NZ615742B2 - Methods for increasing efficacy of folr1 cancer therapy - Google Patents
Methods for increasing efficacy of folr1 cancer therapy Download PDFInfo
- Publication number
- NZ615742B2 NZ615742B2 NZ615742A NZ61574212A NZ615742B2 NZ 615742 B2 NZ615742 B2 NZ 615742B2 NZ 615742 A NZ615742 A NZ 615742A NZ 61574212 A NZ61574212 A NZ 61574212A NZ 615742 B2 NZ615742 B2 NZ 615742B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- folr1
- staining
- seq
- sample
- cancer
- Prior art date
Links
- 230000001965 increasing effect Effects 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims description 103
- 238000011275 oncology therapy Methods 0.000 title description 3
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 245
- 238000010186 staining Methods 0.000 claims abstract description 214
- 239000000523 sample Substances 0.000 claims abstract description 149
- 230000014509 gene expression Effects 0.000 claims abstract description 130
- 201000011510 cancer Diseases 0.000 claims abstract description 101
- 238000001514 detection method Methods 0.000 claims abstract description 75
- 230000027455 binding Effects 0.000 claims abstract description 69
- 101001023230 Homo sapiens Folate receptor alpha Proteins 0.000 claims abstract description 68
- 102100035139 Folate receptor alpha Human genes 0.000 claims abstract description 67
- 229940127121 immunoconjugate Drugs 0.000 claims abstract description 56
- 102000036639 antigens Human genes 0.000 claims abstract description 50
- 108091007433 antigens Proteins 0.000 claims abstract description 50
- 239000000427 antigen Substances 0.000 claims abstract description 48
- 239000003814 drug Substances 0.000 claims abstract description 32
- 239000012634 fragment Substances 0.000 claims abstract description 30
- 239000013074 reference sample Substances 0.000 claims abstract description 27
- 229940127089 cytotoxic agent Drugs 0.000 claims abstract description 23
- 231100000599 cytotoxic agent Toxicity 0.000 claims abstract description 23
- 239000002254 cytotoxic agent Substances 0.000 claims abstract description 20
- 238000004519 manufacturing process Methods 0.000 claims abstract description 7
- 230000003432 anti-folate effect Effects 0.000 claims abstract description 3
- 229940127074 antifolate Drugs 0.000 claims abstract description 3
- 239000004052 folic acid antagonist Substances 0.000 claims abstract description 3
- 238000003364 immunohistochemistry Methods 0.000 claims description 46
- 238000011282 treatment Methods 0.000 claims description 44
- 206010033128 Ovarian cancer Diseases 0.000 claims description 41
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 32
- 239000008188 pellet Substances 0.000 claims description 28
- 239000003153 chemical reaction reagent Substances 0.000 claims description 26
- 239000003795 chemical substances by application Substances 0.000 claims description 26
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims description 26
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 24
- -1 N-maleimidopropionamido Chemical group 0.000 claims description 22
- 150000002500 ions Chemical class 0.000 claims description 22
- 230000002611 ovarian Effects 0.000 claims description 22
- 102000004190 Enzymes Human genes 0.000 claims description 19
- 108090000790 Enzymes Proteins 0.000 claims description 19
- 208000020816 lung neoplasm Diseases 0.000 claims description 17
- 229960002685 biotin Drugs 0.000 claims description 12
- 235000020958 biotin Nutrition 0.000 claims description 12
- 239000011616 biotin Substances 0.000 claims description 12
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 11
- 239000002253 acid Substances 0.000 claims description 11
- 201000005202 lung cancer Diseases 0.000 claims description 11
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 claims description 8
- 206010014759 Endometrial neoplasm Diseases 0.000 claims description 7
- 150000002148 esters Chemical class 0.000 claims description 7
- UOWVMDUEMSNCAV-WYENRQIDSA-N rachelmycin Chemical compound C1([C@]23C[C@@H]2CN1C(=O)C=1NC=2C(OC)=C(O)C4=C(C=2C=1)CCN4C(=O)C1=CC=2C=4CCN(C=4C(O)=C(C=2N1)OC)C(N)=O)=CC(=O)C1=C3C(C)=CN1 UOWVMDUEMSNCAV-WYENRQIDSA-N 0.000 claims description 7
- JSHOVKSMJRQOGY-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-(pyridin-2-yldisulfanyl)butanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCSSC1=CC=CC=N1 JSHOVKSMJRQOGY-UHFFFAOYSA-N 0.000 claims description 6
- GTBCXYYVWHFQRS-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-(pyridin-2-yldisulfanyl)pentanoate Chemical compound C=1C=CC=NC=1SSC(C)CCC(=O)ON1C(=O)CCC1=O GTBCXYYVWHFQRS-UHFFFAOYSA-N 0.000 claims description 6
- AMRJKAQTDDKMCE-UHFFFAOYSA-N dolastatin Chemical compound CC(C)C(N(C)C)C(=O)NC(C(C)C)C(=O)N(C)C(C(C)C)C(OC)CC(=O)N1CCCC1C(OC)C(C)C(=O)NC(C=1SC=CN=1)CC1=CC=CC=C1 AMRJKAQTDDKMCE-UHFFFAOYSA-N 0.000 claims description 6
- VQNATVDKACXKTF-XELLLNAOSA-N duocarmycin Chemical compound COC1=C(OC)C(OC)=C2NC(C(=O)N3C4=CC(=O)C5=C([C@@]64C[C@@H]6C3)C=C(N5)C(=O)OC)=CC2=C1 VQNATVDKACXKTF-XELLLNAOSA-N 0.000 claims description 6
- 229940002612 prodrug Drugs 0.000 claims description 6
- 239000000651 prodrug Substances 0.000 claims description 6
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 claims description 6
- 229960005532 CC-1065 Drugs 0.000 claims description 5
- 206010014733 Endometrial cancer Diseases 0.000 claims description 5
- YZCKVEUIGOORGS-NJFSPNSNSA-N Tritium Chemical compound [3H] YZCKVEUIGOORGS-NJFSPNSNSA-N 0.000 claims description 5
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 5
- 230000002285 radioactive effect Effects 0.000 claims description 5
- 229910052722 tritium Inorganic materials 0.000 claims description 5
- NNPUPCNWEHWRPW-UHFFFAOYSA-N 4-(pyridin-2-yldisulfanyl)-2-sulfobutanoic acid Chemical compound OC(=O)C(S(O)(=O)=O)CCSSC1=CC=CC=N1 NNPUPCNWEHWRPW-UHFFFAOYSA-N 0.000 claims description 4
- 229940049706 benzodiazepine Drugs 0.000 claims description 4
- 229930195731 calicheamicin Natural products 0.000 claims description 4
- 229960005501 duocarmycin Drugs 0.000 claims description 4
- 229930184221 duocarmycin Natural products 0.000 claims description 4
- YACHGFWEQXFSBS-RJXCBBHPSA-N leptomycin Chemical class OC(=O)/C=C(C)/C[C@H](C)[C@@H](O)[C@H](C)C(=O)[C@H](C)/C=C(\C)/C=C/C[C@@H](C)\C=C(/CC)\C=C\[C@@H]1OC(=O)C=C[C@@H]1C YACHGFWEQXFSBS-RJXCBBHPSA-N 0.000 claims description 4
- 229920001223 polyethylene glycol Polymers 0.000 claims description 4
- SVUOLADPCWQTTE-UHFFFAOYSA-N 1h-1,2-benzodiazepine Chemical compound N1N=CC=CC2=CC=CC=C12 SVUOLADPCWQTTE-UHFFFAOYSA-N 0.000 claims description 3
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 claims description 3
- CSLUPULZHVYVJA-UHFFFAOYSA-N OC(C(C=CC=C1C(CI)=O)=C1NN(C(CC1)=O)C1=O)=O Chemical compound OC(C(C=CC=C1C(CI)=O)=C1NN(C(CC1)=O)C1=O)=O CSLUPULZHVYVJA-UHFFFAOYSA-N 0.000 claims description 3
- 108010044540 auristatin Proteins 0.000 claims description 3
- HXCHCVDVKSCDHU-LULTVBGHSA-N calicheamicin Chemical compound C1[C@H](OC)[C@@H](NCC)CO[C@H]1O[C@H]1[C@H](O[C@@H]2C\3=C(NC(=O)OC)C(=O)C[C@](C/3=C/CSSSC)(O)C#C\C=C/C#C2)O[C@H](C)[C@@H](NO[C@@H]2O[C@H](C)[C@@H](SC(=O)C=3C(=C(OC)C(O[C@H]4[C@@H]([C@H](OC)[C@@H](O)[C@H](C)O4)O)=C(I)C=3C)OC)[C@@H](O)C2)[C@@H]1O HXCHCVDVKSCDHU-LULTVBGHSA-N 0.000 claims description 3
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 claims description 3
- SHIBSTMRCDJXLN-KCZCNTNESA-N digoxigenin Chemical compound C1([C@@H]2[C@@]3([C@@](CC2)(O)[C@H]2[C@@H]([C@@]4(C)CC[C@H](O)C[C@H]4CC2)C[C@H]3O)C)=CC(=O)OC1 SHIBSTMRCDJXLN-KCZCNTNESA-N 0.000 claims description 3
- 108091005418 scavenger receptor class E Proteins 0.000 claims description 3
- UQVNRKBFAXNOGA-LWTNMJDUSA-N (E)-tomaymycin Chemical class CO[C@H]1NC2=CC(O)=C(OC)C=C2C(=O)N2C\C(=C\C)C[C@@H]12 UQVNRKBFAXNOGA-LWTNMJDUSA-N 0.000 claims description 2
- ORKSBKSSOSUJOY-UHFFFAOYSA-N 2,5-dioxo-1-[4-(pyridin-2-yldisulfanyl)pentanoyloxy]pyrrolidine-3-sulfonic acid Chemical compound C=1C=CC=NC=1SSC(C)CCC(=O)ON1C(=O)CC(S(O)(=O)=O)C1=O ORKSBKSSOSUJOY-UHFFFAOYSA-N 0.000 claims description 2
- 229930188854 dolastatin Natural products 0.000 claims description 2
- 230000002163 immunogen Effects 0.000 claims description 2
- 102000050083 Class E Scavenger Receptors Human genes 0.000 claims 2
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 claims 1
- 101000652736 Homo sapiens Transgelin Proteins 0.000 claims 1
- 102100031013 Transgelin Human genes 0.000 claims 1
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 claims 1
- SXNQAQBMEWMYAU-UHFFFAOYSA-N sulfo butanoate Chemical compound CCCC(=O)OS(O)(=O)=O SXNQAQBMEWMYAU-UHFFFAOYSA-N 0.000 claims 1
- VKBKSLDOBKBYSW-UHFFFAOYSA-N sulfo pentanoate Chemical compound CCCCC(=O)OS(O)(=O)=O VKBKSLDOBKBYSW-UHFFFAOYSA-N 0.000 claims 1
- 102000005962 receptors Human genes 0.000 abstract description 3
- 108020003175 receptors Proteins 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 209
- 210000001519 tissue Anatomy 0.000 description 121
- 238000003556 assay Methods 0.000 description 44
- 125000005842 heteroatom Chemical group 0.000 description 37
- 239000011230 binding agent Substances 0.000 description 32
- 108090000765 processed proteins & peptides Proteins 0.000 description 30
- 210000004881 tumor cell Anatomy 0.000 description 28
- 108090000623 proteins and genes Proteins 0.000 description 25
- 238000012360 testing method Methods 0.000 description 25
- 241001465754 Metazoa Species 0.000 description 24
- 102000004169 proteins and genes Human genes 0.000 description 24
- 241000699670 Mus sp. Species 0.000 description 23
- 230000000694 effects Effects 0.000 description 22
- 102000004196 processed proteins & peptides Human genes 0.000 description 21
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 20
- 229940079593 drug Drugs 0.000 description 20
- 239000003446 ligand Substances 0.000 description 19
- 210000004072 lung Anatomy 0.000 description 19
- 229920001184 polypeptide Polymers 0.000 description 19
- 230000004614 tumor growth Effects 0.000 description 19
- 229940088598 enzyme Drugs 0.000 description 18
- 238000010253 intravenous injection Methods 0.000 description 18
- 230000035945 sensitivity Effects 0.000 description 18
- 210000004379 membrane Anatomy 0.000 description 17
- 239000012528 membrane Substances 0.000 description 17
- 239000000203 mixture Substances 0.000 description 17
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 16
- 201000010099 disease Diseases 0.000 description 15
- 239000000758 substrate Substances 0.000 description 15
- 230000000259 anti-tumor effect Effects 0.000 description 14
- 208000009956 adenocarcinoma Diseases 0.000 description 13
- 238000004458 analytical method Methods 0.000 description 13
- 150000001875 compounds Chemical class 0.000 description 13
- 201000009030 Carcinoma Diseases 0.000 description 12
- 239000012472 biological sample Substances 0.000 description 11
- 230000004044 response Effects 0.000 description 11
- 230000001225 therapeutic effect Effects 0.000 description 11
- 208000008839 Kidney Neoplasms Diseases 0.000 description 10
- 238000000684 flow cytometry Methods 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 238000007447 staining method Methods 0.000 description 10
- 230000004083 survival effect Effects 0.000 description 10
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 9
- 239000002246 antineoplastic agent Substances 0.000 description 9
- 231100000673 dose–response relationship Toxicity 0.000 description 9
- 238000009396 hybridization Methods 0.000 description 9
- 108060003951 Immunoglobulin Proteins 0.000 description 8
- GPKUGWDQUVWHIC-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine tetrahydrochloride Chemical compound Cl.Cl.Cl.Cl.NNC1=CC=C(C=C1)C1=CC=C(NN)C=C1 GPKUGWDQUVWHIC-UHFFFAOYSA-N 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 8
- 102000018358 immunoglobulin Human genes 0.000 description 8
- 238000001727 in vivo Methods 0.000 description 8
- 210000003734 kidney Anatomy 0.000 description 8
- 210000000496 pancreas Anatomy 0.000 description 8
- 239000013641 positive control Substances 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 238000011579 SCID mouse model Methods 0.000 description 7
- 150000001413 amino acids Chemical class 0.000 description 7
- 238000009826 distribution Methods 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 150000007523 nucleic acids Chemical class 0.000 description 7
- 239000012188 paraffin wax Substances 0.000 description 7
- 229920000642 polymer Polymers 0.000 description 7
- 108091033319 polynucleotide Proteins 0.000 description 7
- 102000040430 polynucleotide Human genes 0.000 description 7
- 239000002157 polynucleotide Substances 0.000 description 7
- 230000008685 targeting Effects 0.000 description 7
- 238000002560 therapeutic procedure Methods 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 6
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 6
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 6
- 102220497176 Small vasohibin-binding protein_T47D_mutation Human genes 0.000 description 6
- 230000004071 biological effect Effects 0.000 description 6
- 210000000481 breast Anatomy 0.000 description 6
- 239000007795 chemical reaction product Substances 0.000 description 6
- 230000002357 endometrial effect Effects 0.000 description 6
- 235000019152 folic acid Nutrition 0.000 description 6
- 239000011724 folic acid Substances 0.000 description 6
- 238000009472 formulation Methods 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 208000037841 lung tumor Diseases 0.000 description 6
- 238000007920 subcutaneous administration Methods 0.000 description 6
- 108090001008 Avidin Chemical class 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 102000010451 Folate receptor alpha Human genes 0.000 description 5
- 108050001931 Folate receptor alpha Proteins 0.000 description 5
- 239000004480 active ingredient Substances 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 239000005557 antagonist Substances 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 230000000875 corresponding effect Effects 0.000 description 5
- 230000001472 cytotoxic effect Effects 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 208000035475 disorder Diseases 0.000 description 5
- 230000001747 exhibiting effect Effects 0.000 description 5
- 238000003384 imaging method Methods 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- 230000000670 limiting effect Effects 0.000 description 5
- 208000014018 liver neoplasm Diseases 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 210000000056 organ Anatomy 0.000 description 5
- 208000004019 papillary adenocarcinoma Diseases 0.000 description 5
- 230000036961 partial effect Effects 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 210000003079 salivary gland Anatomy 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 229940124597 therapeutic agent Drugs 0.000 description 5
- 230000009258 tissue cross reactivity Effects 0.000 description 5
- LVSPDZAGCBEQAV-UHFFFAOYSA-N 4-chloronaphthalen-1-ol Chemical compound C1=CC=C2C(O)=CC=C(Cl)C2=C1 LVSPDZAGCBEQAV-UHFFFAOYSA-N 0.000 description 4
- 208000026310 Breast neoplasm Diseases 0.000 description 4
- 208000003170 Bronchiolo-Alveolar Adenocarcinoma Diseases 0.000 description 4
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 4
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical group C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 4
- 102000008300 Mutant Proteins Human genes 0.000 description 4
- 108010021466 Mutant Proteins Proteins 0.000 description 4
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 4
- 239000004698 Polyethylene Substances 0.000 description 4
- 208000034841 Thrombotic Microangiopathies Diseases 0.000 description 4
- 230000000890 antigenic effect Effects 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 238000001574 biopsy Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 210000000349 chromosome Anatomy 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 231100000433 cytotoxic Toxicity 0.000 description 4
- 229940014144 folate Drugs 0.000 description 4
- 238000011532 immunohistochemical staining Methods 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 229910052751 metal Inorganic materials 0.000 description 4
- 239000002184 metal Substances 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 238000003127 radioimmunoassay Methods 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 4
- LLXVXPPXELIDGQ-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(2,5-dioxopyrrol-1-yl)benzoate Chemical compound C=1C=CC(N2C(C=CC2=O)=O)=CC=1C(=O)ON1C(=O)CCC1=O LLXVXPPXELIDGQ-UHFFFAOYSA-N 0.000 description 3
- FUHCFUVCWLZEDQ-UHFFFAOYSA-N 1-(2,5-dioxopyrrolidin-1-yl)oxy-1-oxo-4-(pyridin-2-yldisulfanyl)butane-2-sulfonic acid Chemical compound O=C1CCC(=O)N1OC(=O)C(S(=O)(=O)O)CCSSC1=CC=CC=N1 FUHCFUVCWLZEDQ-UHFFFAOYSA-N 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical class OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 3
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 3
- 206010006187 Breast cancer Diseases 0.000 description 3
- 206010009944 Colon cancer Diseases 0.000 description 3
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 3
- 101710112752 Cytotoxin Proteins 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 206010027476 Metastases Diseases 0.000 description 3
- 241001529936 Murinae Species 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 102000003992 Peroxidases Human genes 0.000 description 3
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 3
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 3
- 108010090804 Streptavidin Chemical class 0.000 description 3
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 3
- 230000001476 alcoholic effect Effects 0.000 description 3
- 238000011319 anticancer therapy Methods 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 208000019065 cervical carcinoma Diseases 0.000 description 3
- 238000002648 combination therapy Methods 0.000 description 3
- 239000002619 cytotoxin Substances 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 231100000517 death Toxicity 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 239000000834 fixative Substances 0.000 description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 3
- 208000005017 glioblastoma Diseases 0.000 description 3
- 150000003278 haem Chemical class 0.000 description 3
- 208000014829 head and neck neoplasm Diseases 0.000 description 3
- 229940072221 immunoglobulins Drugs 0.000 description 3
- 230000002055 immunohistochemical effect Effects 0.000 description 3
- 238000007901 in situ hybridization Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 201000007270 liver cancer Diseases 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000009401 metastasis Effects 0.000 description 3
- 238000002493 microarray Methods 0.000 description 3
- 238000000386 microscopy Methods 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 201000002528 pancreatic cancer Diseases 0.000 description 3
- 108040007629 peroxidase activity proteins Proteins 0.000 description 3
- 239000008194 pharmaceutical composition Substances 0.000 description 3
- 239000000049 pigment Substances 0.000 description 3
- 230000000069 prophylactic effect Effects 0.000 description 3
- 230000004043 responsiveness Effects 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 230000002195 synergetic effect Effects 0.000 description 3
- 231100000588 tumorigenic Toxicity 0.000 description 3
- 230000000381 tumorigenic effect Effects 0.000 description 3
- NKUZQMZWTZAPSN-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 2-bromoacetate Chemical compound BrCC(=O)ON1C(=O)CCC1=O NKUZQMZWTZAPSN-UHFFFAOYSA-N 0.000 description 2
- VRDGQQTWSGDXCU-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 2-iodoacetate Chemical compound ICC(=O)ON1C(=O)CCC1=O VRDGQQTWSGDXCU-UHFFFAOYSA-N 0.000 description 2
- WGMMKWFUXPMTRW-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-[(2-bromoacetyl)amino]propanoate Chemical compound BrCC(=O)NCCC(=O)ON1C(=O)CCC1=O WGMMKWFUXPMTRW-UHFFFAOYSA-N 0.000 description 2
- CXNVOWPRHWWCQR-UHFFFAOYSA-N 4-Chloro-ortho-toluidine Chemical compound CC1=CC(Cl)=CC=C1N CXNVOWPRHWWCQR-UHFFFAOYSA-N 0.000 description 2
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 2
- 206010001197 Adenocarcinoma of the cervix Diseases 0.000 description 2
- 208000034246 Adenocarcinoma of the cervix uteri Diseases 0.000 description 2
- 206010005003 Bladder cancer Diseases 0.000 description 2
- 208000003174 Brain Neoplasms Diseases 0.000 description 2
- 206010058354 Bronchioloalveolar carcinoma Diseases 0.000 description 2
- FERIUCNNQQJTOY-UHFFFAOYSA-M Butyrate Chemical compound CCCC([O-])=O FERIUCNNQQJTOY-UHFFFAOYSA-M 0.000 description 2
- 206010008342 Cervix carcinoma Diseases 0.000 description 2
- 208000030808 Clear cell renal carcinoma Diseases 0.000 description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- 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 2
- 108090000371 Esterases Proteins 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 2
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 2
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Natural products OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 206010060862 Prostate cancer Diseases 0.000 description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 2
- 102000001708 Protein Isoforms Human genes 0.000 description 2
- 108010029485 Protein Isoforms Proteins 0.000 description 2
- 206010038389 Renal cancer Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 229940124650 anti-cancer therapies Drugs 0.000 description 2
- 210000004436 artificial bacterial chromosome Anatomy 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 239000000090 biomarker Substances 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 201000006662 cervical adenocarcinoma Diseases 0.000 description 2
- 201000010881 cervical cancer Diseases 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000002738 chelating agent Substances 0.000 description 2
- 125000003636 chemical group Chemical group 0.000 description 2
- 206010073251 clear cell renal cell carcinoma Diseases 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 210000002808 connective tissue Anatomy 0.000 description 2
- 239000007822 coupling agent Substances 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 201000003914 endometrial carcinoma Diseases 0.000 description 2
- 210000004696 endometrium Anatomy 0.000 description 2
- 208000023965 endometrium neoplasm Diseases 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 210000000981 epithelium Anatomy 0.000 description 2
- 210000003238 esophagus Anatomy 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000010195 expression analysis Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 229960000304 folic acid Drugs 0.000 description 2
- 210000004907 gland Anatomy 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 201000010536 head and neck cancer Diseases 0.000 description 2
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 2
- 210000004754 hybrid cell Anatomy 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- JORABGDXCIBAFL-UHFFFAOYSA-M iodonitrotetrazolium chloride Chemical compound [Cl-].C1=CC([N+](=O)[O-])=CC=C1N1[N+](C=2C=CC(I)=CC=2)=NC(C=2C=CC=CC=2)=N1 JORABGDXCIBAFL-UHFFFAOYSA-M 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 150000002540 isothiocyanates Chemical group 0.000 description 2
- 201000010982 kidney cancer Diseases 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 201000005249 lung adenocarcinoma Diseases 0.000 description 2
- 208000016992 lung adenocarcinoma in situ Diseases 0.000 description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
- 238000001531 micro-dissection Methods 0.000 description 2
- 208000024191 minimally invasive lung adenocarcinoma Diseases 0.000 description 2
- JPXMTWWFLBLUCD-UHFFFAOYSA-N nitro blue tetrazolium(2+) Chemical compound COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC(=CC=2)[N+]([O-])=O)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=C([N+]([O-])=O)C=C1 JPXMTWWFLBLUCD-UHFFFAOYSA-N 0.000 description 2
- 231100001221 nontumorigenic Toxicity 0.000 description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000013610 patient sample Substances 0.000 description 2
- 150000002989 phenols Chemical class 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 210000004043 pneumocyte Anatomy 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000003825 pressing Methods 0.000 description 2
- 208000037821 progressive disease Diseases 0.000 description 2
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 2
- 235000019833 protease Nutrition 0.000 description 2
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 description 2
- 210000001533 respiratory mucosa Anatomy 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 206010041823 squamous cell carcinoma Diseases 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 2
- 238000013414 tumor xenograft model Methods 0.000 description 2
- 210000003932 urinary bladder Anatomy 0.000 description 2
- 201000005112 urinary bladder cancer Diseases 0.000 description 2
- 208000024719 uterine cervix neoplasm Diseases 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- TYKASZBHFXBROF-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 2-(2,5-dioxopyrrol-1-yl)acetate Chemical compound O=C1CCC(=O)N1OC(=O)CN1C(=O)C=CC1=O TYKASZBHFXBROF-UHFFFAOYSA-N 0.000 description 1
- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 1
- PVGATNRYUYNBHO-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-(2,5-dioxopyrrol-1-yl)butanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O PVGATNRYUYNBHO-UHFFFAOYSA-N 0.000 description 1
- YMXHPSHLTSZXKH-RVBZMBCESA-N (2,5-dioxopyrrolidin-1-yl) 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoate Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)ON1C(=O)CCC1=O YMXHPSHLTSZXKH-RVBZMBCESA-N 0.000 description 1
- VLARLSIGSPVYHX-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-(2,5-dioxopyrrol-1-yl)hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCN1C(=O)C=CC1=O VLARLSIGSPVYHX-UHFFFAOYSA-N 0.000 description 1
- OJQSISYVGFJJBY-UHFFFAOYSA-N 1-(4-isocyanatophenyl)pyrrole-2,5-dione Chemical compound C1=CC(N=C=O)=CC=C1N1C(=O)C=CC1=O OJQSISYVGFJJBY-UHFFFAOYSA-N 0.000 description 1
- DIYPCWKHSODVAP-UHFFFAOYSA-N 1-[3-(2,5-dioxopyrrol-1-yl)benzoyl]oxy-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)C1=CC=CC(N2C(C=CC2=O)=O)=C1 DIYPCWKHSODVAP-UHFFFAOYSA-N 0.000 description 1
- ZOHXWSHGANNQGO-DSIKUUPMSA-N 1-amino-4-[[5-[[(2S)-1-[[(1S,2R,3S,5S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl]oxy]-1-oxopropan-2-yl]-methylamino]-2-methyl-5-oxopentan-2-yl]disulfanyl]-1-oxobutane-2-sulfonic acid Chemical compound CO[C@@H]([C@@]1(O)C[C@H](OC(=O)N1)[C@@H](C)[C@@H]1O[C@@]1(C)[C@@H](OC(=O)[C@H](C)N(C)C(=O)CCC(C)(C)SSCCC(C(N)=O)S(O)(=O)=O)CC(=O)N1C)\C=C\C=C(C)\CC2=CC(OC)=C(Cl)C1=C2 ZOHXWSHGANNQGO-DSIKUUPMSA-N 0.000 description 1
- YNXICDMQCQPQEW-UHFFFAOYSA-N 1-naphthyl dihydrogen phosphate Chemical class C1=CC=C2C(OP(O)(=O)O)=CC=CC2=C1 YNXICDMQCQPQEW-UHFFFAOYSA-N 0.000 description 1
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 1
- HKAVADYDPYUPRD-UHFFFAOYSA-N 1h-pyrazine-2-thione Chemical compound SC1=CN=CC=N1 HKAVADYDPYUPRD-UHFFFAOYSA-N 0.000 description 1
- FJXJIUHGLVUXQP-UHFFFAOYSA-N 2',7'-difluoro-3',6'-dihydroxyspiro[2-benzofuran-3,9'-xanthene]-1-one Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(F)=C(O)C=C1OC1=C2C=C(F)C(O)=C1 FJXJIUHGLVUXQP-UHFFFAOYSA-N 0.000 description 1
- VGIRNWJSIRVFRT-UHFFFAOYSA-N 2',7'-difluorofluorescein Chemical compound OC(=O)C1=CC=CC=C1C1=C2C=C(F)C(=O)C=C2OC2=CC(O)=C(F)C=C21 VGIRNWJSIRVFRT-UHFFFAOYSA-N 0.000 description 1
- HSTOKWSFWGCZMH-UHFFFAOYSA-N 3,3'-diaminobenzidine Chemical compound C1=C(N)C(N)=CC=C1C1=CC=C(N)C(N)=C1 HSTOKWSFWGCZMH-UHFFFAOYSA-N 0.000 description 1
- AGAYQLZJBWFZQV-UHFFFAOYSA-N 3-acetyloxolane-2,5-dione Chemical compound CC(=O)C1CC(=O)OC1=O AGAYQLZJBWFZQV-UHFFFAOYSA-N 0.000 description 1
- QRYXYRQPMWQIDM-UHFFFAOYSA-N 3-benzoyl-3-(2,5-dioxopyrrol-1-yl)-1-hydroxypyrrolidine-2,5-dione Chemical compound O=C1N(O)C(=O)CC1(C(=O)C=1C=CC=CC=1)N1C(=O)C=CC1=O QRYXYRQPMWQIDM-UHFFFAOYSA-N 0.000 description 1
- JNWSNQJRTXEHIV-UHFFFAOYSA-N 4-(pyridin-2-yldisulfanyl)-2-sulfopentanoic acid Chemical compound OC(=O)C(S(O)(=O)=O)CC(C)SSC1=CC=CC=N1 JNWSNQJRTXEHIV-UHFFFAOYSA-N 0.000 description 1
- WTWSDDKGKIGSJE-UHFFFAOYSA-N 5-(4-aminophenyl)cyclohexa-2,4-diene-1,1,2-triamine;tetrahydrochloride Chemical compound Cl.Cl.Cl.Cl.C1C(N)(N)C(N)=CC=C1C1=CC=C(N)C=C1 WTWSDDKGKIGSJE-UHFFFAOYSA-N 0.000 description 1
- IDLISIVVYLGCKO-UHFFFAOYSA-N 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein Chemical compound O1C(=O)C2=CC=C(C(O)=O)C=C2C21C1=CC(OC)=C(O)C(Cl)=C1OC1=C2C=C(OC)C(O)=C1Cl IDLISIVVYLGCKO-UHFFFAOYSA-N 0.000 description 1
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 1
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 1
- JIEINYQEXWLMCU-UHFFFAOYSA-N 7-bromo-3-hydroxy-n-(2-methoxyphenyl)naphthalene-2-carboxamide Chemical compound COC1=CC=CC=C1NC(=O)C1=CC2=CC(Br)=CC=C2C=C1O JIEINYQEXWLMCU-UHFFFAOYSA-N 0.000 description 1
- CJIJXIFQYOPWTF-UHFFFAOYSA-N 7-hydroxycoumarin Natural products O1C(=O)C=CC2=CC(O)=CC=C21 CJIJXIFQYOPWTF-UHFFFAOYSA-N 0.000 description 1
- OXEUETBFKVCRNP-UHFFFAOYSA-N 9-ethyl-3-carbazolamine Chemical compound NC1=CC=C2N(CC)C3=CC=CC=C3C2=C1 OXEUETBFKVCRNP-UHFFFAOYSA-N 0.000 description 1
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 1
- 241001193964 Ameles Species 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 235000011330 Armoracia rusticana Nutrition 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Natural products CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 1
- PCDQPRRSZKQHHS-XVFCMESISA-N CTP Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 PCDQPRRSZKQHHS-XVFCMESISA-N 0.000 description 1
- 206010006895 Cachexia Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000006332 Choriocarcinoma Diseases 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 239000004971 Cross linker Substances 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 208000002699 Digestive System Neoplasms Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 229910052693 Europium Inorganic materials 0.000 description 1
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 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
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- XKMLYUALXHKNFT-UUOKFMHZSA-N Guanosine-5'-triphosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XKMLYUALXHKNFT-UUOKFMHZSA-N 0.000 description 1
- 206010019695 Hepatic neoplasm Diseases 0.000 description 1
- 101001099381 Homo sapiens Peroxisomal biogenesis factor 19 Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical class C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 229930010555 Inosine Natural products 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
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- HLFSDGLLUJUHTE-SNVBAGLBSA-N Levamisole Chemical compound C1([C@H]2CN3CCSC3=N2)=CC=CC=C1 HLFSDGLLUJUHTE-SNVBAGLBSA-N 0.000 description 1
- 206010025323 Lymphomas Diseases 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
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 229930126263 Maytansine Natural products 0.000 description 1
- NXTVQNIVUKXOIL-UHFFFAOYSA-N N-chlorotoluene-p-sulfonamide Chemical compound CC1=CC=C(S(=O)(=O)NCl)C=C1 NXTVQNIVUKXOIL-UHFFFAOYSA-N 0.000 description 1
- 108091006036 N-glycosylated proteins Proteins 0.000 description 1
- AWZJFZMWSUBJAJ-UHFFFAOYSA-N OG-514 dye Chemical compound OC(=O)CSC1=C(F)C(F)=C(C(O)=O)C(C2=C3C=C(F)C(=O)C=C3OC3=CC(O)=C(F)C=C32)=C1F AWZJFZMWSUBJAJ-UHFFFAOYSA-N 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102100025386 Oxidized low-density lipoprotein receptor 1 Human genes 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102100038883 Peroxisomal biogenesis factor 19 Human genes 0.000 description 1
- 108010053210 Phycocyanin Proteins 0.000 description 1
- 108010004729 Phycoerythrin Proteins 0.000 description 1
- 208000002151 Pleural effusion Diseases 0.000 description 1
- 241001510071 Pyrrhocoridae Species 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 101001039269 Rattus norvegicus Glycine N-methyltransferase Proteins 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 206010061934 Salivary gland cancer Diseases 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 206010041067 Small cell lung cancer Diseases 0.000 description 1
- 231100000632 Spindle poison Toxicity 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- GYDJEQRTZSCIOI-UHFFFAOYSA-N Tranexamic acid Chemical compound NCC1CCC(C(O)=O)CC1 GYDJEQRTZSCIOI-UHFFFAOYSA-N 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- PGAVKCOVUIYSFO-XVFCMESISA-N UTP Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 PGAVKCOVUIYSFO-XVFCMESISA-N 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- 208000002495 Uterine Neoplasms Diseases 0.000 description 1
- 241000812633 Varicus Species 0.000 description 1
- 206010047741 Vulval cancer Diseases 0.000 description 1
- PUDCZUQFOPHIGU-UHFFFAOYSA-N [2-methyl-4-[(2-methylphenyl)diazenyl]phenyl]azanium;chloride Chemical compound Cl.C1=C(N)C(C)=CC(N=NC=2C(=CC=CC=2)C)=C1 PUDCZUQFOPHIGU-UHFFFAOYSA-N 0.000 description 1
- SHTFMUPUBYXPCD-UHFFFAOYSA-N [3-[(4-chloro-2-methylphenyl)carbamoyl]naphthalen-2-yl] dihydrogen phosphate Chemical compound CC1=CC(Cl)=CC=C1NC(=O)C1=CC2=CC=CC=C2C=C1OP(O)(O)=O SHTFMUPUBYXPCD-UHFFFAOYSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 229910052946 acanthite Inorganic materials 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 150000008065 acid anhydrides Chemical class 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000001919 adrenal effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 125000003158 alcohol group Chemical group 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- WLDHEUZGFKACJH-UHFFFAOYSA-K amaranth Chemical compound [Na+].[Na+].[Na+].C12=CC=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(O)=C1N=NC1=CC=C(S([O-])(=O)=O)C2=CC=CC=C12 WLDHEUZGFKACJH-UHFFFAOYSA-K 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000012805 animal sample Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 230000006023 anti-tumor response Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 239000012122 aqueous mounting media Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- XKRFYHLGVUSROY-UHFFFAOYSA-N argon Substances [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 239000013584 assay control Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- VSRXQHXAPYXROS-UHFFFAOYSA-N azanide;cyclobutane-1,1-dicarboxylic acid;platinum(2+) Chemical compound [NH2-].[NH2-].[Pt+2].OC(=O)C1(C(O)=O)CCC1 VSRXQHXAPYXROS-UHFFFAOYSA-N 0.000 description 1
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 1
- 239000000987 azo dye Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960000686 benzalkonium chloride Drugs 0.000 description 1
- 229960003872 benzethonium Drugs 0.000 description 1
- 150000001557 benzodiazepines Chemical class 0.000 description 1
- 235000019445 benzyl alcohol Nutrition 0.000 description 1
- CADWTSSKOVRVJC-UHFFFAOYSA-N benzyl(dimethyl)azanium;chloride Chemical compound [Cl-].C[NH+](C)CC1=CC=CC=C1 CADWTSSKOVRVJC-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 201000000053 blastoma Diseases 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000010504 bond cleavage reaction Methods 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- LRHPLDYGYMQRHN-UHFFFAOYSA-N butyl alcohol Substances CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 208000035269 cancer or benign tumor Diseases 0.000 description 1
- 229960004562 carboplatin Drugs 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000015861 cell surface binding Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 210000002987 choroid plexus Anatomy 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- HPXRVTGHNJAIIH-UHFFFAOYSA-N cyclohexanol Chemical compound OC1CCCCC1 HPXRVTGHNJAIIH-UHFFFAOYSA-N 0.000 description 1
- 238000012303 cytoplasmic staining Methods 0.000 description 1
- 239000000824 cytostatic agent Substances 0.000 description 1
- 230000001085 cytostatic effect Effects 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 125000001295 dansyl group Chemical group [H]C1=C([H])C(N(C([H])([H])[H])C([H])([H])[H])=C2C([H])=C([H])C([H])=C(C2=C1[H])S(*)(=O)=O 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 229960000975 daunorubicin Drugs 0.000 description 1
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 239000012954 diazonium Substances 0.000 description 1
- 150000001989 diazonium salts Chemical class 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- SIYLLGKDQZGJHK-UHFFFAOYSA-N dimethyl-(phenylmethyl)-[2-[2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethoxy]ethyl]ammonium Chemical compound C1=CC(C(C)(C)CC(C)(C)C)=CC=C1OCCOCC[N+](C)(C)CC1=CC=CC=C1 SIYLLGKDQZGJHK-UHFFFAOYSA-N 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 1
- 125000002228 disulfide group Chemical group 0.000 description 1
- 239000003534 dna topoisomerase inhibitor Substances 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 239000006196 drop Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 210000000804 eccrine gland Anatomy 0.000 description 1
- 201000008184 embryoma Diseases 0.000 description 1
- 201000003908 endometrial adenocarcinoma Diseases 0.000 description 1
- 208000029382 endometrium adenocarcinoma Diseases 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 102000006815 folate receptor Human genes 0.000 description 1
- 108020005243 folate receptor Proteins 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000003633 gene expression assay Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 description 1
- 229940093915 gynecological organic acid Drugs 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 229950002932 hexamethonium Drugs 0.000 description 1
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 1
- 239000012145 high-salt buffer Substances 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 102000053180 human FOLR1 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 210000003917 human chromosome Anatomy 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 239000002563 ionic surfactant Substances 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- 239000012948 isocyanate Substances 0.000 description 1
- 150000002513 isocyanates Chemical class 0.000 description 1
- 229940043355 kinase inhibitor Drugs 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 229960001614 levamisole Drugs 0.000 description 1
- ZNOVTXRBGFNYRX-ABLWVSNPSA-N levomefolic acid Chemical compound C1NC=2NC(N)=NC(=O)C=2N(C)C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 ZNOVTXRBGFNYRX-ABLWVSNPSA-N 0.000 description 1
- 235000007635 levomefolic acid Nutrition 0.000 description 1
- 239000011578 levomefolic acid Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 235000014666 liquid concentrate Nutrition 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 210000004880 lymph fluid Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- WKPWGQKGSOKKOO-RSFHAFMBSA-N maytansine Chemical compound CO[C@@H]([C@@]1(O)C[C@](OC(=O)N1)([C@H]([C@@H]1O[C@@]1(C)[C@@H](OC(=O)[C@H](C)N(C)C(C)=O)CC(=O)N1C)C)[H])\C=C\C=C(C)\CC2=CC(OC)=C(Cl)C1=C2 WKPWGQKGSOKKOO-RSFHAFMBSA-N 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- DYQNRMCKBFOWKH-UHFFFAOYSA-N methyl 4-hydroxybenzenecarboximidate Chemical compound COC(=N)C1=CC=C(O)C=C1 DYQNRMCKBFOWKH-UHFFFAOYSA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 1
- 229960002216 methylparaben Drugs 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002991 molded plastic Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000012120 mounting media Substances 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- CNXZLZNEIYFZGU-UHFFFAOYSA-N n-(4-amino-2,5-diethoxyphenyl)benzamide Chemical compound C1=C(N)C(OCC)=CC(NC(=O)C=2C=CC=CC=2)=C1OCC CNXZLZNEIYFZGU-UHFFFAOYSA-N 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 108091014756 nucleotide binding proteins Proteins 0.000 description 1
- 102000026415 nucleotide binding proteins Human genes 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 150000002895 organic esters Chemical class 0.000 description 1
- 239000012285 osmium tetroxide Substances 0.000 description 1
- 229910000489 osmium tetroxide Inorganic materials 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 210000003101 oviduct Anatomy 0.000 description 1
- LXCFILQKKLGQFO-UHFFFAOYSA-N p-hydroxybenzoic acid methyl ester Natural products COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 1
- 239000006174 pH buffer Substances 0.000 description 1
- 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 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000005298 paramagnetic effect Effects 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 201000002628 peritoneum cancer Diseases 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 239000003757 phosphotransferase inhibitor Substances 0.000 description 1
- 239000003504 photosensitizing agent Substances 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 229920000889 poly(m-phenylene isophthalamide) Polymers 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 208000023958 prostate neoplasm Diseases 0.000 description 1
- 210000000512 proximal kidney tubule Anatomy 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 239000002213 purine nucleotide Substances 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 229910052761 rare earth metal Inorganic materials 0.000 description 1
- 150000002910 rare earth metals Chemical class 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 208000015347 renal cell adenocarcinoma Diseases 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 201000003804 salivary gland carcinoma Diseases 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 238000013077 scoring method Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 208000019694 serous adenocarcinoma Diseases 0.000 description 1
- 208000004548 serous cystadenocarcinoma Diseases 0.000 description 1
- 229940056910 silver sulfide Drugs 0.000 description 1
- XUARKZBEFFVFRG-UHFFFAOYSA-N silver sulfide Chemical compound [S-2].[Ag+].[Ag+] XUARKZBEFFVFRG-UHFFFAOYSA-N 0.000 description 1
- 208000000587 small cell lung carcinoma Diseases 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- VUFNRPJNRFOTGK-UHFFFAOYSA-M sodium;1-[4-[(2,5-dioxopyrrol-1-yl)methyl]cyclohexanecarbonyl]oxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)C1CCC(CN2C(C=CC2=O)=O)CC1 VUFNRPJNRFOTGK-UHFFFAOYSA-M 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000012128 staining reagent Substances 0.000 description 1
- SFVFIFLLYFPGHH-UHFFFAOYSA-M stearalkonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 SFVFIFLLYFPGHH-UHFFFAOYSA-M 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 210000001138 tear Anatomy 0.000 description 1
- 210000004876 tela submucosa Anatomy 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 238000011285 therapeutic regimen Methods 0.000 description 1
- 125000000101 thioether group Chemical group 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 229940044693 topoisomerase inhibitor Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- ZSDSQXJSNMTJDA-UHFFFAOYSA-N trifluralin Chemical compound CCCN(CCC)C1=C([N+]([O-])=O)C=C(C(F)(F)F)C=C1[N+]([O-])=O ZSDSQXJSNMTJDA-UHFFFAOYSA-N 0.000 description 1
- PYIHTIJNCRKDBV-UHFFFAOYSA-L trimethyl-[6-(trimethylazaniumyl)hexyl]azanium;dichloride Chemical compound [Cl-].[Cl-].C[N+](C)(C)CCCCCC[N+](C)(C)C PYIHTIJNCRKDBV-UHFFFAOYSA-L 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- ORHBXUUXSCNDEV-UHFFFAOYSA-N umbelliferone Chemical compound C1=CC(=O)OC2=CC(O)=CC=C21 ORHBXUUXSCNDEV-UHFFFAOYSA-N 0.000 description 1
- HFTAFOQKODTIJY-UHFFFAOYSA-N umbelliferone Natural products Cc1cc2C=CC(=O)Oc2cc1OCC=CC(C)(C)O HFTAFOQKODTIJY-UHFFFAOYSA-N 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- 208000012991 uterine carcinoma Diseases 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 201000005102 vulva cancer Diseases 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000010148 water-pollination Effects 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- HBOMLICNUCNMMY-XLPZGREQSA-N zidovudine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-XLPZGREQSA-N 0.000 description 1
- 229960002555 zidovudine Drugs 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
- A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
- A61K47/6817—Toxins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
- A61K47/6857—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from lung cancer cell
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
- A61K47/6869—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
Abstract
Disclosed is the use of an anti-Folate receptor 1 (FOLR1) immunoconjugate in the manufacture of a medicament for treating cancer in a subject, wherein increased expression of FOLR1 in a cancer sample from said subject has been detected using a detection method that distinguishes between staining intensity or staining uniformity in a FOLR1 expressing cancer sample as compared to staining intensity or staining uniformity in one or more reference sample, wherein said anti-FOLR1 immunoconjugate has the formula (A) – (L) – (C) or (C) – (L) – (A), wherein: (A) is an anti-FOLR1 antibody or antigen binding fragment thereof comprising: (a) a heavy chain CDR1 comprising GYFMN (SEQ ID NO:6); a heavy chain CDR2 comprising RIHPYDGDTFYNQKFQG (SEQ ID NO:7); and a heavy chain CDR3 comprising YDGSRAMDY (SEQ ID NO:8); and (b) a light chain CDR1 comprising KASQSVSFAGTSLMH (SEQ ID NO:9); a light chain CDR2 comprising RASNLEA (SEQ ID NO:10); and a light chain CDR3 comprising QQSREYPYT (SEQ ID NO:11), (L) is a linker, and (C) is a cytotoxic agent, and wherein the linker (L) links (A) to (C). tensity or staining uniformity in a FOLR1 expressing cancer sample as compared to staining intensity or staining uniformity in one or more reference sample, wherein said anti-FOLR1 immunoconjugate has the formula (A) – (L) – (C) or (C) – (L) – (A), wherein: (A) is an anti-FOLR1 antibody or antigen binding fragment thereof comprising: (a) a heavy chain CDR1 comprising GYFMN (SEQ ID NO:6); a heavy chain CDR2 comprising RIHPYDGDTFYNQKFQG (SEQ ID NO:7); and a heavy chain CDR3 comprising YDGSRAMDY (SEQ ID NO:8); and (b) a light chain CDR1 comprising KASQSVSFAGTSLMH (SEQ ID NO:9); a light chain CDR2 comprising RASNLEA (SEQ ID NO:10); and a light chain CDR3 comprising QQSREYPYT (SEQ ID NO:11), (L) is a linker, and (C) is a cytotoxic agent, and wherein the linker (L) links (A) to (C).
Description
METHODS FOR INCREASING EFFICACY OF FOLRl CANCER THERAPY CROSS-REFERENCE TO RELATED APPLICATIONS {ENG}; This application claims the benefit of US Provisional App? No. ,007, filed April 1, 2011 which is herein incorporated by reference.
BACKGROUND OF THE ION Field of the Invention The field of invention lly relates to increasing the efficacy of the treatment of cancers characterized by the overexpression of human folate receptor 1 (FOLRl). More specifically, the ion concerns more effective treatment of patients susceptible to or diagnosed with cancer, in which the tumor cells overexpress FOLRl as determined by a gene expression assay, with a FOLRl antagonist, e.g., a FOLRl immunoconjugate.
Background Art Cancer is one of the leading causes of death in the developed world, with over one n people diagnosed with cancer and 500,000 deaths per year in the United States alone. Overall it is estimated that more than 1 in 3 people will develop some form of cancer during their lifetime. There are more than 200 different types of cancer, four of which— breast, lung, ctal, and prostate—account for over half of all new cases (Jemal et al., 2003, Cancer]. Clin. 5325-26).
Folate Receptor 1 (FOLRI), also known as Folate Receptor-alpha, or Folate Binding Protein, is an N-glycosylated protein expressed on plasma membrane of cells. FOLRl has a high y for folic acid and for several d folic acid derévatives. FOLRl mediates delivery of the physiological folate, 5-methyltetrahydrofolate, to the interior of cells.
FOLRl is overexpressed in vast majority of ovarian cancers, as well as in many uterine, endometrial, pancreatic, renal, lung, and breast cancers, While the sion of FOLRl on normal s is restricted to the apical membrane of epithelial cells in the kidney proximal tubules, alveolar pneumocytes of the lung, bladder, testes, choroid plexus, and thyroid (Weitman SD, 62‘ al., Cancer Res 52: 3396-3401 (1992); Antony AC, Annu Rev Nutr 16: 501-521 (1996); Kalli KR, et a1. Gynecol Oncol 108: 6 (2008)). This expression pattern of FOLRl makes it a desirable target for FOLRl —directed cancer therapy.
Because ovarian cancer is typically asymptomatic until advanced stage, it is often diagnosed at a late stage and has poor prognosis when treated with currently available procedures, lly chemotherapeutic drugs after al de-bulking (von Gruenigen V et al., Cancer 112: 2221-2227 (2008); Ayhan A et al., Am J Obstet Gynecol 196: 81 e81-86 (2007); Harry VN et 61]., Obstet Gynecol Surv 64: 548-560 (2009)). Thus, there is a clear unmet medical need for more effective eutics for ovarian cancers.
SUMMARY OF THE INVENTION The present invention is based on the discovery of a dynamic range of expression of FOLRI in tumor tissue and the discovery that tumors with increased levels of FOLRl expression are more responsive to treatment with anti-FOLRl antibodies or anti-FOLRl immunoconjugates. The present invention advantageously permits treatment of patients who have a greater likelihood of responding to ent by administering therapeutic agents, i.e., anti-FOLRl antibodies or anti-FOLRl immunoconjugates, to patients who are found to have an increased expression level of FOLRl.
The present invention provides a method for identifying a subject pre-disposed to respond favorably to a Folate Receptor 1 (FOLRl)—targeting anti-cancer therapeutic, the method comprising detecting FOLRl expression in a tissue sample from the subject.
The present ion also provides a method for increasing the likelihood of effectiveness of a cancer treatment, the method comprising stering a therapeutically effective dose of a FOLRl-targeting anti-cancer eutic to a subject, wherein FOLRI expression in a tissue sample from the subject has been found to be increased.
The present invention also provides a method for predicting effectiveness of a low— dose cancer treatment, the method comprising administering a therapeutically effective dose of a FOLRl—targeting anti-cancer therapeutic to a subject, wherein said subject has been found to have sed sion of FOLRl in a sample.
In one embodiment, the methods are directed to ovarian carcinoma, non-small cell lung adenocarcinoma ding bronchioloalveolar carcinoma), renal omas, and trial carcinomas.
In one embodiment, the extent and uniformity of FOLRI expression is detected by immunohistochemistry (IHC), flow cytometry, or c acid hybridization. in another embodiment, the level of FOLRl expression is detected by immunohistochemistry. Non- limiting examples of IHC include IHC methods that distinguish between varying levels of FOLRI and calibrated IHC methods, such as those described herein. The FOLRl expression can be scored using an appropriate scoréng system, including but not limited to the scoring methods described herein. For example, FOLRI sion can be scored using a calibrated IHC method that es a range of 0, 1, 2, 3, and 3+ for staining intensity with 0 being the lowest level of staining intensity and 3+ being the highest level of staining intensity. Alternatively or additionally, FOLRI sion can be scored using a calibrated IHC method that includes a staining mity that ranges from focal (<25% of cells stained), to heterogeneous (25-75% of cells stained), to homogenous (>75% of cells stained), where focal staining is the least uniform staining and neous is the most uniform staining.
In a further embodiment, the FOLRI expression in a sample (e.g., a tumor tissue sample) is measured and compared to one or more reference samples and the FOLRl expression in the tissue sample from a subject tumor, xenograft tumor, or cell line has a FOLRI specific score correlating to extent and uniformity of expression as compared to the one or more reference samples. In various examples, a tissue sample or cell with a level 1, 2, 3 or 3+ FOLRl staining intensity with a homogeneous staining pattern is considered to have increased FOLRl expression; a tissue sample or cell with a level 3 FOLRl staining intensity with heterogeneous or focal staining patterns is considered to have increased FOLRl expression. In r embodiment the FOLRI expression in a sample is measured and compared to one or more reference samples to identify a comparable level of staining.
In one embodiment, the reference sample has a pre-assigned IHC score and/or a pre- determined antigen per cell (or ABC) number and the antigen or ABC number for the sample tissue can be ined based on the comparison.
In one embodiment, the FOLRl expression in a sample (e.g., a tumor tissue sample) is measured and ed to one or more control samples and the FOLRl expression in the tissue sample from a t tumor, xenograft tumor, or cell line has a FOLRl ic score correlating to extent and mity of expression as compared to the one or more control samples. In one embodiment, the FOLRl expression in the sample is compared to a negative control sample which demonstrates no or low detectable FOLRI expression. In another embodiment, the FGLR'E expression in the sample is compared to a positive control sample having increased FOLRl expressinn {level i, 2, 3. or 3+). In some embodin‘ien‘ts, the control samples include, but are not limited in Namalwa, SW2, SWéZO, T47D, IGRG‘V’J, 300,19 FRI, i-Ielga, or KB cells. In particular r‘nents, the control samples include cells or cell pellets from cells transfected with folate receptor (e.g., 300.19 FRl).
In one embodiment, the FOLRl-targeting anti-cancer therapeutic is a FOLRI conjugate. In one embodiment, the immunoconjugate ses an anti-FOLRI antibody, a linker, and a cytotoxin.
In a further embodiment, the anti-FOLRI antibody is huMOVl9. In another embodiment, the linker is selected from the group consisting of a cleavable linker, a non~ cleavable linker, a hilic linker, and a dicarboxylic acid based linker. in another embodiment, the linker is ed from the group consisting: N—succinimidyl 4-(2— pyridyldithio)pentanoate (SPP) or N—succinimidyl 4-(2-pyridyldithio)-2—sulfopentanoate (sulfo—SPP); inimidyl 4-(2-pyn'dyldithio)butanoate (SPDB) or N-succinimidyl 4-(2— pyridyldithio)-2—sulfobutanoate (sulfo-SPDB); inimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC); N—sulfosuccinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC); N—succinimidyl(iodoacetyl)-aminobenzoate (SIAB); and N—succinimidyl—[(N-maleimidopropionamido)—tetraethyleneglycol] ester (NHS-PEG4-maleimide). In another ment, the linker is N—succinimidyl 4-(2- pyridyldithio)-2—sulfobutanoate (sulfo-SPDB). In another ment, the xic agent is selected from the group consisting of a maytansinoid, maytansinoid analog, benzodiazepine, taxoid, CC—1065, CC-1065 analog, duocarmycin, mycin analog, calicheamicin, atin, dolastatin analog, auristatin, ycin derivative, and leptomycin derivative or a prodrug of the agent. In another embodiment, the cytotoxic agent is a maytansinoid. In another embodiment, the cytotoxic agent is N(2')-deacetyl- N(2')—(3 -mercaptooxopropyl)-maytansine or N(2')—deacetyl-NZ-(4-mercapto—4-methyl— l - oxopentyl)—maytansine. In another embodiment, the cytotoxic agent is N(2')-deacetyl-N2— (4-mercaptomethyl-l-oxopentyl)—maytansine (DM4). In a further embodiment, the immunoconjugate comprises the antibody HUMOV19, SPDB, and DM4 (IMGN853).
The invention is also directed to a kit for ing FOLRI expression in a subject comprising a FOLRl detection reagent, and instructions for use. In one embodiment, the FOLRl ion reagent comprises a FOLRl binding peptide, protein or a molecular probe (i.e. nucleic acid). In another embodiment, the FOLRl detection reagent is an anti-FOLRI antibody. In another embodiment, the kit further comprises a secondary antibody which binds the anti-FOLRl antibody. In one embodiment the antibody is ed at a tration of 0.5 to 7.5 ug/ml, desirably 0.9 to 3.8 +/- 0.5 ug/ml. In various embodiments, the antibody is included at a concentration of 1.0 +/— 0.5 ug/ml, 1.5 +/— 0.5 ug/ml, 1.9 +/- 0.5 ug/ml, 2.5 +/— 0.5 ug/ml, 3.0 +/- 0.5 rig/ml, 3.5 +/- 0.5 pig/ml, 3.8 +/- 0.5 ug/ml, or up to 4.2 ug/ml. In another embodiment, the antibody is included in concentrated solution with instructions for ons to e a final concentration of 0.9 to 3.8 +/— 0.5 rig/ml. In another embodiment, the kit further comprises a detection reagent selected from the group consisting of: an enzyme, a fluorophore, a radioactive label, and a luminophore.
In another embodiment, the detection reagent is selected from the group consisting of: biotin, digo-xigenin, fluorescein, tritium, and rhodamine.
The kit can also include instructions for detection and scoring of FOLRl expression.
The kit can also include control or reference samples. Non-limiting examples of control or reference samples include tissue samples, cell pellets or cells. The controi or reference samples may be derived from tissue culture cell lines (normal or tumor), normal tissue (normal control) or tumor tissues (positive control) samples. Exemplary cell lines include SW620, T47D, IGROV-l, HELA, KB, JEG-3 and cell lines stably or transiently ected with an expression vector that ses FOLRl (e.g., FR1). ary tissues that detection s are may be used as normal reference tissues in the FOLRl expression bed herein and include normal lung, salivary gland, and pancreas.
The invention is also directed to a method for identifying a cancer likely to respond to an anti-FOLRl antibody, or OLRl conjugate comprising: (a) contacting a biological sample comprising cells from said cancer with an agent that binds FOLRl protein on the cell surface; (b)detecting binding of said agent that binds FOLRl protein on the cell surface of said biological sample of (a); (c) assigning a score to said binding of step (b), wherein said score is assigned based on comparison to one or more reference samples; and (d) comparing said score in step (c) to the score of a nce tissue or cell, wherein a score for said cancer FOLRl level that is greater than the score for a normal or low P‘O'LRI expressing reference sample or a score for said cancer FOLRl level that is equal to or greater than the score for a high FOLRl expressing reference sample identifies said cancer as likely to respond to an anti—FOLRI antibody or anti-FOLRl immunoconjugate. In certain ments, the cancer is ovarian or lung cancer.
The ion is also directed to a method of identifying a tumor as sensitive to ent with an anti-FOLRI antibody, or anti—FOLRI immunoconjugate, said method comprising: (a) ing the level of FOLRI expression in a tumor tissue sample obtained from said tumor, wherein said ing comprises the use of a detection method that distinguishes n staining intensity or ng uniformity in a FOLRI expressing cancer sample as compared to staining intensity or staining uniformity in one or more reference samples; (b) determining a FOLRI staining intensity score for said tumor tissue sample; and (c) ing the FOLRI staining ity score determined in step (b) to a relative value determined by measuring FOLRI protein sion in at least one reference sample, wherein said at least one reference sample is a tissue, cell, or cell pellet sample which is not sensitive to treatment with an anti—FOLRI antibody, or anti-FOLRl immunoconjugate, and wherein a FOLRI staining intensity score for said sample determined in step (b) that is higher than said ve value identifies said tumor as being sensitive to treatment with an anti—FOLRl dy, or anti—FOLRI immunoconjugate. In certain embodiments, the detection method is performed manually or using an automated system. In one embodiment, the ion method is IHC. In another embodiment, the IHC is calibrated IHC that can distinguish different levels of FOLRl expression.
The invention is also directed to a method of optimizing a therapeutic regimen with an anti-FOLRI antibody or an anti-FOLRI immunoconjugate for a subject having lung or ovarian cancer, said method comprising: (a) contacting said sample from said subject with an antibody that cally binds cell surface FOLRI; (b) measuréng the binding of said antibody in (a) to said cell surface FOLRl in said sample using a detection method that can distinguish between staining intensity or staining uniformity in a FOLRI expressing cancer sample as compared to staining intensity or staining uniformity in one or more reference samples and assigning a staining score to said sample; and (c) administering a high dose of an anti-FOLRl immunoconjugate when the score in step (b) is less than or equal to the score for a normal or low FOLRl expressing nce sample or administering a low dose of an anti—FOLRI immunoconjugate when the score is greater than the score for a normal or low FOLRl expressing reference sample.
The invention is also directed to a method of detecting the expression of cell surface FOLRl on cancer cells in a tumor tissue sample from a subject, said method comprising: (a) obtaining tumor tissue sample, wherein said cancer sample is formalin-fixed paraffin ed; (b) contacting said sample with an antibody that specifically binds cell surface FOLRl; (c) measuring the binding of said antibody in (b) to said cell e FOLRl in said tumor tissue sample using a detection method that can distinguish between staining intensity or staining mity in a FOLRl expressing cancer sample as compared to staining intensity or staining uniformity in one or more reference samples; and (d) assigning a FOLRl sion score to said FOLRl after comparing the level of cell surface FOLRl staining intensity or staining uniformity in said tumor tissue sample to one or more reference samples.
The invention is also directed to a method of identifying a subject having a lung or ovarian cancer as likely to d to a low dose anti-FOLRl antibody or anti-FOLRI immunoconjugate treatment regimen, said method comprising: (a) contacting a ical sample comprising cells from said ovarian or lung cancer with an agent that binds cell surface FOLRI n; (b) detecting binding of said agent to said biological sample of (a); (c) assigning a score to said binding of step (b), wherein said score is assigned based on comparison to one or more reference samples; and (d) comparing said score in step (c) to the score of a reference tissue or cell, wherein a score for said ovarian or lung cancer FOLRl level that is greater than the score for a normal or low FOLRl expressing nce sample or a score for said ovarian or lung cancer FOLRl level that is equal to or greater than the score for a high FOLRl expressing reference sample identifies said ovarian or lung cancer as likely to respond to a low dose anti-FOLRl antibody or anti-FOLRl oconjugate. In n embodiments, the method further comprises stering a therapeutically effective amount of a humanized anti-FOLRl antibody or an anti-FOILRI immunoconjugate to said subject.
BRIEF DESCRIPTIONS OF THE DRAWINGS [0024} Figure 1. Manual Staining : Anti-FOLRl antibodies detect FOLRl expression in transfected cells. 300.19 cells were transfected with a polynucleotide that encodes human FOLRl. FOLRl protein expression was detected using the murine antibody BN3.2. Smith AE et a], oma (Larchmt). 2007 Oct;26(5):281-8.
Figure 2. Manual Staining Method: Anti-FOLRl antibodies can distinguish different levels of FOLRl expression. Antibody BN3.2 was used to detect FOLR] expression in various xenograft cells. The limit of detection for the BN3.2 antibody was approximately 4000 antibodies bound per cell (ABC).
Figure 3. Manual Staining Method: Anti-FOLRl antibodies can distinguish different levels of FOLRI expression in tissue samples. BN3.2 was used to detect FOLRl expression in both ovarian tumors (A), as well as non-small cell lung cancer tumors (B).
Figure 4. Manual Staining : Uniform FOLRl sion in ovarian and NSCLC tumors. FOLRl expression was high in many of the ovaréan carcinomas, as well as lung adenocarcinomas and bronchioloalveolar carcinomas tested. The majority of ovarian carcinoma samples had the highest intensity staining in serous or endometrioid cells. In the NSCLC tumors, the t ABC values were found in bronchioloalveolar carcinoma and papillary adenocarcinoma.
Figure 5. Manual Staining Method: FOLRl expression is generally confined to the membrane of NSCLC cells. High resolution microscopy revealed that the majority of FOLRl staining was restricted to the membrane in NSCLC tumors.
Figure 6. Manual ng : FOLRl expression is generally confined to the membrane of ovarian cancer cells. High resolution microscopy revealed that the majority of FOLRl staining was restricted to the membrane in ovarian tumors.
Figure 7. In vivo efficacy of 9-targeted conjugates in a KB xenograft model. FOLRl-targeting cleavable conjugate huMovl9-SPDB—DM4 (B) in comparison with non-FOLRl—targeting -SPDB-DM4 (D), and non-cleavable conjugate huMovl9-PEG4-Mal-DM4 (C) in comparison with non-targeting huC242-PEG4Mal-DM4 (B) were tested using an established xenograft model of KB cells ted aneous into SCID mice. Targeting of FOLRl by huMov19 resulted in significant ion in mean tumor volume.
Figure 8. Dose-response anti-tumor activity of IMGN853 treatment in OVCAR—3 human n carcinoma xenografts. Mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg/kg. A control group of animals received a single intravenous injection of PBS.
Figure 9. Dose—response anti-tumor activity of IMGN853 treatment in lGROV—l human ovarian carcinoma xenografts. Mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg/kg. A control group of animals received a single intravenous injection of PBS.
Figure 10. Dose-response anti-tumor activity of IMGN853 ent in OV-90 human ovarian carcinoma xenografts. Mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg/kg. A control group of animals received a single intravenous injection of PBS.
Figure 11. Dose-response umor activity of IMGN853 treatment in SKOV—3 human ovarian carcinoma xenografts. Mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg/kg. A control group of animals received a single intravenous injection of PBS.
Figure 12. Dose-response umor activity of IMGN853 treatment in KB human cervical adenocarcinoma xenografts. Mice were treated with a single intravenous injection of IMGN853 at 1.0, 2.5 or 5.0 mg/kg. A control group of animals received a single intravenous injection of PBS.
Figure 13. Automated Staining s: entative Photographs and Histograms depicting FOLRl Expression in Cell Lines by IHC and Flow Cytometry.
SW620, T47D, Igrov-l, 300.19/FR1, HeLa, and KB cells were all scored for FOLRl staining intensity and uniformity. SWti30 and IGROV-l cells were scored 1—3 hetero, T47D was scored 1-2 hetero, HeLa was scored 2-3 hetero, while 300.19/FR1 and KB were scored 3 homo.
Figure 14. Automated Staining Methods: Representative FOLR] Staining in Serous n Cancer. Staining patterns demonstrating 3 homo, 2-3 homo, 2 homo, and 2 hetero staining are shown for tissue sections from serous ovarian cancer by IHC. [0038} Figure 15. Automated Staining Methods: Representative FOLRl Staining in Endometrioid Ovarian Cancer. Staining ns demonstrating 3 homo, 2—3 homo, 3 focal, and 1-2 hetero ng are shown for tissue sections from endometroid cancer by IHC.
Figure 16. Automated ng Methods: entative FOLRl Staining in NSCLC of the Adenocarcinoma Subtype (excluding bronchioloalveolar carcinomas).
Staining patterns demonstrating 3 homo, 2-3 homo, 2 , 2 homo, and 1-2 hetero staining are shown for tissue sections from non-small cell lung cancer, adenocarcinoma subtype by IHC. {0040} Figure 17. Automated Staining Methods: Representative FOLRl Staining in Endometrial arcinoma. Staining patterns demonstrating 3 hetero, 2 hetero, and 1 hetero staining are shown for tissue sections from endometrial adenocarcinoma by IHC. {0041} Figure 18. Automated ng Methods: Representative FOLRl Staining in Renal Clear Cell Carcinoma. ng patterns demonstrating 2 homo, 2 hetero, and l heteto staining are shown for tissue sections from renal cell cancer by IHC. {@423} Figure l9. The cytotoxic aetivity 0f lMGNSfiB in vitro. Five FtfliRiwpnsitive cell lines (KB? lGROV—l, IE i~3, SKOVG and OVCAR.~3} and two FOLlenegative cell, lines (Namalwa and SW2) were ed for their sensitivity to the cytotoxic effects of liviGN8530 Cells were d tn ll‘VlGNSSB {solid line) or tn lMGNtiSB plus 0.5 trM unceninga‘ted VlQ (M9346A) (dashed iine) fer 5 days, and the cell survival was determined by WSlmg-based assay, Representative data are Shawn, The percent at surviving cells was plotted against base it) legatitlnn Of'il‘ltj concentration of lMGN853. {#3343} Figure 20. The ivity ni’tlie litflQRl-pnsitive cell lines to EMGNXSB versus the level of itiOLRl expression. Peteney and city of lMG‘NSSTj was analyzed t FGLRl—pnsitive cell lines with a wide range of FOLRl expression. Cell lines were incubated Win iMGN853 and KB, lgruV—L and 3eg~3 were sneeifieally sensitive to lMGNts‘Sfi while uneonjugatetl link/lnvlil (:lVi9346A) snowed decreased activity of the ennjugate. Sam—3 and ()V‘earfi were not sensitiVe to lMtiNSSS and unconjugaied vlil A} (lid not change the activity of the conjugate.
EMMA; Figure 21. Automated Staining Methods: Ovarian Carcinema Xenegraft aey models stained for FOLRl. ng nattenis demonstrating ls} hetero (Swat '3), i—3 heme (igrov l), i~2 hetere (CV 90) and negative (SKOV 3:) are slinwn fer tissue seetiens frnni Ovarian cancer nenegrafts by ll—lC. {($45} Figure ‘22. Automated Staining Methods: Mouse Xenograft Medela Staining patterns for FOLRl in xenngrai‘ts tin" NSCLC (A), Entienietriuni Carcinoma (B) and Cervical Carcinoma ((3) Cell Lines are shown. NSCLC samples demonstrated 23 home at 2 heme ng, endornetrinni carcinoma demonstrated 2 lietern/E fecal ng, and cervical carcinoma demonstrated 3 home staining. {fitflié} Figure 23. Assay control tissues automated staining guide. Staining patterns for negative (esenliagns ti} and positive control s (salivary gland l~2 hetero? lung 21 borne, pancreas 3 home) are shown as determined by anteniated lHC. {($47} Figure- 34. Turner tissues automated staining guide. Representative staining patterns fer level 3,, level. 2, and level i staining are shown an l tissue as detennined by automated iii-iii. {@438} Figure 25. Turner tissues automated staining guide, representative staining patterns fer level 3?, level 2, and level l/negative staining are sltewn on central tissue as determined by anternated ll-lC. [0048a] tions of the specific embodiments of the invention as claimed herein follow.
] According to a first embodiment of the invention, there is provided use of an anti-Folate receptor 1 (FOLR1) immunoconjugate in the manufacture of a medicament for treating cancer in a subject, wherein increased expression of FOLR1 in a cancer sample from said subject has been detected using a detection method that distinguishes between staining intensity or staining uniformity in a FOLR1 sing cancer sample as compared to staining intensity or staining mity in one or more reference sample, wherein said anti-FOLR1 immunoconjugate has the formula (A) – (L) – (C) or (C) – (L) – (A), wherein: (A) is an anti-FOLR1 dy or antigen binding fragment thereof comprising: (a) a heavy chain CDR1 comprising GYFMN (SEQ ID NO:6); a heavy chain CDR2 comprising RIHPYDGDTFYNQKFQG (SEQ ID NO:7); and a heavy chain CDR3 comprising YDGSRAMDY (SEQ ID NO:8); and (b) a light chain CDR1 comprising KASQSVSFAGTSLMH (SEQ ID NO:9); a light chain CDR2 comprising RASNLEA (SEQ ID NO:10); and a light chain CDR3 comprising QQSREYPYT (SEQ ID NO:11), (L) is a linker, and (C) is a cytotoxic agent, and wherein the linker (L) links (A) to (C). [0048c] According to a second embodiment of the ion, there is provided use of an anti-FOLR1 immunoconjugate in the manufacture of a medicament for ng cancer in a subject, wherein a tumor of said subject has been identified as sensitive to treatment with an OLR1 conjugate, wherein the identifying comprises (a) measuring the level of FOLR1 expression in a tumor tissue sample obtained from said tumor using a detection method that distinguishes between ng intensity in a FOLR1 expressing cancer sample as compared to ng intensity in one or more reference samples; (b) determining a FOLR1 staining intensity score for said tumor tissue sample; and (c) comparing the FOLR1 staining intensity score determined in step (b) to a reference value determined by measuring FOLR1 protein expression in at least one reference sample which is not sensitive to treatment with an anti-FOLR1 immunoconjugate, and wherein a FOLR1 staining intensity score for said sample - 10a - determined in step (b) that is equal to or higher than said reference value identifies said tumor as being sensitive to treatment with an anti-FOLR1 immunoconjugate; wherein the anti-FOLR1 immunoconjugate has the formula (A) – (L) – (C) or (C) – (L) – (A), wherein: (A) is an anti-FOLR1 antibody or antigen g fragment thereof comprising: (a) a heavy chain CDR1 comprising GYFMN (SEQ ID NO:6); a heavy chain CDR2 comprising RIHPYDGDTFYNQKFQG (SEQ ID NO:7); and a heavy chain CDR3 comprising YDGSRAMDY (SEQ ID NO:8); and (b) a light chain CDR1 comprising SFAGTSLMH (SEQ ID NO:9); a light chain CDR2 comprising RASNLEA (SEQ ID NO:10); and a light chain CDR3 comprising QQSREYPYT (SEQ ID NO:11), (L) is a linker, and (C) is a cytotoxic agent, and wherein the linker (L) links (A) to (C). [0048d] According to a third embodiment of the invention, there is provided use of an anti-FOLR1 immunoconjugate in the manufacture of a medicament for treating cancer in a subject, wherein a tumor tissue sample from the subject has been identified as having a FOLR1 expression score of 1 or greater, wherein the identifying comprises: (a) contacting a tumor tissue sample from a subject having cancer with a detection antibody or antigen binding nt thereof that specifically binds FOLR1, wherein the sample is in-fixed paraffin embedded; (b) measuring the binding of said dy or n binding fragment thereof in step (a) using a detection method that can distinguish between ng intensity and staining uniformity in a FOLR1 expressing cancer sample as compared to staining intensity and ng uniformity in one or more reference samples; and (c) assigning a FOLR1 expression score to said tumor tissue sample after comparing the level of FOLR1 staining intensity and staining uniformity in said tumor tissue sample to one or more reference samples, wherein the FOLR1 expression score includes a range of 0, 1, 2, 3, and 3+ for staining intensity, wherein 0 is the lowest level of staining intensity, and wherein 3+ is the highest level of staining intensity; n the anti-FOLR1 immunoconjugate has the formula (A) – (L) – (C) or (C) – (L) – (A), wherein: - 10b - (A) is an anti-FOLR1 antibody or antigen binding fragment thereof comprising: (a) a heavy chain CDR1 comprising GYFMN (SEQ ID NO:6); a heavy chain CDR2 comprising RIHPYDGDTFYNQKFQG (SEQ ID NO:7); and a heavy chain CDR3 comprising YDGSRAMDY (SEQ ID NO:8); and (b) a light chain CDR1 comprising KASQSVSFAGTSLMH (SEQ ID NO:9); a light chain CDR2 comprising RASNLEA (SEQ ID NO:10); and a light chain CDR3 sing QQSREYPYT (SEQ ID NO:11), (L) is a linker, and (C) is a cytotoxic agent, and n the linker (L) links (A) to (C).
[TEXT CONTINUES ON PAGE 11] - 10c - DETAILED DESCRIPTION OF THE INVENTION {00419} The present inventien presides methods for increasing the efficacy of er the hood of response to the treatment of cancers characterised by the everexpressien of FOLRI. The present invention is based on the discovery of a dynamic range of expression of FOLRE in tumor tissue as ed to nerrnai tissue and the discovery that tumors with increased. Eevels nf FOLK} expression are more responsive te treatment with OLK} antibodies or antidi‘OlgRi irnmunoconjugate *, We have else discovered differences in sensitity and detection of c ranges between auternated and manual s. Kits comprising one or more reagents useful fer practicing the methods of the invention are further provided 1. Definitions To facilitate an understanding of the present invention, a number of terms and phrases are defined below. {0051} The terms "human folate or 1" or "FOLRI", as used herein, refers to any native human FOLRI, unless otherwise indicated. The term "FOLRI" encompasses "full- length," unprocessed FOLR] as well as any form of FOLRl that results from sing within the cell. The term also encompasses naturally occurring variants of FOLRl, e.g., splice variants, allelic ts and isoforms. The FOLRI polypeptides described herein can be isolated from a variety of sources, such as from human tissue types or from another source, or ed by recombinant or synthetic methods. Examples of FOLRI ces include, but are not limited to NCBI reference numbers P15328, 092242.1, AAX292681, AAXBH 19.}, Ni§>g057937.l, and NP_O57936.1, and those shown in SEQ iii) NOS: 1 and 2.
The term "increased expression" of FOLRI refers to a sample which contains elevated levels of FOLRl expression. In one example, the FOLRI expression is measured by IHC and given a staining intensity score or a staining uniformity score by comparison to controls (e.g., calibrated controls) exhibiting defined scores (e.g. an intensity score of 3 is given to the test sample if the intensity is comparable to the level 3 calibrated control or an intensity of 2 is given to the test sample if the intensity is comparable to the level 2 calibrated l). For example, a score of 1, 2, 3, or 3+ or greater by immunohistochemistry indicates an increased expression of FOLRI. A staining uniformity that is heterogeneous or homogeneous is also indicative of increased FOLRI expression. "11., The staining iretensity and ng uniformity scores can be used alone or in combination (e.g., 2 homo, 2 hetero, 3 homo, 3 hetero, etc.). In another example, an increase in FOLRI expression can be determined by detection of an increase of at least 2-fold, at least 3-fold, or at least 5-fold) relative to control values (e.g., expression level in a tissue or cell from a subject without cancer or with a cancer that does not have ed FOLRI values).
A ence sample" can be used to correlate and compare the results obtained in the methods of the ion from a test sample. Reference samples can be cells (e.g., cell lines, cell pellets) or . The FOLRl levels in the "reference sample" may be an absolute or ve amount, a range of amount, a minimum and/or maximum amount, a mean amount, and/or a median amount of FOLRI. The diagnostic methods of the ion involve a comparison between expression levels of FOLRI in a test sample and a "reference value." In some embodiments, the reference value is the expression level of the FOLRl in a reference sample. A reference value may be a predetermined value and may also be determined from reference samples (e.g., control biological samples) tested in parallel with the test s. A reference value can be a single f value, such as a median or mean or a range of values, such as a confidence interval. Reference values can be established for various subgroups of individuals, such as individuals predisposed to cancer, individuals having early or late stage , male and/or female individuals, or individuals undergoing cancer y. Examples of normal reference samples or values and positive reference samples or values are described herein.
In some embodiments, the reference sample is a sample from a healthy tissue, in ular a corresponding tissue which is not affected by cancer. These types of reference samples are referred to as negative control samples. In other embodiments, the reference sample is a sample from a tumor tissue that expresses FOLRl. These types of nce samples are ed to as positive control samples. Positivie control samples can also be used as a comparative indicator for the uniformity (hetero versus homo) and/or degree (1, 2, 3, 3+) of staining ity, which ates with the level of FOLRl expression. Positive control comparative samples are also referred to as calibrated reference samples which demonstrate a c range of staining intensity or uniformity. As shown in Examples 1- 9, non FOLRl-expressing reference samples include human esophagus tissue; low FOLRI reference includes salivary gland (particularly the intercalated ducts) and lung (particularly respiratory epithelium) tissue; and high FOLRl-expressing tissue includes the pancreas (particularly ductal cells). For cell lines, low expressors include, but are not limited to -12...
OVCAR3 and T471), moderate expressers include, but are not limited in SW‘tSZi), i lRUX/ll, 515.63, and high expressers include, but are not d to, KB and EGROVE. Particularly desirable positive high FOLK} reference is a cell line stably or transiently transt‘eeted with Folate or 1 (e.g., 300.19/FR1). Appropriate ve and negative reference levels of FOLRl for a particular cancer, may be determined by measuring levels of FOLRl in one or more appropriate subjects, and such reference levels may be tailored to c populations of subjects (e.g., a reference level may be age-matched so that comparisons may be made between FOLRl levels in samples from subjects of a certain age and reference levels for a particular disease state, phenotype, or lack thereof in a certain age group). Such reference levels may also be tailored to c techniques that are used to e levels of FOLRl in biological samples (e.g., immunoassays, etc.), where the levels of FOLRl may differ based on the specific technique that is used.
The term "primary antibody" herein refers to an antibody that binds specifically to the target protein antigen in a tissue sample. A primary antibody is generally the first antibody used in an histochemical (IHC) procedure. In one embodiment, the primary antibody is the only dy used in an IHC procedure. The term "secondary antibody" herein refers to an antibody that binds specifically to a y antibody, thereby forming a bridge between the primary antibody and a subsequent reagent, if any. The secondary antibody is generally the second antibody used in an immunohistochemical procedure. {9956} A "sample" or "biological sample" of the present ion is of biological origin, in specific embodiments, such as from eukaryotic organisms. In preferred embodiments, the sample is a human sample, but animal samples may also be used in the practice of the invention. Non-limiting sources of a sample for use in the present invention e solid tissue, biopsy aspirates, s, fluidic ts, blood, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, atory, intestinal, and genitourinary tracts, tears, saliva, milk, tumors, organs, cell cultures and/or cell culture constituents, for example. The present ion is particularly useful for cancer samples which generally comprise solid tissue samples, or other bodily fluids such as ascites, where the amount of available material is small. The method can be used to examine an aspect of expression of FOLRl or a state of a sample, including, but not limited to, comparing ent types of cells or tissues, comparing different developmental stages, and detecting or determining the presence and/or type of disease or abnormality.
For the es herein, a "section" of a tissue sample refers to a single part or piece of a tissue sample, e.g. a thin slice of tissue or cells cut from a tissue sample. It is understood that multiple sections of tissue samples may be taken and subjected to analysis according to the t invention. In some cases, the ed portion or section of tissue ses a homogeneous population of cells. In other cases, the selected portion comprises a region of tissue, e.g. the lumen as a non-limiting example. The selected portion can be small as one cell or two cells, or could represent néany thousands of cells, for example. In most cases, the collection of cells is important, and while the invention has been described for use in the detection of cellular components, the method may also be used for detecting non-cellular components of an organism (e.g. soluble components in the blood as a non— limiting example). {0058] By "correlate" or "correlating" is meant comparing, in any way, the performance and/0r results of a first is with the performance and/or results of a second analysis.
For example, one may use the results of a first analysis in carrying out the second analysis and/or one may use the results of a first analysis to determine whether a second is should be performed and/or one may e the results of a first analysis with the results of a second analysis. In one embodiment, increased expression of FOLRl correlates with increased likelihood of effectiveness of a FOLRl-targeting ancer therapy.
FREQ} The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site Wéthin the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact onal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, ic antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule sing an n ition site so long as the dies exhibit the desired biological activity. An antibody can be of any the five major s of immunoglobulins: EgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgGl, lgGZ, IgG3, lgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, resFectively. The different classes of immunoglobulins have ent and well known subunit structures and three~dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc. {0060} A "blocking" antibody or an "antagonist" antibody is one which inhibits or reduces biological activity of the antigen it binds, such as FOLRl. In a certain embodiment blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. Desirably, the biological activity is reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%. {006;} The term "anti-FOLRI antibody" or "an antibody that binds to FOLRl" refers to an antibody that is capable of binding FOLRl with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting FOLRl. The extent of binding of an anti-FOLRl antibody to an unrelated, non—FOLRl protein is less than about 10% of binding of the antibody to FOLRl as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to FOLRl has a dissociation constant (Kd) of :1 nM, 5100 nM, 510 nM, 51 nM, or 50.1 nM. Examples of OLRI antibodies are known in the art and are disclosed in US Appl. Pub. No. 009181, which is herein incorporated by reference.
The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single chain dies, and multispecifrc antibodies formed from antibody fragments. {0063] A "monoclonal dy" refers to a homogeneous antibody tion involved in the highly specific ition and binding of a single antigenic determinant, or epitope.
This is in contrast to polyclonal antibodies that typically e different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses both intact and ength monoclonal antibodies as well as dy fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion ns comprising an antibody n, and any other modified immunoglobulin molecule comprising an antigen ition site. Furthermore, "monoclonal antibody" refers to such antibodies made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals. {00%} The term "epitope" or "antigenic determinant" are used interchangeably herein and refer to that portion of an n capable of being recognized and specifically bound by a particular antibody. When the n is a polypeptide, epitopes can be formed both from uous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas es formed by ry folding are lly lost upon protein denaturing. An epitope lly includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
"Binding affinity" generally refers to the strength of the sum total of alent ctions between a single binding site of a moiecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated ise, as used herein, "binding affinity" refers to intrinsic binding y which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high— affinity antibodies generally bind antigen faster and tend to remain bound longer. A varéety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative embodiments are described in the following.
"Or better" when used herein to refer to g affinity refers to a stronger binding n a molecule and its g partner. "Or better" when used herein refers to a stronger binding, represented by a smaller numerical Kd value. For example, an antibody which has an affinity for an antigen of "0.6 nM or better", the dy’s affinity for the antigen is <0.6 nM, i.e. 0.59 nM, 0.58 nM, 0.57 nM etc. or any value less than 0.6 nM.
The phrase "substantially similar," or "substantially the same", as used herein, denotes a sufficiently high degree of similarity between two numeric values (generally one associated with an antibody of the invention and the other associated with a reference/comparator dy) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristics measured by said values (e.g., Kd values). The difference between said two values is less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% as a function of the value for the reference/comparator antibody.
» WO 35675 {00603 A polypeptide, antibody, polynucleotide, vector, cell, or composition which is "isolated" is a ptide, antibody, polynucleotide, vector, cell, or ition which is in a form not found in nature. isolated polypeptides, antibodies, polynucleotides, vectors, cell or compositions include those which have been d to a degree that they are no longer in a form in which they are found in nature. In some embodiments, an antibody, polyaucleotide, vector, cell, or composition which is isolated is substantially pure.
As used herein, "substantially pure" refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.
The term "immunoconjugate" or "conjugate" as used herein refers to a compound or a tive thereof that is linked to a cell binding agent (i.e., an anti-FOLRl antibody or fragment thereof) and is defined by a generic formula: C-L-A, wherein C = cytotoxin, L = linker, and A — cell binding agent or anti-FOLRl antibody or antibody fragment. lmmunoconjugates can also be defined by the generic formula in reverse order: A-L-C. [0%71] A "linker" is any chemical moiety that is capable of g a compound, usually a drug, such as a maytansinoid, to a cell-binding agent such as an anti FOLRI antibody or a fragment thereof in a stable, covalent manner. s can be susceptible to or be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active. Suitable linkers are well known in the art and e, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Linkers also include charged linkers, and hilic forms thereof as described herein and know in the art.
The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth.
Examples of cancer include, but are not d to, carcinoma, lymphoma, blastoma, a, and leukemia. More particular examples of such cancers include us cell cancer, small-cell lung cancer, non—small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal , pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast , colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancers. {0073] "Tumor" and "neoplasm" refer to any mass of tissue that result from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including ncerous lesions.
The terms "cancer cell," "tumor cell," and grammatical equivalents refer to the total population of cells deréved from a tumor or a pre—cancerous lesion, including both nontumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells). As used herein, the term "tumor cell" will be modified by the term "non—tumorigenic" when referring solely to those tumor cells lacking the ty to renew and differentiate to distinguish those tumor cells from cancer stem cells.
The term "subject" refers to any animal (e. g., a mammal), including, but not limited to , non-human es, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and nt" are used interchangeably herein in reference to a human subject.
Administration "in combination with" one or more further therapeutic agents includes simultaneous rrent) and consecutive stration in any order.
The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulation can be sterile.
An "effective amount" of an antibody as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An "effective amoun can be determined empirically and in a routine manner, in relation to the stated purpose.
The term "therapeutically effective " refers to an amount of an antibody or other drug effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the tumor size; t (i.e., slow to some extent and in a n embodiment, stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and in a certain embodiment, stop) tumor metastasis; inhibit, to some , tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. See the definition herein of "treating". To the extent the drug can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. In certain ments, identification of -18<— increased FOLRl levels allows for administration of sed amounts of the FOLRl- targeting therapeutic to achieve the same therapeutic effect as seen with higher dosages. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time ary, to achieve the desired prophylactic result. Typically but not necessarély, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically ive amount will be less than the therapeutically effective amount.
The term nd favorably" generally refers to causing a beneficial state in a subject. With respect to cancer treatment, the term refers to providing a eutic effect on the subject. Positive therapeutic effects in cancer can be measured in a number of ways (See, W.A. Weber, J. Nucl. Med. 50:lS-IOS (2009)). For example, tumor growth inhibition, molecular marker expression, serum marker expression, and molecular imaging techniques can all be used to assess therapeutic efficacy of an anti—cancer therapeutic. With to tumor growth inhibition, according to NCI standards, a T/C S 42% is the respect minimum level of anti-tumor activity. A T/C <10% is considered a high anti—tumor activity level, with T/C (%) = Median tumor volume of the treated / Median tumor volume of the control x 100.
The word "label" when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody. The label can be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, can catalyze chemical tion of a ate nd or composition which is detectable.
A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Classes of herapeutic agents include, are not d to: alkyating agents, antimetabolites, spindle poison plant alkaloids, cytoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in "targeted therapy" and conventional herapy.
. Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both 1) eutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or er. Thus, those in need of ent include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. in certain embodiments, a subject is sueeessfitliy "treated" for cane-er ing to the methods of the present invention if the patient shows one or more of the following: reduction in eaehexia, increase in survival time, elongation in time to tumor ssion, ion in tumor mass, reduction in tumor burden and/or a prolongation in time to tumor metastasis, time to tumor recurrence, tumor response, eompiete response, partiai response, stabie e, progressive disease, progression tree survival (FPS), overali survival (08), each as measured by standards set by the Nationai Cancer institute and the US. Food and Drug Administration for the approvai of new drugs. See Johnson et ai, (2003) 3, Che.
Oneoi. 21t?):i404~i41 t.
"Progression free survivai" (PPS), also referred to as or "Time to Tumor ssion" (YEP) indicates the length of time during and after treatment that the cancer does not grow. Progression-free survival es the amount of time patients have experéenced a complete response or a partial response, as well as the amount of time patients have experienced stable disease.
"Disease free survival" (DFS) refers to the length of time during and after treatment that the patient remains free of disease.
"Overall Survival" (OS) refers to a prolongation in life expectancy as ed to naive or untreated individuals or patients.
As used in the present disclosure and claims, the singular forms "a," "an," and "the" include plural forms unless the context clearly dictates otherwise.
It is tood that wherever embodiments are described herein with the ge "comprising," otherwise analogous embodiments described in terms of "consisting of‘ and/or "consisting ially of" are also provided.
The term r" as used in a phrase such as "A and/or B" herein is intended to include both "A and B," "A or B," "A," and "B." Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following ments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). 11. Biological samples Biological samples are often fixed with a fixative. Aldehyde fixatives such as formalin ldehyde) and glutaraldehyde are typically used. Tissue samples fixed using other fixation techniques such as alcohol immersion (Battifora and Kopinski, J. Histochem. _20e 2012/031544 Cytechem. (ll-$86) .?) are also suitable. The samples used may also be embedded in paraffin. in one embodiment, the tissue samples are both lbrmalinafixed and in-- embedded (PEPE). in another embodiment, the FFPE block is hematoxylin and ecsin stained prior to ing ene 01' more portions fer analysis in order to select specific areats) fer the FFPE core sample. Methods of preparéng tissue blocks from these particulate specimens have been used in previous EEC studies cf varicus pregnnstic factors, and/er is well. known to those of skill in the art (see, for example, Abbcndanzo et al, Am 3 Clin Pathol. @990 l‘vlay;93(5):698»702; Allred et al., Arch Surg. l99t) .Ean;l25(l):lll7—l3). {@591} Briefly, any intact organ or tissue may be cut into fairly small pieces and incubated in various fixatives (eg. formalin, alcohol, etc.) for varying s of time until the tissue is "fixed". The samples may be virtually any intact tissue surgically removed from the body.
The samples may be cut into reasonably small piece(s) that fit on the equipment ely used in histopathology laboratories. The size of the cut pieces typically ranges from a few millimeters to a few centimeters.
III. Detection Antibody Conjugates The present invention further provides antibodies against FOLRl, generally of the monoclonal type, that are linked to at least one agent to form a detection antibody conjugate. In order to increase the efficacy of antibody molecules as stic it is conventional to link or ntly bind or x at least one desired molecule or moiety.
Such a molecule or moiety may be, but is not limited to, at least one reporter molecule. A reporter molecule is defined as any moiety that may be detected using an assay. Non- limiting examples of reporter molecules that have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, scent molecules, photoaffinity molecules, colored particles and/or ligands, such as .
Any cell binding agent (e.g., an antibody or polypeptide) of sufficient selectivity, specificity or affinity may be employed as the basis for detection of the FOLRl polypeptide.
Such properties may be evaluated using conventional immunological screening methodology known to those of skill in the art. Sites for binding to biological active molecules in the antibody molecule, in addition to the cal antigen g sites, include sites that reside in the variable domain that can bind the antigen. In addition, the -21". variable domain is involved in antibody self—binding (Kang et al., 1988) and ns epitopes (idiotopes) recognized by anti-antibodies (Kohler et al., 1989).
Certain examples of protein binding (e.g., antibody) conjugates. are those conjugates in which the protein binding agent (e.g., antibody) is linked to a detectable label.
"Detectable labels" are compounds and/or ts that can be detected due to their specific functional properties, and/or chemical teristics, the use of which allows the antibody to which they are attached to be detected, and/or further quantified if desired. {05.395} Many appropriate g agents are known in the art, as are s for their attachment to antibodies (see, for e.g., US. Pat. Nos. 236; 4,938,948; and 509, each incorporated herein by nce). The imaging moieties used can be paramagnetic ions; radioactive isotopes; fluorochromes; NMR—detectable substances; and/or X—ray imaging, for example. ary fluorescent labeis contemplated for use as protein binding (e.g., antibody) conjugates include Alexa 350, Alexa 430, Alexa 488, AMCA, BODIPY 630/650, BODIPY 5, BODIPY—FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Dylight 488, Fluorescein Isothiocyanate, Green fluorescent protein (GFP), HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, Phycoerythrin, REG, Rhodamine Green, Rhodamine Red, tetramethyl rhodamine (TMR), Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, Texas Red, and derivatives of these labels (i.e halogenated analogues, modified with isothiocyanate or other linker for ating, etc), for example. An exemplary radiolabel is tritium. £13097} Protein binding (e.g., antibody) detection conjugates contemplated in the present invention include those for use in Vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will te a colored product upon contact with a genic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and/or glucose oxidase. Preferred secondary binding ligands are biotin and/or avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in US.
Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.
Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene ediates that are generated by low intensity ultraviolet light r & Haley, 1983). In particular, 2- and 8—azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). The 2- and 8- azido nucleotides have also been used to map tide binding domains of purified proteins on et al., 1989; King et al., 1989; and Dholakia et al., 1989) and may be used as antibody binding agents.
Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate x employing, for example, an organic chelating agent such a diethylenetréaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N- chloro-p-toluenesulfonamide; and/or tetrachloro-3a-6d-diphenylglycouril-3 attached to the antibody (US. Pat. Nos. 509 and 4,938,948, each incorporated herein by nce).
Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Protein binding (e. g., antibody) conjugates with fluorescein markers are ed in the presence of these coupling agents or by reaction with an isothiocyanate. In US. Pat. No. 4,938,948, imaging of breast tumors, for example, is achieved using monoclonal dies, and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p-hydroxybenzimidate or N—succinimidyl(4- yphenyl)—propionate.
In other embodiments, derivatization of globulins by selectively introducing sulfliydryl groups in the Fc region of an immunoglobulin using reaction conditions that do not alter the antibody combining site are contemplated. Antibody conjugates produced according to this methodo‘rogy are disclosed to t improved longevity, specificity and sensitivity (US. Pat. No. 5,196,066, orated herein by reference). Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is ated to a carbohydrate residue in the Fc region, have also been disclosed in the literature (O'Shannessy et al., 1987).
In other embodiments of the invention, immunoglobulins are abeled with nuclides such as tritium. In additional embodiments, nanogold particles (such as sizes from about 0.5 nm-40 nm) and/or Quantum Dots (Hayward, Calif.) are employed.
IV. Enzymes and Substrates (Chromagens) The use of substrates and indicators is contemplated for detection of FOLRl, such as the exemplary embodiments ed below, for example. _23, Horseradish peroxidase (HRP) is an enzyme that first forms a complex with hydrogen peroxide and then causes it to decompose, resulting in water and atomic oxygen.
Like many other enzymes, HRP and some HRP—like activities can be inhibited by excess substrate. The complex formed between HRP and excess hydrogen peroxide is catalytically inactive and in the absence of an electron donor (e.g. chromogenic substance) is ibly inhibited. It is the excess hydrogen peroxide and the e of an electron donor that brings about quenching of endogenous HRP activities.
} When used in assays systems, HRP can also be used to convert a defined substrate into its activated chromagen, thus causing a color change. The HRP enzyme may be ated to an antibody, protein, peptide, polymer, or other molecule by a number of methods. Such methods are known in the art. Adding glutaraldehyde to a solution ning an admixture of HRP and antibody will result in more antibody molecules being conjugated to each other than to the . In the two-step procedure, HRP reacts with the bifunctional ts first. In the second stage, only activated HRP is admixed with the antibody, resulting in much more efficient labelling and no polymerization. HRP is also ated to (strept)avidin using the two—step glutaraldehyde procedure. This form is used in ures where LAB and LSAB are substrate, for example. Conjugation with biotin also involves two steps, as biotin must first be derivatized to the biotinyl-N— hydroxysuccinimide ester or to biotin hydrazide before it can be reacted with the epsilonamino groups of the HRP enzyme. 3,3'—Diaminobenzidine (DAB) is a ate for enzymes such as HRP that produces a brown end product that is highly insoluble in l and other organic solvents.
Oxidation of DAB also causes polymerization, resulting in the ability to react with osmium tetroxide, and thus increasing its staining intensity and electron density. Of the several metals and methods used to intensify the optical density of polymerized DAB, gold chloride in combination with silver sulfide appears to be the most successful. 3-Amino—9-ethylcarbazole (ABC) is a substrate for enzymes such as HRP. Upon oxidation, forms a rose-red end product that is alcohol soluble. Therefore, ens processed with ABC must not be immersed in alcohol or alcoholic ons (e.g., Harris' hematoxylin). Instead, an aqueous counterstain and ng medium should be used. ABC is unately susceptible to further oxidation and, when exposed to excessive light, will fade in intensity. Storage in the dark is therefore recommended. 4-Chloro-1—naphthol (CN) is a substrate for enzymes such as HRP and itates as a blue end product. Because CN is soluble in alcohol and other c solvents, the specimen must not be dehydrated, exposed to alcoholic counterstains, or coverslipped with mounting media ning organic solvents. Unlike DAB, CN tends to diffase from the site of precipitation. ylenediamine dihydrochloride/pyrocatechol (Hanker-Yates reagent) is a an electron donor substrate for enzymes such as HRP and gives a lack reaction product that is insoluble in alcohol and other c solvents. Like polymerized DAB, this reaction product can be osmicated. Varying results have been achieved with Hanker-Yates reagent in immunoperoxidase techniques. [0100} Calf intestine alkaline phosphatase (AP) (molecular weight 100 kD) is an enzyme that removes (by hydrolysis) and transfers phosphate groups from organic esters by breaking the P-O bond; an intermediate enzyme-substrate bond is briefly formed. The chief metal activators for AP are Mg++, Mn++ and Ca++. {0101] AP had not been used extensively in immunohistochemistry until publication of the unlabeled alkaline phosphataseantialkaline phosphatase (APAAP) procedure. The soluble immune complexes utilized in this procedure have molecular weights of approximately 560 kD. The major advantage of the APAAP procedure compared to the PAP technique is the lack of interference posed by nous peroxidase activity. Because of the potential distraction of endogenous peroxidase activity on PAP staining, the APAAP technique is recommended for use on blood and bone marrow smears. Endogenous alkaline phosphatase activity from bone, kidney, liver and some white cells can be inhibited by the addition of l min-ft levamisole to the substrate solution, although 5 mM has been found to be more effective. Intestinal ne phosphatases are not adequately inhibited by levamisoie.
In the immunoalkaline phosphatase staining method, the enzyme hydrntyzes naphthol phosphate esters (substrate) to phenolic compounds and ates. The phenols couple to colorless diazonium salts (chromogen) to produce insoluble, d azo dyes.
Several different combinations of substrates and chromogens have been used sfully. ol AS-MX ate can be used in its acid form or as the sodium salt. The chromogens Fast Red TR and Fast Blue BB produce a bright red or blue end product, respectively. Both are e in alcoholic and other organic solvents, so aqueous mounting media must be used. Fast Red TR is preferred when staining cell smears. {area} Additional exemplary substrates e naphthol AS—BI ate, naphthol AS- TR phosphate and 5—bromochloro-3—indoxyl ate . Other possible gens include Fast Red LB, Fast Garnet GBC, Nitro Blue Tetrazolium (NBT) iodonitrotetrazolium Violet (INT), and derivatives of the structures, for example.
V. Immunodetection Methods In still further embodiments, the present invention concerns immunodetection methods for binding, purifying, removing, quantifying and/or otherwése generally detecting biological ents such as a ligand as contemplated by the present invention. The antibodies prepared in accordance with the present invention may be ed to detect wild-type and/or mutant ligand proteins, polypeptides and/or peptides. As described throughout the present application, the use of wild—type and/or mutant ligand specific antibodies is contemplated. Some immunodetection s include flow cytometry, enzyme linked imrnunosorbent assay ), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent useful immunodetection assay, and Western blot to mention a few. The steps of various methods have been described in the scientific literature, such as, e.g., Doolittle M H and Ben-Zeev 0, Methods Mol Biol. 1999;109:215-37; Gulbis B and Galand P, Hum Pathol. 199% Dec;24(12):1271—85; and De Jager R et al., Semin Nucl Med. 1993 (2):l65-79, each incorporated herein by reference.
In general, the immunobinding methods include obtaining a sample suspected of comprising ligand protein, polypeptide and/or peptide, and contacting the sample with a first ligand binding peptide (e.g., an anti-ligand antibody) in accordance with the present invention, as the case may be, under conditions effective to allow the formation of complexes. in terms of antigen ion, the biological sample analyzed may be any sample that is suspected of comprising a wild—type or mutant ligand protein-specific antigen, such as a tissue section or specimen, a homogenized tissue t, biopsy aspirates, a cell, separated and/or purified forms of any of the above wild-type or mutant FOLRl-containing itions, or even any biological fluid that comes into contact with the tissue, including blood and/or serum, although tissue samples or extracts are preferred.
Contacting the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes -26, (prémary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to, any ligand protein antigens present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or n blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the prémary immune complexes to be detected.
In l, the detection of immunocomplex formation is well known in the art and may be achieved through the ation of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, ical and enzymatic tags. US. Patents concerning the use of such labels include US. Pat. Nos. 837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 241, each incorporated herein by reference. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody arid/or a biotin/avidin ligand binding arrangement, as is known in the art.
The anti—ligand antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined.
Alternatively, the first antibody that s bound within the primary immune xes may be detected by means of a second binding agent that has binding y for the antibody. In these cases, the second binding agerét may be linked to a detectable label. The second binding agent is itself often an antibody, which may thus be termed a "secondary" antibody, or a polymer detection system. The primary immune complexes are contacted with the labeled, secondary binding agent, or antibody/polymer detection system, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune xes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected. [0111: Further methods e the detection of primary immune complexes by a two-step ch. A second g agent, such as an antibody, that has binding affinity for the antibody is used to form secondary immune complexes, as bed above. After washing, the secondary immune complexes are contacted with a third binding agent or antibody that has binding y for the second antibody, again under effective conditions and for a .27~ period of time sufficient to ailow the formation of immune complexes {tertiary immune. compiexes). The third ligand or antibody is iinked to a detectabie iabei, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal. itication if this is desired. it}: E2} In another ment, a biotinylated monoclonal or polyclonal antibody is used to detect the target antigen(s), and a second step antibody is then used to detect the biotin attached to the complexed biotin. In that method the sample to be tested is first incubated in a solution comprising the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody/antigen complex. The antibody/antigen x is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or mentary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is ted in a solution comprising the second step dy against biotin. This second step antibody is labeled, as for example wéth an enzyme that can be used to detect the presence of the antibody/antigen x by histoenzymology using a chromogen substrate. With suitable amplification, a protein binding (e.g., antibody) conjugate can be produced that is macroscopically visible.
Another known method of immunodetection takes age of the immuno-PCR (Polymerase Chain Reaction) methodology. The PCR method uses a DNA/biotin/streptavidin/antibody complex that is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate ls. In specific embodiments, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule. Such detection may take place in real-time. For example, the use of quantitative real-time PCR is contemplated. {(3114} In the clinical diagnosis and/or monitoring of patients with various forms of e, the detection of a FOLRI mutant, and/or an tion in the levels of FOLRl, in comparison to the levels in a corresponding biological sample from a normal t is indicative of a patient with the disease. However, as is known to those of skill in the art, such a clinical diagnosis would not necessarily be made on the basis of this method in isolation. Those of skill in the art are very familiar with differentiating between significant differences in types and/or amounts of biomarkers, which ent a positive identification, and/or low level and/or background changes of biomarkers. Indeed, ound expression levels are often used to form a "cut-off" above which increased detection will be scored as significant and/or positive. 39115; In one embodiment, immunological detection (by immunohistochemistry) of FOLRI is scored for both intensity and uniformity (percent of stained cells — membrane only). Comparative scales for FOLRl expression for intensity correlate as 0 — Negative, 0- 1 — te, 2-3 — Moderate to Strong, - Weak to Moderate, 2 - Very Weak, 1 — Weak, 1-2 3 — Strong. Quantitatively, Score 0 represents that no membrane staining is observed in tumor cells. A Score 1 represents a faint/barely perceptible membrane staining in tumor cells. For Score 2, a moderate membrane staining is ed in tumor cells. Lastly, Score 3 or 3+ represents a moderate to strong membrane staining in the tumor cells. Those samples with 0 or 1 score for FOLRI expression may be terized as not pressing FOLRl, whereas those samples with 2 or E scores may be characterized as overexpressing FOLRI. Samples pressing FOLRl may also be rated by immunohistochemical scores corresponding to the number of copies of FOLRI molecules sed per cell, and have been determined biochemically: 0=0-10,000 copies/cell, l=at least about 200,000 copies/cell, 2=at least about 500,000 copies/cell, and 3=at least about 2,000,000 copies/cell. Comparative scales for FOLRl percent cell membrane staining uniformity correlate as follows: 0 — ve, Focal - 25- - <25%, heterogeneous (hetero) 75%, and homogeneous (homo) - >75%.
VI. Nucleic Acid Hybridization In situ ization is generally d out on cells or tissue sections fixed to slides. In situ hybridization may be performed by several conventional methodologies (See for e.g. Leitch et al. In situ Hybridization: a practical guide, Oxford BIOS Scientific Publishers, Microscopy handbooks v. 27 (1994)). In one in situ ure, fluorescent dyes (such as fluorescein isothiocyanate (FITC) that fluoresces green when excited by an Argon ion laser) are used to label a nucleic acid sequence probe that is complementary to a target tide sequence in the cell. Each cell comprising the target nucleotide sequence will bind the labeled probe, producing a fluorescent signal upon exposure of the cells to a tight source of a wavelength appropriate for tion of the specific fluorochrome used.
Various degrees of hybridization stringency can be employed. As the hybridization conditions become more stringent, a greater degree of complementarity is required between -29.. 2012/031544 the probe and target to form and maintain a stable duplex. ency is increased by raising temperature, lowering salt concentration, or raising formamide concentration. Adding dextran sulfate or raising its concentration may also increase the effective concentration of labeled probe to increase the rate of hybridization and ultimate signal intensity. After hybridization, slides are washed in a solution generally comprising reagents similar to those found in the hybridization solution with washing time varying from minutes to hours depending on required stringency. Longer or more ent washes typically lower nonspecific background but run the risk of decreasing overall sensitivity. {aria} Probes used in c hybrédization analysis may be either RNA or DNA oligonucleotides or polynucleotides and may contain not only naturally-occurring nucleotides but their analogs, like digoxygenin dCTP, biotin dcTP 7—azaguanosine, azidothymidine, inosine, or uridine, for example. Other useful probes e e probes and ues thereof, branched gene DNA, peptidometics, peptide c acid (PNA) and/or antibodies, for example. {911§§ Probes should have sufficient mentarity to the target nucleic acid sequence of interest so that stable and specific binding occurs between the target nucleic acid sequence and the probe. The degree of homology required for stable hybridization varies with the stringency of the hybridization medium and/or wash medium. Preferably, completely homologous probes are employed in the present ion, but persons of skill in the art will readily appreciate that probes exhibiting lesser but ent homology can be used in the present invention (see for e.g. Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, (1989)). {@129} Probes may also be generated and chosen by several means including, but not d to, mapping by in situ hybridization, somatic cell hybrid panels, or spot blots of sorted chromosomes; chromosomal linkage analysis; or cloned and isolated from sorted chromosome libraries from human cell lines or somatic cell hybrids with human chromosomes, radiation c cell hybrids, microdissection of a chromosome region, or from yeast artificial somes (YACs) identified by PCR primers specific for a unique chromosome locus or other suitable means like an nt YAC clone. Probes may be genomic DNA, cDNA, or RNA cloned in a plasmid, phage, cosmid, YAC, Bacterial Artificial Chromosomes (BACS), viral vector, or any other le vector. Probes may be cloned or synthesized chemically by conventional methods. When cloned, the isolated probe nucleic acid fragments are typically inserted into a vector, such as lambda phage, pBR322, M13, or vectors containing the SP6 or T7 promoter and cloned as a library in a bacterial host. [See for e.g. ok, J., Fritsch, E. F., Maniatis, T., lar Cloning A tory , Cold Spring Harbor Press, (1989)]. {$122} Probes are preferably labeled, such as with a fluorophor, for example. Examples of fluorophores include, but are not limited to, rare earth chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, Lissamine, umbelliferone, phycocrytherin, phycocyanin, or commercially available fluorophors such SPECTRUM TM and SPECTRUM GREENTM and/or derivatives of any one or more of the above. Multiple probes used in the assay may be labeled with more than one distinguishable fluorescent or pigment color. These color differences provide a means to identify the hybridization positions of specific probes. Moreover, probes that are not separated spatially can be identified by a different color light or pigment resulting from mixing two other colors (e.g., light red+green=yellow) pigment (e.g., blue+yellow=green) or by using a filter set that passes only one color at a time.
Probes can be labeled ly or indirectly with the fluorophor, utilizing conventional methodology known to one with skill in the art.
VII. Detection Kits and itions Also provided by the invention are kits for use in the practice of the present invention as disclosed herein. Such kits may comprise containers, each with one or more of the various reagents (typically in concentrated form) utilized in the s, including, for example, one or more binding agents (antibodies), already attached to a marker or optionally with reagents for coupling a binding agent to an antibody or c acid le (as well as the marker itself); buffers, the appropriate nucleotide triphosphates (e.g. dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP and UTP), e transcriptase, DNA polymerase, RNA polymerase, and one or more sequence-specific or degenerate prémers for use in detection of nucleic acid molecules by amplification; and/or reagents and instrumentation for the isolation (optionally by microdissection) to support the practice of the invention. A label or tor describing, or a set of instructions for use of, kit components in a ligand detection method of the t invention, will also be typically included, Where the instructions may be associated with a package insert and/or the packaging of the kit or the components thereof. -31... 2012/031544 In still further ments, the present invention concerns detection kits for use with the immunodetection methods described above. As the antibodies are generally used to detect wild—type and/or mutant proteins, polypeptides and/or peptides, the antibodies will preferably be included in the kit. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to a ype and/or mutant protein, polypeptide and/or peptide, and/or optionally, an immunodetection reagent and/or further optionally, a ype and/or mutant protein, polypeptide and/or peptide.
The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are ated with and/or linked to the given antibody. Detectable labels that are associated with and/or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies or polymers that have g affinity for the first antibody.
Further suitable immunodetection reagents for use in the present kits include the two—component t that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody or polymer that has binding y for the second antibody, the third antibody being linked to a able label. As noted above, a number of exemplary labels are known in the art and/0r all such labels may be suitably employed in tion wéth the present invention.
The kits may further comprise a suitably aliquoted composition of the wild-type and/or mutant protein, polypeptide and/or polypeptide, Whether labeled and/or unlabeled, as prepare contain may be used to a standard curve for a detection assay. The kits may antibody- or polymer-label conjugates either in fully conjugated form, in the form of intermediates, and/or as separate moieties to be conjugated by the user of the kit. The components of the kits may be ed either in aqueous media and/or in lized form. {0123] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the antibody may be placed, and/0r ably, suitably aliquoted. The kits of the present invention will also typically include a means for containing the antibody, antigen, and/or any other reagent containers in close confinement for commercial sale. Such containers may include injection and/or blow- molded plastic containers into which the desired Vials are retained. gnzw The kits may further comprise one or more therapeutic agents for the treatment of cancer, such as a FOLRl immunoconjugate and/or a chemotherapeutic agent. -32.. terse; The kit may further comprise an a FOLRl detection t used to measure FOLRl expression in a subject comprésing a FOLRl detection reagent, and instructions for use. In one embodiment, the FOLRl detection reagent comprises a FOLR] binding peptide, protein or a lar probe (i.e. nucleic acid). In another embodiment, the FOLRl detection reagent is an anti-FOLRl antibody. In another embodiment, the kit further comprises a secondary antibody which binds the anti-FOLRI antibody. In one embodiment the FOLRl- c antibody is included at a concentration of 0.5 to 7.5 ug/ml, preferably 0.9 to 3.8 +/— 0.5 ug/ml. In r embodiment, the antibody is included at a concentration of 1.0 +/- 0.5 ug/ml, 1.5 +/— 0.5 ug/ml, 1.9 +/- 0.5 ug/ml, 2.5 +/- 0.5 ug/ml, 3.0 +/- 0.5 ug/ml, 3.5 +/— 0.5 ug/ml, 3.8 +/- 0.5 ug/ml, or up to 4.2 ug/nil. In another embodiment, the antibody is included in concentrated solution with instructions for dilutions to achieve a final concentration of 0.9 to 3.8 +/- 0.5 ug/ml. In another embodiment, the kit further comprises a detection reagent selected from the group consisting of: an enzyme, a fluorophore, a radioactive label, and a luminophore. In another embodiment, the detection reagent is selected from the group ting of: , digoxigenin, fluorescein, tritium, and rhodamine. {$131} The kit can also include instructions for detection and scoring of FOLRl expression.
The kit can also include control or reference samples. Non-limiting examples of control or reference s include cell pellets or tissue culture cell lines derived from normal (normal control) or tumor ive l) samples. Exemplary cell lines include KB, NCI-H2llO, Igrov-l, Ishikawa, Jeg-3, Skov-3, Hela, T47D, Caco2, SW620, OAW28, HCC827, Ovcar—8, and Ovcar-3, OV—90, other tumor cell lines known to express FOLRI, and cell lines stably or transiently transfected with an expression vector that expresses FOLRl. Additional es for positive control tissues can also be found in Examples 9- 11. The kit can also comprise a staining guide which ly depicts positive and normal reference samples for staining intensity and uniformity. Such staining guides can have reference s from normal lung, pancreas, and/or salivary gland, and d tumors with standardized scores (e.g., ovarian, lung, renal, and endometrial cancers, as well as those described in the es and in Figures 23—25) VIII, FOLifilubinding agents Any antibodies that bind FOLRI can be used in the detection methods of the present invention. Examples oftherapeuticaily effective anti—FOLRI antibodies can be tbund in US Appl. Pub. No. US 009181 which is herein incorporated by reference. The fulllength amino acid (aa) and nucleotide (nt) sequences for FOLR1 are known in the art and also provided herein as represented by SEQ ID NOs: 1 and 2, respectively. A specifically useful antibody for detection of FOLR1 is the mouse monoclonal antihuFOLR1 clone BN3.2 (Leica # NCL-L-FRalpha). An e of a therapeutically effective anti-FOLR1 antibody is huMov19 (M9346A). The polypeptides of SEQ ID NOs: 3-5 comprise the variable domain of the heavy chain of huMov19 (M9346A), and the variable domain light chain version 1.00, the variable domain light chain version 1.60 of huMov19, respectively. The huMov19 (M9346A) antibody comprises: (a) a heavy chain CDR1 comprising GYFMN (SEQ ID NO:6); a heavy chain CDR2 comprising GDTFYNQKFQG (SEQ ID NO:7); and a heavy chain CDR3 comprising YDGSRAMDY (SEQ ID NO:8); and (b) a light chain CDR1 comprising KASQSVSFAGTSLMH (SEQ ID NO:9); a light chain CDR2 comprising A (SEQ ID NO:10); and a light chain CDR3 comprising QQSREYPYT (SEQ ID NO:11).
In certain embodiments, the huMov19 (M9346A) antibody is encoded by the plasmids deposited with the American Type Culture tion (ATCC), located at 10801 University Boulevard, Manassas, VA 20110 on April 7, 2010 under the terms of the Budapest Treaty and having ATCC t nos. PTA-10772 and PTA-10773 or 10774.
Examples of FOLR1 immunoconjugates useful in the therapeutic methods of the invention are provided below.
IX. FOLR1 Immunoconjugates The present invention also es methods for increasing the cy of conjugates (also referred to herein as immunoconjugates), comprising the anti-FOLR1 antibodies, antibody fragments, onal equivalents, improved antibodies and their aspects as disclosed herein, linked or conjugated to a cytotoxin (drug) or g.
Exemplary FOLR1 immunoconjugates can be found in US Appl. Pub. No. US 2012/0009181, which is herein incorporated by reference. A particularly effective therapeutic immunoconjugate of the invention comprises the huMov19 antibody described above.
Suitable drugs or prodrugs are known in the art. In certain embodiments, drugs or prodrugs are cytotoxic agents. The xic agent used in the xic conjugate of the present invention can be any compound that results in the death of a cell, or induces cell death, or in some manner decreases cell ity, and includes, for example, maytansinoids and maytansinoid analogs, benzodiazepines, taxoids, CC-1065 and CC- 1065 analogs, duocarmycins and duocarmycin analogs, enediynes, such as calicheamicins, dolastatin and dolastatin analogs including auristatins, tomaymycin derivatives, leptomycin derivatives, rexate, cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, stine, [TEXT CONTINUES ON PAGE 35] - 34a - ian, mitoh’tyciri C, chiorambucii and iirio doxombicin, In certain embodiments, the cytotoxic agents are maytansinoids and maytansirioids analogs, {0135} The drug or prodrug cart, for exampie, be limited to the anti—FOLK antibody, such as hul‘vtoviéi, or fragment thereof through a disulfide bond. The iinirer molecule or inking agent comprises a reactive chemical group that can react with the airtix-FOLRl antibody or tragn‘tent thereof, in certain embodiments, reactive chemicai groups for reaction with the cell—binding agent are ,NLSticciitimidyi esters and. Neuifosuccinimidyi esters. Additionaliy the linker molecule comprises a reactive chemical group, in certain embodiments a ditiiiopyridyl group that can react with the drug to form a disuitide bond. in certain embodiments, ticker molecules inciude, for e, Al’nsuccinimidyi 3«{2~ pyridyidithio} propionate (SPDP) (see, cg, on et ai‘, Biochem. J, {173: 23337 )g A’T—SUCCiHitnidyi 4-«(2:pyridyidithiokutaiioate (SPDB) (see, eg US: Patent No. 4,563,304): i\7l~3ttccininiidyi yridyidithioiflm-suifobutanoate (sulfo-SPDB) (see US Publication No. 74713) , N—succinimidyl 4—(2—pyridyldithio) pentanoate (SPP) (see, e.g., CAS Registry number 341498-08—6), othiolane, or acetylsuccinic anhydride.
Antibody-maytansinoid conjugates with non-cleavable links can also be prepared.
Such crosslinkers are described in the art (see Scientific Pierce Crosslinking Technical Handbook and US Patent Application Publication No. 2005/0169933) and include but are not limited to, N—succinimidyl 4-(maleimidomethyl) exanecarboxylate (SMCC), N—succinimidyl—4-(N-maleimidomethyl)-cyclohexane—l- carboxy—(6-amidocaproate), which is a "long chain" analog of SMCC (LC—SMCC), K- maleimidoundecanoic acid N—succinimidyl ester (KMUA), B-maleimidopropanoic acid N- imidyl ester (BMPS), y—maleimidobutyric acid N-succinimidyl ester (GMBS), 8- maleimidocaproic acid N—hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl—N- hydroxysuccinimide ester (MBS), N-(oc-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6—(B-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4—(p- maleimidophenyl)~butyrate (SMPB), and N—(p-maleimidophenyl)isocyanate (PMPI), N- succinimidyl(iodoacetyl)—aminobenzoate (SIAB), N-succinimidyl iodoacetate (SEA), N— succinimidyl bromoacetate (SBA), and N-succinimidyl 3-(bromoacetamido)propionate (SBAP). In certain embodiments, the antibody is modified with crosslinking ts such as succinimidyl 4—(N-maleimidomethyl)—cyclohexane—l-carboxylate (SMCC), sulfo—SMCC, maleimidobenzoyl-N—hydroxysuccinimide ester (MBS), sulfo—MBS or succinimidyl- etate, as described in the literature, to uce l-lO reactive groups (Yoshitake et a1, Eur. J. Biochem, 101:395-399 (1979); Hashida et al, J. Applied Biochem., 56—63 (1984); and Liu et at, Biochem, 182690-697 (1979)).
The present invention includes aspects wherein about 2 to about 8 drug molecules ("drug load"), for example, maytansinoid, are linked to an anti-FOLRl antibody or fragment thereof, the anti-tumor effect of the conjugate is much more efficacious as compared to a drug load of a lesser or higher number of drugs linked to the same cell binding agent.
"Drug load", as used herein, refers to the number of drug molecules (e.g., a sinoid) that can be attached to a cell binding agent (e.g., an anti-FOLRl antibody or fragment thereof). In one aspect the number of drug les that can be attached to a cell binding agent can average from about 2 to about 8 (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, .0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1). In n embodiments, the drug is N21 deacetyl—NT-(3—mercapto-l-oxopropyl)—maytansine (DMl) or N27—deacetyl—N2’-(4-mercapto- 4-methyl-l-oxopentyl) maytansine (DM4). Thus, in a certain embodiment, the antibody huMovl9 is conjugated to DMI or DM4. In another embodiment, the antibody FR—l-21 is conjugated to DMl or DM4. In another embodiment, the antibody FR-l-48 is conjugated to DMl or DM4. In another embodiment, the antibody FR—1-49 is ated to DMl or DM4. In another ment, the antibody FR—l—57 is conjugated to DMl or DM4. In another embodiment, the antibody FR—l-65 is ated to DMl or DM4.
X. Correlation of FOLRl expression and therapeutic efficacy ’ In certain embodiments, the invention provides a method for fying subjects with an increased likelihood for responding to FOLRl-targeting anti-cancer therapies. The ion is based, in part, on the discovery that elevated FOLRl expression levels correlates with efficacy of FOLR-l -targeting anti-cancer therapeutics.
Evaluation of patient samples and ation to in vivo efficacy using xenograft models demonstrates the power of the expression analysis for selecting ts more likely to respond to treatment. IHC provides a score for FOLRl expression on tumor cells: 0 (no expression) to 3+ (very high levels of expression). In vivo data using xenograft models demonstrates that samples scoring 1, 2, 3, or 3+ for FOLRl expression, ably a score of 2, 3, or 3+, have an sed likelihood to respond to FOLR—l-targeted anti-cancer therapies at clinically-relevant doses of FOLRl immunoconjugates (e.g., 5 mg/kg xenograft -36~ dose of a FOLRI immunoconjugate can approximate a 185 mg/m2 in patients). Thus, identification of duals having an elevated FOLRl score would help fy those individuals who might respond to a ally relevant . As described in more detail below, sensitivity to FOLRI therapeutics correlated with FOLRl scoring of 2 or higher, especially with level 3 scoring. Moreover, expression of more uniform levels of FOLRl es better correlation with therapeutic benefit. Thus, a homogeneous staining uniformity is preferred but combinations of increased staining intensity with heterogeneous staining uniformity are also indicative of sed FOLRl expression. For example, scores of greater than 2 hetero is a patient selection criterion for treatment with a FOLRl therapeutic agent. {(31%} FOLRl expression analysis also identifies patients in whom decreased levels of a FOLRl-targeting anti-cancer therapy ("low dose therapy") can be effective to cause anti- tumor responses. As is appreciated in the art, compounds are generally administered at the smallest dosage that achieves the desired eutic response. This is specifically important for therapeutics that cause clinical, and often undesired, side effects. The ability to recognize those subjects with elevated FOLRl expression levels allows for minimization of the dosage of the FOLR-l—targeting therapeutic, thus decreasing possible side effects, while maintaining eutic y.
{M41} As shown herein, FOLRl sion scores of 2 hetero or greater correlate with increased responsiveness to anti—FOLRI immunoconjugates. In certain embodiments, the increased responsiveness is cachexia, increase in survival time, elongation in time to tumor ssion, reduction in tumor mass, reduction in tumor burden and/or a prolongation in time to tumor metastasis, time to tumor recurrerice, tumor response, complete response, l response, stable disease, progressive disease, progression free survival (PFS), or overall survival (OS). In certain embodiments, FOLRl expression scores of 2 hetero or greater correlate with increasing PFS, DFS, or OS.
Kits for use in the detection s and correlation to reference/control samples can se control (positive and/or negative) or reference samples. The positive control or positive reference samples can be derived from tissue culture cell lines, normal tissue tumor tissue. Positive and negative reference samples can be derived from cell lines including SW620, T47D, IGROV-l, HeLa, KB, EEG-3, other tumor cell lines, and cell lines stably or transiently ected with an expression vector that encodes FOLRl. Normal or WO 35675 turner tissue samples and tissue culture eeii Eines can also be used as a negative central reference samples. Fer additienai sampies, see Examples 9—} I and Figures 22.3»25.
XI. Pharmaceutical compositions and therapeutic methods {@143} binding agents (including antibodies, immunoconjugates, and polypeptides) are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as the ent of . In certain embodiments, the agents are useful for inhibiting tumor , inducing differentiation, reducing tumor volume, and/or reducing the tumorigenicity of a tumor. The methods of use may be in vilro, ex vivo, or in viva methods. In certain embodiments, the FOLRl-binding agent or dy or immunoconjugate, or polypeptide is an antagonist of the human FOLRI to which it binds. {$144} In certain embodiments, the disease treated with the binding agent or antagonist (e.g., a huMovl9 antibody or immunoconjugate) is a cancer. In n embodiments, the cancer is characterized by tumors expressing folate receptor 1 to which the FOLRl-binding agent (e.g., antibody) binds.
The present ion provides for methods of treating cancer comprising administering a therapeuticaliy effective amount of a FOLRLbinding agent to a subject (e.g., a subject in need of treatment). In certain embodiments, the cancer is a cancer selected from the group consisting of colorectal cancer, pancreatic cancer, lung cancer, ovarian , liver cancer, breast cancer, brain cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma, and head and neck cancer. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is lung cancer. In certain embodiments, the subject is a human.
The present invention further provides methods for inhibiting tumor growth using the antibodies or other agents described herein. In certain embodiments, the method of inhibiting the tumor growth ses contacting the cell with a FOLRl-binding agent (e.g., dy) in Vitro. For example, an immortalized cell line or a cancer cell line that expresses FOLRI is cultured in medium to which is added the dy or other agent to inhibit tumor growth. In some embodiments, tumor cells are ed from a patient sample such as, for example, a tissue biopsy, pleural effusion, or blood sample and ed in medium to which is added an FOLRl -binding agent to inhibit tumor growth.
In some embodiments, the method of inhibiting tumor growth comprises contacting the tumor or tumor cells with the FOLRl-binding agent (e.g., antibody) in Vivo. In certain embodiments, contacting a tumor or tumor cell with a FOLRl-binding agent is undertaken in an animal model. For example, binding agents can be administered to xenografts expressing one or more FOLRls that have been grown in immunocompromised mice (e.g.
NOD/SCID mice) to inhibit tumor growth. In some embodiments, the FOLRE—binding agent is administered at the same time or shortly after introduction of tumorigenic cells into the animal to prevent tumor growth. In some embodiments, the FOLRl-binding agent is administered as a eutic after the tumorigenic cells have grown to a specified size. {$148} In certain embodiments, the method of inhibiting tumor growth compréses administering to a t an eutically effective amount of a FOLRl-binding agent.
In certain embodiments, the subject is a human. In certain ments, the subject has a tumor or has had a tumor removed.
In n embodiments, the tumor is a tumor selected from the group ting of brain tumor, colorectal tumor, pancreatic tumor, lung tumor, n tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor. In certain embodiments, the tumor is an ovarian tumor. {915%} In certain embodiments, the invention provides s of inhibiting tumor growth using low doses of a FOLRl-binding agent. The term "low dose" as used herein refers to the therapeutically effective dose of a FOLRl—binding agent which is less than the usual or the conventional dose ed to produce the therapeutic effect.
Thus, in certain embodiments the inventions es methods of treating cancer using huMovl9 antibody and immunoconjugates. In certain embodiments, the huMovl9 immunoconjugate is huMovl9-SPDB-DM4; huMovl9-sulfo-SPP-DM1; huMovl9—SPF- DMI; or huMovl9-PEG4-Mal-DM4. In a certain embodiment, the huMovl9 immunconjuage is huMovl9-SPDB-DM4, which is also referred to as IMGN853. {$152} In certain embodiments, formulations are prepared for storage and use by combining a purified antibody or agent of the present invention with a pharrnaceutically acceptable vehicle (e.g. carrier, excipient) gton, The Science and Practice of Pharmacy 20th Edition Mack Publishing, 2000). Suitable pharmaceutically acceptable vehicles include, but: are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (e.g. octadecyldimethylbenzyl ammonium chloride; hexamethonium chioride; benzalkonium chloride; benzethonium de; phenol, butyl or benzyl alcohol; alkyl parahens, such as methyl or propyl paraben; cateehol; resorcinol; cyclohexanol; 3~pentanoh and ItiaCI‘t‘fiSQl/i; low molecular weight polypeptides (cg. less than about it} amino acid residues); proteins such as serum albumin, n, or irnmunoglohulins; hydrophilie polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, gine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, niarniose, or destrins; chelating agents such as Eli)’l‘A; sugars such as sucrose, mannitol, trehalose or sorbitol; saltmt‘onning counter—ions such as sodium; metal complexes (cg. Lin—protein complexes); and non~ionic surfactants such as 'l‘W'lEElEN or polyethylene glycol (PEG). {eras} The ceutical compositions of the present invention, can be administered in Administration can be topical any number ot‘ ways for either local or systen'iic treatment. {such as to mucous membranes including vaginal and rectal delivery) such as transdermal patches, ointntents, lotions, creams, gels, drops, suppositories, sprays, s and powders; pulmonary (cg, by inhalation or lation of powders or aerosols, including by nehulizer; intratracheal, intranasal, epidermal and transdennal); oral; or parenteral including intravenous, intraarterial, subcutaneous, eritoneal or intramuscular injection or infusion; or intracranial leg, intrathecai or intraventricnlar) stration. {@154} An antibody or iimnunoconiugate of the invention can be combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with a second compound having anti—cancer properties. The second nd of the pharmaceutical combination tonnulation or dosing regimen preferably has complementary activities to the ADC of the combination such that they do not adversely affect each other.
Pharmaceutical compositions comprising the FOLRl-hinding a;\4 ant and the second anti» cancer agent are also provided.
U} U! For the treatment of the disease, the riate dosage of an antihody or agent of the present invention depends on the type of disease to he treated, the severity and course of the disease, the responsiveness of the disease, r the dy or agent is administered for therapeutic or tative es, previous therapy, patient’s clinical y, and so on all at the discretion of the treating physician. "l‘he antibody or agent can he administered one time or over a series oftreatinents g from l days to several months, or until a cure is effected or a diminution of the disease state is achieved {eg reduction in turner sire). Optimal dosing schedules can he calculated from measurements of drug accumulation in the hotly of the patient and will vary depending on the relative potency of -49" an individual antibody or agent. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. In certain ments, dosage is from 0.0] ug to 100 mg per kg of body weight, and can be given once or more daily, weekly, monthly or yearly. In n embodiments, the antibody or other FOLRl-binding agent is given once every two weeks or once every three weeks. In certain embodiments, the dosage of the antibody or other FOLRl-binding agent is from about 0.1 mg to about 20 mg per kg of body weight. The treating physician can estimate tion rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. {eissi The combination therapy can provide "synergy" and prove "synergistic", i.e. the effect achieved when the active ingredients used together is greater than the sum of the effects that s from using the compounds separately. A synergistic effect can be attained when the active ingredients are: (l) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect can be attained when the compounds are administered or delivered sequentially, e.g. by different injections in separate es. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e. serially, whereas in combination therapy, effective s of two or more active ingredients are administered er. {3157} Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples, which describe in detail preparation of n antibodies of the present disclosure and methods for using antibodies of the present disclosure. It will be apparent to those skilled in the art that many cations, both to materials and methods, can be practiced without ing from the scope of the present disclosure.
EXAMPLES It is understood that the examples and ments described herein are for rative s only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and w of this application.
The Folate Receptor-l (FOLRl) has been reported to be highly expressed in ovarian tumors and expressed at high to moderate levels in brain, breast, bladder, endometrioid, -41, lung, pancreatic, and renei emes. I‘Iowever, the expression of FOLK} is limited in normal tissues and includes kidney, lung, eiiereid plexus, pancreas, breast, d? every, prestate, and lung. {316%} Methods have been ed that quantify FOLRI using fresh frezen tissue homogenates. Wheie "tissue homogenates cannot distinguish eyteplasniie from membrane asseeiated expression and freshly frozen sampies are not amenable in the clinical g.
However, fennaiin fixed paraffin embedded {FFPE} sanipies can be archived for patients in the clinic.
Exempie I {8161} immunoizistoehemieai staining 0f FQLRI in cell samples ~ manual methods in fixed paraffin embedded eeii pellets and tissues were used as test s with {he feiiewing staining reagents and conditions. iHC Antibodies FFPE assay conditions Iesi Amele r: E Ste e mouse mnneeiei‘ai anti-huFOLRi AnnéenRetrieval eiene BN3.4 (Leia-a # NC I.I_._, i1‘3.piia) ng SIeps Immuneflen: Piekaiyetie reeem‘einant pretein corresponding :0 189 amine TeWIZOEE/mL acids efthe external domain efthe felate Article irieeepiei aphamoieeuie D11uent hnrtfiznnin02%i {4011110} ANNIE: :jhorspserum Secondary Ant1body 10 ug/rnL Sewndary OdV ‘Detection System* A :AVidin-Biotin- I‘WIIAA,‘1____..A ieBietinViated heist: antinieiise (Vet01, i :iperoxidase- i :CamKéiseW ‘Complex ABC W(X?_9t03}32§2wm.i a213£9magen TDA13192912111311.3331mi DAB development > 5 min E 39162} Fennaiin—fixed paraffinuembedded (FFPE) t. ovarian tumor biopsies and ovarian xenegrafi tumors were stained with the iniiiine anti—FILILRI antibody eiene BNEQ, (Leice Cat #NIIIIRaipiie) and a, eeritrei muigGI fieni Ceslter. ing antigen retrieval in pH 9.5 , slides were blocked with 2% horse serum plus avidin. Slides were washed in PBS, and incubated at reein temperature fer {if} minutes with the attti~FOlRl er eentrel mitigGl antibetiy, followed by 30 minutes with laintinylated anti—meme lgsfi and 4G minntee with avidinbiotinperoxidase eemplex to detect bound ary antibeiyi incubation for 5 minutes with DAB (33 {lldlnxfifibtjl’lv’lflllle tetralmtreehlerid.) resultedin the color signal. Slides were eeenterstained with hematexyiin Ri staining ity and distrihution patterns were seated relative to central lgG staining {_nenepeeifie). Intensity was sceie01 en a scale of O 1103 (O-==no staining, 1 : weak, 2 = moderate and 3 strong) and distribution was scored as focal (<25% ei’ cells stained), heterogeneous (25-75% of cells stained) and homogeneous (>75% of cells stained).
FFPE samples were derived from tumor micro arrays, as well as human tissue blocks from seven different tumors, as ed below.
EFF}? Test Samples ,,,1;‘_\\"\"\\"\""" i"lltrnan Tenet Miere \nays DthllpilOfi Commerc1al Source 5 il"A" 3—3_________________________________________________ eMued tumet 96__cores from "3 types of cancer BiomaxCat# MC961 eOt-ariantame} _6_4_ cores _M iB10cha1nCat# 25t i$38le _ 58.0.99Ie__s_ B10maxCat#L% eCeleimtal ma :85 duplicate cores B10maXCat#BC000110 "——"T‘"" 1.3mmMeme sNILraher Commercial So ce LeBreast 'l'nmeru etlel ereetal Carei """"""" CHTN {$1653 The FOLRl test article, marine OLRi atone BN32, was tested to determine binding speeiticit‘v tn the huFOLRl antigi.1}. Using tne reperted IHC staining methods FFPE sections of 30049 and 300—19 transfeeted with huFCfiLRi (3004(Ii/FO'LRL) cell pellets were stained and ted fer FOUU. The FOLRl test article specifically stained 300~l9/FOLR1+ cells and retumed tie staining in 38049 cells (3 home and negative, -43, respectively). These results demonstrate that clone BN3.2 specifically targets the huFOLRl antigen. (Figure l).
The BN3.2 antibody was also used to detect FOLRl expression on tissue samples.
The immunoreactivity of each test and control article with s and cell pellets was determined by the consulting pathologist, Dr. David Dorfman. The cell pellet controls were first ted ed by tissue samples. For each tissue evaluated, a description of the staining ity and staining uniformity was reported. The staining intensity score and uniformity scales are described below. The final reported score for each tissue sample evaluated is the score of the test article minus the score of the respective control article.
The ABC level for each sample was estimated by comparing the staining score to the calibrated cell pellet controls.
InLenSIty(amountofstaln) l Tlnil‘urmiiy (nmnberafstained cells} : \ \"i 0 Negative \ : lwwweak ,__M_, twe 2 Moderate E<2V Strong Heterogeneous(hetero)E2575/ """" """""""""veryStronrlHomogeneous(homrww>75/ {0157} Antibodies bound per cell (ABC) values were determined for the FOLRl-positive tumor cell lines (KB, IGROVl, JEG3, and OVCAR3) using anti-FOLRl-Phycoerythyn'n, BD Quantibrite Beads, and flow cytometry and were shown to have different ABC values.
(Figure 2). Staining conditions were optimized for FOLRl so that the cell pellets prepared from the FOLRl-positive tumor cell lines showed varying levels of staining intensity by IHC. The KB cell pellet exhibited very strong (3+) homogeneous staining with high intensity, the IGROVl cell pellet showed strong (3) staining, the JEG3 cell pellet showed moderate (2-3) heterogeneous staining, while staining of the OVCAR3 cell pellet showed low intensity (1—2) heterogeneous staining. The FOLRl ng intensity trends observed from the cell pellets correspond to the ed ABC values, where KB cells ted the highest ABC value of 1,700,000, IGROV—l cells exhibited the next t ABC value of 260,000, JEG-3 exhibited a lower ABC value or 41,000 ABC, while OVCAR3 cells -44_ showed the lowest ABC: value of 4,000. The staining results and respective ABt‘IZ vaiues are iisted in the table below.
ABC values and respective staining results for huFOLRl on cell lines and tive cell pellets.
FQLR i KB 1,709,300 EGROV} 260,000 3 heme a'B'EGS 4i ,sus 2~3 hetero UV’CARE E 4,000 Additional cell lines, including Capan-l, Jar, Hec—l-A, Hec—l—B, wa, NCI H292, BT474EEI, PA-l, OV—90, CaOv-4, CaOv—E, A2780, Ovcar-S, Ovcar—4, HCT—lS, 786-0, NCI H838, NCI H 522, NCl H2110, NCI H1734, NCI H228, and FU.OV-3 were tested and found to be FOLRl positive but FOLRl expression levels and sensitivity to the anti-FOLRl immunoconjugate ty varied. For reference purposes, cell lines having consistent FOLRl expression and sensitivity to anti—FOLRI immunoconjugates are preferred. {0168} Particularly important was that the lHC method was able to reliable detected FOLRl expression in ovarian carcinomas and all cell lung cancer (NSCLC) tissue samples.
As shown in Figure 3, FOLRl expression could reliably be detected in ovarian carcinoma and NSCLC samples scored 2 hetero to 3 homo. ABC values for these samples ranged from approximately 41,000 for samples scored 2 hetero, to greater than 260,000 for samples scored 3 homo. As shown in Figure 4, high staining intensity and uniformity of ng was also observed in ovarian carcinomas, lung adenocarcinomas, and bronchioloalveloar carcinomas. Furthermore, expression of FOLRI in NSCLC samples e 5) and in ovarian carcinomas (Figure 6) were further found to be predominantly localized to the membrane in tissue samples. Expression was detected across multiple samples from the same, as well as different tumor s. stingly, none of the s from colorectal, breast, or small cell lung tumors were scored r than 2 hetero.
Example 2 In vivo efficacy ofhuMov19-PEG4Mal-DM4 and huMov19—SPDB-DM4 ates in comparison with similar non-targeting ates in a KB xenograft model FOLRl—targeting cleavable conjugate huMov19-SPDB—DM4 in comparison with non—targeting huC242—SPDB-DM4, and non-cleavable conjugate huMov19-PEG4-Mal- DM4 in ison with rgeting huC242—PEG4Mal-DM4 were tested using an established xenograft model of KB cells (very high FOLRl expression, 3+ homozygous by manual IHC) implanted subcutaneous into SCID mice. Mice were randomized by body weight into treatment groups and treated either singly (SPDB conjugates) on day 3 post cell inoculation, or three times weekly on days 3, 10, and 17 post cell inoculation with 5 and 10 mg/kg of a conjugate, respectively. The median tumor volume of the ent ent groups is d in Figure 7. The treatments with either huMov19—SPDB-DM4, or huMov19-PEG4Mal-DM4 resulted in a decrease in median tumor volume as compared to the PBS control, while the treatments with either of the respective non-targeting conjugate did not produce any significant effect.
Example 3 Dose-response anti—tumor activity of IMGN853 treatment in OVCAR-3 human ovarian carcinoma xenografts {317%} The umor effect of IMGN853 was evaluated in an established aneous xenograft model of ovarian carcinoma. SCID mice were ated with OVCAR—3 ovarian carcinoma cells (1 x 107 cells/animal) injected subcutaneously into the right flank. When the tumors reached about 100 m3 in size (21 days after tumor cell inoculation), the mice were randomly d into four groups (6 animals per group). Mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg/kg. A control group of animals received a single intravenous injection of PBS. Tumor growth was monitored by measuring tumor size twice per week. Tumor size was calculated with the formula: length x width x height x 1/2.
IMGN853 was highly active against OVCAR—3 tumors (IHC score of 3 homozygous using manual IHC methods) in terms of tumor growth inhibition (T/C = 0 %) at both the 2.5 and 5.0 mg/kg dose levels (Figure 8). There were complete tumor regressions (CR) in 6/6 mice treated with IMGN853 at 5.0 mg/kg. There were partial tumor regressions (PR) in 6/6 -46..
WO 35675 mice and CR in 4/6 mice treated with IMGN853 at the 2.5 mg/kg dose level. IMGN853 was active at the 1.2 mg/kg dose level, resulting in a T/C of 18%, with 2/6 PR and 1/6 CR.
According to NCI standards the T/C values ranging from 10% to 42% are considered to be active, T/C of less than 10% are considered to be highly active.
Example 4 Dose-response anti—tumor activity of 3 treatment in IGROV-l human ovarian oma xenografts. {$172} The anti—tumor effect of IMGN853 was evaluated in an established subcutaneous xenograft model of ovarian carcinoma. SCID mice were inoculated with IGROV-l ovarian carcinoma cells (1 X 107 cells/animal) injected subcutaneously into the right flank. When the tumors reached about 100 mm3 in size (7 days after tumor cell inoculation), the mice were randomly divided into four groups (6 animals per group). Mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg/kg. A control group of animals received a single enous injection of PBS. Tumor growth was monitored by ing tumor size twice per week. Tumor size was calculated with the formula: length X width x height X 1/2.
IMGN853 was highly active against IGROV-l tumors (IHC score of 3 homozygous by manual methods) at the 2.5 and 5.0 mg/kg dose levels, resulting in T/C values of 5% for both dose levels (Figure 9). There were partial tumor regressions in 5/6 and 6/6 mice in the 2.5 and 5.0 mg/kg groups, respectively. IMGN853 was inactive at the 1.2 mg/kg dose (T/C = 47%).
Example 5 Dose-response anti-tumor activity of 3 treatment in OV-90 human ovarian carcinoma xenografts.
The anti-tumor effect of 3 was evaluated in an established subcutaneous xenograft model of ovarian oma. SCID mice were inoculated with OV-90 ovarian carcinoma cells (1 X 107 cells/animal) injected subcutaneously into the right flank. When the tumors d about 100 m3 in size (13 days after tumor cell ation), the mice were randomly divided into four groups (6 animals per group). Mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg/kg. A control group of -47r animals received a single intravenous insection of PBS. A, l group of s ed PBS administered intravenously a". the same schedule. Tumor growth was monitored lay measuring tumor size twice per week. 'iE‘umor size was ated with the formula: length x width x height X 1/3..
{M75} lMGNSSZS was active against OV~9C¥ tumors {ii-EC score of 3 lretero~homo by manual methods) at the 2,5 and 5.0 rag/kg dose , resulting in T/C values of 36 and. 18%.», respectively e l0). ‘l‘wo animals had partial, tumor regressions in the :10 trig/leg the treatment groups. EMGNSSB was group; there were no other tumor regressions in any of inactive at the 1.2 mg/kg dose (T/C = 77%).
Exanqfieo Dose-response anti-tumor ty of IMGN853 treatment in SKOV-3 human ovarian carcinoma xenografts. [0176} The anti-tumor effect of IMGN853 was evaluated in an established subcutaneous xenograft model of ovarian carcinoma. SCID mice were inoculated with SKOV—3 ovarian oma cells (1 x 107 cells/animal) injected subcutaneously into the right flank. When the tumors d about 100 mm3 in size (26 days after tumor cell inoculation), the mice were randomly divided into four groups (6 animals per group). Mice were treated with a single intravenous injection of IMGN853 at 1.2, 2.5 or 5.0 mg/kg. A control group of animals received a single intravenous injection of PBS. Tumor growth was monitored by measuring tumor size twice per week. Tumor size was calculated with the formula: length X width X height x 1/2. lMGNSfiS was inactive against SKOV—B tumors (lllC score of 1—3 focal by manual methods) at all closes, with growth of ihlilGNiéSfi—treated tumors paralleling the PBS control group (Figure ii). "there was no data analysis performed; and the study was terminated early based on the inactivity of lMGNSSCi in this model.
Exanqfle7 Dose~response antiutumor activity of llMGNSfi treatment in KB human cervical adenocarcinoma xenografts.
{M78} The antiwtumor effect of lMGNSSEl was evaluated in an established subcutaneous xenegraft al adenoearcinoma model. SCED miee were inoculated with KB cervical ,4gr adenocarcinoma cells (1 X 107 cells/animal) injected subcutaneously into the right flank.
When the tumors reached about 100 mm3 in size (7 days after tumor cell ation), the mice were randomly divided into four groups (6 animals per group). Mice were treated with a single intravenous injection of IMGN853 at 1.0, 2.5 or 5.0 mg/kg. A control group of animals received a single intravenous injection of PBS. Tumor growth was monitored by measuring tumor size twice per week. Tumor size was ated with the formula: length X width X height X 1/2.
IMGN853 was highly active against KB tumors in terms of tumor growth inhibition (T/C = 0 %) at both the 2.5 and 5.0 mg/kg dose levels (Figure 12). Six of six mice in the 5.0 mg/kg and five of siX mice in the 2.5 mg/kg treatment group had CR5, and remained tumor- free to the end of the study (day 120). The 1.0 mg/kg dose was , resulting in a T/C of 37%, but there were no partial or complete regressions. lmmunohistochemical staining of FOLRl in in fixed paraffin embedded (FFPE) samples— automated methods. {0180} The IHC staining assay uses IVD class I reagents including the Novocastra FOLRl antibody astra/Leica Cat # NCI-L-FRalpha, clone BN3.2) as the test article and the Leica Bond RX automated stainer. Bound test or control article were detected by incubation wéth the Leica Bond Refine detection system which includes a post primary reagent (rabbit anti—mouse IgG), followed by a polyneer reagent (goat anti—rabbit polymer) and 3,3- Diaminobenzidine tetrahydrochloride (DAB) chromogen. FFPE samples were stained with the specified concentration(s) of y antibody (prepared by diluting in Leica diluent FOLRl) as outlined below.
IHC Antibodies wFolate Rec$8}'Kififig‘ZfiBQSEEQEE/Leica‘E‘éiQiiiicil‘fffiiigifiii? Test article" Lot ), murine, clone BN3.2, liquid concentrate: 75 ug/mL IgGl (Beckman Coulter Cat.# 6602872, LotsZS7SPSO4-23 Control article" and 2S7SPSO4-26) stock concentrationzl mg/mL, murine, clone 2T8—2F5 _49" FFPE Assay Method using Leica Bond RX Amman Time minutes 1333515112 20maid?" -— w:Perokide(Refine mdooenous Peiexjidasc Blank kit component)m5m1nuteswwm fest Arti‘cie F:0LR1 at 1.9 ug/mL1n Leica 15 suwm diluent ""llw"mm""V";N‘m‘mmmmary,, Reag€n¥kRefine‘ """"""""""""" ‘ E kit) POWEW E'Iriiiiiitégmj lVIlXedDAB (Refinekit)" W10mlnutesmm: :(JouriterstammwHematoxylm (Refine kit) "5mhiutes WM {$181} All stained samples were evaluated and scored. Control s were first evaluated followed by test samples (whole sections and individual cores from the TMAs).
For each tumor tissue or cell pellet evaluated, a description of the staining ity and respective proportion of tumor cells d was ed. Membrane associated staining was recorded for every . When duplicate scores were evaluated from one patient, only the higher score was included in the analysis. If the score described only cytoplasmic staining then the final score was reported as zero (0). Intensity and uniformity were given to each sample as described in the table outlined below. Staining intensity and bution patterns were scored relative to control IgG staining (non-specific). Intensity was scored on a scale of 0 to 3 (0 = no staining, 1 = weak, 2 t=== moderate and 3 E strong) and distrébution was scored as focal (<25% of cells stained), heterogeneous (25-75% of cells stained) and homogeneous (>75% of cells stained). In normal tissue, only the defined substructures were evaluated when calculating intensity and proportion.
IHC Scoring System Consisting of Intensity and Uniformity Scales Intensrty (brlghtness of stain) f intensity wed Intens1ty Category IntenSIty Reported ‘ l O Negatlve {H Very Weak intensity (brivhmess QfSEain) RRoak to lviodercrte 2 Moderate 2—3 Moderateto Strong E9182} FFPE tumor samples were derived from tumor micro arrays, as well as human tissue blocks from seven different tumors, as outlined below.
FFPE Test s: TMAs ic Site Number of Total # of Cores per Patients Patient E Kidney Pantomics KIC1501 P-T-ARR-KID- 2 69 122711-1 1 Pantomics i 1 RR-LNG— E 2 70 122711 1 E a "‘"EW" W ‘ 777777 Tristar 69571059/TA1249 E-PTARR-OVA- E ‘ E122711 1 : T8235725-5 EL)T-ARR-OVA- E 122111 1 a E OVClSOl EP-T—ARR—OVA- E 12271 l- l ...... "WWW..." ...m WE." E;...........».... 6957109l/TA1322 E-PT-ARR-OVA- E010912- l l m."-—_ I ._...........«......" ,, m""W E Uterus (Endo— Pantomics EMClSOl PT-ARR-EME- Emetrium) MM1227ll—l.. +._m"......".....
Various Pantomics MTUZESTW P—T-ARR— E I 12271l—mlVVVVVVVVVV w...;..;.;(._;«.‘........... ...7...7....r.r....n..»""""an.muu...................._ «SE, FFPE Test Samples: Whole Sections Organ iCode Source EDiagnosis (per Source Documentation) ..........7.7.7.7.7........................ ,,,,, _.......... 77..7.7.7....................m........... ........................_._.
Ovary ,,,,,,,,,,,¥,..,,,,.,,N C§§ endometroid adenocarcinoma ........7.7.7.7.....7 ...... ...........m.............m...\~uu.. endometroid adenocarcinoma.7.............7. ............... Proteogenex ................M.M....u.mu.uu.............._..........7 .............
,.,,,,,...,.. L;) CHTN adenocarcinoma, mixed with features of endomehod serous and clear ' ‘ " s 1 4 CHTN adenocarcmoma high grade w/ mixed papillary, serous, g § endometrloidandclear cell areas E CHTN serous papillaryadenocarcinoma , 5 . .... .—.MW.
Proteogenex 33 serous adenocarcinoma m....................m.....muuu.uu".u............................ ........... genex serous papillary adenocarcinoma 77._7.7.7.7..."‘_"‘_..»""..W.M.....uuc...u"..............................7.7.7.7.7............"..rrrm. ,_....
Proteogenex 22w"... serous papillary adenocarcinoma ........._..7.7...............
CHTN serous papillary adenocarcinoma u...7.......................+v.....7...................
Proteogenex serous papillary arcinoma 1 : ............................................................................4 11i.i adenocarcmoma, poorly differentiated adenocarcinoma, acinar fferentiatedwith 1 bronchioloalveolar features .5 "r. ....u..... . . ......w.......__7.....................11 . .............7.7.7.7.7................... adenocarcinoma, us features adenocarcinoma CHTN adenocarcinoma mm» W Mm .. w» mm» m... 777777 adenocarcirioma (bronchioloalveolar) carcinoma 7.7.7.........E ‘ ..
WW ' """ E .
CHTN g adenocarcmoma CHTN adenocarcinoma, moderately differentiated, with clear cell features 7.........;....... ..7...............W»m»»»uuu «.............._.7. \£3 CHTN iadenocarcinoma 44"in,..7...............W_...........u.mnn.u.u..."n" i l O CHTN i E E»... squamous cell carcinoma ..c. "u. ............v.w..~....................WAMA Cells (tumor cells or ected cells) were formalin fixed and paraffin embedded (FFPE). FFPE cell pellet samples shown to exhibit varying ranges of FOLRI expression by flow cytometry and normal human s were used in this study to characterize positive and negative controls and for analysis of specificity. The cell s exhibiting varying levels of FOLRl and the respective scores are reported below. There is a poor correlation between staining scores and the respective FOLRl expression levels (antibodies bound per cell, ABC, determined by calibrated flow cytometry) in the cell pellets. For example a score of 1—3 hetero is given for the SW620 and lGROV-l exhibiting 40,098 and 565,481 ABC values, respectively. Additionally, Hela cells showing an ABC value of 1.5 n resulted in a score of 2-3 hetero while 300.19/FR1 exhibiting 830,003 ABC returned a higher score of 3 homo.
Final Scores for Celi Pellets at a Test Article Concentration of 1.9 ug/mL § {39.31 Line aABC Value rrrrrr "1:"Staining Score .rv»"»»""" Wu""u....n~"~"vw_»__rrrrrrrrrr w—v—_ § 3 83137621} 411,888 I-3 hetero T471) l -2 hetero —(nun........"WW"."way."u.»»»‘ ".Tmfié Wuuuu.«....VVwwwww«Whe‘""»»»"‘Wu—— _ 1URUV 1 L565,481 1-3 hetero g 30019/ FRl 830003 3homo E HBEA E1 500587 :2:3hetero KB "14,000,000 3homo aThe reported ABC Valueis anaverageofant1bod1es boundpercell1n thecellpopulation and" was determined as follows: a concentration of 1.0 X 108M of anti—FOLRl ~PE (1 :1) was used determine ABC values on the respective cell line using flow cytometry methods and Quantibrite TM Beads (BD Biosciences).
- The flow cytometry histograms represent the bution of cells versus the number of anti-FOLRl bound the and per cell (FOLRl expression level). Both histograms respective IHC staining results indicate that each of these cells lines contain a geneous population of cells having a broad range of FOLRl expression. The exception is the 300.19/FR1 cell line g botl: a uniform flow cytometry histogram and IHC staining score. This data suggests that cell lines each expressing a more uniform level of FOLRl may provide a better correlation between ABC values and tive staining scores. Although the assay demonstrated positive staining in all positive cell pellet controls, there is a poor correlation between staining scores and the respective FOLRl expression levels from most of these cell pellets. ore, cell pellets from this group could not be identified as high-, medium—, and low-expressing controls. Representative photographs and histograms irzg FOLRl sion in cell lines by IHC and flow cytometry are shown in FIGURE 13. 3 To determine assay ions, a range of dilutions of test and control article were tested to select conditions that exhibit an appropriate level of sensitivity. Experiments were -53.. performed on a panel of FFPE samples including FOLRl-positive cell pellets and a TMA consisting of FOLRl positive and negative normal tissues (adrenal (cortex/medulla), breast (ducts and lobules/connective tissue), fallopian tube (surface epithelium/muscle wall), kidney (tubules/ glomeruli), lung (type I/Il pneumocytes/interalveolar connective tissues), pancreas (ducts/islets of Langerhans), ry gland (ducts/stroma), skin (eccrine glands/epidermis), stomach (surface epithelium/submucosa)), and whole sections of tumor tissues (10 ovarian tumor samples and 10 lung tumor samples). Each sample was stained with a serial dilution of test article concentrations (0.25, 0.5, 0.9, 1.9, 3.8, and 7.5 ug/mL) or control article concentration of 1.9 ug/mL or 9.8 ug/mL. The relative staining intensities for each dilution were compared for each sample to identify the optimal dilution. The criteria for optimal dilution was a on which 1) caused no background staining in samples stained with e control 2) caused no staining in negative tissue controls stained with test article and 3) entiated between g levels of membrane-associated FOLRl expression among test samples representing the indication of interest (ovarian tumor, endometrial tumor, NSCLC tumor, and kidney tumor FFPE tissues). Of the five ons of test e evaluated, the concentration of 1.9 ug/mL showed the best dynamic ted protocols (Bake and Dewax range in staining results using the Leica Bond RX ol, HIER using the ER2 for 20 minutes protocol, and the staining protocol IHC F- With Extra Rinses).
Example 9 Identification and characterization of controls that characterize the dynamic range of the assayautomated staining s y controls: Human normal ry gland, lung and pancreas were identified as positive tissue controls to be employed in each assay to verify that the staining procedure performed as expected. Human normal esophagus was identified as a negative control.
These controls were characterized as follows: in order to establish controls which cover the c range of the assay, a tissue microarray (TMA) consisting of several FOLRl positive and negative normal tissue samples expected to exhibit the c range of the assay was used as an assay verification control during the optimization and validation phases. Four normal tissues with identified structures in this TMA were identified as suitable assay controls as follows: respiratory epithelium of normal human lung (score of 2 homo); ducts of normal human pancreas (score of " homo ); intercalated ducts of normal human salivary gland {score of l~2 hetero); and normal human gus (score of 0) Over a total of 5; assay runs the identified suitable assay controls from this TMA gave cal results. These s indicate that the selected controls give consistent results and span the dynamic range of the assay.
Structures in Normal ’if'issues identified as ls that Span the Dynamic Range of the Assay ENtirin;E iii}Structure StainingScoremiim Staining Scorefiest‘i E'G:gen : (controi article) article) EEsophagiix kill stmctnre; 0 (negative) Efiiiicganm; livaryGland lotei‘calated ducts NEW0 (negative) WE i~2 here i WWW RespiratoryEpitheimvo‘iElhomo NPantria:---~WEDncts (E) {negati"3)meMMwE"honiowapitai [\rpicaE staining is definedas polarized nonuinfmm membrane staining Example 10 Perfom‘iance analysis oftite automated staining method. {$187} The intended use of this assay is to cally detect FOLRl reproducibly and with the appropriate sensitivity to differentiate varying levels and varying uniformity of membrane—associated FOLRI expression (optimal dynamic range) in ovarian, endometrial, NSCLC, and kidney FFPE tumor tissues. Therefore, specificity, reproducibility, and sensitivity were considered as performance criteria.
The specificity and sensitivity of the study assay was evaluated by comparison of normal tissue staining with the study assay to previously reported results. Staining results frorn this study were compared with corresponding staining results from Scorer et al 2010 (A Fall Immunohistochemical Evaluation of a Novel Monoclonal Antibody to Folate Receptor alpha. The Novocastra Journal of Histopathology, TS: 2010(3):8-12, describing the same antibody clone BN3.2) with FFPE normal tissue and from the Tissue Cross Reactivity (TCR) study using IMGN853 (huMovl9 (M9E46A) dy) on fresh. frozen normal tissue (ImmunoGen Report lMH28—OO3). ison of the staining results from each method te that the three assays showed generally similar normal tissue ng profiles with differing ve sensitivities, with the Scorer assay being least sensitive, the study assay (IMH28-011) having ediate sensitivity, and the TCR study method being the most sensitive. Some structures showed positive staining in the two more ive methods (study assay and TCR assay) only. There were no examples of positive staining in the least sensitive assay used by Scorer that were not also positive in the study assay and TCR method. These results demonstrate that the specificity and sensitivity of the study assay is appropriate for the tion of FOLRl expression in normal tissues.
The specificity and sensitivity of the study assay was further characterized by staining and evaluating a panel of tumor TMAs consisting of ovarian, endometrial, NSCLC, and kidney tumors (a sample set representative of the assay’s intended al use).
Positive staining was consistently localized to the tumor tissue with normal adjacent tissue components including stroma, blood vessels, lymphocytes and normal organ tissue staining negative or positive as expected. For each subtype of either ovarian carcinoma or NSCLC, the distribution of staining scores among TMAs from the different vendors showed a similar distribution of scores suggesting this method is not sensitive to various fixation and processing conditions. Because the distribution patterns were similar among the TMAS, the data from the different arrays was ed and scores were rized. A summary of these scores for tumor subtypes that contained 20 or more samples per subtype are listed in the following tables. As ized in these tables, a dynamic range of scores is noted for each tumor type and indicates that this assay shows the appropriate sensitivity to distinguish varying levels and varying mity of membrane—associated FOLRl expression in ovarian, endometrial, NSCLC, and kidney FFPE tumor tissues. Representative photos of serous ovarian, endometroid ovarian, NSCLC, endometrial carcinoma, and renal clear cell carcinoma are provided in Figures 14-18. Additional representatitive photos useful, for example, in a staining guide or diagnostic kit, are shown in Figures 23—25. These studies te the assay is specific and has the appropriate sensitivity for use as a diagnostic or companion stic t.
Summary of Staining Scores for Predominant Subtypes of Ovarian Tumors Subg""p"";""" "E‘Silifiglvumser 1 We) ~56- 1-3 Any focal Positivity Eff} l§§ l)Focal staining ns were excluded Summary of Staining Scores for NSCLC Tumors Sample Number §Tota1 23 722 A gNegative hetero" heteroa = ‘ Positiww EAdeno- A11b s 47 20 oma $100) (25) . (58) Spec1fied 7 Bronchi010-~~ (100)(29) l (71) ;alveolar ! """""+ vwvvv-v'v"~"""‘»»»»»~~" 7777777777 tm-u-uu"""V W = W"? Squamous All 74 l 3 l1W..."u"("innunwwww w cell "ml 100 l 5 carcinoma i )j:3} ) ( ) ) Focal stalnlng patterns were excluded All adenocarcinoma samples were included except the specified ioloalveolar carcinoma samples _ 57 2012/031544 Summary of ng Scores for Adenocarcinoma ofthe Endometrium and Clear Cell Tumors of the Kidney WW, "uh..."‘e Tumor/Subflpe i Sample Number 1:%) i Exec"..‘.\w.m.n.}..~m......m..V"w.wV__vmv.r.""""ee» m" i tT."nmfimmzzzrfiteeee i a Total E 23 _2 2 1 1-3 focal k 3 Any Negative i : Positivity E heteroa heteroa hetero" l : ‘ We." .ze..""‘""»""" a»... .w...wmvw.‘wr ._._.......... "finnnvwfivw eeeeecee‘" i Endometnum/ 58 5 3 E 30 10 40 18 i Aden0_ u..u"~§un~~finwwwmea »»»»»""""" "nun"......u"ui,u."w"vVV__-;;;=A.x»§\»»\\\."W (100) (9) 40) carcinoma E (52) E (17) (69) (31) {gvvvvvv,_ w ,,,,,, t 3 Cell (100) (0) E (68) (18) (85) (15) WWW "WW" FoEai";i;1‘Hng§aEEfi wereieileiiiied The precision of the study assay was investigated by evaluating intra—run and inter- run reproducibility of the assay using three FFPE tumor tissue samples of n, NSCLC, or kidney tumor where each sample exhibits either a high, , or low score. For intra~ run reproducibility, nine slides each containing a section of lung, ovarian, and renal tumor were placed at nine random locations on the Leica Bond RX. For run reproducibility, three slides containing sections from the same sample were stained on three different days.
All slides from both intra-run and inter—run reproducibility experiments were evaluated and showed equivalent staining results for each respective sample: lung tumor (high: 3 homo), ovarian tumor (medium: 2 hetero), and renal tumor (low: 1-2 hetero). This data demonstrated reproducibility across tissue types with low, medium and high level of expression.
Example 1 l A FOLRl expression score ofZ 2 heterogeneous by IHC is a patient selection ion for treatment with IMGN853.
The levels of FOLRl-expression in tumor cell lines were determined using the an antibody-l)E conjugate (FRIPE) and the BRlTE system. Three ovarian carcinoma cell lines (Igrov—l, Skov-3 and Ovear-3), a choriocarcinoma cell line leg-3 and a cervical carcinoma cell line KB were included in the study. In order to obtain reliable ABC values, the g experiments with an antibody—PE conjugate should be performed at a -58— WO 35675 2012/031544 saturating concentration (concentration, at which all available binding sites are occupied by the conjugate). To determine such concentration for the FRIPE conjugate, we med binding experiments on a panel of FOLRl—positive cell lines with various FOLRl expression. The cells were incubated with a wide concentration, range of FRl—24-PE conjugate for two hours on ice, washed with FACS buffer (PBS with 1% BSA), fixed with 1% formaldehyde in PBS and analyzed on a FACSCalibur flow cytometer. At a concentration of 1x10'8 M the conjugate ted cell surface binding sites on all tested cell lines lgrov-l, Jeg-3, Skov-3, Ovcar-3, and KB. In subsequent binding ABC-experiments FRIPE conjugate was used at concentration of 1x10"8 M. Each sample was analyzed in triplicates; several ndent experéments were performed on each cell line. The highest expression was found on KB cells with the approximate ABC value of 4,000,000i300,000, followed by Igrov-l and Jeg-3 cell lines with the ABC values of 400,000i85,000 and 150,000i75,000, respectively. Two cell lines, Skov—3 and Ovcar—3, had low FOLRl expression, 20,000i10,000 and 7,00014,000 ABC, respectively. A significant experimentto-experément variation of ABC values was observed for Jeg—3 cells, where the ABC—values varied from 40,000 to 0. This varéability likely reflected some biological properties of the cell line rather than the assay variability, since ABC values ed for the other analyzed cell lines were much less variable (see table .
"Celi‘line l i ABC Experiment--toexPehEIehi‘QQIii {Mean i ‘53» it)" The highest ABC The lowest ABC , ,1 i 1 registered registered KB 00 3:300000,4 4,500,000"" WW3800000W ,, ‘i‘ Igrov-l % 400,000j:85000 5 480,000 280,000 Jeg-3 150,000 i:75,000, 14 imwwm 40w000w Skov—3" r ,000"3: 10,000 2m 10,OOOWWWWa SDStandarddev1ation n- number ofindependent experiments ABC values were determined by a FACS based assay with FR1-24 PE—labeled antibody and QuantiBRITE system. Mear: i rd Deviation (SD) was calculated for independent experiments.
E8192} Potency and specificity of 1MN853wwas analyzed against FOLRE ~positive teii lines with a wide range of FOUR} expression (the ABC values of the cell lines are provided above) in on FOL-R1negative ceiii lines a and SWZ were included in the experiments. IMGN853 was highly xic against cells with high FOLRl sion KB (4,000,000i300,000 ABC), Igrov—l (400,000zt85,000 ABC) and Jeg—3 (150900171000 ABC), with the ICso values of 0.10i0.01 nM, 0.50i0.07 nM and 1.00i0.05 nM, respectively. The cell-killing activity against all three cell lines was FOLRl-dependent, since an excess of unmodified 9 (M9346A) antibody (0.5 uM) markedly decreased potency of the conjugate to the l non-specific levels (from 10 to 20~fold). 1MGN853 was only ally active against the low FOLRl expressors Skov-3 and Ovcar-3 cells (20,000::10,000 and 7,000i4,000 ABC, respectively), and against FOLRl-negative cells Namalwa and SW2, with the IC50 values greater than 2 nM. The cytotoxic activity of IMGN853 t these cell lines was low and not FOLRl-dependent, as blocking with huMovl9 (M9346A) did not affect it. See Figures 19 and 20.
FFPE samples prepared from mouse aft tumor models were evaluated for FOLRl positivity using the optimized and validated assay described above. No staining was seen in tumor cells of any xenograft s stained with control article. FFPE mouse xenograft tissues derived from the following cell lines showed the following staining patterns: lgrov-l, KB, and NCI-H2110 showed homogeneous staining patterns with level 3 intensity; Ishikawa and Ovcar 3 showed heterogeneous staining patterns with level 3 intensity; LXFA737 showed homogeneous staining patterns with level 2 ity; OV-90 showed heterogenous patterns with level 2 intensity; and SKOV3 was negative.
Representative photos of tumor xenografts are provided in Figures 21 and 22. -60— {ParentalN DiseaseE EFlnalbcore "Sigma Cell Line E Indication E Cagegmy or Tumor E Fragment": i IGROV— l EOvarian cancer E l —3 homo E 3 homo E l-3homo V E E1-3 homo Ishikawam"‘EndometnumE2-3 hetero 7777 1E3 hetero cancer 1 2‘hetero/3 E E 2 hetero/3 focal E E2 hetero/3 focal 1 1 ""E‘éFVMiMo Ehomo cancer E 3WW new we 1'2 hdfiiB'Ziiiiii........ 2 homo .1ENsEic NCI— 2—3 homo" EE‘homo H"Ovarian OV-90 cancer 1-2 hetero 5:16:30 OVCAR3 63mm cancer 111-113‘ii‘eui‘éi'6""""""WghetemE hetero " E12 Ovarian cancer Negatrve NegatEémi Negatlve E A staining threshold (22 heterogeneous) requires both a minimal level of expression (staining intensity) and minimum distribution of staining (percentage of tumor cells expressing FOLRl). Pre-clinical data provides justification for this old in ovarian carcinoma. Mouse xenograft tumor samples with IHC scores of 2 2 geneous exhibit ivity to IMGN 853 in vivo. FFPE samples prepared from mouse xenograft ovarian tumor models were evaluated for FOLRl positivity using the optimized and validated assay described above. Two ovarian carcinoma xenograft models OVCAR-3, and IGROV— 1 showed a heterogeneous or neous staining pattern with level 3 intensity. The xenograft model derived from OV-9O ovarian carcinoma cells showed a heterogeneous ng n with level 2 intensity; the Skov-3 ovarian carcinoma model was FOLRl- negative. lMGN853 was highly active in the two ovarian models with level 3 FOLRl intensity and active in the OV—9O model with level 2 FOLRl intensity. No ty was observed in the SKOV-3 model. Xenograft models were also evaluated for other diseseas ~61— indications including lung, endometrium, and cervical tumors and, although correlations were detected between activity and FOLRl staining scores, additional s must be tested. {@195} The sensitivity of ovarian tumor xenograft models to IMGN853 versus the Eevei of FOLRI expression Xenograft In vivo activity (5 mg/kg of Intensity score, distribution IMGN853, single dose) w, Mew..." ,, OVCAR3 Highly active E - 3 heterogeneous E "mmmm... ,, u...mummw{momenmtme E IGROV-l Highly active E 3 homogeneous OV-90 Active 2 heterogeneous E SKOV-3 s Inactive ve 1 E i E The sensitivity of other tumor xenograft models to IMGN853 versus the level of FOLRl expression In vivo activity (5 Eng/kg 0f Intensity score, E IMGN853, single dose) distribution E Highly active 3 homogeneous lshikawa Eindometrium Inactive 2 homogeneous E E: ooooo WWW KB ical Highly active 3 homogeneous E :"WWMMMWWHW All ations, s, patent applications, intemet sites, and accession numbers/database sequences (including both polynucleotide and polypeptide sequences) cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession /database sequence were cally and individually indicated to be so incorporated by reference.
- SEQEENCES SEQ ID NQt’i — huxtnan foiate recc‘pmr 1 TI‘QLLLLLV"WAX/WGEESAQTR{AWAR'I‘ELLNVCMNAKBHKEKPGPEZEQKLHEQCR PWRKNACCS’ENTSQBAHKD‘VSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNE...GE’Wifi QQVDQSWRKERVLNVPLCKEEIXIEQWWEDCRTSYTCKSNWHKGWNW/"1‘86ENKCAVGA ACQPFHFY}???PTVLC’NEIWTHSYKVSNYSR‘Ci‘SG-KCEQMWFDRKQGNPNEEVAKE1‘Yl’fL/XAl‘s/i SGAGPWAA‘WPF1,,I,,S’3L,.AE.,MILLWLLS SEQ ID N02 ~ human fi‘fiate receptor 1 nudeic acid Sequsnce cagcggatgacaacacagc;tgGigdeem:tagtgigggtggctgtagtaggggaggctcagacaaggattgcatgggccaggact gagcttcicaaigtctgcatgaacgccaagcaccacaaggaaaageGag,gccccgaggacaagttgcatgagcagtgEcgamctggagga agaatgectge‘igttctavmaaaaccagecaggaagcccataaggatgtttfict3cata‘iatagaficaactggaaccactgtggagagatggca. cctgcctgcaaacggcamcatcc21ggacacctgcctctacgagtgctcccacaacfiggggccctggatccagcaggtggatcagagctgg cgcaaagagcgggtactgaacgtgcccctgigcaaagaggactgtgagcaatggtgggaagafigtatgcacc‘tcctacacctgcaagagcaa ctggcacaagggctggaactggacttcagggtttaacaagtgcgcagigggagctgcctgccaacctficcatfictacficcccacacccactg fictgtgcaatgaaatctggactcacit.etacaaggtcagcaactacagccgagg{Iagtggccgctgcatccagatgtggficgacccagccca gggcaacaccaatgaggaggtggcgaggm:iatgctgcagccatgagtggggctgggccctgggcagcctggcc{itcctgcttagcctgg ccciaatgctgctgtggctgctcagc SEQ 11} N03 — huMOViQ VHC QVQLVQSGAE‘VVKPGASVKESCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFY NQKFQGKA‘E‘LTVDKSSNTAHMELLSLXIT‘SEDEAVYYCTRYDGSKAMDYWC:QG'I‘TVTVSS SEQ 113N024 ~ huMevw VLCV} .00 DEVLTQSPLSLAVSLGQFAHSCKASQSVSPA Ts’l‘S LMH'WYHQKPGQQPRLLIYRASNLEA(3V SGSKTDETLN 3 SP VELA'EEEILFAxtXT‘x’YCQQSREYPYTFGGGI‘KJLLEEIKR SEQ ID NOIS ~ huMQv19 VLCVLSO DI V1.30:8 3?1.;S’LAVSLGQPAHSCKASQSVSFAG’E‘SLMHWYHQKPGQQPRIZJiEJYKASNLEAGV PDRFSGSGSK3715)FTLHSPVEAEDAATYYCQQSREY}?YI'FGGGTKLEEKR
Claims (78)
1. Use of an anti-Folate or 1 (FOLR1) immunoconjugate in the manufacture of a medicament for treating cancer in a subject, wherein increased expression of FOLR1 in a cancer sample from said subject has been detected using a detection method that distinguishes between staining intensity or staining uniformity in a FOLR1 expressing cancer sample as compared to staining intensity or staining uniformity in one or more reference sample, wherein said anti-FOLR1 immunoconjugate has the formula (A) – (L) – (C) or (C) – (L) – (A), wherein: (A) is an anti-FOLR1 antibody or antigen binding fragment thereof sing: (a) a heavy chain CDR1 comprising GYFMN (SEQ ID NO:6); a heavy chain CDR2 comprising RIHPYDGDTFYNQKFQG (SEQ ID NO:7); and a heavy chain CDR3 comprising YDGSRAMDY (SEQ ID NO:8); and (b) a light chain CDR1 comprising SFAGTSLMH (SEQ ID NO:9); a light chain CDR2 comprising RASNLEA (SEQ ID NO:10); and a light chain CDR3 sing QQSREYPYT (SEQ ID NO:11), (L) is a linker, and (C) is a cytotoxic agent, and n the linker (L) links (A) to (C).
2. The use of claim 1, wherein the detection method is immunohistochemistry (IHC).
3. The use of claim 2, wherein said IHC can distinguish different levels of FOLR1 expression.
4. The use of any one of claims 1-3, n said detection method produces a range of ng intensity for samples having weak FOLR1 expression, moderate FOLR1 expression, or strong FOLR1 expression.
5. The use of any one of claims 1-4, wherein said detection method distinguishes between staining ity and staining uniformity in a FOLR1 expressing cancer sample as compared to a reference sample.
6. The use of any one of claims 1-5, n the cancer sample has a staining intensity score of 1 or greater for FOLR1 expression by IHC using a scoring system that includes a range of 0, 1, 2, 3, and 3+ for staining intensity, wherein 0 is the lowest level of staining ity, and wherein 3+ is the highest level of staining intensity.
7. The use of claim 6, wherein the cancer sample has a staining intensity score of 2 or greater for FOLR1 expression by IHC.
8. The use of claim 6 or claim 7, wherein the cancer sample has a staining uniformity for FOLR1 expression that is homogenous.
9. The use of claim 6 or claim 7, wherein the cancer sample has a staining uniformity that is heterogeneous.
10. The use of claim 6, wherein 25-75% of cells of the cancer sample have a staining intensity score of 1 or greater.
11. The use of claim 6, wherein greater than 75% of cells of the cancer sample have a staining intensity score of 1 or greater.
12. The use of claim 7, wherein 25-75% of cells of the cancer sample have a staining intensity score of 2 or greater.
13. The use of claim 7, wherein greater than 75% of cells of the cancer sample have a staining intensity score of 2 or greater.
14. The use of any one of claims 2-13, wherein the IHC is performed manually.
15. The use of any one of claims 2-13, n the IHC is performed using an automated system.
16. The use of any one of claims 1-15, wherein said reference sample is a negative reference sample.
17. The use of any one of claims 1-16, wherein the reference sample comprises cells, cell pellets, or .
18. The use of any one of claims 1-17, wherein the subject has non-small cell lung cancer (NSCLC).
19. The use of any one of claims 1-17, wherein the subject has endometrial cancer.
20. The use of any one of claims 1-17, wherein the subject has ovarian cancer.
21. The use of any one of claims 1-20, wherein the detection method ses detecting FOLR1 expression with a detection antibody or antigen binding fragment f that specifically binds FOLR1.
22. Use of an anti-FOLR1 immunoconjugate in the manufacture of a ment for treating cancer in a t, n a tumor of said subject has been fied as sensitive to treatment with an anti-FOLR1 immunoconjugate, wherein the identifying comprises (a) measuring the level of FOLR1 expression in a tumor tissue sample obtained from said tumor using a ion method that distinguishes n staining intensity in a FOLR1 expressing cancer sample as compared to staining intensity in one or more reference samples; (b) determining a FOLR1 staining intensity score for said tumor tissue sample; and (c) comparing the FOLR1 ng intensity score determined in step (b) to a nce value determined by measuring FOLR1 protein expression in at least one reference sample which is not sensitive to treatment with an anti-FOLR1 conjugate, and wherein a FOLR1 staining intensity score for said sample determined in step (b) that is equal to or higher than said reference value identifies said tumor as being sensitive to treatment with an anti-FOLR1 immunoconjugate; wherein the anti-FOLR1 immunoconjugate has the formula (A) – (L) – (C) or (C) – (L) – (A), wherein: (A) is an anti-FOLR1 antibody or antigen g fragment f comprising: (a) a heavy chain CDR1 comprising GYFMN (SEQ ID NO:6); a heavy chain CDR2 comprising RIHPYDGDTFYNQKFQG (SEQ ID NO:7); and a heavy chain CDR3 comprising YDGSRAMDY (SEQ ID NO:8); and (b) a light chain CDR1 comprising KASQSVSFAGTSLMH (SEQ ID NO:9); a light chain CDR2 comprising RASNLEA (SEQ ID NO:10); and a light chain CDR3 comprising QQSREYPYT (SEQ ID NO:11), (L) is a linker, and (C) is a cytotoxic agent, and wherein the linker (L) links (A) to (C).
23. The use of claim 22, wherein the reference sample comprises tissue, cells, or cell pellets.
24. The use of claim 22 or claim 23, wherein the reference sample is a negative reference
25. The use of any one of claims 22-24, wherein said detection method is performed manually or using an automated system.
26. The use of claim 25, wherein said detection method is automated.
27. The use of any one of claims 22-26, wherein the detection method is IHC.
28. The use of any one of claims 22-27, wherein said detection method produces a range of staining intensity for samples having weak FOLR1 expression, moderate FOLR1 expression, or strong FOLR1 expression.
29. The use of any one of claims 22-28, wherein said detection method distinguishes between staining intensity and ng mity in a FOLR1 expressing cancer sample as compared to a reference sample.
30. The use of any one of claims 22-29, wherein the tumor tissue sample has a staining intensity score of 1 or greater for FOLR1 expression by IHC using a scoring system that includes a range of 0, 1, 2, 3, and 3+ for staining intensity, wherein 0 is the lowest level of staining intensity, and wherein 3+ is the highest level of staining intensity.
31. The use of claim 30, wherein the tumor tissue sample has a ng intensity score of 2 or greater for FOLR1 expression by IHC.
32. The use of claim 30 or claim 31, wherein the tumor tissue sample has a staining uniformity for FOLR1 expression that is homogenous.
33. The use of claim 30 or claim 31, wherein the tumor tissue sample has a staining uniformity that is heterogeneous.
34. The use of claim 30, wherein 25-75% of cells of the cancer sample have a staining intensity score of 1 or greater.
35. The use of claim 30, wherein greater than 75% of cells of the cancer sample have a staining ity score of 1 or greater.
36. The use of claim 31, wherein 25-75% of cells of the cancer sample have a staining intensity score of 2 or greater.
37. The use of claim 31, n greater than 75% of cells of the cancer sample have a staining intensity score of 2 or greater.
38. The use of any one of claims 22-37, wherein the subject has all cell lung cancer (NSCLC).
39. The use of any one of claims 22-37, wherein the subject has endometrial cancer.
40. The use of any one of claims 22-37, wherein the subject has ovarian cancer.
41. The use of any one of claims 22-40, wherein the detection method comprises detecting FOLR1 expression with a detection antibody or n binding fragment thereof that specifically binds FOLR1.
42. Use of an OLR1 immunoconjugate in the manufacture of a ment for treating cancer in a subject, wherein a tumor tissue sample from the subject has been identified as having a FOLR1 expression score of 1 or greater, wherein the identifying comprises: (a) contacting a tumor tissue sample from a subject having cancer with a detection antibody or antigen binding fragment thereof that specifically binds FOLR1, wherein the sample is formalin-fixed in embedded; (b) measuring the binding of said antibody or n binding fragment thereof in step (a) using a ion method that can distinguish between staining intensity and staining uniformity in a FOLR1 expressing cancer sample as compared to staining intensity and staining uniformity in one or more nce samples; and (c) ing a FOLR1 expression score to said tumor tissue sample after comparing the level of FOLR1 staining intensity and staining uniformity in said tumor tissue sample to one or more reference samples, wherein the FOLR1 expression score includes a range of 0, 1, 2, 3, and 3+ for staining intensity, wherein 0 is the lowest level of staining intensity, and wherein 3+ is the highest level of staining intensity; wherein the anti-FOLR1 immunoconjugate has the formula (A) – (L) – (C) or (C) – (L) – (A), wherein: (A) is an anti-FOLR1 antibody or antigen binding fragment thereof comprising: (a) a heavy chain CDR1 comprising GYFMN (SEQ ID NO:6); a heavy chain CDR2 sing RIHPYDGDTFYNQKFQG (SEQ ID NO:7); and a heavy chain CDR3 comprising YDGSRAMDY (SEQ ID NO:8); and (b) a light chain CDR1 comprising KASQSVSFAGTSLMH (SEQ ID NO:9); a light chain CDR2 comprising A (SEQ ID NO:10); and a light chain CDR3 comprising QQSREYPYT (SEQ ID NO:11), (L) is a linker, and (C) is a xic agent, and wherein the linker (L) links (A) to (C).
43. The use of claim 42, wherein the tumor tissue sample has an FOLR1 expression score of 2 or greater.
44. The use of claim 43, wherein the tumor tissue sample has an FOLR1 expression score of 3 or greater.
45. The use of any one of claims 42-44, n the tumor tissue sample has a staining uniformity for FOLR1 expression that is heterogeneous.
46. The use of any one of claims 42-44, n the tumor tissue sample has a staining uniformity for FOLR1 expression that is homogenous.
47. The use of claim 42, wherein 25-75% of cells of the cancer sample have a staining intensity score of 1 or greater.
48. The use of claim 42, wherein greater than 75% of cells of the cancer sample have a staining intensity score of 1 or greater.
49. The use of claim 43, wherein 25-75% of cells of the cancer sample have a staining intensity score of 2 or greater.
50. The use of claim 43, n greater than 75% of cells of the cancer sample have a ng intensity score of 2 or greater.
51. The use of claim 44, wherein 25-75% of cells of the cancer sample have a staining intensity score of 3 or greater.
52. The use of claim 44, wherein greater than 75% of cells of the cancer sample have a staining intensity score of 3 or greater.
53. The use of any one of claims 42-52, wherein the subject has non-small cell lung cancer (NSCLC).
54. The use of any one of claims 42-52, wherein the subject has endometrial cancer.
55. The use of any one of claims 42-52, wherein the subject has ovarian .
56. The use of any one of claims 42-55, wherein the detection method is performed using an automated system.
57. The use of any one of claims 42-56, wherein said reference sample is a negative reference sample.
58. The use of any one of claims 42-57, wherein the reference sample comprises cells, cell pellets, or tissue.
59. The use of any one of claims 21 and 41-58, wherein said detection antibody or antigen binding fragment thereof comprises: (a) a heavy chain CDR1 comprising GYFMN (SEQ ID NO:6); a heavy chain CDR2 comprising GDTFYNQKFQG (SEQ ID NO:7); and a heavy chain CDR3 comprising YDGSRAMDY (SEQ ID NO:8); and (b) a light chain CDR1 comprising KASQSVSFAGTSLMH (SEQ ID NO:9); a light chain CDR2 comprising RASNLEA (SEQ ID NO:10); and a light chain CDR3 comprising QQSREYPYT (SEQ ID
60. The use of claim 59, wherein said detection antibody or antigen g fragment thereof comprises the heavy chain variable domain of SEQ ID NO:3 and the light chain variable domain of SEQ ID NO:4 or 5.
61. The use of claim 60, wherein said detection antibody or antigen binding fragment f is the dy huMov19 (M9346A).
62. The use of any one of claims 21 and 41-58, n said detection antibody or antigen binding fragment thereof is the antibody BN3.2.
63. The use of any one of claims 21 and 41-62, wherein said detection antibody or antigen binding fragment thereof further ses a detection reagent selected from the group consisting of: an enzyme, a phore, a radioactive label, and a luminophore.
64. The use of claim 63, wherein said detection t is selected from the group consisting of: biotin, digoxigenin, fluorescein, tritium, and rhodamine.
65. The use of any one of claims 21 and 41-64, wherein the concentration of said detection antibody or antigen binding fragment thereof is about 0.9 to about 3.8 µg/ml.
66. The use of any one of claims 1-65, wherein said OLR1 antibody or antigen binding fragment thereof of said immunoconjugate comprises the heavy chain le domain of SEQ ID NO:3 and the light chain variable domain of SEQ ID NO:4 or 5.
67. The use of claim 66, wherein said anti-FOLR1 antibody or antigen binding fragment thereof of said conjugate is huMov19 (M9346A).
68. The use of any one of claims 1-67, wherein said linker is selected from the group ting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, and a oxylic acid based linker.
69. The use of claim 68, wherein said linker is selected from the group consisting of: N- succinimidyl 4-(2-pyridyldithio)pentanoate (SPP); N-succinimidyl 4-(2-pyridyldithio) sulfopentanoate (sulfo-SPP); N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB); N- succinimidyl 4-(2-pyridyldithio)sulfobutanoate (sulfo-SPDB); inimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC); N-sulfosuccinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC); N-succinimidyl(iodoacetyl)- aminobenzoate (SIAB); and N-succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester (NHS-PEG4-maleimide).
70. The use of claim 69, wherein said linker is N-succinimidyl 4-(2-pyridyldithio) sulfobutanoate (sulfo-SPDB).
71. The use of any one of claims 1-70, wherein said cytotoxic agent is selected from the group consisting of a maytansinoid, maytansinoid analog, benzodiazepine, taxoid, CC-1065, CC-1065 , duocarmycin, duocarmycin analog, calicheamicin, dolastatin, dolastatin analog, auristatin, ycin derivative, and leptomycin derivative or a prodrug of the agent.
72. The use of claim 71, wherein said cytotoxic agent is selected from the group ting of a maytansinoid, maytansinoid analog, benzodiazepine, taxoid, CC-1065, CC-1065 analog, duocarmycin, duocarmycin analog, calicheamicin, atin, dolastatin analog, auristatin, tomaymycin derivative, and leptomycin derivative or a prodrug of the agent.
73. The use of claim 72, n said cytotoxic agent is a maytansinoid.
74. The use of claim 73, wherein said cytotoxic agent is N(2')-deacetyl-N(2')-(3-mercapto- 1-oxopropyl)-maytansine (DM1) or N(2')-deacetyl-N(2')-(4-mercaptomethyloxopentyl)- sine (DM4).
75. The use of claim 74, wherein said cytotoxic agent is N(2')-deacetyl-N(2')-(4-mercapto- 4-methyl-l-oxopentyl)-maytansine (DM4).
76. The use of any one of claims 1-75, wherein said linker is N-succinimidyl 4-(2- pyridyldithio)sulfobutanoate (sulfo-SPDB), and wherein said cytotoxic agent is N(2')- deacetyl-N(2')-(4-mercaptomethyl-l-oxopentyl)-maytansine (DM4).
77. The use of any one of claims 1-75, wherein said anti-FOLR1 antibody or n binding fragment thereof of said conjugate comprises the heavy chain variable domain of SEQ ID NO:3 and the light chain le domain of SEQ ID NO:4 or 5, wherein said linker is N-succinimidyl 4-(2-pyridyldithio)sulfobutanoate (sulfo-SPDB), and wherein said cytotoxic agent is N(2')-deacetyl-N(2')-(4-mercaptomethyl-l-oxopentyl)-maytansine (DM4).
78. The use of any one of claims 1-75, wherein said anti-FOLR1 antibody or antigen binding fragment thereof of said conjugate is huMov19 (M9346A), wherein said linker is sulfo-SPDB, and wherein said cytotoxic agent is N(2')-deacetyl-N(2')-(4-mercaptomethyll-oxopentyl )-maytansine (DM4). ImmunoGen, Inc. By the patent attorneys for the applicant CULLENS W0
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NZ711375A NZ711375B2 (en) | 2011-04-01 | 2012-03-30 | Methods for increasing efficacy of folr1 cancer therapy |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161471007P | 2011-04-01 | 2011-04-01 | |
US61/471,007 | 2011-04-01 | ||
PCT/US2012/031544 WO2012135675A2 (en) | 2011-04-01 | 2012-03-30 | Methods for increasing efficacy of folr1 cancer therapy |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ615742A NZ615742A (en) | 2015-09-25 |
NZ615742B2 true NZ615742B2 (en) | 2016-01-06 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2019202611B2 (en) | Methods for increasing efficacy of FOLR1 cancer therapy | |
US20150093397A1 (en) | Methods for Increasing Efficacy of CD37-Based Therapy | |
NZ615742B2 (en) | Methods for increasing efficacy of folr1 cancer therapy | |
NZ711375B2 (en) | Methods for increasing efficacy of folr1 cancer therapy | |
EA041061B1 (en) | METHODS FOR IDENTIFYING CANCER AS LIKELY ANTI-FOLR1 REACTIVE IMMUNOCONJUGATE |