids
- lower case denotes D amino acids
- Dip is diphenylalanine
- Bip is biphenylalanine
- Orn is ornithine
- Dap is 2,3-diaminopropionic acid
- Dab is 2,4-diaminobutyric acid
- 1-Nal is 1-naphthylalanine
- 2-Nal is 2-naphthylalanine
- Ath is 2-amino-3-(anthracen-9-yl)propanoic acid
- Phe(4,4′Bip) is 2-amino-3-[1,1′:4′,1″-terphenyl-4-yl]propionic acid
- Compound LTX-315 is most preferred.
- All of the molecules described herein may be in salt, ester or amide form.
- The molecules are preferably peptides and preferably have a modified, particularly an amidated, C-terminus. Amidated peptides may themselves be in salt form and acetate forms are preferred. Suitable physiologically acceptable salts are well known in the art and include salts of inorganic or organic acids, and include trifluoracetate as well as acetate and salts formed with HCl.
- The peptidic compounds described herein are amphipathic in nature, their 2° structure, which may or may not tend towards the formation of an α-helix, provides an amphipathic molecule in physiological conditions.
- The combination therapies defined herein are for the treatment of tumours, in particular solid tumours and thus for the treatment of cancer.
- The peptidic compounds of the invention destabilise and/or permeabilise the membranes of tumour cell organelles, e.g. mitochondria, the nucleus or lysomome, in particular the mitochondria.
- By ‘destabilising’ is meant a perturbation of the normal lipid bi-layer configuration including but not limited to membrane thinning, increased membrane permeability to water, ions or metabolites etc.
- Immune checkpoints are known in the art (Naidoo et al. and Pardoll et al supra) and the term is well understood in the context of cancer therapy. The most well-known are CTLA-4, PD-1 and its ligand PDL-1. Others include TIM-3, KIR, LAG-3, VISTA, BTLA. Inhibitors of immune checkpoints inhibit their normal immunosuppressive function, for example by down regulation of expression of the checkpoint molecules or by binding thereto and blocking normal receptor/ligand interactions. As the immune checkpoints put brakes on the immune system response to an antigen, so an inhibitor thereof reduces this immunosuppressive effect and enhances the immune response. Inhibitors of immune checkpoints are known in the art and preferred are anti-immune checkpoint antibodies, such as anti-CTLA-4 antibodies (e.g. ipilimumab and tremelimumab), anti-PD-1 antibodies (e.g. nivolumab, pembrolizumab, formerly known as lambrolizumab, pidilizumab and RG7446 (Roche)) and anti-PDL-1 antibodies (e.g. BMS-936559 (Bristol-Myers Squibb), MPDL3280A (Genentech), MSB0010718C (EMD-Serono) and MED14736 (AstraZeneca)).
- Thus preferred ICIs are antibodies that bind to a specific immune checkpoint molecule, whether that immune checkpoint molecule is itself a receptor or a ligand therefor. Preferred immune checkpoint molecules are listed above. Receptors which form part of an immune checkpoint are typically found on the surface of T cells.
- With knowledge of an immune checkpoint target, a skilled man is able to develop an inhibitor thereof. Inhibitors may be selected from proteins, peptides, peptidomimetics, peptoids, antibodies, antibody fragments, small inorganic molecules, small non-nucleic acid organic molecules or nucleic acids such as anti-sense nucleic acids, small interfering RNA (siRNA) molecules or oligonucleotides. Thus the inhibitor may act to down regulate expression of an immune checkpoint molecule. The inhibitor may for example be a modified version of the natural ligand (e.g. for CTLA-4, CD80 (B7-1) and CD86 (B7-2)), such as a truncated version of one of the ligands. They may be naturally occurring, recombinant or synthetic.
- Preferably the inhibitor is either an antibody, a modified ligand or an antisense nucleic acid molecule such as siRNA designed to inhibit a particular immune checkpoint molecule. Preferably the siRNA is capable of preventing or prevents the translation of the immune checkpoint, thus preventing the expression of the protein. Given that the genomic sequence of many immune checkpoints are known, the skilled person would be able to use routine methods to design suitable inhibitory antisense nucleic acid molecules.
- Inhibitors of CTLA-4, PD-1 and PD-L1 are preferred and antibodies thereto are particularly preferred. Such antibodies are known as immune checkpoint-blocking antibodies and can invigorate the antitumour immune response.
- The invention provides methods of treating a tumour and a method of treating tumour cells. The combination therapy should be effective to kill all or a proportion of the target tumour cells or to prevent or reduce their rate of multiplication, or to inhibit metastasis or otherwise to lessen the harmful effect of the tumour on the patient. The clinician or patient should observe improvement in one or more of the parameters or symptoms associated with the tumour. Administration may also be prophylactic and this is encompassed by the term “treatment”. The patient will typically be a human patient but non-human animals, such as domestic or livestock animals may also be treated.
- Cancer targets include melanomas, sarcomas, lymphomas, leukemias, neuroblastomas and glioblastomas (e.g. from the brain), carcinomas and adenocarcinomas. Cancers of the breast, colon, bladder, kidney, liver (e.g. hepatocellular carcinoma), lung, ovary, pancreas, prostate and skin) are preferred targets. Head and neck cancers are also preferred targets. Melanomas, sarcomas and lymphomas are preferred targets. Tumours for treatment are typically solid tumours and may be metastatic lesions that are accessible for transdermal injection.
- The peptides may be synthesised in any convenient way. Generally the reactive groups present (for example amino, thiol and/or carboxyl) will be protected during overall synthesis. The final step in the synthesis will thus be the deprotection of a protected derivative of the invention. In building up the peptide, one can in principle start either at the C-terminal or the N-terminal although the C-terminal starting procedure is preferred. Methods of peptide synthesis are well known in the art but for the present invention it may be particularly convenient to carry out the synthesis on a solid phase support, such supports being well known in the art. A wide choice of protecting groups for amino acids which are used in the synthesis of peptides are known.
- References and techniques for synthesising peptidomimetic compounds and the other bioactive molecules of the invention are described herein and are well known in the art.
- While it is possible for the peptidic compounds (including salts, esters or amides thereof) to be administered as pure compounds, it is preferable to present them as pharmaceutical formulations, i.e. incorporating one or more pharmaceutically acceptable diluents, carriers or excipients.
- The active agents according to the invention may be presented, for example, in a form suitable for oral, topical, nasal, parenteral, intravenal, intratumoral, rectal or regional (e.g. isolated limb perfusion) administration. Unless otherwise stated, administration is typically by a parenteral route, preferably by injection subcutaneously, intramuscularly, intracapsularly, intraspinally. intraperitoneally. intratumourally. transdermally or intravenously. For the peptidic compound, administration is preferably intratumoural. Particularly preferred are intratumoural injections of the peptidic compound of the invention once a day for several consecutive days, e.g. 2, 3, 4, 5, 6 or 7 days, preferably on 2-4 consecutive days or at 2, 3, 4, 5, 6 or 7 daily intervals, e.g. 2-4 times at 5, 6, 7, 8 or 9 daily intervals.
- For the checkpoint inhibitor, administration is preferably intravenous or intralesional.
- The peptidic compound may be administered with or after the immune checkpoint inhibitor, as has been shown to be effective with respect to anti-PD1 and anti-CTLA4 antibodies. Alternatively the peptidic compound may be administered with or before the immune checkpoint inhibitor, as has been shown to be effective with respect to anti-CTLA4 and anti-PD-L1 antibodies. There are preferably multiple administrations. The immune system is preferably stimulated over time. For example, administrations of the peptidic compound and/or the checkpoint inhibitor may be carried out over the course of eight months, preferably four months, more preferably two months; in such regimens administration is preferably weekly. Thus there may be 2 to 40, preferably 3 to 30, e.g. 6 to 30 or 6 to 20 administrations.
- The active compounds defined herein may be presented in the conventional pharmacological forms of administration, such as tablets, coated tablets, nasal sprays, solutions, emulsions, liposomes, powders, capsules or sustained release forms. Conventional pharmaceutical excipients as well as the usual methods of production may be employed for the preparation of these forms. Simple solutions are preferred.
- Organ specific carrier systems may also be used.
- Injection solutions may, for example, be produced in the conventional manner, such as by the addition of preservation agents, such as p-hydroxybenzoates, or stabilizers, such as EDTA. The solutions are then filled into injection vials or ampoules.
- Preferred formulations are those in which the molecules are in saline. Such formulations being suitable for use in preferred methods of administration, especially local administration, i.e. intratumoural, e.g. by injection.
- Unless otherwise stated, dosage units containing the peptidic molecules preferably contain 0.1-10 mg, for example 1-5 mg. The formulation may additionally comprise further active ingredients, including other cytotoxic agents such as other anti-tumour peptides. Other active ingredients may include different types of cytokines e.g. IFN-γ, TNF, CSF and growth factors, immunomodulators, chemotherapeutics e.g. cisplatin or antibodies or cancer vaccines.
- Also provided according to the present invention is the use of a peptidic compound as defined above in the manufacture of a medicament for the treatment of a tumour, wherein said peptidic compound is co-administered with a checkpoint inhibitor as defined above.
- Preferably, the medicament is for the treatment of multidrug resistant (MDR) tumours.
- Also provided according to the present invention is a pharmaceutical pack or composition comprising:
-
- (i) a peptidic compound as defined herein; and
- (ii) a checkpoint inhibitor as described herein.
- With pharmaceutical packs, the components can be for administration separately. The pharmaceutical pack can of course also comprise instructions for administration. The pack and composition are for use in the treatment of a tumour.
- Also provided according to the present invention is a method of treatment of a tumour, comprising the step of administering a peptidic compound as defined herein and a checkpoint inhibitor as described herein, together in pharmaceutically effective amounts, to a patient in need of same.
- As discussed above, the peptidic compounds of the invention are able to destabilise mitochondrial membranes and cause release of DAMPs and antigenic material. This can have a powerful positive effect on the immune system's response to cancer cells. In certain cancer treatments, the immune response is of primary importance, e.g. to treat unidentified secondary tumours, to prevent formation of metastatic tumours, when surgery or other direct intervention is not possible. Different cancers are more or less immunogenic and therefore in some scenarios boosting the immune response to cancer is vital.
- Thus, in a further aspect, the present invention provides a peptidic compound as defined herein for use in the destabilisation of a mitochondrial membrane, wherein said use is in the treatment of a tumour. This can be regarded as an immunotherapeutic use or treatment and the tumour will typically be cancerous. Preferred features and embodiments discussed elsewhere in relation to the combination therapies apply, mutatis mutandis, to this aspect.
- In a further aspect the invention provides a composition comprising or consisting of a peptidic compound of the invention and an immunotherapeutic agent. In a further aspect the invention provides a method of treating tumours in a patient, said method comprising administration of an effective amount of a peptidic compound of the invention and simultaneous or sequential administration of an effective amount of an immunotherapeutic agent.
- Alternatively viewed, there is provided a peptidic compound of the invention and an immunotherapeutic agent for use in the treatment of tumours.
- By “immunotherapeutic agent” is meant an agent which modulates the immune response. Preferably, the immunotherapeutic agent enhances the immune response against one or more tumor antigens, for example by suppressing (preferably selectively) Treg cells and or MDSCs and/or by blocking cytotoxic T lymphocyte antigen-4 (CTLA-4), an inhibitory receptor expressed on T cells. In all aspects and embodiments of the invention, the immunotherapeutic agent is preferably an anti-CTLA-4 agent.
- The skilled person would be able to select suitable dosages of the immunotherapeutic agent.
- The immunotherapeutic agent, e.g. the immune checkpoint inhibitor such as an anti-CTLA-4 agent, is preferably administered prior to or simultaneously with the peptidic compound of the invention, it is most preferred that it is administered prior to the first administration of the peptidic compound of the invention. Preferably, it is administered prior to the peptidic compound of the invention, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to the peptidic compound of the invention. Preferably the first peptidic administration is 1 or 2 days after the last ICI administration. There may be 1 to 8 ICI administrations, e.g. 2 to 4 administrations given 2, 3 or 4 days apart. Multiple doses may be used, which may be e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days apart.
- For example, the anti-CTLA-4 agent ipilimumab is typically administered once every three weeks for a total of four doses. The anti-PD1 agent nivolumab is typically administered every second week, and the anti-PD1 agent pembrolizumab, formerly known as lambrolizumab, is typically administered every third week. Such dosage regimens are preferable.
- Anti-PD-L1 is preferably administered at the same time or after the peptidic compound.
- In some embodiments the peptidic compound is administered on the same day as the first dose of the immunotherapeutic agent, followed by weekly administrations for four to eight weeks, e.g. six weeks, as part of an induction phase. The peptidic compound may then be administered every second week thereafter as part of a maintenance phase.
- Co-administration may be simultaneous or sequential and by the same or different routes of administration, e.g. oral and/or parenteral, or i.p. for the ICI and intra-tumoural for the peptidic compound.
- There is further provided a product containing a peptidic compound of the invention and an immunotherapeutic agent as a combined preparation for separate, simultaneous or sequential use in treating tumours.
- The inventors have also surprisingly found that the treatment of a tumour with a peptidic compound of the invention in conjunction with an immunotherapeutic agent can induce an adaptive immunity against further tumours. The above methods, uses and products (compositions) may therefore optionally extend to the induction of an adaptive immunity against further tumours. Thus, for example, the invention also provides a peptidic compound of the invention and an immunotherapeutic agent for use in the treatment of tumours in a patient and inducing adaptive immunity against tumour growth, development or establishment in said patient.
- Thus, in a further aspect, there is provided a method of inducing adaptive immunity against tumour growth, development or establishment in a patient, said method comprising administration of an effective amount of a peptidic compound of the invention and simultaneous or sequential administration of an effective amount of an immunotherapeutic agent.
- Alternatively viewed, the invention provides a peptidic compound of the invention and an immunotherapeutic agent for use in inducing an adaptive immunity against tumour growth, development or establishment.
- Alternatively viewed, there is provided the use of a peptidic compound of the invention and an immunotherapeutic agent in the manufacture of a medicament for use as a vaccine against tumour growth, development or establishment.
- Thus, there is provided a product containing a peptidic compound of the invention and an immunotherapeutic agent as a combined preparation for separate, simultaneous or sequential use in inducing an adaptive immunity against tumour growth, development or establishment.
- The invention also provides a method of vaccinating a subject against tumour growth, development or establishment through administration of an effective amount of a peptidic compound of the invention and an immunotherapeutic agent to said patient. Reference to a ‘vaccine’ and ‘vaccinating’ both imply a prophylactic effect, thus while there may be beneficial direct treatment of existing tumours, a significant motivation in this aspect of the invention is the prevention or reduction in future tumour growth or development.
- The invention will now be further described in the following Examples and with reference to the figures in which:
-
FIG. 1 is a graph showing the percentage of red blood cell death in a series of experiments to test peptide LTX-315 at varying concentrations. X-axis shows peptide concentration (μg/ml). Y-axis shows % cell death; -
FIG. 2 shows tumour growth in mice re-inoculated with murine A20 B cell lymphoma cells compared with growth in the control animals from the initial study. Diamonds indicate controls from primary studies. Solid squares indicate re-inoculated mice; -
FIG. 3 shows tumour growth in individual mice re-inoculated with murine A20 B cell lymphoma cells having been initially treated with LTX-315. Squares indicateMouse 1. Triangles (base at bottom) indicate Mouse 2. Triangles (base at top) indicateMouse 3. Diamonds indicateMouse 4; -
FIG. 4 shows tumour growth in mice re-inoculated with murine CT26WT colon carcinoma cells compared with growth in the control animals. Diamonds indicate controls from primary studies. Solid squares indicate re-inoculated mice; -
FIG. 5 shows tumour growth in individual mice re-inoculated with murine CT26WT colon carcinoma cells having been initially treated with LTX-315. Small squares indicateMouse 1. Small triangles (base at bottom) indicate Mouse 2. Small triangles (base at top) indicateMouse 3. Small diamonds indicateMouse 4; Circles indicateMouse 5. Large squares indicateMouse 6. Large triangles (base at bottom) indicateMouse 7. Large triangles (base at top) indicateMouse 8. Large diamonds indicateMouse 9; -
FIG. 6 shows growth of A20 B-cell lymphomas in irradiated mice that received splenocytes from donor mice showing complete tumour regression following treatment with LTX-315 (Group 1) or control mice (Group 2) that received splenocytes from naïve donor mice. Squares indicate Group 1 (mice that received splenocytes from donors showing complete regression). Diamonds indicate Group 2 (mice that received splenocytes from naive donors); -
FIG. 7 shows anti-cancer effect of two different treatment regimes on solid murine A20 tumours (Groups 1 and 2) as compared to non-treated controls (Group 3). Inverted solid triangles (base at top) indicate Group 1 (treatment). Open squares indicate Group 2 (treatment +adjuvant). Open triangles (base at bottom) indicate Group 3 (control). Order of tumour size (mm2) atDay 21 is (largest to smallest):Group 3,Group 1, Group 2. -
FIGS. 8 a-8 b LTX-315 causes rapid cell death in human melanoma cells. In vitro cell-killing kinetics of LTX-315 against human melanoma cell line A375 measured with MTT. Results from three experiments are presented for each time point as mean±SD.FIG. 8 a shows IC50 values at different incubation times.FIG. 8 b shows percentage cell viability at different concentrations of L TX-315. -
FIG. 9 LTX-315 internalizes and accumulates close to the mitochondria. A375 cells treated 30 minutes with 1.5 μM fluorescence-labeled LTX-315, and with labeled mitochondria and nucleus. The peptide was internalized and detected in close proximity to the mitochondria. A: overlay channels, B: close up, C: mitochondria. D: peptide -
FIG. 10 Internalization occurs only in lytic 9-mer compounds such as LTX-315 and not in the non-lytic mock peptide LTX-328. A375 cells treated with 3 μM LTX-315 or LTX-328 peptide for 60 min. LTX-315 was detected in the cytoplasm, while LTX-328 was not internalized. A: LTX-315 60 min incubation, B: LTX-328 60 min incubation. -
FIG. 11 LTX-315 treatment causes ultrastructural changes. TEM images of A375 cells treated with LTX-315 for 60 minutes compared to control cells. A&D: untreated control cells, B&E: cells treated with 3,5 μM, C:&F cells treated with 17 μM.Magnification 10 000X A-C, 30 000 D-F, scale bar 5 μm. -
FIG. 12 ROS generation in LTX-315 induced cell death. A375 cells were treated with LTX-315 at different concentrations for 15 minutes. After peptide treatment, carboxy-H2DCFDA was added to the samples and fluorescence was analyzed with a fluorescence plate reader. The experiment was conducted in duplicate, with bars representing mean fluorescence +−S. D. -
FIG. 13 Human melanoma cells treated with LTX-315 release cytochrome-C in the supernatant. Cytochrome-C release in the supernatant after LTX-315 treatment of A375 after designated time points (5, 15, 45 min) were determined by ELISA assay. -
FIG. 14 HMGB1 is released in the supernatant after LTX-315 treatment. A375 human melanoma cells were treated with 35 μM LTX-315 (top) or LTX-328 (bottom), and cell lysate (L) and supernatant (S) were analyzed with Western blot, and the LTX-315-treated cells showed a gradual translocation from the cell lysate to the cell supernatant. Control cells were treated with media alone, and showed no translocation after 60 minutes. -
FIG. 15 Extracellular ATP levels following LTX-315 treatment: A375 cells were treated with LTX-315 for 5 minutes at different concentrations or maintained under controlled conditions, and the supernatant was analyzed for the quantification of ATP secretion by luciferase bioluminescence. Quantitative data (mean +−S. D.) for one representative experiment are reported. -
FIG. 16 LTX-315 disintegrates the mitochondria membrane. TEM images ofFIG. 16 a : human A547 melanoma cells treated with LTX-315 (10 μg/ml) for 60 minutes compared toFIG. 16 b : control cells. -
FIGS. 17 a-17 e Experimental setting in a MCA205 sarcoma model starting with LTX-315 first and boosting with either anti-programmed cell death protein 1 (PD-1) antibodies or anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibodies (a). Tumour regression is seen with LTX-315 combined with CTLA-4 Ab and with LTX-315 combined with PD-1 Ab ((b) and (d)). -
FIGS. 18 a-18 e Experimental setting in a MCA205 sarcoma model starting with either anti-PD-1 antibodies or anti-CTLA-4 antibodies and boosting with weekly LTX-315 (a). Tumour regression is seen with LTX-315 combined with CTLA-4 Ab and LTX-315 combined with PD-1 Ab ((b) and (d)). -
FIGS. 19 a-19 e Experimental setting in a MCA205 sarcoma model starting with either anti-PD-1 antibodies or anti-CTLA-4 antibodies followed by iterative LTX-315 local inoculations (a). Tumour regression is seen with LTX-315 combined with CTLA-4 Ab and LTX-315 combined with PD-1 Ab ((b) and (d)). -
FIGS. 20 a-20 k Adaptive immunity effects of the combination of anti-CTLA-4 antibodies with LTX-315 in a MCA205 sarcoma model. - (a) Experimental setting showing timing of administrations. MCA205 cells are inoculated into the animal's right flank at day −8 and into the animal's left flank at day −4. Anti-CTLA-4 antibody is administered s.c. in the right flank on
days days -
FIG. 21 Combined direct antitumour and adaptive immunity effects of the combination of anti-PD-L1 antibodies with LTX-315 in a EMT-6 murine mammary carcinoma model. - Example 1—that LTX-315 is the most potent of the 5 tested compounds in an in vitro cytotoxic activity study against a panel of 37 human cancer cell lines.
- Example 2—that LTX-315 is the most potent of the 5 tested compounds in an in vitro cytotoxic activity study against a panel of 10 lymphoma cell lines.
- Example 3—that
LTX 315 has a mean EC50 value greater than 1200 μg/ml (833 pM) against human red blood cells. - Example 4—that the anti-tumour activity of LTX-315 resulted in a complete tumour response in 3 of 7 treated mice for the Group receiving the optimal dose (Group 1) in an investigation into the effect of LTX-315 at different dose levels on a murine A20 B-cell lymphoma in mice.
- Example 5—that four different LTX-315 treatment regimes demonstrated a strong anti tumour effect against murine CT26WT (multidrug resistant) tumours.
- Example 6—that LTX-315 has a broad spectrum of activity against various multidrug resistant cancer cell lines and, significantly, a much weaker cytotoxic effect on normal human cells.
- Example 7—that complete tumour regression following initial treatment of solid murine tumours with LTX-315 resulted in a form of endogenous long-term protection against growth of the same tumours following re-inoculation.
- Example 8 —that treatment with LTX-315 may confer long term protection against specific tumours by eliciting an immune response.
- Example 9—that an anti A20 cell immune response have been induced by the injection of the cocktail of LTX-315 and lysed A20 cells.
- Example 10—that treatment with LTX-315 induces hallmarks of immunogenic cell death by mitochondria distortion in human melanoma cells.
- Example 11—that treatment with LTX-315 in combination with an anti-CTLA-4 antibody caused a complete and long-lasting tumor regression in a high proportion of test subjects and induced an adaptive immune response. Anti-PD-1 antibody also showed an ability to act in combination with LTX-315 to inhibit tumour growth.
- Example 12—that treatment with LTX-315 in combination with an anti-PD-L1 antibody caused tumour regression in a high proportion of test subjects and induced an adaptive immune response.
- To determine the concentrations of five novel compounds to obtain a 50% inhibition of proliferation (IC50) Snowfallagainst a panel of 37 human cancer cell lines.
- Test substances, LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329 (see Table 1) provided in powder form.
- Triton X-100 was used as positive control, supplied by Oncodesign (Dijon, France) from Sigma (Saint Quentin Fallavier, France).
- Compounds were stored at 4° C. Powder was first dissolved in serum free culture medium (RPMI 1640, Lonza, Verviers, Belgium) and further diluted using serum-free culture medium to reach appropriate dilutions. Stock solution was not stored and was prepared fresh the day of experiment.
1% (final concentration) Triton X-100 was obtained by dilution using culture medium. - Cancer cell lines and culture media were purchased and provided by Oncodesign. The details of the cell lines is presented in Table 1 below.
-
TABLE 1 Cell lines Origin Source BLOOD CCRF-CEM acute lymphoblastic leukemia, T cells Pharmacell a CCRF- acute lymphoblastic leukemia, T cells Pharmacell CEM/VLB HL-60 acute promyelocytic leukemia, AML, ATCC b pluripotent differentiation HL-60/ADR acute promyelocytic leukemia, AML Pharmacell K-562 chronic myeloid leukemia, pleural ATCC effusion metastasis K-562/Gleevec chronic myeloid leukemia, pleural Oncodesign effusion metastasis RPMI 8226 myeloma, B cells, Igl-type Pharmacell BRAIN SH-SY5Y neuroblastoma, bone marrow ATCC metastasis SK-N-AS neuroblastoma, bone marrow ATCC metastasis U-87 MG glioblastoma, astrocytoma ATCC BREAST MCF-7 invasive ductal carcinoma, pleural Pharmacell effusion metastasis MCF7/mdr adenocarcinoma, pleural effusion Pharmacell metastasis MDA-MB-231 invasive ductal carcinoma, pleural Pharmacell effusion metastasis MDA-MB- invasive ductal carcinoma, pleural ATCC 435S effusion metastasis T-47D invasive ductal carcinoma, pleural ATCC effusion metastasis COLON COLO 205 colorectal adenocarcinoma, ascites ATCC metastasis HCT 116 colorectal carcinoma ATCC HCT-15 colorectal adenocarcinoma ATCC HT-29 colorectal adenocarcinoma ATCC ENDOTHELIUM HUV-EC-C normal ATCC KIDNEY 786-O renal cell adenocarcinoma ATCC A-498 carcinoma ATCC LIVER Hep G2 hepatocellular carcinoma ATCC SK-HEP-1 adenocarcinoma, ascites metastasis ATCC LUNG A549 carcinoma Pharmacell Calu-6 anaplastic carcinoma ATCC NCI-H460 carcinoma, pleural effusion ATCC metastasis OVARY IGROV-1 carcinoma Pharmacell IGROV- carcinoma Pharmacell 1/CDDP NIH:OVCAR-3 adenocarcinoma, ascites metastasis Pharmacell SK-OV-3 adenocarcinoma, ascites metastasis Pharmacell PANCREAS BxPC-3 adenocarcinoma ATCC PANC-1 carcinoma ATCC PROSTATE DU 145 carcinoma, brain metastasis Pharmacell PC-3 adenocarcinoma, bone metastasis ATCC SKIN A-431 epidermoid carcinoma ATCC Malme-3M Malignant melanoma ATCC SK-MEL-2 malignant melanoma, skin metastasis ATCC a - Pharmacell, Paris b - ATCC, Manassas, Virginia, USA - Tumor cells were grown as adherent monolayers or as suspensions at 37° C. in a humidified atmosphere (5% CO2, 95% air). The culture medium was RPMI 1640 containing 2 mM L-glutamine (Lonza, Belgium) and supplemented with 10% fetal bovine serum (FBS, Lonza). For experimental use, the adherent cells were detached from the culture flask by a 5-minute treatment with trypsin-versene (Lonza), diluted in Hanks' medium without calcium or magnesium (Lonza) and neutralized by addition of complete culture medium. Cells were counted in a hemocytometer and their viability was assessed by 0.25% trypan blue exclusion.
- Mycoplasma detection was performed using the MycoAlert (RTM) Mycoplasma Detection Kit (Lonza) in accordance with the manufacturers instructions. All tested cells were found to be negative for mycoplasma contamination.
- Tumor cells were plated in 96-well flat-bottom microtitration plates (Nunc, Dutscher, Brumath, France) and incubated at 37° C. for 24 hours before treatment in 190 μl of drug-free culture medium supplemented or not with 10% FBS for adherent or suspension growing cell lines, respectively.
- Implantation densities for each cell lines are summarized in Table 2 below:
-
TABLE 2 Implantation Implantation densities densities Cell lines (cells/well) Cell lines (cells/well) CCRF-CEM 25,000 HUV-EC-C 20,000 CCRF-CEM/VLB 25,000 786-O 15,000 HL-60 20,000 A-498 15,000 HL-60/ADR 20,000 Hep G2 15,000 K-562 20,000 SK-HEP-1 15,000 K-562/IMR 20,000 A549 15,000 RPMI 8226 20,000 Calu-6 15,000 SH-SY5Y 20,000 NCI-H460 15,000 SK-N-AS 15,000 IGROV-1 15,000 U-87 MG 15,000 IGROV-1/CDDP 15,000 MCF-7 20,000 NIH:OVCAR-3 15,000 MCF7/mdr 20,000 SK-OV-3 15,000 MDA-MB-231 15,000 BxPC-3 15,000 MDA-MB-435S 20,000 PANC-1 15,000 T-47D 15,000 DU 145 15,000 COLO 205 15,000 PC-3 15,000 HCT 116 15,000 A-431 15,000 HCT-15 15,000 Malme-3M 15,000 HT-29 20,000 SK-MEL-2 15,000 - The adherent cell lines were washed once with 200 μl FBS-free culture medium before treatment. Tumor cells were incubated for 4 hours with 10 concentrations of compounds in ¼ dilution step with a top dose of 400 μM (range 4×10−4 to 4×10−10 M), with 1% (final concentration) Triton X-100 as positive control and FBS-free culture medium as negative control. The cells (190 μl) were incubated in a 200 μl final volume of FBS-free culture medium containing test substances at 37° C. under 5% CO2.
- Three independent experiments were performed, each concentration being tested in quadruplicate. Control cells were treated with vehicle alone. At the end of treatments, the cytotoxic activity was evaluated by a MTS assay (see § 3.3.).
- Dilutions of tested compound as well as distribution to plates containing cells were performed using a
Sciclone ALH 3000 liquid handling system (Caliper Life Sciences S. A.). According to automate use, a single range of concentrations was tested whatever the cell lines to be tested. The range was not adapted for each cell line. - The in vitro cytotoxic activity of the test substance was revealed by a MTS assay (BALTROP J. A. et al., Bioorg. Med. Chem. Lett. 1991, 1:611-614) using a novel tetrazolium compound (MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and an electron coupling reagent named PMS (phenazine methosulfate). Like MTT, MTS is bioreduced by cells into a formazan product that is directly soluble in culture medium without processing, unlike MTT.
- At the end of the cells treatment, 40 μl of a 0.22 μm filtered freshly combined solution of MTS (20 ml at 2 mg/ml, Ref G1111, Batch 235897, Exp 03/2009, Promega, Charbonnières, France) and PMS (1 ml at 0.92 mg/ml, Ref P9625, Batch 065K0961, Sigma) in Dulbecco's Phosphate Buffered Saline (DPBS, Ref 17-513Q, Batch 6MB0152, Cambrex), were added in each well. Culture plates were incubated for 2 h at 37° C. Absorbency (OD) were measured at 490 nm in each well using VICTOR3TM 1420 multilabeled counter (Wallac, PerkinElmer, Courtaboeuf, France).
- The dose response inhibition of proliferation (IC) was expressed as follows:
-
- The OD values are the mean of 4 experimental measurements.
- IC50: drug concentration to obtain a 50% inhibition of cell proliferation.
The dose-response curves were plotted using XLFit 3 (IDBS, United Kingdom). The IC50 determination values were calculated using theXLFit 3 software from semi-log curves. Individual IC50 determination values as well as mean and SD values were generated. - Resistance index was calculated using the following formula:
-
- Resistance index was calculated for each compound for each couple of sensitive and resistant cell lines. Individual resistance index was calculated when IC50 values of both sensitive and corresponding resistant cell lines were determined within same experiment. In addition, resistance index was also calculated ratio of mean IC50 values obtained during three independent experiments.
- All thirty seven human tumor cell lines tested were sensitive to LTX-302 compound with IC50 values ranging from 4.83±0.96 μM to 20.09±4.07 μM for T-47D and Hep G2 cell lines, respectively.
- Mean IC50 value for LTX-302 compound obtained on the 37 tumor cell lines was 12.05±4.27 μM with a median value of 11.70 μM. Mean IC50 value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.
- Hematological and lung cancer cell lines were the most sensitive to LTX-302 compound (median IC50 values 7.96 μM (n=7) and 9.02 μM (n=3) for hematological and lung cancer cell lines, respectively) whereas liver cancer cell lines were the most resistant (median IC50 value 17.84 μM, n=2).
- Activity of LTX-302 compound seemed to be slightly decreased by acquired resistance towards doxorubicin as exhibited by the RI values of both HL-60/ADR and MCF-7/mdr cell lines (1.31 and 1.23 for HL-60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, activity of LTX-302 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a RI value of 0.33 for IGROV-1/CDDP cell line.
- All thirty seven (37) human tumor cell lines tested were sensitive to LTX-313 compound with IC50 values ranging from 4.01±0.39 μM to 18.49±4.86 μM for RPMI 8226 and U-87 MG cell lines, respectively.
- Mean IC50 value for LTX-313 compound obtained on the 37 tumor cell lines was 9.60±3.73 μM with a median value of 8.83 μM. Mean IC50 value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.
- Hematological cancer cell lines were the most sensitive to LTX-313 compound (median IC50 value 7.04 μM, n=7) whereas liver cancer cell lines were the most resistant (median IC50 value 13.71 μM, n=2).
- Activity of LTX-313 compound seemed not to be modified by acquired resistance towards doxorubicin as exhibited by the RI values of CCRF-CEM/VLB, HL-60/ADR and MCF-7/mdr cell lines (0.76, 1.16 and 1.24 for CCRF-CEM/VLB, HL-60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, activity of LTX-313 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a RI value of 0.49 for IGROV-1/CDDP cell line.
- All thirty seve human tumor cell lines tested were sensitive to LTX-315 compound with IC50 values ranging from 1.18±0.25 μM to 7.16±0.99 μM for T-47D and SK-OV-3 cell lines, respectively.
- Mean IC50 value for LTX-315 compound obtained on the 37 tumor cell lines was 3.63±1.45 μM with a median value of 3.27 μM. Mean IC50 value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.
- Breast, hematological and lung cancer cell lines were the most sensitive to LTX-315 compound (median IC50 values 2.45 μM (n=5), 2.60 μM (n=7) and 2.83 μM (n=3) for breast, hematological and lung cancer cell lines respectively) whereas liver cancer cell lines were the most resistant (median IC50 value 5.86 μM, n=2).
- Activity of LTX-315 compound seemed to be slightly decreased by acquired resistance towards doxorubicin as exhibited by the RI values of HL-60/ADR and MCF-7/mdr cell lines (1.45 and 1.12 for HL-60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, activity of LTX-315 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a RI value of 0.50 for IGROV-1/CDDP cell line.
- All thirty seven human tumor cell lines tested were sensitive to LTX-320 compound with IC50 values ranging from 3.46±0.22 μM to 16.64±3.15 μM for T-47D and Hep G2 cell lines, respectively.
- Mean IC50 value for LTX-320 compound obtained on the 37 tumor cell lines was 7.58±2.79 μM with a median value of 6.92 μM. Mean IC50 value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.
- Hematological, breast, kidney and brain cancer cell lines were the most sensitive to LTX-320 compound (median IC50 values 6.04 μM (n=7), 6.60 μM (n=5), 6.60 μM (n=2) and 6.92 μM (n=3) for hematological, breast, kidney and brain cancer cell lines respectively) whereas liver cancer cell lines were the most resistant (median IC50 value 11.46 μM, n=2).
- Activity of LTX-320 compound seemed not to be modified by acquired resistance towards doxorubicin as exhibited by the RI values of HL-60/ADR and MCF-7/mdr cell lines (0.90 and 1.19 for HL-60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, activity of LTX-320 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a RI value of 0.49 for IGROV-1/CDDP cell line.
- All thirty seven human tumor cell lines tested were sensitive to LTX-329 compound with IC50 values ranging from 2.43±0.34 μM to 16.90±1.18 μM for T-47D and U-87 MG cell lines, respectively.
- Mean IC50 value for LTX-329 compound obtained on the 37 tumor cell lines was 8.17±3.20 μM with a median value of 7.89 μM. Mean IC50 value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.
- Breast and hematological cancer cell lines were the most sensitive to LTX-329 compound (median IC50 values 4.92 μM (n=5) and 5.26 μM (n=7) for breast and hematological cancer cell lines respectively) whereas ovarian cancer cell lines were the most resistant (median IC50 value 13.37 μM, n=4).
- Activity of LTX-329 compound seemed not to be modified by acquired resistance towards doxorubicin as exhibited by the RI values of CCRF-CEM/VLB, HL-60/ADR and MCF-7/mdr cell lines (0.76, 0.80 and 1.07 for CCRF-CEM/VLB, HL-60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, activity of LTX-329 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a RI value of 0.46 for IGROV-1/CDDP cell line.
- T-47D breast cancer cell line is the most sensitive cell line whatever the LTX compound tested.
- Hematological cancer cell lines are the most sensitive histological type for all five compounds tested, liver and ovarian cancer cell lines being within the most resistant cell lines.
- All five compounds tested exhibited highest activity on IGROV-1/CDDP cell line (resistant to cisplatin) than on parental IGROV-1 ovarian cancer cell line. Doxorubicin resistance seemed to slightly decrease activity of LTX compounds.
- LTX-315 compound is the most potent compound from the five compounds tested.
- All five compounds tested (i.e. LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329) exhibited cytolytic activity against 37 human cancer cell lines tested with IC50 values in micromolar to ten micromolar range.
LTX-315 compound is the most potent compound tested with IC50 values between 1 and 5 micromolar on all 37 human cancer cell lines tested. - To determine the concentrations of five novel compounds to obtain a 50% inhibition of proliferation (IC50) against a panel of 10 lymphoma cell lines.
- Test substances, LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329 (see Table 1) provided in powder form.
- Triton X-100 was used as positive control and supplied by Oncodesign (Dijon, France) from Sigma (Saint Quentin Fallavier, France).
- Compounds were stored at 4° C. Powder was first dissolved in serum free culture medium (RPMI 1640, Lonza, Verviers, Belgium) and further diluted using serum-free culture medium to reach appropriate dilutions. Stock solution was not stored and was prepared fresh the day of experiment.
1% (final concentration) Triton X-100 was obtained by dilution using culture medium. - Cancer cell lines and culture media were purchased and provided by Oncodesign. The details of the cells lines are presented in Table 3 below.
-
TABLE 3 N° Cell lines Origin Source BLOOD 1 Daudi Burkitt's lymphoma, B cells, peripheral ATCC a blood 2 Hs 445 Hodgkin's lymphoma, lymph node ATCC 3 KARPAS- Anaplastic large cell lymphoma, T cells, DSMZ b 299 peripheral blood 4 Mino Mantle cell lymphoma, peripheral blood ATCC 5 NAMALWA Burkitt's lymphoma, B cells, peripheral ATCC blood 6 Raji Burkitt's lymphoma, B cells, peripheral DSMZ blood 7 Ramos Burkitt's lymphoma, B cells, peripheral ATCC blood 8 SU-DHL-1 Anaplastic large cell lymphoma, DSMZ pleural effusion 9 Toledo Non-Hodgkin's B cell lymphoma, ATCC peripheral blood 10 U-937 Lymphoma, histiocytic, macrophage ATCC differentiation, pleural effusion a American Type Culture Collection, Manassas, Virginia, USA b Deutsche Sammlung von Mikroorganismen und Zellkuturen Gmbh, Braunschweig, Germany - Tumor cells were grown as suspensions at 37° C. in a humidified atmosphere (5% CO2, 95% air). The culture medium for each cell line is described in Table 4 below. For experimental use, cells were counted in a hemocytometer and their viability was assessed by 0.25% trypan blue exclusion.
-
TABLE 4 Additives Culture FBS Glucose Glutamine NaPyr Hepes Cell lines medium (%) (g/l) (mM) (mM) (mM) Daudi RPMI 1640 10 — 2 1 10 Hs 445 RPMI 1640 20 4.5 2 1 10 KARPAS- RPMI 1640 20 — 2 — — 299 Mino RPMI 1640 15 4.5 2 1 10 NAMALWA RPMI 1640 10 2.5 2 1 10 Raji RPMI 1640 10 — 2 1 10 Ramos RPMI 1640 10 — 2 1 10 SU-DHL-1 RPMI 1640 10 — 2 — — Toledo RPMI 1640 15 4.5 2 1 10 U-937 RPMI 1640 10 — 2 — — - Mycoplasma detection was performed using the MycoAlert (RTM) Mycoplasma Detection Kit (Lonza) in accordance with the manufacturers instructions. All tested cells were found to be negative for mycoplasma contamination.
- 3.1. Cell lines Amplification and Plating
- Tumor cells were plated in 96-well flat-bottom microtitration plates (Nunc, Dutscher, Brumath, France) and incubated at 37° C. for 24 hours before treatment in 190 μl of drug-free and FBS-free culture medium.
- Implantation densities for each cell lines are summarized in Table 5 below:
-
TABLE 5 Implantation densities N° Cell lines (cells/well) 1 Daudi 25,000 2 Hs 445 25,000 3 KARPAS-299 25,000 4 Mino 25,000 5 NAMALWA 15,000 6 Raji 20,000 7 Ramos 20,000 8 SU-DHL-1 25,000 9 Toledo 25,000 10 U-937 15,000
3.2. IC50 determination - Tumor cells were incubated for 4 hours with 10 concentrations of compounds in ¼ dilution step with a top dose of 400 μM (range 4×10−4 to 4×10−10 M), with 1% (final concentration) Triton X-100 as positive control and FBS-free culture medium as negative control. The cells (190 μl) were incubated in a 200 μl final volume of FBS-free culture medium containing test substances at 37° C. under 5% CO2.
- Three independent experiments were performed, each concentration being issued from quadruplicate. Control cells were treated with vehicle alone. At the end of treatments, the cytotoxic activity was evaluated by a MTS assay (see § 3.3 below).
- Dilutions of tested compound as well as distribution to plates containing cells were performed using a
Sciclone ALH 3000 liquid handling system (Caliper Life Sciences S. A.). According to automate use, a single range of concentrations was tested whatever the cell lines to be tested. The range was not adapted for each cell line. - The in vitro cytotoxic activity of the test substance was revealed by a MTS assay (Baltorp et al.) using a novel tetrazolium compound (MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and an electron coupling reagent named PMS (phenazine methosulfate). Like MTT, MTS is bioreduced by cells into a formazan product that is directly soluble in culture medium without processing, unlike MTT.
- At the end of the cells treatment, 40 μl of a 0.22 μm filtered freshly combined solution of MTS (20 ml at 2 mg/ml, Ref G1111, Batch 235897, Exp 03/2009, Promega, Charbonnières, France) and PMS (1 ml at 0.92 mg/ml, Ref P9625, Batch 065K0961, Sigma) in Dulbecco's Phosphate Buffered Saline (DPBS, Ref 17-513Q, Batch 6MB0152, Cambrex), were added in each well. Culture plates were incubated for 2 h at 37° C. Absorbency (OD) were measured at 490 nm in each well using VICTOR3TM 1420 multilabeled counter (Wallac, PerkinElmer, Courtaboeuf, France).
- All ten human lymphoma cell lines tested were sensitive to LTX-302 compound with IC50 values ranging from 5.30±2.02 μM to 12.54±3.52 μM for U-937 and Raji cell lines, respectively.
- Mean IC50 value for LTX-302 compound obtained on 10 sensitive cell lines was 8.11±2.44 μM with a median value of 7.53 μM.
- All ten human lymphoma cell lines tested were sensitive to LTX-313 compound with IC50 values ranging from 3.21±2.81 μM to 16.08±4.86 μM for Ramos and Raji cell lines, respectively.
- Mean IC50 value for LTX-313 compound obtained on 10 sensitive cell lines was 7.05±3.91 μM with a median value of 5.89 μM.
- All ten human lymphoma cell lines tested were sensitive to LTX-315 compound with IC50 values ranging from 1.15±0.42 μM to 4.93±1.03 μM for U-937 and Raji cell lines, respectively.
- Mean IC50 value for LTX-315 compound obtained on 10 sensitive cell lines was 3.01±1.36 μM with a median value of 2.93 μM.
- All ten human lymphoma cell lines tested were sensitive to LTX-320 compound with IC50 values ranging from 2.22±NA μM to 11.26±3.42 μM for Hs 445 and Raji cell lines, respectively.
- Mean IC50 value for LTX-320 compound obtained on 10 sensitive cell lines was 5.03±2.82 μM with a median value of 4.84 μM.
- All ten human lymphoma cell lines tested were sensitive to LTX-329 compound with IC50 values ranging from 2.46±NA μM to 8.70±1.70 μM for Hs 445 and Raji cell lines, respectively.
- Mean IC50 value for LTX-329 compound obtained on 10 sensitive cell lines was 5.76±2.27 μM with a median value of 5.72 μM.
- 5.6. General comments
- KARPAS-299 and Raji cell lines are the most resistant cell lines whatever the LTX compound tested.
- Hs 445, Ramos and U-937 cell lines are the most sensitive cell lines whatever the LTX compound tested.
- LTX-315 compound is the most potent compound from the five compounds tested.
- All five compounds tested (i.e. LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329) exhibited cytolytic activity against the 10 human lymphoma cell lines tested with IC50 values in micromolar range.
LTX-315 compound is the most potent compound tested with IC50 values between 1 and 5 micromolar on all 10 human lymphoma cell lines tested. - The haemolytic activity of the peptide LTX-315 against human red blood cells was measured.
- Freshly collected human blood was centrifuged at 1500 rpm for 10 minutes in order to isolate the red blood cells. The red blood cells (RBC) were washed three times with PBS [35 mM phosphate buffer with 150 mM NaCl, pH 7.4] by centrifugation at 1500 rpm for 10 minutes, and adjusted to 10% haematocrit with PBS. LTX-315 solutions were added to give a final concentration range of the peptide from 1200 μg/ml to 1 μg/ml and an RBC concentration of 1%. The resulting suspension was incubated with agitation for one hour at 37° C. After incubation the suspension was centrifuged at 4000 rpm for 5 minutes, and the released haemoglobin were monitored by measuring the absorbance of the supernatant at 405 nm. PBS was used as negative control and assumed to cause no haemolysis. 0.1% Triton was used as positive control and assumed to cause complete haemolysis.
- Test substance: LTX-315
- Reference substances: PBS (negative control) and Triton X-100 (positive control). Components of reaction mixtures: LTX-315, 10% Triton X-100, PBS and RBC (10% haematocrit). Details regarding these substances is presented in Table 6 below.
-
TABLE 6 PBS RBC LTX-315/Triton Concentration (μl) (μl) X-100 (μl) Neg. Control 630 70 — Pos. Control 623 70 7 1200 150 50 300 (2 mg/ml stock) 1000 200 50 250 (2 mg/ml stock) 500 325 50 125 (2 mg/ml stock) 100 595 70 35 (2 mg/ml stock) 50 612.5 70 17.5 (2 mg/ml stock) 10 560 70 70 (0.1 mg/ml stock) 1 623 70 7 (0.1 mg/ml stock) - Released haemoglobin was monitored by measuring the absorbance of the supernatant at 405 nm, and percent haemolysis was calculated by the equation:
-
% Haemolysis=[(A 405 LTX-315−A 405PBS)/(A - LTX-315 concentration corresponding to 50% haemolysis (EC50) was determined from a dose-response curve.
- Mean value of five different experiments with standard deviation are presented in Table 7 below.
-
TABLE 7 LTX-315 Mean cell Number Concentration death Standard of (μg/ml) (%) Deviation parallels 1200 37.7 8.1445 3 1000 38.2 9.5760 5 500 20.4 7.8613 5 100 3.6 1.1402 5 50 1.6 0.5477 5 10 0.6 0.8944 5 1 0.0 0.000 5 - The data are also represented in
FIG. 1 .FIG. 1 shows that LTX-315 has a mean value of EC50 higher than 1200 μg/ml (833 μM). - The aim of the study was to investigate the effect of LTX-315 at different dose levels on a murine A20 B-cell lymphoma in mice.
- The administration took place by intratumoural injection of LTX-315 dissolved in sterile saline.
- Female mice were inoculated subcutaneously in the abdomen with 5 million murine A20 cells (ATCC, LGC Promochem AB, Middlesex, England) in a volume of 50 μl. The mice were divided into four groups (see Table 8 below for details). The intratumoural treatment was initiated when the tumours had reached the desired size of approximately 5 mm in diameter (minimum of 20 mm2). Three dose levels of LTX-315, 1 mg (Group 1), 0.5 mg (Group 2) and 0.25 mg (Group 3) per injection, were investigated. The volume was 50 μl for all injections. LTX-315 was dissolved in sterile 0.9% NaCl water solution. This vehicle was used as control (Group 4). All four groups received three injections.
- The mice were monitored during the study by measuring the tumours and weighing the animals regularly. The mice were followed until the maximum tumour burden of 125 mm2 was reached, or until serious adverse events occurred (i.e. wound formation upon repeated treatments during the follow up period), then the mice were sacrificed. A calliper was used for tumour size measurements and weighing and physical examination were used as health control.
- Animals: Specific pathogen-free female Balb/c mice, 6-8 weeks old, supplied form Harlan (England, UK)
- Conditioning of animals: Animals were kept on standard laboratory chow and water.
- Mean body weight, dose, route and treatment schedule is given in Table 8 below.
-
TABLE 8 Number Initial body of weight (g; Schedule Group animals mean ± SE) Treatment Dose Route (Day*) 1 7 20.36 ± 0.56 Once 1 mg in Intra 1, 2, 3 daily 50 μl (20 tumour mg/ml) 2 7 19.96 ± 0.38 Once 0.5 mg in Intra 1, 2, 3 daily 50 μl (10 tumour mg/ml) 3 9 20.11 ± 0.33 Once 0.25 mg Intra 1, 2, 3 daily in 50 μl tumour (5 mg/ml) 4 7 19.73 ± 0.40 Once 50 μl Intra 1, 2, 3 daily 0.9% tumour NaCl in H 20* Day 1 is first day of treatment - The anti-tumour effect of the various treatments is presented as mean tumour size in Table 9 below.
-
TABLE 9 Mean Mean Mean Mean tumour tumour tumour tumour size (mm2) size (mm2) size (mm2) size (mm2) Treatment at day 1*on day 4on day 9on day 14Group 125.82 ± 0.80 0 3.70 ± 2.40 12.43 ± 7.87 Group 2 22.03 ± 0.63 0 11.41 ± 4.69 61.08 ± 23.84 Group 321.25 ± 0.64 20.60 ± 5.71 68.49 ± 12.74 69.42 ± 17.70 Group 422.79 ± 0.68 45.51 ± 5.27 57.79 ± 4.39 84.70 ± 7.35 *Tumour size prior to start of treatment at first day of treatment - The degree of tumour response in the different treatment groups is summarised in Table 10 below.
-
TABLE 10 Free of Tumour Response Relapse Tumour at Animal no partial complete of end of Group response response response Tumour Follow- Up 1 0 42.8% 57.2% 25% 42.8% (3/7) (4/7) (3/7) 2 0 71.42% 28.57% 0% 28.57% (2/7) (0/2) (2/7) 3 77.77% 22.22% 0% NA 0 (0/9) 4 100% NA NA NA NA - In
Group 3, receiving the lowest LTX-315 dose (0.25 mg/dose), a small inhibitory effect is observed during the first days. InGroup 1 and Group 2, receiving LTX-315 doses of 1.0 mg/dose and 0.5 mg/dose respectively, all animals showed partial or complete tumour response. It was found that the anti-tumour activity resulted in a complete tumour response in 3 of 7 treated mice for the Group receiving the optimal dose (Group 1). - Generally stronger necrosis and more wound formation were observed in
Group 1 compared to the other two groups. Except from the wound formation no other adverse events or toxic effects were observed in either of the groups of animals. - Both 1 mg and 0.5 mg of LTX-315 demonstrated a strong and rapid anti tumour effect in the first period of the study. However, as the study progresses more animals in Group 2 relapses than in
Group 1. - The administration takes place by intra-tumoural injection of LTX-315 dissolved in sterile saline (0.9% NaCl in sterile water).
- Each of a total of 40 female mice was inoculated with five million murine CT26WT cells (ATCC, LGC Promochem AB, Boras, Sweden) subcutaneously on the abdomen surface in a volume of 50 μl. The mice were divided into five groups, 8 mice in each group. When the tumours reached the desired size of 20 mm2 the treatment by intra tumoural injection was initiated. Group one was treated solely on
day 1, Group two onday 1 and 2, Group three onday day - The mice were monitored during the study by measuring the tumours (digital calliper) and weighing the animals regularly. The mice were followed until the maximum tumour burden of 125 mm2 was reached, or until serious adverse events occurred (i.e. wound formation due to repeated injections), then the mice were sacrificed. Weighing and physical examination were used as health controls.
- Animals: Specific pathogen-free female Balb/c mice, 6-8 weeks old, supplied form Harlan (England, UK)
- Conditioning of animals: Standard animal facility conditions. Mean body weight, dose, route and treatment schedule is given in Table 11 below.
-
TABLE 11 Num- Initial body ber of weight (g; Treat- Schedule Group animals mean ± SE) ment Dose Route (Day*) 1 8 19.00 ± 1.087 Once 1 mg in Intra 1 daily 50 μl (20 tumour mg/ml) 2 8 19.56 ± 1.087 Once 1 mg in Intra 1, 2 daily 50 μl (20 tumour mg/ml) 3 8 19.41 ± 0.8999 Once 1 mg in Intra 1, 3 daily 50 μl (20 tumour mg/ml) 4 8 19.00 ± 0.9396 Once 1 mg in Intra 1, 2, 3 daily 50 μl (20 tumour mg/ml) 5 8 18.71 ± 0.7868 Once 50 μl Intra 1, 2, 3 (con- daily 0.9% tumour trol) NaCl in H 20* Day 1 is first day of treatment - The anti-tumour effect of the various treatments is presented as mean tumour size in Table 12 below.
-
TABLE 12 Mean tumour Mean Mean Mean size tumour tumour tumour (mm2) at size (mm2) size (mm2) size (mm2) Treatment day 1* on day 6on day 10on day 17Group 122.69 ± 0.4070 4.343 ± 2.295 7.171 ± 4.035 3.712 ± 3.712 Group 2 22.90 ± 1.155 1.458 ± 1.458 5.058 ± 4.014 6.644 ± 3.430 Group 321.43 ± 1.141 2.983 ± 2.983 10.85 ± 7.553 0.00 ± 0.00 Group 424.09 ± 1.653 0.00 ± 0.00 0.00 ± 0.00 1.308 ± 1.308 Group 521.39 ± 1.683 33.77 ± 3.168 48.37 ± 7.035 40.64 ± 19.77 *Tumour size prior to start of treatment at first day of treatment - Complete tumour response was observed in the vast majority of all animals treated with LTX-315. The degree of tumour response in the different treatment groups is summarised in Table 13 below.
-
TABLE 13 Free of Tumour Response Relapse Tumour at Animal no partial complete of end of Group response response response Tumour Follow- Up 1 0 27.5% 62.5% 20% 50% (1/5) (4/8) 2 0 12.5% 87.5% 71% 25% (5/7) (2/8) 3 12.5% 0 87.5% 29% 62.5% (2/7) (5/8) 4 0 0 100% 37.5% 62.5% (8/8) (5/8) 5 100% NA NA NA NA (8/8) - The treatment was started when the tumours had reached the desired size of a minimum of 20 mm2 and animals were sacrificed when the tumours reached the maximum tumour burden of 125 mm2.
- End of study was defined as
day 17 when six out of eight control animals (Group 5) were sacrificed. - All LTX-315 treatment regimes resulted in a strong anti CT26WT-tumour effect.
- Totally 27 of the 32 treated animals were observed with a complete tumour response and four with a partial response. Only one animal (in Group 3) did not have a response to the treatment. The results presented show that all four treated groups have very similar overall tumour response, the data also indicate that the degree of relapse of tumour was higher in Group 2 than in
Group FIG. 2 ). - Necrosis and complete tumour response was observed in all the treated groups. In
Group 1 four out of eight animals, in Group 2 two out of eight animals, inGroup 3 five out of eight animals, and inGroup 4 five out of eight animals showed complete tumour response. At this stage the tumour was completely necrotic and a wound crust formed at the location of the tumour. - Necrosis at the tumour site was seen in all treatment groups. Generally, animals in
Group Group 1 that were given only one injection of LTX-315.Group 4 animals, which were given three injections, showed the most necrosis, wound and crust formation. The difference in necrosis betweenGroup 1 andGroup 4 was quite large but the animals given the highest number of treatments seemed to cope well. No toxic or other adverse effects besides local necrotic tissue and wound formation were observed in either of the treated groups of animals. - All four treatment regimes of LTX-315 tested demonstrated a strong anti tumour effect against murine CT26WT tumours.
- The amount of necrosis, wound and crust formation was proportional to the number of LTX-315 treatments given.
- Characteristics of the cell lines tested are presented in Table 14 below.
-
TABLE 14 Drug IC50 Cell line susceptibility Origin μM HL-60 Sensitive Acute promyelocytic leukemia 2.07 HL-60/ADR Resistant Acute promyelocytic leukemia 3.01 MCF-7 Sensitive Breast carcinoma 1.94 MCF-7/mdr Resistant Breast carcinoma 1.96 IGROV-1 Sensitive Ovary carcinoma 6.37 IGROV- Resistant Ovary carcinoma 3.19 1/CDDP K-562 Sensitive Chronic myeloid leukemia 3.27 K5627/ Resistant Chronic myeloid leukemia 2.98 Gleevec HUV-EC-C — Normal endothelial cells 23 RBC — Red blood cells 833 - The above data shows the broad spectrum of activity of LTX-315 against various cancer cell lines and, significantly, a much weaker cytotoxic effect on normal human cells.
- Re-challenge with murine A20 B-cell lymphoma and murine CT26WT colon carcinoma cells in mice with complete tumour regression.
- This study sought to investigate the effects of tumour growth in animals that had previously shown complete tumour regression following treatment with LTX-315.
- Methods: Female Balb-c mice (n=4), previously treated with LTX-315, 1 mg) or (n=9); previously treated with LTX-315 0.5 or 1 mg) were re-inoculated (s.c. in the abdominal area) with either murine A20 B cell lymphoma cells or CT26WT colon carcinoma cells (5 million) respectively 6 weeks following initial treatment with LTX-315. Tumour growth was monitored for up to 36 days following re-inoculation.
- Significant inhibition (P<0.006) of tumour growth was observed in all 4 mice treated previously with LTX-315 (1 mg) in study R315-03 compared with control animals (
FIG. 2 ) and while relapse was seen in 1 animal, 3 weeks later, complete tumour regression was observed in the other 3 mice (FIG. 3 ). - In 9 mice previously treated with LTX-315 (0.5 or 1 mg) inhibition (P<0.01) of tumour growth was observed in comparison with control animals (
FIG. 3 ). The sudden drop in tumour size inFIG. 20 , afterDay 18, is explained by the death of 6 animals bearing large tumours. Inhibition was observed in 7 mice and complete regression in 2 of the animals (FIG. 5 ). - Taken together these data suggest that complete tumour regression following initial treatment of solid murine tumours (murine A20 B cell lymphoma or CT26WT colon carcinoma) with LTX-315 resulted in a form of endogenous long-term protection against growth of the same tumours following re-inoculation. Inhibition of tumour growth was more pronounced in animals bearing A20 B cell lymphoma tumours when compared with animals bearing CT26WT colon tumours.
- Immunological effects of LTX-315 in a murine A20 B-cell lymphoma model. An in vivo adoptive spleen cell transfer pilot study.
- This study was undertaken to investigate whether the long-term protection against growth of the same tumours following re-inoculation in animals observed in study R315-33 could be passively transferred to naive recipients via spleen cells taken from LTX-315 -treated donor animals.
- Ten female Balb/c mice (n=32) were each inoculated with A20 cells (5 million in 50 μL s.c.) on the abdominal surface. Once tumours had reached 20mm2 they were injected with LTX-315 (1 mg) injected intratumourally, once daily for 3 days, in a volume of 50 μL. Tumour size (mm2) and body weight were subsequently monitored and a further injection of LTX-315 was given if any tumour re-growth was observed. Subsequently, mice showing complete tumour regression were sacrificed and used as donors for transfer of splenocytes while naive donor mice were used as controls. Spleens from donor mice were excised and cells isolated. Naive receiver mice were irradiated and divided into 2 groups.
Group 1 received isolated splenocytes from cured mice, whereas group 2 received isolated splenocytes from naive mice. Freshly prepared cells were injected (20×106 per 100 μl) via the tail vein. Twenty four hours later receiver mice were inoculated with 5 million murine A20 B-cell lymphoma cells on the abdominal surface as described above. Tumour size and body weight were monitored until the maximum tumour burden of ˜125mm2 was reached, or a serious adverse events occurred (i.e. wound formation due to tumour tissue necrosis) at which point mice were sacrificed. - Inhibition of tumour growth was observed in irradiated mice that received splenocytes isolated from animals that had shown complete tumour regression following treatment with LTX-315 when compared with control animals that received splenocytes from naive donors (
FIG. 6 ). It was also noted that there was a difference in the colour and texture of the tumours in recipients of splenocytes from LTX-315-treated mice suggesting an immediate inflammatory response. - Based on these observations, the data provides evidence for an adaptive immune response in the animals that received splenocytes from animals that previously showed complete regression of A20-B lymphoma tumours following treatment with LTX-315. This data suggests that treatment with LTX-315 may confer long term protection against specific tumours by eliciting an immune response.
- The objective of the study was to investigate the anti-cancer effect of prophylactic vaccination with A20 lymphoma cells lysed by 10 mg/ml LTX-315:
- (i) alone; and
- (ii) in combination with 20 mg/ml LTX-315 injected at the vaccination site prior to the vaccine.
- In total, two different treatment regimens were used.
- Administration was by subcutaneous injection of LTX-315 dissolved in growth media containing A20 lymphoma cells. The cell-LTX-315 “cocktail” was left for 30 min prior to injection in order to assure complete lysis of the cancer cells.
- Group 1 (“vaccine”) mice were injected subcutaneously on the abdomen surface with 50 μl of a “cocktail” of ten million murine A20 cells (ATCC, LGC Promochem AB, Boras, Sweden) and 10 mg/ml LTX-315 (“A20 lysate”). Group 2 (“vaccine +adjuvant”) mice were treated as per
Group 1, but in addition were given 25 μl of 20 mg/ml LTX-315 subcutaneously at the site ofvaccination 5 minutes prior to the A20 lysate injection. Group 3 (“control”) mice received no treatment. - Six weeks after the treatment, all mice were inoculated with 5 million viable A20 B-cell lymphoma cells subcutaneously on the abdomen surface in a volume of 50 μl.
- The mice were monitored during the study by measuring the tumour size and weighing the animals regularly. The mice were followed until the maximum tumour burden of ˜130 mm2 was reached, at which point the mice were sacrificed.
- Animals: Specific pathogen-free female Balb/c mice, 6-8 weeks old, supplied from Harlan Laboratories (England, UK; www.harlan.com)
- Conditioning of animals: Standard animal facility conditions at the University of Tromsø.
- Test substance: Murine A20 cells lysed by LTX-315 (Lot 1013687), and LTX-315 (Lot 1013687) alone
- Test substance preparation: 10×106 A20 cells were added to a 50 μl 10mg/ml LTX-315/vehicle (“A20 lysate”). The test substance was ready for
use 30 minutes after mixing. LTX-315 alone was dissolved in 0.9% NaCl in sterile H2O - Vehicle: RPMI-1640 w/2mM L-glutamine or 0.9% NaCl in sterile H2O
- Reference substances: Not applicable
- Treatment of controls: Not applicable
- Method of evaluation: Tumour size measurements and health control by weighing and examination
- Additional data regarding method: A digital calliper was used for tumour size measurements and weighing and physical examination were used as health control
- Mean body weight, dose, route and treatment schedule are shown in Table 15 (below).
-
TABLE 15 Initial body No of weight (g; Treat- Cell numbers Group animals mean ± SE) ment and dose Route 1 8 17.31 ± Once 10 × 106 A20 Sub- 0.3815 cells in cutaneous 50 μl LTX-315 (10 mg/ml) 2 8 17.14 ± Once 0.25 μl LTX-315 Sub- 0.4633 20 mg/ml) + cutaneous 10 × 106 A20 cells in 50 μl LTX-315 (10 mg/ml) 3 7 17.29 ± Not Not applicable Not 0.3020 treated applicable - The anti cancer effect of the various treatments is presented as mean tumour size in Table 16 below and a graphical presentation of the data is provided in
FIG. 7 . In Table 16,Day 1 was the day of inoculation of viable A20 cells six weeks post-vaccination. -
TABLE 16 Mean tumour Mean tumour Mean tumour Mean tumour size (mm2) size (mm2) size (mm2) size (mm2) Treatment at day 4on day 11 on day 16 on day 21Group 19.515 ± 1.528 20.44 ± 6.191 36.21 ± 10.30 55.89 ± 15.27 Group 2 7.315 ± 2.231 17.13 ± 5.078 29.13 ± 7.903 47.16 ± 13.54 Group 310.25 ± 3.100 34.49 ± 8.298 56.04 ± 8.339 82.89 ± 14.06 - The inoculation of viable A20 B-cell lymphoma cells was accomplished 6 weeks after the treatment was given (day 1) and the animals were sacrificed when the tumours reached the maximum allowed tumour burden of ˜130 mm2.
- The results show that the tumours developed more slowly in both LTX-315/A20-lysate treatment Groups as compared to the control Group. The median survival of
Group 1 was 28 days, 33 days forGroup 2, and 25 days for the control group (Group 3). Increase in median survival was 12% forGroup - The data indicate a prolonged survival of the treated groups compared to the untreated control group. On day 34, when the last animal in the control group was sacrificed, 50% of the animals in Group 2 were still alive while 37.5% of the animals in
Group 1 were still alive. End of study was defined asday 60. At this time-point, a total of 3 of the 16 treated animals had a complete regression of an initially developing tumour and were tumour free. At the end of thestudy 25% of animals fromGroup 1, and 12.5% of animals from Group 2 were observed to be tumour free. - Macroscopically there were morphological differences between the treated groups (
Group 1 and 2) compared to the non-treated control group (Group 3). The developing tumours in the two treatment groups were observed to be whiter and harder than the tumours observed in the control group. This finding together with the slower growth rate of the tumours indicates that an anti-A20 cell immune response was induced by the vaccination with the cocktail of LTX-315 and lysed A20 cells. - Hence, LTX-315 may have a dual use by lysing the tumour cells and inducing release of danger signals from normal cells at the injection site.
- In this study, we investigated the tumoricidal effect of LTX-315 on human melanoma cells. The peptide internalized and was shown in association with mitochondria, ultimately leading to a lytic cell death. The LTX-315 peptide was designed to treat solid tumors with intratumoral injections through a two-stage mode of action: the first is the collapse of the tumor itself, while the second is the released damage-associated molecular pattern molecules (DAMPs) from the dying tumor cell, which can induce a subsequent immune protection against recurrences and metastastis.
- LTX-315 and LTX-328 (K-A-Q-Dip-Q-K-Q-A-W-NH2) were made on request by Bachem AG (Bubendorf, Switzerland) and Innovagen (Lund, Sweden), respectively. LTX-315 Pacific Blue and LTX-328 Pacific Blue were purchased on request from Innovagen (Lund, Sweden) Norud (Tromso, Norway), respectively.
- The A375 cell line A375 (ECACC, 88113005) is a human malignant melanoma derived from patient material, and was purchased from Public Health England (PHE Culture Collections, Porton Down, Salisbury,UK). Cells were maintained as monolayer cultures in high glucose 4.5% DMEM supplemented with 10% FBS and 1% L-glutamine, but not as antibiotics (complete media). The cell line was grown in a humidified 5% CO2 atmosphere at 37° C., and was regularly tested for the presence of mycoplasma with MycoAlert (Lonza).
- The cytotoxic effect of LTX-315 was investigated using the colorimetric MTT viability assay as described in Eliassen et al. (2002), 22(5): pp2703-10. The A375 cells were seeded at a concentration of 1×105 cells/ml in a volume of 0.1 ml in 96-well plates, and allowed to adhere in a complete growth media overnight. The media was then removed and the cells were washed twice in serum-free, RPMI-1650 media, before adding LTX-315 dissolved in serum-free RPMI at concentrations ranging from 2.5-300 μg/ml, and incubated for 5-180 minutes. Cells treated with a serum-free RPMI were used as negative control cells, while cells treated with 1% Triton X-100 in serum-free media were used as a positive control. The final results were calculated using the mean of three experiments, each with triplicate wells.
- Live cell imaging with unlabeled cells—A375 cells were seeded at 10,000 cells/well in a complete media in Nunc Lab-Tec 8-wells chambered covered glass (Sigma) precoated with 25 μg/ml human fibronectin (Sigma) that were allowed to adhere overnight. Cells were washed twice with a serum-free RPMI, treated with peptide dissolved in RPMI and investigated using Bright on a Leica TCS SP5 confocal microscope, with a 63X/1.2W objective. The microscope was equipped with an incubation chamber with CO2 and temperature control.
- Fixed cells, mitotracker—Cells were seeded as for live cell imaging, and treated with Mitotracker CMH2XROS (Invitrogen) at 100 nm for 15 minutes prior to peptide treatment. Cells were treated with 17 μM LTX-315, with negative control serum-free RPMI only. After 60 min of incubation, cells were analyzed using a Zeiss microscope. All confocal imaging experiments were subsequently conducted at least twice with similar results.
- Fixed cells, fluorescence-labeled peptide—Subconfluential A375 cells were seeded at 8,000 cells/well as above, and transfected on the second day using the Lipofectamine LTX with Plus transfection reagents (Invitrogen) following the manufacturer's protocol. The mitochondria were labeled using the pDsRed2-Mito, and the nucleus was labeled using the GFP-Histon2B plasmid (Imaging Platform, University of Tromso). A day after transfection, cells were washed twice with serum-free RPMI, and treated at different concentration and incubation periods with LTX-315 Pacific Blue or LTX-328 Pacific Blue. LTX-315 PB exhibited a similar cytotoxic profile as the unlabeled LTX-315 as determined by MTT assay. Control cells were treated with unlabeled LTX-315 and also with serum-free RPMI only. After incubation, cells were fixed with 4% paraformaldehyde in PBS, and the wells were covered with Prolong Gold antifade (Invitrogen). Cells were further analyzed by use of a Leica TCS SP5 confocal microscope, with a 693, 1.2 W objective. Pacific Blue, GFP and Ds Red were excited using UV, with 488 and 561 lasers, and fluorescence channels were sequentially detected using the following band passes: UV: 420-480nm (with attenuation), 488: 501-550nm and 561: 576-676nm.
- A375 cells were seeded at 1×105 cells per well in 6-well plates and allowed to grow for three days to optimize membrane structures in the culture, and the media was changed on the second day. Cells were washed twice in serum-free RPMI before being treated with LTX-315 dissolved in serum-free RPMI at 5, 10 and 25 μg/ml, with serum-free RPMI as a negative control. Cells were then washed with PBS twice before fixation for 24 hours in 4° C. with 4% formaldehyde and 1% gluteralaldehyde in a Hepes buffer at pH 7.8. Dehydration and post-fixation protocols included incubation in a 5% buffered tannic acid and incubation in a 1% osmium-reduced ferrocyanide. Ultrathin sections were prepared, and uranyl acetate (5%) and Reynolds's lead citrate were used for staining and contrasting. Samples were examined on a JEOL JEM-1010 transmission electron microscope, and images were taken with an Olympus Morada side-mounted TEM CCD camera (Olympus soft imaging solutions, GmbH, Germany).
- A DCFDA cellular reactive oxygen species detection assay kit was purchased from abcam®, and A375 cells seeded in a 96-well Costar black clear bottom plate with 20,000 cells per well incubated in 37° C. 16 hours prior to DCFDA assay. Cells were washed with a 100 μL/well of pre-warmed PBS one time, and incubated with 20 μM of DCFDA in a buffer solution supplied with the kit at 37° C. in a cell culture incubator for 45 min, and then washed again with a buffer solution of 100 μL/well. The cells were then stimulated with a 100 μL/well LTX-315 peptide dissolved in a buffer solution at concentrations of 17 μM for 30 min, and cells not treated were used as a negative control. The fluorescence intensity was determined at an excitation wavelength of 485nm and an emission wavelength of 530 nm on a FLUOstar Galaxy plate reader.
- A375 cells were seeded with 3×105 cells/well in 6-well plates in a complete media, and allowed to adhere overnight. Cells were treated with LTX-315 or LTX-328 at 35 μM, and incubated at 37° C. and 5% CO2 for different time points (5, 10, 15, 30, 60 min), and negative controls were serum-free RPMI-1650. Supernatants (S) were collected and centrifuged at 1,400g for five minutes, and cell lysates (L) were harvested after washing with PBS twice and then subsequently lysed using a 4X Sample buffer (Invitrogen, number), 0.1 M DTT (Sigma number) and water. Supernatants were concentrated using Amicon Ultra 50K centrifugal filters (Millipore UFC505024), and the cell lysate was sonicated. Both supernatants and lysate were boiled and resolved in a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then electro transferred to a polyvindiline difluoride (PVDF) membrane (Millipore). The membrane was blocked in 5% milk and incubated with the HMGB1 antibody (rabbit, polyclonal, abcam ab 18256); the membrane was then rinsed several times with TBST, incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (abcam ab6721), rinsed again with TBST and then developed using WB Luminol Reagent (Santa Cruz Biotechnology, Heidelberg, Germany).
- A375 cells were seeded as with HMGB1 studies, and treated with 35 μM for different time points (5, 15, 45). Supernatants were collected and concentrated as with HMGB1 studies, and samples from the supernatants were analyzed using a 4.5 hour solid form Cytochrome C- Elisa kit (R&D Systems, USA, #DCTCO) following the manufacturer's description. Shortly thereafter, a 50% diluted sample was analyzed and the optical density was determined using a microplate reader set at 450 nm, and this reading was then subtracted from the reading at 540 nm. A standard curve was generated for each set of samples assayed. Samples were run in four parallels, and the cytochrome-c released into the supernatant was expressed as a fold over the level of cytochrome-c in the supernatant of untreated cells.
- The supernatant of LTX-315-treated A375 cells was analyzed using an Enliten ATP luciferase assay kit (Promega, USA). Cells were then seeded as with an ROS assay, and treated with LTX-315 in different incubation times, from 1 to 15 minutes with two parallels, which was then conducted three times. Negative controls were untreated A375 cells exposed to serum-free media alone. Samples were diluted at 1:50 and 1:100, and analyzed with a Luminoscan RT luminometer according to the manufacturer's protocol.
- All data represent at least two independent experiments with at least two parallels, which were expressed as the mean ±SD. Cytochrome-C release and ATP release data was compared using one-way ANOVA and a multiple comparison test, and we considered the P-value <0.05 to indicate statistical significance.
- To investigate the effect of LTX-315 on A735 melanoma cells in vitro, we determined the IC-50 values for the peptide by a cell viability assay MTT at different incubation times. The IC-50 value was 30 μM after only five minutes of incubation, and progressed to 14 μM after 90 minutes. Further incubation up to 180 minutes did not offer any additional effect (
FIG. 8 ). - We next wanted to assess the cell morphology of A735 melanoma cells treated with LTX-315. Cells were treated with LTX-315 at an IC-50 value, and investigated by bright field confocal microscopy. Treated cells displayed a rapid change from a normal epithelial morphology to a total collapse of the cells with an extrusion of cytoplasmic content, which was preceded by a rounding up of the cell (data not shown). These changes typically occurred within 15-60 minutes at an IC-50 value in the majority of the cells.
- To investigate the internalization and fate of the peptide within the cells, LTX-315 was labeled with Pacific Blue and incubated with cells at concentrations of 3 μM and 1.5 μM, respectively. The labeled LTX-315 rapidly penetrated the plasma membrane and at 1.5 μM, the peptide showed an accumulation around the mitochondria after 30 minutes of incubation but was not detected in the cell nucleus (
FIG. 9 ). The labeled non-lytic mock-sequence peptide LTX-328 did not demonstrate any internalization at any concentration or incubation time tested (FIG. 10 ). - We further evaluated the ultrastructural changes in treated cells by performing transmission electron microscopy (TEM), in which A375 cells were treated with either peptides dissolved directly in media or in media alone. A significant number of the cells treated with a low concentration (3.5 μM) of the LTX-315 peptide for 60 minutes showed vacuolization, as well as some altering of the mitochondrial morphology (
FIG. 11 ). The mitochondria appeared to be less electron-dense, also exhibiting some degree of reorganization, with the cristae lying further apart or not visible at all. The number of necrotic cells in these samples was less than 5%. In these low concentrations, vacuolization of the cytoplasm was observed. Another common finding in these samples were peripherally placed vacuoles, which were lined with a single membrane layer containing a homogenous material (FIG. 11B ). When cells were treated with higher concentration (17 μM) for 60 min, approximately 40% of them displayed a necrotic morphology with a loss of plasma membrane integrity (FIG. 11C &E). The cells that were still intact displayed a great heterogeneity, from a normal appearance with microvilli to a round appearance, with mitochondria clearly affected. In this high concentration, only 4% of the cells investigated displayed vacuolization, and chromatin condensation was not visible in this material at any peptide concentration tested. These results demonstrate that LTX-315 kills the tumor cells with a lytic mode of action, while lower concentrations cause the cells to undergo ultrastructure changes, such as vacuolization and an altered mitochondrial morphology. Moreover, no significant morphological changes suggestive of apoptotic cell death were observed. - In a separate experiment, exposure of LTX-315 at 10 μg/ml to human A547 cells (an ovarian melanoma cell line) led to disintegration of the mitochondrial membrane (
FIG. 16 ). - DAMPs are molecules that are released from intracellular sources during cellular damage. DAMPs can initiate and perpetuate an immune response through binding to Pattern Recognition Receptors (PRRs) on Antigen Presenting Cells (APCs). Among commonly known DAMPs are ATP, HMGB1, Calreticulin, Cytochrome C, mitochondrial DNA and Reactive oxygen species (ROS). We next wanted to investigate whether ATP was released into the supernatant from cells treated with LTX-315. Hence, the supernatant from treated and non-treated cells analyzed using luciferase detection assay. As shown in
FIG. 15 , ATP was detected in the supernatant as early as after 5 minutes of treatment with LTX-315, and the release was concentration-dependent. - To assess whether LTX-315-treated cells released cytochrome-C into the medium, A375 cells were treated with LTX-315 at 35 μM at different time points (5, 15, 45 min). The supernatant was subsequently analyzed using an ELISA assay. Cells treated with 35 μM value had three times more cytochrome-C in the supernatant compared to untreated control cells. The increase in cytochrome-C was detected after only five minutes of treatment, and there was also an increase after 15 and 45 minutes of peptide treatment, respectively (
FIG. 13 ). - HMGB1 is a non-histone, chromatin-binding nuclear protein. Once passively released from necrotic cells, HMGB1 is able to trigger the functional maturation of dendritic cells, cytokine stimulation and chemotaxis among several immunopotentiating effects.
- HMGB1 is normally found in the cell nucleus and would be expected in a cell lysate of healthy cells, though not in the culture media (supernatant). In order to assess the release of HMGB1 from LTX-315-treated cells, we measured the translocation and free HMGB1 from the nuclear compartment within the cell lysate into the cell supernatant.
- Both cell lysate and the cell supernatant of LTX-315- and LTX-328-treated A375 melanoma cells were analyzed using a Western blot. Cells were treated with 35 μM of either LTX-315 or LTX-328, with a gradual translocation from the cell lysate to the supernatant detected in the LTX-315-treated melanoma cells, but not in the cells treated with the mock sequence peptide LTX-328 or a serum-free medium only (
FIG. 14 ). - The ROS generation following LTX-315 treatment was measured by CH2DCFDA fluorometric assay. Significant amounts of ROS were generated after 15 minutes of incubation with LTX-315, and the ROS levels were concentration-dependent (
FIG. 12 ). - LTX-315 labeled with the fluorescent molecule Pacific Blue was internalized within minutes after incubation with A375 melanoma cells, and was distributed in the cytoplasm (
FIG. 9 ). At low concentrations, accumulation of the peptide around the mitochondria was evident, whereas at higher concentrations the peptide was more spread within the cytoplasm and accumulated in circular structures closer to the cell membrane (FIG. 10 ). If the peptide attacks the mitochondrial membrane, a decrease or even a total collapse of the mitochondrial membrane potential would be expected. A confocal imaging of cells with the membrane potential-dependent mitochondrial stain Mitotracker CMXh2ROS showed a loss of mitochondrial signal a short time after peptide treatment (data not shown). The loss of the signal shows that the peptide interaction with the mitochondria causes a loss of mitochondrial membrane potential, which is crucial for the mitochondria's most important cellular functions. An altered mitochondrial morphology was also demonstrated with TEM. Cells treated with LTX-315 for 60 minutes had less electron-dense mitochondria with an altered organization of the cristae, as well as vacuolization within the mitochondria compared to untreated cells (FIG. 11 ). Furthermore, vacuolization was evident in approximately 20% of cells treated with 3.5 μM of LTX-315. When the mitochondria are dysfunctional, free oxygen radicals (ROS) may be formed, and by using fluorometric assays we demonstrated ROS formation within a few minutes after peptide treatment (FIG. 12 ). - In this study, we demonstrate that treatment with the LTX-315 peptide causes an increase in ROS levels in A375 melanoma cells after treatment. One explanation for these higher levels of ROS following peptide treatment could be that the peptide enters the cells and targets the mitochondria, and the dysfunctional mitochondria then releases ROS. Through an ELISA assay, we detected the release of cytochrome-C in the supernatant of peptide-treated cells after only a few minutes of treatment (
FIG. 13 ). Cytochrome-C is a mitochondrial protein released from the intermembrane space and into the cytosol when the outer mitochondrial membrane is perturbed, and by binding to the apoptotic protease activating factor-1 (Apaf-1) it is also a part of the apoptotic cascade that eventually leads to cell death by apoptosis. However, if cytochrome-C is found in the extracellular space, it has been reported to act as a pro-inflammatory mediator, thus activating NF-kB and inducing cytokine and chemokine production. The transition of HMGB1 from the cellular compartment to the extracellular compartment was detected using a western blot (FIG. 14 ). When the nuclear protein HMBG1 is released into the extracellular fluid, it functions as a DAMP, and can bind to both the PRR TLRs and to the RAGE receptors; the activation of these may lead to a number of inflammatory responses such as the transcription of pro-inflammatory cytokines. We also detected ATP released in the supernatant after peptide incubation (FIG. 15 ), and presented extracellularly it functions as a DAMP by activating the purinerg P2RX7 receptors on the DC. This receptor not only functions as a pore that opens for small cationic and later bigger molecules after binding to ATP, its activation also causes the processing and release of the pro-inflammatory cytokine IL-1β. - In summary, our data suggests that LTX-315 induces lytic cell death in cancer cells, not only by direct attack on the plasma membrane, but also as a result of an injury to vital intracellular organelles after the internalization of the peptide at concentrations too low to cause an immediate loss of plasma membrane integrity. We demonstrate that the peptide treatment causes the release of several DAMPs such as CytC, ATP, HMGB1 and ROS. The DAMPs may affect the cellular integrity of the damaged cells in several ways, but are also associated with so-called immunogenic cell death. The release of tumor-specific antigens into the extracellular compartment, together with potent immune stimulatory molecules (DAMPs) such as ATP, CytC and HMGB1, can give a strong immune response. In turn, these factors will lead to a maturation and activation of DCs and other accessory cells of the adaptive immune system.
- The combination of LTX-315 with either an anti-programmed cell death protein 1 (PD-1) antibody or an anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody was assayed in a mouse MCA205 sarcoma model. Seven animals were studied per group. For each study, two independent experiments were performed yielding identical results. Anova statistical analyses were performed.
- The anti-PD-1 antibody used in this study was a murine IgG isotype. The anti-CTLA-4 antibody was a rat IgG isotype. Both antibodies were purchased from eBioscience.
-
FIGS. 17 a, 18 a, 19 a and 20 a show the timings and administration routes of LTX-315 and the antibodies in the various studies. Thus, at day −8 mice were inoculated with MCA205 cancer cells and palpable tumours were allowed to form. LTX-315 was then administered intratumorally on the days shown in the Figures and the anti-PD-1 or anti- CTLA-4 antibodies were administered i.p. on the days shown. - An adaptive immunity study using the combination of anti-CTLA-4 antibody and LTX-315 was carried out in a similar manner to that previously discussed.
- The findings of these studies are discussed in the legend of
FIGS. 17 to 20 . As the effects of anti-PD1 antibodies occur in the local tumour microenvironment (anti-CTLA-4 works in lymphatic tissue), it is predicted that the combination of the peptidic compound with anti-PD1 antibodies would be more pronounced in a clinical setting in comparison to this single tumour model which is not representative of metastatic disease. - The combination of LTX-315 with an anti-programmed
cell death protein 1 ligand (PD-L1) antibody was assayed in a mouse EMT-6 mammary carcinoma model. - The anti-PD-L1 antibody had the following characteristics: ref: BE0101, Bioxcell; clone 10F.9G2; reactivity: mouse; isotype: Rat IgG2b. LTX-315 was prepared at a dose of 0.5 mg/50 μL in 0.9% sodium chloride solution. Anti-PD-L1 antibody was prepared at a concentration in phosphate-buffered saline and was administered at a dose of 10 mg/kg. LTX-315 was injected into the tumour grafted on the right flank of the mice, anti-PD-L1 antibody was injected into the peritoneal cavity of the mice.
- The EMT-6 cell line was established from a transplantable murine mammary carcinoma that arose in a BALB/cCRGL mouse after implantation of a hyperplastic mammary alveolar nodule (VOLENEC FJ., et al., J Surg Oncol. 13(1):39-44, 1980).
- Cell culture conditions: EMT-6 tumor cells were grown as a monolayer at 37° C. in a humidified atmosphere (5% CO2, 95% air). The culture medium was RPMI 1640 containing 2 mM L-glutamine (ref: BE12-702F, Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (ref: 3302, Lonza). EMT-6 tumor cells are adherent to plastic flasks. For experimental use, tumor cells were detached from the culture flask by a 5-minute treatment with trypsin-versene (ref: BE17-161E, Lonza), in Hanks' medium without calcium or magnesium (ref: BE10-543F, Lonza) and neutralized by addition of complete culture medium. The cells were counted in a hemocytometer and their viability were assessed by 0.25% trypan blue exclusion assay.
- Use of mice: Healthy female Balb/C mice, 6-8 weeks old at reception, were obtained from CHARLES RIVER (L'Arbresles, France) and from Janvier (France). Mice were maintained in SPF health status according to the FELASA guidelines. Mouse housing and experimental procedures were realized according to the French and European Regulations (Principe d'éthique de I'expérimentation animale, Directive n°2010/63 CEE du 22 septembre 2010, Décret n°2013-118 du 01 février 2013) and the NRC Guide for the Care and Use of Laboratory Animals (NRC Guide for the Care and Use of Laboratory Animals). The animal facility was authorized by the French authorities (Agreement
N° B 21 231 011 EA). All procedures using mice were submitted to the Animal Care and Use Committee of Oncodesign (Oncomet) agreed by French authorities (CNREEA agreement N° 91). Mice were individually identified with a RFID transponder and each cage was labeled with a specific code. - Housing conditions: Mice were maintained in housing rooms under controlled environmental conditions: Temperature: 22±2° C., Humidity 55±10%, Photoperiod (12 h light/12 h dark), HEPA filtered air, 15 air exchanges per hour with no recirculation. Mice enclosures provided sterile and adequate space with bedding material, food and water, environmental and social enrichment (group housing) as described: Top filter polycarbonate Eurostandard Type III or IV cages, Corn cob bedding (ref:
LAB COB 12, SERLAB, France), 25 kGy Irradiated diet (Ssniff® Soest, Germany), Complete food for immunecompetent rodents—R/M-H Extrudate, Sterile, filtrated at 0.2 μm water, and Environmental enrichment (SIZZLE-dri kraft—D20004 SERLAB, France). - Induction of MET-6 tumours in mice: A first tumor was induced by subcutaneous injection of 1×106 of EMT-6 cells in 200 μL of RPMI 1640 into the right flank of Balb/C female mice. The day of tumor cell injection in the right flank was considered as D0. A second tumor was induced by subcutaneous injection of 1×105 of EMT-6 cells in 200 μL of RPMI 1640 into the left flank of Balb/C female mice. The day of tumor cell injection in the left flank was considered as D3.
- Treatment schedule: Mice were randomized according to their body weight on D3 into four groups each of 5 mice (group 1) or 10 mice (
groups - The treatment schedule was as follows: the mice from
group 1 were not treated; the mice fromgroup 3 received a total of 3 intratumoral injections of LTX135; the mice fromgroup 5 received a total of 6 IP injections of anti-PD-L1; and the mice fromgroup 7 received a total of 3 intratumoral injections of LTX315 and a total of 6 IP injections of anti-PD-L1. - The treatment schedule is summarized in Table 17 below:
-
TABLE 17 No. Treatment Group Mice Treatment Schedule G1 5 Untreated — G3 10 LTX315 Q1Dx3 G5 10 Anti-PD- L1 Q2Dx6 G7 10 LTX315 Q1Dx3 Anti-PD-L1 Q2Dx6 Q1Dx3 = one injection for 3 consecutive days (total of 3 injections). Q2DX6 = one injection every 2 days (total of 6 injections). - Mouse monitoring: All study data, including mouse body weight measurements, tumour volume, clinical and mortality records, and treatment were scheduled and recorded on Vivo Manager® database (Biosystemes, Dijon, France). The viability and behaviour was recorded every day. Body weights were measured thrice a week. The length and width of the tumor was measured three times a week with calipers and the volume of the tumour was estimated by the following formula: tumour volume=(width2 ×length)/2 (SIMPSON-HERREN L. et al. Cancer Chemotherapy Rep., 54: 143, 1970).
- Statistical Tests: All statistical analyses were performed using Vivo manager® software (Biosystemes, Couternon, France). Statistical analyses of mean body weights, MBWC, mean tumor volumes at randomization, mean tumor volumes, mean times to reach mean tumour volumes and mean tumor doubling times were performed using ANOVA and pairwise tests were performed using the Bonferroni/Dunn correction in case of significant ANOVA results. A p value <0.05 was considered as significant.
- As shown in
FIG. 21 , 50% of the test mice survived for more than 50 days after tumour induction when administered with both LTX-315 and anti-PD-L1 antibody, compared to 40% of the test mice treated with anti-PD-L1 antibody alone, 30% of the test mice treated with LTX-315 alone and none of the untreated mice. This shows that the combination therapy is more effective at both directly treating tumours (treating the tumour in the right flank of the mouse) and inducing an adaptive immunity response (treating the tumour in the left flank of the mouse). - Details regarding the percentage of mice that show total tumour regression in the right and/or left flank are shown in Table 18 below. Mice were tested at day 52 after inoculation or when sacrificed.
-
TABLE 18 Proportion of Group Flank Mice G1 Right 0% Left 0% G3 Right 30% Left 10% G5 Right 20% Left 20% G7 Right 20% Left 40% - The table shows that the combination of LTX-315 and anti-PD-L1 antibody is particularly effective in inducing adaptive immunity, as 40% of the mice treated with the combination (G7) showed total regression of the tumour in the left flank, compared to 10% of mice treated with LTX-315 alone (G3), 20% of mice treated with anti-PD-L1 antibody alone (G5) and none of the untreated mice (G1).
- Median and mean tumour volume for each cohort was measured and while G3 and G5 always gave significantly lower volumes than G1, G7 was always clearly the best performing cohort.
- It will be appreciated that it is not intended to limit the present invention to the above specific embodiments only, numerous embodiments, modifications and improvements being readily apparent to one of ordinary skill in the art without departing from the scope of the appended claims.
Claims (14)
1. A method of treating a tumour in a subject in need thereof, comprising combined, sequential or separate administration to said subject of an immune checkpoint inhibitor and a compound having the following characteristics:
a) consisting of 9 amino acids in a linear arrangement;
b) of those 9 amino acids, 5 are cationic and 4 have a lipophilic R group; and
c) at least one of said 9 amino acids is a non-genetically coded amino acid;
wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.
2. The method of claim 1 , wherein the compound is of formula:
wherein C represents a cationic amino acid and L represents an amino acid with a lipophilic R group and in which one of the amino acids having a lipophilic R group is a non-genetically coded amino acid, said compound optionally in the form of a salt, ester or amide.
3. The method of claim 1 , wherein each lipophilic R group has at least 9 non-hydrogen atoms.
4. The method of claim 1 , wherein each lipophilic R group has at least one cyclic group.
5. The method of claim 1 , in which 1 to 3 of the amino acids with lipophilic R groups are tryptophan.
6. The method of claim 1 , wherein the compound incorporates a non-genetically coded amino acid selected from the group consisting of:
2-amino-3-(biphenyl-4-yl)propanoic acid (biphenylalanine), 2-amino-3,3-diphenylpropanoic acid (diphenylalanine), 2-amino-3-(anthracen-9-yl)propanoic acid, 2-amino-3-(naphthalen-2-yl)propanoic acid, 2-amino-3-(naphthalen-1-yl) propanoic acid, 2-amino-3-[1,1′:4′,1″-terphenyl-4-yl]propionic acid, 2-amino-3-(2,5,7-tri-tert-butyl-1H-indol-3-yl)propanoic acid, 2-amino-3-[1,1′:3′,1″-terphenyl -4-yl]-propionic acid, 2-amino-3-[1,1′:2′,1″-terphenyl-4-yl]propionic acid, 2-amino-3-(4-naphthalen-2-yl-phenyl)-propionic acid, 2-amino-3-(4′-butylbiphenyl -4-yl)propanoic acid, 2-amino-3-[1,1′:3′,1″-terphenyl-5′-yl]propionic acid and 2-amino-3-(4-(2,2-diphenylethyl)phenyl)propanoic acid.
7. The method of claim 1 , wherein the compound has the formula of SEQ ID NO: 23, or a salt, ester or amide thereof.
8. The method of claim 1 , wherein the immune checkpoint inhibitor is selected from the group consisting of nivolumab, pembrolizumab and pidilizumab.
9. The method of claim 8 , wherein the immune checkpoint inhibitor is pembrolizumab.
11. The method of claim 1 , wherein the compound is a peptide.
12. The method of claim 1 , wherein the compound has the further characteristic that lipophilic and cationic residues are arranged such that there are no more than two of either type of residue adjacent to one another.
13. The method of claim 1 , wherein the compound has the further characteristic that the molecule comprises two pairs of adjacent cationic amino acids and one or two pairs of adjacent lipophilic residues.
14. The method of claim 7 , wherein the immune checkpoint inhibitor is selected from the group consisting of nivolumab, pembrolizumab and pidilizumab.
15. The method of claim 7 , wherein the immune checkpoint inhibitor is pembrolizumab.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/366,463 US20230201305A9 (en) | 2014-12-11 | 2021-07-02 | Immune checkpoint inhibitor combinations |
Applications Claiming Priority (11)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB201422084 | 2014-12-11 | ||
GB1422084.2 | 2014-12-11 | ||
GB2015-04-10 | 2014-12-11 | ||
GB1506127.8 | 2015-04-10 | ||
GBGB1506127.8A GB201506127D0 (en) | 2015-04-10 | 2015-04-10 | Immune checkpoint inhibitor combinations |
JP2015-118495 | 2015-06-11 | ||
JP2015118495A JP6767096B2 (en) | 2014-12-11 | 2015-06-11 | Combination of immune checkpoint inhibitors |
PCT/EP2015/075722 WO2016091487A1 (en) | 2014-12-11 | 2015-11-04 | Immune checkpoint inhibitor combinations |
JP2015-11-04 | 2015-11-04 | ||
US201715534800A | 2017-06-09 | 2017-06-09 | |
US17/366,463 US20230201305A9 (en) | 2014-12-11 | 2021-07-02 | Immune checkpoint inhibitor combinations |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/534,800 Continuation US11083774B2 (en) | 2014-12-11 | 2015-11-04 | Immune checkpoint inhibitor combinations |
PCT/EP2015/075722 Continuation WO2016091487A1 (en) | 2014-12-11 | 2015-11-04 | Immune checkpoint inhibitor combinations |
Publications (2)
Publication Number | Publication Date |
---|---|
US20210401933A1 US20210401933A1 (en) | 2021-12-30 |
US20230201305A9 true US20230201305A9 (en) | 2023-06-29 |
Family
ID=56384106
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/534,800 Active US11083774B2 (en) | 2014-12-11 | 2015-11-04 | Immune checkpoint inhibitor combinations |
US17/366,463 Pending US20230201305A9 (en) | 2014-12-11 | 2021-07-02 | Immune checkpoint inhibitor combinations |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/534,800 Active US11083774B2 (en) | 2014-12-11 | 2015-11-04 | Immune checkpoint inhibitor combinations |
Country Status (11)
Country | Link |
---|---|
US (2) | US11083774B2 (en) |
EP (1) | EP3230318B1 (en) |
JP (1) | JP6767096B2 (en) |
AU (3) | AU2015359881B2 (en) |
DK (1) | DK3230318T3 (en) |
ES (1) | ES2819282T3 (en) |
HU (1) | HUE051268T2 (en) |
LT (1) | LT3230318T (en) |
PL (1) | PL3230318T3 (en) |
PT (1) | PT3230318T (en) |
WO (1) | WO2016091487A1 (en) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP6767096B2 (en) * | 2014-12-11 | 2020-10-14 | リティックス バイオファーマ エイエス | Combination of immune checkpoint inhibitors |
US11666646B2 (en) | 2016-12-26 | 2023-06-06 | National University Corporation Kobe University | Cancer therapy utilizing combination of oral tumor vaccine and immunosuppression inhibitor |
WO2018188761A1 (en) * | 2017-04-13 | 2018-10-18 | Lytix Biopharma As | Method of reducing population size of tregs and/or mdscs |
JPWO2019240218A1 (en) | 2018-06-14 | 2021-07-01 | 株式会社明治 | Compositions for facilitating immune checkpoint inhibition therapy |
KR20220024701A (en) | 2019-06-20 | 2022-03-03 | 케모센트릭스, 인크. | Compounds for the treatment of PD-L1 disease |
CN110317245B (en) * | 2019-08-02 | 2021-04-09 | 郑州大学 | LAG-3 protein affinity cyclic peptide and application thereof |
PE20221764A1 (en) | 2019-10-16 | 2022-11-11 | Chemocentryx Inc | HETEROARYL-BIPHENYL AMINES FOR THE TREATMENT OF PD-L1 DISEASES |
WO2021142088A1 (en) * | 2020-01-07 | 2021-07-15 | The Trustees Of The University Of Pennsylvania | Source specific exosomes for determining avoidance of cancer treatment and avoidance of checkpoint inhibitor therapies |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5811097A (en) | 1995-07-25 | 1998-09-22 | The Regents Of The University Of California | Blockade of T lymphocyte down-regulation associated with CTLA-4 signaling |
CN1309569A (en) | 1998-05-04 | 2001-08-22 | 托马斯杰斐逊大学 | Composition comprising tumor cells and extracts and method for using thereof |
GB0605685D0 (en) | 2006-03-21 | 2006-05-03 | Lytix Biopharma As | Inhibition of tumour growth |
DK2206517T3 (en) * | 2002-07-03 | 2023-11-06 | Ono Pharmaceutical Co | Immunopotentiating compositions comprising anti-PD-L1 antibodies |
GB0821616D0 (en) * | 2008-11-26 | 2008-12-31 | Lytix Biopharma As | Compounds |
EP3375791A1 (en) * | 2009-09-30 | 2018-09-19 | Memorial Sloan Kettering Cancer Center | Combination immunotherapy for the treatment of cancer |
US9410206B2 (en) * | 2011-11-30 | 2016-08-09 | John Wayne Cancer Institute | Long noncoding RNA (lncRNA) as a biomarker and therapeutic marker in cancer |
US10202454B2 (en) * | 2013-10-25 | 2019-02-12 | Dana-Farber Cancer Institute, Inc. | Anti-PD-L1 monoclonal antibodies and fragments thereof |
PE20161209A1 (en) * | 2014-02-21 | 2016-11-10 | Abbvie Stemcentrx Llc | CONJUGATES OF ANTI-DROSOPHILA SIMILAR ANTIBODIES TO DELTA 3 (ANTI-DLL3) AND DRUGS FOR USE IN THE TREATMENT OF MELANOMA |
EP3230498B1 (en) * | 2014-12-09 | 2023-01-18 | Merck Sharp & Dohme LLC | System and methods for deriving gene signature biomarkers of response to pd-1 antagonists |
JP6767096B2 (en) * | 2014-12-11 | 2020-10-14 | リティックス バイオファーマ エイエス | Combination of immune checkpoint inhibitors |
JP6813258B2 (en) * | 2014-12-11 | 2021-01-13 | リティックス バイオファーマ エイエス | Chemotherapy combination |
-
2015
- 2015-06-11 JP JP2015118495A patent/JP6767096B2/en active Active
- 2015-11-04 LT LTEP15793746.7T patent/LT3230318T/en unknown
- 2015-11-04 HU HUE15793746A patent/HUE051268T2/en unknown
- 2015-11-04 WO PCT/EP2015/075722 patent/WO2016091487A1/en active Application Filing
- 2015-11-04 PL PL15793746T patent/PL3230318T3/en unknown
- 2015-11-04 PT PT157937467T patent/PT3230318T/en unknown
- 2015-11-04 ES ES15793746T patent/ES2819282T3/en active Active
- 2015-11-04 AU AU2015359881A patent/AU2015359881B2/en active Active
- 2015-11-04 EP EP15793746.7A patent/EP3230318B1/en active Active
- 2015-11-04 US US15/534,800 patent/US11083774B2/en active Active
- 2015-11-04 DK DK15793746.7T patent/DK3230318T3/en active
-
2019
- 2019-05-07 AU AU2019203199A patent/AU2019203199B2/en active Active
-
2021
- 2021-06-25 AU AU2021204356A patent/AU2021204356A1/en active Pending
- 2021-07-02 US US17/366,463 patent/US20230201305A9/en active Pending
Also Published As
Publication number | Publication date |
---|---|
AU2019203199B2 (en) | 2021-04-01 |
US11083774B2 (en) | 2021-08-10 |
AU2019203199A1 (en) | 2019-05-30 |
HUE051268T2 (en) | 2021-03-01 |
LT3230318T (en) | 2020-10-12 |
WO2016091487A1 (en) | 2016-06-16 |
AU2015359881B2 (en) | 2019-02-07 |
PL3230318T3 (en) | 2021-03-08 |
ES2819282T3 (en) | 2021-04-15 |
AU2015359881A1 (en) | 2017-07-20 |
US20210401933A1 (en) | 2021-12-30 |
EP3230318A1 (en) | 2017-10-18 |
DK3230318T3 (en) | 2020-08-31 |
PT3230318T (en) | 2020-09-21 |
AU2021204356A1 (en) | 2021-07-22 |
EP3230318B1 (en) | 2020-06-24 |
JP2016128399A (en) | 2016-07-14 |
JP6767096B2 (en) | 2020-10-14 |
US20180264079A1 (en) | 2018-09-20 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20230201305A9 (en) | Immune checkpoint inhibitor combinations | |
US9241968B2 (en) | Nonapeptide with anti-tumour activity | |
EP4292606A2 (en) | Compositions for treating cancer | |
US11123398B2 (en) | Chemotherapeutic combinations of cationic antimicrobial peptides and chemotherapeutics | |
WO2016091490A1 (en) | Chemotherapeutic combinations of cationic antimicrobial peptides and chemotherapeutics | |
AU2017214321B2 (en) | Methods of generating populations of tumour-infiltrating T cells |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE AFTER FINAL ACTION FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: ADVISORY ACTION MAILED |