WO2021037319A1 - Immunohistochemical assessment of b7-h3 expression - Google Patents
Immunohistochemical assessment of b7-h3 expression Download PDFInfo
- Publication number
- WO2021037319A1 WO2021037319A1 PCT/DK2020/050236 DK2020050236W WO2021037319A1 WO 2021037319 A1 WO2021037319 A1 WO 2021037319A1 DK 2020050236 W DK2020050236 W DK 2020050236W WO 2021037319 A1 WO2021037319 A1 WO 2021037319A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- antibody
- minutes
- antigen
- binding fragment
- cancer
- Prior art date
Links
- 230000014509 gene expression Effects 0.000 title claims description 54
- 230000002055 immunohistochemical effect Effects 0.000 title description 25
- 238000000034 method Methods 0.000 claims abstract description 580
- 239000000427 antigen Substances 0.000 claims abstract description 283
- 102000036639 antigens Human genes 0.000 claims abstract description 283
- 108091007433 antigens Proteins 0.000 claims abstract description 283
- 238000009739 binding Methods 0.000 claims abstract description 265
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 244
- 239000012634 fragment Substances 0.000 claims abstract description 237
- 201000011510 cancer Diseases 0.000 claims abstract description 175
- 239000012188 paraffin wax Substances 0.000 claims abstract description 47
- 239000000090 biomarker Substances 0.000 claims abstract description 36
- 241001529936 Murinae Species 0.000 claims abstract description 31
- 239000012520 frozen sample Substances 0.000 claims abstract description 10
- 210000001519 tissue Anatomy 0.000 claims description 193
- 238000010186 staining Methods 0.000 claims description 192
- 239000000523 sample Substances 0.000 claims description 174
- 210000004027 cell Anatomy 0.000 claims description 140
- 102100038078 CD276 antigen Human genes 0.000 claims description 103
- 101710185679 CD276 antigen Proteins 0.000 claims description 93
- 239000002953 phosphate buffered saline Substances 0.000 claims description 68
- 210000004881 tumor cell Anatomy 0.000 claims description 66
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical group P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 61
- 206010033128 Ovarian cancer Diseases 0.000 claims description 60
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 58
- 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 claims description 52
- 230000000903 blocking effect Effects 0.000 claims description 49
- 208000009956 adenocarcinoma Diseases 0.000 claims description 48
- 230000008901 benefit Effects 0.000 claims description 44
- 206010017758 gastric cancer Diseases 0.000 claims description 44
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 42
- 201000009030 Carcinoma Diseases 0.000 claims description 40
- 108090000623 proteins and genes Proteins 0.000 claims description 37
- 235000018102 proteins Nutrition 0.000 claims description 35
- 102000004169 proteins and genes Human genes 0.000 claims description 35
- 102000003992 Peroxidases Human genes 0.000 claims description 34
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 34
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 34
- 201000011549 stomach cancer Diseases 0.000 claims description 34
- 210000004379 membrane Anatomy 0.000 claims description 31
- 239000012528 membrane Substances 0.000 claims description 31
- 102000004190 Enzymes Human genes 0.000 claims description 30
- 108090000790 Enzymes Proteins 0.000 claims description 30
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 30
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 30
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 30
- 229910019142 PO4 Inorganic materials 0.000 claims description 28
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 28
- 239000010452 phosphate Substances 0.000 claims description 28
- 239000000126 substance Substances 0.000 claims description 25
- 238000011534 incubation Methods 0.000 claims description 23
- 206010039491 Sarcoma Diseases 0.000 claims description 22
- 239000003795 chemical substances by application Substances 0.000 claims description 22
- 239000011534 wash buffer Substances 0.000 claims description 22
- 238000003745 diagnosis Methods 0.000 claims description 21
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 20
- 206010029260 Neuroblastoma Diseases 0.000 claims description 20
- 238000011156 evaluation Methods 0.000 claims description 20
- HSTOKWSFWGCZMH-UHFFFAOYSA-N 3,3'-diaminobenzidine Chemical group C1=C(N)C(N)=CC=C1C1=CC=C(N)C(N)=C1 HSTOKWSFWGCZMH-UHFFFAOYSA-N 0.000 claims description 18
- 239000003153 chemical reaction reagent Substances 0.000 claims description 18
- 230000002496 gastric effect Effects 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 18
- 239000005018 casein Substances 0.000 claims description 17
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical group NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 17
- 235000021240 caseins Nutrition 0.000 claims description 17
- 230000001086 cytosolic effect Effects 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 15
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 15
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 14
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 claims description 14
- 208000005017 glioblastoma Diseases 0.000 claims description 14
- 229920000642 polymer Polymers 0.000 claims description 14
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims description 13
- WDLRUFUQRNWCPK-UHFFFAOYSA-N Tetraxetan Chemical compound OC(=O)CN1CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC1 WDLRUFUQRNWCPK-UHFFFAOYSA-N 0.000 claims description 13
- 239000000872 buffer Substances 0.000 claims description 13
- 208000004548 serous cystadenocarcinoma Diseases 0.000 claims description 13
- 208000000172 Medulloblastoma Diseases 0.000 claims description 12
- 239000002738 chelating agent Substances 0.000 claims description 12
- 150000001875 compounds Chemical class 0.000 claims description 12
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 claims description 12
- 201000001441 melanoma Diseases 0.000 claims description 12
- 101000884279 Homo sapiens CD276 antigen Proteins 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 11
- 208000002445 cystadenocarcinoma Diseases 0.000 claims description 10
- 238000009826 distribution Methods 0.000 claims description 10
- 208000010749 gastric carcinoma Diseases 0.000 claims description 10
- 201000000498 stomach carcinoma Diseases 0.000 claims description 10
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 9
- 230000002018 overexpression Effects 0.000 claims description 9
- 208000004019 papillary adenocarcinoma Diseases 0.000 claims description 9
- 208000019694 serous adenocarcinoma Diseases 0.000 claims description 9
- JHALWMSZGCVVEM-UHFFFAOYSA-N 2-[4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl]acetic acid Chemical compound OC(=O)CN1CCN(CC(O)=O)CCN(CC(O)=O)CC1 JHALWMSZGCVVEM-UHFFFAOYSA-N 0.000 claims description 8
- VLEIUWBSEKKKFX-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid Chemical compound OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O VLEIUWBSEKKKFX-UHFFFAOYSA-N 0.000 claims description 8
- 208000005234 Granulosa Cell Tumor Diseases 0.000 claims description 8
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 8
- 206010025323 Lymphomas Diseases 0.000 claims description 8
- UBQYURCVBFRUQT-UHFFFAOYSA-N N-benzoyl-Ferrioxamine B Chemical compound CC(=O)N(O)CCCCCNC(=O)CCC(=O)N(O)CCCCCNC(=O)CCC(=O)N(O)CCCCCN UBQYURCVBFRUQT-UHFFFAOYSA-N 0.000 claims description 8
- 206010043276 Teratoma Diseases 0.000 claims description 8
- 208000012018 Yolk sac tumor Diseases 0.000 claims description 8
- HFACYLZERDEVSX-UHFFFAOYSA-N benzidine Chemical group C1=CC(N)=CC=C1C1=CC=C(N)C=C1 HFACYLZERDEVSX-UHFFFAOYSA-N 0.000 claims description 8
- 208000009060 clear cell adenocarcinoma Diseases 0.000 claims description 8
- 208000001991 endodermal sinus tumor Diseases 0.000 claims description 8
- 201000006585 gastric adenocarcinoma Diseases 0.000 claims description 8
- 230000002285 radioactive effect Effects 0.000 claims description 8
- 238000011363 radioimmunotherapy Methods 0.000 claims description 8
- 238000002603 single-photon emission computed tomography Methods 0.000 claims description 8
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 claims description 7
- 238000001816 cooling Methods 0.000 claims description 7
- 201000007270 liver cancer Diseases 0.000 claims description 7
- 208000014018 liver neoplasm Diseases 0.000 claims description 7
- 201000005249 lung adenocarcinoma Diseases 0.000 claims description 7
- 230000002611 ovarian Effects 0.000 claims description 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 6
- 210000005166 vasculature Anatomy 0.000 claims description 6
- 201000009051 Embryonal Carcinoma Diseases 0.000 claims description 5
- 206010027476 Metastases Diseases 0.000 claims description 5
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 5
- 238000005520 cutting process Methods 0.000 claims description 5
- 230000009401 metastasis Effects 0.000 claims description 5
- 229920002866 paraformaldehyde Polymers 0.000 claims description 5
- 238000002560 therapeutic procedure Methods 0.000 claims description 5
- 206010006187 Breast cancer Diseases 0.000 claims description 4
- 208000026310 Breast neoplasm Diseases 0.000 claims description 4
- 208000007690 Brenner tumor Diseases 0.000 claims description 4
- 206010007279 Carcinoid tumour of the gastrointestinal tract Diseases 0.000 claims description 4
- 201000000274 Carcinosarcoma Diseases 0.000 claims description 4
- 208000006332 Choriocarcinoma Diseases 0.000 claims description 4
- 208000001154 Dermoid Cyst Diseases 0.000 claims description 4
- 208000007033 Dysgerminoma Diseases 0.000 claims description 4
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 claims description 4
- 208000021309 Germ cell tumor Diseases 0.000 claims description 4
- 208000034176 Neoplasms, Germ Cell and Embryonal Diseases 0.000 claims description 4
- 208000026149 Primary peritoneal carcinoma Diseases 0.000 claims description 4
- 208000006930 Pseudomyxoma Peritonei Diseases 0.000 claims description 4
- 206010038111 Recurrent cancer Diseases 0.000 claims description 4
- 208000000097 Sertoli-Leydig cell tumor Diseases 0.000 claims description 4
- 208000002669 Sex Cord-Gonadal Stromal Tumors Diseases 0.000 claims description 4
- 238000002659 cell therapy Methods 0.000 claims description 4
- 208000037828 epithelial carcinoma Diseases 0.000 claims description 4
- 201000011243 gastrointestinal stromal tumor Diseases 0.000 claims description 4
- 230000036571 hydration Effects 0.000 claims description 4
- 238000006703 hydration reaction Methods 0.000 claims description 4
- 238000009169 immunotherapy Methods 0.000 claims description 4
- 201000005950 juvenile type testicular granulosa cell tumor Diseases 0.000 claims description 4
- 208000032839 leukemia Diseases 0.000 claims description 4
- 201000005202 lung cancer Diseases 0.000 claims description 4
- 201000005296 lung carcinoma Diseases 0.000 claims description 4
- 208000020816 lung neoplasm Diseases 0.000 claims description 4
- 230000003211 malignant effect Effects 0.000 claims description 4
- 201000000271 mature teratoma Diseases 0.000 claims description 4
- 201000010225 mixed cell type cancer Diseases 0.000 claims description 4
- 208000029638 mixed neoplasm Diseases 0.000 claims description 4
- 201000010879 mucinous adenocarcinoma Diseases 0.000 claims description 4
- 208000022669 mucinous neoplasm Diseases 0.000 claims description 4
- 201000002524 peritoneal carcinoma Diseases 0.000 claims description 4
- 229920003023 plastic Polymers 0.000 claims description 4
- 239000004033 plastic Substances 0.000 claims description 4
- 208000024246 polyembryoma Diseases 0.000 claims description 4
- 102200153160 rs1131691322 Human genes 0.000 claims description 4
- 102200143804 rs121434229 Human genes 0.000 claims description 4
- 102220023085 rs185212943 Human genes 0.000 claims description 4
- 102200108034 rs41311087 Human genes 0.000 claims description 4
- 102220049370 rs587783484 Human genes 0.000 claims description 4
- 208000028467 sex cord-stromal tumor Diseases 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 206010041823 squamous cell carcinoma Diseases 0.000 claims description 4
- 238000006467 substitution reaction Methods 0.000 claims description 4
- 206010044412 transitional cell carcinoma Diseases 0.000 claims description 4
- 150000001298 alcohols Chemical class 0.000 claims description 3
- 230000001413 cellular effect Effects 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000002202 Polyethylene glycol Substances 0.000 claims description 2
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 2
- 238000002512 chemotherapy Methods 0.000 claims description 2
- 230000018044 dehydration Effects 0.000 claims description 2
- 238000006297 dehydration reaction Methods 0.000 claims description 2
- 239000004615 ingredient Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 238000001356 surgical procedure Methods 0.000 claims description 2
- 238000009966 trimming Methods 0.000 claims description 2
- 238000012151 immunohistochemical method Methods 0.000 abstract description 6
- 230000003118 histopathologic effect Effects 0.000 description 43
- 238000003364 immunohistochemistry Methods 0.000 description 36
- 238000011532 immunohistochemical staining Methods 0.000 description 22
- 230000002792 vascular Effects 0.000 description 13
- 238000010212 intracellular staining Methods 0.000 description 11
- 230000003511 endothelial effect Effects 0.000 description 10
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 9
- 230000001613 neoplastic effect Effects 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 210000000981 epithelium Anatomy 0.000 description 8
- 238000012744 immunostaining Methods 0.000 description 8
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000013642 negative control Substances 0.000 description 7
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 6
- 238000000386 microscopy Methods 0.000 description 6
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 5
- 208000006265 Renal cell carcinoma Diseases 0.000 description 5
- 210000000805 cytoplasm Anatomy 0.000 description 5
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 5
- 210000004877 mucosa Anatomy 0.000 description 5
- 201000002528 pancreatic cancer Diseases 0.000 description 5
- 208000008443 pancreatic carcinoma Diseases 0.000 description 5
- 239000013641 positive control Substances 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 102000001708 Protein Isoforms Human genes 0.000 description 4
- 108010029485 Protein Isoforms Proteins 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 235000019441 ethanol Nutrition 0.000 description 4
- 238000013401 experimental design Methods 0.000 description 4
- 230000003902 lesion Effects 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 210000001672 ovary Anatomy 0.000 description 4
- 230000008685 targeting Effects 0.000 description 4
- 210000004876 tela submucosa Anatomy 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 201000010260 leiomyoma Diseases 0.000 description 3
- 230000003389 potentiating effect Effects 0.000 description 3
- 230000002062 proliferating effect Effects 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000003656 tris buffered saline Substances 0.000 description 3
- 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 description 2
- 208000035404 Autolysis Diseases 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 206010057248 Cell death Diseases 0.000 description 2
- 208000030808 Clear cell renal carcinoma Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 208000008771 Lymphadenopathy Diseases 0.000 description 2
- 208000034841 Thrombotic Microangiopathies Diseases 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 239000007979 citrate buffer Substances 0.000 description 2
- 206010073251 clear cell renal cell carcinoma Diseases 0.000 description 2
- 238000007398 colorimetric assay Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 201000011330 nonpapillary renal cell carcinoma Diseases 0.000 description 2
- 230000004768 organ dysfunction Effects 0.000 description 2
- 208000011932 ovarian sarcoma Diseases 0.000 description 2
- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical compound OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 230000028043 self proteolysis Effects 0.000 description 2
- 208000028647 spindle cell neoplasm Diseases 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 230000009092 tissue dysfunction Effects 0.000 description 2
- 229950002929 trinitrophenol Drugs 0.000 description 2
- 108010047303 von Willebrand Factor Proteins 0.000 description 2
- 102100036537 von Willebrand factor Human genes 0.000 description 2
- 229960001134 von willebrand factor Drugs 0.000 description 2
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 101710172562 Cobra venom factor Proteins 0.000 description 1
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 206010061968 Gastric neoplasm Diseases 0.000 description 1
- 208000032612 Glial tumor Diseases 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 101000777646 Homo sapiens Uncharacterized protein encoded by LINC01587 Proteins 0.000 description 1
- 101150009057 JAK2 gene Proteins 0.000 description 1
- 206010051696 Metastases to meninges Diseases 0.000 description 1
- 206010051676 Metastases to peritoneum Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 101150099493 STAT3 gene Proteins 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 206010064390 Tumour invasion Diseases 0.000 description 1
- 102100031590 Uncharacterized protein encoded by LINC01587 Human genes 0.000 description 1
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000001745 anti-biotin effect Effects 0.000 description 1
- 230000005809 anti-tumor immunity Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000009400 cancer invasion Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 208000027744 congestion Diseases 0.000 description 1
- 229920000547 conjugated polymer Polymers 0.000 description 1
- 210000004246 corpus luteum Anatomy 0.000 description 1
- 230000000139 costimulatory effect Effects 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- 208000028919 diffuse intrinsic pontine glioma Diseases 0.000 description 1
- 208000026144 diffuse midline glioma, H3 K27M-mutant Diseases 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 208000014616 embryonal neoplasm Diseases 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 230000009760 functional impairment Effects 0.000 description 1
- 210000001156 gastric mucosa Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 125000000267 glycino group Chemical group [H]N([*])C([H])([H])C(=O)O[H] 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002962 histologic effect Effects 0.000 description 1
- 238000007489 histopathology method Methods 0.000 description 1
- 102000048770 human CD276 Human genes 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000002991 immunohistochemical analysis Methods 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 230000002637 immunotoxin Effects 0.000 description 1
- 229940051026 immunotoxin Drugs 0.000 description 1
- 239000002596 immunotoxin Substances 0.000 description 1
- 231100000608 immunotoxin Toxicity 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000004923 pancreatic tissue Anatomy 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000003439 radiotherapeutic effect Effects 0.000 description 1
- 230000009528 severe injury Effects 0.000 description 1
- 208000000649 small cell carcinoma Diseases 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/42—Low-temperature sample treatment, e.g. cryofixation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
- G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
- G01N1/06—Devices for withdrawing samples in the solid state, e.g. by cutting providing a thin slice, e.g. microtome
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- the present invention relates to a method for providing or measuring a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof using immunohistochemical methods.
- the present invention further relates to an immunohistochemical-method for detecting and optionally quantifying overexpression of B7-H3 for the selection of patients that may benefit from B7-H3 antibody or antigen binding fragment thereof based radioimmunotherapy, immunotherapy or cell therapy. More specifically, the invention relates to a method for providing or measuring a biomarker for assessing the efficacy and/or safety of an antibody or antigen-binding fragment thereof based on B7-H3 expression in the treatment of cancer in an individual, or in the treatment of a specific indication of cancer, such as ovarian cancer.
- the invention further discloses a method for measuring or qualifying the clinical benefit of a B7-H3 antibody or antigen- binding fragment thereof. Additionally, the invention discloses a method for detecting B7-H3 expression in a tissue sample comprising cancer cells by use of a murine 8H9 antibody. In addition, the invention relates to a kit of parts adapted to carry out the inventive method. Moreover, the invention relates to a method of treating a cancer, comprising providing or measuring a biomarker for measuring and/or verifying the suitability, efficacy and/or safety of an antibody or antigen-binding fragment thereof, wherein said antibody is used in said treatment.
- a particular utility comes from demonstrated clinical benefit with radio immunotherapies for high expressing B7H3 tumors and the need to determine benefit and select patients with heterogeneous expression (among patients and among tumor tissues, as well as localization in tumor tissue).
- Anti-B7-H3 mouse monoclonal antibody 8H9 has been successfully used for radioimmunotherapy for patients with B7-H3 positive tumors.
- Murine 8H9 antibody targeting B7-H3 protein is broadly reactive with human solid tumors, including embryonal tumors and carcinomas. It has shown favorable tumor uptake for both sarcomas and brain tumors in xenograft models. When conjugated to cobra venom factor, it induces efficient complement-mediated tumor lysis. In its single chain Fv (scFv) format, it targets a potent immunotoxin to sarcoma and glioma in preclinical models. As a chimeric antigen receptor, it redirects natural killer cells to kill B7-H3 positive tumor cells.
- scFv single chain Fv
- radiolabeled murine 8H9 antibody was shown to prolong survival among high risk patients with solid tumors suffering from central nervous system (CNS) metastasis. It is a promising target for radioimmunotherapy of leptomeningeal metastases, diffuse intrinsic pontine glioma and peritoneal metastases (Ahmed et al. Humanized Affinity-matured Monoclonal Antibody 8H9 Has Potent Antitumor Activity and Binds to FG Loop of Tumor Antigen B7-H3, The Journal of Biological Chemistry Vol. 290, NO. 50, pp. 30018 - 30029, December 11, 2015).
- Murine mAb 8H9 has been shown to bind to a unique epitope on both the 2lg- and 4lg-B7-H3 isoforms of B7-H3, namely the FG-loop that includes the "I RDF " sequence motif (Ahmed et al. Humanized Affinity-matured Monoclonal Antibody 8H9 Has Potent Antitumor Activity and Binds to FG Loop of Tumor Antigen B7-H3, The Journal of Biological Chemistry Vol. 290, NO.
- B7-H3 is a new cancer-specific endothelial marker in clear cell renal cell carcinoma, OncoTargets and Therapy 2013:6 1667-1673, 2013
- RCC clear cell renal cell carcinoma
- B7-H3 antibody goat anti-human B7-H3 monoclonal antibody from R&D Systems, Minneapolis, MN, USA
- Expression patterns of B7-H3 were compared with that of the pan-endothelial cell specific marker CD34. Paired normal renal parenchyma specimens from all patients and four corpus luteum specimens were also analyzed.
- Formalin-fixed paraffin-embedded tissues were cut into 5 mm sections, deparaffinized and rehydrated in a graded series of ethanols.
- Antigen retrieval was done heating tissue sections in ethylenediaminetetraacetic acid (EDTA) 1 mmol/L, pH 8 to 121 °C using a Digital Decloing Chamber, cooling to 90 °C, and incubating for 5 minutes, and treating with peroxidase blocking reagent. Sections were incubated with a biotin-labeled polyclonal antibody against B7-H3 and CD34 followed by a horseradish peroxidase-conjugated anti- biotin antibody and visualization by diaminobenzidine staining.
- EDTA ethylenediaminetetraacetic acid
- Sections were lightly counterstained with hematoxylin.
- the study detected B7-H3 expression in cancer cells in 19% of clear cell RCC cases, and B7-H3 expression in tumor vasculature was confirmed in 98% of cases.
- the study states that the total probability of cancer cell-specific B7-H3 expression in the clear cell RCC population was ⁇ 20%, suggesting that induction of cytotoxicity or antitumor immunity by agents against B7-H3 expressed in cancers cells may not be sufficient in clinical practice.
- B7-H3 is related to tumor progression in ovarian cancer
- Oncology reports 38, 2017 shows that B7-H3 affect ovarian cancer progression through the Jak2/Stat3 pathway, indicating that B7-H3 has the potential to be a useful prognostic marker.
- Paraffin embedded samples from biopsies have been used in immunohistochemical (IHC) methods in combination with goat anti-human B7-H3 antibody as described in Qin et al, and with mouse anti-human B7-H3 antibody as described in Loos et al. Further, frozen samples have been used in immunohistochemical methods. However, paraffin embedded samples in combination with a murine 8H9 antibody, such as an antibody comprising a heavy chain according to SEQ ID No. 1 and a light chain according to SEQ ID No. 2, have not been used successfully in this context. Because frozen tissue samples have limited storage stability, and because paraffin embedded tissue samples in general preserve tissue better, there is a need for a method that works on paraffin embedded tissue samples. The present invention discloses a method that can use both frozen samples and paraffin embedded samples, and which may find general use within the field of IHC.
- An IHC technique may be used for each individual patient. Multiple samples may be collected from an individual patient and analyzed for the expression of B7-H3.
- B7-H3 is highly expressed.
- B7-H3 is not necessarily highly expressed in other types of cancer.
- Zang et al. has shown that ovarian borderline tumors and carcinomas aberrantly express B7-H3.
- the test of the invention may help to determine the highest possible dose for a population or individual who may benefit from treatment targeting B7-H3.
- an immunohistochemical method which allows measurement, detection and/or evaluation of a biomarker for the efficacy and/or the safety and/or clinical benefit of specific antibodies, such as B7-H3 antibodies.
- a method to detect which patient might benefit from B7-H3 antibody or antigen binding fragment thereof based radioimmunotherapy, immunotherapy or cell therapy there is a need for such a method, which may be used on at least one paraffin embedded tissue sample or a sample formerly embedded in paraffin.
- the anti-B7-H3 antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof disclosed in International Publication No. WO 02/32375, WO 2003/033670, WO 03/075846, WO 2008/116219, WO 2016/033225 and WO 2018/209346, all of which are incorporated by reference in their entireties.
- the invention concerns a method for detecting expression and/or overexpression of an antigen in at least one tissue sample comprising cancer cells, or potentially comprising cancer cells, wherein said sample is selected among a thawed sample, a frozen sample, a fresh sample, a paraffin embedded sample, a formalin embedded sample, and/or a sample which has formerly been embedded in formalin and/or paraffin.
- a “thawed sample” is a sample which has been frozen, but is no longer frozen.
- a “paraffin embedded sample” is a sample that is embedded in paraffin.
- a “formalin embedded sample” is a sample that is embedded in formalin.
- a sample may belong to more than one category, e.g. a paraffin embedded sample may also be a thawed sample, and a sample that is embedded in formalin may also be embedded in paraffin.
- the invention concerns a method for detecting expression and/or overexpression of an epitope in a tissue sample comprising cancer cells, or potentially comprising cancer cells, wherein said sample preferably is a sample which has formerly been embedded in formalin and/or paraffin, and wherein said method comprises the steps of: i. Enzyme blocking with an enzyme blocking substance, ii. Protein blocking with a protein blocking substance, iii. Binding with a primary antibody, iv. Incubation with a secondary antibody, and v. Staining with a staining agent.
- the invention concerns the method, wherein said epitope is an epitope of B7-H3 antigen. It has been shown by indirect evidence that 8H9 binds to 'IRDF' sequence in B7H3. 4lg-B7H3 (more common isoform) has 2 IRDF motifs (since 4lg B7H3 is a duplicate of two 2lg-B7H3 sequences). The 'IRDF' sequence is located on the FG-loop, a region thought to be critical to the function of B7-family of proteins.
- the invention concerns a method for providing and/or measuring a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of cancer in an individual, wherein the method is performed on a tissue sample comprising cancer cells.
- the invention concerns a method for detecting the clinical benefit of an antibody or antigen-binding fragment thereof in the treatment of cancer in an individual, wherein the method is performed on a tissue sample comprising cancer cells.
- the individual is a specific individual or a specific cancer patient, as the method is to be used for each individual patient.
- the invention concerns a method for providing and/or measuring a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of an indication of cancer, wherein the method is performed on tissue samples comprising cancer cells.
- the invention concerns a chimeric antigen receptor (CAR) comprising an antibody or antigen-binding fragment thereof, wherein said chimeric antigen receptor is used according to a method of the invention.
- CAR chimeric antigen receptor
- the invention concerns a CAR-T cell expressing the CAR.
- the invention concerns a population of the CAR-T cells.
- the invention concerns a composition comprising the population of CAR-T cells.
- the invention concerns a method of treating cancer in an individual, wherein the method comprises providing of a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof, and wherein said antibody or antigen-binding fragment thereof is used in said treatment.
- the invention concerns a method for treating cancer in an individual, wherein the method comprises use of an antibody or antigen binding fragment thereof, and wherein the indication, such as ovarian cancer, for treatment of said cancer with said antibody or antigen-binding fragment thereof is determined or confirmed by a method of the invention.
- the invention concerns a method for treating cancer in an individual, wherein the method comprises evaluation of the clinical benefit of an antibody or antigen-binding fragment thereof, comprising use of a method of the invention, and wherein the clinical benefit is the clinical benefit for a patient, and wherein said antibody or antigen- binding fragment thereof is used in said treatment.
- the invention concerns a method for treating cancer in an individual, wherein the method comprises providing a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in a tissue sample comprising cancer cells, comprising use of a method of the invention, and wherein said antibody or antigen-binding fragment thereof is used in said treatment.
- the invention concerns a method of diagnosing cancer in an individual, wherein the method comprises providing a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof, and wherein said antibody or antigen-binding fragment thereof is used in said diagnosis.
- the invention concerns a method for diagnosing cancer in an individual, wherein the method comprises use of an antibody or antigen binding fragment thereof, and wherein the diagnosis of said cancer with said antibody or antigen-binding fragment thereof is determined or confirmed by a method of the invention.
- the invention concerns a method for diagnosing cancer in an individual, wherein the method comprises evaluation of the clinical benefit of an antibody or antigen-binding fragment thereof, comprising use of a method of the invention, and wherein the clinical benefit is the clinical benefit for a patient, and wherein said antibody or antigen- binding fragment thereof is used in said diagnosis.
- the invention concerns a method for diagnosing cancer in an individual, wherein the method comprises providing a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in a tissue sample comprising cancer cells, comprising use of a method according to the invention, and wherein said antibody or antigen-binding fragment thereof is used in said diagnosis.
- the invention concerns an antibody or antigen-binding fragment thereof for use in the treatment of cancer, wherein the use comprises providing a biomarker of the efficacy and/or safety of the antibody or antigen-binding fragment thereof.
- the invention concerns an antibody or antigen-binding fragment thereof for use in the treatment of cancer, wherein the treatment of said cancer with said antibody or antigen-binding fragment thereof is determined or confirmed by a method of the invention.
- the invention concerns an antibody or antigen-binding fragment thereof for use in the treatment of cancer, wherein the use comprises evaluation of the clinical benefit of the antibody or antigen-binding fragment thereof, comprising use of a method of the invention, and wherein the clinical benefit is the clinical benefit for a patient.
- the invention concerns an antibody or antigen-binding fragment thereof for use in the treatment of cancer, wherein the use comprises providing a biomarker of the efficacy and/or safety of the antibody or antigen-binding fragment thereof in a tissue sample comprising cancer cells, comprising use of a method of the invention.
- the invention concerns an antibody or antigen-binding fragment thereof for use in a method of diagnosis of cancer, wherein the use comprises providing a biomarker of the efficacy and/or safety of the antibody or antigen-binding fragment thereof.
- the invention concerns an antibody or antigen-binding fragment thereof for use in a method of diagnosis of cancer, and wherein the diagnosis of said cancer with said antibody or antigen-binding fragment thereof is determined or confirmed by a method according to the invention.
- the invention concerns an antibody or antigen-binding fragment thereof for use in a method of diagnosis of cancer, wherein the use comprises evaluation of the clinical benefit of the antibody or antigen-binding fragment thereof, comprising use of a method according to the invention, and wherein the clinical benefit is the clinical benefit for a patient.
- the invention concerns an antibody or antigen-binding fragment thereof for use in a method of diagnosis of cancer, wherein the use comprises providing a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in a tissue sample comprising cancer cells, comprising use of a method of the invention.
- the invention concerns a kit of parts, adapted to carry out a method of the invention.
- the invention concerns a kit of parts, wherein said kit is to be used to determine or qualify a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof is to be used in the treatment of a cancer.
- the invention concerns a kit of parts, wherein said kit is to be used to determine or qualify the clinical benefit a patient has or will have from treatment with an antigen or antigen-binding fragment thereof, and wherein said patient is a cancer patient.
- the invention concerns a kit of parts, wherein said kit is to be used to determine or qualify a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of an indication of cancer.
- the invention concerns a kit of parts, wherein said kit is used in detecting B7-H3 expression in a tissue sample comprising cancer cells, and wherein said sample is selected among a sample that have been embedded in paraffin and a sample have been frozen.
- the invention concerns a method for treating cancer in an individual, wherein the method comprises use of a kit of the invention.
- the invention concerns a method of treating ovarian cancer, comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to the invention.
- the invention concerns a method of treating gastric cancer, comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to the invention.
- the invention concerns an antibody or antigen binding fragment thereof, wherein said antibody or antigen-binding fragment comprises at least one of the sequences selected among a heavy chain variable region CDR1 according to SEQ ID No. 3, a heavy chain variable region CDR2 according to SEQ IN No. 4 or 19, a heavy chain variable region CD3 according to SEQ ID No. 5, a light chain variable region CDR1 according to SEQ ID No. 6, a light chain variable region CDR2 according to SEQ ID No. 7 and a light chain variable region CDR3 according to SEQ ID No. 8.
- the invention concerns an antibody or antigen binding fragment thereof, wherein said antibody or antigen-binding fragment comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2.
- the invention concerns an antibody or antigen binding fragment thereof, wherein said antibody or antigen binding fragment comprises a heavy chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No.
- a light chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No. 2.
- the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of ovarian cancer. According to another aspect, the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of gastric cancer.
- the invention concerns a method for detecting expression and/or overexpression of an antigen in at least one tissue sample comprising cancer cells, or potentially comprising cancer cells, wherein said sample is selected among a thawed sample, a frozen sample, a fresh sample, a paraffin embedded sample, a formalin embedded sample, and/or a sample which has formerly been embedded in formalin and/or paraffin.
- a "thawed sample” is a sample which has been frozen, but is no longer frozen.
- a "paraffin embedded sample” is a sample that is embedded in paraffin.
- a “formalin embedded sample” is a sample that is embedded in formalin.
- a sample may belong to more than one category, e.g.
- a paraffin embedded sample may also be a thawed sample, and a sample that is embedded in formalin may also be embedded in paraffin.
- the invention concerns a method for detecting expression and/or overexpression of an epitope in a tissue sample comprising cancer cells, or potentially comprising cancer cells, wherein said sample preferably is a sample which has formerly been embedded in formalin and/or paraffin, and wherein said method comprises the steps of: i. Enzyme blocking with an enzyme blocking substance, ii. Protein blocking with a protein blocking substance, iii. Binding with a primary antibody, iv. Incubation with a secondary antibody, and v. Staining with a staining agent.
- the invention concerns the method, wherein said epitope is an epitope of B7-H3 antigen.
- the invention concerns the method, wherein said primary antibody has a concentration of about 30 mg/ml, and wherein said binding with a primary antibody is carried out at about 4 °C, and/or wherein said binding is carried out for about 4- 24 hours.
- the invention concerns the method , wherein said method further comprises the following step: i. Antigen retrieval with Tris-EDTA, wherein said Tris-EDTA has a pH value of about 9 and/or a temperature of about 100 °C
- the invention concerns the method, wherein said antigen retrieval is carried out for about 1-30 minutes, 5-25 minutes, 10-20 minutes or preferably about 15-20 minutes.
- the invention concerns the method, wherein said antigen retrieval is carried out for about 15 minutes.
- the invention concerns the method, wherein said antigen retrieval is carried out for about 20 minutes.
- the invention concerns the method, wherein said method further comprises a step of cooling down said tissue sample at room temperature, wherein said cooling down is performed after said antigen retrieval.
- the invention concerns the method, wherein said cooling down is performed for about 1-30 minutes, about 10-25 minutes, about 15-20 minutes or preferably about 20 minutes.
- the invention concerns the method, wherein said tissue sample is from an individual, and wherein said method is used for evaluating whether said individual may benefit from a therapy, wherein said therapy is selected among radioimmunotherapy, immunotherapy or cell therapy.
- the invention concerns the method, wherein said antigen is a B7-H3 antigen.
- B7-H3 is also referred to as CD276.
- the invention concerns the method, wherein said B7-H3 antigen comprises the sequence of SEQ ID No. 17 and/or 18.
- the invention concerns the method, wherein said B7-H3 antigen comprises at least one peptide selected among the peptide according to SEQ ID No. 15 and the peptide according to SEQ ID No. 16.
- the invention concerns the method, wherein said method uses antibodies to detect proteins in cells of said tissue sample.
- the invention concerns the method, wherein said primary antibody is an anti-B7H3 antibody.
- the invention concerns the method, wherein an anti-B7-H3 antibody or antigen-binding fragment thereof is used to detect B7-H3 expression.
- the invention concerns the method, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof comprises at least one of the sequences selected among a heavy chain variable region CDR1 according to SEQ ID No. 3, a heavy chain variable region CDR2 according to SEQ IN No. 4 or 19, a heavy chain variable region CD3 according to SEQ ID No. 5, a light chain variable region CDR1 according to SEQ ID No. 6, a light chain variable region CDR2 according to SEQ ID No. 7 and a light chain variable region CDR3 according to SEQ ID No. 8.
- the CDRs refers to the complementarity determining region amino acid sequences of an antibody.
- the invention concerns the method, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2.
- the invention concerns the method, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof comprises a heavy chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No.
- a light chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No. 2.
- the invention concerns the method, wherein said antigen- binding fragment thereof is a single chain variable fragment (scFv).
- scFv single chain variable fragment
- the invention concerns the method, wherein said scFv comprises a portion of the amino acid sequence set forth in SEQ ID No. 9, SEQ ID No. 13 or SEQ ID No. 14.
- the 8H9 scFv gene sequence, sense strand is represented in SEQ ID No. 10, the complementary strand is represented in SEQ ID No. 11 and the cDNA is represented in SEQ ID No. 12.
- the invention concerns the method, wherein said scFv comprises at least one of the amino acid substitutions K13E, R18Q, R45Q, K103E and K107E.
- the invention concerns the method, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof is a murine 8H9 antibody or an antigen- binding fragment thereof.
- the invention concerns the method, wherein said antibody or antigen-binding fragment thereof is humanized.
- the invention concerns the method, wherein said antibody or antigen-binding fragment thereof is a chimeric antibody or an antigen-binding fragment thereof.
- the invention concerns the method, wherein said antibody or antigen-binding fragment thereof is radiolabeled.
- the invention concerns the method, wherein said radiolabel is selected among an alpha or beta emitting radiotherapeutic label, a PET label and a SPECT label.
- the invention concerns the method, wherein said alpha or beta emitting label is selected among 131 l, 177 Lu, 228 Th, 225 Ac, 227 Ra.
- the invention concerns the method, wherein said PET label is selected among 124 l, 18 F, 64 Cu, 68 Ga, 11 C 225 Ac and 89 Zr.
- the invention concerns the method, wherein said SPECT label is selected among 131 l, 177 Lu, 99 mTc, 64 Cu and 99 mTc. According to an embodiment, the invention concerns the method, wherein said antibody or antigen-binding fragment thereof this conjugated to a chelator compound.
- the invention concerns the method, wherein said chelator compound is bound to a radioactive isotope.
- the invention concerns the method, wherein said radioactive isotope is selected among 124 l, 131 l and 177 Lu or 99 mTc, 64 Cu (chelated to NOTA) and 89 Zr (chelated to DFO).
- the invention concerns the method, wherein said chelator compound is selected among DOTA, DTPA, NOTA and DFO.
- the invention concerns the method, wherein said DOTA is a variant of DOTA.
- the invention concerns the method, wherein said DTPA is a variant of DTPA.
- DOTA is also referred to as l,4,7,10-tetraazacyclododecane-l,4,7 10-tetraacetic acid, and has the formula (CH2CH2NCH2C02H)4 DTPA is also referred to with the lUPAC name 2-[bis[2-
- DTPA has the molecular formula C14H23N3O10.
- the invention concerns the method, wherein said method may be used on at least a sample that has formerly been embedded in paraffin, as well as a sample which has formerly been frozen.
- the invention concerns the method, wherein said method further comprises one or more steps of rinse, preferably wherein at least one rinsing step is with water.
- the invention concerns the method, wherein said water is d2H20. d2H20 is also referred to as doubles-distilled water.
- the invention concerns the method, wherein one or more or all of said steps are separated by wash with at least one wash buffer.
- the invention concerns the method, wherein one or more of all said steps are separated by at least one wash with at least one wash buffer.
- a wash may be defined as adding an adequate volume of a soluble reagent, preferably a buffer, to a tissue sample and removing the soluble reagent, preferably buffer, again.
- the invention concerns the method, wherein one or more of all said steps are separated by at least two washes with at least one wash buffer.
- the invention concerns the method, wherein one or more of all said steps are separated by at least three washes with at least one wash buffer. According to an embodiment, the invention concerns the method, wherein each wash is carried out for about 1-5 minutes, about 2-4 minutes or preferably about 3 minutes.
- the invention concerns the method, wherein each wash is carried out for about 0.1 minute - 2 minutes, 0.5 minutes - 1.5 minutes or preferably about 1 minute.
- the invention concerns the method, wherein said wash buffer is phosphate-buffered saline.
- the invention concerns the method, wherein said wash buffer is 0.01M phosphate-buffered saline-0,05 % polyoxyethylene sorbitan monolaurate.
- the invention concerns the method, wherein said enzyme blocking substance is a peroxidase block.
- said invention concerns the method, wherein said peroxidase block is 3% H 2 O 2 in d2H20.
- the invention concerns the method, wherein said protein blocking substance is casein solution.
- said invention concerns the method, wherein said enzyme blocking is carried out for 1-60 minutes, 2-55 minutes, 3-50 minutes, 4-45 minutes, 5-40 minutes, 6-35 minutes, 7-30 minutes, 8-25 minutes, 9- 20 minutes 10-15 minutes or about 10 minutes.
- said invention concerns the method, wherein said protein blocking is carried out for 1-60 minutes, 5-55 minutes, 10-50 minutes, 15-45 minutes, 20-40 minutes, 25-35 minutes, or about 30 minutes.
- the invention concerns the method, wherein said method further comprises a step of incubation with a reagent that amplify the signal of the primary antibody
- a reagent that amplify the signal of the primary antibody may be EnvisionTM Flex + Mouse (linker).
- the invention concerns the method, wherein said reagent that amplify the signal of the primary antibody is an antibody polymer. According to an embodiment, the invention concerns the method, wherein said incubation with a reagent that amplify the signal of the primary antibody is carried out for about 1 - 30 minutes, about 5 - 25 minutes, about 10 - 20 minutes or preferably about 15 minutes.
- the invention concerns the method, wherein said reagent that amplify the signal of the primary antibody, enhance the signal at least 2 times, at least 3 times, at least 4 times or preferably at least 5 times.
- the invention concerns the method, wherein said binding with a primary antibody is carried out for 1-48 hours, 4- 44 hours, 8 - 40 hours, 12 - 36 hours, 16 - 32 hours, 20 - 28 hours or about 24 hours.
- the invention concerns the method, wherein said binding with a primary antibody is carried out for 1 - 240 minutes, 30 - 110 minutes, 60 - 180 minutes,
- the invention concerns the method, wherein said binding with a primary antibody is carried out for about 120 minutes or about 24 hours. According to an embodiment, the invention concerns the method, wherein said binding with a primary antibody is carried out for a period of time allowing at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 99% of the antigens to be bound by the primary antibody. According to an embodiment, the invention concerns the method, wherein said primary antibody is any of the previously mentioned antibodies or an IgGl antibody.
- the invention concerns the method, wherein said IgGl antibody is a mouse IgGl antibody.
- the invention concerns the method, wherein said primary antibody has a concentration of 1 - 60 mg/ml, 5 - 55 mg/ml, 10 - 50 mg/ml, 15 - 45 mg/ml, 20 - 40 mg/ml, 25 - 35 mg/ml or about 30 mg/ml.
- the invention concerns the method, wherein said primary antibody has a concentration of 1-40 mg/ml, 3-35 mg/ml, 10-30 mg/ml, 15-25 mg/ml or preferably about 20 mg/ml.
- the invention concerns the method, wherein said secondary antibody is conjugated to a labeled polymer.
- the invention concerns the method, wherein said labeled polymer is a horseradish peroxidase labeled polymer.
- the invention concerns the method, wherein said secondary antibody is an anti-mouse antibody.
- the invention concerns the method, wherein said incubation with a secondary antibody is carried out for 1 - 120 minutes, 10 - 110 minutes, 20 - 100 minutes, 30- 90 minutes, 40 - 80 minutes, 50 - 70 minutes or about 60 minutes.
- the invention concerns the method, wherein said incubation with a secondary antibody is carried out for 1 - 240 minutes, 10 - 230 minutes, 20 - 220 minutes, 30 - 210 minutes, 40 - 200 minutes, 50 - 190 minutes, 60 - 180 minutes, 70 - 170 minutes, 80 - 160 minutes, 90 - 150 minutes, 100 - 140 minutes, 110 - 130 minutes or preferably about 120 minutes.
- said staining agent is a benzidine.
- the invention concerns the method, wherein said benzidine is 3,3'-diaminobenzidine.
- the invention concerns the method, wherein said incubating with a staining agent is carried out for 1-6 minutes, 1,5 -5,5 minutes, 2-5 minutes, 2,5 - 4,5 minutes, 3 -4 minutes or about 3 minutes.
- the invention concerns the method, wherein said incubating with a staining agent is carried out for about 1 - 10 minutes, about 1 - 9 minutes, about 2 - 7 minutes or preferably about 3 - 5 minutes.
- the invention concerns the method, comprising a step, wherein said tissue sample is counterstained with a counterstaining agent.
- a counterstaining agent is a substance used to provide contrast so the antibody-stained cells stand out more than it does without said counterstaining agent.
- the invention concerns the method, wherein said counterstaining agent is hematoxylin and optionally eosin.
- the invention concerns the method, wherein said sample is dehydrated after counterstaining.
- the invention concerns the method, wherein said tissue sample is coverslipped after dehydration.
- the invention concerns the method, wherein one or more or all of said steps are separated by at least one wash according to the invention.
- the invention concerns the method, wherein said steps are performed in the following order:
- Blocking of enzymes with an enzyme blocking substance ii. Blocking of proteins with a protein blocking substance, iii. Binding with a primary antibody, iv. Incubation with a secondary antibody, and v. Staining with a staining agent.
- the invention concerns the method, wherein the preparation of said tissue sample comprises one or more or all of the following steps:
- the invention concerns the method, wherein the method comprises one or more or all of the following steps:
- the invention concerns the method, wherein said paraffin embedded sample is microtome-sectioned to a thickness of 1 to 7 mm thick, 2 to 6 mm thick or about 3 to 5 mm thickness.
- the invention concerns the method, wherein said tissue sample is slide-mounted.
- the invention concerns the method, wherein the preparation for staining and staining comprises one or more or all of the following steps:
- the invention concerns the method, wherein said sample has formerly been embedded in paraffin. According to an embodiment, the invention concerns the method, wherein said sample has been or is embedded in paraffin and/or formalin fixed.
- the invention concerns the method, wherein said sample is deparaffinized. According to an embodiment, the invention concerns the method, wherein said antigen retrieval is carried out by a buffer.
- the invention concerns the method, wherein said buffer is Tris-EDTA buffer.
- EDTA is also referred to as ethylenediaminetetraacetic acid.
- the invention concerns the method, wherein said Tris-EDTA buffer has a pH value of 7 - 10, 7.5 - 9.5, 8 - 9 or about 9.
- the invention concerns the method, wherein said Tris-EDTA buffer is 60 - 140 °C, 65 - 135 °C, 70 - 130 °C, 75 - 125 °C, 80 - 120 °C, 85 - 115 °C, 90 - 110 °C, 95 - 105 °C or about 100 °C.
- the invention concerns the method, wherein said antigen retrieval is carried out for 1-60 minutes, 2-55 minutes, 3-50 minutes, 4-45 minutes, 5-40 minutes, 6-35 minutes, 7-30 minutes, 8-25 minutes, 9- 20 minutes, 10-15 minutes or about 15 minutes.
- the invention concerns the method, wherein said steps are performed at 0 - 8 °C, 0.5 - 7.5 °C, 1-7 °C, 1.5 - 6.5 °C, 2-6 °C, 2.5 -5.5 °C, 3 - 5 °C, 3.5 - 4.5 °C or about 4 °C.
- the invention concerns the method, wherein said steps are performed for 1-48 hours, 4- 44 hours, 8 - 40 hours, 12 - 36 hours, 16 - 32 hours, 20 - 28 hours or about 24 hours. According to an embodiment, the invention concerns the method, wherein the steps are followed by one or more or all of the steps described above.
- the invention concerns the method, wherein said steps are performed in the following order:
- the invention concerns the method, wherein the steps are performed in the following order: Blocking, binding, incubation, staining.
- the invention concerns the method, wherein the preparation of said tissue sample comprises one or more or all of the following steps:
- tissue sample is a frozen sample or a thawed sample.
- the invention concerns the method, wherein said tissue sample is a thawed sample.
- the invention concerns the method, wherein said optimal cutting temperature medium comprises one or more or all of the following: polyvinyl alcohol, polyethylene glycol or non-reactive ingredients.
- the invention concerns the method, wherein said sample is maintained below -30 °C, - 35 °C, -40 °C, -45 °C, -50 °C, -55 °C, -60 °C, -65 °C, -70 °C, -75 °C, - 80 °C, -85 °C, -90 °C, -95 °C or below -100 °C
- the invention concerns the method, wherein said sample is sectioned to a thickness of 1-10 mm thick, 1.5 - 9.5 mm thick, 2 - 9 mm thick, 2.5 - 8.5 mm thick, 3 - 8 mm thick, 3.5 - 7.5 mm thick, 4 - 7 mm thick, 4.5 - 6.5 mm thick, 5 - 6 mm thick or about 5 mm thicknesses.
- the invention concerns the method, wherein said sample is sectioned using cryostat. According to an embodiment, the invention concerns the method, wherein said sample is transferred onto a charged slide.
- the invention concerns the method, wherein said sample is air-dried. According to an embodiment, the invention concerns the method, wherein said sample is air-dried at room temperature, wherein said room temperature is 10 °C - 40 °C.
- the invention concerns the method, wherein said room temperature is selected among 10 - 40 °C, 15 - 35 °C, 17 - 33 °C, 18 - 30 °C, 19 - 27 °C or about 20 - 25 °C.
- the invention concerns the method, wherein said sample is air-dried for 1-48 hours, 4- 44 hours, 8 - 40 hours, 12 - 36 hours, 16 - 32 hours, 20 - 28 hours or about 24 hours.
- the invention concerns the method, wherein the method comprises one or more or all of the following steps: B.l Exposure of a tissue sample to room temperature, wherein said room temperature is 10 - 40 °C.
- the invention concerns the method, wherein said room temperature is selected among 10 - 40 °C, 15 - 35 °C, 17 - 33 °C, 18 - 30 °C, 19 - 27 °C or about 20 - 25 °C.
- the invention concerns the method, wherein said exposure to room temperature is carried out for 1 - 60 minutes, 5 - 55 minutes, 10 - 50 minutes, 15 - 45 minutes, 20 - 40 minutes, 25 - 35 minutes or about 30 minutes.
- the invention concerns the method, wherein said sample is fixated in paraformaldehyde.
- the invention concerns the method, wherein said sample is fixated in acetone.
- the invention concerns the method, wherein said acetone is - 10 - 20 °C, -5 - 15 °C, 0 - 10 °C, 0.5 - 7 °C, 1 - 6 °C or 2 - 5 °C.
- the invention concerns the method, wherein said fixation is carried out for 1-20 minutes, 5 - 15 minutes or about 10 minutes.
- the invention concerns the method, wherein said steps are followed by one or more or all of the steps previously described.
- the invention concerns the method, wherein said cancer cells are cancer cells from any cancer type that express or may express B7-H3.
- the invention concerns the method, wherein said cancer cells are selected among carcinoma, sarcoma, lymphoma and leukemia cancer cells.
- the invention concerns the method, wherein said cancer cells are selected among neuroblastoma, medulloblastoma, glioblastoma, small celled lung cancer, non-small celled carcinoma, a pediatric sarcoma, an adult sarcoma, breast cancer, liver cancer, melanoma, non-small celled lung carcinoma, lung adenocarcinoma or gastrointestinal cancer cells.
- said cancer cells are selected among neuroblastoma, medulloblastoma, glioblastoma, small celled lung cancer, non-small celled carcinoma, a pediatric sarcoma, an adult sarcoma, breast cancer, liver cancer, melanoma, non-small celled lung carcinoma, lung adenocarcinoma or gastrointestinal cancer cells.
- the invention concerns the method, wherein said cancer cells are ovarian cancer cells.
- the invention concerns the method, wherein said ovarian cancer cells are adenocarcinoma cells.
- the invention concerns the method, wherein said adenocarcinoma cells are serous adenocarcinoma cells.
- the invention concerns the method, wherein said adenocarcinoma cells are serous papillary adenocarcinoma cells.
- the invention concerns the method, wherein said cancer cells are gastric cancer cells.
- the invention concerns the method, wherein said gastric cancer cells are gastric carcinoma cells.
- the invention concerns the method, wherein said gastric carcinoma cells are gastric adenocarcinoma cells. According to an embodiment, the invention concerns the method, wherein said gastric carcinoma cells are gastric cystadenocarcinoma cells.
- the invention concerns the method, wherein said cancer cells are neuroblastoma cancer cells. According to an embodiment, the invention concerns the method, wherein said cancer cells are from a metastasis, a high-risk cancer and/or a recurrent cancer.
- the invention concerns the method, wherein said method comprises the following steps performed on said tissue sample:
- Binding with a primary antibody wherein said primary antibody comprises a heavy chain sequence according to SEQ ID No. 1, and a light chain sequence according to SEQ ID No. 2 or is an MslgG1 antibody, and wherein said antibody has a concentration of 30ug/ml, and wherein said exposure is to be carried out for about 24 hours, and wherein said exposure is to be carried out in about in 4°C ix.
- EDTA is also referred to as ethylenediaminetetraacetic acid.
- PBS is also referred to as phosphate-buffered saline.
- CAS-protein block is also referred to as casein solution.
- DAB is also referred to as 3,3'-diaminobenzidine.
- d2H20 is also referred to as double-distilled water.
- the invention concerns the method, wherein said method comprises the following steps performed on said tissue sample: i. Remove the frozen sample from -80 °C and keep in room temperature, wherein said room temperature is selected among any of the temperatures described above as room temperatur 110, and wherein said samples are kept in room temperature for about 30 minutes ii.
- the invention concerns the method, wherein said method comprises the following steps performed on said tissue sample: i. Deparaffinization ii. Antigen retrieval with Tris-EDTA buffer, wherein said buffer has a pH value of 9 and wherein said antigen retrieval is carried out for about 20 minutes iii. Cool down for about 20 minutes iv. First rinse in d2H20 for about 1 minute v. Second rinse in d2H20 for about 1 minute vi. First wash with 0.01M phosphate-buffered saline for about 2 minutes vii. Second wash with 0.01M phosphate-buffered saline for about 2 minutes viii. Third wash with 0.01M phosphate-buffered saline for about 2 minutes ix.
- Blocking enzymes with peroxidase block wherein said peroxidase block is 3% H 2 O 2 in d2H20, and wherein said exposure is carried out for about 10 minutes x.
- the invention concerns the method, wherein said tissue sample is an ovarian tissue sample.
- the invention concerns the method, wherein said method comprises the following steps performed on said tissue sample: i. Deparaffinization ii. Antigen retrieval with Tris-EDTA buffer, wherein said buffer has a pH value of 9 and wherein said antigen retrieval is carried out for about 20 minutes iii. Cool down for about 20 minutes iv. First rinse in d2H20 for about 1 minute v. Second rinse in d2H20 for about 1 minute vi. First wash with 0.01M phosphate-buffered saline for about 2 minutes vii. Second wash with 0.01M phosphate-buffered saline for about 2 minutes viii. Third wash with 0.01M phosphate-buffered saline for about 2 minutes ix.
- Blocking enzymes with peroxidase block wherein said peroxidase block is 3% H 2 O 2 in d2H20, and wherein said exposure is carried out for about 10 minutes x.
- the invention concerns the method, wherein said tissue sample is a gastric tissue sample.
- the invention concerns a method for providing and/or measuring a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of cancer in an individual, wherein the method is performed on a tissue sample comprising cancer cells.
- the invention concerns a method for detecting the clinical benefit of an antibody or antigen-binding fragment thereof in the treatment of cancer in an individual, wherein the method is performed on a tissue sample comprising cancer cells.
- the individual is a specific individual or a specific cancer patient, as the method is to be used for each individual patient.
- the invention concerns a method for providing and/or measuring a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of an indication of cancer, wherein the method is performed on tissue samples comprising cancer cells.
- the invention concerns a chimeric antigen receptor (CAR) comprising an antibody or antigen-binding fragment thereof, wherein said chimeric antigen receptor is used according to a method of the invention.
- the invention concerns a CAR-T cell expressing the chimeric antigen receptor.
- the invention concerns a population of CAR-T cells.
- the invention concerns a composition comprising the population of CAR-T cells.
- the invention concerns a method of treating cancer in an individual, wherein the method comprises use of a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof, and wherein said antibody or antigen- binding fragment thereof is used in said treatment.
- the invention concerns a method for treating cancer in an individual, wherein the method comprises use of an antibody or antigen binding fragment thereof, and wherein the indication, such as ovarian cancer, for treatment of said cancer with said antibody or antigen-binding fragment thereof is determined or confirmed by a method of the invention.
- the invention concerns a method for treating cancer in an individual, wherein the method comprises evaluation of the clinical benefit of an antibody or antigen-binding fragment thereof, comprising use of a method of the invention, and wherein the clinical benefit is the clinical benefit for a patient, and wherein said antibody or antigen- binding fragment thereof is used in said treatment.
- the invention concerns a method for treating cancer in an individual, wherein the method comprises evaluation of a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in a tissue sample comprising cancer cells, comprising use of a method of the invention, and wherein said antibody or antigen-binding fragment thereof is used in said treatment.
- the invention concerns the method, wherein said tissue sample is a tissue sample from said individual.
- the invention concerns the method, wherein said method evaluates overexpression of B7-H3.
- the invention concerns the method, wherein said cancer cells are selected among any of the cancer cells as described above.
- the invention concerns the method, wherein said antibody or antigen-binding fragment thereof is any of the antibodies or antigen-binding fragments as described above.
- the invention concerns the method, wherein said cancer is any cancer type that express or may express B7-H3.
- the invention concerns the method, wherein said cancer is selected among a carcinoma, a sarcoma, a lymphoma and a leukemia.
- the invention concerns the method, wherein said cancer is selected among neuroblastoma, medulloblastoma, glioblastoma, small celled lung cancer, non-small celled carcinoma, a pediatric sarcoma, an adult sarcoma, breast cancer, liver cancer, melanoma, non-small celled lung carcinoma, lung adenocarcinoma or a gastrointestinal cancer
- said cancer is selected among neuroblastoma, medulloblastoma, glioblastoma, small celled lung cancer, non-small celled carcinoma, a pediatric sarcoma, an adult sarcoma, breast cancer, liver cancer, melanoma, non-small celled lung carcinoma, lung adenocarcinoma or a gastrointestinal cancer
- the invention concerns the method, wherein said cancer is an ovarian cancer.
- the invention concerns the method, wherein said ovarian cancer is an adenocarcinoma.
- the invention concerns the method, wherein said adenocarcinoma is a serous adenocarcinoma. According to an embodiment, the invention concerns the method, wherein said adenocarcinoma is a serous papillary adenocarcinoma.
- the invention concerns the method, wherein said cancer is a gastric cancer.
- the invention concerns the method, wherein said gastric cancer is a carcinoma.
- the invention concerns the method, wherein said carcinoma is an adenocarcinoma.
- the invention concerns the method, wherein said carcinoma is a cystadenocarcinoma.
- the invention concerns the method, wherein said cancer is neuroblastoma.
- the invention concerns the method, wherein said cancer is a metastasis, a high-risk cancer and/or a recurrent cancer.
- the invention concerns the method, wherein said treatment is combined with chemotherapy and/or surgery.
- the invention concerns a kit of parts, adapted to carry out a method of the invention.
- the invention concerns a kit of parts, wherein said kit is to be used to determine or qualify a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof is to be used in the treatment of a cancer.
- the invention concerns a kit of parts, wherein said kit is to be used to determine or qualify the clinical benefit a patient has or will have from treatment with an antigen or antigen-binding fragment thereof, and wherein said patient is a cancer patient.
- the invention concerns a kit of parts, wherein said kit is to be used to determine or qualify a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of an indication of cancer.
- the invention concerns a kit of parts, wherein said kit is used in detecting B7-H3 expression in a tissue sample comprising cancer cells, and wherein said sample is selected among a sample that have been embedded in paraffin and a sample have been frozen.
- the invention concerns the kit, comprising an anti-B7-H3 antibody or antigen-binding fragment thereof used to detect B7-H3 expression.
- the invention concerns the kit, wherein said kit comprises a primary antibody.
- the invention concerns the kit, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof, or said primary antibody, is any of the antibodies or antigen-binding fragments thereof as described above.
- the invention concerns the kit, wherein said kit uses antibodies to detect proteins in cells of said tissue sample.
- the invention concerns the kit, wherein said kit comprises a secondary antibody.
- the invention concerns the kit, wherein said secondary antibody is an anti-mouse antibody.
- the invention concerns the kit, wherein said secondary antibody is conjugated to an enzyme.
- the invention concerns the kit, wherein said enzyme is a peroxidase.
- the invention concerns the kit, wherein said peroxidase is Horseradish peroxidase.
- the invention concerns the kit, wherein said kit comprises an enzyme block.
- the invention concerns the kit, wherein said enzyme block is a peroxidase block.
- the invention concerns the kit, where said peroxidase block is 3% H 2 O 2 in d2H20.
- the invention concerns the kit, wherein said kit comprises a protein block.
- the invention concerns the kit, wherein said protein block is casein solution.
- the invention concerns the kit, wherein said kit comprises a substance for chromogenic staining.
- the invention concerns the kit, wherein said substance for chromogenic staining is a benzidine.
- the invention concerns the kit, wherein said benzidine is 3,3'- Diaminobenzidine.
- the invention concerns the kit, wherein said substance for chromogenic staining is a counterstaining agent.
- the invention concerns the kit, wherein said counterstaining agent is hematoxylin and optionally eosin.
- the invention concerns the kit, wherein said kit further comprises a reagent that amplify the signal of the primary antibody.
- the invention concerns the kit, wherein said reagent that amplify the signal of the primary antibody is an antibody polymer.
- the invention concerns the kit, wherein said kit comprises a substance used to wash.
- the invention concerns the kit, wherein said substance used to wash is a wash buffer.
- the invention concerns the kit, wherein said wash buffer is phosphate-buffered saline.
- the invention concerns the kit, wherein said wash buffer is 0,01M phosphate-buffered saline-0,05 % polyoxyethylene sorbitan monolaurate.
- the invention concerns the kit, wherein the kit further comprises water.
- the invention concerns the kit, wherein said water is d2H20.
- the invention concerns the kit, wherein said cancer cells are selected among any of the cancer cells as described above.
- the invention concerns the kit, wherein said cancer is selected among any of the cancers as described above.
- the invention concerns a method for treating cancer in an individual, wherein the method comprises use of a kit according to the invention.
- the invention concerns the method, wherein said cancer is selected among any of the cancers as described above. According to an embodiment, the invention concerns the method, wherein said cancer is a gastric cancer.
- the invention concerns the method, wherein said gastric cancer is a carcinoma.
- the invention concerns the method, wherein said carcinoma is an adenocarcinoma.
- the invention concerns the method, wherein said carcinoma is a cystadenocarcinoma.
- the invention concerns the method, wherein said gastric cancer is selected from the group consisting of adenocarcinoma, lymphoma, gastrointestinal stromal tumor (GIST), carcinoid tumor, and other cancers.
- said gastric cancer is selected from the group consisting of adenocarcinoma, lymphoma, gastrointestinal stromal tumor (GIST), carcinoid tumor, and other cancers.
- the invention concerns the method, wherein said cancer is an ovarian cancer.
- the invention concerns the method, wherein said ovarian cancer is an adenocarcinoma.
- the invention concerns the method, wherein said adenocarcinoma is a serous adenocarcinoma.
- the invention concerns the method, wherein said adenocarcinoma is a serous papillary adenocarcinoma.
- the invention concerns the method, wherein said ovarian cancer is selected from the group consisting of epithelial carcinoma, serous carcinoma, primary peritoneal carcinoma, clear-cell carcinoma, clear-cell adenocarcinoma, endometrioid, malignant mixed mijllerian tumor (carcinosarcoma), mucinous tumors, mucinous adenocarcinoma, pseudomyxoma peritonei, undifferentiated epithelial cancers, malignant Brenner tumor, transitional cell carcinoma, sex cord-stromal tumor, granulosa cell tumor, adult granulosa cell tumor, juvenile granulosa cell tumor, sertoli-Leydig cell tumor, sclerosing stromal tumors, germ cell tumor, dysgerminoma, choriocarcinoma, immature (solid) teratoma, mature teratoma (dermoid cyst), yolk sac tumor/endodermal sinus tumor, embryonal carcinoma, polye
- the invention concerns the method, wherein said cancer is a neuroblastoma.
- the invention concerns the method, wherein the distribution of cells is selected from the group consisting of diffuse and multifocal, preferably as determined by the methods of Example 2.
- the invention concerns the method, wherein the incidence is 1, preferably 2, more preferred 3, preferably 4, preferably as determined by the methods of Example 2.
- the invention concerns the method, wherein the incidence is 2-4, preferably 3-4, preferably as determined by the methods of Example 2.
- the invention concerns the method, wherein the staining intensity is 1, preferably 2, more preferred 3, preferably 4, preferably as determined by the methods of Example 2.
- the invention concerns the method, wherein the staining intensity is 2-4, preferably 3-4, preferably as determined by the methods of Example 2.
- the invention concerns the method, wherein the staining is granular, preferably as determined by the methods of Example 2. According to an embodiment, the invention concerns the method, wherein the staining is located on the membrane or cytoplasmic, preferably as determined by the methods of Example 2.
- the invention concerns the method, wherein the staining is located on the membrane, preferably as determined by the methods of Example 2.
- the invention concerns the method, wherein the subcellular location is selected from the group consisting of Granular (G), Membrane (M), Cytoplasmic (C), Extracellular (E), Vasculature (V), Intranuclear (In), Apical (A) and Basal (B), preferably as determined by the methods of Example 3.
- the invention concerns the method, wherein the amount of positive tumor cells in percentage is selected from the group consisting of up to 29%, 30- 49%, 50-74%, and 75-100%, preferably as determined by the methods of Example 3.
- the invention concerns the method, wherein the amount of positive tumor cells in percentage is above 10%, above 20%, preferably above 30%, preferably as determined by the methods of Example 3.
- the invention concerns the method, wherein the incidence of positive tumor cells is selected from the group consisting of normal, minimal, mild, moderate and severe, preferably as determined by the methods of Example 3.
- the invention concerns the method, wherein the intensity of positive staining at cellular level is selected from the group consisting of equivocal, mild (weak), moderate, strong and intense, preferably as determined by the methods of Example 3.
- the invention concerns the method, wherein the incidence of staining represents a mild to severe distribution of positive tumor cells, preferably as determined by the methods of Example 3.
- the invention concerns an antibody or antigen binding fragment thereof, wherein said antibody or antigen-binding fragment comprises at least one of the sequences selected among a heavy chain variable region CDR1 according to SEQ ID No. 3, a heavy chain variable region CDR2 according to SEQ IN No. 4 or 19, a heavy chain variable region CD3 according to SEQ ID No. 5, a light chain variable region CDR1 according to SEQ ID No. 6, a light chain variable region CDR2 according to SEQ ID No. 7 and a light chain variable region CDR3 according to SEQ ID No. 8.
- the invention concerns an antibody or antigen binding fragment thereof, wherein said antibody or antigen-binding fragment comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2.
- the invention concerns an antibody or antigen binding fragment thereof, wherein said antibody or antigen binding fragment comprises a heavy chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No.
- a light chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No. 2.
- the invention concerns an antibody or antigen binding fragment according to the invention, wherein said antigen-binding fragment thereof is a single chain variable fragment (scFv).
- scFv single chain variable fragment
- the invention concerns an antibody or antigen binding fragment according to the invention, wherein said scFv comprises a portion of the amino acid sequence set forth in SEQ ID No. 9, SEQ ID No. 13 or SEQ ID No. 14.
- the invention concerns an antibody or antigen binding fragment according to the invention, wherein said scFv comprises at least one of the amino acid substitutions K13E, R18Q, R45Q, K103E and K107E.
- the invention concerns an antibody or antigen binding fragment according to the invention, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof is a murine 8H9 antibody or an antigen binding fragment thereof. According to an embodiment, the invention concerns an antibody or antigen binding fragment according to the invention, wherein said antibody or antigen-binding fragment thereof is humanized.
- the invention concerns an antibody or antigen binding fragment according to the invention, wherein said antibody or antigen-binding fragment thereof is a chimeric antibody or an antigen-binding fragment thereof.
- the invention concerns an antibody or antigen binding fragment according to the invention, wherein said antibody or antigen-binding fragment thereof is radiolabeled.
- the invention concerns an antibody or antigen binding fragment according to the invention, wherein said radiolabel is selected among a PET label and a SPECT label.
- the invention concerns an antibody or antigen binding fragment according to the invention, wherein said PET label is selected among 124 l, 225 Ac and 89 Zr.
- the invention concerns an antibody or antigen binding fragment according to the invention, wherein said SPECT label is selected among 131 l, 177 Lu, 99 mTc, 64 Cu and 89 Zr.
- the invention concerns an antibody or antigen binding fragment according to the invention, wherein said antibody or antigen-binding fragment thereof is conjugated to a chelator compound.
- the invention concerns an antibody or antigen binding fragment according to the invention, wherein said chelator compound is bound to a radioactive isotope.
- the invention concerns an antibody or antigen binding fragment according to the invention, wherein said radioactive isotope is selected among 124 l, 131 l and 177 Lu or 99 mTc, 64 Cu (chelated to NOTA) and 89 Zr (chelated to DFO).
- said radioactive isotope is selected among 124 l, 131 l and 177 Lu or 99 mTc, 64 Cu (chelated to NOTA) and 89 Zr (chelated to DFO).
- the invention concerns an antibody or antigen binding fragment according to the invention, wherein said chelator compound is selected among DOTA, DTPA, NOTA and DFO.
- the invention concerns an antibody or antigen binding fragment according to the invention, wherein said DOTA is a variant of DOTA.
- the invention concerns an antibody or antigen binding fragment according to the invention, wherein said DTPA is a variant of DTPA.
- the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of ovarian cancer, wherein said ovarian cancer is an adenocarcinoma.
- the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of ovarian cancer, wherein said adenocarcinoma is a serous adenocarcinoma.
- the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of ovarian cancer, wherein said adenocarcinoma is a serous papillary adenocarcinoma.
- the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of ovarian cancer
- said ovarian cancer is selected from the group consisting of epithelial carcinoma, serous carcinoma, primary peritoneal carcinoma, clear-cell carcinoma, clear-cell adenocarcinoma, endometrioid, malignant mixed müllerian tumor (carcinosarcoma), mucinous tumors, mucinous adenocarcinoma, pseudomyxoma peritonei, undifferentiated epithelial cancers, malignant Brenner tumor, transitional cell carcinoma, sex cord-stromal tumor, granulosa cell tumor, adult granulosa cell tumor, juvenile granulosa cell tumor, sertoli-Leydig cell tumor, sclerosing stromal tumors, germ cell tumor, dysgerminoma, choriocarcinoma, immature (solid) teratoma, mature teratoma (
- the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of gastric cancer, wherein said gastric cancer is a carcinoma.
- the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of gastric cancer, wherein said carcinoma is an adenocarcinoma.
- the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of gastric cancer, wherein said carcinoma is a cystadenocarcinoma.
- the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of gastric cancer, wherein said gastric cancer is selected from the group consisting of adenocarcinoma, lymphoma, gastrointestinal stromal tumor (GIST), carcinoid tumor, and other cancers.
- gastric cancer is selected from the group consisting of adenocarcinoma, lymphoma, gastrointestinal stromal tumor (GIST), carcinoid tumor, and other cancers.
- Fig. 1 provides a flow chart of a method for detection related to an embodiment of the invention.
- Fig. 2A and 2B show the immunohistochemistry of FFPE sections of non-small cell lung carcinoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
- the slides were stained with said antibody, at a concentration of 30 mg/mL, overnight at 4 °C. There is severe granular cell surface and intracellular staining of tumor cells.
- Fig. 2A shows a field of view with 20 times magnification
- Fig. 2B shows a field of view with 40 times magnification.
- the histopathologic score was 4/4.
- Fig. 3A and 3B show the immunohistochemistry of FFPE sections of non-small cell lung carcinoma with isotype control mouse IgG1 antibody ( MslgG1). The slides were stained with 30 mg/mL MslgG1 overnight at 4 °C. There is no staining of tumor cells.
- Fig. 3A shows a field of view with 20 times magnification and Fig. 2B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
- Fig. 4A and 4B show the immunohistochemistry of FFPE sections of neuroblastoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
- the slides were stained with said antibody, at a concentration of 30 mg/mL, overnight at 4 °C. There is strong to intense granular cell surface and some intracellular staining of tumor cells.
- Fig. 4A shows a field of view with 20 times magnification
- Fig. 4B shows a field of view with 40 times magnification.
- the histopathologic score was 4/3.
- Fig. 5A and 5B show the immunohistochemistry of FFPE sections of neuroblastoma with MslgG1. The slides were stained with 30 mg/mL MslgG1 overnight at 4 °C. There is no staining.
- Fig. 5A shows a field of view with 20 times magnification and Fig. 5B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
- Fig. 6A and 6B show the immunohistochemistry of FFPE sections of medulloblastoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
- Fig. 6A shows a field of view with 20 times magnification
- Fig. 6B shows a field of view with 40 times magnification.
- the histopathologic score was 4/4.
- Fig. 7A and 7B show the immunohistochemistry of FFPE sections of medulloblastoma with MslgG1. The slides were stained with 30 ⁇ g/mL MslgG1 overnight at 4 °C. There is no staining.
- Fig. 7A shows a field of view with 20 times magnification and Fig. 7B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
- Fig. 8A and 8B show the immunohistochemistry of FFPE sections of glioblastoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
- the slides were stained with said antibody, at a concentration of 30 ⁇ g/mL, overnight at 4 °C. There is intense granular cell surface and intracellular staining of tumor cells.
- Fig. 8A shows a field of view with 20 times magnification
- Fig. 8B shows a field of view with 40 times magnification.
- the histopathologic score was 4/4.
- Fig. 9A and 9B show the immunohistochemistry of FFPE sections of glioblastoma with MslgG1. The slides were stained with 30 ⁇ g/mL MslgG1 overnight at 4 °C. There is no staining.
- Fig. 9A shows a field of view with 20 times magnification and Fig. 9B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
- Fig. 10A and 10B show the immunohistochemistry of FFPE sections of ovarian cancer with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
- the slides were stained with said antibody, at a concentration of 30 ⁇ g/mL, overnight at 4 °C. There is moderate multifocal granular cell surface and intracellular and vascular staining of a subset of tumor cells.
- Fig. 10A shows a field of view with 20 times magnification
- Fig. 10B shows a field of view with 40 times magnification.
- the histopathologic score was 2/2.
- Fig. 11A and 11B show the immunohistochemistry of FFPE sections of ovarian cancer with MslgG1. The slides were stained with 30 ⁇ g/mL MslgG1 overnight at 4 °C. There is no staining.
- Fig. 11A shows a field of view with 20 times magnification and Fig. 11B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
- Fig. 12A and 12B show the immunohistochemistry of FFPE sections of sarcoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
- the slides were stained with said antibody, at a concentration of 30 ⁇ g/mL, overnight at 4 °C. There is moderate multifocal granular cell surface and intracellular staining of a subset of tumor cells.
- Fig. 12A shows a field of view with 20 times magnification
- Fig. 12B shows a field of view with 40 times magnification.
- the histopathologic score was 2/2.
- Fig. 13A and 13B show the immunohistochemistry of FFPE sections of sarcoma with MslgG1. The slides were stained with 30 ⁇ g/mL MslgG1 overnight at 4 °C. There is no staining. Fig.
- FIG. 13A shows a field of view with 20 times magnification and Fig. 13B shows a field of view with 40 times magnification.
- the histopathologic score was 0/0.
- Fig. 14A and 14B show the immunohistochemistry of FFPE sections of melanoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
- the slides were stained with said antibody, at a concentration of 30 ⁇ g/mL, overnight at 4 °C. There is severe granular cell surface and intracellular staining of tumor cells.
- Fig. 14A shows a field of view with 20 times magnification
- Fig. 14B shows a field of view with 40 times magnification.
- the histopathologic score was 4/4.
- Fig. 15A and 15B show the immunohistochemistry of FFPE sections of melanoma with MslgG1. The slides were stained with 30 ⁇ g/mL MslgG1 overnight at 4 °C. There is no staining of tumor cells. It shows moderate staining of macrophages.
- Fig. 15A shows a field of view with 20 times magnification and Fig. 15B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
- Fig. 16A and 16B show the immunohistochemistry of FFPE sections of liver cancer with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
- the slides were stained with said antibody, at a concentration of 30 ⁇ g/mL, overnight at 4 °C. There is strong granular cell surface and intracellular staining of tumor cells.
- Fig. 16A shows a field of view with 20 times magnification
- Fig. 16B shows a field of view with 40 times magnification.
- the histopathologic score was 3/3.
- Fig. 17A and 17B show the immunohistochemistry of FFPE sections of normal liver with an antibody comprising a heavy chain according to SEQ ID No.
- Fig. 17A shows a field of view with 20 times magnification
- Fig. 17B shows a field of view with 40 times magnification.
- the histopathologic score was 0/0.
- Fig. 18A shows the immunohistochemistry of frozen sections of non-small cell lung carcinoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
- the slides were stained with said antibody, at a concentration of 2 ⁇ g/mL. There is a moderate number of strong positive stained tumor cells.
- Fig. 18A shows a field of view with 40 times magnification. The histopathologic score was 2/3.
- Fig. 18B shows the immunohistochemistry of frozen sections of non-small cell lung carcinoma with MslgG1. The slides were stained with 2 ⁇ g/mL MslgG1. There is no staining. Fig. 18B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
- Fig. 19A shows the immunohistochemistry of frozen sections of lung adenocarcinoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
- the slides were stained with said antibody, at a concentration of 2 ⁇ g/mL. There is a strong positive stained subset of tumor cells.
- Fig. 19A shows a field of view with 40 times magnification. The histopathologic score was 3/3.
- Fig. 19B shows the immunohistochemistry of frozen sections of lung adenocarcinoma with MslgG1.
- the slides were stained with 2 ⁇ g/mL MslgG1. There is no staining.
- Fig. 19B shows a field of view with 40 times magnification.
- the histopathologic score was 0/0.
- Fig. 20A shows the immunohistochemistry of frozen sections of melanoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
- the slides were stained with said antibody, at a concentration of 2 ⁇ g/mL. There are strong multifocal stained tumor cells.
- Fig. 20A shows a field of view with 40 times magnification.
- Fig. 20B shows the immunohistochemistry of frozen sections of melanoma with MslgG1. The slides were stained with 2 mg/mL MslgG1. There is no staining. Fig. 20B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
- Fig. 21A shows the immunohistochemistry of frozen sections with Sarcoma with an antibody comprising a heavy chain according to SEQ ID no. 1, and a light chain according to SEQ ID no. 2.
- the slides were stained with said antibody, at a concentration of 2 mg/mL. There is strong to intense stained tumor cells.
- Fig. 21A shows a field of view with 40 times magnification. The histopathologic score was 4/4.
- Fig. 21B shows the immunohistochemistry of frozen sections of Sarcoma with MslgG1. The slides were stained with 2 mg/mL MslgG1. There is no staining. Fig. 21B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
- Fig. 22A shows the immunohistochemistry of frozen sections of ovarian cancer with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
- the slides were stained with said antibody, at a concentration of 2 mg/mL. There is strong to intense multifocal stained tumor cells.
- Fig. 22A shows a field of view with 40 times magnification. The histopathologic score was 4/3.
- Fig. 22B shows the immunohistochemistry of frozen sections of ovarian cancer with MslgG1. The slides were stained with 2 mg/mL MslgG1. There is no staining. Fig. 22B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
- Fig. 23A shows the immunohistochemistry of frozen sections of glioblastoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2.
- the slides were stained with said antibody, at a concentration of 2 mg/mL. There is a strong stained subset of tumor cells.
- Fig. 23A shows a field of view with 40 times magnification. The histopathologic score was 3/3.
- Fig. 23B shows the immunohistochemistry of frozen sections of glioblastoma with MslgG1. The slides were stained with 2 mg/mL MslgG1. There is no staining. Fig. 23B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
- Fig. 24A and 24B show the immunohistochemistry of FFPE sections of non-small cell lung carcinoma with commercially available anti-B7-H3 antibody. The slides were stained with 2 mg/mL B7-H3 antibody overnight at 4 °C. There is severe granular cell surface and intracellular staining of tumor cells.
- Fig. 24A shows a field of view with 20 times magnification and Fig. 24B shows a field of view with 40 times magnification. The histopathologic score was 4/4.
- Fig. 25A and 25B show the immunohistochemistry of FFPE sections of neuroblastoma with commercially available anti-B7-H3 antibody. The slides were stained with 2 mg/mL B7-H3 antibody overnight at 4 °C. There is severe granular cell surface staining of tumor cells.
- Fig. 25A shows a field of view with 20 times magnification and Fig. 25B shows a field of view with 40 times magnification. The histopathologic score was 4/3.
- Fig. 26A and 26B show the immunohistochemistry of FFPE sections of medulloblastoma with commercially available anti-B7-H3 antibody. The slides were stained with 2 mg/mL B7-H3 antibody overnight at 4 °C. There is strong granular cell surface and intracellular staining of tumor cells.
- Fig. 26A shows a field of view with 20 times magnification and Fig. 26B shows a field of view with 40 times magnification. The histopathologic score was 3/4.
- Fig. 27A and 27B show the immunohistochemistry of FFPE sections of glioblastoma with commercially available a nti-B7-H3 antibody. The slides were stained with 2 mg/mL B7-H3 antibody overnight at 4 °C. There is strong granular cell surface and intracellular staining of tumor cells.
- Fig. 27A shows a field of view with 20 times magnification and Fig. 27B shows a field of view with 40 times magnification. The histopathologic score was 4/4.
- Fig. 28A and 28B show the immunohistochemistry of FFPE sections of ovarian cancer with commercially available anti-B7-H3 antibody.
- the slides were stained with 2 mg/mL B7-H3 antibody overnight at 4 °C. There is moderate granular cell surface, intracellular and vascular staining of tumor cells.
- Fig. 28A shows a field of view with 20 times magnification and Fig. 28B shows a field of view with 40 times magnification.
- the histopathologic score was 3/3.
- Fig. 29A and 29B show the hematoxylin and eosin counter-staining of FFPE section of non- small cell lung carcinoma. Well preserved tumor tissue is shown.
- Fig. 29A shows a field of view with 20 times magnification and
- Fig. 29B shows a field of view with 40 times magnification.
- Fig. 30A and 30B show the hematoxylin and eosin counter-staining of FFPE section of neuroblastoma.
- Well preserved tumor tissue is shown.
- Fig. 30A shows a field of view with 20 times magnification and
- Fig. 30B shows a field of view with 40 times magnification.
- Fig. 31A and 31B show the hematoxylin and eosin counter-staining of FFPE section of medulloblastoma.
- Well preserved tumor tissue is shown.
- Fig. 31A shows a field of view with 20 times magnification
- Fig. 31B shows a field of view with 40 times magnification.
- Fig. 32A and 32B show the hematoxylin and eosin counter-staining of FFPE section of ovarian cancer. Well preserved tumor tissue is shown.
- Fig. 32A shows a field of view with 20 times magnification and
- Fig. 32B shows a field of view with 40 times magnification.
- Fig. 33A and 33B show the hematoxylin and eosin counter-staining of FFPE section of sarcoma.
- Well preserved tumor tissue is shown.
- Fig. 33A shows a field of view with 20 times magnification and
- Fig. 33B shows a field of view with 40 times magnification.
- Fig. 34A and 34B show the hematoxylin and eosin counter-staining of FFPE section of sarcoma.
- Well preserved tumor tissue is shown.
- Fig. 34A shows a field of view with 20 times magnification
- Fig. 34B shows a field of view with 40 times magnification.
- Fig. 35A and 35B show the hematoxylin and eosin counter-staining of FFPE section of glioblastoma. Well preserved tumor tissue is shown.
- Fig. 35A shows a field of view with 20 times magnification and
- Fig. 35B shows a field of view with 40 times magnification.
- Fig. 36-40 shows IHC staining on 25 TMA Ovarian Cancer Cases. Donor ID as in table 7.
- Fig. 41 shows H&E and IHC staining of patient's gastric tissue (ID: 001, Spindle cell tumor, possible leiomyoma, benign).
- ID 001, Spindle cell tumor, possible leiomyoma, benign.
- H&E image shows a large mass in the muscularis layer (A, black star).
- the tumor consists of spindle cells which showed no immunoreactivity with 8H9 antibody (B and D) or MslgG (C and E).
- Fig. 42 shows IHC staining of patient's gastric tissue (ID: 001-2, Gastric Adenocarcinoma).
- ID 001-2, Gastric Adenocarcinoma
- 8H9 antibody is expressed on the membrane of 80% of tumor cells with grade 3 staining intensity (A). Capillaries are often filled with neutrophils and lymphocytes as well as tumor cells (B, C). At 20 mg/ml, membrane staining with grade 2-3 staining intensity is observed in about 50% of tumor cells (D). The expression of 8H9 antibody is detected on membrane of most tumor cells (E, F). No immunoreactivity is found with MslgG control isotype (Data not shown).
- Fig. 43A shows H&E and IHC staining of patient's gastric tissue (ID 002; gastric adenocarcinoma).
- H&E image shows a protruding proliferative mass in the mucosa (A, doted box).
- the tumor consists of signet ring cells (B, yellow arrows).
- the immunoreactivity with 8H9 antibody was detected in the membrane and cytoplasm of neoplastic epithelium (B and D). No immunostaining was found in sections treated with MslgG (C and E).
- Fig. 43B shows at 20 mg/ml, there is occasional weak granular membrane staining on less than 10% of the tumor cells (F and G, red arrow). Vascular endothelial staining was observed in tumor areas (H).
- Fig. 44A shows H&E and IHC staining of patient's gastric tissue (ID: 003, Gastric Carcinoma).
- H&E image shows a nodule of tumor with no gastric structure (A, doted circle).
- the immunoreactivity with 8H9 antibody was detected in about half of all neoplastic epithelium (B) with a diffuse and cytoplasmic pattern (D, yellow arrows). No immunostaining was found in sections treated with MslgG (C and E).
- Fig. 44B shows at 20 mg/ml, there is little to no positive membrane staining (F and G). Vascular endothelial staining was present in tumor areas (H).
- Fig. 45A shows H&E and IHC staining of patient's gastric tissue (ID: 004, Gastric Adencarcinoma).
- H&E image shows a polypoid proliferative mass in the mucosa and submucosa including numerous small lymphoid nodules (A, starts represents lymphoid nodules).
- the immunoreactivity with 8H9 antibody was detected in nearly all neoplastic epithelium with a diffuse and cytoplasmic pattern (B and D). No immunostaining was found in sections treated with MslgG (C and E).
- Fig. 45 B shows at 20 mg/ml, there is moderate granular membrane staining on about 20% of the tumor cells (F and G, red arrows). Vascular endothelial staining was present in tumor areas (H).
- Fig. 46A shows H&E and IHC staining of patient's gastric tissue (ID: 005, Gastric Adenocarcinoma).
- H&E image shows a large diffuse mass in the muscularis and serosa with severe inflammation, congestion, and hemorrhage (A).
- the immunoreactivity with 8H9 antibody was detected in nearly all neoplastic epithelium with a diffuse and cytoplasmic pattern (B and D). No immunostaining was found in sections treated with MslgG (C and E).
- Fig. 46B shows At 20 mg/ml, there is moderate granular membrane staining on about 30% of the tumor cells in areas of less differentiated tumor cells (F and G, red arrows).
- Fig. 47A shows H&E and IHC staining of patient's gastric tissue (ID: 006, Gastric Adenocarcinoma).
- H&E image shows a large diffuse infiltrative mass in the mucosa infiltrating the submucosa, muscularis, and serosa and serosa (A).
- the immunoreactivity with 8H9 antibody was detected in nearly all neoplastic epithelium with a diffuse and cytoplasmic pattern (B and D). No immunostaining was found in sections treated with MslgG (C and E).
- Fig. 47B shows at 20 ug/ml, there is moderate granular membrane staining on about 30% of the tumor cells (F and G, red arrows). Vascular endothelial staining was present in tumor areas (H).
- Fig. 48A shows H&E and IHC staining of patient's gastric tissue (ID: 007, Gastric Cystadenocarcinoma).
- H&E image shows a large diffuse infiltrative mass invading mucosa, submucosa, muscularis, and serosa (A).
- the immunoreactivity with 8H9 antibody was detected in nearly all neoplastic epithelium with a diffuse and cytoplasmic pattern (B and D). No immunostaining was found in sections treated with MslgG (C and E).
- Fig. 48B shows at 20 ug/ml, there is occasional granular membrane staining on less than 10% of the tumor cells (F and G, red arrows). Vascular endothelial staining was present in tumor areas (H).
- Fig. 49A shows H&E and IHC staining of patient's gastric tissue (ID: 008, Gastric Adenocarcinoma).
- H&E image shows a full thickness of gastric mucosa with a protruding proliferative mass and expansion of submucosa (A).
- the immunoreactivity with 8H9 antibody was detected in nearly all neoplastic epithelium with a diffuse and cytoplasmic pattern (B and D). No immunostaining was found in sections treated with MslgG (C and E).
- Fig. 49B shows at 20 mg/ml, the majority of tumor cells demonstrated granular membrane staining (F and G, red arrows). Vascular endothelial staining was present in tumor areas (H).
- Fig. 50A shows H&E and IHC staining of patient's gastric tissue (ID: 009, Gastric Adenocarcinoma).
- H&E image shows normal and neoplastic gastric tissues (A).
- the immunoreactivity with 8H9 antibody was detected in about 50% of neoplastic epithelium with a diffuse and cytoplasmic pattern (B and D). No immunostaining was found in sections treated with MslgG (C and E).
- Fig. 50B shows At 20 mg/ml, there is weak granular membrane staining on about 30% of the tumor cells (F and G, red arrows). Vascular endothelial staining was present in tumor areas (H).
- Immunohistochemical staining is in general a challenging assay and the selection of an optimal condition requires manipulating a variety of parameters.
- the following conditions did not provide optimal conditions for immunohistochemical staining of formalin fixed paraffin embedded (FFPE) samples with murine 8H9 antibody comprising a heavy chain according to SEQ ID No. 1 and a light chain according to SEQ ID No. 2:
- Carcinoma Ovarian Cancer, Medulloblastoma, Neuroblastoma, Glioblastoma, Sarcoma and Melanoma.
- the preset study has determined whether a murine 8H9 antibody comprising a heavy chain according to SEQ ID No. 1 and a light chain according to SEQ ID No. 2 targeting human immunoregulatory B7-H3 protein, binds to any human tumor tissues.
- the present study has assessed potential binding of a murine 8H9 antibody to neuroblastoma, medulloblastoma, ovarian cancer, non-small cell lung cancer (NSCLS), sarcoma, melanoma and glioblastoma.
- NSCS non-small cell lung cancer
- sarcoma melanoma
- glioblastoma Specific positive tissue staining results suggest cancer types that may be targeted by the antibody in vivo in humans.
- the Isotype Control Mouse lgGl binds to the immunogen: TNP (Trinitrophenol) + KLH (keyhole limpet haemocyanin).
- All tissues were gross trimmed, processed through a graded series of alcohols, oriented and embedded in paraffin, microtome-sectioned at 3 to 5 mm thicknesses, slide-mounted, immunohistochemically stained with mu8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2, and control antibodies, and cover-slipped by standard methodology.
- the unfixed frozen tissues were embedded in optimal cutting temperature medium (OCT) and blocks were prepared.
- OCT tissue blocks were placed in sealed plastic bags or other appropriate containers, and maintained below -70 °C pending sectioning.
- Sections were stained with mu8H9 antibody comprising a heavy chain sequence according to SEQ ID No.l and a light chain sequence according to SEQ ID No. 2 in order to assess expression of the B7-H3 in these tissues. Staining were performed on both formalin fixed paraffin embedded sections and frozen sections.
- the staining consisted of the following steps, separated by rinses with wash buffer: peroxidase block for 10 minutes; CAS block (casein solution) for 30 minutes; primary antibody overnight; horseradish peroxidase labeled polymer conjugated to an anti-mouse antibody for 60 minutes; 3,3'-diaminobenzidine for 3 minutes.
- the slides then were counterstained with hematoxylin, dehydrated, and coverslipped by standard methodologies.
- 0 Normal - Tissue considered to be normal under the conditions of the study and considering the age, sex and strain of the animal concerned. Alterations may be present which, under other circumstances, would be considered deviations from normal.
- the pathologist examined all of the submitted tissue sections using light microscopy and recorded the findings directly into tables. The data generated was used by the pathologist in assessment of the histopathologic findings associated with epitope expression. Results
- Mu8H9 staining was detected diffusely and multifocally in a subset of tumor cells in all tissues. The staining was granular, generally strong to intense (Grade 3-4) and located on the membrane and occasionally cytoplasmic, although there was some vasculature and intracellular staining as well. The staining of most of the tumor tissues was subjected to immunohistochemical staining with commercially available anti-BH-73 antibody. The condition of staining and staining pattern was similar to that of mu8H9, in particular for the IHC staining of frozen sections.
- Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 recognized the target molecule B7-H3 in FFPE samples of different tumor types under following condition: - Antigen retrieval Tris-EDTA pH 9 at 100°C (without any pressure which avoided tissue damage).
- Mu8H9 comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 stained the tumor cells in all tumor tissues diffusely and in some cases multifocally.
- the staining pattern was generally intense, granular and located on the membrane, although there was cytoplasmic, intracellular, and vascular staining as well. Positive staining was evident in all tumor types, however appeared to be more extensive in the FFPE sections of non-small cell carcinoma, neuroblastoma, medulloblastoma, glioblastoma, melanoma and liver cancer. Staining was weak to moderate in ovarian cancer and sarcoma. There was strong positive staining in all frozen tumors, with the exception of moderate staining of lung adenocarcinoma.
- the objective of this study was to determine whether Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 binds to formalin fixed paraffin embedded ovarian cancer tissues using immunohistochemical techniques. Specific staining was detected diffusely and multifocally in the tumor cells in a majority of the ovarian cancer tissues. Generally, 80% of tumor samples showed positive staining for the antibody representing the expression of B7-H3 in 40-100% of tumor cells. The staining was granular, generally mild to intense (Grade 1-4) with cytoplasmic/membranous pattern. The positive membrane staining was found in majority of tumor cells (50-100%) with overall intensity of +2 to +4.
- the study protocol from example 2 was used for IHC staining, however, additional steps were taken to optimize the intensity of the signal in the ovarian cancer tissues as described in the section Immunohistochemical Staining Methodology. Initially the staining protocol was optimized for three concentrations of Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 (10, 20 and 30 mg/ml) and the concentration of 20 mg/ml was selected as the optimal concentration for the rest of the study. Von Willebrand Factor (vWF) was used as a marker for quality control of the testing system. The modification and reproducibility of the staining protocol was validated through eight validation runs using the experimental design presented in table 4.
- vWF Von Willebrand Factor
- ovarian normal and cancer FFPF tissues from individual donors were microtome- sectioned at 5 mm thicknesses and mounted onto charged slides.
- Commercially available ovarian cancer at 5 tissue microarray (TMA) slides of 25 cases (50 cores, 25 cores for testing Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 and 25 cores for testing Ms IgG) were used to validate the results of the staining in a large number of tissue samples.
- Sections were stained with murine 8H9 antibody in order to assess expression of the B7-H3 in these tissues.
- FFPE tissue sections on slides were deparaffinized, hydrated, and subjected to heated Tris-EDTA buffer (pH9, 100 °C) antigen retrieval. After PBS wash, sections were treated with peroxide block solution for 10 minutes, washed with PBS, and incubated with protein blocking buffer for 30 min at room temperature (RT). Then, the tissues were incubated with Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 overnight.
- the second day of staining was conducted with a modified version of the staining protocol used in Example 2 where the sections were treated with a signal enhancer (EnvisionTM FLEX + Mouse (linker)) for 15 minutes prior to adding HRP conjugated polymer for 60 minutes. After three runs of PBS wash, the sections were exposed to a chromogen reagens (EnVisionTM FLEX DAB+ Chromogen) for 1-2 minutes and the intensity of the signal was evaluated under microscope. The slides then were counterstained with hematoxylin, dehydrated, and coverslipped by standard methodologies. IHC methodology for ovarian FFPE tissue:
- results of histopathologic scoring with Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 are detailed in table 6 and table 7.
- Table 5 showing the histopathological scoring terms. Representative photomicrographs are shown in Fig. 36-40.
- the objective of this study was to assess the staining of Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2, a therapeutic agent when radiolabeled as a tool to detect B7-H3, to FFPE ovarian cancer tissue using immunohistochemical techniques. Specific staining was detected diffusely and multifocally in the tumor cells in a majority of the ovarian cancer tissues. The control tissue (normal ovary) dit not show any reaction with 8H9 antibody. The staining was granular, generally mild to intense (Grade 1-4) with cytoplasmic/membranous pattern.
- Example 4 Histopathological Evaluation of Gastric Cancer Tissues for Expression of B7-H3 (CD276) Using Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2.
- the objective of this study was to evaluate histopathologic and immunohistochemical findings of gastric carcinomas from nine patients that were stained with the monoclonal Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2.
- This immunohistochemistry study assessed potential binding of the murine 8H9 antibody to human gastric cancer tissues derived from nine patients. Specific positive staining results suggest tumor cell types that may be targeted by 8H9 antibody in vivo in humans.
- Tissues were then evaluated via light microscopy by a board-certified veterinary pathologist.
- Sections were stained with murine 8H9 antibody to assess expression of the B7-H3 in these tissues.
- the staining technique was developed at CBI. The detailed methodology is shown below.
- PBS (IX) is used as washing buffer for this study.
- PBS could be replaced by other washing buffer e.g. Tris-EDTA or PBS with Tween-20 (0.05%).
- Tissues were qualitatively examined by light microscopy by a veterinary pathologist certified by the American College of Veterinary Pathologists. The pathologist prepared a report describing the histologic features and confirming the diagnosis or the purposes of confirming tissue identity. If any numerical semiquantitative assessment was needed, lesions were scored using the industry standard 5-point scoring system as described in example 2 (Mann, et al., 2012). Immunohistochemical Assessment
- Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 for the gastric cancer tissues in comparison with neuroblastoma (positive control), human heart and liver (negative control) tissues, and commercially available B7-H3, respectively.
- the specificity of the 8H9 antibody was compared with MslgG control.
- the sensitivity of the antibody was optimized at 30 mg/ml only for IHC staining uses.
- M Membrane (M), Nuclear (N), Perinuclear(P) with a granular pattern.
- H&E Control Characterizing cellular morphology and identifying microstructural abnormality including inflammation, vascular leakage, fibrosis, tumor invasion, ect. Masked assessment for description and diagnosis of patient tissues were applied.
- H&E Summary histopathologic staining assessment of patient tissues is presented in table 8.
- Table 9 Summary immunohistochemical staining assessment of patients is presented in Table 9. Representative photomicrographs are presented in Fig. 41-50. Table 9 ICH staining with Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 results for each tissue tested
- Tumor cells were clearly and strongly positive for 8H9 antibody staining. The staining was specific and limited to the tumor cells. Normal cells such as smooth muscle, vessels, nerves, normal mucosa, and vessels were not stained.
- Results indicate that there is strong binding of 8H9 specifically to the cytoplasm and membrane of the gastric carcinoma tumor cells in the tissues submitted.
- the leiomyoma (ID 001) was not stained.
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Oncology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hospice & Palliative Care (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a method for providing a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof using immunohistochemical methods. Former immunohistochemical methods using paraffin embedded samples in combination with a murine 8H9 antibody, have not provided satisfactory results. The present invention discloses a method that can use both frozen samples as well as paraffin embedded samples. Further, the present invention relates to a kit of parts adapted to carry out the inventive method. Moreover, the invention relates to a method of treating a cancer, comprising measuring or verifying a biomarker of the efficacy, the suitability and/or safety of an antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof is used for treating said cancer.
Description
Immunohistochemical Assessment of B7-H3 Expression
The present invention relates to a method for providing or measuring a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof using immunohistochemical methods. The present invention further relates to an immunohistochemical-method for detecting and optionally quantifying overexpression of B7-H3 for the selection of patients that may benefit from B7-H3 antibody or antigen binding fragment thereof based radioimmunotherapy, immunotherapy or cell therapy. More specifically, the invention relates to a method for providing or measuring a biomarker for assessing the efficacy and/or safety of an antibody or antigen-binding fragment thereof based on B7-H3 expression in the treatment of cancer in an individual, or in the treatment of a specific indication of cancer, such as ovarian cancer. The invention further discloses a method for measuring or qualifying the clinical benefit of a B7-H3 antibody or antigen- binding fragment thereof. Additionally, the invention discloses a method for detecting B7-H3 expression in a tissue sample comprising cancer cells by use of a murine 8H9 antibody. In addition, the invention relates to a kit of parts adapted to carry out the inventive method. Moreover, the invention relates to a method of treating a cancer, comprising providing or measuring a biomarker for measuring and/or verifying the suitability, efficacy and/or safety of an antibody or antigen-binding fragment thereof, wherein said antibody is used in said treatment. A particular utility comes from demonstrated clinical benefit with radio immunotherapies for high expressing B7H3 tumors and the need to determine benefit and select patients with heterogeneous expression (among patients and among tumor tissues, as well as localization in tumor tissue).
Technical Background
Anti-B7-H3 mouse monoclonal antibody 8H9 has been successfully used for radioimmunotherapy for patients with B7-H3 positive tumors. Murine 8H9 antibody targeting B7-H3 protein is broadly reactive with human solid tumors, including embryonal tumors and carcinomas. It has shown favorable tumor uptake for both sarcomas and brain tumors in xenograft models. When conjugated to cobra venom factor, it induces efficient complement-mediated tumor lysis. In its single chain Fv (scFv) format, it targets a potent immunotoxin to sarcoma and glioma in preclinical models. As a chimeric antigen receptor, it
redirects natural killer cells to kill B7-H3 positive tumor cells. In early phase human clinical trials, radiolabeled murine 8H9 antibody was shown to prolong survival among high risk patients with solid tumors suffering from central nervous system (CNS) metastasis. It is a promising target for radioimmunotherapy of leptomeningeal metastases, diffuse intrinsic pontine glioma and peritoneal metastases (Ahmed et al. Humanized Affinity-matured Monoclonal Antibody 8H9 Has Potent Antitumor Activity and Binds to FG Loop of Tumor Antigen B7-H3, The Journal of Biological Chemistry Vol. 290, NO. 50, pp. 30018 - 30029, December 11, 2015).
Human B7-H3, also named CD276, exists in two isoforms, 2lg-B7-H3 and 4lg-B7-H3. It is highly expressed in neuroblastoma, but not necessarily highly expressed in other types of cancer. Murine mAb 8H9 has been shown to bind to a unique epitope on both the 2lg- and 4lg-B7-H3 isoforms of B7-H3, namely the FG-loop that includes the "I RDF " sequence motif (Ahmed et al. Humanized Affinity-matured Monoclonal Antibody 8H9 Has Potent Antitumor Activity and Binds to FG Loop of Tumor Antigen B7-H3, The Journal of Biological Chemistry Vol. 290, NO. 50, pp. 30018 - 30029, December 11, 2015). Given the clinical utility of radioimmunotherapy of B7-H3 positive tumors with murine 8H9 antibody, there is a need for a test to detect which patients will benefit from a B7-H3 directed treatment. To date, known immunohistochemical techniques to detect B7-H3 expression using a murine 8H9 antibody, have not provided satisfactory results on samples such as paraffin embedded samples.
Loos et al. (Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer, BMC Cancer 9, 2009) has examined B7-H3 expression in human patients with pancreatic cancer, using immunohistochemical analyses with a different anti-B7-H3 antibody (MAB 1027, R&D Systems, Minneapolis, MN). Antigen retrieval was achieved by microwave pre-treatment in citrate buffer. Paraffin-embedded pancreatic tissue sections, 3 mm thick, were deparaffinized with xylene and rehydrated through graded alcohol into distilled water. After washing in Tris-buffered saline, endogenous peroxidase activity was quenched by incubating the slides in 3% hydrogen peroxide in methanol. Slides were treated with TBS/3%BSA, to block unspecific activity of secondary antibodies. After overnight incubation at 4 °C with the primary antibody, a mouse anti-human B7-H3 antibody, slides were washed with Tris-buffered saline and 0.05% polyoxyethylene sorbitan
monolaurate, treated with anti-mouse horseradish peroxidase-labeled secondary antibody for 1 hour and counterstained with hematoxylin. Among the examined 68 pancreatic cancer tissues, 60 showed positive B7-H3 protein expression in pancreatic cancer cells. The study demonstrated that tumor cell-associated B7-H3 expression significantly correlates with prolonged postoperative survival. The study suggests that treatment with B7-H3, e.g. by gene transfer, might represent a promising approach to improve the outcome of pancreatic cancer patients.
Qin et al. (B7-H3 is a new cancer-specific endothelial marker in clear cell renal cell carcinoma, OncoTargets and Therapy 2013:6 1667-1673, 2013) has examined B7-H3 expression by immunohistochemistry in paraffin-embedded specimens of clear cell renal cell carcinoma (RCC), using a different anti-B7-H3 antibody (goat anti-human B7-H3 monoclonal antibody from R&D Systems, Minneapolis, MN, USA). Expression patterns of B7-H3 were compared with that of the pan-endothelial cell specific marker CD34. Paired normal renal parenchyma specimens from all patients and four corpus luteum specimens were also analyzed. Formalin-fixed paraffin-embedded tissues were cut into 5 mm sections, deparaffinized and rehydrated in a graded series of ethanols. Antigen retrieval was done heating tissue sections in ethylenediaminetetraacetic acid (EDTA) 1 mmol/L, pH 8 to 121 °C using a Digital Decloing Chamber, cooling to 90 °C, and incubating for 5 minutes, and treating with peroxidase blocking reagent. Sections were incubated with a biotin-labeled polyclonal antibody against B7-H3 and CD34 followed by a horseradish peroxidase-conjugated anti- biotin antibody and visualization by diaminobenzidine staining. Sections were lightly counterstained with hematoxylin. The study detected B7-H3 expression in cancer cells in 19% of clear cell RCC cases, and B7-H3 expression in tumor vasculature was confirmed in 98% of cases. The study indicated that diffuse vascular expression of B7-H3 was associated with multiple adverse clinical and pathological features, such as higher TNM stage and nuclear grade. The study states that the total probability of cancer cell-specific B7-H3 expression in the clear cell RCC population was <20%, suggesting that induction of cytotoxicity or antitumor immunity by agents against B7-H3 expressed in cancers cells may not be sufficient in clinical practice.
Zang et al. (Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas, Mod Pathol. 2010) shows that ovarian borderline tumors and carcinomas
aberrantly express B7-H3 and B7x, and that B7-H3-positive tumor vasculature is associated with high-grade serous histological subtype, increased recurrence and reduced survival. B7- H4 expression in tumor vasculature may be a reflection of tumor aggressiveness and has diagnostic and immunotherapeutic implications in ovarian carcinomas. Zhang et al. (B7-H3 is related to tumor progression in ovarian cancer, Oncology reports 38, 2017) shows that B7-H3 affect ovarian cancer progression through the Jak2/Stat3 pathway, indicating that B7-H3 has the potential to be a useful prognostic marker.
Summary of the invention Paraffin embedded samples from biopsies have been used in immunohistochemical (IHC) methods in combination with goat anti-human B7-H3 antibody as described in Qin et al, and with mouse anti-human B7-H3 antibody as described in Loos et al. Further, frozen samples have been used in immunohistochemical methods. However, paraffin embedded samples in combination with a murine 8H9 antibody, such as an antibody comprising a heavy chain according to SEQ ID No. 1 and a light chain according to SEQ ID No. 2, have not been used successfully in this context. Because frozen tissue samples have limited storage stability, and because paraffin embedded tissue samples in general preserve tissue better, there is a need for a method that works on paraffin embedded tissue samples. The present invention discloses a method that can use both frozen samples and paraffin embedded samples, and which may find general use within the field of IHC.
An IHC technique may be used for each individual patient. Multiple samples may be collected from an individual patient and analyzed for the expression of B7-H3.
In neuroblastoma B7-H3 is highly expressed. B7-H3 is not necessarily highly expressed in other types of cancer. For example, Zang et al. has shown that ovarian borderline tumors and carcinomas aberrantly express B7-H3. Thus, there is a need for a test to distinguish populations or individuals who may benefit from treatment targeting B7-H3.
As the B7-H3 expression on normal tissue is low, the test of the invention may help to determine the highest possible dose for a population or individual who may benefit from treatment targeting B7-H3.
There is a need for an immunohistochemical method, which allows measurement, detection and/or evaluation of a biomarker for the efficacy and/or the safety and/or clinical benefit of specific antibodies, such as B7-H3 antibodies. Further there is a need for a method to detect which patient might benefit from B7-H3 antibody or antigen binding fragment thereof based radioimmunotherapy, immunotherapy or cell therapy. In particular, there is a need for such a method, which may be used on at least one paraffin embedded tissue sample or a sample formerly embedded in paraffin.
In certain embodiments, the anti-B7-H3 antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof disclosed in International Publication No. WO 02/32375, WO 2003/033670, WO 03/075846, WO 2008/116219, WO 2016/033225 and WO 2018/209346, all of which are incorporated by reference in their entireties.
According to an aspect, the invention concerns a method for detecting expression and/or overexpression of an antigen in at least one tissue sample comprising cancer cells, or potentially comprising cancer cells, wherein said sample is selected among a thawed sample, a frozen sample, a fresh sample, a paraffin embedded sample, a formalin embedded sample, and/or a sample which has formerly been embedded in formalin and/or paraffin.
A "thawed sample" is a sample which has been frozen, but is no longer frozen. A "paraffin embedded sample" is a sample that is embedded in paraffin. A "formalin embedded sample" is a sample that is embedded in formalin. A sample may belong to more than one category, e.g. a paraffin embedded sample may also be a thawed sample, and a sample that is embedded in formalin may also be embedded in paraffin.
According to another aspect, the invention concerns a method for detecting expression and/or overexpression of an epitope in a tissue sample comprising cancer cells, or potentially comprising cancer cells, wherein said sample preferably is a sample which has formerly been embedded in formalin and/or paraffin, and wherein said method comprises the steps of: i. Enzyme blocking with an enzyme blocking substance, ii. Protein blocking with a protein blocking substance, iii. Binding with a primary antibody, iv. Incubation with a secondary antibody, and
v. Staining with a staining agent.
According to an embodiment, the invention concerns the method, wherein said epitope is an epitope of B7-H3 antigen. It has been shown by indirect evidence that 8H9 binds to 'IRDF' sequence in B7H3. 4lg-B7H3 (more common isoform) has 2 IRDF motifs (since 4lg B7H3 is a duplicate of two 2lg-B7H3 sequences). The 'IRDF' sequence is located on the FG-loop, a region thought to be critical to the function of B7-family of proteins.
According to another aspect, the invention concerns a method for providing and/or measuring a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of cancer in an individual, wherein the method is performed on a tissue sample comprising cancer cells.
According to another aspect, the invention concerns a method for detecting the clinical benefit of an antibody or antigen-binding fragment thereof in the treatment of cancer in an individual, wherein the method is performed on a tissue sample comprising cancer cells.
According to this aspect the individual is a specific individual or a specific cancer patient, as the method is to be used for each individual patient.
According to another aspect, the invention concerns a method for providing and/or measuring a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of an indication of cancer, wherein the method is performed on tissue samples comprising cancer cells.
According to another aspect, the invention concerns a chimeric antigen receptor (CAR) comprising an antibody or antigen-binding fragment thereof, wherein said chimeric antigen receptor is used according to a method of the invention.
According to another aspect, the invention concerns a CAR-T cell expressing the CAR.
According to another aspect, the invention concerns a population of the CAR-T cells.
According to another aspect, the invention concerns a composition comprising the population of CAR-T cells.
According to another aspect, the invention concerns a method of treating cancer in an individual, wherein the method comprises providing of a biomarker of the efficacy and/or
safety of an antibody or antigen-binding fragment thereof, and wherein said antibody or antigen-binding fragment thereof is used in said treatment.
According to another aspect, the invention concerns a method for treating cancer in an individual, wherein the method comprises use of an antibody or antigen binding fragment thereof, and wherein the indication, such as ovarian cancer, for treatment of said cancer with said antibody or antigen-binding fragment thereof is determined or confirmed by a method of the invention.
According to another aspect, the invention concerns a method for treating cancer in an individual, wherein the method comprises evaluation of the clinical benefit of an antibody or antigen-binding fragment thereof, comprising use of a method of the invention, and wherein the clinical benefit is the clinical benefit for a patient, and wherein said antibody or antigen- binding fragment thereof is used in said treatment.
According to another aspect, the invention concerns a method for treating cancer in an individual, wherein the method comprises providing a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in a tissue sample comprising cancer cells, comprising use of a method of the invention, and wherein said antibody or antigen-binding fragment thereof is used in said treatment.
According to another aspect, the invention concerns a method of diagnosing cancer in an individual, wherein the method comprises providing a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof, and wherein said antibody or antigen-binding fragment thereof is used in said diagnosis.
According to another aspect, the invention concerns a method for diagnosing cancer in an individual, wherein the method comprises use of an antibody or antigen binding fragment thereof, and wherein the diagnosis of said cancer with said antibody or antigen-binding fragment thereof is determined or confirmed by a method of the invention.
According to another aspect, the invention concerns a method for diagnosing cancer in an individual, wherein the method comprises evaluation of the clinical benefit of an antibody or antigen-binding fragment thereof, comprising use of a method of the invention, and wherein the clinical benefit is the clinical benefit for a patient, and wherein said antibody or antigen- binding fragment thereof is used in said diagnosis.
According to another aspect, the invention concerns a method for diagnosing cancer in an individual, wherein the method comprises providing a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in a tissue sample comprising cancer cells, comprising use of a method according to the invention, and wherein said antibody or antigen-binding fragment thereof is used in said diagnosis.
According to another aspect, the invention concerns an antibody or antigen-binding fragment thereof for use in the treatment of cancer, wherein the use comprises providing a biomarker of the efficacy and/or safety of the antibody or antigen-binding fragment thereof.
According to another aspect, the invention concerns an antibody or antigen-binding fragment thereof for use in the treatment of cancer, wherein the treatment of said cancer with said antibody or antigen-binding fragment thereof is determined or confirmed by a method of the invention.
According to another aspect, the invention concerns an antibody or antigen-binding fragment thereof for use in the treatment of cancer, wherein the use comprises evaluation of the clinical benefit of the antibody or antigen-binding fragment thereof, comprising use of a method of the invention, and wherein the clinical benefit is the clinical benefit for a patient.
According to another aspect, the invention concerns an antibody or antigen-binding fragment thereof for use in the treatment of cancer, wherein the use comprises providing a biomarker of the efficacy and/or safety of the antibody or antigen-binding fragment thereof in a tissue sample comprising cancer cells, comprising use of a method of the invention.
According to another aspect, the invention concerns an antibody or antigen-binding fragment thereof for use in a method of diagnosis of cancer, wherein the use comprises providing a biomarker of the efficacy and/or safety of the antibody or antigen-binding fragment thereof.
According to another aspect, the invention concerns an antibody or antigen-binding fragment thereof for use in a method of diagnosis of cancer, and wherein the diagnosis of said cancer with said antibody or antigen-binding fragment thereof is determined or confirmed by a method according to the invention.
According to another aspect, the invention concerns an antibody or antigen-binding fragment thereof for use in a method of diagnosis of cancer, wherein the use comprises evaluation of the clinical benefit of the antibody or antigen-binding fragment thereof, comprising use of a method according to the invention, and wherein the clinical benefit is the clinical benefit for a patient.
According to another aspect, the invention concerns an antibody or antigen-binding fragment thereof for use in a method of diagnosis of cancer, wherein the use comprises providing a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in a tissue sample comprising cancer cells, comprising use of a method of the invention.
According to another aspect, the invention concerns a kit of parts, adapted to carry out a method of the invention.
According to another aspect, the invention concerns a kit of parts, wherein said kit is to be used to determine or qualify a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof is to be used in the treatment of a cancer.
According to another aspect, the invention concerns a kit of parts, wherein said kit is to be used to determine or qualify the clinical benefit a patient has or will have from treatment with an antigen or antigen-binding fragment thereof, and wherein said patient is a cancer patient.
According to another aspect, the invention concerns a kit of parts, wherein said kit is to be used to determine or qualify a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of an indication of cancer.
According to another aspect, the invention concerns a kit of parts, wherein said kit is used in detecting B7-H3 expression in a tissue sample comprising cancer cells, and wherein said sample is selected among a sample that have been embedded in paraffin and a sample have been frozen.
According to another aspect, the invention concerns a method for treating cancer in an individual, wherein the method comprises use of a kit of the invention.
According to another aspect, the invention concerns a method of treating ovarian cancer, comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to the invention.
According to another aspect, the invention concerns a method of treating gastric cancer, comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to the invention.
According to another aspect, the invention concerns an antibody or antigen binding fragment thereof, wherein said antibody or antigen-binding fragment comprises at least one of the sequences selected among a heavy chain variable region CDR1 according to SEQ ID No. 3, a heavy chain variable region CDR2 according to SEQ IN No. 4 or 19, a heavy chain variable region CD3 according to SEQ ID No. 5, a light chain variable region CDR1 according to SEQ ID No. 6, a light chain variable region CDR2 according to SEQ ID No. 7 and a light chain variable region CDR3 according to SEQ ID No. 8.
According to another aspect, the invention concerns an antibody or antigen binding fragment thereof, wherein said antibody or antigen-binding fragment comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2.
According to another aspect, the invention concerns an antibody or antigen binding fragment thereof, wherein said antibody or antigen binding fragment comprises a heavy chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No. 1 and/or a light chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No. 2.
According to another aspect, the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of ovarian cancer.
According to another aspect, the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of gastric cancer.
Detailed Disclosure
According to an embodiment, the invention concerns a method for detecting expression and/or overexpression of an antigen in at least one tissue sample comprising cancer cells, or potentially comprising cancer cells, wherein said sample is selected among a thawed sample, a frozen sample, a fresh sample, a paraffin embedded sample, a formalin embedded sample, and/or a sample which has formerly been embedded in formalin and/or paraffin. A "thawed sample" is a sample which has been frozen, but is no longer frozen. A "paraffin embedded sample" is a sample that is embedded in paraffin. A "formalin embedded sample" is a sample that is embedded in formalin. A sample may belong to more than one category, e.g. a paraffin embedded sample may also be a thawed sample, and a sample that is embedded in formalin may also be embedded in paraffin. According to an embodiment, the invention concerns a method for detecting expression and/or overexpression of an epitope in a tissue sample comprising cancer cells, or potentially comprising cancer cells, wherein said sample preferably is a sample which has formerly been embedded in formalin and/or paraffin, and wherein said method comprises the steps of: i. Enzyme blocking with an enzyme blocking substance, ii. Protein blocking with a protein blocking substance, iii. Binding with a primary antibody, iv. Incubation with a secondary antibody, and v. Staining with a staining agent.
According to an embodiment, the invention concerns the method, wherein said epitope is an epitope of B7-H3 antigen.
It has been shown by indirect evidence that 8H9 binds to 'IRDF' sequence in B7H3. 4lg-B7H3 (more common isoform) has 2 IRDF motifs (since 4lg B7H3 is a duplicate of two 2lg-B7H3
sequences). The 'IRDF' sequence is located on the FG-loop, a region thought to be critical to the function of B7-family of proteins.
According to an embodiment, the invention concerns the method, wherein said primary antibody has a concentration of about 30 mg/ml, and wherein said binding with a primary antibody is carried out at about 4 °C, and/or wherein said binding is carried out for about 4- 24 hours.
According to an embodiment, the invention concerns the method , wherein said method further comprises the following step: i. Antigen retrieval with Tris-EDTA, wherein said Tris-EDTA has a pH value of about 9 and/or a temperature of about 100 °C
According to an embodiment, the invention concerns the method, wherein said antigen retrieval is carried out for about 1-30 minutes, 5-25 minutes, 10-20 minutes or preferably about 15-20 minutes.
According to an embodiment, the invention concerns the method, wherein said antigen retrieval is carried out for about 15 minutes.
According to an embodiment, the invention concerns the method, wherein said antigen retrieval is carried out for about 20 minutes.
According to an embodiment, the invention concerns the method, wherein said method further comprises a step of cooling down said tissue sample at room temperature, wherein said cooling down is performed after said antigen retrieval.
According to an embodiment, the invention concerns the method, wherein said cooling down is performed for about 1-30 minutes, about 10-25 minutes, about 15-20 minutes or preferably about 20 minutes.
According to an embodiment, the invention concerns the method, wherein said tissue sample is from an individual, and wherein said method is used for evaluating whether said individual may benefit from a therapy, wherein said therapy is selected among radioimmunotherapy, immunotherapy or cell therapy.
According to an embodiment, the invention concerns the method, wherein said antigen is a B7-H3 antigen.
B7-H3 is also referred to as CD276.
According to an embodiment, the invention concerns the method, wherein said B7-H3 antigen comprises the sequence of SEQ ID No. 17 and/or 18.
According to an embodiment, the invention concerns the method, wherein said B7-H3 antigen comprises at least one peptide selected among the peptide according to SEQ ID No. 15 and the peptide according to SEQ ID No. 16.
According to an embodiment, the invention concerns the method, wherein said method uses antibodies to detect proteins in cells of said tissue sample.
According to an embodiment, the invention concerns the method, wherein said primary antibody is an anti-B7H3 antibody.
According to an embodiment, the invention concerns the method, wherein an anti-B7-H3 antibody or antigen-binding fragment thereof is used to detect B7-H3 expression.
According to an embodiment, the invention concerns the method, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof comprises at least one of the sequences selected among a heavy chain variable region CDR1 according to SEQ ID No. 3, a heavy chain variable region CDR2 according to SEQ IN No. 4 or 19, a heavy chain variable region CD3 according to SEQ ID No. 5, a light chain variable region CDR1 according to SEQ ID No. 6, a light chain variable region CDR2 according to SEQ ID No. 7 and a light chain variable region CDR3 according to SEQ ID No. 8. The CDRs refers to the complementarity determining region amino acid sequences of an antibody.
According to an embodiment, the invention concerns the method, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2. According to an embodiment, the invention concerns the method, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof comprises a heavy chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to
the sequence set forth in SEQ ID No. 1 and/or a light chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No. 2.
According to an embodiment, the invention concerns the method, wherein said antigen- binding fragment thereof is a single chain variable fragment (scFv).
According to an embodiment, the invention concerns the method, wherein said scFv comprises a portion of the amino acid sequence set forth in SEQ ID No. 9, SEQ ID No. 13 or SEQ ID No. 14.
The 8H9 scFv gene sequence, sense strand is represented in SEQ ID No. 10, the complementary strand is represented in SEQ ID No. 11 and the cDNA is represented in SEQ ID No. 12.
According to an embodiment, the invention concerns the method, wherein said scFv comprises at least one of the amino acid substitutions K13E, R18Q, R45Q, K103E and K107E.
According to an embodiment, the invention concerns the method, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof is a murine 8H9 antibody or an antigen- binding fragment thereof.
According to an embodiment, the invention concerns the method, wherein said antibody or antigen-binding fragment thereof is humanized.
According to an embodiment, the invention concerns the method, wherein said antibody or antigen-binding fragment thereof is a chimeric antibody or an antigen-binding fragment thereof.
According to an embodiment, the invention concerns the method, wherein said antibody or antigen-binding fragment thereof is radiolabeled.
According to an embodiment, the invention concerns the method, wherein said radiolabel is selected among an alpha or beta emitting radiotherapeutic label, a PET label and a SPECT label.
According to an embodiment, the invention concerns the method, wherein said alpha or beta emitting label is selected among 131l, 177Lu, 228Th, 225Ac, 227Ra.
According to an embodiment, the invention concerns the method, wherein said PET label is selected among 124l, 18F, 64Cu, 68Ga, 11C225Ac and 89Zr.
According to an embodiment, the invention concerns the method, wherein said SPECT label is selected among 131l, 177Lu, 99mTc, 64Cu and 99mTc. According to an embodiment, the invention concerns the method, wherein said antibody or antigen-binding fragment thereof this conjugated to a chelator compound.
According to an embodiment, the invention concerns the method, wherein said chelator compound is bound to a radioactive isotope.
According to an embodiment, the invention concerns the method, wherein said radioactive isotope is selected among 124l, 131l and 177Lu or 99mTc, 64Cu (chelated to NOTA) and 89Zr (chelated to DFO).
According to an embodiment, the invention concerns the method, wherein said chelator compound is selected among DOTA, DTPA, NOTA and DFO.
According to an embodiment, the invention concerns the method, wherein said DOTA is a variant of DOTA.
According to an embodiment, the invention concerns the method, wherein said DTPA is a variant of DTPA.
DOTA is also referred to as l,4,7,10-tetraazacyclododecane-l,4,7 10-tetraacetic acid, and has the formula (CH2CH2NCH2C02H)4 DTPA is also referred to with the lUPAC name 2-[bis[2-
[bis(carboxymethyl)amino]ethyl]amino]acetic acid. DTPA has the molecular formula C14H23N3O10.
According to an embodiment, the invention concerns the method, wherein said method may be used on at least a sample that has formerly been embedded in paraffin, as well as a sample which has formerly been frozen.
According to an embodiment, the invention concerns the method, wherein said method further comprises one or more steps of rinse, preferably wherein at least one rinsing step is with water.
According to an embodiment, the invention concerns the method, wherein said water is d2H20. d2H20 is also referred to as doubles-distilled water. According to an embodiment, the invention concerns the method, wherein one or more or all of said steps are separated by wash with at least one wash buffer.
According to an embodiment, the invention concerns the method, wherein one or more of all said steps are separated by at least one wash with at least one wash buffer.
A wash may be defined as adding an adequate volume of a soluble reagent, preferably a buffer, to a tissue sample and removing the soluble reagent, preferably buffer, again. According to an embodiment, the invention concerns the method, wherein one or more of all said steps are separated by at least two washes with at least one wash buffer.
According to an embodiment, the invention concerns the method, wherein one or more of all said steps are separated by at least three washes with at least one wash buffer. According to an embodiment, the invention concerns the method, wherein each wash is carried out for about 1-5 minutes, about 2-4 minutes or preferably about 3 minutes.
According to an embodiment, the invention concerns the method, wherein each wash is carried out for about 0.1 minute - 2 minutes, 0.5 minutes - 1.5 minutes or preferably about 1 minute.
According to an embodiment, the invention concerns the method, wherein said wash buffer is phosphate-buffered saline.
According to an embodiment, the invention concerns the method, wherein said wash buffer is 0.01M phosphate-buffered saline-0,05 % polyoxyethylene sorbitan monolaurate.
According to an embodiment, the invention concerns the method, wherein said enzyme blocking substance is a peroxidase block. According to an embodiment, the invention concerns the method, wherein said peroxidase block is 3% H2O2 in d2H20.
According to an embodiment, the invention concerns the method, wherein said protein blocking substance is casein solution.
According to an embodiment, the invention concerns the method, wherein said enzyme blocking is carried out for 1-60 minutes, 2-55 minutes, 3-50 minutes, 4-45 minutes, 5-40 minutes, 6-35 minutes, 7-30 minutes, 8-25 minutes, 9- 20 minutes 10-15 minutes or about 10 minutes. According to an embodiment, the invention concerns the method, wherein said protein blocking is carried out for 1-60 minutes, 5-55 minutes, 10-50 minutes, 15-45 minutes, 20-40 minutes, 25-35 minutes, or about 30 minutes.
According to an embodiment, the invention concerns the method, wherein said method further comprises a step of incubation with a reagent that amplify the signal of the primary antibody
A reagent that amplify the signal of the primary antibody may be Envision™ Flex + Mouse (linker).
According to an embodiment, the invention concerns the method, wherein said reagent that amplify the signal of the primary antibody is an antibody polymer. According to an embodiment, the invention concerns the method, wherein said incubation with a reagent that amplify the signal of the primary antibody is carried out for about 1 - 30 minutes, about 5 - 25 minutes, about 10 - 20 minutes or preferably about 15 minutes.
According to an embodiment, the invention concerns the method, wherein said reagent that amplify the signal of the primary antibody, enhance the signal at least 2 times, at least 3 times, at least 4 times or preferably at least 5 times. According to an embodiment, the invention concerns the method, wherein said binding with a primary antibody is carried out for 1-48 hours, 4- 44 hours, 8 - 40 hours, 12 - 36 hours, 16 - 32 hours, 20 - 28 hours or about 24 hours.
According to an embodiment, the invention concerns the method, wherein said binding with a primary antibody is carried out for 1 - 240 minutes, 30 - 110 minutes, 60 - 180 minutes,
90 - 150 minutes or about 120 minutes.
According to an embodiment, the invention concerns the method, wherein said binding with a primary antibody is carried out for about 120 minutes or about 24 hours.
According to an embodiment, the invention concerns the method, wherein said binding with a primary antibody is carried out for a period of time allowing at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 99% of the antigens to be bound by the primary antibody. According to an embodiment, the invention concerns the method, wherein said primary antibody is any of the previously mentioned antibodies or an IgGl antibody.
According to an embodiment, the invention concerns the method, wherein said IgGl antibody is a mouse IgGl antibody.
According to an embodiment, the invention concerns the method, wherein said primary antibody has a concentration of 1 - 60 mg/ml, 5 - 55 mg/ml, 10 - 50 mg/ml, 15 - 45 mg/ml, 20 - 40 mg/ml, 25 - 35 mg/ml or about 30 mg/ml.
According to an embodiment, the invention concerns the method, wherein said primary antibody has a concentration of 1-40 mg/ml, 3-35 mg/ml, 10-30 mg/ml, 15-25 mg/ml or preferably about 20 mg/ml. According to an embodiment, the invention concerns the method, wherein said secondary antibody is conjugated to a labeled polymer.
According to an embodiment, the invention concerns the method, wherein said labeled polymer is a horseradish peroxidase labeled polymer.
According to an embodiment, the invention concerns the method, wherein said secondary antibody is an anti-mouse antibody.
According to an embodiment, the invention concerns the method, wherein said incubation with a secondary antibody is carried out for 1 - 120 minutes, 10 - 110 minutes, 20 - 100 minutes, 30- 90 minutes, 40 - 80 minutes, 50 - 70 minutes or about 60 minutes.
According to an embodiment, the invention concerns the method, wherein said incubation with a secondary antibody is carried out for 1 - 240 minutes, 10 - 230 minutes, 20 - 220 minutes, 30 - 210 minutes, 40 - 200 minutes, 50 - 190 minutes, 60 - 180 minutes, 70 - 170 minutes, 80 - 160 minutes, 90 - 150 minutes, 100 - 140 minutes, 110 - 130 minutes or preferably about 120 minutes.
According to an embodiment, the invention concerns the method, wherein said staining agent is a benzidine.
According to an embodiment, the invention concerns the method, wherein said benzidine is 3,3'-diaminobenzidine. According to an embodiment, the invention concerns the method, wherein said incubating with a staining agent is carried out for 1-6 minutes, 1,5 -5,5 minutes, 2-5 minutes, 2,5 - 4,5 minutes, 3 -4 minutes or about 3 minutes.
According to an embodiment, the invention concerns the method, wherein said incubating with a staining agent is carried out for about 1 - 10 minutes, about 1 - 9 minutes, about 2 - 7 minutes or preferably about 3 - 5 minutes.
According to an embodiment, the invention concerns the method, comprising a step, wherein said tissue sample is counterstained with a counterstaining agent.
A counterstaining agent is a substance used to provide contrast so the antibody-stained cells stand out more than it does without said counterstaining agent. According to an embodiment, the invention concerns the method, wherein said counterstaining agent is hematoxylin and optionally eosin.
According to an embodiment, the invention concerns the method, wherein said sample is dehydrated after counterstaining.
According to an embodiment, the invention concerns the method, wherein said tissue sample is coverslipped after dehydration.
According to an embodiment, the invention concerns the method, wherein one or more or all of said steps are separated by at least one wash according to the invention.
According to an embodiment, the invention concerns the method, wherein said steps are performed in the following order:
Blocking of enzymes with an enzyme blocking substance, ii. Blocking of proteins with a protein blocking substance, iii. Binding with a primary antibody, iv. Incubation with a secondary antibody, and
v. Staining with a staining agent.
According to an embodiment, the invention concerns the method, wherein the preparation of said tissue sample comprises one or more or all of the following steps:
1.1 Gross trimming,
1.2 Processing through graded series of alcohols,
1.3 Tissue orientation, and
1.4 Embedding in paraffin.
Wherein said steps are followed by any of the steps according to the invention.
According to an embodiment, the invention concerns the method, wherein the method comprises one or more or all of the following steps:
2.1 Microtome-sectioning of a paraffin embedded sample,
2.2 Slide-mounting,
2.3 Preparing for staining and staining for an antibody, and
2.4 Counterstaining.
According to an embodiment, the invention concerns the method, wherein said paraffin embedded sample is microtome-sectioned to a thickness of 1 to 7 mm thick, 2 to 6 mm thick or about 3 to 5 mm thickness.
According to an embodiment, the invention concerns the method, wherein said tissue sample is slide-mounted.
According to an embodiment, the invention concerns the method, wherein the preparation for staining and staining comprises one or more or all of the following steps:
3.1 Deparaffinization,
3.2 Hydration,
3.3 Antigen retrieval, and
Wherein said steps are followed by blocking, binding, incubation and staining.
According to an embodiment, the invention concerns the method, wherein said sample has formerly been embedded in paraffin.
According to an embodiment, the invention concerns the method, wherein said sample has been or is embedded in paraffin and/or formalin fixed.
According to an embodiment, the invention concerns the method, wherein said sample is deparaffinized. According to an embodiment, the invention concerns the method, wherein said antigen retrieval is carried out by a buffer.
According to an embodiment, the invention concerns the method, wherein said buffer is Tris-EDTA buffer.
EDTA is also referred to as ethylenediaminetetraacetic acid. According to an embodiment, the invention concerns the method, wherein said Tris-EDTA buffer has a pH value of 7 - 10, 7.5 - 9.5, 8 - 9 or about 9.
According to an embodiment, the invention concerns the method, wherein said Tris-EDTA buffer is 60 - 140 °C, 65 - 135 °C, 70 - 130 °C, 75 - 125 °C, 80 - 120 °C, 85 - 115 °C, 90 - 110 °C, 95 - 105 °C or about 100 °C. According to an embodiment, the invention concerns the method, wherein said antigen retrieval is carried out for 1-60 minutes, 2-55 minutes, 3-50 minutes, 4-45 minutes, 5-40 minutes, 6-35 minutes, 7-30 minutes, 8-25 minutes, 9- 20 minutes, 10-15 minutes or about 15 minutes.
According to an embodiment, the invention concerns the method, wherein said steps are performed at 0 - 8 °C, 0.5 - 7.5 °C, 1-7 °C, 1.5 - 6.5 °C, 2-6 °C, 2.5 -5.5 °C, 3 - 5 °C, 3.5 - 4.5 °C or about 4 °C.
According to an embodiment, the invention concerns the method, wherein said steps are performed for 1-48 hours, 4- 44 hours, 8 - 40 hours, 12 - 36 hours, 16 - 32 hours, 20 - 28 hours or about 24 hours. According to an embodiment, the invention concerns the method, wherein the steps are followed by one or more or all of the steps described above.
According to an embodiment, the invention concerns the method, wherein said steps are performed in the following order:
4.1 Deparaffinization
4.2 Hydration
4.3 Antigen retrieval
Followed by blocking, binding, incubation and staining.
According to an embodiment, the invention concerns the method, wherein the steps are performed in the following order: Blocking, binding, incubation, staining.
According to an embodiment, the invention concerns the method, wherein the preparation of said tissue sample comprises one or more or all of the following steps:
A.1 Embedding said tissue sample in optimal cutting temperature medium, A.2 Preparation of a block of said sample,
A.3 Placement of said sample in a sealed plastic bag or other appropriate container, and
A.4 Maintaining said sample below -20 °C,
Wherein said tissue sample is a frozen sample or a thawed sample. According to an embodiment, the invention concerns the method, wherein said tissue sample is a thawed sample.
According to an embodiment, the invention concerns the method, wherein said optimal cutting temperature medium comprises one or more or all of the following: polyvinyl alcohol, polyethylene glycol or non-reactive ingredients. According to an embodiment, the invention concerns the method, wherein said sample is maintained below -30 °C, - 35 °C, -40 °C, -45 °C, -50 °C, -55 °C, -60 °C, -65 °C, -70 °C, -75 °C, - 80 °C, -85 °C, -90 °C, -95 °C or below -100 °C
According to an embodiment, the invention concerns the method, wherein said sample is sectioned to a thickness of 1-10 mm thick, 1.5 - 9.5 mm thick, 2 - 9 mm thick, 2.5 - 8.5 mm thick, 3 - 8 mm thick, 3.5 - 7.5 mm thick, 4 - 7 mm thick, 4.5 - 6.5 mm thick, 5 - 6 mm thick or about 5 mm thicknesses.
According to an embodiment, the invention concerns the method, wherein said sample is sectioned using cryostat.
According to an embodiment, the invention concerns the method, wherein said sample is transferred onto a charged slide.
According to an embodiment, the invention concerns the method, wherein said sample is air-dried. According to an embodiment, the invention concerns the method, wherein said sample is air-dried at room temperature, wherein said room temperature is 10 °C - 40 °C.
According to an embodiment, the invention concerns the method, wherein said room temperature is selected among 10 - 40 °C, 15 - 35 °C, 17 - 33 °C, 18 - 30 °C, 19 - 27 °C or about 20 - 25 °C. According to an embodiment, the invention concerns the method, wherein said sample is air-dried for 1-48 hours, 4- 44 hours, 8 - 40 hours, 12 - 36 hours, 16 - 32 hours, 20 - 28 hours or about 24 hours.
According to an embodiment, the invention concerns the method, wherein the method comprises one or more or all of the following steps: B.l Exposure of a tissue sample to room temperature, wherein said room temperature is 10 - 40 °C.
B.2 Fixation of the sample, wherein said steps are followed by the step previously mentioned.
According to an embodiment, the invention concerns the method, wherein said room temperature is selected among 10 - 40 °C, 15 - 35 °C, 17 - 33 °C, 18 - 30 °C, 19 - 27 °C or about 20 - 25 °C.
According to an embodiment, the invention concerns the method, wherein said exposure to room temperature is carried out for 1 - 60 minutes, 5 - 55 minutes, 10 - 50 minutes, 15 - 45 minutes, 20 - 40 minutes, 25 - 35 minutes or about 30 minutes. According to an embodiment, the invention concerns the method, wherein said sample is fixated in paraformaldehyde.
According to an embodiment, the invention concerns the method, wherein said sample is fixated in acetone.
According to an embodiment, the invention concerns the method, wherein said acetone is - 10 - 20 °C, -5 - 15 °C, 0 - 10 °C, 0.5 - 7 °C, 1 - 6 °C or 2 - 5 °C.
According to an embodiment, the invention concerns the method, wherein said fixation is carried out for 1-20 minutes, 5 - 15 minutes or about 10 minutes.
According to an embodiment, the invention concerns the method, wherein said steps are followed by one or more or all of the steps previously described.
According to an embodiment, the invention concerns the method, wherein said cancer cells are cancer cells from any cancer type that express or may express B7-H3.
According to an embodiment, the invention concerns the method, wherein said cancer cells are selected among carcinoma, sarcoma, lymphoma and leukemia cancer cells.
According to an embodiment, the invention concerns the method, wherein said cancer cells are selected among neuroblastoma, medulloblastoma, glioblastoma, small celled lung cancer, non-small celled carcinoma, a pediatric sarcoma, an adult sarcoma, breast cancer, liver cancer, melanoma, non-small celled lung carcinoma, lung adenocarcinoma or gastrointestinal cancer cells.
According to an embodiment, the invention concerns the method, wherein said cancer cells are ovarian cancer cells.
According to an embodiment, the invention concerns the method, wherein said ovarian cancer cells are adenocarcinoma cells.
According to an embodiment, the invention concerns the method, wherein said adenocarcinoma cells are serous adenocarcinoma cells.
According to an embodiment, the invention concerns the method, wherein said adenocarcinoma cells are serous papillary adenocarcinoma cells.
According to an embodiment, the invention concerns the method, wherein said cancer cells are gastric cancer cells.
According to an embodiment, the invention concerns the method, wherein said gastric cancer cells are gastric carcinoma cells.
According to an embodiment, the invention concerns the method, wherein said gastric carcinoma cells are gastric adenocarcinoma cells.
According to an embodiment, the invention concerns the method, wherein said gastric carcinoma cells are gastric cystadenocarcinoma cells.
According to an embodiment, the invention concerns the method, wherein said cancer cells are neuroblastoma cancer cells. According to an embodiment, the invention concerns the method, wherein said cancer cells are from a metastasis, a high-risk cancer and/or a recurrent cancer.
According to an embodiment, the invention concerns the method, wherein said method comprises the following steps performed on said tissue sample:
Deparaffinization ii. Antigen retrieval with Tris-EDTA buffer, wherein said buffer has a pH value of 9 and a temperature of 100°C, and wherein said antigen retrieval is carried out for about 15 minutes iii. Cool down for about 30 minutes iv. Rinse in d2H20 for about 1 minute v. Blocking enzymes with peroxidase block, wherein said peroxidase block is 3% H O in d2H20, and wherein said exposure is carried out for about 10 minutes vi. Rinsing with 0.01M phosphate-buffered saline for about 1 minute vii. Blocking of proteins with casein solution for about 30 minutes viii. Binding with a primary antibody, wherein said primary antibody comprises a heavy chain sequence according to SEQ ID No. 1, and a light chain sequence according to SEQ ID No. 2 or is an MslgG1 antibody, and wherein said antibody has a concentration of 30ug/ml, and wherein said exposure is to be carried out for about 24 hours, and wherein said exposure is to be carried out in about in 4°C ix. Rinsing with 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for about 2 minutes x. Rinsing with 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for about 2 minutes
xi. Incubating with a secondary antibody conjugated to a horseradish peroxidase, wherein said secondary antibody is an anti-mouse antibody, and wherein said exposure is carried out for about 60 minutes xii. Rinsing with 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for about 2 minutes xiii. Rinsing with 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for about 2 minutes xiv. Staining with 3,3'-diaminobenzidine for about 3 minutes xv. Rinsing with water for about 1 minute xvi. Hematoxylin counter staining.
EDTA is also referred to as ethylenediaminetetraacetic acid. PBS is also referred to as phosphate-buffered saline. CAS-protein block is also referred to as casein solution. DAB is also referred to as 3,3'-diaminobenzidine. d2H20 is also referred to as double-distilled water. According to an embodiment, the invention concerns the method, wherein said method comprises the following steps performed on said tissue sample: i. Remove the frozen sample from -80 °C and keep in room temperature, wherein said room temperature is selected among any of the temperatures described above as room temperatur 110, and wherein said samples are kept in room temperature for about 30 minutes ii. Fixation in 4% paraformaldehyde or acetone for about 10 minutes iii. Rinse in d2H20 for about 1 minute iv. Blocking of enzymes with peroxidase block, wherein said peroxidase block is 3% H2O2 in d2H20, and wherein said exposure is carried out for about 10 minutes v. Rinsing with 0.01M phosphate-buffered saline for about 1 minute vi. Blocking of proteins with casein solution for about 30 minutes
vii. Binding with primary antibody, wherein said primary antibody comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 or is an MslgG1 antibody, and wherein said antibody has a concentration of 30 mg/ml, and wherein said exposure is to be carried out for about 120 minutes. viii. Rinsing with 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for about 2 minutes ix. Rinsing with 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for about 2 minutes x. Incubating with a secondary antibody conjugated to a horseradish peroxidase, wherein said secondary antibody is an anti-mouse antibody, and wherein said exposure is carried out for about 60 minutes xi. Rinsing with 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for about 2 minutes xii. Rinsing with 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for about 2 minutes xiii. Staining with 3,3'-Diaminobenzidine for about 3 minutes xiv. Rinsing with water for about 1 minute xv. Hematoxylin counter staining.
According to an embodiment, the invention concerns the method, wherein said method comprises the following steps performed on said tissue sample: i. Deparaffinization ii. Antigen retrieval with Tris-EDTA buffer, wherein said buffer has a pH value of 9 and wherein said antigen retrieval is carried out for about 20 minutes iii. Cool down for about 20 minutes iv. First rinse in d2H20 for about 1 minute v. Second rinse in d2H20 for about 1 minute vi. First wash with 0.01M phosphate-buffered saline for about 2 minutes
vii. Second wash with 0.01M phosphate-buffered saline for about 2 minutes viii. Third wash with 0.01M phosphate-buffered saline for about 2 minutes ix. Blocking enzymes with peroxidase block, wherein said peroxidase block is 3% H2O2 in d2H20, and wherein said exposure is carried out for about 10 minutes x. First wash with 0.01M phosphate-buffered saline for about 2 minutes xi. Second wash with 0.01M phosphate-buffered saline for about 2 minutes xii. Third wash with 0.01M phosphate-buffered saline for about 2 minutes xiii. Blocking of proteins with casein solution for about 30 minutes xiv. Binding with a primary antibody, wherein said primary antibody comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 or is an MslgG1 antibody, and wherein said antibody has a concentration of 20 mg/ml, and wherein said exposure is to be carried out for about 24 hours, and wherein said exposure is to be carried out in about in 4°C xv. First wash with 0.01M phosphate-buffered saline for about 2 minutes xvi. Second wash with 0.01M phosphate-buffered saline for about 2 minutes xvii. Third wash with 0.01M phosphate-buffered saline for about 2 minutes xviii. Incubation with a reagent that amplify the signal of the primary antibody, wherein said incubation is carried out for about 15 minutes xix. Incubating with a secondary antibody conjugated to a horseradish peroxidase, wherein said secondary antibody is an anti-mouse antibody, and wherein said exposure is carried out for about 60 minutes
XX. First wash with 0.01M phosphate-buffered saline for about 2 minutes xxi. Second wash with 0.01M phosphate-buffered saline for about 2 minutes xxii. Third wash with 0.01M phosphate-buffered saline for about 2 minutes xxiii. Staining with 3,3'-diaminobenzidine for about 3 - 5 minutes xxiv. Rinse with 0.01M phosphate-buffered saline for about 2 minutes
xxv. Hematoxylin counter staining.
Accordng to an embodiment, the invention concerns the method, wherein said tissue sample is an ovarian tissue sample.
According to an embodiment, the invention concerns the method, wherein said method comprises the following steps performed on said tissue sample: i. Deparaffinization ii. Antigen retrieval with Tris-EDTA buffer, wherein said buffer has a pH value of 9 and wherein said antigen retrieval is carried out for about 20 minutes iii. Cool down for about 20 minutes iv. First rinse in d2H20 for about 1 minute v. Second rinse in d2H20 for about 1 minute vi. First wash with 0.01M phosphate-buffered saline for about 2 minutes vii. Second wash with 0.01M phosphate-buffered saline for about 2 minutes viii. Third wash with 0.01M phosphate-buffered saline for about 2 minutes ix. Blocking enzymes with peroxidase block, wherein said peroxidase block is 3% H2O2 in d2H20, and wherein said exposure is carried out for about 10 minutes x. First wash with 0.01M phosphate-buffered saline for about 2 minutes xi. Second wash with 0.01M phosphate-buffered saline for about 2 minutes xii. Third wash with 0.01M phosphate-buffered saline for about 2 minutes xiii. Blocking of proteins with casein solution for about 30 minutes xiv. Binding with a primary antibody, wherein said primary antibody comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 or is an MslgG1 antibody, and wherein said antibody has a concentration of 30 mg/ml, and wherein said exposure is to be carried out for about 24 hours, and wherein said exposure is to be carried out in about in 4°C xv. First wash with 0.01M phosphate-buffered saline for about 2 minutes
xvi. Second wash with 0.01M phosphate-buffered saline for about 2 minutes xvii. Third wash with 0.01M phosphate-buffered saline for about 2 minutes xviii. Incubating with a secondary antibody conjugated to a horseradish peroxidase, wherein said secondary antibody is an anti-mouse antibody, and wherein said exposure is carried out for about 120 minutes xix. First wash with 0.01M phosphate-buffered saline for about 2 minutes xx. Second wash with 0.01M phosphate-buffered saline for about 2 minutes xxi. Third wash with 0.01M phosphate-buffered saline for about 2 minutes xxii. Staining with 3,3'-diaminobenzidine for about 3 - 5 minutes i. Rinsing with water for about 1 minute xxiii. Hematoxylin counter staining.
According to an embodiment, the invention concerns the method, wherein said tissue sample is a gastric tissue sample. According to an embodiment, the invention concerns a method for providing and/or measuring a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of cancer in an individual, wherein the method is performed on a tissue sample comprising cancer cells.
According to an embodiment, the invention concerns a method for detecting the clinical benefit of an antibody or antigen-binding fragment thereof in the treatment of cancer in an individual, wherein the method is performed on a tissue sample comprising cancer cells. According to this embodiment the individual is a specific individual or a specific cancer patient, as the method is to be used for each individual patient.
According to an embodiment, the invention concerns a method for providing and/or measuring a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of an indication of cancer, wherein the method is performed on tissue samples comprising cancer cells.
According to an embodiment, the invention concerns a chimeric antigen receptor (CAR) comprising an antibody or antigen-binding fragment thereof, wherein said chimeric antigen receptor is used according to a method of the invention.
According to an embodiment, the invention concerns a CAR-T cell expressing the chimeric antigen receptor.
According to an embodiment, the invention concerns a population of CAR-T cells.
According to an embodiment, the invention concerns a composition comprising the population of CAR-T cells.
According to an embodiment, the invention concerns a method of treating cancer in an individual, wherein the method comprises use of a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof, and wherein said antibody or antigen- binding fragment thereof is used in said treatment.
According to an embodiment, the invention concerns a method for treating cancer in an individual, wherein the method comprises use of an antibody or antigen binding fragment thereof, and wherein the indication, such as ovarian cancer, for treatment of said cancer with said antibody or antigen-binding fragment thereof is determined or confirmed by a method of the invention.
According to an embodiment, the invention concerns a method for treating cancer in an individual, wherein the method comprises evaluation of the clinical benefit of an antibody or antigen-binding fragment thereof, comprising use of a method of the invention, and wherein the clinical benefit is the clinical benefit for a patient, and wherein said antibody or antigen- binding fragment thereof is used in said treatment.
According to an embodiment, the invention concerns a method for treating cancer in an individual, wherein the method comprises evaluation of a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in a tissue sample comprising cancer cells, comprising use of a method of the invention, and wherein said antibody or antigen-binding fragment thereof is used in said treatment.
According to an embodiment, the invention concerns the method, wherein said tissue sample is a tissue sample from said individual.
According to an embodiment, the invention concerns the method, wherein said method evaluates overexpression of B7-H3.
According to an embodiment, the invention concerns the method, wherein said cancer cells are selected among any of the cancer cells as described above.
According to an embodiment, the invention concerns the method, wherein said antibody or antigen-binding fragment thereof is any of the antibodies or antigen-binding fragments as described above.
According to an embodiment, the invention concerns the method, wherein said cancer is any cancer type that express or may express B7-H3.
According to an embodiment, the invention concerns the method, wherein said cancer is selected among a carcinoma, a sarcoma, a lymphoma and a leukemia.
According to an embodiment, the invention concerns the method, wherein said cancer is selected among neuroblastoma, medulloblastoma, glioblastoma, small celled lung cancer, non-small celled carcinoma, a pediatric sarcoma, an adult sarcoma, breast cancer, liver cancer, melanoma, non-small celled lung carcinoma, lung adenocarcinoma or a gastrointestinal cancer
According to an embodiment, the invention concerns the method, wherein said cancer is an ovarian cancer. According to an embodiment, the invention concerns the method, wherein said ovarian cancer is an adenocarcinoma.
According to an embodiment, the invention concerns the method, wherein said adenocarcinoma is a serous adenocarcinoma. According to an embodiment, the invention concerns the method, wherein said adenocarcinoma is a serous papillary adenocarcinoma.
According to an embodiment, the invention concerns the method, wherein said cancer is a gastric cancer.
According to an embodiment, the invention concerns the method, wherein said gastric cancer is a carcinoma.
According to an embodiment, the invention concerns the method, wherein said carcinoma is an adenocarcinoma.
According to an embodiment, the invention concerns the method, wherein said carcinoma is a cystadenocarcinoma.
According to an embodiment, the invention concerns the method, wherein said cancer is neuroblastoma. According to an embodiment, the invention concerns the method, wherein said cancer is a metastasis, a high-risk cancer and/or a recurrent cancer.
According to an embodiment, the invention concerns the method, wherein said treatment is combined with chemotherapy and/or surgery.
According to an embodiment, the invention concerns a kit of parts, adapted to carry out a method of the invention.
According to an embodiment, the invention concerns a kit of parts, wherein said kit is to be used to determine or qualify a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof is to be used in the treatment of a cancer.
According to an embodiment, the invention concerns a kit of parts, wherein said kit is to be used to determine or qualify the clinical benefit a patient has or will have from treatment with an antigen or antigen-binding fragment thereof, and wherein said patient is a cancer patient.
According to an embodiment, the invention concerns a kit of parts, wherein said kit is to be used to determine or qualify a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of an indication of cancer.
According to an embodiment, the invention concerns a kit of parts, wherein said kit is used in detecting B7-H3 expression in a tissue sample comprising cancer cells, and wherein said sample is selected among a sample that have been embedded in paraffin and a sample have been frozen.
According to an embodiment, the invention concerns the kit, comprising an anti-B7-H3 antibody or antigen-binding fragment thereof used to detect B7-H3 expression. According to an embodiment, the invention concerns the kit, wherein said kit comprises a primary antibody.
According to an embodiment, the invention concerns the kit, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof, or said primary antibody, is any of the antibodies or antigen-binding fragments thereof as described above.
According to an embodiment, the invention concerns the kit, wherein said kit uses antibodies to detect proteins in cells of said tissue sample.
According to an embodiment, the invention concerns the kit, wherein said kit comprises a secondary antibody. According to an embodiment, the invention concerns the kit, wherein said secondary antibody is an anti-mouse antibody.
According to an embodiment, the invention concerns the kit, wherein said secondary antibody is conjugated to an enzyme.
According to an embodiment, the invention concerns the kit, wherein said enzyme is a peroxidase.
According to an embodiment, the invention concerns the kit, wherein said peroxidase is Horseradish peroxidase.
According to an embodiment, the invention concerns the kit, wherein said kit comprises an enzyme block. According to an embodiment, the invention concerns the kit, wherein said enzyme block is a peroxidase block.
According to an embodiment, the invention concerns the kit, where said peroxidase block is 3% H2O2 in d2H20.
According to an embodiment, the invention concerns the kit, wherein said kit comprises a protein block.
According to an embodiment, the invention concerns the kit, wherein said protein block is casein solution.
According to an embodiment, the invention concerns the kit, wherein said kit comprises a substance for chromogenic staining. According to an embodiment, the invention concerns the kit, wherein said substance for chromogenic staining is a benzidine.
According to an embodiment, the invention concerns the kit, wherein said benzidine is 3,3'- Diaminobenzidine.
According to an embodiment, the invention concerns the kit, wherein said substance for chromogenic staining is a counterstaining agent.
According to an embodiment, the invention concerns the kit, wherein said counterstaining agent is hematoxylin and optionally eosin. According to an embodiment, the invention concerns the kit, wherein said kit further comprises a reagent that amplify the signal of the primary antibody.
According to an embodiment, the invention concerns the kit, wherein said reagent that amplify the signal of the primary antibody is an antibody polymer.
According to an embodiment, the invention concerns the kit, wherein said kit comprises a substance used to wash.
According to an embodiment, the invention concerns the kit, wherein said substance used to wash is a wash buffer.
According to an embodiment, the invention concerns the kit, wherein said wash buffer is phosphate-buffered saline. According to an embodiment, the invention concerns the kit, wherein said wash buffer is 0,01M phosphate-buffered saline-0,05 % polyoxyethylene sorbitan monolaurate.
According to an embodiment, the invention concerns the kit, wherein the kit further comprises water.
According to an embodiment, the invention concerns the kit, wherein said water is d2H20. According to an embodiment, the invention concerns the kit, wherein said cancer cells are selected among any of the cancer cells as described above.
According to an embodiment, the invention concerns the kit, wherein said cancer is selected among any of the cancers as described above.
According to an embodiment, the invention concerns a method for treating cancer in an individual, wherein the method comprises use of a kit according to the invention.
According to an embodiment, the invention concerns the method, wherein said cancer is selected among any of the cancers as described above.
According to an embodiment, the invention concerns the method, wherein said cancer is a gastric cancer.
According to an embodiment, the invention concerns the method, wherein said gastric cancer is a carcinoma.
According to an embodiment, the invention concerns the method, wherein said carcinoma is an adenocarcinoma.
According to an embodiment, the invention concerns the method, wherein said carcinoma is a cystadenocarcinoma.
According to an embodiment, the invention concerns the method, wherein said gastric cancer is selected from the group consisting of adenocarcinoma, lymphoma, gastrointestinal stromal tumor (GIST), carcinoid tumor, and other cancers.
According to an embodiment, the invention concerns the method, wherein said cancer is an ovarian cancer.
According to an embodiment, the invention concerns the method, wherein said ovarian cancer is an adenocarcinoma.
According to an embodiment, the invention concerns the method, wherein said adenocarcinoma is a serous adenocarcinoma.
According to an embodiment, the invention concerns the method, wherein said adenocarcinoma is a serous papillary adenocarcinoma.
According to an embodiment, the invention concerns the method, wherein said ovarian cancer is selected from the group consisting of epithelial carcinoma, serous carcinoma, primary peritoneal carcinoma, clear-cell carcinoma, clear-cell adenocarcinoma, endometrioid, malignant mixed mijllerian tumor (carcinosarcoma), mucinous tumors, mucinous adenocarcinoma, pseudomyxoma peritonei, undifferentiated epithelial cancers, malignant Brenner tumor, transitional cell carcinoma, sex cord-stromal tumor, granulosa cell tumor, adult granulosa cell tumor, juvenile granulosa cell tumor, sertoli-Leydig cell tumor, sclerosing stromal tumors, germ cell tumor, dysgerminoma, choriocarcinoma, immature (solid) teratoma, mature teratoma (dermoid cyst), yolk sac tumor/endodermal sinus tumor,
embryonal carcinoma, polyembryoma, squamous cell carcinoma, mixed tumors, secondary ovarian cancer, and borderline tumors.
According to an embodiment, the invention concerns the method, wherein said cancer is a neuroblastoma. According to an embodiment, the invention concerns the method, wherein the distribution of cells is selected from the group consisting of diffuse and multifocal, preferably as determined by the methods of Example 2.
According to an embodiment, the invention concerns the method, wherein the incidence is 1, preferably 2, more preferred 3, preferably 4, preferably as determined by the methods of Example 2.
According to an embodiment, the invention concerns the method, wherein the incidence is 2-4, preferably 3-4, preferably as determined by the methods of Example 2.
According to an embodiment, the invention concerns the method, wherein the staining intensity is 1, preferably 2, more preferred 3, preferably 4, preferably as determined by the methods of Example 2.
According to an embodiment, the invention concerns the method, wherein the staining intensity is 2-4, preferably 3-4, preferably as determined by the methods of Example 2.
According to an embodiment, the invention concerns the method, wherein the staining is granular, preferably as determined by the methods of Example 2. According to an embodiment, the invention concerns the method, wherein the staining is located on the membrane or cytoplasmic, preferably as determined by the methods of Example 2.
According to an embodiment, the invention concerns the method, wherein the staining is located on the membrane, preferably as determined by the methods of Example 2. According to an embodiment, the invention concerns the method, wherein the subcellular location is selected from the group consisting of Granular (G), Membrane (M), Cytoplasmic (C), Extracellular (E), Vasculature (V), Intranuclear (In), Apical (A) and Basal (B), preferably as determined by the methods of Example 3.
According to an embodiment, the invention concerns the method, wherein the amount of positive tumor cells in percentage is selected from the group consisting of up to 29%, 30- 49%, 50-74%, and 75-100%, preferably as determined by the methods of Example 3.
According to an embodiment, the invention concerns the method, wherein the amount of positive tumor cells in percentage is above 10%, above 20%, preferably above 30%, preferably as determined by the methods of Example 3.
According to an embodiment, the invention concerns the method, wherein the incidence of positive tumor cells is selected from the group consisting of normal, minimal, mild, moderate and severe, preferably as determined by the methods of Example 3.
According to an embodiment, the invention concerns the method, wherein the intensity of positive staining at cellular level is selected from the group consisting of equivocal, mild (weak), moderate, strong and intense, preferably as determined by the methods of Example 3.
According to an embodiment, the invention concerns the method, wherein the incidence of staining represents a mild to severe distribution of positive tumor cells, preferably as determined by the methods of Example 3.
According to an embodiment, the invention concerns an antibody or antigen binding fragment thereof, wherein said antibody or antigen-binding fragment comprises at least one of the sequences selected among a heavy chain variable region CDR1 according to SEQ ID No. 3, a heavy chain variable region CDR2 according to SEQ IN No. 4 or 19, a heavy chain variable region CD3 according to SEQ ID No. 5, a light chain variable region CDR1 according to SEQ ID No. 6, a light chain variable region CDR2 according to SEQ ID No. 7 and a light chain variable region CDR3 according to SEQ ID No. 8.
According to an embodiment, the invention concerns an antibody or antigen binding fragment thereof, wherein said antibody or antigen-binding fragment comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2.
According to an embodiment, the invention concerns an antibody or antigen binding fragment thereof, wherein said antibody or antigen binding fragment comprises a heavy chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %,
about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No. 1 and/or a light chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No. 2.
According to an embodiment, the invention concerns an antibody or antigen binding fragment according to the invention, wherein said antigen-binding fragment thereof is a single chain variable fragment (scFv).
According to an embodiment, the invention concerns an antibody or antigen binding fragment according to the invention, wherein said scFv comprises a portion of the amino acid sequence set forth in SEQ ID No. 9, SEQ ID No. 13 or SEQ ID No. 14.
According to an embodiment, the invention concerns an antibody or antigen binding fragment according to the invention, wherein said scFv comprises at least one of the amino acid substitutions K13E, R18Q, R45Q, K103E and K107E.
According to an embodiment, the invention concerns an antibody or antigen binding fragment according to the invention, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof is a murine 8H9 antibody or an antigen binding fragment thereof. According to an embodiment, the invention concerns an antibody or antigen binding fragment according to the invention, wherein said antibody or antigen-binding fragment thereof is humanized.
According to an embodiment, the invention concerns an antibody or antigen binding fragment according to the invention, wherein said antibody or antigen-binding fragment thereof is a chimeric antibody or an antigen-binding fragment thereof.
According to an embodiment, the invention concerns an antibody or antigen binding fragment according to the invention, wherein said antibody or antigen-binding fragment thereof is radiolabeled.
According to an embodiment, the invention concerns an antibody or antigen binding fragment according to the invention, wherein said radiolabel is selected among a PET label and a SPECT label.
According to an embodiment, the invention concerns an antibody or antigen binding fragment according to the invention, wherein said PET label is selected among 124l, 225Ac and 89Zr.
According to an embodiment, the invention concerns an antibody or antigen binding fragment according to the invention, wherein said SPECT label is selected among 131l, 177Lu, 99mTc, 64Cu and 89Zr.
According to an embodiment, the invention concerns an antibody or antigen binding fragment according to the invention, wherein said antibody or antigen-binding fragment thereof is conjugated to a chelator compound.
According to an embodiment, the invention concerns an antibody or antigen binding fragment according to the invention, wherein said chelator compound is bound to a radioactive isotope.
According to an embodiment, the invention concerns an antibody or antigen binding fragment according to the invention, wherein said radioactive isotope is selected among124l, 131l and 177Lu or 99mTc, 64Cu (chelated to NOTA) and 89Zr (chelated to DFO).
According to an embodiment, the invention concerns an antibody or antigen binding fragment according to the invention, wherein said chelator compound is selected among DOTA, DTPA, NOTA and DFO.
According to an embodiment, the invention concerns an antibody or antigen binding fragment according to the invention, wherein said DOTA is a variant of DOTA.
According to an embodiment, the invention concerns an antibody or antigen binding fragment according to the invention, wherein said DTPA is a variant of DTPA.
According to an embodiment, the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of ovarian cancer, wherein said ovarian cancer is an adenocarcinoma.
According to an embodiment, the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of ovarian cancer, wherein said adenocarcinoma is a serous adenocarcinoma.
According to an embodiment, the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of ovarian cancer, wherein said adenocarcinoma is a serous papillary adenocarcinoma.
According to an embodiment, the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of ovarian cancer, wherein said ovarian cancer is selected from the group consisting of epithelial carcinoma, serous carcinoma, primary peritoneal carcinoma, clear-cell carcinoma, clear-cell adenocarcinoma, endometrioid, malignant mixed müllerian tumor (carcinosarcoma), mucinous tumors, mucinous adenocarcinoma, pseudomyxoma peritonei, undifferentiated epithelial cancers, malignant Brenner tumor, transitional cell carcinoma, sex cord-stromal tumor, granulosa cell tumor, adult granulosa cell tumor, juvenile granulosa cell tumor, sertoli-Leydig cell tumor, sclerosing stromal tumors, germ cell tumor, dysgerminoma, choriocarcinoma, immature (solid) teratoma, mature teratoma (dermoid cyst), yolk sac tumor/endodermal sinus tumor, embryonal carcinoma, polyembryoma, squamous cell carcinoma, mixed tumors, secondary ovarian cancer, and borderline tumors.
According to an embodiment, the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of gastric cancer, wherein said gastric cancer is a carcinoma.
According to an embodiment, the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of gastric cancer, wherein said carcinoma is an adenocarcinoma.
According to an embodiment, the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of gastric cancer, wherein said carcinoma is a cystadenocarcinoma.
According to an embodiment, the invention concerns an antibody or antigen-binding fragment thereof according to the invention for use in the treatment of gastric cancer,
wherein said gastric cancer is selected from the group consisting of adenocarcinoma, lymphoma, gastrointestinal stromal tumor (GIST), carcinoid tumor, and other cancers.
Use of an antibody or antigen-binding fragment thereof, according to the invention, for the manufacture of a medicament for treatment of an indication according to the invention.
Figures
Fig. 1 provides a flow chart of a method for detection related to an embodiment of the invention.
Fig. 2A and 2B show the immunohistochemistry of FFPE sections of non-small cell lung carcinoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2. The slides were stained with said antibody, at a concentration of 30 mg/mL, overnight at 4 °C. There is severe granular cell surface and intracellular staining of tumor cells. Fig. 2A shows a field of view with 20 times magnification and Fig. 2B shows a field of view with 40 times magnification. The histopathologic score was 4/4.
Fig. 3A and 3B show the immunohistochemistry of FFPE sections of non-small cell lung carcinoma with isotype control mouse IgG1 antibody ( MslgG1). The slides were stained with 30 mg/mL MslgG1 overnight at 4 °C. There is no staining of tumor cells. Fig. 3A shows a field of view with 20 times magnification and Fig. 2B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
Fig. 4A and 4B show the immunohistochemistry of FFPE sections of neuroblastoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2. The slides were stained with said antibody, at a concentration of 30 mg/mL, overnight at 4 °C. There is strong to intense granular cell surface and some intracellular staining of tumor cells. Fig. 4A shows a field of view with 20 times magnification and Fig. 4B shows a field of view with 40 times magnification. The histopathologic score was 4/3.
Fig. 5A and 5B show the immunohistochemistry of FFPE sections of neuroblastoma with MslgG1. The slides were stained with 30 mg/mL MslgG1 overnight at 4 °C. There is no staining. Fig. 5A shows a field of view with 20 times magnification and Fig. 5B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
Fig. 6A and 6B show the immunohistochemistry of FFPE sections of medulloblastoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2. The slides were stained with said antibody, at a concentration of 30 μg/mL, overnight at 4 °C. There is intense granular cell surface and intracellular staining of tumor cells. Fig. 6A shows a field of view with 20 times magnification and Fig. 6B shows a field of view with 40 times magnification. The histopathologic score was 4/4.
Fig. 7A and 7B show the immunohistochemistry of FFPE sections of medulloblastoma with MslgG1. The slides were stained with 30 μg/mL MslgG1 overnight at 4 °C. There is no staining. Fig. 7A shows a field of view with 20 times magnification and Fig. 7B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
Fig. 8A and 8B show the immunohistochemistry of FFPE sections of glioblastoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2. The slides were stained with said antibody, at a concentration of 30 μg/mL, overnight at 4 °C. There is intense granular cell surface and intracellular staining of tumor cells. Fig. 8A shows a field of view with 20 times magnification and Fig. 8B shows a field of view with 40 times magnification. The histopathologic score was 4/4.
Fig. 9A and 9B show the immunohistochemistry of FFPE sections of glioblastoma with MslgG1. The slides were stained with 30 μg/mL MslgG1 overnight at 4 °C. There is no staining. Fig. 9A shows a field of view with 20 times magnification and Fig. 9B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
Fig. 10A and 10B show the immunohistochemistry of FFPE sections of ovarian cancer with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2. The slides were stained with said antibody, at a concentration of 30 μg/mL, overnight at 4 °C. There is moderate multifocal granular cell surface and intracellular and vascular staining of a subset of tumor cells. Fig. 10A shows a field of view with 20 times magnification and Fig. 10B shows a field of view with 40 times magnification. The histopathologic score was 2/2.
Fig. 11A and 11B show the immunohistochemistry of FFPE sections of ovarian cancer with MslgG1. The slides were stained with 30 μg/mL MslgG1 overnight at 4 °C. There is no
staining. Fig. 11A shows a field of view with 20 times magnification and Fig. 11B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
Fig. 12A and 12B show the immunohistochemistry of FFPE sections of sarcoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2. The slides were stained with said antibody, at a concentration of 30 μg/mL, overnight at 4 °C. There is moderate multifocal granular cell surface and intracellular staining of a subset of tumor cells. Fig. 12A shows a field of view with 20 times magnification and Fig. 12B shows a field of view with 40 times magnification. The histopathologic score was 2/2.
Fig. 13A and 13B show the immunohistochemistry of FFPE sections of sarcoma with MslgG1. The slides were stained with 30 μg/mL MslgG1 overnight at 4 °C. There is no staining. Fig.
13A shows a field of view with 20 times magnification and Fig. 13B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
Fig. 14A and 14B show the immunohistochemistry of FFPE sections of melanoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2. The slides were stained with said antibody, at a concentration of 30 μg/mL, overnight at 4 °C. There is severe granular cell surface and intracellular staining of tumor cells. Fig. 14A shows a field of view with 20 times magnification and Fig. 14B shows a field of view with 40 times magnification. The histopathologic score was 4/4.
Fig. 15A and 15B show the immunohistochemistry of FFPE sections of melanoma with MslgG1. The slides were stained with 30 μg/mL MslgG1 overnight at 4 °C. There is no staining of tumor cells. It shows moderate staining of macrophages. Fig. 15A shows a field of view with 20 times magnification and Fig. 15B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
Fig. 16A and 16B show the immunohistochemistry of FFPE sections of liver cancer with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2. The slides were stained with said antibody, at a concentration of 30 μg/mL, overnight at 4 °C. There is strong granular cell surface and intracellular staining of tumor cells. Fig. 16A shows a field of view with 20 times magnification and Fig. 16B shows a field of view with 40 times magnification. The histopathologic score was 3/3.
Fig. 17A and 17B show the immunohistochemistry of FFPE sections of normal liver with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2. The slides were stained with said antibody, at a concentration of 30 μg/mL, overnight at 4 °C. There is no staining. Fig. 17A shows a field of view with 20 times magnification and Fig. 17B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
Fig. 18A shows the immunohistochemistry of frozen sections of non-small cell lung carcinoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2. The slides were stained with said antibody, at a concentration of 2 μg/mL. There is a moderate number of strong positive stained tumor cells. Fig. 18A shows a field of view with 40 times magnification. The histopathologic score was 2/3.
Fig. 18B shows the immunohistochemistry of frozen sections of non-small cell lung carcinoma with MslgG1. The slides were stained with 2 μg/mL MslgG1. There is no staining. Fig. 18B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
Fig. 19A shows the immunohistochemistry of frozen sections of lung adenocarcinoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2. The slides were stained with said antibody, at a concentration of 2 μg/mL. There is a strong positive stained subset of tumor cells. Fig. 19A shows a field of view with 40 times magnification. The histopathologic score was 3/3.
Fig. 19B shows the immunohistochemistry of frozen sections of lung adenocarcinoma with MslgG1. The slides were stained with 2 μg/mL MslgG1. There is no staining. Fig. 19B shows a field of view with 40 times magnification. The histopathologic score was 0/0. Fig. 20A shows the immunohistochemistry of frozen sections of melanoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2. The slides were stained with said antibody, at a concentration of 2 μg/mL. There are strong multifocal stained tumor cells. Fig. 20A shows a field of view with 40 times magnification. The histopathologic score was 3/3.
Fig. 20B shows the immunohistochemistry of frozen sections of melanoma with MslgG1. The slides were stained with 2 mg/mL MslgG1. There is no staining. Fig. 20B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
Fig. 21A shows the immunohistochemistry of frozen sections with Sarcoma with an antibody comprising a heavy chain according to SEQ ID no. 1, and a light chain according to SEQ ID no. 2. The slides were stained with said antibody, at a concentration of 2 mg/mL. There is strong to intense stained tumor cells. Fig. 21A shows a field of view with 40 times magnification. The histopathologic score was 4/4.
Fig. 21B shows the immunohistochemistry of frozen sections of Sarcoma with MslgG1. The slides were stained with 2 mg/mL MslgG1. There is no staining. Fig. 21B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
Fig. 22A shows the immunohistochemistry of frozen sections of ovarian cancer with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2. The slides were stained with said antibody, at a concentration of 2 mg/mL. There is strong to intense multifocal stained tumor cells. Fig. 22A shows a field of view with 40 times magnification. The histopathologic score was 4/3.
Fig. 22B shows the immunohistochemistry of frozen sections of ovarian cancer with MslgG1. The slides were stained with 2 mg/mL MslgG1. There is no staining. Fig. 22B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
Fig. 23A shows the immunohistochemistry of frozen sections of glioblastoma with an antibody comprising a heavy chain according to SEQ ID No. 1, and a light chain according to SEQ ID No. 2. The slides were stained with said antibody, at a concentration of 2 mg/mL. There is a strong stained subset of tumor cells. Fig. 23A shows a field of view with 40 times magnification. The histopathologic score was 3/3.
Fig. 23B shows the immunohistochemistry of frozen sections of glioblastoma with MslgG1. The slides were stained with 2 mg/mL MslgG1. There is no staining. Fig. 23B shows a field of view with 40 times magnification. The histopathologic score was 0/0.
Fig. 24A and 24B show the immunohistochemistry of FFPE sections of non-small cell lung carcinoma with commercially available anti-B7-H3 antibody. The slides were stained with 2 mg/mL B7-H3 antibody overnight at 4 °C. There is severe granular cell surface and
intracellular staining of tumor cells. Fig. 24A shows a field of view with 20 times magnification and Fig. 24B shows a field of view with 40 times magnification. The histopathologic score was 4/4.
Fig. 25A and 25B show the immunohistochemistry of FFPE sections of neuroblastoma with commercially available anti-B7-H3 antibody. The slides were stained with 2 mg/mL B7-H3 antibody overnight at 4 °C. There is severe granular cell surface staining of tumor cells. Fig. 25A shows a field of view with 20 times magnification and Fig. 25B shows a field of view with 40 times magnification. The histopathologic score was 4/3.
Fig. 26A and 26B show the immunohistochemistry of FFPE sections of medulloblastoma with commercially available anti-B7-H3 antibody. The slides were stained with 2 mg/mL B7-H3 antibody overnight at 4 °C. There is strong granular cell surface and intracellular staining of tumor cells. Fig. 26A shows a field of view with 20 times magnification and Fig. 26B shows a field of view with 40 times magnification. The histopathologic score was 3/4.
Fig. 27A and 27B show the immunohistochemistry of FFPE sections of glioblastoma with commercially available a nti-B7-H3 antibody. The slides were stained with 2 mg/mL B7-H3 antibody overnight at 4 °C. There is strong granular cell surface and intracellular staining of tumor cells. Fig. 27A shows a field of view with 20 times magnification and Fig. 27B shows a field of view with 40 times magnification. The histopathologic score was 4/4.
Fig. 28A and 28B show the immunohistochemistry of FFPE sections of ovarian cancer with commercially available anti-B7-H3 antibody. The slides were stained with 2 mg/mL B7-H3 antibody overnight at 4 °C. There is moderate granular cell surface, intracellular and vascular staining of tumor cells. Fig. 28A shows a field of view with 20 times magnification and Fig. 28B shows a field of view with 40 times magnification. The histopathologic score was 3/3.
Fig. 29A and 29B show the hematoxylin and eosin counter-staining of FFPE section of non- small cell lung carcinoma. Well preserved tumor tissue is shown. Fig. 29A shows a field of view with 20 times magnification and Fig. 29B shows a field of view with 40 times magnification.
Fig. 30A and 30B show the hematoxylin and eosin counter-staining of FFPE section of neuroblastoma. Well preserved tumor tissue is shown. Fig. 30A shows a field of view with 20 times magnification and Fig. 30B shows a field of view with 40 times magnification.
Fig. 31A and 31B show the hematoxylin and eosin counter-staining of FFPE section of medulloblastoma. Well preserved tumor tissue is shown. Fig. 31A shows a field of view with 20 times magnification and Fig. 31B shows a field of view with 40 times magnification.
Fig. 32A and 32B show the hematoxylin and eosin counter-staining of FFPE section of ovarian cancer. Well preserved tumor tissue is shown. Fig. 32A shows a field of view with 20 times magnification and Fig. 32B shows a field of view with 40 times magnification.
Fig. 33A and 33B show the hematoxylin and eosin counter-staining of FFPE section of sarcoma. Well preserved tumor tissue is shown. Fig. 33A shows a field of view with 20 times magnification and Fig. 33B shows a field of view with 40 times magnification. Fig. 34A and 34B show the hematoxylin and eosin counter-staining of FFPE section of sarcoma. Well preserved tumor tissue is shown. Fig. 34A shows a field of view with 20 times magnification and Fig. 34B shows a field of view with 40 times magnification.
Fig. 35A and 35B show the hematoxylin and eosin counter-staining of FFPE section of glioblastoma. Well preserved tumor tissue is shown. Fig. 35A shows a field of view with 20 times magnification and Fig. 35B shows a field of view with 40 times magnification.
Fig. 36-40 shows IHC staining on 25 TMA Ovarian Cancer Cases. Donor ID as in table 7.
Fig. 41 shows H&E and IHC staining of patient's gastric tissue (ID: 001, Spindle cell tumor, possible leiomyoma, benign). H&E image shows a large mass in the muscularis layer (A, black star). The tumor consists of spindle cells which showed no immunoreactivity with 8H9 antibody (B and D) or MslgG (C and E).
Fig. 42 shows IHC staining of patient's gastric tissue (ID: 001-2, Gastric Adenocarcinoma). At 30 mg/ml, 8H9 antibody is expressed on the membrane of 80% of tumor cells with grade 3 staining intensity (A). Capillaries are often filled with neutrophils and lymphocytes as well as tumor cells (B, C). At 20 mg/ml, membrane staining with grade 2-3 staining intensity is observed in about 50% of tumor cells (D). The expression of 8H9 antibody is detected on membrane of most tumor cells (E, F). No immunoreactivity is found with MslgG control isotype (Data not shown).
Fig. 43A shows H&E and IHC staining of patient's gastric tissue (ID 002; gastric adenocarcinoma). H&E image shows a protruding proliferative mass in the mucosa (A, doted
box). The tumor consists of signet ring cells (B, yellow arrows). The immunoreactivity with 8H9 antibody was detected in the membrane and cytoplasm of neoplastic epithelium (B and D). No immunostaining was found in sections treated with MslgG (C and E). Fig. 43B shows at 20 mg/ml, there is occasional weak granular membrane staining on less than 10% of the tumor cells (F and G, red arrow). Vascular endothelial staining was observed in tumor areas (H).
Fig. 44A shows H&E and IHC staining of patient's gastric tissue (ID: 003, Gastric Carcinoma). H&E image shows a nodule of tumor with no gastric structure (A, doted circle). The immunoreactivity with 8H9 antibody was detected in about half of all neoplastic epithelium (B) with a diffuse and cytoplasmic pattern (D, yellow arrows). No immunostaining was found in sections treated with MslgG (C and E). Fig. 44B shows at 20 mg/ml, there is little to no positive membrane staining (F and G). Vascular endothelial staining was present in tumor areas (H).
Fig. 45A shows H&E and IHC staining of patient's gastric tissue (ID: 004, Gastric Adencarcinoma). H&E image shows a polypoid proliferative mass in the mucosa and submucosa including numerous small lymphoid nodules (A, starts represents lymphoid nodules).The immunoreactivity with 8H9 antibody was detected in nearly all neoplastic epithelium with a diffuse and cytoplasmic pattern (B and D). No immunostaining was found in sections treated with MslgG (C and E). Fig. 45 B shows at 20 mg/ml, there is moderate granular membrane staining on about 20% of the tumor cells (F and G, red arrows). Vascular endothelial staining was present in tumor areas (H).
Fig. 46A shows H&E and IHC staining of patient's gastric tissue (ID: 005, Gastric Adenocarcinoma). H&E image shows a large diffuse mass in the muscularis and serosa with severe inflammation, congestion, and hemorrhage (A).The immunoreactivity with 8H9 antibody was detected in nearly all neoplastic epithelium with a diffuse and cytoplasmic pattern (B and D). No immunostaining was found in sections treated with MslgG (C and E). Fig. 46B shows At 20 mg/ml, there is moderate granular membrane staining on about 30% of the tumor cells in areas of less differentiated tumor cells (F and G, red arrows). Vascular endothelial staining was present in tumor areas (H).
Fig. 47A shows H&E and IHC staining of patient's gastric tissue (ID: 006, Gastric Adenocarcinoma). H&E image shows a large diffuse infiltrative mass in the mucosa infiltrating the submucosa, muscularis, and serosa and serosa (A). The immunoreactivity with 8H9 antibody was detected in nearly all neoplastic epithelium with a diffuse and cytoplasmic pattern (B and D). No immunostaining was found in sections treated with MslgG (C and E). Fig. 47B shows at 20 ug/ml, there is moderate granular membrane staining on about 30% of the tumor cells (F and G, red arrows). Vascular endothelial staining was present in tumor areas (H).
Fig. 48A shows H&E and IHC staining of patient's gastric tissue (ID: 007, Gastric Cystadenocarcinoma). H&E image shows a large diffuse infiltrative mass invading mucosa, submucosa, muscularis, and serosa (A).The immunoreactivity with 8H9 antibody was detected in nearly all neoplastic epithelium with a diffuse and cytoplasmic pattern (B and D). No immunostaining was found in sections treated with MslgG (C and E). Fig. 48B shows at 20 ug/ml, there is occasional granular membrane staining on less than 10% of the tumor cells (F and G, red arrows). Vascular endothelial staining was present in tumor areas (H).
Fig. 49A shows H&E and IHC staining of patient's gastric tissue (ID: 008, Gastric Adenocarcinoma). H&E image shows a full thickness of gastric mucosa with a protruding proliferative mass and expansion of submucosa (A).The immunoreactivity with 8H9 antibody was detected in nearly all neoplastic epithelium with a diffuse and cytoplasmic pattern (B and D). No immunostaining was found in sections treated with MslgG (C and E). Fig. 49B shows at 20 mg/ml, the majority of tumor cells demonstrated granular membrane staining (F and G, red arrows). Vascular endothelial staining was present in tumor areas (H).
Fig. 50A shows H&E and IHC staining of patient's gastric tissue (ID: 009, Gastric Adenocarcinoma). H&E image shows normal and neoplastic gastric tissues (A).The immunoreactivity with 8H9 antibody was detected in about 50% of neoplastic epithelium with a diffuse and cytoplasmic pattern (B and D). No immunostaining was found in sections treated with MslgG (C and E). Fig. 50B shows At 20 mg/ml, there is weak granular membrane staining on about 30% of the tumor cells (F and G, red arrows). Vascular endothelial staining was present in tumor areas (H).
All cited references are incorporated by reference.
The accompanying Figures and Examples are provided to explain rather than limit the present invention. It will be clear to the person skilled in the art that aspects, embodiments, claims and any items of the present invention may be combined.
Unless otherwise mentioned, all percentages are in weight/weight. Unless otherwise mentioned, all measurements are conducted under standard conditions (ambient temperature and pressure). Unless otherwise mentioned, test conditions are according to European Pharmacopoeia 8.0.
Examples The following examples are intended to illustrate the present invention and are in no way to be understood as limiting.
Example 1: Immunohistochemical Evaluation of B7-H3 Expression
Immunohistochemical staining is in general a challenging assay and the selection of an optimal condition requires manipulating a variety of parameters. The following conditions did not provide optimal conditions for immunohistochemical staining of formalin fixed paraffin embedded (FFPE) samples with murine 8H9 antibody comprising a heavy chain according to SEQ ID No. 1 and a light chain according to SEQ ID No. 2:
Antigen retrieval Citrate Buffer pH6,
Antigen retrieval Tris-EDTA Buffer pH8, - Concentration of the primary antibody of 1-20 mg/ml, and/or
Incubation at room temperature less than 3 hours.
High and Low pressure within the Tris-EDTA pH 9 treatment caused severe damage to tissues. Example 2: Immunohistochemical Evaluation of B7-H3 Expression in Non-small Cell
Carcinoma, Ovarian Cancer, Medulloblastoma, Neuroblastoma, Glioblastoma, Sarcoma and Melanoma.
The preset study has determined whether a murine 8H9 antibody comprising a heavy chain according to SEQ ID No. 1 and a light chain according to SEQ ID No. 2 targeting human
immunoregulatory B7-H3 protein, binds to any human tumor tissues. The present study has assessed potential binding of a murine 8H9 antibody to neuroblastoma, medulloblastoma, ovarian cancer, non-small cell lung cancer (NSCLS), sarcoma, melanoma and glioblastoma. Specific positive tissue staining results suggest cancer types that may be targeted by the antibody in vivo in humans.
The experimental design is presented in Table 1 Table 1:
The Isotype Control Mouse lgGl binds to the immunogen: TNP (Trinitrophenol) + KLH (keyhole limpet haemocyanin).
All tissues were obtained by the Comparative Bioscience, Inc Histology Laboratory from approved vendors.
Tissue preparation
All tissues were gross trimmed, processed through a graded series of alcohols, oriented and embedded in paraffin, microtome-sectioned at 3 to 5 mm thicknesses, slide-mounted, immunohistochemically stained with mu8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2, and control antibodies, and cover-slipped by standard methodology. The unfixed frozen tissues were embedded in optimal cutting temperature medium (OCT) and blocks were prepared. The OCT tissue blocks were placed in sealed plastic bags or other appropriate containers, and maintained below -70 °C pending sectioning. Unfixed, OCT-embedded tissue samples were sectioned at ~5-mm thicknesses using a cryostat, transferred onto charged slides, and allowed to air-dry at room temperature overnight. Slides were placed in a plastic bag- enclosed slide box with desiccant and stored below -70°C pending use. Immunohistochemical Staining Methodology
Sections were stained with mu8H9 antibody comprising a heavy chain sequence according to SEQ ID No.l and a light chain sequence according to SEQ ID No. 2 in order to assess
expression of the B7-H3 in these tissues. Staining were performed on both formalin fixed paraffin embedded sections and frozen sections.
IHC methodology FFPE sections
Primary antibodies, mu8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 or Isotype control Mouse IgGl, were recognized via a horseradish peroxidase labeled polymer conjugated to an anti-mouse antibody, permitting detection by a colorimetric assay, for target molecules expressing cells. Tissues on slides were deparaffinized, hydrated, and subjected to antigen retrieval by heated Tris-EDTA buffer with a value of pH 9, and a temperature of 100°C, for 15 minutes. Tissues were stained at 4°C overnight. The staining consisted of the following steps, separated by rinses with wash buffer: peroxidase block for 10 minutes; CAS block (casein solution) for 30 minutes; primary antibody overnight; horseradish peroxidase labeled polymer conjugated to an anti-mouse antibody for 60 minutes; 3,3'-diaminobenzidine for 3 minutes. The slides then were counterstained with hematoxylin, dehydrated, and coverslipped by standard methodologies.
The method was performed in the following steps:
• Deparaffinization
• Antigen retrieval (Tris-EDTA, pH 9, 100 °C) for 15 minutes
• Cool down for 30 minutes
1. Rinse in d2H20 for 1 minute
2. Peroxidase Block (3% H2O2 in d2H20) for 10 minutes
3. 0.01M phosphate-buffered saline for 1 minute
4. Casein solution for 30 minutes
5. Primary Antibody (mu8H9 or MslgG1, 30 mg/ml) for about 24 hours, 4°C
6. 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for 2 minutes
7. 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for 2 minutes
8. Secondary antibody conjugated to a horseradish peroxidase for 60 minutes
9. 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for 2 minutes
10. 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for 2 minutes
11. 3,3'-diaminobenzidine for 3 minutes
12. Water for 1 minute 13. Hematoxylin counter staining
IHC methodology frozen sections
Primary antibodies (mu8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 or Isotype control Mouse IgGl) were recognized via a horseradish peroxidase labeled polymer conjugated to an anti-mouse antibody, permitting detection by a colorimetric assay, for target molecules expressing cells. Tissues, on slides, were kept at room temperature for 30 minutes followed by fixation in 2 —
5 °C cold acetone for 10 minutes. Tissues were stained at room temperature. The staining consisted of the following steps, separated by rinses with wash buffer: peroxidase block for 10 minutes; CAS block (casein solution) for 30 minutes; primary antibody for 120 min; horseradish peroxidase labeled polymer conjugated to an anti-mouse antibody for 60 minutes for 60 minutes; 3,3'-diaminobenzidine for 3 minutes. The slides were then counterstained with hematoxylin, dehydrated, and coverslipped by standard methodologies.
• Remove the frozen sections from -80 and keep it in room temperature for 30 minutes · Fixation in 4% paraformaldehyde for 10 minutes
1. Rinse in d2H2Q for 1 minute
2. Peroxidase Block (3% H2O2 in d2H20) for 10 minutes
3. 0.01M phosphate-buffered saline for 1 minute
4. Casein solution for 30 minutes
5. Primary Antibody (mu8H9 or MslgG1, 30 mg/ml) for 120 minutes 6. 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for 2 minutes
7. 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for 2 minutes
8. Secondary antibody conjugated to a horseradish peroxidase for 60 minutes 9. 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for 2 minutes
10. 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for 2 minutes
11. 3,3'-diaminobenzidine for 3 minutes 12. Water for 1 minute
13. Hematoxylin counter staining
Tissue evaluation
For routine histopathologic evaluation, all tissues were qualitatively examined by light microscopy by a veterinary pathologist certified by the American College of Veterinary Pathologists for the purposes of confirming tissue identity, normal morphology and lack of significant autolysis.
Histopathologic Scoring System
0 = Normal - Tissue considered to be normal under the conditions of the study and considering the age, sex and strain of the animal concerned.
Alterations may be present which, under other circumstances, would be considered deviations from normal.
1 = Minimal - The amount of change barely exceeds that which is considered to be within normal limits. 2 = Mild — In general, the lesion is easily identified but of limited severity. The lesion probably does not produce any functional impairment.
3 = Moderate - The lesion is prominent but there is significant potential for increased severity. Limited tissue or organ dysfunction is possible.
4 = Severe - The degree is either as complete as considered possible or great enough in intensity or extent to expect significant tissue or organ dysfunction.
For immunohistochemical assessment and scoring, all stained tissues were evaluated by the Study Pathologist, including both the identity of any tissue or cell type stained and the intensity of staining. All slides were judged for adequacy of tissue elements and staining. Adequate positive and negative controls were also included.
Immunohistochemical Scoring System
• The intensity of the staining was scored on a multi-point scale as follows:
± (equivocal), 1+ (weak), 2+ (moderate), 3+ (strong), 4+ (intense), or Neg (negative). Statistical analysis was not used for the data generated. · Any staining judged specific by comparison to corresponding control slides and nature of staining was scored for intensity, frequency (number and incidence of stained cells), and staining affinity where appropriate.
• Any staining was judged to be specific or nonspecific based upon known expression of target antigen. · The sensitivity, specificity, and reproducibility of the particular assay was assessed based on specific reactions of the test article with positive control materials; lack of specific activity with negative control materials; and lack of reactivity with negative control article.
• All slides were judged for adequacy of tissue elements and staining. Slides deemed inadequate were repeated.
Photomicroscopy
Representative photomicrographs are presented in figures 2 - 35. Data
The pathologist examined all of the submitted tissue sections using light microscopy and recorded the findings directly into tables. The data generated was used by the pathologist in assessment of the histopathologic findings associated with epitope expression. Results
Microscopic Findings and Scoring
The scoring is presented in Table 2. Representative photomicrographs are presented in figures 2 - 35. Mu8H9 staining was detected diffusely and multifocally in a subset of tumor cells in all tissues. The staining was granular, generally strong to intense (Grade 3-4) and located on the membrane and occasionally cytoplasmic, although there was some vasculature and intracellular staining as well. The staining of most of the tumor tissues was subjected to immunohistochemical staining with commercially available anti-BH-73 antibody. The condition of staining and staining pattern was similar to that of mu8H9, in particular for the IHC staining of frozen sections.
Human tumor tissues
Positive staining with mu8H9 was present in all FFPE and frozen tissues. Incidence of expression appeared to be more extensive in NSCLC, neuroblastoma, medulloblastoma, glioblastoma, melanoma, and liver cancer, and moderate in ovarian cancer and sarcoma.
Conclusion
Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 recognized the target molecule B7-H3 in FFPE samples of different tumor types under following condition: - Antigen retrieval Tris-EDTA pH 9 at 100°C (without any pressure which avoided tissue damage).
Incubation at 30 mg/ml overnight at 4°C.
Mu8H9 comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 stained the tumor cells in all tumor tissues diffusely and in some cases multifocally. The staining pattern was generally intense, granular and located on the membrane, although there was cytoplasmic, intracellular, and vascular staining as well. Positive staining was evident in all tumor types, however appeared to be more extensive in the FFPE sections of non-small cell carcinoma, neuroblastoma, medulloblastoma, glioblastoma, melanoma and liver cancer. Staining was weak to moderate in ovarian cancer and sarcoma. There was strong positive staining in all frozen tumors, with the exception of moderate staining of lung adenocarcinoma.
Example 3 Histopathological Evaluation of Ovarian Cancer Tissues for Expression of B7-H3 (CD276) Using Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2.
The objective of this study was to determine whether Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 binds to formalin fixed paraffin embedded ovarian cancer tissues using immunohistochemical techniques. Specific staining was detected diffusely and multifocally in the tumor cells in a majority of the ovarian cancer tissues. Generally, 80% of tumor samples showed positive staining for the antibody representing the expression of B7-H3 in 40-100% of tumor cells. The staining was granular, generally mild to intense (Grade 1-4) with cytoplasmic/membranous pattern. The positive membrane staining was found in majority of tumor cells (50-100%) with overall intensity of +2 to +4. The incidence of staining represented a mild to severe (+2 to +4) distribution of positive tumor cells in the entire section. There was no staining with mouse isotype control (Ms IgG) at any concentrations. The control tissue (normal ovary) did not show any reaction with the 8H9 antibody.
Experimental design
The study protocol from example 2 was used for IHC staining, however, additional steps were taken to optimize the intensity of the signal in the ovarian cancer tissues as described in the section Immunohistochemical Staining Methodology. Initially the staining protocol was optimized for three concentrations of Murine 8H9 antibody comprising a heavy chain
sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 (10, 20 and 30 mg/ml) and the concentration of 20 mg/ml was selected as the optimal concentration for the rest of the study. Von Willebrand Factor (vWF) was used as a marker for quality control of the testing system. The modification and reproducibility of the staining protocol was validated through eight validation runs using the experimental design presented in table 4.
Table 4 Experimental design
Histopathology methods Tissues submitted
All tissues were obtained by the Comparative Bioscience, Inc Histology Laboratory from approved vendors.
Tissue preparation
The ovarian normal and cancer FFPF tissues from individual donors were microtome- sectioned at 5 mm thicknesses and mounted onto charged slides. Commercially available ovarian cancer at 5 tissue microarray (TMA) slides of 25 cases (50 cores, 25 cores for testing Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 and 25 cores for testing Ms IgG) were used to validate the results of the staining in a large number of tissue samples. Immunohistochemical Staining Methodology
Sections were stained with murine 8H9 antibody in order to assess expression of the B7-H3 in these tissues. In brief, FFPE tissue sections on slides were deparaffinized, hydrated, and subjected to heated Tris-EDTA buffer (pH9, 100 °C) antigen retrieval. After PBS wash, sections were treated with peroxide block solution for 10 minutes, washed with PBS, and incubated with protein blocking buffer for 30 min at room temperature (RT). Then, the tissues were incubated with Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 overnight. The second day of staining was conducted with a modified version of the staining protocol used in Example 2 where the sections were treated with a signal enhancer (Envision™ FLEX + Mouse (linker)) for 15 minutes prior to adding HRP conjugated polymer for 60 minutes. After three runs of PBS wash, the sections were exposed to a chromogen reagens (EnVision™ FLEX DAB+ Chromogen) for 1-2 minutes and the intensity of the signal was evaluated under microscope. The slides then were counterstained with hematoxylin, dehydrated, and coverslipped by standard methodologies. IHC methodology for ovarian FFPE tissue:
Tissue Evaluation
For routine histopathologic evaluation, all tissues were qualitatively examined by light microscopy by a veterinary pathologist certified by the American College af Veterinary Pathologists for the purposes of confirming tissue identity, normal morphology and lack of significant autolysis.
Immunohistochemical Scoring System
For immunohistochemical assessment and scoring, all stained tissues were evaluated by the Study Director and Study Pathologists, including both the identity of any tissue or cell type stained and the intensity of staining. All slides were judged for adequacy of tissue elements and staining. Adequate positive and negative controls were also included. The scoring was provided by considering the following criteria:
• The intensity of the staining was scored on a multi-point scale as follows:
± (equivocal), 1+ (weak), 2+ (moderate), 3+ (strong), 4+ (intense), or Neg (negative). Statistical analysis was not used for the data generated. · Any staining judged specific by comparison to corresponding control slides and nature of staining was scored for intensity, frequency (number and incidence of stained cells), and staining affinity where appropriate. The incidence of staining represented the distribution of positive tumor cells in the entire section. If positive tumor cells were localized in distinct areas of sections, a range of scoring (e.g. +2-3) was provided reflecting the difference between the incidences of positive cells in those areas.
• Any staining was judged to be specific or nonspecific based upon known expression of target antigen.
• The sensitivity, specificity, and reproducibility of the particular assay was assessed based on specific reactions of the test antibody with positive control materials; lack of specific activity with negative control materials; and lack of reactivity with negative control article.
• All slides were judged for adequacy of tissue elements and staining. Slides deemed inadequate were repeated. Table 5 Histopathological Scoring Terms
Photomicroscopy
Representative photomicrographs are presented in Fig. 36-40
Data All of the tissue sections were evaluated using light microscopy and the findings were recorded directly into tables. The data generated was used in assessment of the histopathologic findings associated with epitope expression. The quantitative measurement of the staining intensity was conducted using image processing software for the selected representative images of the individual donor tissue samples. The intensity reflects the darkness of brown DAB(3,3 Diaminobenzidine) reaction which is measured in different areas of sections and presented as an average. This quantitative intensity measurement is for didactic purposes only and should not be utilized for making clinical decisions. The intensity of staining (darkness vs brightness) may not represent the concentration of B7-H3 in the targeted organ as DAB staining does not follow Beer-Lambert law. Results
Microscopic findings and scoring
Results of histopathologic scoring with Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 are
detailed in table 6 and table 7. Table 5 showing the histopathological scoring terms. Representative photomicrographs are shown in Fig. 36-40.
Table 6
*The tissues are obtained from donors with confirmed clinical diagnosis. Occasionally, tissues are harvested from areas that may not represent the diagnosis due to heterogeneity of the tumors.
Table 7 IHC Scoring of 25 TMA Ovarian Cancer Cases
Specific staining was detected diffusely and multifocally in the tumor cells in the majority of ovarian cancer tissues. The control tissue (normal ovary) did not show any reaction with the 8H9 antibody. The staining was granular, generally mild to intense (Grade 1-4) with cytoplasmic/membranous pattern in both individual donors and TMAs. Generally, 80% of tumor samples (24 of 30 tumor samples from 5 individual donors and 25 TMAs) showed positive staining for the antibody representing the expression of B7-H3 in 40-100% of tumor cells. The percentage of membrane staining was evaluated in three random areas of each section under high-power field (HPF) magnification (400X and 1000X). The percentage of the membrane staining was calculated using the below formula
% of membrane staining
An average of those three readings was provided as the percentage of membrane staining in the tumor cells which showed positive membrane staining in majority of tumor cells with overall intensity of +2 to +4. The incidence of staining represented a mild to severe (+2 to +4) distribution of positive tumor cells in the entire section. The quantitative analysis of digital
images demonstrated different staining intensity for the samples of each individual donor. There was no staining with mouse isotype control at any concentrations.
Conclusion and discussion
The objective of this study was to assess the staining of Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2, a therapeutic agent when radiolabeled as a tool to detect B7-H3, to FFPE ovarian cancer tissue using immunohistochemical techniques. Specific staining was detected diffusely and multifocally in the tumor cells in a majority of the ovarian cancer tissues. The control tissue (normal ovary) dit not show any reaction with 8H9 antibody. The staining was granular, generally mild to intense (Grade 1-4) with cytoplasmic/membranous pattern. Generally, 80% of tumor samples showed positive staining for the antibody representing the expression of B7-H3 in 40-100% of tumor cells. The staining was granular, generally mild to intense (Grade 1-4) with cytoplasmic/membranous pattern. The positive membrane staining was found in majority of tumor cells (50-100%) with overall intensity of +2 to +4. The incidence of staining represented a mild to severe (+2 to +4) distribution of positive tumor cells in the entire section. There was no staining with mouse isotype control at any concentration. The control tissue (normal ovary) did not show any reaction with the 8H9 antibody. There was no staining with mouse isotype control at any concentrations. The results of this study suggest a variation in expression of B7-H3 in different types of ovarian cancer which could be targeted by a 8H9 antibody for therapeutic and/or diagnostic purposes.
Example 4 Histopathological Evaluation of Gastric Cancer Tissues for Expression of B7-H3 (CD276) Using Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2.
The objective of this study was to evaluate histopathologic and immunohistochemical findings of gastric carcinomas from nine patients that were stained with the monoclonal Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2. This immunohistochemistry study assessed potential binding of the murine 8H9 antibody to human gastric cancer tissues
derived from nine patients. Specific positive staining results suggest tumor cell types that may be targeted by 8H9 antibody in vivo in humans.
Tissues Submitted Glass slides from formalin fixed paraffin embedded (FFPE) blocks derived from patients were submitted to the CBI Histology Laboratory. The tissues were gastric carcinomas and were examined both histopathologically and immunohistochemically, using the monoclonal murine 8H9 antibody to assess specific 8H9 antibody binding and expression.
FFPE Tissue Preparation Human gastric cancer tissue sections were stained with hematoxylin and eosin (H&E).
Tissues were then evaluated via light microscopy by a board-certified veterinary pathologist.
Immunohistochemical Staining Methodology
Sections were stained with murine 8H9 antibody to assess expression of the B7-H3 in these tissues. The staining technique was developed at CBI. The detailed methodology is shown below.
IHC Protocol for Human gastric cancer tissues (FFPE)
*PBS (IX) is used as washing buffer for this study. However PBS could be replaced by other washing buffer e.g. Tris-EDTA or PBS with Tween-20 (0.05%).
FFPE Tissue Evaluation
Tissues were qualitatively examined by light microscopy by a veterinary pathologist certified by the American College of Veterinary Pathologists. The pathologist prepared a report describing the histologic features and confirming the diagnosis or the purposes of confirming tissue identity. If any numerical semiquantitative assessment was needed, lesions were scored using the industry standard 5-point scoring system as described in example 2 (Mann, et al., 2012). Immunohistochemical Assessment
For immunohistochemical assessment and scoring, all stained tissues were evaluated by the Study Pathologist, including the identity of any tissue or cell type stained, as well as the intensity and distribution of staining. All slides were judged for adequacy of tissue elements and staining. In addition, the results of the previous studies (e.g. example 1 and 2 among others) were used as a reference to evaluate the specificity of Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 for the gastric cancer tissues in comparison with neuroblastoma
(positive control), human heart and liver (negative control) tissues, and commercially available B7-H3, respectively. The specificity of the 8H9 antibody was compared with MslgG control. The sensitivity of the antibody was optimized at 30 mg/ml only for IHC staining uses.
In particular, positive staining on the cell membrane or within the cell cytoplasm was assessed. Positive staining for 8H9 antibody may be observed both in the tumor cell cytoplasm and on the cell membrane.
Immunohistochemical Scoring system
The system as can be seen in example 2 was applied.
Interpretation of staining was conducted with: · Positive tissue control: Expression of 8H9 antibody in Cytoplasm (C), Extracellular (E),
Membrane (M), Nuclear (N), Perinuclear(P) with a granular pattern.
• Negative tissue control: Absence of specific staining.
• H&E Control: Characterizing cellular morphology and identifying microstructural abnormality including inflammation, vascular leakage, fibrosis, tumor invasion, ect. Masked assessment for description and diagnosis of patient tissues were applied.
Western Blot Analysis
Western blot was performed on two sections of each patient FFPE tissue sample. The digital image achieved was analyzed and the quantified data of the detected protein were reported as molecular weight, signal/peak, intensity, ect. Results
Histopathologic findings
Summary histopathologic (H&E) staining assessment of patient tissues is presented in table 8.
Table 8 Summary of histopathologic and Immunohistochemical findings
I = Intensity, D = Distribution
Immunohistochemistry findings
Summary immunohistochemical staining assessment of patients is presented in Table 9. Representative photomicrographs are presented in Fig. 41-50. Table 9 ICH staining with Murine 8H9 antibody comprising a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 results for each tissue tested
Simple Western Blot Analysis findings Western blot analysis for the 8H9 antibody showed multiple non-specific bands due to crosslinking and degradation of proteins. Thus the expression of the target antigen could not be evaluated using this method.
Patient findings
Results indicate that there is strong binding of 8H9 specifically to the cytoplasm and membrane of the gastric carcinoma tumor cells in the tissues submitted. The leiomyoma (ID 001) was not stained.
Conclusion
Specific staining was detected in the tumor cells in a majority of the gastric cancer tissues. 1/9 was negative namely the Spindle cell tumor, possible leiomyoma, benign.
Down titration from 30 ug/ml to 20 ug/ml was necessary to discern membrane staining. There was a high heterogeneity in intensity, however the majority of gastric tumor express membrane staining.
The results of this study suggest a variation in expression of B7-H3 in different types of gastric cancer which could be targeted by a 8H9 antibody for therapeutic and/or diagnostic purposes.
Claims
1. A method for detecting expression and/or overexpression of an antigen in at least one tissue sample comprising cancer cells, or potentially comprising cancer cells, wherein said sample is selected among a thawed sample, a frozen sample, a fresh sample, a paraffin embedded sample, a formalin embedded sample, and/or a sample which has formerly been embedded in formalin and/or paraffin.
2. A method for detecting expression and/or overexpression of an epitope in a tissue sample comprising cancer cells, or potentially comprising cancer cells, wherein said sample preferably is a sample which has formerly been embedded in formalin and/or paraffin, and wherein said method comprises the steps of: i. Enzyme blocking with an enzyme blocking substance, ii. Protein blocking with a protein blocking substance, iii. Binding with a primary antibody, iv. Incubation with a secondary antibody, and v. Staining with a staining agent.
3. The method of claim 2, wherein said epitope is an epitope of B7-H3 antigen.
4. The method of any of the preceding claims, wherein said primary antibody has a concentration of about 30 mg/ml, and wherein said binding with a primary antibody is carried out at about 4 °C, and/or wherein said binding is carried out for about 4 - 24 hours.
5. The method of any of the preceding claims, wherein said method further comprises the following step:
Antigen retrieval with Tris-EDTA, wherein said Tris-EDTA has a pH value of about 9 and/or a temperature of about 100 °C.
6. The method of any of the preceding claims, wherein said antigen retrieval is carried out for about 1 - 30 minutes, 5 - 25 minutes, 10 - 20 minutes or preferably about 15
- 20 minutes.
7. The method of any of the preceding claims, wherein said antigen retrieval is carried out for about 15 minutes.
8. The method of any of the preceding claims, wherein said antigen retrieval is carried out for about 20 minutes.
9. The method of any of the preceding claims, wherein said method further comprises a step of cooling down said tissue sample at room temperature, wherein said cooling down is performed after said antigen retrieval.
10. The method of any of the preceding claims, wherein said cooling down is performed for about 1 - 30 minutes, about 10 - 25 minutes, about 15 - 20 minutes or preferably about 20 minutes.
11. The method of any of the preceding claims, wherein said tissue sample is from an individual, and wherein said method is used for evaluating whether said individual may benefit from a therapy, wherein said therapy is selected among radioimmunotherapy, immunotherapy or cell therapy.
12. The method of any of the preceding claims, wherein said antigen is a B7-H3 antigen.
13. The method of claim 12, wherein said B7-H3 antigen comprises the sequence of SEQ
ID No. 17 or SEQ ID No. 18
14. The method of any of the claims 12 - 13, wherein said B7-H3 antigen comprises at least one peptide selected among the peptide according to SEQ ID No. 15 and the peptide according to SEQ ID No. 16.
15. The method of any of the preceding claims, wherein said method uses antibodies to detect proteins in cells of said tissue sample.
16. The method of any of the preceding claims, wherein said primary antibody is an anti- B7H3 antibody.
17. The method of any of the preceding claims, wherein an anti-B7-H3 antibody or antigen-binding fragment thereof is used to detect B7-H3 expression.
18. The method of claim 17, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof comprises at least one of the sequences selected among a heavy chain variable region CDR1 according to SEQ ID No. 3, a heavy chain variable region CDR2 according to SEQ IN No. 4 or 19, a heavy chain variable region CD3 according to
SEQ ID No. 5, a light chain variable region CDR1 according to SEQ ID No. 6, a light chain variable region CDR2 according to SEQ ID No. 7 and a light chain variable region CDR3 according to SEQ ID No. 8.
19. The method of claim 17, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2.
20. The method of claim 17, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof comprises a heavy chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No. 1 and/or a light chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No. 2.
21. The method of any of the claims 17 - 20, wherein said antigen-binding fragment thereof is a single chain variable fragment (scFv).
22. The method of claim 21, wherein said scFv comprises a portion of the amino acid sequence set forth in SEQ ID No. 9, SEQ ID No. 13 or SEQ ID No. 14.
23. The method of claim 21 - 22, wherein said scFv comprises at least one of the amino acid substitutions K13E, R18Q, R45Q, K103E and K107E.
24. The method of any of the claims 17 -23, wherein said anti-B7-H3 antibody or antigen- binding fragment thereof is a murine 8H9 antibody or an antigen-binding fragment thereof.
25. The method of any of the claims 17 -24, wherein said antibody or antigen-binding fragment thereof is humanized.
26. The method of any of the claims 17 - 25, wherein said antibody or antigen-binding fragment thereof is a chimeric antibody or an antigen-binding fragment thereof.
27. The method of any of the claims 17 -26, wherein said antibody or antigen-binding fragment thereof is radiolabeled.
28. The method of claim 27, wherein said radiolabel is selected among a PET label and a
SPECT label.
29. The method of claim 28, wherein said PET label is selected among 124l, 225Ac and 89Zr.
30. The method of claim 28, wherein said SPECT label is selected among 131l, 177Lu, 99mTc,
64Cu and 89Zr.
31. The method of any of the claims 17 -30, wherein said antibody or antigen-binding fragment thereof is conjugated to a chelator compound.
32. The method of claim 31, wherein said chelator compound is bound to a radioactive isotope.
33. The method of claim 32, wherein said radioactive isotope is selected among124l, 131l and 177Lu or 99mTc, 64Cu (chelated to NOTA) and 89Zr (chelated to DFO).
34. The method of any of the claims 31 - 33, wherein said chelator compound is selected among DOTA, DTPA, NOTA and DFO.
35. The method of claim 34, wherein said DOTA is a variant of DOTA.
36. The method of claim 34, wherein said DTPA is a variant of DTPA.
37. The method of any of the preceding claims, wherein said method may be used on at least a sample that has formerly been embedded in paraffin, as well as a sample which has formerly been frozen.
38. The method of any of the preceding claims, wherein said method further comprises one or more steps of rinse, preferably wherein at least one rinsing step is with water.
39. The method of claim 38, wherein said water is d2H20.
40. The method of any of the preceding claims, wherein one or more or all of said steps are separated by wash with at least one wash buffer.
41. The method of any of the preceding claims, wherein one or more or all of said steps are separated by at least one wash with at least one wash buffer.
42. The method of any of the preceding claims, wherein one or more or all of said steps are separated by at least two washes with at least one wash buffer.
43. The method of any of the preceding claims, wherein one or more or all of said steps are separated by at least three washes with at least one wash buffer.
44. The method of any of the preceding claims, wherein each wash is carried out for about 1 - 5 minutes, about 2 -4 minutes or preferably about 3 minutes.
45. The method of any of the preceding claims, wherein each wash is carried out for about 0.1 minute - 2 minutes, 0.5 minutes - 1.5 minutes or preferably about 1 minute.
46. The method of claim 40, wherein said wash buffer is phosphate-buffered saline.
47. The method of any of the claims 40 - 46, wherein said wash buffer is 0.01M phosphate-buffered saline-0.05 % polyoxyethylene sorbitan monolaurate.
48. The method of any of the preceding claims, wherein said enzyme blocking substance is a peroxidase block.
49. The method of claim 48, wherein said peroxidase block is 3% H2O2 in d2H20.
50. The method of any of the preceding claims, wherein said protein blocking substance is casein solution.
51. The method of any of the preceding claims, wherein said enzyme blocking is carried out for 1 - 60 minutes, 2 - 55 minutes, 3 - 50 minutes, 4 - 45 minutes, 5 - 40 minutes, 6 - 35 minutes, 7 - 30 minutes, 8 - 25 minutes, 9 - 20 minutes 10 - 15 minutes or about 10 minutes.
52. The method of any of the preceding claims, wherein said protein blocking is carried out for 1 - 60 minutes, 5 - 55 minutes, 10 - 50 minutes, 15 - 45 minutes, 20 - 40 minutes, 25 - 35 minutes, or about 30 minutes.
53. The method of any of the preceding claims, wherein said method further comprises a step of incubation with a reagent that amplify the signal of the primary antibody.
54. The method of any of the preceding claims, wherein said reagent that amplify the signal of the primary antibody is an antibody polymer.
55. The method of any of the preceding claims, wherein said incubation with a reagent that amplify the signal of the primary antibody is carried out for about 1 - 30 minutes, about 5 - 25 minutes, about 10 - 20 minutes or preferably about 15 minutes.
56. The method of any of the preceding claims, wherein said reagent that amplify the signal of the primary antibody, enhance the signal at least 2 times, at least 3 times, at least 4 times or preferably at least 5 times.
57. The method of any of the preceding claims, wherein said binding with a primary antibody is carried out for 1- 48 hours, 4 - 44 hours, 8 - 40 hours, 12 - 36 hours, 16 - 32 hours, 20 - 28 hours or about 24 hours.
58. The method of any of the preceding claims, wherein said binding with a primary antibody is carried out for 1 - 240 minutes, 30 - 110 minutes, 60 - 180 minutes, 90 - 150 minutes or about 120 minutes.
59. The method of any of the preceding claims, wherein said binding with a primary antibody is carried out for about 120 minutes or about 24 hours.
60. The method of any of the preceding claims, wherein said binding with a primary antibody is carried out for a period of time allowing at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 99% of the antigens to be bound by the primary antibody.
61. The method of any of the preceding claims, wherein said primary antibody is any of the antibodies of claim 15 - 36 or an IgGl antibody.
62. The method of claim 61, wherein said IgGl antibody is a mouse IgGl antibody.
63. The method of any of the preceding claims, wherein said primary antibody has a concentration of 1 - 60 mg/ml, 5 - 55 mg/ml, 10 - 50 mg/ml, 15 -45 mg/ml, 20 - 40 mg/ml, 25 - 35 mg/ml or about 30 mg/ml.
64. The method of any of the preceding claims, wherein said primary antibody has a concentration of 1 - 40 mg/ml, 5 - 35 mg/ml, 10 - 30 mg/ml, 15 - 25 mg/ml or preferably about 20 mg/ml.
65. The method of any of the preceding claims, wherein said secondary antibody is conjugated to a labeled polymer.
66. The method of claim 65, wherein said labeled polymer is a horseradish peroxidase labeled polymer.
67. The method of any of the preceding claims, wherein said secondary antibody is an anti-mouse antibody.
68. The method of any of the preceding claims, wherein said incubation with a secondary antibody is carried out for 1 - 120 minutes, 10 - 110 minutes, 20 - 100 minutes, 30- 90 minutes, 40 - 80 minutes, 50 - 70 minutes or about 60 minutes.
69. The method of any of the preceding claims, wherein said incubation with a secondary antibody is carried out for 1 - 240 minutes, 10 - 230 minutes, 20 - 220 minutes, 30 - 210 minutes, 40 - 200 minutes, 50 - 190 minutes, 60 - 180 minutes, 70 - 170
minutes, 80 - 160 minutes, 90 - 150 minutes, 100 - 140 minutes, 110 - 130 minutes or preferably about 120 minutes.
70. The method of any of the preceding claims, wherein said staining agent is a benzidine.
71. The method of claim 70, wherein said benzidine is 3,3'-diaminobenzidine.
72. The method of any of the preceding claims, wherein said incubating with a staining agent is carried out for 1 - 6 minutes, 1.5 - 5.5 minutes, 2 - 5 minutes, 2.5 - 4.5 minutes, 3 -4 minutes or about 3 minutes.
73. The method of any of the preceding claims, wherein said incubating with a staining agent is carried out for about 1 - 10 minutes, about 1 - 9 minutes, about 2 - 7 minutes or preferably about 3 -5 minutes.
74. The method of any of the preceding claims, comprising a step, wherein said tissue sample is counterstained with a counterstaining agent.
75. The method of claim 74, wherein said counterstaining agent is hematoxylin and optionally eosin.
76. The method of any of the claims 74 - 75, wherein said sample is dehydrated after counterstaining.
77. The method of any of the claims 74 - 76, wherein said tissue sample is coverslipped after dehydration.
78. The method of any of the preceding claims, wherein one or more or all of said steps are separated by at least one wash according to any of the preceding claims.
79. The method of any of the preceding claims, wherein said steps are performed in the following order: i. Blocking of enzymes with an enzyme blocking substance, ii. Blocking of proteins with a protein blocking substance, iii. Binding with a primary antibody, iv. Incubation with a secondary antibody, and v. Staining with a staining agent.
80. The method of any of the preceding claims, wherein the preparation of said tissue sample comprises one or more or all of the following steps:
1.1 Gross trimming, 1.2 Processing through graded series of alcohols,
1.3 Tissue orientation, and
1.4 Embedding in paraffin.
Wherein said steps are followed by any of the steps according to any of the preceding claims.
81. The method of any of the preceding claims, wherein the method comprises one or more or all of the following steps:
2.1 Microtome-sectioning of a paraffin embedded sample,
2.2 Slide-mounting, 2.3 Preparing for staining and staining for an antibody, and
2.4 Counterstaining.
82. The method of any of the claims 80 - 81, wherein said paraffin embedded sample is microtome-sectioned to a thickness of 1 to 7 mm thick, 2 to 6 mm thick or about 3 to 5 mm thickness.
83. The method of any of the claims 80 - 82, wherein said tissue sample is slide-mounted.
84. The method of any of the claims 81 - 83, wherein the preparation for staining and staining comprises one or more or all of the following steps: 3.1 Deparaffinization,
3.2 Hydration,
3.3 Antigen retrieval, and
Wherein said steps are followed by the steps of claim 2.
85. The method of any of the preceding claims, wherein said sample has formerly been embedded in paraffin.
86. The method of any of the preceding claims, wherein said sample has been or is embedded in paraffin and/or formalin fixed.
87. The method of claim 86, wherein said sample is deparaffinized.
88. The method of any of the preceding claims, wherein said antigen retrieval is carried out by a buffer.
89. The method of any of the preceding claims, wherein said buffer is Tris-EDTA buffer.
90. The method of claim 89, wherein said Tris-EDTA buffer has a pH value of 7 - 10, 7.5 -
9.5, 8 - 9 or about 9.
91. The method of any of the claims 89 - 90, wherein said Tris-EDTA buffer is 60 - 140 °C, 65 - 135 °C, 70 - 130 °C, 75 - 125 °C, 80 - 120 °C, 85 - 115 °C, 90 - 110 °C, 95 -
105 °C or about 100 °C.
92. The method of any of the preceding claims, wherein said antigen retrieval is carried out for 1 - 60 minutes, 2 - 55 minutes, 3 - 50 minutes, 4 - 45 minutes, 5 - 40 minutes, 6 - 35 minutes, 7 - 30 minutes, 8 - 25 minutes, 9 - 20 minutes, 10 - 15 minutes or about 15 minutes.
93. The method of any of the preceding claims, wherein said steps are performed at 0 - 8 °C, 0.5 - 7.5 °C, 1-7 °C, 1.5 - 6.5 °C, 2-6 °C, 2.5 -5.5 °C, 3 - 5 °C, 3.5 - 4.5 °C or about 4 °C.
94. The method of any of the preceding claims, wherein said steps are performed for 1- 48 hours, 4 - 44 hours, 8 - 40 hours, 12 - 36 hours, 16 - 32 hours, 20 - 28 hours or about 24 hours.
95. The method of any of the claims 80 - 94, wherein the steps are followed by one or more or all of the steps of any of the claims 37 -79.
96. The method of any of the claims 84 - 95, wherein said steps are performed in the following order:
4.1 Deparaffinization
4.2 Hydration
4.3 Antigen retrieval
4.4 The steps according to claim 2.
97. The method of claim 80 - 96, wherein the steps are performed in the order according to claim 79.
98. The method of any of the preceding claims, wherein the preparation of said tissue sample comprises one or more or all of the following steps:
A.l Embedding said tissue sample in optimal cutting temperature medium, A.2 Preparation of a block of said sample,
A.3 Placement of said sample in a sealed plastic bag or other appropriate container, and
A.4 Maintaining said sample below -20 °C,
Wherein said tissue sample is a frozen sample or a thawed sample.
99. The method of any of the preceding claims, wherein said tissue sample is a thawed sample.
100. The method of any of the claims 98 - 99, wherein said optimal cutting temperature medium comprises one or more or all of the following: polyvinyl alcohol, polyethylene glycol or non-reactive ingredients.
101. The method of any of the claims 98 - 100, wherein said sample is maintained below -30 °C, - 35 °C, -40 °C, -45 °C, -50 °C, -55 °C, -60 °C, -65 °C, -70 °C, -75 °C, -80 °C, -85 °C, -90 °C, -95 °C or below -100 °C
102. The method of any of the claims 98 - 101, wherein said sample is sectioned to a thickness of 1-10 mm thick, 1.5 - 9.5 mm thick, 2 - 9 mm thick, 2.5 - 8.5 mm thick, 3 - 8 mm thick, 3.5 - 7.5 mm thick, 4 - 7 mm thick, 4.5 - 6.5 mm thick, 5 - 6 mm thick or about 5 mm thicknesses.
103. The method of any of the claims 98 - 102, wherein said sample is sectioned using cryostat.
104. The method of any of the claims 98 - 103, wherein said sample is transferred onto a charged slide.
105. The method of any of the claims 98 - 104, wherein said sample is air-dried.
106. The method of any one of the claims 98 - 105, wherein said sample is air-dried at room temperature, wherein said room temperature is 10 °C - 40 °C.
107. The method of claim 106, wherein said room temperature is selected among
10 - 40 °C, 15 - 35 °C, 17 - 33 °C, 18 - 30 °C, 19 - 27 °C or about 20 - 25 °C.
108. The method of any of the claims 98 - 107, wherein said sample is air-dried for
1-48 hours, 4 - 44 hours, 8 - 40 hours, 12 - 36 hours, 16 - 32 hours, 20 - 28 hours or about 24 hours.
109. The method of any of the claims 98 - 108, wherein the method comprises one or more or all of the following steps:
B.l Exposure of a tissue sample to room temperature, wherein said room temperature is 10 - 40 °C.
B.2 Fixation of the sample,
wherein said steps are followed by the steps of claim 2.
110. The method of claim 109, wherein said room temperature is selected among 10 - 40 °C, 15 - 35 °C, 17 - 33 °C, 18 - 30 °C, 19 - 27 °C or about 20 - 25 °C.
111. The method of any of the claims 109 - 110, wherein said exposure to room temperature is carried out for 1 - 60 minutes, 5 - 55 minutes, 10 - 50 minutes, 15 - 45 minutes, 20 - 40 minutes, 25 - 35 minutes or about 30 minutes.
112. The method of any of the claims 109 - 111, wherein said sample is fixated in paraformaldehyde.
113. The method of any of the claims 109 - 111, wherein said sample is fixated in acetone.
114. The method of claim 113, wherein said acetone is -10 - 20 °C, -5 - 15 °C, 0 -
10 "I :, 0.5 - 7 °C, 1 - 6 °C or 2 - 5 °C.
115. The method of any of the claims 109 - 114, wherein said fixation is carried out for 1-20 minutes, 5 - 15 minutes or about 10 minutes.
116. The method of any of the claims 109 - 115, wherein said steps are followed by one or more or all of the steps of any of the claims 37 -79.
117. The method of any of the preceding claims, wherein said cancer cells are cancer cells from any cancer type that express or may express B7-H3.
118. The method of any of the preceding claims, wherein said cancer cells are selected among carcinoma, sarcoma, lymphoma and leukemia cancer cells.
119. The method of any of the preceding claims, wherein said cancer cells are selected among neuroblastoma, medulloblastoma, glioblastoma, small celled lung cancer, non-small celled carcinoma, a pediatric sarcoma, an adult sarcoma, breast cancer, liver cancer, melanoma, non-small celled lung carcinoma, lung adenocarcinoma or gastrointestinal cancer cells.
120. The method of any of the preceding claims, wherein said cancer cells are ovarian cancer cells.
121. The method of any of the preceding claims, wherein said ovarian cancer cells are adenocarcinoma cells.
122. The method of any of the preceding claims, wherein said adenocarcinoma cells are serous adenocarcinoma cells.
123. The method of any of the preceding claims, wherein said adenocarcinoma cells are serous papillary adenocarcinoma cells.
124. The method of any of the preceding claims, wherein said cancer cells are gastric cancer cells.
125. The method of any of the preceding claims, wherein said gastric cancer cells are gastric carcinoma cells.
126. The method of any of the preceding claims, wherein said gastric carcinoma cells are gastric adenocarcinoma cells.
127. The method of any of the preceding claims, wherein said gastric carcinoma cells are gastric cystadenocarcinoma cells.
128. The method of any of the preceding claims, wherein said cancer cells are neuroblastoma cancer cells.
129. The method of any of the preceding claims, wherein said cancer cells are from a metastasis, a high-risk cancer and/or a recurrent cancer.
130. The method of any of the preceding claims, wherein said method comprises the following steps performed on said tissue sample: i. Deparaffinization ii. Antigen retrieval with Tris-EDTA buffer, wherein said buffer has a pH value of 9 and a temperature of 100°C, and wherein said antigen retrieval is carried out for about 15 minutes iii. Cool down for about 30 minutes iv. Rinse in d2H20 for about 1 minute v. Blocking enzymes with peroxidase block, wherein said peroxidase block is 3% H2O2 in d2H20, and wherein said exposure is carried out for about 10 minutes vi. Rinsing with 0.01M phosphate-buffered saline for about 1 minute vii. Blocking of proteins with casein solution for about 30 minutes viii. Binding with a primary antibody, wherein said primary antibody comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 or is an MslgG1 antibody, and wherein said antibody has a concentration of 30 mg/ml, and wherein said exposure is to be carried out for about 24 hours, and wherein said exposure is to be carried out in about in 4°C
ix. Rinsing with 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for about 2 minutes x. Rinsing with 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for about 2 minutes xi. Incubating with a secondary antibody conjugated to a horseradish peroxidase, wherein said secondary antibody is an anti-mouse antibody, and wherein said exposure is carried out for about 60 minutes xii. Rinsing with 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for about 2 minutes xiii. Rinsing with 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for about 2 minutes xiv. Staining with 3,3'-diaminobenzidine for about 3 minutes xv. Rinsing with water for about 1 minute xvi. Hematoxylin counter staining.
131. The method of any of the preceding claims, wherein said method comprises the following steps performed on said tissue sample: i. Remove the frozen sample from -80 °C and keep in room temperature, wherein said room temperature is selected among any of the temperatures of claim 110, and wherein said samples are kept in room temperature for about 30 minutes ii. Fixation in 4% paraformaldehyde or acetone for about 10 minutes iii. Rinse in d2H20 for about 1 minute iv. Blocking of enzymes with peroxidase block, wherein said peroxidase block is 3% H2O2 in d2H20, and wherein said exposure is carried out for about 10 minutes v. Rinsing with 0.01M phosphate-buffered saline for about 1 minute vi. Blocking of proteins with casein solution for about 30 minutes
vii. Binding with primary antibody, wherein said primary antibody comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 or is an MslgG1 antibody, and wherein said antibody has a concentration of 30 mg/ml, and wherein said exposure is to be carried out for about 120 minutes. viii. Rinsing with 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for about 2 minutes ix. Rinsing with 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for about 2 minutes x. Incubating with a secondary antibody conjugated to a horseradish peroxidase, wherein said secondary antibody is an anti-mouse antibody, and wherein said exposure is carried out for about 60 minutes xi. Rinsing with 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for about 2 minutes xii. Rinsing with 0.01M phosphate-buffered saline-0.05% polyoxyethylene sorbitan monolaurate for about 2 minutes xiii. Staining with 3,3'-Diaminobenzidine for about 3 minutes xiv. Rinsing with water for about 1 minute xv. Hematoxylin counter staining.
132. The method of any of the preceding claims, wherein said method comprises the following steps performed on said tissue sample: i. Deparaffinization ii. Antigen retrieval with Tris-EDTA buffer, wherein said buffer has a pH value of 9 and wherein said antigen retrieval is carried out for about 20 minutes iii. Cool down for about 20 minutes iv. First rinse in d2H20 for about 1 minute v. Second rinse in d2H2Q for about 1 minute
vi. First wash with 0.01M phosphate-buffered saline for about 2 minutes vii. Second wash with 0.01M phosphate-buffered saline for about 2 minutes viii. Third wash with 0.01M phosphate-buffered saline for about 2 minutes ix. Blocking enzymes with peroxidase block, wherein said peroxidase block is 3% H2O2 in d2H20, and wherein said exposure is carried out for about 10 minutes x. First wash with 0.01M phosphate-buffered saline for about 2 minutes xi. Second wash with 0.01M phosphate-buffered saline for about 2 minutes xii. Third wash with 0.01M phosphate-buffered saline for about 2 minutes xiii. Blocking of proteins with casein solution for about 30 minutes xiv. Binding with a primary antibody, wherein said primary antibody comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 or is an MslgG1 antibody, and wherein said antibody has a concentration of 20 mg/ml, and wherein said exposure is to be carried out for about 24 hours, and wherein said exposure is to be carried out in about in 4°C xv. First wash with 0.01M phosphate-buffered saline for about 2 minutes xvi. Second wash with 0.01M phosphate-buffered saline for about 2 minutes xvii. Third wash with 0.01M phosphate-buffered saline for about 2 minutes xviii. Incubation with a reagent that amplify the signal of the primary antibody, wherein said incubation is carried out for about 15 minutes xix. Incubating with a secondary antibody conjugated to a horseradish peroxidase, wherein said secondary antibody is an anti-mouse antibody, and wherein said exposure is carried out for about 60 minutes xx. First wash with 0.01M phosphate-buffered saline for about 2 minutes xxi. Second wash with 0.01M phosphate-buffered saline for about 2 minutes xxii. Third wash with 0.01M phosphate-buffered saline for about 2 minutes xxiii. Staining with 3,3'-diaminobenzidine for about 3 - 5 minutes
xxiv. Rinse with 0.01M phosphate-buffered saline for about 2 minutes xxv. Hematoxylin counter staining.
133. The method of claim 132, wherein said tissue sample is an ovarian tissue sample.
134. The method of any of the preceding claims, wherein said method comprises the following steps performed on said tissue sample: i. Deparaffinization ii. Antigen retrieval with Tris-EDTA buffer, wherein said buffer has a pH value of 9 and wherein said antigen retrieval is carried out for about 20 minutes iii. Cool down for about 20 minutes iv. First rinse in d2H20 for about 1 minute v. Second rinse in d2H20 for about 1 minute vi. First wash with 0.01M phosphate-buffered saline for about 2 minutes vii. Second wash with 0.01M phosphate-buffered saline for about 2 minutes viii. Third wash with 0.01M phosphate-buffered saline for about 2 minutes ix. Blocking enzymes with peroxidase block, wherein said peroxidase block is 3% H2O2 in d2H20, and wherein said exposure is carried out for about 10 minutes x. First wash with 0.01M phosphate-buffered saline for about 2 minutes xi. Second wash with 0.01M phosphate-buffered saline for about 2 minutes xii. Third wash with 0.01M phosphate-buffered saline for about 2 minutes xiii. Blocking of proteins with casein solution for about 30 minutes xiv. Binding with a primary antibody, wherein said primary antibody comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2 or is an MslgG1 antibody, and wherein said antibody has a concentration of 30 mg/ml, and wherein said exposure is to be
carried out for about 24 hours, and wherein said exposure is to be carried out in about in 4°C xv. First wash with 0.01M phosphate-buffered saline for about 2 minutes xvi. Second wash with 0.01M phosphate-buffered saline for about 2 minutes xvii. Third wash with 0.01M phosphate-buffered saline for about 2 minutes xviii. Incubating with a secondary antibody conjugated to a horseradish peroxidase, wherein said secondary antibody is an anti-mouse antibody, and wherein said exposure is carried out for about 120 minutes xix. First wash with 0.01M phosphate-buffered saline for about 2 minutes xx. Second wash with 0.01M phosphate-buffered saline for about 2 minutes xxi. Third wash with 0.01M phosphate-buffered saline for about 2 minutes xxii. Staining with 3,3'-diaminobenzidine for about 3 - 5 minutes xvi. Rinsing with water for about 1 minute xxiii. Flematoxylin counter staining.
135. The method of claim 134, wherein said tissue sample is a gastric tissue sample.
136. A method for providing and/or measuring a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of cancer in an individual, wherein said method comprises use of the method according to any of the preceding claims, and wherein said method is performed on a tissue sample comprising cancer cells.
137. A method for detecting the clinical benefit of an antibody or antigen-binding fragment thereof in the treatment of cancer in an individual, wherein said method comprises use of the method according to any of the preceding claims, and wherein said method is performed on a tissue sample comprising cancer cells.
138. A method for providing and/or measuring a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of an indication of cancer, wherein said method comprises use of the method according to any of the preceding claims, and wherein said method is performed on tissue samples comprising cancer cells.
139. A chimeric antigen receptor (CAR) comprising an antibody or antigen-binding fragment thereof according to any of the claims 17 - 36, wherein said chimeric antigen receptor is used in the method according to any of the preceding claims.
140. A CAR-T cell expressing a CAR according to claim 139.
141. A population of CAR-T cells according to claim 140.
142. A composition comprising the population of CAR-T cells of claim 141.
143. A method of treating cancer in an individual, wherein the method comprises providing a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof according to any of the preceding claims, and wherein said antibody or antigen-binding fragment thereof is used in said treatment.
144. A method for treating cancer in an individual, wherein the method comprises use of an antibody or antigen binding fragment thereof, and wherein the indication, such as ovarian cancer, for treatment of said cancer with said antibody or antigen- binding fragment thereof is determined or confirmed by the method according to any one of the preceding claims.
145. A method for treating cancer in an individual, wherein the method comprises evaluation of the clinical benefit of an antibody or antigen-binding fragment thereof, comprising use of the method according to any of the preceding claims, and wherein the clinical benefit is the clinical benefit for a patient, and wherein said antibody or antigen-binding fragment thereof is used in said treatment.
146. A method for treating cancer in an individual, wherein the method comprises providing a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in a tissue sample comprising cancer cells, comprising use of the method according to any of the preceding claims, and wherein said antibody or antigen-binding fragment thereof is used in said treatment.
147. A method of diagnosing cancer in an individual, wherein the method comprises providing a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof according to any of the preceding claims, and wherein said antibody or antigen-binding fragment thereof is used in said diagnosis.
148. A method for diagnosing cancer in an individual, wherein the method comprises use of an antibody or antigen binding fragment thereof, and wherein the diagnosis of said cancer with said antibody or antigen-binding fragment thereof is determined or confirmed by the method according to any one of the preceding claims.
149. A method for diagnosing cancer in an individual, wherein the method comprises evaluation of the clinical benefit of an antibody or antigen-binding fragment thereof, comprising use of the method according to any of the preceding claims, and wherein the clinical benefit is the clinical benefit for a patient, and wherein said antibody or antigen-binding fragment thereof is used in said diagnosis.
150. A method for diagnosing cancer in an individual, wherein the method comprises providing a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in a tissue sample comprising cancer cells, comprising use of the method according to any of the preceding claims, and wherein said antibody or antigen-binding fragment thereof is used in said diagnosis.
151. The method of claim 146 or 150, wherein said tissue sample is a tissue sample from said individual.
152. The method of any of the claims 143 -151, wherein said method evaluates overexpression of B7-H3.
153. The method of any of the claims 146 or 150, wherein said cancer cells are selected among any of the cancer cells of claim 117- 129.
154. The method of any of the claims 143 -153, wherein said antibody or antigen- binding fragment thereof is any of the antibodies or antigen-binding fragments of any of the claims 17 - 36.
155. The method of any of the claims 143 -154, wherein said cancer is any cancer type that express or may express B7-H3.
156. The method of any of the claims 143 -155, wherein said cancer is selected among a carcinoma, a sarcoma, a lymphoma and a leukemia.
157. The method of any of the claims 143 -156, wherein said cancer is selected among neuroblastoma, medulloblastoma, glioblastoma, small celled lung cancer, non-small celled carcinoma, a pediatric sarcoma, an adult sarcoma, breast cancer,
liver cancer, melanoma, non-small celled lung carcinoma, lung adenocarcinoma or a gastrointestinal cancer
158. The method of any of the claims 143 -157, wherein said cancer is an ovarian cancer.
159. The method according to any of the preceding claims, wherein said ovarian cancer is an adenocarcinoma.
160. The method according to any of the preceding claims, wherein said adenocarcinoma is a serous adenocarcinoma.
161. The method according to any of the preceding claims, wherein said adenocarcinoma is a serous papillary adenocarcinoma.
162. The method of any of the preceding claims, wherein said cancer is a gastric cancer.
163. The method of any of the preceding claims, wherein said gastric cancer is a carcinoma.
164. The method according to any of the preceding claims, wherein said carcinoma is an adenocarcinoma.
165. The method according to any of the preceding claims, wherein said carcinoma is a cystadenocarcinoma.
166. The method of any of the preceding claims, wherein said cancer is neuroblastoma.
167. The method of any of the claims 143 - 158, wherein said cancer is a metastasis, a high-risk cancer and/or a recurrent cancer.
168. The method of any of the claims 143 - 167, wherein said treatment is combined with chemotherapy and/or surgery.
169. A kit of parts, adapted to carry out the method according to any one or more of the preceding claims.
170. A kit of parts, wherein said kit is to be used to determine or qualify a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof is to be used in the treatment of a cancer.
171. A kit of parts, wherein said kit is to be used to determine or qualify the clinical benefit a patient has or will have from treatment with an antigen or antigen-binding fragment thereof, and wherein said patient is a cancer patient.
172. A kit of parts, wherein said kit is to be used to determine or qualify a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in the treatment of an indication of cancer.
173. A kit of parts, wherein said kit is used in detecting B7-H3 expression in a tissue sample comprising cancer cells, and wherein said sample is selected among a sample that have been embedded in paraffin and a sample have been frozen.
174. The kit according to any of the preceding claims, wherein said kit comprises instructions to carry out the method according to any of the preceding claims.
175. The kit according to any one of claims 169 -173, comprising an anti-B7-H3 antibody or antigen-binding fragment thereof used to detect B7-H3 expression.
176. The kit according to any of the claims 169 - 175, wherein said kit comprises a primary antibody.
177. The kit according to claim 169 - 176, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof, or said primary antibody, is any of the antibodies or antigen-binding fragments thereof of claim 17 - 36.
178. The kit according to any one of claims 169 - 177, wherein said kit uses antibodies to detect proteins in cells of said tissue sample.
179. The kit according to any one of claims 169 -178, wherein said kit comprises a secondary antibody.
180. The kit according to claim 179, wherein said secondary antibody is an anti- mouse antibody.
181. The kit according to any one of claims 179 - 180, wherein said secondary antibody is conjugated to an enzyme.
182. The kit according to claim 181, wherein said enzyme is a peroxidase.
183. The kit according to claim 182, wherein said peroxidase is Horseradish peroxidase.
184. The kit according to any one of claims 169 -183, wherein said kit comprises an enzyme block.
185. The kit according to claim 184, wherein said enzyme block is a peroxidase block.
186. The kit according to claim 185, where said peroxidase block is 3% H2O2 in d2H20.
187. The kit according to any of claims 169 - 186, wherein said kit comprises a protein block.
188. The kit according to claim 187, wherein said protein block is casein solution.
189. The kit according to any of the claims 169 - 188, wherein said kit comprises a substance for chromogenic staining.
190. The kit according to claim 189, wherein said substance for chromogenic staining is a benzidine.
191. The kit according to claim 190, wherein said benzidine is 3,3'-
Diaminobenzidine.
192. The kit according to claim 189 - 191, wherein said substance for chromogenic staining is a counterstaining agent.
193. The kit according to claim 192, wherein said counterstaining agent is hematoxylin and optionally eosin.
194. The kit according to any of the preceding claims, wherein said kit further comprises a reagent that amplify the signal of the primary antibody.
195. The kit according to any of the preceding claims, wherein said reagent that amplify the signal of the primary antibody is an antibody polymer.
196. The kit according to any of the claims 169 - 193, wherein said kit comprises a substance used to wash.
197. The kit according to any of the claims 196, wherein said substance used to wash is a wash buffer.
198. The kit according to claim 197, wherein said wash buffer is phosphate-buffered saline.
199. The kit according to any of the claims 196 - 198, wherein said wash buffer is 0.01M phosphate-buffered saline-0,05 % polyoxyethylene sorbitan monolaurate.
200. The kit according to any of the claims 169 - 199, wherein the kit further comprises water
201. The kit according to claim 200, wherein said water is d2H20.
202. The kit according to any of the claims 169 - 201, wherein said cancer cells are selected among any of the cancer cells of claim 117 - 129.
203. The kit according to any of the claims 169 -202, wherein said cancer is selected among any of the cancers of claim 155 - 167.
204. A method for treating cancer in an individual, wherein the method comprises use of a kit according to any one of claims 169 - 203.
205. The method according to claim 204, wherein said cancer is selected among any of the cancers of claim 155 - 167.
206. The method according to any of the preceding claims, wherein said cancer is a gastric cancer.
207. The method of any of the preceding claims, wherein said gastric cancer is a carcinoma.
208. The method according to any of the preceding claims, wherein said carcinoma is an adenocarcinoma.
209. The method according to any of the preceding claims, wherein said carcinoma is a cystadenocarcinoma.
210. The method according to any of the preceding claims, wherein said cancer is an ovarian cancer.
211. The method according to any of the preceding claims, wherein said ovarian cancer is an adenocarcinoma.
212. The method according to any of the preceding claims, wherein said adenocarcinoma is a serous adenocarcinoma.
213. The method according to any of the preceding claims, wherein said adenocarcinoma is a serous papillary adenocarcinoma.
214. The method according to any of the preceding claims, wherein said cancer is a neuroblastoma.
215. The method according to any of the preceding claims, wherein the distribution of cells is selected from the group consisting of diffuse and multifocal, preferably as determined by the methods of Example 2.
216. The method according to any of the preceding claims, wherein the incidence is 1, preferably 2, more preferred 3, preferably 4, preferably as determined by the methods of Example 2.
217. The method according to any of the preceding claims, wherein the incidence is 2-4, preferably 3-4, preferably as determined by the methods of Example 2.
218. The method according to any of the preceding claims, wherein the staining intensity is 1, preferably 2, more preferred 3, preferably 4, preferably as determined by the methods of Example 2.
219. The method according to any of the preceding claims, wherein the staining intensity is 2-4, preferably 3-4, preferably as determined by the methods of Example 2.
220. The method according to any of the preceding claims, wherein the staining is granular, preferably as determined by the methods of Example 2.
221. The method according to any of the preceding claims, wherein the staining is located on the membrane or cytoplasmic, preferably as determined by the methods of Example 2.
222. The method according to any of the preceding claims, wherein the staining is located on the membrane, preferably as determined by the methods of Example 2.
223. The method according to any of the preceding claims, wherein the subcellular location is selected from the group consisting of Granular (G), Membrane (M), Cytoplasmic (C), Extracellular (E), Vasculature (V), Intranuclear (In), Apical (A) and Basal (B), preferably as determined by the methods of Example 3.
224. The method according to any of the preceding claims, wherein the amount of positive tumor cells in percentage is selected from the group consisting of up to 29%, 30-49%, 50-74%, and 75-100%, preferably as determined by the methods of Example
3.
225. The method according to any of the preceding claims, wherein the amount of positive tumor cells in percentage is above 10%, above 20%, preferably above 30%, preferably as determined by the methods of Example 3.
226. The method according to any of the preceding claims, wherein the incidence of positive tumor cells is selected from the group consisting of normal, minimal, mild, moderate and severe, preferably as determined by the methods of Example 3.
227. The method according to any of the preceding claims, wherein the intensity of positive staining at cellular level is selected from the group consisting of equivocal, mild (weak), moderate, strong and intense, preferably as determined by the methods of Example 3.
228. The method according to any of the preceding claims, wherein the incidence of staining represents a mild to severe distribution of positive tumor cells, preferably as determined by the methods of Example 3.
229. An antibody or antigen-binding fragment thereof according to any of the preceding claims for use in the treatment of cancer, wherein the use comprises providing a biomarker of the efficacy and/or safety of the antibody or antigen- binding fragment thereof according to any of the preceding claims.
230. An antibody or antigen-binding fragment thereof according to any of the preceding claims for use in the treatment of cancer, wherein the treatment of said cancer with said antibody or antigen-binding fragment thereof is determined or confirmed by the method according to any one of the preceding claims.
231. An antibody or antigen-binding fragment thereof according to any of the preceding claims for use in the treatment of cancer, wherein the use comprises evaluation of the clinical benefit of the antibody or antigen-binding fragment thereof, comprising use of the method according to any of the preceding claims, and wherein the clinical benefit is the clinical benefit for a patient.
232. An antibody or antigen-binding fragment thereof according to any of the preceding claims for use in the treatment of cancer, wherein the use comprises providing a biomarker of the efficacy and/or safety of the antibody or antigen- binding fragment thereof in a tissue sample comprising cancer cells, comprising use of the method according to any of the preceding claims.
233. An antibody or antigen-binding fragment thereof according to any of the preceding claims for use in a method of diagnosis of cancer, wherein the use comprises providing a biomarker of the efficacy and/or safety of the antibody or antigen-binding fragment thereof according to any of the preceding claims.
234. An antibody or antigen-binding fragment thereof according to any of the preceding claims for use in a method of diagnosis of cancer, and wherein the diagnosis of said cancer with said antibody or antigen-binding fragment thereof is determined or confirmed by the method according to any one of the preceding claims.
235. An antibody or antigen-binding fragment thereof according to any of the preceding claims for use in a method of diagnosis of cancer, wherein the use comprises evaluation of the clinical benefit of the antibody or antigen-binding fragment thereof, comprising use of the method according to any of the preceding claims, and wherein the clinical benefit is the clinical benefit for a patient.
236. An antibody or antigen-binding fragment thereof according to any of the preceding claims for use in a method of diagnosis of cancer, wherein the use comprises providing a biomarker of the efficacy and/or safety of an antibody or antigen-binding fragment thereof in a tissue sample comprising cancer cells, comprising use of the method according to any of the preceding claims.
237. A method of treating ovarian cancer, comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any of the preceding claims.
238. The method according to claim 237, wherein said ovarian cancer is an adenocarcinoma.
239. The method according to claim 238, wherein said adenocarcinoma is a serous adenocarcinoma.
240. The method according to any of claims 238 - 239, wherein said adenocarcinoma is a serous papillary adenocarcinoma.
241. The method according to claim 237, wherein said ovarian cancer is selected from the group consisting of epithelial carcinoma, serous carcinoma, primary peritoneal carcinoma, clear-cell carcinoma, clear-cell adenocarcinoma, endometrioid, malignant mixed mijllerian tumor (carcinosarcoma), mucinous tumors, mucinous adenocarcinoma, pseudomyxoma peritonei, undifferentiated epithelial cancers, malignant Brenner tumor, transitional cell carcinoma, sex cord-stromal tumor, granulosa cell tumor, adult granulosa cell tumor, juvenile granulosa cell tumor, sertoli-Leydig cell tumor, sclerosing stromal tumors, germ cell tumor, dysgerminoma, choriocarcinoma, immature (solid) teratoma, mature teratoma (dermoid cyst), yolk sac tumor/endodermal sinus tumor, embryonal carcinoma, polyembryoma, squamous cell carcinoma, mixed tumors, secondary ovarian cancer, and borderline tumors.
242. A method of treating gastric cancer, comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any of the preceding claims.
243. The method according to claim 242, wherein said gastric cancer is a carcinoma.
244. The method according to claim 243, wherein said carcinoma is an adenocarcinoma.
245. The method according to any of claims 243 - 244, wherein said carcinoma is a cystadenocarcinoma.
246. The method according to claim 242, wherein said gastric cancer is selected from the group consisting of adenocarcinoma, lymphoma, gastrointestinal stromal tumor (GIST), carcinoid tumor, and other cancers.
247. An antibody or antigen binding fragment thereof, wherein said antibody or antigen-binding fragment comprises at least one of the sequences selected among a heavy chain variable region CDR1 according to SEQ ID No. 3, a heavy chain variable region CDR2 according to SEQ IN No. 4 or 19, a heavy chain variable region CD3 according to SEQ ID No. 5, a light chain variable region CDR1 according to SEQ ID No. 6, a light chain variable region CDR2 according to SEQ ID No. 7 and a light chain variable region CDR3 according to SEQ ID No. 8.
248. An antibody or antigen binding fragment thereof, wherein said antibody or antigen-binding fragment comprises a heavy chain sequence according to SEQ ID No. 1 and a light chain sequence according to SEQ ID No. 2.
249. An antibody or antigen binding fragment thereof, wherein said antibody or antigen binding fragment comprises a heavy chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about
93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No. 1 and/or a light chain sequence that is at least about 80 %, about 81 %, about 82 %, about 83 %, about 84 %, about 85 %, about 86 %, about 87 %, about 88 %, about 89 %, about 90 %, about 91 % about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about
97 %, about 98 % or about 99% sequence identity to the sequence set forth in SEQ ID No. 2.
250. The antibody or antigen binding fragment according to any of the preceding claims, wherein said antigen-binding fragment thereof is a single chain variable fragment (scFv).
251. The antibody or antigen binding fragment according to any of the preceding claims, wherein said scFv comprises a portion of the amino acid sequence set forth in SEQ ID No. 9, SEQ ID No. 13 or SEQ ID No. 14.
252. The antibody or antigen binding fragment according to any of the preceding claims, wherein said scFv comprises at least one of the amino acid substitutions K13E, R18Q, R45Q, K103E and K107E.
253. The antibody or antigen binding fragment according to any of the preceding claims, wherein said anti-B7-H3 antibody or antigen-binding fragment thereof is a murine 8H9 antibody or an antigen binding fragment thereof.
254. The antibody or antigen binding fragment according to any of the preceding claims, wherein said antibody or antigen-binding fragment thereof is humanized.
255. The antibody or antigen binding fragment according to any of the preceding claims, wherein said antibody or antigen-binding fragment thereof is a chimeric antibody or an antigen-binding fragment thereof.
256. The antibody or antigen binding fragment according to any of the preceding claims, wherein said antibody or antigen-binding fragment thereof is radiolabeled.
257. The antibody or antigen binding fragment according to any of the preceding claims, wherein said radiolabel is selected among a PET label and a SPECT label.
258. The antibody or antigen binding fragment according to any of the preceding claims, wherein said PET label is selected among 124l, 225Ac and 89Zr.
259. The antibody or antigen binding fragment according to any of the preceding claims, wherein said SPECT label is selected among 131l, 177Lu, 99mTc, 64Cu and 89Zr.
260. The antibody or antigen binding fragment according to any of the preceding claims, wherein said antibody or antigen-binding fragment thereof is conjugated to a chelator compound.
261. The antibody or antigen binding fragment according to any of the preceding claims, wherein said chelator compound is bound to a radioactive isotope.
262. The antibody or antigen binding fragment according to any of the preceding claims, wherein said radioactive isotope is selected among124l, 131l and 177Lu or 99mTc,
64Cu (chelated to NOTA) and 89Zr (chelated to DFO).
263. The antibody or antigen binding fragment according to any of the preceding claims, wherein said chelator compound is selected among DOTA, DTPA, NOTA and DFO.
264. The antibody or antigen binding fragment according to any of the preceding claims, wherein said DOTA is a variant of DOTA.
265. The antibody or antigen binding fragment according to any of the preceding claims, wherein said DTPA is a variant of DTPA.
266. An antibody or antigen-binding fragment thereof according to any of the preceding claims for use in the treatment of ovarian cancer.
267. An antibody or antigen-binding fragment thereof according to any of the preceding claims for use according to claim 266, wherein said ovarian cancer is an adenocarcinoma.
268. An antibody or antigen-binding fragment thereof according to any of the preceding claims for use according to claim 267, wherein said adenocarcinoma is a serous adenocarcinoma.
269. An antibody or antigen-binding fragment thereof according to any of the preceding claims for use according to any of claims 267 - 268, wherein said adenocarcinoma is a serous papillary adenocarcinoma.
270. An antibody or antigen-binding fragment thereof according to any of the preceding claims for use according to claim 266, wherein said ovarian cancer is selected from the group consisting of epithelial carcinoma, serous carcinoma, primary peritoneal carcinoma, clear-cell carcinoma, clear-cell adenocarcinoma,
endometrioid, malignant mixed mijllerian tumor (carcinosarcoma), mucinous tumors, mucinous adenocarcinoma, pseudomyxoma peritonei, undifferentiated epithelial cancers, malignant Brenner tumor, transitional cell carcinoma, sex cord-stromal tumor, granulosa cell tumor, adult granulosa cell tumor, juvenile granulosa cell tumor, sertoli-Leydig cell tumor, sclerosing stromal tumors, germ cell tumor, dysgerminoma, choriocarcinoma, immature (solid) teratoma, mature teratoma (dermoid cyst), yolk sac tumor/endodermal sinus tumor, embryonal carcinoma, polyembryoma, squamous cell carcinoma, mixed tumors, secondary ovarian cancer, and borderline tumors.
271. An antibody or antigen-binding fragment thereof according to any of the preceding claims for use in the treatment of gastric cancer.
272. An antibody or antigen-binding fragment thereof according to any of the preceding claims for use according to claim 271, wherein said gastric cancer is a carcinoma.
273. An antibody or antigen-binding fragment thereof according to any of the preceding claims for use according to claim 272, wherein said carcinoma is an adenocarcinoma
274. An antibody or antigen-binding fragment thereof according to any of the preceding claims for use according to any of claims 272 - 273, wherein said carcinoma is a cystadenocarcinoma.
275. An antibody or antigen-binding fragment thereof according to any of the preceding claims for use according to claim 271, wherein said gastric cancer is selected from the group consisting of adenocarcinoma, lymphoma, gastrointestinal stromal tumor (GIST), carcinoid tumor, and other cancers.
76. Use of an antibody or antigen-binding fragment thereof, according to any of the preceding claims, for the manufacture of a medicament for treatment of an indication according to any of the preceding claims.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP20765196.9A EP4022313A1 (en) | 2019-08-30 | 2020-08-20 | Immunohistochemical assessment of b7-h3 expression |
| TW110105983A TW202208855A (en) | 2019-08-30 | 2021-02-20 | Immunohistochemical assessment of b7-h3 expression |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201962893883P | 2019-08-30 | 2019-08-30 | |
| US62/893,883 | 2019-08-30 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2021037319A1 true WO2021037319A1 (en) | 2021-03-04 |
Family
ID=72340165
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/DK2020/050236 WO2021037319A1 (en) | 2019-08-30 | 2020-08-20 | Immunohistochemical assessment of b7-h3 expression |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP4022313A1 (en) |
| TW (1) | TW202208855A (en) |
| WO (1) | WO2021037319A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021213601A1 (en) * | 2020-04-24 | 2021-10-28 | Y-Mabs Therapeutics, Inc. | B7h3 antibodies with chelators |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002032375A2 (en) | 2000-10-18 | 2002-04-25 | Sloan-Kettering Institute For Cancer Research | Uses of monoclonal antibody 8h9 |
| WO2003033670A2 (en) | 2001-10-17 | 2003-04-24 | Sloan-Kettering Institute For Cancer Research | Method for preparation of single chain antibodies |
| WO2003075846A2 (en) | 2002-03-08 | 2003-09-18 | Sloan-Kettering Institute For Cancer Research | Uses of monoclonal antibody 8h9 |
| WO2008116219A2 (en) | 2007-03-22 | 2008-09-25 | Sloan-Kettering Institute For Cancer Research | Uses of monoclonal antibody 8h9 |
| WO2016033225A2 (en) | 2014-08-27 | 2016-03-03 | Memorial Sloan Kettering Cancer Center | Antibodies, compositions, and uses |
| WO2018209346A1 (en) | 2017-05-12 | 2018-11-15 | Memorial Sloan-Kettering Cancer Center | Use of anti-b7h3 antibodies for treating cancer in the central nervous system |
-
2020
- 2020-08-20 WO PCT/DK2020/050236 patent/WO2021037319A1/en unknown
- 2020-08-20 EP EP20765196.9A patent/EP4022313A1/en not_active Withdrawn
-
2021
- 2021-02-20 TW TW110105983A patent/TW202208855A/en unknown
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002032375A2 (en) | 2000-10-18 | 2002-04-25 | Sloan-Kettering Institute For Cancer Research | Uses of monoclonal antibody 8h9 |
| WO2003033670A2 (en) | 2001-10-17 | 2003-04-24 | Sloan-Kettering Institute For Cancer Research | Method for preparation of single chain antibodies |
| WO2003075846A2 (en) | 2002-03-08 | 2003-09-18 | Sloan-Kettering Institute For Cancer Research | Uses of monoclonal antibody 8h9 |
| WO2008116219A2 (en) | 2007-03-22 | 2008-09-25 | Sloan-Kettering Institute For Cancer Research | Uses of monoclonal antibody 8h9 |
| WO2016033225A2 (en) | 2014-08-27 | 2016-03-03 | Memorial Sloan Kettering Cancer Center | Antibodies, compositions, and uses |
| WO2018209346A1 (en) | 2017-05-12 | 2018-11-15 | Memorial Sloan-Kettering Cancer Center | Use of anti-b7h3 antibodies for treating cancer in the central nervous system |
Non-Patent Citations (12)
| Title |
|---|
| AHMED ET AL.: "Humanized Affinity-matured Monoclonal Antibody 8H9 Has Potent Antitumor Activity and Binds to FG Loop of Tumor Antigen B7-H3", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 290, no. 50, 11 December 2015 (2015-12-11), pages 30018 - 30029, XP055396132, DOI: 10.1074/jbc.M115.679852 |
| ANONYMOUS: "Dako EnVision + Dual Link System-HRP (DAB+) Code K4065", 10 August 2008 (2008-08-10), XP055750418, Retrieved from the Internet <URL:https://www.agilent.com/cs/library/packageinsert/public/PD04048EFG_01.pdf> [retrieved on 20201113] * |
| D. LOO ET AL: "Development of an Fc-Enhanced Anti-B7-H3 Monoclonal Antibody with Potent Antitumor Activity", CLINICAL CANCER RESEARCH, vol. 18, no. 14, 15 July 2012 (2012-07-15), pages 3834 - 3845, XP055092714, ISSN: 1078-0432, DOI: 10.1158/1078-0432.CCR-12-0715 * |
| JINGJING ZHANG ET AL: "B7-H3 is related to tumor progression in ovarian cancer", ONCOLOGY REPORTS, vol. 38, no. 4, 1 April 2017 (2017-04-01), pages 2426 - 2434, XP055748414, ISSN: 1021-335X, DOI: 10.3892/or.2017.5858 * |
| LOOS ET AL.: "Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer", BMC CANCER, 2009, pages 9 |
| LOOS MARTIN ET AL: "Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer", BMC CANCER, BIOMED CENTRAL, LONDON, GB, vol. 9, no. 1, 26 December 2009 (2009-12-26), pages 463, XP021067030, ISSN: 1471-2407, DOI: 10.1186/1471-2407-9-463 * |
| N. MAEDA ET AL: "Expression of B7-H3, a Potential Factor of Tumor Immune Evasion in Combination with the Number of Regulatory T Cells, Affects Against Recurrence-Free Survival in Breast Cancer Patients", ANNALS OF SURGICAL ONCOLOGY., vol. 21, no. S4, 1 December 2014 (2014-12-01), US, pages 546 - 554, XP055710370, ISSN: 1068-9265, DOI: 10.1245/s10434-014-3564-2 * |
| QIN ET AL.: "B7-H3 is a new cancer-specific endothelial marker in clear cell renal cell carcinoma", ONCOTARGETS AND THERAPY, vol. 2013, no. 6, 2013, pages 1667 - 1673, XP055219361, DOI: 10.2147/OTT.S53565 |
| XIAO QIN ET AL: "B7-H3 is a new cancer-specific endothelial marker in clear cell renal cell carcinoma", ONCOTARGETS AND THERAPY, vol. 6, 1 November 2013 (2013-11-01), pages 1667 - 1673, XP055219361, DOI: 10.2147/OTT.S53565 * |
| XINGXING ZANG ET AL: "Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas", MODERN PATHOLOGY, vol. 23, no. 8, 21 May 2010 (2010-05-21), GB, pages 1104 - 1112, XP055749749, ISSN: 0893-3952, DOI: 10.1038/modpathol.2010.95 * |
| ZANG ET AL.: "Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas", MOD PATHOL., 2010 |
| ZHIPING ZHOU ET AL: "B7-H3, a potential therapeutic target, is expressed in diffuse intrinsic pontine glioma", JOURNAL OF NEURO-ONCOLOGY, vol. 111, no. 3, 1 February 2013 (2013-02-01), pages 257 - 264, XP055127403, ISSN: 0167-594X, DOI: 10.1007/s11060-012-1021-2 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021213601A1 (en) * | 2020-04-24 | 2021-10-28 | Y-Mabs Therapeutics, Inc. | B7h3 antibodies with chelators |
Also Published As
| Publication number | Publication date |
|---|---|
| EP4022313A1 (en) | 2022-07-06 |
| TW202208855A (en) | 2022-03-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Arigami et al. | B7–h3 ligand expression by primary breast cancer and associated with regional nodal metastasis | |
| EP3018146A1 (en) | Monoclonal antibodies and diagnostic uses thereof | |
| Barrier et al. | Characterization of human leukocyte antigen-G (HLA-G) expression in endometrial adenocarcinoma | |
| WO2012141285A1 (en) | Biomarker for breast cancer | |
| US20120052071A1 (en) | Tumor markers and methods of use thereof | |
| US20170227543A1 (en) | Breast Tumor Markers And Methods Of Use Thereof | |
| US20150301059A1 (en) | B7-h3 as a biomarker for diagnosing the progression and early lymph node metastasis of cancer | |
| EP2494361B2 (en) | Ovary tumor markers and methods of use thereof | |
| US9625461B2 (en) | Method of detecting cancer using delta-catenin | |
| WO2021037319A1 (en) | Immunohistochemical assessment of b7-h3 expression | |
| JPWO2019159825A1 (en) | Biomarker predicting the effect of anti-PD-1 antibody / anti-PD-L1 antibody therapy | |
| EP2102655B1 (en) | In vitro method for diagnosing prostate cancer | |
| US20100081192A1 (en) | Early prostate cancer antigens (epca), polynucleotide sequences encoding them, and their use | |
| WO2020075325A1 (en) | Biomarker for predicting prognosis of endometrial cancer patient | |
| Nassar et al. | Estimation of hormone receptor status and HER2 in cytologic cell blocks from breast cancer using the novel rabbit monoclonal antibodies (SP1, SP2, and SP3) | |
| JP2006520912A (en) | Cancer prognosis detection method | |
| WO2013116537A1 (en) | A method to distinguish benign from malignant oncocytic cell tissue |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 20765196 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| ENP | Entry into the national phase |
Ref document number: 2020765196 Country of ref document: EP Effective date: 20220330 |