WO2022199555A1 - Protéine de liaison à un antigène multispécifique se liant à cd3 et son utilisation - Google Patents
Protéine de liaison à un antigène multispécifique se liant à cd3 et son utilisation Download PDFInfo
- Publication number
- WO2022199555A1 WO2022199555A1 PCT/CN2022/082129 CN2022082129W WO2022199555A1 WO 2022199555 A1 WO2022199555 A1 WO 2022199555A1 CN 2022082129 W CN2022082129 W CN 2022082129W WO 2022199555 A1 WO2022199555 A1 WO 2022199555A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- seq
- variable region
- chain variable
- amino acid
- antigen binding
- Prior art date
Links
- 230000027455 binding Effects 0.000 title claims abstract description 224
- 102000025171 antigen binding proteins Human genes 0.000 title claims abstract description 134
- 108091000831 antigen binding proteins Proteins 0.000 title claims abstract description 134
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 55
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 49
- 239000003814 drug Substances 0.000 claims abstract description 10
- 229940079593 drug Drugs 0.000 claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 230000003213 activating effect Effects 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 232
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 216
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 claims description 81
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 claims description 80
- 239000000427 antigen Substances 0.000 claims description 70
- 108091007433 antigens Proteins 0.000 claims description 67
- 102000036639 antigens Human genes 0.000 claims description 67
- 150000007523 nucleic acids Chemical class 0.000 claims description 54
- 239000013598 vector Substances 0.000 claims description 44
- 108020004707 nucleic acids Proteins 0.000 claims description 41
- 102000039446 nucleic acids Human genes 0.000 claims description 41
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 claims description 40
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 claims description 40
- 238000000034 method Methods 0.000 claims description 36
- 210000004881 tumor cell Anatomy 0.000 claims description 27
- 239000008194 pharmaceutical composition Substances 0.000 claims description 20
- 210000004408 hybridoma Anatomy 0.000 claims description 19
- -1 MAGE-1 Proteins 0.000 claims description 16
- 201000011510 cancer Diseases 0.000 claims description 12
- 239000003550 marker Substances 0.000 claims description 11
- 206010073071 hepatocellular carcinoma Diseases 0.000 claims description 9
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 claims description 8
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 claims description 8
- 230000004913 activation Effects 0.000 claims description 7
- 230000001580 bacterial effect Effects 0.000 claims description 7
- 230000002538 fungal effect Effects 0.000 claims description 7
- 102100024210 CD166 antigen Human genes 0.000 claims description 6
- 102100028757 Chondroitin sulfate proteoglycan 4 Human genes 0.000 claims description 6
- 206010009944 Colon cancer Diseases 0.000 claims description 6
- 101000916489 Homo sapiens Chondroitin sulfate proteoglycan 4 Proteins 0.000 claims description 6
- 101710120463 Prostate stem cell antigen Proteins 0.000 claims description 6
- 102100036735 Prostate stem cell antigen Human genes 0.000 claims description 6
- 102100029986 Receptor tyrosine-protein kinase erbB-3 Human genes 0.000 claims description 6
- 101710100969 Receptor tyrosine-protein kinase erbB-3 Proteins 0.000 claims description 6
- 231100000844 hepatocellular carcinoma Toxicity 0.000 claims description 6
- 230000003993 interaction Effects 0.000 claims description 6
- 208000014018 liver neoplasm Diseases 0.000 claims description 6
- 210000004962 mammalian cell Anatomy 0.000 claims description 6
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims description 6
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 claims description 6
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 claims description 5
- 230000006044 T cell activation Effects 0.000 claims description 5
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 5
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims description 5
- 230000002401 inhibitory effect Effects 0.000 claims description 5
- 230000008685 targeting Effects 0.000 claims description 5
- 210000005253 yeast cell Anatomy 0.000 claims description 5
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 4
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 claims description 4
- 206010006187 Breast cancer Diseases 0.000 claims description 4
- 208000026310 Breast neoplasm Diseases 0.000 claims description 4
- 241000196324 Embryophyta Species 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 claims description 4
- 241000700605 Viruses Species 0.000 claims description 4
- 239000002671 adjuvant Substances 0.000 claims description 4
- 150000002270 gangliosides Chemical class 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- BGFTWECWAICPDG-UHFFFAOYSA-N 2-[bis(4-chlorophenyl)methyl]-4-n-[3-[bis(4-chlorophenyl)methyl]-4-(dimethylamino)phenyl]-1-n,1-n-dimethylbenzene-1,4-diamine Chemical compound C1=C(C(C=2C=CC(Cl)=CC=2)C=2C=CC(Cl)=CC=2)C(N(C)C)=CC=C1NC(C=1)=CC=C(N(C)C)C=1C(C=1C=CC(Cl)=CC=1)C1=CC=C(Cl)C=C1 BGFTWECWAICPDG-UHFFFAOYSA-N 0.000 claims description 3
- 108010075348 Activated-Leukocyte Cell Adhesion Molecule Proteins 0.000 claims description 3
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 claims description 3
- 206010061424 Anal cancer Diseases 0.000 claims description 3
- 102100024003 Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 1 Human genes 0.000 claims description 3
- 108010008014 B-Cell Maturation Antigen Proteins 0.000 claims description 3
- 102000006942 B-Cell Maturation Antigen Human genes 0.000 claims description 3
- 102100027205 B-cell antigen receptor complex-associated protein alpha chain Human genes 0.000 claims description 3
- 101710095183 B-cell antigen receptor complex-associated protein alpha chain Proteins 0.000 claims description 3
- 102100027203 B-cell antigen receptor complex-associated protein beta chain Human genes 0.000 claims description 3
- 101710166261 B-cell antigen receptor complex-associated protein beta chain Proteins 0.000 claims description 3
- 102100038080 B-cell receptor CD22 Human genes 0.000 claims description 3
- 206010005003 Bladder cancer Diseases 0.000 claims description 3
- 102100035875 C-C chemokine receptor type 5 Human genes 0.000 claims description 3
- 101710149870 C-C chemokine receptor type 5 Proteins 0.000 claims description 3
- 102100036305 C-C chemokine receptor type 8 Human genes 0.000 claims description 3
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 claims description 3
- 108700012439 CA9 Proteins 0.000 claims description 3
- 102100027207 CD27 antigen Human genes 0.000 claims description 3
- 102100032912 CD44 antigen Human genes 0.000 claims description 3
- 108010058905 CD44v6 antigen Proteins 0.000 claims description 3
- 102100025222 CD63 antigen Human genes 0.000 claims description 3
- 102100027221 CD81 antigen Human genes 0.000 claims description 3
- 102000000905 Cadherin Human genes 0.000 claims description 3
- 108050007957 Cadherin Proteins 0.000 claims description 3
- 102100024423 Carbonic anhydrase 9 Human genes 0.000 claims description 3
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 3
- 108010009685 Cholinergic Receptors Proteins 0.000 claims description 3
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 3
- 241000689227 Cora <basidiomycete fungus> Species 0.000 claims description 3
- 102100034573 Desmoglein-4 Human genes 0.000 claims description 3
- 101710183213 Desmoglein-4 Proteins 0.000 claims description 3
- 102100025012 Dipeptidyl peptidase 4 Human genes 0.000 claims description 3
- 102000012804 EPCAM Human genes 0.000 claims description 3
- 101150084967 EPCAM gene Proteins 0.000 claims description 3
- 206010014733 Endometrial cancer Diseases 0.000 claims description 3
- 206010014759 Endometrial neoplasm Diseases 0.000 claims description 3
- 102100038083 Endosialin Human genes 0.000 claims description 3
- 101710144543 Endosialin Proteins 0.000 claims description 3
- 108010055196 EphA2 Receptor Proteins 0.000 claims description 3
- 102100030340 Ephrin type-A receptor 2 Human genes 0.000 claims description 3
- 208000000461 Esophageal Neoplasms Diseases 0.000 claims description 3
- 102100027844 Fibroblast growth factor receptor 4 Human genes 0.000 claims description 3
- 102100039554 Galectin-8 Human genes 0.000 claims description 3
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 claims description 3
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 claims description 3
- 102100020948 Growth hormone receptor Human genes 0.000 claims description 3
- 102100033365 Growth hormone-releasing hormone receptor Human genes 0.000 claims description 3
- 102000003693 Hedgehog Proteins Human genes 0.000 claims description 3
- 108090000031 Hedgehog Proteins Proteins 0.000 claims description 3
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 claims description 3
- 101000716063 Homo sapiens C-C chemokine receptor type 8 Proteins 0.000 claims description 3
- 101000922348 Homo sapiens C-X-C chemokine receptor type 4 Proteins 0.000 claims description 3
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 claims description 3
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 claims description 3
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 claims description 3
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 claims description 3
- 101000914324 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 5 Proteins 0.000 claims description 3
- 101000914321 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 7 Proteins 0.000 claims description 3
- 101000908391 Homo sapiens Dipeptidyl peptidase 4 Proteins 0.000 claims description 3
- 101000917134 Homo sapiens Fibroblast growth factor receptor 4 Proteins 0.000 claims description 3
- 101000608769 Homo sapiens Galectin-8 Proteins 0.000 claims description 3
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 claims description 3
- 101001075287 Homo sapiens Growth hormone receptor Proteins 0.000 claims description 3
- 101000997535 Homo sapiens Growth hormone-releasing hormone receptor Proteins 0.000 claims description 3
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 claims description 3
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 claims description 3
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 claims description 3
- 101000628547 Homo sapiens Metalloreductase STEAP1 Proteins 0.000 claims description 3
- 101001133056 Homo sapiens Mucin-1 Proteins 0.000 claims description 3
- 101001133081 Homo sapiens Mucin-2 Proteins 0.000 claims description 3
- 101000972284 Homo sapiens Mucin-3A Proteins 0.000 claims description 3
- 101000972286 Homo sapiens Mucin-4 Proteins 0.000 claims description 3
- 101000972282 Homo sapiens Mucin-5AC Proteins 0.000 claims description 3
- 101000972276 Homo sapiens Mucin-5B Proteins 0.000 claims description 3
- 101000972273 Homo sapiens Mucin-7 Proteins 0.000 claims description 3
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 claims description 3
- 101000617725 Homo sapiens Pregnancy-specific beta-1-glycoprotein 2 Proteins 0.000 claims description 3
- 101000880769 Homo sapiens Protein SSX1 Proteins 0.000 claims description 3
- 101000880770 Homo sapiens Protein SSX2 Proteins 0.000 claims description 3
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 claims description 3
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 claims description 3
- 101000851018 Homo sapiens Vascular endothelial growth factor receptor 1 Proteins 0.000 claims description 3
- 101000851007 Homo sapiens Vascular endothelial growth factor receptor 2 Proteins 0.000 claims description 3
- 102100037852 Insulin-like growth factor I Human genes 0.000 claims description 3
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 claims description 3
- 102100037792 Interleukin-6 receptor subunit alpha Human genes 0.000 claims description 3
- 102100037795 Interleukin-6 receptor subunit beta Human genes 0.000 claims description 3
- 101710152369 Interleukin-6 receptor subunit beta Proteins 0.000 claims description 3
- 208000008839 Kidney Neoplasms Diseases 0.000 claims description 3
- 102100022430 Melanocyte protein PMEL Human genes 0.000 claims description 3
- 102000003735 Mesothelin Human genes 0.000 claims description 3
- 108090000015 Mesothelin Proteins 0.000 claims description 3
- 102100026712 Metalloreductase STEAP1 Human genes 0.000 claims description 3
- 102000036436 Metzincins Human genes 0.000 claims description 3
- 108091007161 Metzincins Proteins 0.000 claims description 3
- 102100034256 Mucin-1 Human genes 0.000 claims description 3
- 102100023123 Mucin-16 Human genes 0.000 claims description 3
- 102100034263 Mucin-2 Human genes 0.000 claims description 3
- 102100022497 Mucin-3A Human genes 0.000 claims description 3
- 102100022693 Mucin-4 Human genes 0.000 claims description 3
- 102100022494 Mucin-5B Human genes 0.000 claims description 3
- 102100022492 Mucin-7 Human genes 0.000 claims description 3
- 101100046559 Mus musculus Tnfrsf12a gene Proteins 0.000 claims description 3
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 claims description 3
- KUIFHYPNNRVEKZ-VIJRYAKMSA-N O-(N-acetyl-alpha-D-galactosaminyl)-L-threonine Chemical compound OC(=O)[C@@H](N)[C@@H](C)O[C@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1NC(C)=O KUIFHYPNNRVEKZ-VIJRYAKMSA-N 0.000 claims description 3
- 206010030155 Oesophageal carcinoma Diseases 0.000 claims description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 3
- 206010033128 Ovarian cancer Diseases 0.000 claims description 3
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 3
- 102000012850 Patched-1 Receptor Human genes 0.000 claims description 3
- 108010065129 Patched-1 Receptor Proteins 0.000 claims description 3
- 102100021768 Phosphoserine aminotransferase Human genes 0.000 claims description 3
- 108010069381 Platelet Endothelial Cell Adhesion Molecule-1 Proteins 0.000 claims description 3
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 claims description 3
- 102100022019 Pregnancy-specific beta-1-glycoprotein 2 Human genes 0.000 claims description 3
- 206010060862 Prostate cancer Diseases 0.000 claims description 3
- 108010072866 Prostate-Specific Antigen Proteins 0.000 claims description 3
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 3
- 102100037687 Protein SSX1 Human genes 0.000 claims description 3
- 102100037686 Protein SSX2 Human genes 0.000 claims description 3
- 102100029981 Receptor tyrosine-protein kinase erbB-4 Human genes 0.000 claims description 3
- 101710100963 Receptor tyrosine-protein kinase erbB-4 Proteins 0.000 claims description 3
- 208000015634 Rectal Neoplasms Diseases 0.000 claims description 3
- 206010038389 Renal cancer Diseases 0.000 claims description 3
- 206010061934 Salivary gland cancer Diseases 0.000 claims description 3
- 206010041067 Small cell lung cancer Diseases 0.000 claims description 3
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 3
- 101800001271 Surface protein Proteins 0.000 claims description 3
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 claims description 3
- 101150057140 TACSTD1 gene Proteins 0.000 claims description 3
- 102100033455 TGF-beta receptor type-2 Human genes 0.000 claims description 3
- 102000016946 TWEAK Receptor Human genes 0.000 claims description 3
- 108010014401 TWEAK Receptor Proteins 0.000 claims description 3
- 101150117918 Tacstd2 gene Proteins 0.000 claims description 3
- 208000024770 Thyroid neoplasm Diseases 0.000 claims description 3
- 108010082684 Transforming Growth Factor-beta Type II Receptor Proteins 0.000 claims description 3
- 102100033733 Tumor necrosis factor receptor superfamily member 1B Human genes 0.000 claims description 3
- 101710187830 Tumor necrosis factor receptor superfamily member 1B Proteins 0.000 claims description 3
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 claims description 3
- 102100027212 Tumor-associated calcium signal transducer 2 Human genes 0.000 claims description 3
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 3
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 3
- 208000002495 Uterine Neoplasms Diseases 0.000 claims description 3
- 102100033178 Vascular endothelial growth factor receptor 1 Human genes 0.000 claims description 3
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 claims description 3
- 206010047741 Vulval cancer Diseases 0.000 claims description 3
- 102000034337 acetylcholine receptors Human genes 0.000 claims description 3
- SRHNADOZAAWYLV-XLMUYGLTSA-N alpha-L-Fucp-(1->2)-beta-D-Galp-(1->4)-[alpha-L-Fucp-(1->3)]-beta-D-GlcpNAc Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@H]2[C@@H]([C@@H](NC(C)=O)[C@H](O)O[C@@H]2CO)O[C@H]2[C@H]([C@H](O)[C@H](O)[C@H](C)O2)O)O[C@H](CO)[C@H](O)[C@@H]1O SRHNADOZAAWYLV-XLMUYGLTSA-N 0.000 claims description 3
- 201000007538 anal carcinoma Diseases 0.000 claims description 3
- 201000010881 cervical cancer Diseases 0.000 claims description 3
- 208000029742 colonic neoplasm Diseases 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 108010087914 epidermal growth factor receptor VIII Proteins 0.000 claims description 3
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 claims description 3
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 claims description 3
- 201000004101 esophageal cancer Diseases 0.000 claims description 3
- 230000001605 fetal effect Effects 0.000 claims description 3
- 210000002950 fibroblast Anatomy 0.000 claims description 3
- 125000002446 fucosyl group Chemical group C1([C@@H](O)[C@H](O)[C@H](O)[C@@H](O1)C)* 0.000 claims description 3
- 206010017758 gastric cancer Diseases 0.000 claims description 3
- 208000005017 glioblastoma Diseases 0.000 claims description 3
- 201000010536 head and neck cancer Diseases 0.000 claims description 3
- 208000014829 head and neck neoplasm Diseases 0.000 claims description 3
- 108040006858 interleukin-6 receptor activity proteins Proteins 0.000 claims description 3
- 201000010982 kidney cancer Diseases 0.000 claims description 3
- 201000007270 liver cancer Diseases 0.000 claims description 3
- 201000005249 lung adenocarcinoma Diseases 0.000 claims description 3
- 201000005243 lung squamous cell carcinoma Diseases 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 210000004379 membrane Anatomy 0.000 claims description 3
- QCQYVCMYGCHVMR-AAZUGDAUSA-N n-[(2r,3r,4s,5r)-4,5,6-trihydroxy-1-oxo-3-[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyhexan-2-yl]acetamide Chemical compound CC(=O)N[C@@H](C=O)[C@H]([C@@H](O)[C@H](O)CO)O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O QCQYVCMYGCHVMR-AAZUGDAUSA-N 0.000 claims description 3
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 claims description 3
- 210000000496 pancreas Anatomy 0.000 claims description 3
- 208000030940 penile carcinoma Diseases 0.000 claims description 3
- 201000008174 penis carcinoma Diseases 0.000 claims description 3
- 201000002628 peritoneum cancer Diseases 0.000 claims description 3
- 210000004180 plasmocyte Anatomy 0.000 claims description 3
- 229920001481 poly(stearyl methacrylate) Polymers 0.000 claims description 3
- 108040000983 polyphosphate:AMP phosphotransferase activity proteins Proteins 0.000 claims description 3
- 239000002243 precursor Substances 0.000 claims description 3
- 230000001737 promoting effect Effects 0.000 claims description 3
- 102000005962 receptors Human genes 0.000 claims description 3
- 108020003175 receptors Proteins 0.000 claims description 3
- 238000003259 recombinant expression Methods 0.000 claims description 3
- 206010038038 rectal cancer Diseases 0.000 claims description 3
- 201000001275 rectum cancer Diseases 0.000 claims description 3
- 201000003804 salivary gland carcinoma Diseases 0.000 claims description 3
- 208000000587 small cell lung carcinoma Diseases 0.000 claims description 3
- 206010041823 squamous cell carcinoma Diseases 0.000 claims description 3
- 201000011549 stomach cancer Diseases 0.000 claims description 3
- 101150047061 tag-72 gene Proteins 0.000 claims description 3
- 201000002510 thyroid cancer Diseases 0.000 claims description 3
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 3
- 230000002485 urinary effect Effects 0.000 claims description 3
- 206010046766 uterine cancer Diseases 0.000 claims description 3
- 201000005102 vulva cancer Diseases 0.000 claims description 3
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 claims 19
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 claims 19
- 101000946863 Homo sapiens T-cell surface glycoprotein CD3 delta chain Proteins 0.000 description 92
- 102100035891 T-cell surface glycoprotein CD3 delta chain Human genes 0.000 description 92
- 108090000765 processed proteins & peptides Proteins 0.000 description 71
- 102000004196 processed proteins & peptides Human genes 0.000 description 70
- 229920001184 polypeptide Polymers 0.000 description 69
- 239000012634 fragment Substances 0.000 description 51
- 108090000623 proteins and genes Proteins 0.000 description 46
- 229940022353 herceptin Drugs 0.000 description 33
- 239000006228 supernatant Substances 0.000 description 33
- 229960004641 rituximab Drugs 0.000 description 31
- 229940127174 UCHT1 Drugs 0.000 description 30
- 239000000203 mixture Substances 0.000 description 27
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 26
- 230000014509 gene expression Effects 0.000 description 25
- 235000001014 amino acid Nutrition 0.000 description 24
- 230000002147 killing effect Effects 0.000 description 24
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 24
- 102000004169 proteins and genes Human genes 0.000 description 24
- 241000699666 Mus <mouse, genus> Species 0.000 description 22
- 150000001413 amino acids Chemical class 0.000 description 22
- 239000013612 plasmid Substances 0.000 description 22
- 241000700159 Rattus Species 0.000 description 21
- 235000018102 proteins Nutrition 0.000 description 21
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 16
- 108090001005 Interleukin-6 Proteins 0.000 description 15
- 102000004889 Interleukin-6 Human genes 0.000 description 15
- 230000000694 effects Effects 0.000 description 13
- 238000000684 flow cytometry Methods 0.000 description 13
- 239000012980 RPMI-1640 medium Substances 0.000 description 12
- 230000022534 cell killing Effects 0.000 description 12
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 238000010186 staining Methods 0.000 description 12
- 108020004414 DNA Proteins 0.000 description 11
- 102000001398 Granzyme Human genes 0.000 description 11
- 108060005986 Granzyme Proteins 0.000 description 11
- 102000004503 Perforin Human genes 0.000 description 11
- 108010056995 Perforin Proteins 0.000 description 11
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 11
- 239000003153 chemical reaction reagent Substances 0.000 description 11
- 206010052015 cytokine release syndrome Diseases 0.000 description 11
- 239000002609 medium Substances 0.000 description 11
- 229930192851 perforin Natural products 0.000 description 11
- 102000004127 Cytokines Human genes 0.000 description 10
- 108090000695 Cytokines Proteins 0.000 description 10
- 229920002873 Polyethylenimine Polymers 0.000 description 10
- 238000001890 transfection Methods 0.000 description 9
- 102100035360 Cerebellar degeneration-related antigen 1 Human genes 0.000 description 8
- 108060003951 Immunoglobulin Proteins 0.000 description 8
- 108091008874 T cell receptors Proteins 0.000 description 8
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 8
- 102000018358 immunoglobulin Human genes 0.000 description 8
- 230000001404 mediated effect Effects 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 229950010131 puromycin Drugs 0.000 description 8
- 238000006467 substitution reaction Methods 0.000 description 8
- 238000001262 western blot Methods 0.000 description 8
- 238000001994 activation Methods 0.000 description 7
- 231100000433 cytotoxic Toxicity 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 239000013604 expression vector Substances 0.000 description 7
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 6
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 6
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 6
- 230000003013 cytotoxicity Effects 0.000 description 6
- 231100000135 cytotoxicity Toxicity 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 239000002773 nucleotide Substances 0.000 description 6
- 125000003729 nucleotide group Chemical group 0.000 description 6
- 230000002829 reductive effect Effects 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 239000006152 selective media Substances 0.000 description 6
- 101000946860 Homo sapiens T-cell surface glycoprotein CD3 epsilon chain Proteins 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 102100035794 T-cell surface glycoprotein CD3 epsilon chain Human genes 0.000 description 5
- 210000004899 c-terminal region Anatomy 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 230000001472 cytotoxic effect Effects 0.000 description 5
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 5
- 239000000833 heterodimer Substances 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 4
- 108010019160 Pancreatin Proteins 0.000 description 4
- 239000004698 Polyethylene Substances 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 230000009227 antibody-mediated cytotoxicity Effects 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 238000002737 cell proliferation kit Methods 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 210000004698 lymphocyte Anatomy 0.000 description 4
- 229940055695 pancreatin Drugs 0.000 description 4
- 108091033319 polynucleotide Proteins 0.000 description 4
- 102000040430 polynucleotide Human genes 0.000 description 4
- 239000002157 polynucleotide Substances 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000012800 visualization Methods 0.000 description 4
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 238000008157 ELISA kit Methods 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 3
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 3
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 206010035226 Plasma cell myeloma Diseases 0.000 description 3
- 108010028230 Trp-Ser- His-Pro-Gln-Phe-Glu-Lys Proteins 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- 210000001185 bone marrow Anatomy 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000012149 elution buffer Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 201000000050 myeloid neoplasm Diseases 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 230000003285 pharmacodynamic effect Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- 230000035899 viability Effects 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
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 2
- 208000003950 B-cell lymphoma Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 102100029098 Hypoxanthine-guanine phosphoribosyltransferase Human genes 0.000 description 2
- 238000012404 In vitro experiment Methods 0.000 description 2
- 102100037850 Interferon gamma Human genes 0.000 description 2
- 108010074328 Interferon-gamma Proteins 0.000 description 2
- 108090000174 Interleukin-10 Proteins 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 239000012515 MabSelect SuRe Substances 0.000 description 2
- 101710160107 Outer membrane protein A Proteins 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 108010022394 Threonine synthase Proteins 0.000 description 2
- 108010020764 Transposases Proteins 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 229960003896 aminopterin Drugs 0.000 description 2
- 230000030741 antigen processing and presentation Effects 0.000 description 2
- 210000004436 artificial bacterial chromosome Anatomy 0.000 description 2
- 210000004507 artificial chromosome Anatomy 0.000 description 2
- 210000001106 artificial yeast chromosome Anatomy 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 125000000151 cysteine group Chemical class N[C@@H](CS)C(=O)* 0.000 description 2
- 102000004419 dihydrofolate reductase Human genes 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000012982 microporous membrane Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- MFBOGIVSZKQAPD-UHFFFAOYSA-M sodium butyrate Chemical compound [Na+].CCCC([O-])=O MFBOGIVSZKQAPD-UHFFFAOYSA-M 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 210000000225 synapse Anatomy 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 239000013603 viral vector Substances 0.000 description 2
- AUTOLBMXDDTRRT-JGVFFNPUSA-N (4R,5S)-dethiobiotin Chemical compound C[C@@H]1NC(=O)N[C@@H]1CCCCCC(O)=O AUTOLBMXDDTRRT-JGVFFNPUSA-N 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- 108010032595 Antibody Binding Sites Proteins 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- VPAXJOUATWLOPR-UHFFFAOYSA-N Conferone Chemical compound C1=CC(=O)OC2=CC(OCC3C4(C)CCC(=O)C(C)(C)C4CC=C3C)=CC=C21 VPAXJOUATWLOPR-UHFFFAOYSA-N 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 101150029707 ERBB2 gene Proteins 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 241001524679 Escherichia virus M13 Species 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101001076408 Homo sapiens Interleukin-6 Proteins 0.000 description 1
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 1
- 101100495232 Homo sapiens MS4A1 gene Proteins 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 1
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 1
- 102000012745 Immunoglobulin Subunits Human genes 0.000 description 1
- 108010079585 Immunoglobulin Subunits Proteins 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 208000009052 Precursor T-Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 230000005867 T cell response Effects 0.000 description 1
- 208000029052 T-cell acute lymphoblastic leukemia Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 201000011186 acute T cell leukemia Diseases 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000003452 antibody preparation method Methods 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003429 antifungal agent Substances 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
- 239000000872 buffer Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000005859 cell recognition Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000012412 chemical coupling Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- JECGPMYZUFFYJW-UHFFFAOYSA-N conferone Natural products CC1=CCC2C(C)(C)C(=O)CCC2(C)C1COc3cccc4C=CC(=O)Oc34 JECGPMYZUFFYJW-UHFFFAOYSA-N 0.000 description 1
- 230000004940 costimulation Effects 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 239000000412 dendrimer Substances 0.000 description 1
- 229920000736 dendritic polymer Polymers 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000002612 dispersion medium Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 108700020302 erbB-2 Genes Proteins 0.000 description 1
- 239000012527 feed solution Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002489 hematologic effect Effects 0.000 description 1
- 102000051957 human ERBB2 Human genes 0.000 description 1
- 102000052611 human IL6 Human genes 0.000 description 1
- 210000004754 hybrid cell Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 102000028557 immunoglobulin binding proteins Human genes 0.000 description 1
- 108091009323 immunoglobulin binding proteins Proteins 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 210000004882 non-tumor cell Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000000269 nucleophilic effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- 150000003017 phosphorus Chemical class 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 239000012521 purified sample Substances 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003153 stable transfection Methods 0.000 description 1
- 238000012414 sterilization procedure Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 230000005909 tumor killing Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Images
Classifications
-
- 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
- C07K16/2809—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 the T-cell receptor (TcR)-CD3 complex
-
- 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
- C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
-
- 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
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- T cell engager bispecific antibody is a typical bispecific antibody. It may form an immune synapse by simultaneously binding a T cell surface antigen (such as CD3) and a tumor cell surface antigen (such as CD19) to form an immune synapse. The distance between the tumor cells and T cells may be shortened by the bispecific antibody, followed by direct activation and proliferation of T cells, and activated T cells directly kill tumor cells and/or release cytotoxins to kill tumor cells. Hence, activation process of T cell by bispecific antibody does not involve presentation of tumor antigens to T cells to generate specific T lymphocyte clones. Therefore, the process is not restricted by MHC or HLA and is highly applicable in clinical use.
- CD3 Cluster of Differentiation 3
- CD3 Cluster of Differentiation 3
- CD3 Cluster of Differentiation 3
- the first generation of anti-CD3 drugs has achieved great success.
- improvements are needed to reasonably control activation of T cells and/or release of cytokines in order to improve safety (for example, to avoid the cytokine release syndrome (CRS) ) .
- CRS cytokine release syndrome
- Reduced incidence of CRS will not only promote therapeutic application of T cell engager bispecific antibody in hematological cancers, but achieve breakthrough in solid tumor applications.
- the present application provides a multispecific antigen binding protein comprising a binding portion against CD3, and the binding portion against CD3 comprises HCDR1, HCDR2 and HCDR3 regions from the heavy chain variable region VH of SEQ ID NO: 8 and/or LCDR1, LCDR2 and LCDR3 regions from the light chain variable region VH of SEQ ID NO: 7.
- the multispecific antigen binding protein may further comprise a binding portion against a tumor associated antigen (TAA) .
- TAA tumor associated antigen
- the multispecific antigen binding protein of present application may be used in manufacture of a drug for preventing, relieving and/or treating tumor.
- the multispecific antigen binding protein of present application may exhibit more effective killing of tumor cells.
- the multispecific antigen binding protein of present application may exhibit improved safety profile for the administrated subject, for example, may induce less T-cell mediated cytokines release.
- the multispecific antigen binding protein of present application and/or the binding portion against CD3 exhibit significantly enhanced binding affinity to CD3.
- the present application provides a multispecific antigen binding protein comprising a CD3 binding portion, wherein the CD3 binding portion comprises an amino acid sequence having at least 95%identity to heavy chain variable region HCDR1, HCDR2 and HCDR3, and the HCDR1 has an amino acid sequence as set forth in any one of SEQ ID NO: 4, 37, 45 and 53; the HCDR2 has an amino acid sequence as set forth in any one of SEQ ID NO: 5, 38, 46 and 54; the HCDR3 has an amino acid sequence as set forth in any one of SEQ ID NO: and 6, 39, 47 and 55, and/or, the CD3 binding portion comprises an amino acid sequence having at least 95%identity to light chain variable region LCDR1, LCDR2 and LCDR3, and the LCDR1 has an amino acid sequence as set forth in any one of SEQ ID NO: 1, 40, 48 and 56; the LCDR2 has an amino acid sequence as set forth in any one of SEQ ID NO: 2, 41, 49 and 57; the LCDR3 has an amino acid sequence as
- the CD3 binding portion comprises heavy chain variable region HCDR1, HCDR2 and HCDR3, and the HCDR1 has an amino acid sequence as set forth in any one of SEQ ID NO: 4, 37, 45 and 53; the HCDR2 has an amino acid sequence as set forth in any one of SEQ ID NO: 5, 38, 46 and 54; the HCDR3 has an amino acid sequence as set forth in any one of SEQ ID NO: 6, 39, 47 and 55, and/or, the CD3 binding portion comprises light chain variable region LCDR1, LCDR2 and LCDR3, and the LCDR1 has an amino acid sequence as set forth in any one of SEQ ID NO: 1, 40, 48 and 56; the LCDR2 has an amino acid sequence as set forth in any one of SEQ ID NO: 2, 41, 49 and 57; the LCDR3 has an amino acid sequence as set forth in any one of SEQ ID NO: 3, 42, 50 and 58.
- the heavy chain variable region HCDR1, HCDR2 and HCDR3 have the sequences as set forth in SEQ ID NO: 4, 5 and 6 respectively, and/or light chain variable region LCDR1, LCDR2 and LCDR3 have the sequences as set forth in SEQ ID NO: 1, 2 and 3 respectively;
- the heavy chain variable region HCDR1, HCDR2 and HCDR3 have the sequences as set forth in SEQ ID NO: 37, 38 and 39 respectively, and/or light chain variable region LCDR1, LCDR2 and LCDR3 have the sequences as set forth in SEQ ID NO: 40, 41 and 42 respectively;
- the heavy chain variable region HCDR1, HCDR2 and HCDR3 have the sequences as set forth in SEQ ID NO: 45, 46 and 47 respectively, and/or light chain variable region LCDR1, LCDR2 and LCDR3 have the sequences as set forth in SEQ ID NO: 48, 49 and 50 respectively; or, the heavy chain variable region HCDR1, HCDR2 and HCDR3 have the sequences as set forth
- the CD3 binding portion comprises an amino acid sequence having at least 80%identity to heavy chain variable region, and the heavy chain variable region has an amino acid sequence as set forth in any one of SEQ ID NO: 8, 35, 43 and 51; and/or, the CD3 binding portion comprises an amino acid sequence having at least 80%identity to light chain variable region, and the light chain variable region has an amino acid sequence as set forth in any one of SEQ ID NO: 7, 36, 44 and 52.
- the heavy chain variable region has an amino acid sequence as set forth in any one of SEQ ID NO: 8, 35, 43 and 51; and/or, the light chain variable region has an amino acid sequence as set forth in any one of SEQ ID NO: 7, 36, 44 and 52.
- the CD3 binding portion comprises an amino acid sequence having at least 80%identity to heavy chain variable region of SEQ ID NO: 8, and/or light chain variable region of SEQ ID NO: 7; the CD3 binding portion comprises an amino acid sequence having at least 80%identity to heavy chain variable region of SEQ ID NO: 35, and/or light chain variable region of SEQ ID NO: 36; the CD3 binding portion comprises an amino acid sequence having at least 80%identity to heavy chain variable region of SEQ ID NO: 43, and/or light chain variable region of SEQ ID NO: 44; or, the CD3 binding portion comprises an amino acid sequence having at least 80%identity to heavy chain variable region of SEQ ID NO: 51, and/or light chain variable region of SEQ ID NO: 52.
- the CD3 binding portion comprises a heavy chain variable region of SEQ ID NO: 8. and/or a light chain variable region of SEQ ID NO: 7; the CD3 binding portion comprises a heavy chain variable region of SEQ ID NO: 35, and/or a light chain variable region of SEQ ID NO: 36; the CD3 binding portion comprises a heavy chain variable region of SEQ ID NO: 43, and/or a light chain variable region of SEQ ID NO: 44; or, the CD3 binding portion comprises a heavy chain variable region of SEQ ID NO: 51, and/or a light chain variable region of SEQ ID NO: 52.
- the VH comprises framework regions H-FR1, H-FR2, H-FR3 and H-FR4.
- the binding portion against CD3 comprises an antibody heavy chain constant region.
- the binding portion against CD3 comprises an antibody heavy chain constant region derived from IgA, IgG, IgD, IgE or IgM antibody.
- the antibody heavy chain constant region comprises an IgG constant region.
- the antibody heavy chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 18 or SEQ ID NO: 26.
- the VL comprises framework regions L-FR1, L-FR2, L-FR3 and L-FR4.
- the binding portion against CD3 comprises an antibody light chain constant region.
- the binding portion against CD3 comprises an antibody light chain constant region derived from IgA, IgG, IgD, IgE or IgM antibody.
- the antibody light chain constant region comprises an Ig ⁇ constant region.
- the antibody light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 17 OR SEQ ID NO: 25.
- the binding portion against CD3 comprises an antibody or an antigen binding fragment thereof.
- the CD3 binding portion of the multispecific antigen binding protein is humanized.
- the humanized binding portion against CD3 comprises an amino acid sequence having at least 80%identity to the heavy chain variable region of SEQ ID NO: 27, and/or the light chain variable region of SEQ ID NO: 31.
- the humanized binding portion against CD3 comprises an amino acid sequence having at least 80%identity to the heavy chain variable region of SEQ ID NO: 28, and/or the light chain variable region of SEQ ID NO: 31.
- the humanized binding portion against CD3 comprises a heavy chain variable region of SEQ ID NO: 27, and/or a light chain variable region of SEQ ID NO: 31.
- the humanized binding portion against CD3 comprises a heavy chain variable region of SEQ ID NO: 28, and/or a light chain variable region of SEQ ID NO: 31.
- the antigen binding fragment comprises Fab, Fab’, F (ab) 2, Fv fragment, F (ab’) 2, scFv, di-scFv and/or dAb.
- the antibody is selected from a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.
- the multispecific antigen binding protein is produced by recombinant expression of host cell.
- the host cell expressing the multispecific antigen binding protein is selected from a bacterial cell, a fungal cell, a plant cell, a mammalian cell or a virus.
- the bacterial cell is E. coli.
- the fungal cell is yeast cell.
- the mammalian cell is selected from CHO, NS0, BHK, or HEK293 cell.
- the multispecific antigen binding protein is produced by hybridoma cells.
- the hybridoma cell is selected from mouse, rat, or rabbit.
- the format of multispecific antigen binding protein is selected from a bispecific antibody, a bispecific diabody, a bispecific scFv, a TandAb, a trivalent binding molecule, or a tetravalent binding molecule.
- the multispecific antigen binding protein comprises a binding portion against at least one tumor associated antigen (TAA) .
- TAA tumor associated antigen
- the TAA of the multispecific antigen binding protein is selected from the group comprising EPCAM, CCR5, CD19, HER2, HER3 neu, HER3, HER4, EGFR, PSMA, CEA, MUC1, MUC2, MUC3, MUC4, MUC5, MUC7, ⁇ hCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3, 9-O-Acetyl-GD3, GM2, globo H, fucosyl GM1, Poly SA, GD2, RON, c-Met, CEACAM-6, PCTA-1, PSA, PAP, ALCAM (CD166) , PECAM-1, CD151, MAGE-1, TROP2, IGF1 R.
- EPCAM EPCAM
- CCR5 CD19
- HER2, HER3 neu HER3, HER4, EGFR
- PSMA PSMA
- CEA MUC1, MUC2, MUC3, MUC4, MUC5, MUC7, ⁇ hCG, Lewis-Y, CD20, CD33, CD30
- the multispecific antigen binding protein comprises a binding portion against HER2.
- the binding portion against HER2 of the multispecific antigen binding protein comprises an amino acid sequence having at least 95%identity to heavy chain variable region HCDR1, HCDR2 and HCDR3 having the sequences as set forth in SEQ ID NO: 12, 13 and 14 respectively, and/or light chain variable region LCDR1, LCDR2 and LCDR3 having the sequences as set forth in SEQ ID NO: 9, 10 and 11 respectively.
- the binding portion against HER2 of the multispecific antigen binding protein comprises heavy chain variable region HCDR1, HCDR2 and HCDR3 having the sequences as set forth in SEQ ID NO: 12, 13 and 14 respectively, and/or light chain variable region LCDR1, LCDR2 and LCDR3 having the sequences as set forth in SEQ ID NO: 9, 10 and 11 respectively, wherein the CD3 binding portion induce T cell activation.
- the binding portion against HER2 of the multispecific antigen binding protein comprises an amino acid sequence having at least 80%identity to heavy chain variable region of SEQ ID NO: 16, and/or a light chain variable region of SEQ ID NO: 15.
- the binding portion against HER2 of the multispecific antigen binding protein comprises a heavy chain variable region of SEQ ID NO: 16, and/or a light chain variable region of SEQ ID NO: 15.
- the binding portion against HER2 comprises an antibody heavy chain constant region.
- the binding portion against HER2 comprises an antibody heavy chain constant region derived from IgA, IgG, IgD, IgE or IgM antibody.
- the antibody heavy chain constant region comprises an IgG constant region.
- the antibody heavy chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 18 or SEQ ID NO: 26.
- the binding portion against HER2 comprises an antibody light chain constant region.
- the binding portion against HER2 comprises an antibody light chain constant region derived from IgA, IgG, IgD, IgE or IgM antibody.
- the HER2 antibody light chain constant region comprises an Ig ⁇ constant region.
- the HER2 antibody light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 17 or SEQ ID NO: 25.
- the binding portion against HER2 comprises an antibody or an antigen binding fragment thereof.
- the antigen binding fragment against HER2 comprises Fab, Fab’, F (ab) 2, Fv fragment, F (ab’) 2, scFv, di-scFv and/or dAb.
- the antibody against HER2 is selected from a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.
- the multispecific antigen binding protein comprises a binding portion against CD20.
- the heavy chain variable region VH of the binding portion against CD20 comprises an amino acid sequence as set forth in SEQ ID NO: 24.
- the light chain variable region VL of the binding portion against CD20 comprises an amino acid sequence as set forth in SEQ ID NO: 23.
- the binding portion against CD3 comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a heavy chain variable region VH of the antibody that binds to CD3, and the second polypeptide chain comprises a light chain variable region VL of the antibody that binds to CD3.
- the binding portion against a TAA comprises a third polypeptide chain and a fourth polypeptide chain, wherein the third polypeptide chain comprises a heavy chain variable region VH of the antibody that binds to the TAA, and the fourth polypeptide chain comprises a light chain variable region VL of the antibody that binds to the TAA.
- the third polypeptide chain comprises a heavy chain variable region VH of the antibody that binds to the HER2, and the fourth polypeptide chain comprises a light chain variable region VL of the antibody that binds to the HER2.
- the heavy chain variable region VH of the antibody that binds to the HER2 comprises an amino acid sequence as set forth in SEQ ID NO: 16
- the light chain variable region VL of the antibody that binds to the HER2 comprises an amino acid sequence as set forth in SEQ ID NO: 15, or
- the heavy chain variable region VH of the antibody that binds to the HER2 comprises an amino acid sequence as set forth in SEQ ID NO: 20
- the light chain variable region VL of the antibody that binds to the HER2 comprises an amino acid sequence as set forth in SEQ ID NO: 19.
- the third polypeptide chain comprises a heavy chain variable region VH of the antibody that binds to the CD20
- the fourth polypeptide chain comprises a light chain variable region VL of the antibody that binds to the CD20.
- the heavy chain variable region VH of the antibody that binds to the CD20 comprises an amino acid sequence as set forth in SEQ ID NO: 24, and the light chain variable region VL of the antibody that binds to the CD20 comprises an amino acid sequence as set forth in SEQ ID NO: 23.
- the present application provides one or more isolated nucleic acid molecules which encode the multispecific antigen binding protein of the present application.
- the present application provides a vector which comprises the isolated nucleic acid molecule of the present application.
- the present application provides a cell which comprises the isolated nucleic acid molecule of the present application and/or the vector of the present application.
- the present application provides a method of preparing the multispecific antigen binding protein of the present application, wherein the method comprises culturing the cell of the present application under conditions enable expression of the multispecific antigen binding protein of the present application.
- the present application provides a pharmaceutical composition, comprising the multispecific antigen binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, and/or cell of the present application, and optionally a pharmaceutically acceptable adjuvant.
- the present application provides with use of the multispecific antigen binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, cell of the present application, and/or the pharmaceutical composition of the present application in manufacture of a drug for preventing, relieving and/or treating tumor.
- the present application provides a multispecific antigen binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, cell of the present application, and/or the pharmaceutical composition of the present application, for use in preventing, relieving and/or treating tumor.
- the present application provides a method of preventing, relieving and/or treating tumor, comprising administrating the multispecific antigen binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, cell of the present application, and/or the pharmaceutical composition of the present application to a subject in need.
- the present application provides a method of preventing, relieving and/or treating tumor in a subject, wherein the tumor comprises squamous cell carcinoma, small-cell lung cancer, non-small cell lung cancer (NSCLC) , lung adenocarcinoma, squamous cell lung cancer, peritoneum cancer, hepatocellular cancer, stomach cancer, gastrointestinal cancer, esophageal cancer, pancreas tic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland carcinoma, renal cancer, prostate cancer, vulval cancer, thyroid cancer, urinary cancer, hepatocellular carcinoma (HCC) , anal carcinoma, penile carcinoma, head and neck cancer or other type of cancer.
- NSCLC non-small cell lung cancer
- HCC hepatocellular carcinoma
- the present application provides a method of activating a T cell, targeting the CD3 expressed on a T cell, and/or promoting an interaction between a T cell and a tumor cell, comprising administrating the multispecific antigen binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, the cell of the present application, and/or the pharmaceutical composition of the present application.
- FIG. 1 illustrates structure of the multispecific antigen binding protein of the present application.
- FIG. 2 illustrates result of Western blot of the multispecific antigen binding protein of the present application.
- FIG. 3 illustrates result of SDS-PAGE of the multispecific antigen binding protein of the present application.
- FIG. 4 illustrates F (ab) ’2 fragment structure of the multispecific antigen binding protein of the present application.
- FIG. 5 illustrates F (ab) ’2 fragment structure of the multispecific antigen binding protein of the present application.
- FIGs. 6-7 illustrate result of SDS-PAGE of the multispecific antigen binding protein of the present application.
- FIG. 8 illustrates binding affinity of the multispecific antigen binding protein of the present application to Jurkat cells.
- FIG. 9 illustrates cytotoxicity of hHER2 ⁇ CD3 and rHER2 ⁇ CD3 to SK-BR-3 cells.
- FIG. 10 illustrates cytotoxicity of the multispecific antigen binding protein of the present application to SK-BR-3 cells.
- FIG. 11 illustrates cytotoxicity of the multispecific antigen binding protein of the present application to SU-DHL-8 cells.
- FIG. 12 illustrates diagram of a pTT5 vector.
- FIG. 13A-13F illustrate results of western blot and SDS-PAGE of anti-CD3 mAb.
- FIG. 13A, FIG. 13C and FIG. 13E illustrate results of western blot of C3000, C3002-1 and C3002-2 respectively.
- Line 1 of FIG. 13B, FIG. 13D and FIG. 13F illustrate results of reduced SDS-PAGE of C3000, C3002-1 and C3002-2 respectively.
- Line 2 of FIG. 13B, FIG. 13D and FIG. 13F illustrate results of non-reduced SDS-PAGE of C3000, C3002-1 and C3002-2 respectively.
- FIG. 14 illustrates binding affinity of the anti-CD3 mAb C3000, C3002-1 and C3002-2 to CD3 ⁇ and CD3 ⁇ Heterodimer Protein.
- FIG. 15 illustrates cytotoxicity of T cells on SK-BR-3 cells mediated by hHER2 ⁇ CD3 and rHER2 ⁇ CD3.
- antigen binding protein generally refers to a protein comprising a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the antigen binding protein to the antigen.
- antigen binding proteins may comprise antibodies, antibody fragments (e.g., an antigen binding portion of an antibody) , antibody derivatives, and antibody analogs.
- An antigen binding protein may have, for example, the structure of a naturally occurring immunoglobulin.
- An “immunoglobulin” is a tetrameric molecule.
- each tetramer is composed of two identical pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC) .
- the amino-terminal portion of each chain may comprise a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
- the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.
- Human light chains may be classified as ⁇ or ⁇ light chains.
- Heavy chains may be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and define the antibody's isotype as IgM, IgG, IgD, IgA, and IgE, respectively.
- variable regions of naturally occurring immunoglobulin chains may exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. From N-terminus to C-terminus, both light and heavy chains may comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
- FR relatively conserved framework regions
- CDRs complementarity determining regions
- CDR generally refers to the complementarity determining region amino acid sequences of an antigen binding protein. These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. It will be apparent to those skilled in the art that there are various numbering systems for CDR sequences, e.g., Chothia (Chothia et al. (1989) Nature 342: 877-883) , Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987 and 1991) ) . The Chothia numbering system is used for numbering the residues in an antibody of the present disclosure.
- an antigen-binding fragment generally refers to a portion of an immunoglobulin molecule.
- An antigen-binding fragment may comprise one light chain and part of a heavy chain with a single antigen-binding site.
- An antigen-binding fragment may be obtained by enzyme digestion of an immunoglobulin molecule.
- an antigen-binding fragment may be composed of one constant and one variable domain of each of the heavy and the light chain.
- the variable domain may contain the paratope (the antigen-binding site) , comprising a set of the complementarity determining regions, at the amino-terminal end of the immunoglobulin molecule.
- variable fragment generally refers to all or part of the variable regions of the heavy and light chains, and absent of the constant regions of the heavy and light chains.
- the variable regions of the heavy and light chains may comprise CDRs.
- ScFv generally refers to a single-chain antibody fragment.
- An ScFv may be a recombinant single chain polypeptide molecule in which light and heavy chain variable regions of an antibody are connected, either directly or via a peptide linker.
- Fab generally refers to antibody fragments comprising fragments which comprise two N-terminal portions of the heavy chain polypeptide joined by at least one disulfide bridge in the hinge region and two complete light chain polypeptides.
- each light chain may be complexed with one N-terminal portion of a heavy chain.
- Fab may also comprise Fab fragments which comprise all or a large portion of a light chain polypeptide (e.g., VLCL) complexed with the N-terminal portion of a heavy chain polypeptide (e.g., VHCH1) .
- Fab generally refers to an antibody fragment that is the reduction product of an F (ab’) 2 fragment.
- Fab’ fragments may differ from Fab fragments by having several additional residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region.
- Fab’-SH may be the designation herein for Fab’ where the cysteine residue (s) of the constant domain have a free thiol group.
- F (ab) 2 generally refers to an antibody structure that is capable of binding to antigens but is monovalent with no Fc portion.
- the F (ab) 2 may be an antibody digested by the enzyme papain yielding two F (ab) fragments of about 50 kDa each and an Fc fragment.
- the term “F (ab’) 2” generally refers to an antibody fragment comprising two Fab fragments and a portion of the hinge region joined by a disulfide bond.
- the F (ab’) 2 may be produced by pepsin digestion of an intact antibody.
- the F (ab’) 2 fragment may have a bivalent antigen-binding activity and may be capable of crosslinking the antigen.
- the term “fully human antibody” generally refers to an antibody with fully human amino acid sequence derived antibody where antigen specificity has been selected either in vivo by the use of genetically modified mice or by antibody engineering processes combined with screening.
- the term "monoclonal antibody” generally refers to a group of antibodies that are substantially homologous, that is, each antibody included in the group is the same except for possible naturally occurring mutations in trace amounts.
- Monoclonal antibody is understood as antibody made by identical cells that are all clones of a unique parent cell, in contrast to polyclonal antibodies which are made from several different immune cells.
- the monoclonal antibodies may be prepared by hybridoma technology or produced in bacteria, eukaryotic animals or plant cells by recombinant DNA methods.
- the monoclonal antibodies may be also obtained from a phage antibody library, by using a technology as described in, e.g., Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., Mol. Biol., 222: 581-597 (1991) .
- chimeric antibody generally refers to an antibody in which a portion of the amino acid sequence of each heavy or light chain is homologous to the corresponding amino acid sequence in an antibody from a specific species, or belongs to a specific class, and the rest of the chain is homologous to the corresponding sequence in another species.
- the variable regions of both the light chain and the heavy chain may be derived from the variable region of an antibody from one animal species (e.g., mouse, rat, etc. ) , while the constant part is homologous to an antibody sequence from another species (e.g., human) .
- non-human B cells or hybridoma cells may be used to generate variable regions, and the constant regions combined therewith are derived from humans.
- the variable region may have an easy-to-prepare advantage, and its specificity may not be affected by the source of the constant region combined therewith.
- the constant region of the chimeric antibody since the constant region of the chimeric antibody may be derived from humans, the possibility of the chimeric antibody inducing an immune response when injected may be lower than that of an antibody of which the constant region is derived from a non-human origin.
- humanized antibody generally refers to an engineered antibody obtained by reducing the immunogenicity of the antibodies, immunoglobulin binding proteins and polypeptides derived from non-human species (e.g., mice or rats) to humans, while still retaining the antigen-binding properties of the original antibody.
- CD3 generally refers to a TCR complex comprising CD3.
- CD3 is a protein complex composed of four chains in mammals (i.e. CD3 ⁇ , CD3 ⁇ and two CD3 ⁇ chains) , which may associate with molecules known as the T cell receptor (TCR) and with the ⁇ -chain and ⁇ -chain (as homo-or heterodimers) to generate an activation signal in T lymphocytes.
- TCR T cell receptor
- ⁇ -chain and ⁇ -chain as homo-or heterodimers
- HER2 generally refers to ErbB-2, NEU, HER-2 or CD340, may refer to epidermis Growth factor acceptor 2 (for example, human HER2, SwissProt P04626) , and any variant, isoform and species homologue thereof.
- the HER2 may be natively expressed in the cells including tumor cells, or may be expressed through the transfection of HER2 genes or cDNA in the cells.
- CD20 generally refers to bone-marrow-derived lymphocyte antigens CD20, also referred to as bone-marrow-derived lymphocyte surface antigen B1 or leukocyte surface are anti-Former Leu-16, and may comprise any natural CD20 from any vertebrate origin, including mammal, such as primate is dynamic Object (such as people) , non-human primate and rodent (such as mouse and rat) .
- CD20 is a kind of hydrophobic transmembrane protein, and the molecular weight with about 35kD may be expressed on preceding B and mature bone-marrow-derived lymphocyte.
- tumor associated antigen generally refers to a molecule or complex which may be expressed at a higher frequency or density by tumor cells than by non-tumor cells of the same tissue type.
- the tumor associated antigen may be antigens not normally expressed by the host; and they may be mutated, truncated, misfolded, or otherwise abnormal manifestation of molecules normally expressed by the host; they may be identical to molecules normally expressed but expressed at abnormally high levels; or they may be expressed in a context or milieu that is abnormal.
- the tumor associated antigen may be, for example, proteins or protein fragments, complex carbohydrates, gangliosides, haptens, nucleic acids, or any combination of these or other biological molecules.
- the tumor associated antigen may comprise HER2 and/or CD20.
- the term “activating” as used herein generally refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event.
- a stimulatory molecule e.g., a TCR/CD3 complex
- the activating may comprise a signal transduction via the TCR/CD3 complex.
- the term “interaction between a T cell and a tumor cell” as used herein, generally refers to a reaction between the T cell and the tumor cell leading to a negative selection pressure on the tumor cell that is being recognized by the immune system (for example, by the T cell) .
- the T cell recognition may be therefore reduced in the tumor cells by downregulation of molecules important for antigen processing and presentation or costimulation.
- the term “treating” as used herein generally refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated.
- the treating may be performed either for prophylaxis or during the course of clinical pathology.
- Desirable effects may include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, lowering the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
- the term “subject” as used herein generally refers to a mammal.
- the subject may be a human.
- the subject may be a non-human mammal.
- the non-human mammal may comprise farm animals, sport animals, and pets.
- multispecific generally refers to a feature of bispecific, tri-specific or multispecific antigen-binding.
- a multispecific antigen binding protein may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for epitopes of more than one target polypeptide.
- a multispecific antigen binding protein may be a single multifunctional polypeptide, or it may be a multimeric complex of two or more polypeptides that are covalently or non-covalently associated with one another.
- the term “multispecific antigen binding protein” may comprise an antibody of present application and/or an antigen binding fragment of the present application.
- the antibody and/or the antigen binding fragment of present application be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as a protein or fragment thereof to produce a bi-specific or a multispecific antigen binding protein with a second binding specificity.
- the term “multispecific antigen binding protein” may also comprise a bi-specific, tri-specific or multispecific antibody or an antigen-binding fragment thereof.
- isolated nucleic acid molecule or molecules generally refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, isolated from its native environment, or that is artificially synthesized.
- vector or vectors generally refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and expressed.
- the genetic material elements carried in the vector can be expressed in a host cell by transforming, transducing, or transfecting the host cell with the vector.
- Embodiments of vectors include: plasmids; phagemids; cosmid; artificial chromosomes such as yeast artificial chromosomes (YACs) , bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs) ; phages such as ⁇ phage or M13 phage and animal viruses.
- a vector may contain a variety of elements that control expression, including promoter sequences, transcriptional initiation sequences, enhancer sequences, selection elements, and reporter genes.
- the vector may also contain an origin of replication. It is also possible that the vector may include components that assist its entry into the cell, such as viral particles, liposomes or protein shells, but not only these substances.
- the term “cell” as used herein, generally refers to a cell that may be used to carry the vector or vectors of the present disclosure, or be used to express or produce the antibody, the antigen binding fragment or variant of the present disclosure.
- a cell of the present disclosure may be a host cell.
- the cell may be a prokaryotic cell such as Escherichia coli and Bacillus subtilis, a fungal cell such as yeast cell or Aspergillus cell, an insect cell such as S2 Drosophila cell or Sf9, or another cell such as a CHO cell, a COS cell, an NSO cell, a HEK293 cell, or other cells suitable for antibody expression.
- condition enabling expression generally refers to conditions enabling the expression of the multispecific antigen binding protein of the present disclosure.
- the conditions may comprise incubation time, temperature, culture medium, and may depend on cell type and may be readily determined by a person skilled in the art.
- T cell engager generally refers to a bispecific or multispecific antibody directed against a constant-component of the T-cell/CD3 complex and a tumor-associated antigen (TAA) .
- TAA tumor-associated antigen
- the T cell engager may show two benefits: 1) may be able to elicit a polyclonal T-cell response 2) may be not affected by escape mechanisms involving downregulation of antigen presentation.
- the T cell engager may refer to Methods 154 (2019) 102–117.
- the term “about” generally refers to a variation in a range of the given value ⁇ 0.5%- ⁇ 10%, such as, a variation in a range of the given value ⁇ 0.5%, ⁇ 1%, ⁇ 1.5%, ⁇ 2%, ⁇ 2.5%, ⁇ 3%, ⁇ 3.5%, ⁇ 4%, ⁇ 4.5%, ⁇ 5%, ⁇ 5.5%, ⁇ 6%, ⁇ 6.5%, ⁇ 7%, ⁇ 7.5%, ⁇ 8%, ⁇ 8.5%, ⁇ 9%, ⁇ 9.5%, or ⁇ 10%.
- the binding portion against CD3 may comprise an antibody or an antigen binding fragment thereof.
- the binding portion against CD3 may be specifically against CD3.
- the binding portion against CD3 may be an anti-CD3 antibody or an antigen binding fragment against CD3.
- binding portion against CD3 and CD3 binding portion are used interchangeably in the present application.
- the antibody may be selected from a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.
- the antigen binding fragment may comprise Fab, Fab’, F (ab) 2, Fv fragment, F (ab’) 2, scFv, di-scFv and/or dAb.
- the binding portion against CD3 may comprise any one of HCDR1, HCDR2 and/or HCDR3 regions from the heavy chain variable region VH of SEQ ID NO: 8.
- the binding portion against CD3 may comprise HCDR1-3.
- the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 4, the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 5, and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 6.
- the binding portion against CD3 may comprise any one of HCDR1, HCDR2 and/or HCDR3 regions from the heavy chain variable region VH of SEQ ID NO: 35.
- the binding portion against CD3 may comprise HCDR1-3.
- the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 37
- the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 38
- the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 39.
- the binding portion against CD3 may comprise any one of HCDR1, HCDR2 and/or HCDR3 regions from the heavy chain variable region VH of SEQ ID NO: 43.
- the binding portion against CD3 may comprise HCDR1-3.
- the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 45
- the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 46
- the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 47.
- the binding portion against CD3 may comprise any one of HCDR1, HCDR2 and/or HCDR3 regions from the heavy chain variable region VH of SEQ ID NO: 51.
- the binding portion against CD3 may comprise HCDR1-3.
- the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 53
- the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 54
- the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 55.
- the binding portion against CD3 may comprise a VH.
- the VH of the binding portion against CD3 may comprise framework regions H-FR1, H-FR2, H-FR3 and H-FR4.
- the VH of the binding portion against CD3 may comprise an amino acid sequence as set forth in SEQ ID NO: 8. In the present application, the VH of the binding portion against CD3 may comprise an amino acid sequence as set forth in SEQ ID NO: 35. In the present application, the VH of the binding portion against CD3 may comprise an amino acid sequence as set forth in SEQ ID NO: 43. In the present application, the VH of the binding portion against CD3 may comprise an amino acid sequence as set forth in SEQ ID NO: 51.
- the binding portion against CD3 is humanized.
- the VH of the binding portion against CD3 may comprise an amino acid sequence as set forth in SEQ ID NO: 27.
- the VH of the binding portion against CD3 may comprise an amino acid sequence as set forth in SEQ ID NO: 28.
- the binding portion against CD3 may comprise an antibody heavy chain constant region.
- the antibody heavy chain constant region comprises an IgG constant region.
- the antibody heavy chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 18 or SEQ ID NO: 26.
- the binding portion against CD3 may comprise any one of LCDR1, LCDR2 and/or LCDR3 regions from the light chain variable region VL of SEQ ID NO: 7.
- the binding portion against CD3 may comprise LCDR1-3.
- the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 1
- the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 2
- the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 3.
- the binding portion against CD3 may comprise any one of LCDR1, LCDR2 and/or LCDR3 regions from the light chain variable region VL of SEQ ID NO: 36.
- the binding portion against CD3 may comprise LCDR1-3.
- the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 40
- the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 41
- the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 42.
- the binding portion against CD3 may comprise any one of LCDR1, LCDR2 and/or LCDR3 regions from the light chain variable region VL of SEQ ID NO: 44.
- the binding portion against CD3 may comprise LCDR1-3.
- the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 48
- the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 49
- the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 50.
- the binding portion against CD3 may comprise any one of LCDR1, LCDR2 and/or LCDR3 regions from the light chain variable region VL of SEQ ID NO: 52.
- the binding portion against CD3 may comprise LCDR1-3.
- the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 56
- the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 57
- the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 58.
- the binding portion against CD3 may comprise a VL.
- the VL of the binding portion against CD3 may comprise framework regions L-FR1, L-FR2, L-FR3 and L-FR4.
- the VL of the binding portion against CD3 may comprise an amino acid sequence as set forth in SEQ ID NO: 7. In the present application, the VL of the binding portion against CD3 may comprise an amino acid sequence as set forth in SEQ ID NO: 36. In the present application, the VL of the binding portion against CD3 may comprise an amino acid sequence as set forth in SEQ ID NO: 44. In the present application, the VL of the binding portion against CD3 may comprise an amino acid sequence as set forth in SEQ ID NO: 52.
- the binding portion against CD3 is humanized.
- the VL of the binding portion against CD3 may comprise an amino acid sequence as set forth in SEQ ID NO: 31.
- the binding portion against CD3 may comprise an antibody light chain constant region.
- the antibody light chain constant region may comprise an Ig ⁇ constant region.
- the antibody light chain constant region may comprise an amino acid sequence as set forth in SEQ ID NO: 17 or SEQ ID NO: 25.
- the binding portion against CD3 may comprise HCDR1-3 and LCDR1-3.
- the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 4, the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 5, and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 6; the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 1, the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 2, the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 3.
- the binding portion against CD3 may comprise HCDR1-3 and LCDR1-3.
- the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 37
- the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 38
- the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 39
- the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 40
- the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 41
- the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 42.
- the binding portion against CD3 may comprise HCDR1-3 and LCDR1-3.
- the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 45
- the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 46
- the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 47
- the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 48
- the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 49
- the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 50.
- the binding portion against CD3 may comprise HCDR1-3 and LCDR1-3.
- the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 53
- the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 54
- the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 55
- the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 56
- the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 57
- the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 48.
- the binding portion against CD3 may comprise a VH and a VL.
- the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 8, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 7.
- the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 35, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 36.
- the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 43, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 44.
- the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 51, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 52.
- the binding portion against CD3 may comprise a humanized VH and a humanized VL.
- the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 27, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 31.
- the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 28, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 31.
- the multispecific antigen binding protein may comprise constant region derived from IgA, IgG, IgD, IgE or IgM antibody.
- the binding portion against CD3 may compete with a reference antibody for binding to CD3.
- the reference antibody may comprise at least one HCDR region from the heavy chain variable region VH of SEQ ID NO: 8 and at least one LCDR region from the light chain variable region VL of SEQ ID NO: 7. In some cases, the reference antibody may comprise at least one HCDR region from the heavy chain variable region VH of SEQ ID NO: 35 and at least one LCDR region from the light chain variable region VL of SEQ ID NO: 36. In some cases, the reference antibody may comprise at least one HCDR region from the heavy chain variable region VH of SEQ ID NO: 43 and at least one LCDR region from the light chain variable region VL of SEQ ID NO: 44. In some cases, the reference antibody may comprise at least one HCDR region from the heavy chain variable region VH of SEQ ID NO: 51 and at least one LCDR region from the light chain variable region VL of SEQ ID NO: 52.
- the reference antibody may comprise HCDR1-3.
- the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 4, the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 5, and the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 6.
- the reference antibody may comprise LCDR1-3.
- the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 1
- the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 2
- the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 3.
- the reference antibody may comprise HCDR1-3.
- the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 37
- the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 38
- the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 39.
- the reference antibody may comprise LCDR1-3.
- the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 40
- the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 41
- the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 42.
- the reference antibody may comprise HCDR1-3.
- the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 45
- the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 46
- the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 47.
- the reference antibody may comprise LCDR1-3.
- the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 48
- the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 49
- the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 50.
- the reference antibody may comprise HCDR1-3.
- the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 53
- the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 54
- the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 55.
- the reference antibody may comprise LCDR1-3.
- the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 56
- the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 57
- the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 58.
- the reference antibody may comprise a VH and a VL.
- the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 8, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 7.
- the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 35, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 36.
- the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 43, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 44.
- the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 51, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 52.
- the reference antibody may comprise a heavy chain constant region and a light chain constant region.
- the heavy chain constant region may comprise an amino acid sequence as set forth in SEQ ID NO: 18 or SEQ ID NO: 26
- the light chain constant region may comprise an amino acid sequence as set forth in SEQ ID NO: 17 or SEQ ID NO: 25.
- the binding portion against CD3 may also encompass a homology or a variant thereof having substantially the same function/property thereto.
- the homology or variant may be a polypeptide different from the antigen binding protein at least one amino acid.
- the homology or variant may be a polypeptide different from the antigen binding protein by an addition, deletion or substitution of one or more amino acid, such as 1-50, 1-40, 1-30, 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acids.
- TAA tumor associated antigen
- the multispecific antigen binding protein may comprise a binding portion against a tumor associated antigen (TAA) .
- TAA tumor associated antigen
- TAA is selected from the group comprising EPCAM, CCR5, CD19, HER2, HER3 neu, HER3, HER4, EGFR, PSMA, CEA, MUC1, MUC2, MUC3, MUC4, MUC5, MUC7, ⁇ hCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3, 9-O-Acetyl-GD3, GM2, globo H, fucosyl GM1, Poly SA, GD2, RON, c-Met, CEACAM-6, PCTA-1, PSA, PAP, ALCAM (CD166) , PECAM-1, CD151, MAGE-1, TROP2, IGF1 R.
- EPCAM EPCAM
- CCR5 CD19
- HER2, HER3 neu HER3, HER4, EGFR
- PSMA PSMA
- CEA MUC1, MUC2, MUC3, MUC4, MUC5, MUC7, ⁇ hCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3,
- the multispecific antigen binding protein may comprise a binding portion against HER2.
- the binding portion against a HER2 may be an anti-HER2 antibody or an antigen binding fragment against HER2.
- the antibody may be selected from a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.
- the antigen binding fragment may comprise Fab, Fab’, F (ab) 2, Fv fragment, F (ab’) 2, scFv, di-scFv and/or dAb.
- the binding portion against a HER2 may comprise any one of HCDR1, HCDR2 and/or HCDR3 regions from the heavy chain variable region VH of SEQ ID NO: 16 or SEQ ID NO: 20.
- the binding portion against a HER2 may comprise HCDR1-3.
- the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 12
- the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 13
- the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 14.
- the binding portion against a HER2 may comprise a VH.
- the VH of the binding portion against a HER2 may comprise framework regions H-FR1, H-FR2, H-FR3 and H-FR4.
- the VH of the binding portion against a HER2 may comprise an amino acid sequence as set forth in SEQ ID NO: 16 or SEQ ID NO: 20.
- the binding portion against a HER2 may comprise an antibody heavy chain constant region.
- the antibody heavy chain constant region comprises an IgG constant region.
- the antibody heavy chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 18 or SEQ ID NO: 26.
- the binding portion against a HER2 may comprise any one of LCDR1, LCDR2 and/or LCDR3 regions from the light chain variable region VL of SEQ ID NO: 15 or SEQ ID NO: 19.
- the binding portion against a HER2 may comprise LCDR1-3.
- the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 9
- the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 10
- the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 11.
- the binding portion against a HER2 may comprise a VL.
- the VL of the binding portion against a HER2 may comprise framework regions L-FR1, L-FR2, L-FR3 and L-FR4.
- the binding portion against a HER2 may comprise an antibody light chain constant region.
- the antibody light chain constant region may comprise an Ig ⁇ constant region.
- the antibody light chain constant region may comprise an amino acid sequence as set forth in SEQ ID NO: 17 or SEQ ID NO: 25.
- the binding portion against a HER2 may comprise HCDR1-3 and LCDR1-3.
- the HCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 12
- the HCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 13
- the HCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 14
- the LCDR1 may comprise an amino acid sequence as set forth in SEQ ID NO. 9
- the LCDR2 may comprise an amino acid sequence as set forth in SEQ ID NO. 10
- the LCDR3 may comprise an amino acid sequence as set forth in SEQ ID NO. 11.
- the binding portion against a HER2 may comprise a VH and a VL.
- the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 16
- the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 15.
- the binding portion against a HER2 may comprise a VH and a VL.
- the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 20
- the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 19.
- the multispecific antigen binding protein may comprise a binding portion against CD20.
- the binding portion against a CD20 may be an anti-CD20 antibody or an antigen binding fragment against CD20.
- the antibody may be selected from a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.
- the antigen binding fragment may comprise Fab, Fab’, F (ab) 2, Fv fragment, F (ab’) 2, scFv, di-scFv and/or dAb.
- the binding portion against a CD20 may comprise any one of HCDR1, HCDR2 and/or HCDR3 regions from the heavy chain variable region VH of SEQ ID NO: 24.
- the VH of the binding portion against a CD20 may comprise an amino acid sequence as set forth in SEQ ID NO: 24.
- the binding portion against a CD20 may comprise an antibody heavy chain constant region.
- the antibody heavy chain constant region comprises an IgG constant region.
- the antibody heavy chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 18 or SEQ ID NO: 26.
- the binding portion against a CD20 may comprise any one of LCDR1, LCDR2 and/or LCDR3 regions from the light chain variable region VL of SEQ ID NO: 23.
- the binding portion against a CD20 may comprise an antibody light chain constant region.
- the antibody light chain constant region may comprise an Ig ⁇ constant region.
- the antibody light chain constant region may comprise an amino acid sequence as set forth in SEQ ID NO: 17 or SEQ ID NO: 25.
- the binding portion against a CD20 may comprise a VH and a VL.
- the VH may comprise an amino acid sequence as set forth in SEQ ID NO: 24, and the VL may comprise an amino acid sequence as set forth in SEQ ID NO: 23.
- the multispecific antigen binding protein The multispecific antigen binding protein
- the multispecific antigen binding protein may be an antibody or an antigen binding fragment thereof.
- the multispecific antigen binding protein may be an intact antibody (for example, may be a monoclonal antibody, a chimeric antibody, a humanized antibody, and/or a fully human antibody) .
- the multispecific antigen binding protein may be an IgG antibody.
- the multispecific antigen binding protein may be an antigen binding fragment thereof, for example, the multispecific antigen binding protein may comprise Fab, Fab’, F (ab) 2, Fv fragment, F (ab’) 2, scFv, di-scFv and/or dAb.
- the multispecific antigen binding protein may be a F (ab’) 2.
- the binding portion against CD3 may comprise a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain may comprise a heavy chain variable region VH of the antibody that binds to CD3, and the second polypeptide chain may comprise a light chain variable region VL of the antibody that binds to CD3.
- the heavy chain variable region VH of the antibody that binds to CD3 may comprise an amino acid sequence as set forth in SEQ ID NO: 8
- the light chain variable region VL of the antibody that binds to CD3 may comprise an amino acid sequence as set forth in SEQ ID NO: 7.
- the first polypeptide chain may comprise the light chain of the antibody that binds to CD3.
- the second polypeptide chain may comprise the heavy chain of the antibody that binds to CD3.
- the first polypeptide chain may comprise the VL of the antibody that binds to CD3 and the light chain constant region.
- the second polypeptide chain may comprise the VH of the antibody that binds to CD3, CH1 of the heavy chain constant region and hinge.
- the antibody that binds to CD3 may comprise the CD3 antibody of the present application.
- the light chain constant region may be originated from IgG.
- IgG2 may be originated from mouse IgG2 (for example, may be originated from IgG2a) .
- the heavy chain constant region (for example, the CH1) may be originated from IgG.
- IgG may be originated from IgG2, for example, may be originated from rat IgG2 (for example, may be originated from IgG2b) .
- the hinge may be originated from IgG.
- IgG2 for example, may be originated from rat IgG2 (for example, may be originated from IgG2b) .
- the second polypeptide chain may comprise a linker at the C terminal of the hinge.
- the linker may be a flexible linker.
- the linker is GGGGS.
- the second polypeptide chain may comprise a tag at the C terminal thereof.
- the tag may be any tag which is available for protein (for example, the second polypeptide chain, and/or the binding portion against CD3) isolating and/or purification.
- the tag may be a His-tag.
- the binding portion against a TAA may comprise a third polypeptide chain and a fourth polypeptide chain, wherein the third polypeptide chain may comprise a heavy chain variable region VH of the antibody that binds to the TAA, and the fourth polypeptide chain may comprise a light chain variable region VL of the antibody that binds to the TAA.
- the third polypeptide chain may comprise a heavy chain variable region VH of the antibody that binds to the HER2, and the fourth polypeptide chain may comprise a light chain variable region VL of the antibody that binds to the HER2.
- the heavy chain variable region VH of the antibody that binds to the HER2 may comprise an amino acid sequence as set forth in SEQ ID NO: 16
- the light chain variable region VL of the antibody that binds to the HER2 may comprise an amino acid sequence as set forth in SEQ ID NO: 15, or
- the heavy chain variable region VH of the antibody that binds to the HER2 may comprise an amino acid sequence as set forth in SEQ ID NO: 20
- the light chain variable region VL of the antibody that binds to the HER2 may comprise an amino acid sequence as set forth in SEQ ID NO: 19.
- the third polypeptide chain may comprise a heavy chain variable region VH of the antibody that binds to the CD20
- the fourth polypeptide chain may comprise a light chain variable region VL of the antibody that binds to the CD20.
- the heavy chain variable region VH of the antibody that binds to the CD20 may comprise an amino acid sequence as set forth in SEQ ID NO: 24, and the light chain variable region VL of the antibody that binds to the CD20 may comprise an amino acid sequence as set forth in SEQ ID NO: 23.
- the third polypeptide chain may comprise the light chain of the antibody that binds to HER2.
- the fourth polypeptide chain may comprise the heavy chain of the antibody that binds to HER2.
- the third polypeptide chain may comprise the VL of the antibody that binds to HER2 and the light chain constant region.
- the fourth polypeptide chain may comprise the VH of the antibody that binds to HER2, CH1 of the heavy chain constant region and hinge.
- the antibody that binds to HER2 may comprise the HER2 antibody of the present application.
- the third polypeptide chain may comprise the light chain of the antibody that binds to CD20.
- the fourth polypeptide chain may comprise the heavy chain of the antibody that binds to CD20.
- the third polypeptide chain may comprise the VL of the antibody that binds to CD20 and the light chain constant region.
- the fourth polypeptide chain may comprise the VH of the antibody that binds to CD20, CH1 of the heavy chain constant region and hinge.
- the antibody that binds to CD20 may comprise the CD20 antibody of the present application.
- the light chain constant region may be originated from IgG.
- IgG2 may be originated from mouse IgG2 (for example, may be originated from IgG2a) .
- the heavy chain constant region (for example, the CH1) may be originated from IgG.
- IgG may be originated from IgG2, for example, may be originated from mouse IgG2 (for example, may be originated from IgG2a) .
- the hinge may be originated from IgG.
- IgG2 for example, may be originated from mouse IgG2 (for example, may be originated from IgG2a) .
- the fourth polypeptide chain may comprise a linker at the C terminal of the hinge.
- the linker may be a flexible linker.
- the linker is GGGGS.
- the fourth polypeptide chain may comprise a tag at the C terminal thereof.
- the tag may be any tag which is available for protein (for example, the fourth polypeptide chain, and/or the binding portion against HER2 or CD20) isolating and/or purification.
- the tag may be a strep-tag II.
- the multispecific antigen binding protein may be consisted of one first polypeptide chain, one second polypeptide chain, one third polypeptide chain and one fourth polypeptide chain.
- the fourth polypeptide chain may be corresponding to the third polypeptide chain, and the third polypeptide chain as well as the fourth polypeptide chain may be capable of binding to a same TAA.
- the multispecific antigen binding protein may be produced by recombinant expression of host cell.
- the host cell producing the multispecific antigen binding protein may be a bacterial cell, a fungal cell, a plant cell, a mammalian cell or a virus.
- the bacterial cell may be E. coli.
- the fungal cell may be yeast cell.
- the mammalian cell may be CHO, NS0, BHK, or HEK293 cell.
- the multispecific antigen binding protein may be produced by hybridoma cells.
- the hybridoma cell may be from mouse, rat, or rabbit.
- the format of multispecific antigen binding protein may be a bispecific antibody, a bispecific diabody, a bispecific scFv, a TandAb, a trivalent binding molecule, or a tetravalent binding molecule.
- the multispecific antigen binding protein may comprise constant region derived from IgA, IgG, IgD, IgE or IgM antibody.
- the protein may also comprise a polypeptide having a sequence identity of at least 80%with the protein.
- the homology or variant may be a polypeptide having a sequence identity of 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%or higher) to the protein.
- percent (%) sequence identity may generally refer to the percentage of amino acid residues or nucleotides in a query sequence that are identical with the amino acid residues or nucleotides of a second, reference polypeptide sequence or a portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid/nucleotide sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software.
- Percent identity may be measured over the length of an entire defined polypeptide/polynucleotide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide/polynucleotide sequence. It is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
- any of the amino acid sequences of 1-16 may be also substituted by any other amino acid as desired by one of ordinary skill in the art.
- one of skill in the art may make conservative substitutions by replacing particular amino acids with others as shown in Table 1 below.
- the specific amino acid substitution selected may depend on the location of the site selected.
- conservative amino acid substitutions may involve a substitution of a native amino acid residue with a non-native residue such that there is little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position.
- the present application provides one or more isolated nucleic acid molecules which encode the multispecific antigen binding protein of the present application.
- the present application provides a vector which may comprise the isolated nucleic acid molecule of the present application.
- the present application provides a cell which may comprise the isolated nucleic acid molecule of the present application and/or the vector of the present application.
- the present application provides a method of preparing the multispecific antigen binding protein of the present application, wherein the method may comprise culturing the cell of the present application under conditions enables expression of the multispecific antigen binding protein of the present application.
- the isolated nucleic acids may comprise one or more nucleic acid molecules, with each encoding the antigen binding protein (for example, the binding portion against CD3, and/or the binding portion against a TAA) .
- the isolated nucleic acids may comprise at least two nucleic acid molecules, with one encoding the antibody heavy chain or a fragment thereof, and one encoding the antibody light chain or a fragment thereof.
- the isolated nucleic acid or isolated nucleic acids may be synthesized using recombinant techniques well known in the art.
- the isolated nucleic acid or isolated nucleic acids may be synthesized with an automated DNA synthesizer.
- Standard recombinant DNA and molecular cloning techniques include those described by Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by T.J. Silhavy, M.L. Bennan, and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F.M.
- the subject nucleic acids may be prepared from genomic DNA fragments, cDNAs, and RNAs, all of which may be extracted directly from a cell or recombinantly produced by various amplification processes including but not limited to PCR and RT-PCR.
- Direct chemical synthesis of nucleic acids typically involves sequential addition of 3’-blocked and 5’-blocked nucleotide monomers to the terminal 5’-hydroxyl group of a growing nucleotide polymer chain, wherein each addition may be effected by nucleophilic attack of the terminal 5’-hydroxyl group of the growing chain on the 3’-position of the added monomer, which is typically a phosphorus derivative, such as a phosphotriester, phosphoramidite, or the like. See for example, Matteuci et al., Tet. Lett. 521: 719 (1980) ; U.S. Pat. No. 4,500,707 to Caruthers et al.; and U.S. Pat. Nos. 5,436,327 and 5,700,637 to Southern et al.
- the vector may be any linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and the like.
- Non-limiting examples of a viral vector may comprise a retrovirus, an adenovirus and an adeno-associated virus.
- the vector may be an expression vector, e.g., a plasmid.
- An expression vector may be suitable for use in particular types of host cells and not suitable for use in others.
- the expression vector may be introduced into the host organism, which is then monitored for viability and expression of any genes/polynucleotides contained in the vector.
- the expression vector may also comprise one or more selectable marker genes that, upon expression, confer one or more phenotypic traits useful for selecting or otherwise identifying host cells that carry the expression vector.
- selectable markers for eukaryotic cells may comprise dihydrofolate reductase and neomycin resistance.
- the subject vectors may be introduced into a host cell stably or transiently by a variety of established techniques.
- one method may involve a calcium chloride treatment wherein the expression vector is introduced via a calcium precipitate.
- Other salts for example calcium phosphate, may also be used following a similar procedure.
- electroporation that is, the application of current to increase the permeability of cells to nucleic acids
- transformation methods may include microinjection, DEAE dextran mediated transformation, and heat shock in the presence of lithium acetate. Lipid complexes, liposomes, and dendrimers may also be employed to transfect the host cells.
- the cell may express the multispecific antigen binding protein of the present disclosure.
- the cell may be a eukaryotic cell or a prokaryotic cell.
- An appropriate cell may be transformed or transfected with the nucleic acid (s) or vector (s) of the present application and utilized for the expression and/or secretion of the multispecific antigen binding protein of the present application.
- the cell may be HEK293 cells, other bacterial host cells, yeast cells, or various higher eukaryotic cells.
- the method optionally may further comprise harvesting the multispecific antigen binding protein of the present application.
- compositions Use and method
- the present application provides a pharmaceutical composition, comprising the multispecific antigen binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, and/or cell of the present application, and optionally a pharmaceutically acceptable adjuvant.
- the present application provides use of the multispecific antigen binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, cell of the present application, and/or the pharmaceutical composition of the present application in manufacture of a drug for preventing, relieving and/or treating tumor.
- the present application provides a multispecific antigen binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, cell of the present application, and/or the pharmaceutical composition of the present application, for use in preventing, relieving and/or treating tumor.
- the present application provides a method of preventing, relieving and/or treating tumor, comprising administrating the multispecific antigen binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, cell of the present application, and/or the pharmaceutical composition of the present application to a subject in need.
- “pharmaceutically acceptable adjuvant” may comprise any and all preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents. It may also be desirable to include isotonic agents into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.
- compositions typically may be sterile and stable under the conditions of manufacture and storage.
- the composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
- the carrier may be a solvent or dispersion medium comprising, for example, water, ethanol and suitable mixtures thereof.
- the proper fluidity can be maintained, for example, by the use of a coating.
- Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption.
- the pharmaceutical composition of the present application may comprise other compounds, drugs, and/or agents used for the treatment of tumor.
- the tumor may comprise a solid tumor and/or a blood tumor.
- the blood tumor may comprise a B cell lymphoma and/or a leukemia, for example, an acute T cell leukemia, or a human B cell lymphoma.
- the solid tumor may comprise a breast cancer.
- the preventing, relieving and/or treating tumor may comprise inhibiting and/or reducing the growth of the tumor, inhibiting the progress of the tumor.
- the preventing, relieving and/or treating tumor may also comprise administrating another agent, for example, any agent known in the art for treating tumor.
- the pharmaceutical composition of the present application and/or the multispecific antigen binding protein of the present application may be administrated to the subject in need in any reasonable dosage, regimen and/or format, and all these administrating conditions may be adjusted according to the tumor kind, the situation of the subject and the like.
- the present application provides use of the binding portion against CD3 of the present application, and/or the multispecific antigen binding protein of the present application in manufacture of a T cell engager.
- the T cell engager may comprise a Bi-specific T-cell engagers (BiTEs) .
- the T cell engager may direct a host's immune system, and may show the more specifical T cells'cytotoxic activity against the tumor cells.
- the T cell engager may be safer as a medicament for treating tumor. And the T cell engager may exhibit more effective killing of the tumor cell.
- the present application provides a method of activating a T cell, comprising administrating the multispecific antigen binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, the cell of the present application, and/or the pharmaceutical composition of the present application to a subject in need.
- the present application provides a method of targeting the CD3 expressing on a T cell, comprising administrating the multispecific antigen binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, the cell of the present application, and/or the pharmaceutical composition of the present application.
- the present application provides a method of promoting an interaction between a T cell and a tumor cell, comprising administrating the multispecific antigen binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, the cell of the present application, and/or the pharmaceutical composition of the present application.
- the present application provides a method of treating squamous cell carcinoma, small-cell lung cancer, non-small cell lung cancer (NSCLC) , lung adenocarcinoma, squamous cell lung cancer, peritoneum cancer, hepatocellular cancer, stomach cancer, gastrointestinal cancer, esophageal cancer, pancreas tic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland carcinoma, renal cancer, prostate cancer, vulval cancer, thyroid cancer, urinary cancer, hepatocellular carcinoma (HCC) , anal carcinoma, penile carcinoma, head and neck cancer or other type of cancer.
- NSCLC non-small cell lung cancer
- Standard abbreviations may be used, e.g., bp, base pair (s) ; kb, kilobase (s) ; pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i. m., intramuscular (ly) ; i. p., intraperitoneal (ly) ; s. c., subcutaneous (ly) ; and the like.
- compositions comprising HER2 and CD3 antigens were injected into mice and rats respectively in order to stimulate the proliferation of B lymphocytes in the spleen and to secrete antibodies specifically against HER2 and CD3.
- the spleens of mice and rats were surgically removed after multiple immunizations, and polyethylene glycol (PEG) was used for cell fusion with myeloma cells.
- PEG polyethylene glycol
- the fused cells were then screened through HAT selective medium.
- the HAT selective medium comprises three components: hypoxanthine (H) , aminopterin (A) and thymine (T) .
- Aminopterin in the HAT selective medium is an inhibitor of dihydrofolate reductase, which can effectively block the endogenous pathway of DNA synthesis.
- Myeloma cells before fusion are unable to produce antibodies and lack the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene, making them sensitive to the HAT selective medium and therefore unable to survive.
- HGPRT hypoxanthine-guanine phosphoribosyl transferase
- the fused cells can synthesize HGPRT enzyme, inherit dual characteristics of B lymphocytes and myeloma cells, and thus can proliferate indefinitely in the HAT selective medium.
- the fused cells were incubated in the HAT selective medium for approximately 10 to 14 days, and then the medium was diluted into a multi-well plate so that each well comprises only one hybridoma cell. From these single cell clones, the hybrid cell lines that secrete the predetermined specific antibody were selected by ELISA.
- One of the selected hybridoma was a hybridoma that expresses a mouse-derived anti-HER2 antibody (HER2 antibody for short) .
- hybridomas that express rat-derived anti-CD3 antibodies (CD3A antibody, CD3B antibody, CD3C antibody and CD3D antibody for short) .
- hybridoma cells expressing HER2 antibody and hybridoma cells expressing CD3D antibody were fused to obtain a bispecific hybridoma that expresses anti-HER2 ⁇ anti-CD3 bispecific antibody, namely hybridoma HER2-CD3D bispecific antibody, hereinafter referred to as hHER2 ⁇ CD3D.
- Example 2 The binding activity of different CD3 antibodies to human T cell lines
- Human T cell line Jurkat (Clone E6-1, ATCC#TIB-152)
- anti-CD3 antibodies 1. The anti-CD3 antibodies:
- the anti-CD3A antibody (having a VH comprising an amino acid as set forth in SEQ ID No. 35 and a VL comprising an amino acid as set forth in SEQ ID No. 36)
- the anti-CD3B antibody (having a VH comprising an amino acid as set forth in SEQ ID No. 43 and a VL comprising an amino acid as set forth in SEQ ID No. 44)
- the anti-CD3C antibody (having a VH comprising an amino acid as set forth in SEQ ID No. 51 and a VL comprising an amino acid as set forth in SEQ ID No. 52)
- the anti-CD3D antibody (having a VH comprising an amino acid as set forth in SEQ ID No. 8 and a VL comprising an amino acid as set forth in SEQ ID No. 7)
- the concentration of Jurkat cells was adjusted to 1 ⁇ 10 7 cells/ml with PBS; and the concentrations of these two antibodies were adjusted to 80 ⁇ g /ml, 40 ⁇ g /ml, 20 ⁇ g /ml, 10 ⁇ g /ml, 5 ⁇ g /ml, 2.5 ⁇ g /ml, 1.25 ⁇ g /ml, 0.625 ⁇ g /ml, 0.3125 ⁇ g /ml, 0.15625 ⁇ g /ml, 78.125 ng/ml, 39.0625 ng/ml, 19.53125 ng/ml and 0ng/ml.
- the suspension of the Jurkat cell (50 ⁇ l/well) was added, followed by adding these five CD3 antibodies (50 ⁇ l/well) with different concentrations to a 96-well plate with a U-shaped bottom plate.
- Final concentrations of these five CD3 antibodies in mixture were 40 ⁇ g/ml, 20 ⁇ g/ml, 10 ⁇ g/ml, 5 ⁇ g/ml, 2.5 ⁇ g/ml, 1.25 ⁇ g/ml, 0.625 ⁇ g/ml, 0.3125 ⁇ g/ml, 0.15625 ⁇ g/ml, 78.125 ng/ml, 39.0625 ng/ml, 19.53125 ng/ml, 9.765625 ng/ml and 0ng/ml respectively, and the mixture was incubated at 4°C refrigerator for 1h.
- GraphPad Prism 7 software was used to visualize the graph and calculate half maximum effect concentration (EC50) of the binding activity of the antibodies to Jurkat cells.
- EC50 value of CD3A, CD3B, CD3C, CD3D and UCHT-1 antibody were, 25.87 pM, 9.56 pM, 15.23 pM, 10.71 pM, 22.79 pM, respectively.
- CHO cells were genetically modified to express and produce recombinant HER2 ⁇ CD3D bispecific antibody, hereinafter referred to as rHER2 ⁇ CD3D.
- the structure of the rHER2-CD3D bispecific antibody is shown in FIG. 1.
- the antibody comprises two different antigen binding portions, namely a HER2 binding portion and a CD3 binding portion.
- the HER2 binding portion was derived from the mouse anti-HER2 antibody in the example 1, while the CD3 binding portion was derived from the rat anti-CD3D antibody in the example 1.
- the nucleic acid sequences encoding heavy chain and light chain of the anti-HER2 antibody in the example 1 were respectively inserted into two different expression cassettes of p2MPT (comprising the puromycin resistance gene) to obtain a recombinant plasmid p2MPT-EHEL.
- the nucleic acid sequences encoding heavy chain and light chain of the anti-CD3D antibody in the example 1 were respectively inserted into two different expression cassettes of p2MPT (comprising the puromycin resistance gene) to obtain a recombinant plasmid p2MPT-DHDL.
- Two recombinant plasmids were stably co-transfected into CHO cells at 1: 1 ratio to express the rHER2-CD3D bispecific antibody.
- the recombinant plasmid pHT-PBase-ori comprising a transposase gene was used as a reagent for stable transfection.
- the transfection reagent was polyethylenimine (PEI) , and the ratio of the total DNA to PEI was 1: 5.
- DNA and PEI were sequentially added to CHO cells resuspended in the fresh medium, quickly mixed, and the mixture were placed in a 37°C shaker for transfection.
- the supernatant obtained in the step 3 was centrifuged at 2000rpm to remove cell debris, and filted with a 0.45 ⁇ m microporous membrane.
- a Mabselect Sure affinity chromatography column was used to enrich the antibodies having a Fc portion from the supernatant, and pH5.8 sodium citrate buffer was subsequently used for elution of anti-CD3D monoclonal antibody. Then, a gradient linear elution of 20 CVs was performed from pH 5.8 to pH 3.0.
- rHER2-CD3D bispecific antibody Collected sample was adjusted to pH 5.4 with Tris-HCl, followed by separation of rHER2-CD3D bispecific antibody and anti-HER2 monoclonal antibody by Capto S impact ion exchange chromatography column.
- the purified rHER2-CD3D bispecific antibody is a recombinant HER2-CD3D bispecific antibody, hereinafter referred to as rHER2 ⁇ CD3D.
- FIG. 2 represents the results of the Western Blot.
- M represents marker
- Line 1-2 represents rHER2 ⁇ CD3D
- line 3 represents the anti-HER2 monoclonal antibody
- line 4 represents anti-CD3D monoclonal antibody.
- FIG. 3 shows the results of the SDS-PAGE.
- Line 1 represents marker and line 2 represents rHER2 ⁇ CD3D.
- the heavy chain variable regions VH and light chain variable regions VL targeting HER2 and CD20 respectively were obtained from the commercial Herceptin and Rituximab.
- the heavy chain constant region CH1, the light chain constant region of CL and the hinge region thereof were derived from a mouse IgG2a.
- Strep-tag II was linked to the C-terminus of CH1 for purification.
- the heavy chain variable region VH and light chain variable region VL targeting CD3 were derived from the anti-CD3D antibody in the example 1 or commercial UCHT1.
- the heavy chain constant region CH1, the light chain constant region of CL and the hinge region thereof were derived from a rat IgG2b. His-tag was linked to the C-terminus of CH1 for purification.
- the two parts of the rat-derived Fab and the mouse-derived Fab were connected through a disulfide bond in the hinge region to form a F (ab) '2 structure of the recombinant anti-CD3 bispecific antibody.
- the F (ab) '2 fragment of recombinant anti-CD3 bispecific antibody was obtained by co-expressing two different Fab antibody fragments in a host cell.
- a cell pool comprising the F (ab) '2 fragment of recombinant anti-CD3 bispecific antibody was obtained by means of stable gene expression (SGE) .
- SGE stable gene expression
- PEI polymer polyethylenimine
- Genes 1-8 were ligated to expression vectors, transfected into host cell line to produce two different F(ab) ’2 fragments of recombinant anti-CD3 bispecific antibody, and the structures are shown in FIGs. 4-5.
- the antibody structure does not comprise a Fc region.
- the antibody comprises two distinct antigen binding portions, namely a HER2 binding portion and a CD3 binding portion, or a CD20 binding portion and a CD3 binding portion. This structure allows the antibody to simultaneously bind to a tumor-associated antigen (TAA) expressed on tumor cells and to a CD3 molecule on T cells.
- TAA tumor-associated antigen
- the F (ab) ’2 fragment of recombinant anti-CD3 bispecific antibody has both Strep-II and His peptides for purification.
- genes 1-2 or genes 3-4 were separately inserted into two distinct expression cassettes of p2MPT (comprising the puromycin resistance gene) , and recombinant plasmids named p2MPT-HH-HL and p2MPT-RH-RL were obtained.
- the genes 5-6 or genes 7-8 were separately inserted into two distinct expression cassettes of p2MPT (comprising the puromycin resistance gene) , and a recombinant plasmid named p2MPT-CH-CL and p2MPT-UH-UL was obtained.
- CHO-DG44 cells were stably transfected to express the above four F (ab) ’2 fragments of recombinant anti-CD3 bispecific antibody.
- the transfection reagent was polyethylenimine (PEI) , and the ratio of the total DNA to PEI was 1: 3.
- the plasmid p2MPT-HH-HL and p2MPT-CH-CL, p2MPT-RH-RL and p2MPT-CH-CL, p2MPT-HH-HL and p2MPT-UH-UL, and p2MPT-RH-RL and p2MPT-UH-UL were added, and the mass ratio thereof was 1: 1.
- the recombinant plasmid pHT-PBase-ori comprising a transposase gene was added, and the added amount was 1/10 of the total amount of DNA.
- DNA and PEI were sequentially added to CHO-DG44 cells resuspended in the fresh medium, quickly mixed, and then the mixture was placed in a 37°C shaker for transfection.
- the supernatant obtained in step 5 was centrifuged at 2000rpm to remove cell debris, and filted with a 0.45 ⁇ m microporous membrane.
- the His-tagged F (ab') 2 was enriched from the CHO expression supernatant using a Histrap excel affinity chromatography column, followed by elution of enriched F (ab') 2 fragment of recombinant anti-CD3 bispecific antibody from the Histrap affinity column by 300mM imidazole solution. The eluted sample was then enriched with the StrepTrap HP affinity chromatography column.
- FIG. 6 line 1 represents Herceptin ⁇ CD3D F (ab) ’2; line 2 represents Herceptin ⁇ UCHT1 F (ab) ’2; and line 3 represents marker; in FIG. 7, line 1 represents marker; line 2 represents Rituximab ⁇ CD3D F (ab) ’2; and line 3 represents Rituximab ⁇ UCHT1 F (ab) ’2.
- FIG. 4 The structures of the HER2 ⁇ CD F (ab) ’2 and CD20 ⁇ CD F (ab) ’2 are shown in FIG. 4 and FIG. 5.
- PBMC Peripheral blood mononuclear cells
- SK-BR-3 with high HER2 expression on surface was stained by cell stain (Far Red) . Then cells were resuspended in RPMI-1640 medium comprising 10%FBS. Cell concentration was subsequently adjusted to 8x10 5 cell/ml. PBMC from healthy donors were resuspended in RPMI-1640 medium comprising 10%FBS, and cell concentration was adjusted to 8x10 6 cell/ml. Finally, PBMC and SK-BR-3 were mixed thoroughly at a volume ratio of 1: 1.
- the concentrations of rHER2 ⁇ CD3D or hHER2 ⁇ CD3D were serial diluted to 0, 2x10 -4 , 2x10 -3 , 2x10 -2 , 2x10 -1 , 2x10 0 , 2x10 1 , 2x10 2 , 2x10 3 and 2x10 4 pM.
- Cell mixture of PBMC and SK-BR-3 50 ⁇ l/well were added in the 96-wells plates, followed by adding serial diluted rHER2 ⁇ CD3D or hHER2 ⁇ CD3D (50 ⁇ l/well) to the cell mixture.
- the final concentrations of rHER2 ⁇ CD3D or hHER2 ⁇ CD3D were adjusted to 0, 10 -4 , 10 -3 , 10 -2 , 10 -1 , 10 0 , 10 1 , 10 2 , 10 3 and 10 4 pM.
- the mixture of the bispecific antibodies and cells were incubated in an incubator at 37°C for 24 hours, followed by centrifuge at 400g for 5 minutes to remove the supernatant. Cells were digested by pancreatin for 10 minutes, and washed with PBS (200 ⁇ l/well) twice.
- the dead cells were stained with PI, and amount of residual SK-BR-3 cells was analyzed by flow cytometry to determine the killing effect of antibody-mediated cytotoxicity on SK-BR-3 cells.
- the APC channel was used to label SK-BR-3 cells in the cell suspension (Far red staining) , and the PE channel was used to distinguish the live cells from the dead ones (PI staining) . Since PI was capable of staining the dead cell, the number of APC-positive and PI-negative (APC + PI - ) cells analyzed by flow cytometry was finally used to quantify the residual SK-BR-3 cells and to calculate the killing rate.
- GraphPad Prism 7 software was used for graph visualization, and nonlinear regression of the four-parameter fitting curve was used for calculating half-maximum effect concentration (EC50) .
- EC50 value is approximately 3.33pM
- EC50 value is approximately 22.52pM. Therefore, we surprisingly found that rHER2 ⁇ CD3D produced by CHO cell had a significantly better killing activity on tumor cells than that of hHER2 ⁇ CD3D produced by hybridoma.
- FIG. 9 shows killing effect of PBMC on SK-BR-3 cells mediated by hHER2 ⁇ CD3D and rHER2 ⁇ CD3D.
- Example 6 CD3 binding portion contributes to cytotoxicity effect of rHER2 ⁇ CD3D on SK-BR3 cell killing
- SK-BR-3 with high HER2 expression on surface was stained by cell stain (Far Red) . Then cells were resuspended in RPMI-1640 medium comprising 10%FBS. Cell concentration was subsequently adjusted to 2x10 4 cell/ml. T cells purified from PBMC of healthy donors were resuspended in RPMI-1640 medium comprising 10%FBS, and T cell concentration was adjusted to 2x10 5 cell/ml. Finally, T cells purified from PBMC and SK-BR-3 were mixed thoroughly at a volume ratio of 1: 1.
- the concentrations of rHER2 ⁇ CD3D or hHER2 ⁇ CD3D were serial diluted to 0, 2x10 -4 , 2x10 -3 , 2x10 -2 , 2x10 -1 , 2x10 0 , 2x10 1 , 2x10 2 , 2x10 3 and 2x10 4 pM.
- Cell mixture of T cells purified from PBMC and SK-BR-3 50 ⁇ l/well were added in the 96-wells plates, followed by adding serial diluted rHER2 ⁇ CD3D or hHER2 ⁇ CD3D (50 ⁇ l/well) to the cell mixture.
- the final concentrations of rHER2 ⁇ CD3D or hHER2 ⁇ CD3D were adjusted to 0, 10 -4 , 10 -3 , 10 -2 , 10 -1 , 10 0 , 10 1 , 10 2 , 10 3 and 10 4 pM.
- the mixture of the bispecific antibodies and cells were incubated in an incubator at 37°C for 24 hours, followed by centrifuge at 400g for 5 minutes to remove the supernatant. Cells were digested by pancreatin for 10 minutes, and washed with PBS (200 ⁇ l/well) twice.
- the dead cells were stained with PI, and amount of residual SK-BR-3 cells was analyzed by flow cytometry to determine the killing effect of antibody-mediated cytotoxicity on SK-BR-3 cells.
- the APC channel was used to label SK-BR-3 cells in the cell suspension (Far red staining) , and the PE channel was used to distinguish the live cells from the dead ones (PI staining) . Since PI was capable of staining the dead cell, the number of APC-positive and PI-negative (APC + PI - ) cells analyzed by flow cytometry was finally used to quantify the residual SK-BR-3 cells and to calculate the killing rate.
- GraphPad Prism 7 software was used for graph visualization, and nonlinear regression of the four-parameter fitting curve was used for calculating half-maximum effect concentration (EC50) .
- EC50 value for donor 1 is approximately 7.65pM
- EC50 value for donor 1 is approximately 12.75pM
- EC50 value for donor 2 is approximately 3.73pM
- EC50 value for donor 2 is approximately 7.28pM.
- CD3 binding portion contributes to significantly better cytotoxicity effect of rHER2 ⁇ CD3D than hHER2 ⁇ CD3D on SK-BR3 cell killing.
- FIG. 15 shows killing effect of T cells on SK-BR-3 cells mediated by hHER2 ⁇ CD3D and rHER2 ⁇ CD3D.
- Cytokines that induce CRS include TNF- ⁇ , IFN- ⁇ , IL-1 ⁇ , IL-2, IL-6, IL-10 etc, among which IL-6 is the most critical cytokine in inducing CRS.
- rHER2 ⁇ CD3D induces maximum cytotoxic effect at the concentration of 10 2 pM
- hHER2 ⁇ CD3D induces comparable cytotoxic effect as rHER2 ⁇ CD3D at the concentration of 10 4 pM ( Figure. 9) .
- correlation between andibody-induced cytotoxicity and release of cytokine IL-6 at antibody concentrations of 10 2 pM and 10 4 pM were analyzed.
- IL-6 release level was significantly lower (1795.5pg/ml vs 2928.79pg/ml) ; compared to hHER2 ⁇ CD3D, when rHER2 ⁇ CD3D induced slightly increased tumor cell killing at concentration of 10 4 pM (61.9%vs 58.35%) , IL-6 release level was not significantly increased (3837pg/ml vs 3295.82pg/ml) .
- recombinant HER2 ⁇ CD3D induces enhanced PBMC-mediated antitumor effect but lower cytokine release, making it a safer option in clinical use.
- Table 6 Correlation between SK-BR-3 cytotoxicity and release of cytokine IL-6.
- PBMC Healthy Donor #1 (Reid, PBMC-2021010602) ; Healthy Donor #2 (Reid, PBMC-2021011802)
- Herceptin x CD3 bispecific antibody
- SK-BR-3 with high HER2 expression on surface was stained by cell stain (Far Red) . Then cells were resuspended in RPMI-1640 medium comprising 10%FBS. Cell concentration was subsequently adjusted to 8x10 5 cell/ml. PBMC from healthy donors were resuspended in RPMI-1640 medium comprising 10%FBS, and cell concentration was adjusted to 8x10 6 cell/ml. Finally, PBMC and SK-BR-3 were mixed thoroughly at a volume ratio of 1: 1.
- Herceptin ⁇ CD3D F (ab) ’2 or Herceptin ⁇ UCHT1 F (ab) ’2 were serial diluted to 0, 2x10 0 , 2x10 1 , 2x10 2 , 2x10 3 , 2x10 4 , 10 5 and 2x10 5 pM.
- Cell mixture of PBMC and SK-BR-3 (50 ⁇ l/well) were added in the 96-wells plates, followed by adding serial diluted Herceptin ⁇ CD3D F (ab) ’2 or Herceptin ⁇ UCHT1 F (ab) ’2 (50 ⁇ l/well) to the cell mixture.
- Herceptin ⁇ CD3D F (ab) ’2 or Herceptin ⁇ UCHT1 F (ab) ’2 were adjusted to 0, 10 0 , 10 1 , 10 2 , 10 3 , 10 4 , 5x 10 4 and 10 5 pM.
- the mixture of the bispecific antibodies and cells were incubated in an incubator at 37°C for 24 hours, followed by centrifuge at 400g for 5 minutes to remove the supernatant. Cells were digested by pancreatin for 10 minutes, and washed with PBS (200 ⁇ l/well) twice. Finally, the dead cells were stained with PI, and amount of residual SK-BR-3 cells was analyzed by flow cytometry to determine the killing effect of antibody-mediated cytotoxicity on SK-BR-3 cells.
- the APC channel was used to label SK-BR-3 cells in the cell suspension (Far red staining) , and the PE channel was used to distinguish the live cells from the dead ones (PI staining) . Since PI was capable of staining the dead cell, the number of APC-positive and PI-negative (APC + PI - ) cells analyzed by flow cytometry was finally used to quantify the residual SK-BR-3 cells and to calculate the killing rate.
- Herceptin ⁇ CD3D F (ab) ’2 and Herceptin ⁇ UCHT1 F (ab) ’2 were used for graph visualization, and nonlinear regression of the four-parameter fitting curve was used for calculating half-maximum effect concentration (EC50) .
- EC50 value for the first donor is approximately 680.7pM and the second donor is approximately 82.58pM; for Herceptin ⁇ UCHT1 F (ab) ’2, EC50 value for the first donor is approximately 1003pM and the second donor is approximately 941.2pM.
- FIG. 10 shows killing effect of Herceptin ⁇ CD3D F (ab) ’2 and Herceptin ⁇ UCHT1 F (ab) ’2 on SK-BR-3 cells.
- PBMC Healthy Donor #1 (Reid, PBMC-2020122101) ; Healthy Donor #2 (Reid, PBMC-2021010602)
- the concentrations of Rituximab ⁇ CD3D F (ab) ’2 or Rituximab ⁇ UCHT1 F (ab) ’2 were serial diluted.
- Cell mixture of PBMC and SU-DHL-8 (50 ⁇ l/well) were added in the 96-wells plates, followed by adding serial diluted Rituximab ⁇ CD3D F (ab) ’2 or Rituximab ⁇ UCHT1 F (ab) ’2 (50 ⁇ l/well) to the cell mixture.
- the mixture of the bispecific antibodies and cells were incubated in an incubator at 37°C for 24 hours, followed by centrifuge at 400g for 5 minutes to remove the supernatant.
- EC50 value for the first donor is approximately 72.83pM and the second donor is approximately 59.98pM; for Rituximab ⁇ UCHT1 F(ab) ’2, EC50 value for the first donor is approximately 605.5pM and the second donor is approximately 274.4pM.
- FIG. 11 shows killing effect of Rituximab ⁇ CD3D F (ab) ’2 and Rituximab ⁇ UCHT1 F (ab) ’2 on SU-DHL-8 cells.
- the Granzyme B/perforin pathway was considered as a main mechanism for cytotoxic lymphocytes to kill tumor cells.
- F (ab) ’2 fragment of recombinant CD3 bispecific antibody is capable of mediating PBMC to kill the target cell, and the cytotoxic effect attributes to interaction between CD3 and T cells, Release of Granzyme B and perforin were quantified.
- results showed that for the supernatant of Herceptin ⁇ CD3D F (ab) ’2 in example 8, the content of Granzyme B and perforin were 21233.57 pg/ml and 1597.45 pg/ml respectively; for the supernatant of Herceptin ⁇ UCHT1 F (ab) ’2, the content of Granzyme B and perforin were 38655.93 pg/ml and 2647.29 pg/ml respectively.
- Cytokines that induce CRS include TNF- ⁇ , IFN- ⁇ , IL-1 ⁇ , IL-2, IL-6, IL-10 etc, among which IL-6 is the most critical cytokine in inducing CRS.
- IL-6 level in supernatants collected from example 8 and example 9 were measured by the LEGENDplexTM Human CD8/NK Pane Panel kit.
- the contents of IL-6 in the supernatants were measured at the 10 5 pM concentration of anti-CD3 antibody. The results are shown in Table 7.
- anti-CD3D bispecific antibody had lower cytokine release and was safer when mediating tumor cell killing.
- Heavy chain vector insert or light chain vector insert was ligated into expression cassette in a pTT5 vector as depicted in FIG. 12 (designated as pTT5-HC or pTT5-LC) .
- the resulting vector + inserts which contain signal peptides, were also sequenced to confirm correct reading frame and sequence of the coding DNA. Amino acid sequence inserted in each plasmid is listed in Table 8.
- C3000 is an anti-CD3 antibody of rat origin, whereas C3002-1 and C3002-2 are humanized anti-CD3 antibodies.
- Plasmids pTT5-HC and pTT5-LC were transiently transfected into ExpiCHO-S TM cells to obtain anti-CD3 monoclonal antibodies.
- ExpiCHO-S TM cells at a density of 6.0 ⁇ 10 6 cells/ml, were cultured at 37°C in ExpiCHO TM Expression Medium.
- Two solutions were prepared as following: 1) Solution 1: 64 ⁇ l Expifectamine CHO reagent was mixed with 800 ⁇ l OptiPRO TM medium; 2) Solution 2: Mixture of 8 ⁇ g pTT5-HC and 8 ⁇ g pTT5-LC was diluted with 800 ⁇ l Expifectamine CHO reagent.
- Solution 1 and solution 2 were mixed to form ExpiFectamine TM CHO/plasmid DNA mixture. After 5min of incubation, the ExpiFectamine TM CHO/plasmid DNA mixture was added to ExpiCHO-S TM cell culture to form a 20ml culture system. The cell culture mixture was cultured at 37°C, 8%CO 2 .
- the supernatant obtained in step 11.3 was purified by MabSelect SuRe LX column. The supernatant was washed with pH7.4 PBS and eluted by pH3.0 0.1M glycine. Collected samples were neutralized by pH8.0 1M Tris buffer solution, followed by ultrafiltration to replace elution buffer with PBS solution. Purified samples were further identified by SDS-PAGE and western-blot.
- FIG. 13A-13F illustrate results of western blot and SDS-PAGE of anti-CD3 mAb.
- FIG. 13A, FIG. 13C and FIG. 13E illustrate results of western blot of C3000, C3002-1C and 3002-2 respectively. Line 1 of FIG. 13B, FIG. 13D and FIG.
- FIG. 13F illustrate results of reduced SDS-PAGE of C3000, C3002-1C and 3002-2 respectively.
- Line 2 of FIG. 13B, FIG. 13D and FIG. 13F illustrate results of non-reduced SDS-PAGE of C3000, C3002-1C and 3002-2 respectively.
- the ELISA plate was coated with 100ng/well of CD3 ⁇ &CD3 ⁇ Heterodimer Protein overnight at 4°C and further blocked with 3%BSA in PBS for 2 hours, followed by adding serial diluted anti-CD3 mAb C3000, C3002-1C and 3002-2 to the plate and incubated at 37°C for 1h.
- the plate was washed and dried, followed by adding 100 ⁇ L/well of detection antibody THE TM His Tag Antibody [HRP] to the plate and incubated at 37°C for 1h.
- the plate was washed and dried, followed by adding 100 ⁇ L/well of TMB color developing solution to the plate and incubated at 37°C for 10min. When color developed, 50 ⁇ l/well of stop solution was added to terminate color reaction.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
La présente invention concerne une protéine de liaison à un antigène multispécifique, qui comprend une partie de liaison contre CD3. La présente invention concerne également l'utilisation de la protéine de liaison à un antigène multispécifique dans la fabrication d'un médicament pour activer une cellule T et/ou traiter une tumeur.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202280024134.XA CN117120471A (zh) | 2021-03-23 | 2022-03-22 | 结合cd3的多特异性抗原结合蛋白及其用途 |
JP2023558720A JP2024512957A (ja) | 2021-03-23 | 2022-03-22 | Cd3に結合する多重特異性抗原結合タンパク質とその使用方法 |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2021082341 | 2021-03-23 | ||
CNPCT/CN2021/082341 | 2021-03-23 | ||
CNPCT/CN2021/089112 | 2021-04-23 | ||
CN2021089112 | 2021-04-23 | ||
CN2021127043 | 2021-10-28 | ||
CNPCT/CN2021/127043 | 2021-10-28 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2022199555A1 true WO2022199555A1 (fr) | 2022-09-29 |
Family
ID=81386563
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2022/082129 WO2022199555A1 (fr) | 2021-03-23 | 2022-03-22 | Protéine de liaison à un antigène multispécifique se liant à cd3 et son utilisation |
Country Status (3)
Country | Link |
---|---|
JP (1) | JP2024512957A (fr) |
CN (1) | CN117120471A (fr) |
WO (1) | WO2022199555A1 (fr) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2024061350A1 (fr) * | 2022-09-23 | 2024-03-28 | 广州凌腾生物医药有限公司 | Protéine de fusion et son utilisation |
WO2024061352A1 (fr) * | 2022-09-23 | 2024-03-28 | 广州凌腾生物医药有限公司 | Protéine de liaison à l'antigène isolée et son utilisation |
US12006366B2 (en) | 2020-06-11 | 2024-06-11 | Provention Bio, Inc. | Methods and compositions for preventing type 1 diabetes |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117736331B (zh) * | 2024-02-04 | 2024-05-07 | 南昌大学第一附属医院 | 一种特异性结合psma胞外段的单克隆抗体及其应用 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4500707A (en) | 1980-02-29 | 1985-02-19 | University Patents, Inc. | Nucleosides useful in the preparation of polynucleotides |
US5436327A (en) | 1988-09-21 | 1995-07-25 | Isis Innovation Limited | Support-bound oligonucleotides |
US5700637A (en) | 1988-05-03 | 1997-12-23 | Isis Innovation Limited | Apparatus and method for analyzing polynucleotide sequences and method of generating oligonucleotide arrays |
WO2014056783A1 (fr) * | 2012-10-08 | 2014-04-17 | Roche Glycart Ag | Anticorps exempts de fc comprenant deux fragments fab et procédés d'utilisation |
WO2016081490A1 (fr) * | 2014-11-17 | 2016-05-26 | Regeneron Pharmaceuticals, Inc. | Procédés pour le traitement de tumeurs à l'aide de cd3xcd20 anticorps bispécifiques |
WO2016116626A1 (fr) * | 2015-01-23 | 2016-07-28 | Sanofi | Anticorps anti-cd3, anticorps anti-cd123 et anticorps bispécifiques se liant spécifiquement à cd3 et/ou cd123 |
-
2022
- 2022-03-22 CN CN202280024134.XA patent/CN117120471A/zh active Pending
- 2022-03-22 WO PCT/CN2022/082129 patent/WO2022199555A1/fr active Application Filing
- 2022-03-22 JP JP2023558720A patent/JP2024512957A/ja active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4500707A (en) | 1980-02-29 | 1985-02-19 | University Patents, Inc. | Nucleosides useful in the preparation of polynucleotides |
US5700637A (en) | 1988-05-03 | 1997-12-23 | Isis Innovation Limited | Apparatus and method for analyzing polynucleotide sequences and method of generating oligonucleotide arrays |
US5436327A (en) | 1988-09-21 | 1995-07-25 | Isis Innovation Limited | Support-bound oligonucleotides |
WO2014056783A1 (fr) * | 2012-10-08 | 2014-04-17 | Roche Glycart Ag | Anticorps exempts de fc comprenant deux fragments fab et procédés d'utilisation |
WO2016081490A1 (fr) * | 2014-11-17 | 2016-05-26 | Regeneron Pharmaceuticals, Inc. | Procédés pour le traitement de tumeurs à l'aide de cd3xcd20 anticorps bispécifiques |
WO2016116626A1 (fr) * | 2015-01-23 | 2016-07-28 | Sanofi | Anticorps anti-cd3, anticorps anti-cd123 et anticorps bispécifiques se liant spécifiquement à cd3 et/ou cd123 |
Non-Patent Citations (17)
Title |
---|
ANDRES LOPEZ-ALBAITERO ET AL: "Overcoming resistance to HER2-targeted therapy with a novel HER2/CD3 bispecific antibody", ONCOIMMUNOLOGY, vol. 6, no. 3, 4 March 2017 (2017-03-04), pages e1267891, XP055374515, DOI: 10.1080/2162402X.2016.1267891 * |
AUSUBEL, F. M. ET AL.: "Current Protocols in Molecular Biology", 1987, GREENE PUBLISHING ASSOC |
CLACKSON ET AL., NATURE, vol. 352, 1991, pages 624 - 628 |
CO ET AL., J IMMUNOL, vol. 148, 1992, pages 1149 - 1154 |
CO ET AL., PROC NATL ACAD SCI USA, vol. 88, 1991, pages 2869 - 2873 |
JONES ET AL., NATURE, vol. 321, 1986, pages 522 |
MARKS ET AL., MOL. BIOL., vol. 222, 1991, pages 581 - 597 |
MATTEUCI ET AL., TET. LETT., vol. 521, 1980, pages 719 |
P KIEWE: "Ertumaxomab: a trifunctional antibody for breast cancer treatment", EXPERT OPINION ON INVESTIGATIONAL DRUGS, vol. 17, 1 October 2008 (2008-10-01), pages 1553 - 1558, XP055034410, DOI: 10.1517/13543784.17.10.1553 * |
QUEEN ET AL., PROC NATL ACAD SCI USA, vol. 86, 1989, pages 10029 - 10033 |
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 337 |
SAMBROOK, J.FRITSCH, E. F.MANIATIS, T: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS |
STAFLIN KARIN ET AL: "Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody", JCI INSIGHT, vol. 5, no. 7, 9 April 2020 (2020-04-09), XP055889428, Retrieved from the Internet <URL:https://insight.jci.org/articles/view/133757/version/1/pdf/render.pdf> DOI: 10.1172/jci.insight.133757 * |
T. J. SILHAVYM. L. BENNANL. W. ENQUIST: "Experiments with Gene Fusions, Cold Spring Harbor Laboratory", 1984, COLD SPRING HARBOR |
TEMPEST ET AL., BIOLTECHNOL, vol. 9, 1991, pages 266 - 271 |
VERHOEYEN ET AL., SCIENCE, vol. 239, 1988, pages 1534 - 1536 |
WUELLNER ULRICH ET AL: "Bispecific CD3/HER2 Targeting FynomAb Induces Redirected T Cell-Mediated Cytolysis with High Potency and Enhanced Tumor Selectivity", ANTIBODIES, vol. 4, no. 4, 8 December 2015 (2015-12-08), pages 426 - 440, XP055795432, DOI: 10.3390/antib4040426 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US12006366B2 (en) | 2020-06-11 | 2024-06-11 | Provention Bio, Inc. | Methods and compositions for preventing type 1 diabetes |
WO2024061350A1 (fr) * | 2022-09-23 | 2024-03-28 | 广州凌腾生物医药有限公司 | Protéine de fusion et son utilisation |
WO2024061352A1 (fr) * | 2022-09-23 | 2024-03-28 | 广州凌腾生物医药有限公司 | Protéine de liaison à l'antigène isolée et son utilisation |
Also Published As
Publication number | Publication date |
---|---|
CN117120471A (zh) | 2023-11-24 |
JP2024512957A (ja) | 2024-03-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11679127B2 (en) | Antigen binding receptors specific for mutated Fc domains | |
AU2017226510B2 (en) | A pdl-1 antibody, pharmaceutical composition thereof and use thereof | |
WO2022199555A1 (fr) | Protéine de liaison à un antigène multispécifique se liant à cd3 et son utilisation | |
US11660340B2 (en) | Combination therapy using T cell redirection antigen binding molecule against cell having immunosuppressing function | |
WO2019129261A1 (fr) | Anticorps anti-tigit et leur utilisation comme agents thérapeutiques et diagnostiques | |
US9884921B2 (en) | Bispecific heterodimeric diabodies and uses thereof | |
US11505605B2 (en) | T cell-redirected antigen-binding molecule for cells having immunosuppression function | |
US11529425B2 (en) | Immunoconjugates comprising signal regulatory protein alpha | |
WO2020114479A1 (fr) | Molécule de protéine multispécifique | |
WO2020114478A1 (fr) | Anticorps cd3 et son utilisation pharmaceutique | |
CN111787949A (zh) | 变体cd3-结合结构域及其在用于治疗疾病的组合疗法中的用途 | |
CA3200865A1 (fr) | Anticorps bispecifique anti-claudine 18.2 et cd3 et son utilisation | |
WO2022247795A1 (fr) | Nanocorps ciblant la claudine 18.2 et son utilisation | |
US20230322950A1 (en) | Antigen binding receptors | |
TW202302626A (zh) | 用於調節tgf-b信息傳導之細胞療法組成物及方法 | |
WO2023165516A1 (fr) | Anticorps bispécifique anti-pd-l1 et vegf et son utilisation | |
WO2024061352A1 (fr) | Protéine de liaison à l'antigène isolée et son utilisation | |
US20230374132A1 (en) | Anti-cd3 antibody and uses thereof | |
WO2024061350A1 (fr) | Protéine de fusion et son utilisation | |
US11795230B2 (en) | Anti-CD27 antibodies and use thereof | |
CN116997354A (zh) | 用于诊断和治疗用途的针对lilrb1的单克隆抗体 | |
CN117586411A (zh) | 一种结合tnfr2和ccr8的双特异性抗体 | |
TW202334193A (zh) | 靶向γδ T細胞之經修飾IL-2多肽及其用途 | |
CN117120077A (zh) | 用于调节tgf-b信号传导的细胞疗法组合物和方法 | |
CN117881702A (zh) | 一种cd16抗体及其应用 |
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: 22718571 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2023558720 Country of ref document: JP |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 22718571 Country of ref document: EP Kind code of ref document: A1 |