CA2562800A1 - Cross-screening system and methods for detecting a molecule having binding affinity for a target molecule - Google Patents
Cross-screening system and methods for detecting a molecule having binding affinity for a target molecule Download PDFInfo
- Publication number
- CA2562800A1 CA2562800A1 CA002562800A CA2562800A CA2562800A1 CA 2562800 A1 CA2562800 A1 CA 2562800A1 CA 002562800 A CA002562800 A CA 002562800A CA 2562800 A CA2562800 A CA 2562800A CA 2562800 A1 CA2562800 A1 CA 2562800A1
- Authority
- CA
- Canada
- Prior art keywords
- antibody
- ecla
- antibodies
- analyte
- target molecule
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 238000000034 method Methods 0.000 title claims abstract description 118
- 230000027455 binding Effects 0.000 title claims abstract description 89
- 238000012216 screening Methods 0.000 title abstract description 55
- 239000012491 analyte Substances 0.000 claims abstract description 185
- 238000001514 detection method Methods 0.000 claims abstract description 55
- 238000003556 assay Methods 0.000 claims abstract description 52
- 239000000427 antigen Substances 0.000 claims description 57
- 102000036639 antigens Human genes 0.000 claims description 57
- 108091007433 antigens Proteins 0.000 claims description 57
- 230000004044 response Effects 0.000 claims description 48
- 230000001225 therapeutic effect Effects 0.000 claims description 38
- 229960000397 bevacizumab Drugs 0.000 claims description 30
- 239000012634 fragment Substances 0.000 claims description 29
- 230000009870 specific binding Effects 0.000 claims description 14
- 238000004458 analytical method Methods 0.000 claims description 12
- 230000028993 immune response Effects 0.000 claims description 7
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims description 5
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 claims description 3
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 claims description 3
- 238000002965 ELISA Methods 0.000 claims 7
- 230000009824 affinity maturation Effects 0.000 claims 1
- 238000003018 immunoassay Methods 0.000 abstract description 72
- 239000003814 drug Substances 0.000 abstract description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 66
- 108090000765 processed proteins & peptides Chemical group 0.000 description 59
- 102000004196 processed proteins & peptides Human genes 0.000 description 56
- 229920001184 polypeptide Chemical group 0.000 description 54
- 239000003795 chemical substances by application Substances 0.000 description 48
- 210000004408 hybridoma Anatomy 0.000 description 36
- 102000005962 receptors Human genes 0.000 description 27
- 108020003175 receptors Proteins 0.000 description 27
- 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 26
- 239000011859 microparticle Substances 0.000 description 26
- 239000000872 buffer Substances 0.000 description 23
- 239000006228 supernatant Substances 0.000 description 23
- -1 antibodies Proteins 0.000 description 21
- 238000011534 incubation Methods 0.000 description 21
- 239000000523 sample Substances 0.000 description 21
- 150000003384 small molecules Chemical group 0.000 description 19
- 239000011324 bead Substances 0.000 description 18
- 108090000623 proteins and genes Proteins 0.000 description 18
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 16
- 238000010494 dissociation reaction Methods 0.000 description 16
- 230000005593 dissociations Effects 0.000 description 16
- 239000002953 phosphate buffered saline Substances 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 14
- 235000018102 proteins Nutrition 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 14
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 13
- NBXPGTKIFRPXNI-UHFFFAOYSA-N [Ru].CC1(CC(=NC=C1)C1=NC=CC=C1)CCCC(=O)O.N1=C(C=CC=C1)C1=NC=CC=C1.N1=C(C=CC=C1)C1=NC=CC=C1 Chemical compound [Ru].CC1(CC(=NC=C1)C1=NC=CC=C1)CCCC(=O)O.N1=C(C=CC=C1)C1=NC=CC=C1.N1=C(C=CC=C1)C1=NC=CC=C1 NBXPGTKIFRPXNI-UHFFFAOYSA-N 0.000 description 13
- 229960002685 biotin Drugs 0.000 description 13
- 235000020958 biotin Nutrition 0.000 description 13
- 239000011616 biotin Substances 0.000 description 13
- 239000000243 solution Substances 0.000 description 12
- 239000003446 ligand Substances 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 10
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 10
- 229940088598 enzyme Drugs 0.000 description 10
- 239000000203 mixture Substances 0.000 description 9
- 230000035945 sensitivity Effects 0.000 description 9
- 239000000758 substrate Substances 0.000 description 9
- 239000007790 solid phase Substances 0.000 description 8
- 238000005406 washing Methods 0.000 description 7
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 6
- 108060003951 Immunoglobulin Proteins 0.000 description 6
- 229920001213 Polysorbate 20 Polymers 0.000 description 6
- 108010090804 Streptavidin Proteins 0.000 description 6
- 102000018358 immunoglobulin Human genes 0.000 description 6
- 229910052751 metal Inorganic materials 0.000 description 6
- 239000002184 metal Substances 0.000 description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 6
- ROFVEXUMMXZLPA-UHFFFAOYSA-N Bipyridyl Chemical group N1=CC=CC=C1C1=CC=CC=N1 ROFVEXUMMXZLPA-UHFFFAOYSA-N 0.000 description 5
- 241000283707 Capra Species 0.000 description 5
- 238000013019 agitation Methods 0.000 description 5
- 230000000903 blocking effect Effects 0.000 description 5
- 230000005291 magnetic effect Effects 0.000 description 5
- 239000012071 phase Substances 0.000 description 5
- 238000003127 radioimmunoassay Methods 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 241000894007 species Species 0.000 description 5
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 241001529936 Murinae Species 0.000 description 4
- 102100029268 Neurotrophin-3 Human genes 0.000 description 4
- 108090000099 Neurotrophin-4 Proteins 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 4
- 241001494479 Pecora Species 0.000 description 4
- 239000004743 Polypropylene Substances 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 4
- 150000001412 amines Chemical class 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 125000003118 aryl group Chemical group 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 239000013522 chelant Substances 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- 229920001577 copolymer Polymers 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 238000002823 phage display Methods 0.000 description 4
- 239000010452 phosphate Substances 0.000 description 4
- 229920001155 polypropylene Polymers 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 229940126622 therapeutic monoclonal antibody Drugs 0.000 description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 3
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 3
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical class ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 3
- 108010025020 Nerve Growth Factor Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 239000003431 cross linking reagent Substances 0.000 description 3
- 239000003792 electrolyte Substances 0.000 description 3
- 239000003102 growth factor Substances 0.000 description 3
- 238000004020 luminiscence type Methods 0.000 description 3
- 230000005298 paramagnetic effect Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 229960004641 rituximab Drugs 0.000 description 3
- YAYGSLOSTXKUBW-UHFFFAOYSA-N ruthenium(2+) Chemical compound [Ru+2] YAYGSLOSTXKUBW-UHFFFAOYSA-N 0.000 description 3
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- 229940099073 xolair Drugs 0.000 description 3
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 2
- BZRVCSFTQDMJCA-UHFFFAOYSA-N 2-[4-[3-(1,3-dioxolan-2-yl)propyl]pyridin-2-yl]-4-methylpyridine;osmium Chemical compound [Os].CC1=CC=NC(C=2N=CC=C(CCCC3OCCO3)C=2)=C1 BZRVCSFTQDMJCA-UHFFFAOYSA-N 0.000 description 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- 238000011725 BALB/c mouse Methods 0.000 description 2
- 102000007350 Bone Morphogenetic Proteins Human genes 0.000 description 2
- 108010007726 Bone Morphogenetic Proteins Proteins 0.000 description 2
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 2
- KAKZBPTYRLMSJV-UHFFFAOYSA-N Butadiene Chemical compound C=CC=C KAKZBPTYRLMSJV-UHFFFAOYSA-N 0.000 description 2
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 2
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 description 2
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 2
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 2
- 229940045513 CTLA4 antagonist Drugs 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 102000007644 Colony-Stimulating Factors Human genes 0.000 description 2
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- 102000001301 EGF receptor Human genes 0.000 description 2
- 108060006698 EGF receptor Proteins 0.000 description 2
- 108010044063 Endocrine-Gland-Derived Vascular Endothelial Growth Factor Proteins 0.000 description 2
- 241000724791 Filamentous phage Species 0.000 description 2
- 102000006395 Globulins Human genes 0.000 description 2
- 108010044091 Globulins Proteins 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- 108090001117 Insulin-Like Growth Factor II Proteins 0.000 description 2
- 102000048143 Insulin-Like Growth Factor II Human genes 0.000 description 2
- 108010008212 Integrin alpha4beta1 Proteins 0.000 description 2
- 108010063738 Interleukins Proteins 0.000 description 2
- 102000015696 Interleukins Human genes 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 101100335081 Mus musculus Flt3 gene Proteins 0.000 description 2
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 108090000742 Neurotrophin 3 Proteins 0.000 description 2
- 102000003683 Neurotrophin-4 Human genes 0.000 description 2
- 102100033857 Neurotrophin-4 Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 241000609499 Palicourea Species 0.000 description 2
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 2
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 102100040126 Prokineticin-1 Human genes 0.000 description 2
- 101800004937 Protein C Proteins 0.000 description 2
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 2
- 102100029981 Receptor tyrosine-protein kinase erbB-4 Human genes 0.000 description 2
- 101710100963 Receptor tyrosine-protein kinase erbB-4 Proteins 0.000 description 2
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 description 2
- 102400000827 Saposin-D Human genes 0.000 description 2
- 101800001700 Saposin-D Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Natural products C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
- 102100033571 Tissue-type plasminogen activator Human genes 0.000 description 2
- 108010009583 Transforming Growth Factors Proteins 0.000 description 2
- 102000009618 Transforming Growth Factors Human genes 0.000 description 2
- 125000001931 aliphatic group Chemical group 0.000 description 2
- 230000033115 angiogenesis Effects 0.000 description 2
- 230000003466 anti-cipated effect Effects 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 125000006615 aromatic heterocyclic group Chemical group 0.000 description 2
- 125000003710 aryl alkyl group Chemical group 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 229960002319 barbital Drugs 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000013060 biological fluid Substances 0.000 description 2
- 239000002981 blocking agent Substances 0.000 description 2
- 239000003114 blood coagulation factor Substances 0.000 description 2
- 229940112869 bone morphogenetic protein Drugs 0.000 description 2
- 150000001718 carbodiimides Chemical class 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 229940047120 colony stimulating factors Drugs 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 239000010949 copper Substances 0.000 description 2
- 239000007771 core particle Substances 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 238000007876 drug discovery Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 229940022353 herceptin Drugs 0.000 description 2
- 125000000623 heterocyclic group Chemical group 0.000 description 2
- 239000008240 homogeneous mixture Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 108010044426 integrins Proteins 0.000 description 2
- 102000006495 integrins Human genes 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 229940047122 interleukins Drugs 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 210000001165 lymph node Anatomy 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 239000003900 neurotrophic factor Substances 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 229910052762 osmium Inorganic materials 0.000 description 2
- SYQBFIAQOQZEGI-UHFFFAOYSA-N osmium atom Chemical compound [Os] SYQBFIAQOQZEGI-UHFFFAOYSA-N 0.000 description 2
- 102000013415 peroxidase activity proteins Human genes 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 229960000856 protein c Drugs 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 229910052761 rare earth metal Inorganic materials 0.000 description 2
- 150000002910 rare earth metals Chemical class 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 229910052707 ruthenium Inorganic materials 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 238000002764 solid phase assay Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- 229910052723 transition metal Inorganic materials 0.000 description 2
- 150000003624 transition metals Chemical class 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- FXYPGCIGRDZWNR-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-[[3-(2,5-dioxopyrrolidin-1-yl)oxy-3-oxopropyl]disulfanyl]propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSCCC(=O)ON1C(=O)CCC1=O FXYPGCIGRDZWNR-UHFFFAOYSA-N 0.000 description 1
- YTQABVCCMZQUIS-UHFFFAOYSA-N 2,2-bis[2,5-dioxo-3-(2-pyridin-2-ylpyridin-3-yl)pyrrol-1-yl]hexanoic acid Chemical compound O=C1C=C(C=2C(=NC=CC=2)C=2N=CC=CC=2)C(=O)N1C(C(O)=O)(CCCC)N(C1=O)C(=O)C=C1C1=CC=CN=C1C1=CC=CC=N1 YTQABVCCMZQUIS-UHFFFAOYSA-N 0.000 description 1
- KGLPWQKSKUVKMJ-UHFFFAOYSA-N 2,3-dihydrophthalazine-1,4-dione Chemical class C1=CC=C2C(=O)NNC(=O)C2=C1 KGLPWQKSKUVKMJ-UHFFFAOYSA-N 0.000 description 1
- 150000003923 2,5-pyrrolediones Chemical class 0.000 description 1
- MWZADJMZKSVTAF-UHFFFAOYSA-N 2-[4-(4-bromobutyl)pyridin-2-yl]-4-methylpyridine;2-pyridin-2-ylpyridine;ruthenium Chemical compound [Ru].N1=CC=CC=C1C1=CC=CC=N1.N1=CC=CC=C1C1=CC=CC=N1.CC1=CC=NC(C=2N=CC=C(CCCCBr)C=2)=C1 MWZADJMZKSVTAF-UHFFFAOYSA-N 0.000 description 1
- BZSVVCFHMVMYCR-UHFFFAOYSA-N 2-pyridin-2-ylpyridine;ruthenium Chemical compound [Ru].N1=CC=CC=C1C1=CC=CC=N1.N1=CC=CC=C1C1=CC=CC=N1.N1=CC=CC=C1C1=CC=CC=N1 BZSVVCFHMVMYCR-UHFFFAOYSA-N 0.000 description 1
- HJBUBXIDMQBSQW-UHFFFAOYSA-N 4-(4-diazoniophenyl)benzenediazonium Chemical compound C1=CC([N+]#N)=CC=C1C1=CC=C([N+]#N)C=C1 HJBUBXIDMQBSQW-UHFFFAOYSA-N 0.000 description 1
- ZXLYKNKUQNVBPB-UHFFFAOYSA-N 4-[3-(1,3-dioxolan-2-yl)propyl]-4-methyl-2-pyridin-2-yl-3H-pyridine ruthenium Chemical compound [Ru].CC1(CC(=NC=C1)C1=NC=CC=C1)CCCC1OCCO1 ZXLYKNKUQNVBPB-UHFFFAOYSA-N 0.000 description 1
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- NLPWSMKACWGINL-UHFFFAOYSA-N 4-azido-2-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(N=[N+]=[N-])C=C1O NLPWSMKACWGINL-UHFFFAOYSA-N 0.000 description 1
- MBVFRSJFKMJRHA-UHFFFAOYSA-N 4-fluoro-1-benzofuran-7-carbaldehyde Chemical compound FC1=CC=C(C=O)C2=C1C=CO2 MBVFRSJFKMJRHA-UHFFFAOYSA-N 0.000 description 1
- 102100031126 6-phosphogluconolactonase Human genes 0.000 description 1
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 1
- CJIJXIFQYOPWTF-UHFFFAOYSA-N 7-hydroxycoumarin Natural products O1C(=O)C=CC2=CC(O)=CC=C21 CJIJXIFQYOPWTF-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- NLHHRLWOUZZQLW-UHFFFAOYSA-N Acrylonitrile Chemical compound C=CC#N NLHHRLWOUZZQLW-UHFFFAOYSA-N 0.000 description 1
- 108010059616 Activins Proteins 0.000 description 1
- 102000005606 Activins Human genes 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 101710186708 Agglutinin Proteins 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 101000843904 Arabidopsis thaliana Bifunctional phosphatase IMPL2, chloroplastic Proteins 0.000 description 1
- 101800001288 Atrial natriuretic factor Proteins 0.000 description 1
- 102400001282 Atrial natriuretic peptide Human genes 0.000 description 1
- 101800001890 Atrial natriuretic peptide Proteins 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 1
- 108090000363 Bacterial Luciferases Proteins 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 102000013585 Bombesin Human genes 0.000 description 1
- 108010051479 Bombesin Proteins 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 102100031092 C-C motif chemokine 3 Human genes 0.000 description 1
- 101710155856 C-C motif chemokine 3 Proteins 0.000 description 1
- 108010009575 CD55 Antigens Proteins 0.000 description 1
- 102400000113 Calcitonin Human genes 0.000 description 1
- 108060001064 Calcitonin Proteins 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 108010076119 Caseins Proteins 0.000 description 1
- 108010067225 Cell Adhesion Molecules Proteins 0.000 description 1
- 102000016289 Cell Adhesion Molecules Human genes 0.000 description 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- 102100022641 Coagulation factor IX Human genes 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000759568 Corixa Species 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 108090000204 Dipeptidase 1 Proteins 0.000 description 1
- 102000003951 Erythropoietin Human genes 0.000 description 1
- 108090000394 Erythropoietin Proteins 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 108010076282 Factor IX Proteins 0.000 description 1
- 108010054218 Factor VIII Proteins 0.000 description 1
- 102000001690 Factor VIII Human genes 0.000 description 1
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 1
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 1
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 1
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 description 1
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 1
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 1
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 1
- 108010015133 Galactose oxidase Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102400000321 Glucagon Human genes 0.000 description 1
- 108060003199 Glucagon Proteins 0.000 description 1
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 1
- 102100022624 Glucoamylase Human genes 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 102000006771 Gonadotropins Human genes 0.000 description 1
- 108010086677 Gonadotropins Proteins 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 102000018997 Growth Hormone Human genes 0.000 description 1
- 239000000095 Growth Hormone-Releasing Hormone Substances 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 101100495232 Homo sapiens MS4A1 gene Proteins 0.000 description 1
- 101710146024 Horcolin Proteins 0.000 description 1
- 108010000521 Human Growth Hormone Proteins 0.000 description 1
- 102000002265 Human Growth Hormone Human genes 0.000 description 1
- 239000000854 Human Growth Hormone Substances 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 102000009786 Immunoglobulin Constant Regions Human genes 0.000 description 1
- 108010009817 Immunoglobulin Constant Regions Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102100022339 Integrin alpha-L Human genes 0.000 description 1
- 108010064593 Intercellular Adhesion Molecule-1 Proteins 0.000 description 1
- 102100037877 Intercellular adhesion molecule 1 Human genes 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 102000003996 Interferon-beta Human genes 0.000 description 1
- 108090000467 Interferon-beta Proteins 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- 102100038609 Lactoperoxidase Human genes 0.000 description 1
- 108010023244 Lactoperoxidase Proteins 0.000 description 1
- 101710189395 Lectin Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 102000009151 Luteinizing Hormone Human genes 0.000 description 1
- 108010073521 Luteinizing Hormone Proteins 0.000 description 1
- 108010064548 Lymphocyte Function-Associated Antigen-1 Proteins 0.000 description 1
- 108090000542 Lymphotoxin-alpha Proteins 0.000 description 1
- 102000004083 Lymphotoxin-alpha Human genes 0.000 description 1
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102000007651 Macrophage Colony-Stimulating Factor Human genes 0.000 description 1
- 102000009571 Macrophage Inflammatory Proteins Human genes 0.000 description 1
- 108010009474 Macrophage Inflammatory Proteins Proteins 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 101710179758 Mannose-specific lectin Proteins 0.000 description 1
- 101710150763 Mannose-specific lectin 1 Proteins 0.000 description 1
- 101710150745 Mannose-specific lectin 2 Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- 102100030173 Muellerian-inhibiting factor Human genes 0.000 description 1
- 101710122877 Muellerian-inhibiting factor Proteins 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 150000007945 N-acyl ureas Chemical class 0.000 description 1
- 108090000028 Neprilysin Proteins 0.000 description 1
- 102000003729 Neprilysin Human genes 0.000 description 1
- 102000015336 Nerve Growth Factor Human genes 0.000 description 1
- 108090000095 Neurotrophin-6 Proteins 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 102000003982 Parathyroid hormone Human genes 0.000 description 1
- 108090000445 Parathyroid hormone Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108010067902 Peptide Library Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 102000001938 Plasminogen Activators Human genes 0.000 description 1
- 108010001014 Plasminogen Activators Proteins 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 108010076181 Proinsulin Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102100029986 Receptor tyrosine-protein kinase erbB-3 Human genes 0.000 description 1
- 101710100969 Receptor tyrosine-protein kinase erbB-3 Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 208000015634 Rectal Neoplasms Diseases 0.000 description 1
- 102400000834 Relaxin A chain Human genes 0.000 description 1
- 101800000074 Relaxin A chain Proteins 0.000 description 1
- 102400000610 Relaxin B chain Human genes 0.000 description 1
- 101710109558 Relaxin B chain Proteins 0.000 description 1
- 108090000783 Renin Proteins 0.000 description 1
- 102100028255 Renin Human genes 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 102100022831 Somatoliberin Human genes 0.000 description 1
- 101710142969 Somatoliberin Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 102000019197 Superoxide Dismutase Human genes 0.000 description 1
- 108010012715 Superoxide dismutase Proteins 0.000 description 1
- 208000002847 Surgical Wound Diseases 0.000 description 1
- 108091008874 T cell receptors Proteins 0.000 description 1
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 108010000499 Thromboplastin Proteins 0.000 description 1
- 102000002262 Thromboplastin Human genes 0.000 description 1
- 102000011923 Thyrotropin Human genes 0.000 description 1
- 108010061174 Thyrotropin Proteins 0.000 description 1
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 1
- 108050006955 Tissue-type plasminogen activator Proteins 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 102400001320 Transforming growth factor alpha Human genes 0.000 description 1
- 101800004564 Transforming growth factor alpha Proteins 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 108010092464 Urate Oxidase Proteins 0.000 description 1
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 1
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 1
- BZHJMEDXRYGGRV-UHFFFAOYSA-N Vinyl chloride Chemical compound ClC=C BZHJMEDXRYGGRV-UHFFFAOYSA-N 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 108010093894 Xanthine oxidase Proteins 0.000 description 1
- 102100033220 Xanthine oxidase Human genes 0.000 description 1
- 239000000488 activin Substances 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000000910 agglutinin Substances 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 108010050122 alpha 1-Antitrypsin Proteins 0.000 description 1
- 102000015395 alpha 1-Antitrypsin Human genes 0.000 description 1
- 229940024142 alpha 1-antitrypsin Drugs 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 150000001409 amidines Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000001455 anti-clotting effect Effects 0.000 description 1
- RBFQJDQYXXHULB-UHFFFAOYSA-N arsane Chemical class [AsH3] RBFQJDQYXXHULB-UHFFFAOYSA-N 0.000 description 1
- 102000006635 beta-lactamase Human genes 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- DNDCVAGJPBKION-DOPDSADYSA-N bombesin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(N)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC=1NC2=CC=CC=C2C=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1NC(=O)CC1)C(C)C)C1=CN=CN1 DNDCVAGJPBKION-DOPDSADYSA-N 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 108010006025 bovine growth hormone Proteins 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 1
- 229960004015 calcitonin Drugs 0.000 description 1
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 150000003857 carboxamides Chemical class 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 150000007942 carboxylates Chemical class 0.000 description 1
- NSQLIUXCMFBZME-MPVJKSABSA-N carperitide Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 NSQLIUXCMFBZME-MPVJKSABSA-N 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000012412 chemical coupling Methods 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 229910052804 chromium Inorganic materials 0.000 description 1
- 239000011651 chromium Substances 0.000 description 1
- 229940090961 chromium dioxide Drugs 0.000 description 1
- IAQWMWUKBQPOIY-UHFFFAOYSA-N chromium(4+);oxygen(2-) Chemical compound [O-2].[O-2].[Cr+4] IAQWMWUKBQPOIY-UHFFFAOYSA-N 0.000 description 1
- AYTAKQFHWFYBMA-UHFFFAOYSA-N chromium(IV) oxide Inorganic materials O=[Cr]=O AYTAKQFHWFYBMA-UHFFFAOYSA-N 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 239000007822 coupling agent Substances 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 150000001945 cysteines Chemical class 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 125000001295 dansyl group Chemical group [H]C1=C([H])C(N(C([H])([H])[H])C([H])([H])[H])=C2C([H])=C([H])C([H])=C(C2=C1[H])S(*)(=O)=O 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 108700001680 des-(1-3)- insulin-like growth factor 1 Proteins 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000007812 electrochemical assay Methods 0.000 description 1
- 230000005670 electromagnetic radiation Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229940105423 erythropoietin Drugs 0.000 description 1
- 230000005281 excited state Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 229960004222 factor ix Drugs 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 229940126864 fibroblast growth factor Drugs 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 229940028334 follicle stimulating hormone Drugs 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 1
- 229960004666 glucagon Drugs 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000002622 gonadotropin Substances 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- ZRALSGWEFCBTJO-UHFFFAOYSA-O guanidinium Chemical compound NC(N)=[NH2+] ZRALSGWEFCBTJO-UHFFFAOYSA-O 0.000 description 1
- 150000004820 halides Chemical class 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000002607 hemopoietic effect Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- ORTFAQDWJHRMNX-UHFFFAOYSA-N hydroxidooxidocarbon(.) Chemical group O[C]=O ORTFAQDWJHRMNX-UHFFFAOYSA-N 0.000 description 1
- 150000002463 imidates Chemical class 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 229940051026 immunotoxin Drugs 0.000 description 1
- 230000002637 immunotoxin Effects 0.000 description 1
- 239000002596 immunotoxin Substances 0.000 description 1
- 231100000608 immunotoxin Toxicity 0.000 description 1
- 229910052738 indium Inorganic materials 0.000 description 1
- APFVFJFRJDLVQX-UHFFFAOYSA-N indium atom Chemical compound [In] APFVFJFRJDLVQX-UHFFFAOYSA-N 0.000 description 1
- 239000000893 inhibin Substances 0.000 description 1
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 1
- 239000010954 inorganic particle Substances 0.000 description 1
- 102000028416 insulin-like growth factor binding Human genes 0.000 description 1
- 108091022911 insulin-like growth factor binding Proteins 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 229910052741 iridium Inorganic materials 0.000 description 1
- GKOZUEZYRPOHIO-UHFFFAOYSA-N iridium atom Chemical compound [Ir] GKOZUEZYRPOHIO-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 150000002527 isonitriles Chemical class 0.000 description 1
- 229940057428 lactoperoxidase Drugs 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000002761 liquid phase assay Methods 0.000 description 1
- 229940066294 lung surfactant Drugs 0.000 description 1
- 239000003580 lung surfactant Substances 0.000 description 1
- 229940040129 luteinizing hormone Drugs 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 208000037819 metastatic cancer Diseases 0.000 description 1
- 208000011575 metastatic malignant neoplasm Diseases 0.000 description 1
- YCXSYMVGMXQYNT-UHFFFAOYSA-N methyl 3-[(4-azidophenyl)disulfanyl]propanimidate Chemical compound COC(=N)CCSSC1=CC=C(N=[N+]=[N-])C=C1 YCXSYMVGMXQYNT-UHFFFAOYSA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 108010029942 microperoxidase Proteins 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 229910052750 molybdenum Inorganic materials 0.000 description 1
- 239000011733 molybdenum Substances 0.000 description 1
- 229940035032 monophosphoryl lipid a Drugs 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229940053128 nerve growth factor Drugs 0.000 description 1
- 229940032018 neurotrophin 3 Drugs 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 230000002138 osteoinductive effect Effects 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 239000000199 parathyroid hormone Substances 0.000 description 1
- 229960001319 parathyroid hormone Drugs 0.000 description 1
- 239000011236 particulate material Substances 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229940127126 plasminogen activator Drugs 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108010087851 prorelaxin Proteins 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229910052702 rhenium Inorganic materials 0.000 description 1
- WUAPFZMCVAUBPE-UHFFFAOYSA-N rhenium atom Chemical compound [Re] WUAPFZMCVAUBPE-UHFFFAOYSA-N 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 229910052703 rhodium Inorganic materials 0.000 description 1
- 239000010948 rhodium Substances 0.000 description 1
- MHOVAHRLVXNVSD-UHFFFAOYSA-N rhodium atom Chemical compound [Rh] MHOVAHRLVXNVSD-UHFFFAOYSA-N 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 150000001629 stilbenes Chemical class 0.000 description 1
- 235000021286 stilbenes Nutrition 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 229920003048 styrene butadiene rubber Polymers 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 125000000547 substituted alkyl group Chemical group 0.000 description 1
- 125000003107 substituted aryl group Chemical group 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 239000013595 supernatant sample Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229910052713 technetium Inorganic materials 0.000 description 1
- GKLVYJBZJHMRIY-UHFFFAOYSA-N technetium atom Chemical compound [Tc] GKLVYJBZJHMRIY-UHFFFAOYSA-N 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 description 1
- 229910052721 tungsten Inorganic materials 0.000 description 1
- 239000010937 tungsten Substances 0.000 description 1
- ORHBXUUXSCNDEV-UHFFFAOYSA-N umbelliferone Chemical compound C1=CC(=O)OC2=CC(O)=CC=C21 ORHBXUUXSCNDEV-UHFFFAOYSA-N 0.000 description 1
- HFTAFOQKODTIJY-UHFFFAOYSA-N umbelliferone Natural products Cc1cc2C=CC(=O)Oc2cc1OCC=CC(C)(C)O HFTAFOQKODTIJY-UHFFFAOYSA-N 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 229960005356 urokinase Drugs 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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
- C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- 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
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
- C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
- C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
- C07K16/4258—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Abstract
The invention is directed to a cross-screening system and methods of the invention utilizing a combination of an immunoassay (IA) and electrochemiluminescence assay (ECLA) to identify molecules that have binding affinities for a target molecule. The cross-screening system and methods of the invention can detect molecules that have binding affinities for the target molecule below the detection limits of the individual immunoassay or ECLA. The cross-screening system and methods of the invention are useful for generating a pool of candidate analyte molecules enriched in a desired characteristic, such as low binding affinity for a target molecule. Low affinity antibodies identified by the cross-screening system and methods of the invention are useful, for example, in assessing the safety and efficacy of biological therapeutics.
Description
CROSS-SCREENING SYSTEM AND METHODS FOR DETECTING A
MOLECULE HAVING BINDING AFFINITY FOR A TARGET MOLECULE
BACKGROUND OF THE INVENTION
Low affinity antibodies are valuable for therapeutic use, such as combination therapies. Low affinity antibodies are also useful in drug discovery. For example, where high affinity antibodies are difficult to obtain for a specific therapeutic molecule, low affinity antibodies can serve as a starting point for developing useful affinity-matured antibodies.
Anti-therapeutic molecule antibody assays, needed for regulatory approval of therapeutic molecules, require low affinity antibodies. Such assays require sensitive and efficient means to detect unwanted immune responses, important for assessing safety and efficacy of a therapeutic molecule.
Anti-therapeutic molecule antibodies can target different regions of the therapeutic and can exhibit differing binding affinities and isotypes. A panel of varied anti-therapeutic molecule antibodies mimicking the polyclonal nature of an immune response is desirable; to more accurately assess performance of anti-therapeutic molecule antibody assays.
Screening hybridoma clones for high affinity antibodies has traditionally utilized ELISA technology. ELISA, however, is not as effective for screening low affinity antibodies. Although ELISA can identify antibodies that bind an antigen, the assay cannot readily identify antibodies that bind with low affinity. Many low affinity antibodies are lost in the multiple wash steps required to ensure a high signal-to-noise ratio. Minimizing wash steps to retain these low affinity antibodies, however, decreases sensitivity of the assay by decreasing the signal-to-noise ratio.
A minimal number of wash steps are required in electrochemiluminescence assay (ECLA), permitting the ECLA system to detect low affinity antibodies that would be washed away by traditional ELISA methods. Simply replacing ELISA
with ECLA is not a good solution, however. Like ELISA, ECLA cannot readily identify antibodies that bind with low affinity. In addition, labeling agents used in ECLA have the potential to alter binding properties of the antibodies. ECLA
can thus fail to retain antibodies that would otherwise be retained by conventional ELISA methods.
Efficient assay systems and methods are greatly needed for screening a pool of analyte molecules, such as antibodies, to identify those having specific characteristics, including low affinity antibodies, anti-therapeutic molecule antibodies responsive to a variety of epitopes, and the like. In particular, efficient and reliable methods to identify a pool of analyte molecules enriched with those having a desired affinity (low or high) or likely to contain analyte molecules responsive to differing binding sites of a target molecule, would be very useful.
SUMMARY OF THE INVENTION
The present invention provides a cross-screening system and methods for crossing an immunoassay with ECLA to identify analyte molecules, such as antibodies, that have selective binding affinity for a target molecule.
Analyte molecules are identified as having a particular characteristic, such as low or high binding affinity and/or binding to differing binding sites of a target molecule. The cross-screening system and methods of the invention detect analyte molecules that are below the detection limits of an immunoassay (IA) or ECLA individually.
Binding to a target molecule is assayed in both immunoassay methods, such as ELISA, and in ECLA.
A cross-screening system and methods of the invention generally employ the following steps: (1) determining ECLA responses for individual members of a pool of analyte molecules binding to a target molecule; (2) determining IA
responses for individual members of the pool of analyte molecules binding the target molecule;
and (3) generating a pool of candidate analyte molecules enriched in a desired characteristic, such as low or high binding affinity or variety of the antigenic epitopes.
Data from a large pool of analyte molecules is produced and evaluated as IA+
or IA-; ECLA+ or ECLA-. Molecules that are IA+/ECLA+, IA-/ECLA+, or IA+/ECLA- are identified as analyte molecules that specifically bind the target molecule.
The candidate analyte molecule is selected from an enriched pool of analyte molecules generated on the basis of the respective ECLA and IA responses, for . example, IA-/ECLA-, IA+BCLA-, IA-/ECLA+, or IA+/ECLA+. Analyte molecules and/or target molecules can be antibodies. In one embodiment, the target molecules are therapeutic antibodies and the analyte molecules are anti-therapeutic antibodies.
MOLECULE HAVING BINDING AFFINITY FOR A TARGET MOLECULE
BACKGROUND OF THE INVENTION
Low affinity antibodies are valuable for therapeutic use, such as combination therapies. Low affinity antibodies are also useful in drug discovery. For example, where high affinity antibodies are difficult to obtain for a specific therapeutic molecule, low affinity antibodies can serve as a starting point for developing useful affinity-matured antibodies.
Anti-therapeutic molecule antibody assays, needed for regulatory approval of therapeutic molecules, require low affinity antibodies. Such assays require sensitive and efficient means to detect unwanted immune responses, important for assessing safety and efficacy of a therapeutic molecule.
Anti-therapeutic molecule antibodies can target different regions of the therapeutic and can exhibit differing binding affinities and isotypes. A panel of varied anti-therapeutic molecule antibodies mimicking the polyclonal nature of an immune response is desirable; to more accurately assess performance of anti-therapeutic molecule antibody assays.
Screening hybridoma clones for high affinity antibodies has traditionally utilized ELISA technology. ELISA, however, is not as effective for screening low affinity antibodies. Although ELISA can identify antibodies that bind an antigen, the assay cannot readily identify antibodies that bind with low affinity. Many low affinity antibodies are lost in the multiple wash steps required to ensure a high signal-to-noise ratio. Minimizing wash steps to retain these low affinity antibodies, however, decreases sensitivity of the assay by decreasing the signal-to-noise ratio.
A minimal number of wash steps are required in electrochemiluminescence assay (ECLA), permitting the ECLA system to detect low affinity antibodies that would be washed away by traditional ELISA methods. Simply replacing ELISA
with ECLA is not a good solution, however. Like ELISA, ECLA cannot readily identify antibodies that bind with low affinity. In addition, labeling agents used in ECLA have the potential to alter binding properties of the antibodies. ECLA
can thus fail to retain antibodies that would otherwise be retained by conventional ELISA methods.
Efficient assay systems and methods are greatly needed for screening a pool of analyte molecules, such as antibodies, to identify those having specific characteristics, including low affinity antibodies, anti-therapeutic molecule antibodies responsive to a variety of epitopes, and the like. In particular, efficient and reliable methods to identify a pool of analyte molecules enriched with those having a desired affinity (low or high) or likely to contain analyte molecules responsive to differing binding sites of a target molecule, would be very useful.
SUMMARY OF THE INVENTION
The present invention provides a cross-screening system and methods for crossing an immunoassay with ECLA to identify analyte molecules, such as antibodies, that have selective binding affinity for a target molecule.
Analyte molecules are identified as having a particular characteristic, such as low or high binding affinity and/or binding to differing binding sites of a target molecule. The cross-screening system and methods of the invention detect analyte molecules that are below the detection limits of an immunoassay (IA) or ECLA individually.
Binding to a target molecule is assayed in both immunoassay methods, such as ELISA, and in ECLA.
A cross-screening system and methods of the invention generally employ the following steps: (1) determining ECLA responses for individual members of a pool of analyte molecules binding to a target molecule; (2) determining IA
responses for individual members of the pool of analyte molecules binding the target molecule;
and (3) generating a pool of candidate analyte molecules enriched in a desired characteristic, such as low or high binding affinity or variety of the antigenic epitopes.
Data from a large pool of analyte molecules is produced and evaluated as IA+
or IA-; ECLA+ or ECLA-. Molecules that are IA+/ECLA+, IA-/ECLA+, or IA+/ECLA- are identified as analyte molecules that specifically bind the target molecule.
The candidate analyte molecule is selected from an enriched pool of analyte molecules generated on the basis of the respective ECLA and IA responses, for . example, IA-/ECLA-, IA+BCLA-, IA-/ECLA+, or IA+/ECLA+. Analyte molecules and/or target molecules can be antibodies. In one embodiment, the target molecules are therapeutic antibodies and the analyte molecules are anti-therapeutic antibodies.
In an embodiment, IA and ECLA responses are determined within detection limits of the respective assays. An ECLA response equal to or greater than the ECLA detection limit is ECLA+. An ECLA response less than the ECLA detection limit is ECLA-. An IA response equal to or greater than the IA detection limit is IA+. An IA response less than the IA detection limit is IA-. A candidate low affinity analyte molecule is IA-/ECLA+. A candidate high affinity analyte molecule is IA~IECLA+ or IA~/ECLA-. Analyte molecules that are IA~/ECLA- include candidate analyte molecules that bind a target molecule at a binding site that is masked or altered in the ECLA assay, for example, by biotin or a chemical label employed in ECLA.
The cross-screening system and methods of the invention optionally include confirming specific binding affinity of a candidate analyte molecule, for example, by surface plasmon resonance analysis such as Biacore, competitive ELISA, equilibrium dialysis, radioimmunoassay, and the like. Candidate low aff nity analyte molecules demonstrating a Kd;sso~ greater than 10-61/sec or a KD equal to or greater than 10-$ M, for example, can be confirmed as low affinity antibodies.
Low affinity analyte molecules identified by the cross-screening system and methods of the invention generally demonstrate a Kd;sso~ equal to or greater than about 10-51/sec or a KD of about 10-6 M to about 10-8 M.
The cross-screening system and methods of the invention optionally include confirming the isotype of a candidate analyte molecule that is an antibody, for example, by isotyping ELISA.
The cross-screening system and methods of the invention can also be used to detect small amounts of an analyte in a sample. For example, the cross-screening system and methods of the invention can be used to identify a hybridoma producing a low concentration of antibodies that have affinity for the target molecule.
The concentration of antibody in the supernatant can be below the detection limit of the individual immunoassay or ECLA, but not the detection limit of the cross-screening system and methods of the invention, for example, (ECLA-/IA+ or ECLA+/IA-).
The cross-screening system and methods are useful to screen analyte molecules such as small molecules, polypeptides, or polypeptide fragments. The system and methods are particularly useful to screen antibodies, soluble receptors, or fragments thereof. The antibodies can be monoclonal. In an embodiment, the antibodies are monoclonal anti-therapeutic molecule antibodies.
The cross-screening system and methods of the invention optionally include confirming specific binding affinity of a candidate analyte molecule, for example, by surface plasmon resonance analysis such as Biacore, competitive ELISA, equilibrium dialysis, radioimmunoassay, and the like. Candidate low aff nity analyte molecules demonstrating a Kd;sso~ greater than 10-61/sec or a KD equal to or greater than 10-$ M, for example, can be confirmed as low affinity antibodies.
Low affinity analyte molecules identified by the cross-screening system and methods of the invention generally demonstrate a Kd;sso~ equal to or greater than about 10-51/sec or a KD of about 10-6 M to about 10-8 M.
The cross-screening system and methods of the invention optionally include confirming the isotype of a candidate analyte molecule that is an antibody, for example, by isotyping ELISA.
The cross-screening system and methods of the invention can also be used to detect small amounts of an analyte in a sample. For example, the cross-screening system and methods of the invention can be used to identify a hybridoma producing a low concentration of antibodies that have affinity for the target molecule.
The concentration of antibody in the supernatant can be below the detection limit of the individual immunoassay or ECLA, but not the detection limit of the cross-screening system and methods of the invention, for example, (ECLA-/IA+ or ECLA+/IA-).
The cross-screening system and methods are useful to screen analyte molecules such as small molecules, polypeptides, or polypeptide fragments. The system and methods are particularly useful to screen antibodies, soluble receptors, or fragments thereof. The antibodies can be monoclonal. In an embodiment, the antibodies are monoclonal anti-therapeutic molecule antibodies.
The target molecule is typically a small molecule, polypeptide, or polypeptide fragment. The target molecule can be, for example, an antigen if the analyte is an antibody, a receptor or antibody if the analyte is a small molecule or polypeptide, a polypeptide or small molecule if the analyte is a soluble receptor, or phage expressing antibodies, soluble receptors, or fragments thereof if the analyte is a polypeptide or small molecule. The target molecule can be a polypeptide or antibody having therapeutic activity. When the target molecule is a therapeutic antibody or therapeutic polypeptide, the cross-screening system and method can identify low affinity analyte antibodies.
The cross-screening system and methods of the invention have many uses.
The system and methods of the invention can be used to screen serum from a patient who is about to receive or is receiving a therapeutic molecule for antibodies to the therapeutic molecule. The system and methods of the invention can be used to screen libraries of receptors, antibodies, polypeptides, small molecules, and the like, for library members that bind a target molecule with specific binding characteristics.
Low affinity antibodies identified by the cross-screening system and methods of the invention are particularly useful in anti-therapeutic molecule assays for evaluating the efficacy and safety of therapeutic molecules in clinical trials. Low affinity antibodies identified by the cross-screening system and methods of the invention can also serve as a starting point for developing affinity-matured antibodies.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a workflow diagram showing an embodiment of the cross-screening system and methods of the invention applied to identify low affinity antibodies for a specific antigen.
Figure 2 is a plot showing results of cross-screening a pool of anti-2H7 antibodies produced from hybridomas. The ECLA response (ECLU) is plotted against the ELISA response (0.D. at 650 nm). Antibodies in area I (ECLA-/ELISA-) represent antibodies that either do not specifically bind 2H7, or where binding was not detected by either assay. Antibodies in area II (ELISA+/ECLA') and III
(ECLA+/ELISA+) represent candidate high binding affinity anti-2H7 antibodies.
Antibodies in area IV (ECLA+/ELISA-) represent candidate low affinity anti-2H7 antibodies. Antibodies in area IV represent a population of anti-2H7 antibodies not detected by ELISA. Antibodies that are ECLA- /ELISA+ include candidate antibodies that bind the target anti-2H7 at a binding site that is masked or altered in the ECLA assay, for example, by biotin or a chemical label employed in ECLA.
Figure 3 shows equilibrium dissociation constants (Kd;sso~) of select antibodies plotted according to ECLA response and ELISA response. Dissociation rate constants of antibodies in area II (ECLA-/ELISA+) were in the range of about 10-3 to 10-51/sec; those of antibodies in area III (ECLA+/ELISA~) were about llsec or less (Table 1 and Figure 3); those of antibodies in area IV
(ECLA~/ELISA-) were in the range of about 10-2 to 10-4 1/sec (Table 1 and Figure 3).
Antibodies with a Kd;sso~ of about 10-2 1/sec were found only in area IV. The Kd;sso~ of one area IV
antibody was 10-51/sec.
Figure 4 shows heavy chain isotypes of selected antibodies plotted according to ECLA response and ELISA response. All the tested antibodies contained a kappa light chain, except for two antibodies in area II. These two antibodies contained a lambda light chain and are circled in Figure 4. Antibodies in area IT (ECLA-/ELISA+) contained heavy chain isotypes of IgGl or IgG2b. Antibodies in area III
(ECLA+/ELISA+) contained heavy chain isotypes of IgGl or IgG2b. Antibodies in area IV (ECLA''~/ELISA-) contained heavy chain isotypes of IgG, IgG2a, IgG2b, or IgG3.
Figure 5 shows a standard curve for anti-bevacizumab polyclonal antibody binding to bevacizumab in a Biacore assay. The concentration of antibody (nM) is plotted versus response (RU).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions As used herein, the term "Immunoassay"(IA) means a serological assay in which bound analyte is detected by a labeled moiety linked to a detecting agent.
Immunoassay includes, but is not limited to, radioimmunoassay (R.TA), fluoroluminescence assay (FLA), chemiluminescence assay (CLA), and enzyme-linked immunosorbant assay (ELISA). ELISA methods are described, fox example, in WO01/36972. Immunoassays are useful for detecting the presence of analyte molecules, such as antibodies, that bind target molecules, such as antigens.
The term "detecting" is used in the broadest sense to include both qualitative and quantitative measurements of a specific molecule, herein measurements of a specific analyte molecule such as an anti-therapeutic antibody. In one aspect, a detection method as described herein is used to identify the mere presence of an analyte molecule of interest in a sample. In another aspect, the method can be used to quantify an amount of analyte molecule in a sample. In still another aspect, the method can be used to determine the relative binding affinity of an analyte molecule of interest for a target molecule.
The term "detecting agent" refers to an agent that detects an analyte molecule, either directly via a label, such as a fluorescent, enzymatic, radioactive, or chemiluminescent label, that can be linked to the detecting agent, or indirectly via a labeled binding partner, such as an antibody or receptor that specifically binds the detecting agent. Examples of detecting agents include, but are not limited to, an antibody, antibody fragment, soluble receptor, receptor fragment, and the like. In an embodiment, the detecting agent can be expressed on a phage.
The term "label" includes agents that amplify a signal produced by a detecting agent. The label can be a radiologic, photoluminescent, chemiluminescent, or electrochemiluminescent chemical moiety, an enzyme that converts a colorless substrate into a colored product, and the like.
The term "capture reagent" refers to a reagent capable of binding and capturing a target molecule or analyte molecule in a sample. Typically, a capture reagent is immobilized, for example, on a solid substrate, such as a microparticle or bead, microtiter plate, column resin, and the like. The capture reagent can be an antigen, soluble receptor, antibody, a mixture of different antibodies, and the like.
The term "target molecule" refers to a specific binding target of an analyte molecule identified by the cross-screening system and methods of the invention. A
target molecule is typically a small molecule, polypeptide, or polypeptide fragment.
The target molecule can be, for example, an antigen if the analyte molecule is an antibody, a receptor or antibody if the analyte molecule is a small molecule or polypeptide, a polypeptide or small molecule if the analyte molecule is a soluble receptor, a phage expressing antibody, soluble receptor, or fragments thereof if the analyte molecule is a polypeptide or small molecule. The target,molecule can be, for example, a polypeptide or antibody having therapeutic activity. In one embodiment, the target molecule is a therapeutic antibody and the analyte molecule is an anti-therapeutic antibody that binds the therapeutic antibody.
The cross-screening system and methods of the invention have many uses.
The system and methods of the invention can be used to screen serum from a patient who is about to receive or is receiving a therapeutic molecule for antibodies to the therapeutic molecule. The system and methods of the invention can be used to screen libraries of receptors, antibodies, polypeptides, small molecules, and the like, for library members that bind a target molecule with specific binding characteristics.
Low affinity antibodies identified by the cross-screening system and methods of the invention are particularly useful in anti-therapeutic molecule assays for evaluating the efficacy and safety of therapeutic molecules in clinical trials. Low affinity antibodies identified by the cross-screening system and methods of the invention can also serve as a starting point for developing affinity-matured antibodies.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a workflow diagram showing an embodiment of the cross-screening system and methods of the invention applied to identify low affinity antibodies for a specific antigen.
Figure 2 is a plot showing results of cross-screening a pool of anti-2H7 antibodies produced from hybridomas. The ECLA response (ECLU) is plotted against the ELISA response (0.D. at 650 nm). Antibodies in area I (ECLA-/ELISA-) represent antibodies that either do not specifically bind 2H7, or where binding was not detected by either assay. Antibodies in area II (ELISA+/ECLA') and III
(ECLA+/ELISA+) represent candidate high binding affinity anti-2H7 antibodies.
Antibodies in area IV (ECLA+/ELISA-) represent candidate low affinity anti-2H7 antibodies. Antibodies in area IV represent a population of anti-2H7 antibodies not detected by ELISA. Antibodies that are ECLA- /ELISA+ include candidate antibodies that bind the target anti-2H7 at a binding site that is masked or altered in the ECLA assay, for example, by biotin or a chemical label employed in ECLA.
Figure 3 shows equilibrium dissociation constants (Kd;sso~) of select antibodies plotted according to ECLA response and ELISA response. Dissociation rate constants of antibodies in area II (ECLA-/ELISA+) were in the range of about 10-3 to 10-51/sec; those of antibodies in area III (ECLA+/ELISA~) were about llsec or less (Table 1 and Figure 3); those of antibodies in area IV
(ECLA~/ELISA-) were in the range of about 10-2 to 10-4 1/sec (Table 1 and Figure 3).
Antibodies with a Kd;sso~ of about 10-2 1/sec were found only in area IV. The Kd;sso~ of one area IV
antibody was 10-51/sec.
Figure 4 shows heavy chain isotypes of selected antibodies plotted according to ECLA response and ELISA response. All the tested antibodies contained a kappa light chain, except for two antibodies in area II. These two antibodies contained a lambda light chain and are circled in Figure 4. Antibodies in area IT (ECLA-/ELISA+) contained heavy chain isotypes of IgGl or IgG2b. Antibodies in area III
(ECLA+/ELISA+) contained heavy chain isotypes of IgGl or IgG2b. Antibodies in area IV (ECLA''~/ELISA-) contained heavy chain isotypes of IgG, IgG2a, IgG2b, or IgG3.
Figure 5 shows a standard curve for anti-bevacizumab polyclonal antibody binding to bevacizumab in a Biacore assay. The concentration of antibody (nM) is plotted versus response (RU).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions As used herein, the term "Immunoassay"(IA) means a serological assay in which bound analyte is detected by a labeled moiety linked to a detecting agent.
Immunoassay includes, but is not limited to, radioimmunoassay (R.TA), fluoroluminescence assay (FLA), chemiluminescence assay (CLA), and enzyme-linked immunosorbant assay (ELISA). ELISA methods are described, fox example, in WO01/36972. Immunoassays are useful for detecting the presence of analyte molecules, such as antibodies, that bind target molecules, such as antigens.
The term "detecting" is used in the broadest sense to include both qualitative and quantitative measurements of a specific molecule, herein measurements of a specific analyte molecule such as an anti-therapeutic antibody. In one aspect, a detection method as described herein is used to identify the mere presence of an analyte molecule of interest in a sample. In another aspect, the method can be used to quantify an amount of analyte molecule in a sample. In still another aspect, the method can be used to determine the relative binding affinity of an analyte molecule of interest for a target molecule.
The term "detecting agent" refers to an agent that detects an analyte molecule, either directly via a label, such as a fluorescent, enzymatic, radioactive, or chemiluminescent label, that can be linked to the detecting agent, or indirectly via a labeled binding partner, such as an antibody or receptor that specifically binds the detecting agent. Examples of detecting agents include, but are not limited to, an antibody, antibody fragment, soluble receptor, receptor fragment, and the like. In an embodiment, the detecting agent can be expressed on a phage.
The term "label" includes agents that amplify a signal produced by a detecting agent. The label can be a radiologic, photoluminescent, chemiluminescent, or electrochemiluminescent chemical moiety, an enzyme that converts a colorless substrate into a colored product, and the like.
The term "capture reagent" refers to a reagent capable of binding and capturing a target molecule or analyte molecule in a sample. Typically, a capture reagent is immobilized, for example, on a solid substrate, such as a microparticle or bead, microtiter plate, column resin, and the like. The capture reagent can be an antigen, soluble receptor, antibody, a mixture of different antibodies, and the like.
The term "target molecule" refers to a specific binding target of an analyte molecule identified by the cross-screening system and methods of the invention. A
target molecule is typically a small molecule, polypeptide, or polypeptide fragment.
The target molecule can be, for example, an antigen if the analyte molecule is an antibody, a receptor or antibody if the analyte molecule is a small molecule or polypeptide, a polypeptide or small molecule if the analyte molecule is a soluble receptor, a phage expressing antibody, soluble receptor, or fragments thereof if the analyte molecule is a polypeptide or small molecule. The target,molecule can be, for example, a polypeptide or antibody having therapeutic activity. In one embodiment, the target molecule is a therapeutic antibody and the analyte molecule is an anti-therapeutic antibody that binds the therapeutic antibody.
"Analyte" and "analyte molecule," as used herein, refer to a molecule that is analyzed by the cross-screening system and methods of the invention, and includes, but is not limited to, small molecules, polypeptides, polypeptide fragments, antibodies, antibody fragments, phage, displayed polypeptides, and the like.
In the cross-screening system and methods of the invention, an analyte molecule has a binding affinity for the target molecule.
"Polypeptide" refers to a peptide or protein containing two or more amino acids Linked by peptide bonds, and includes peptides, oligomers, proteins, and the like. Polypeptides can contain natural, modified, or synthetic amino acids.
Polypeptides can also be modified naturally, such as by post-translational processing, or chemically, such as amidation acylation, cross-linking, and the like.
As used herein, an "anti-therapeutic antibody" is an antibody that binds a therapeutic antibody. For example, anti-2H7 antibody is an antibody that binds 2H7, a therapeutic antibody.
"Low affinity", as used herein, means an analyte molecule having a dissociation rate constant (Kd;sso~) generally greater then 10-6 I/sec for a target molecule. Preferably the Kd;sso~ of the analyte molecule for the target molecule is 10-5 1/sec or greater, 10-4 1/sec or greater, 10-3 1/sec or greater, or 10-2 1/sec or greater.
Useful low affinity antibodies typically have a dissociation rate constant of about 10-3 to 10-51/sec. A molecule with a high dissociation rate constant (Kd;sso~) is likely to have low affinity, as the equilibrium dissociation constant, KD =
Kd;ssoc/Kassoc. A
molecule with an equilibrium constant (KD) equal to or greater than about 10-8 M
has Low binding affinity. Useful low affinity antibodies can have a KD of about I O-6 M to about 10'8 M, for example.
Electrochemiluminescence assay or "EGLA" is an electrochemical assay in which bound analyte molecule is detected by a label linked to a detecting agent (target molecule). An electrode electrochemically initiates luminescence of a chemical label linked to a detecting agent. Light emitted by the label is measured by a photodetector and indicates the presence or quantity of bound analyte molecule/target molecule complexes. EGLA methods are described, for example, in U.S. Patent Nos. 5,543,112; 5,935,779; and 6,316,607. Signal modulation can be maximized for different analyte molecule concentrations for precise and sensitive measurements.
In the cross-screening system and methods of the invention, an analyte molecule has a binding affinity for the target molecule.
"Polypeptide" refers to a peptide or protein containing two or more amino acids Linked by peptide bonds, and includes peptides, oligomers, proteins, and the like. Polypeptides can contain natural, modified, or synthetic amino acids.
Polypeptides can also be modified naturally, such as by post-translational processing, or chemically, such as amidation acylation, cross-linking, and the like.
As used herein, an "anti-therapeutic antibody" is an antibody that binds a therapeutic antibody. For example, anti-2H7 antibody is an antibody that binds 2H7, a therapeutic antibody.
"Low affinity", as used herein, means an analyte molecule having a dissociation rate constant (Kd;sso~) generally greater then 10-6 I/sec for a target molecule. Preferably the Kd;sso~ of the analyte molecule for the target molecule is 10-5 1/sec or greater, 10-4 1/sec or greater, 10-3 1/sec or greater, or 10-2 1/sec or greater.
Useful low affinity antibodies typically have a dissociation rate constant of about 10-3 to 10-51/sec. A molecule with a high dissociation rate constant (Kd;sso~) is likely to have low affinity, as the equilibrium dissociation constant, KD =
Kd;ssoc/Kassoc. A
molecule with an equilibrium constant (KD) equal to or greater than about 10-8 M
has Low binding affinity. Useful low affinity antibodies can have a KD of about I O-6 M to about 10'8 M, for example.
Electrochemiluminescence assay or "EGLA" is an electrochemical assay in which bound analyte molecule is detected by a label linked to a detecting agent (target molecule). An electrode electrochemically initiates luminescence of a chemical label linked to a detecting agent. Light emitted by the label is measured by a photodetector and indicates the presence or quantity of bound analyte molecule/target molecule complexes. EGLA methods are described, for example, in U.S. Patent Nos. 5,543,112; 5,935,779; and 6,316,607. Signal modulation can be maximized for different analyte molecule concentrations for precise and sensitive measurements.
Microparticles can be suspended in the IA or ECLA sample to concentrate the analyte. For example, the particles can have a diameter of 0.05 ~m to 200 ~,m, 0.1 ~,m to 100 Vim, or 0.5 pm to 10 Vim, and a surface component capable of binding an analyte molecule. In an embodiment, the microparticles have a diameter of about 3 pm. The microparticles can be formed of crosslinked starch, dextran, cellulose, protein, organic polymers, styrene copolymer such as styrene/butadiene copolymer, acrylonitrile/butadiene/styrene copolymer, vinylacetyl acrylate copolymer, vinyl chloride/acrylate copolymer, inert inorganic particles, chromium dioxide, oxides of iron, silica, silica mixtures, proteinaceous matter, or mixtures thereof, including but not limited to sepharose beads, latex beads, shell-core particles, and the like. The microparticles are preferably monodisperse, and can be magnetic, such as paramagnetic beads. See, for example, U.S. Patent Nos. 4,628,037; 4,965,392;
4,695,393; 4,698,302; and 4,554,088. Microparticles can be used in an amount ranging from about 1 to 10,000 ~g/ml, preferably 5 to 1.,000 ~,g/ml.
A "detection limit" for an analyte molecule in a particular assay is a minimum concentration of the analyte molecule that can be detected above background levels for that assay. For example, in IA and ECLA, the detection limit for an analyte molecule that specifically binds a target molecule can be the concentration at which the analyte molecule produces an IA signal or ECLA
signal above that produced by a control antibody that does not bind, or non-specifically binds, the target antigen. Molecules that have an IA response less than the IA
detection limit are IA-. Molecules that have an IA response equal to or greater than the IA detection limit are IA+. Molecules that have an ECLA response Iess than the ECLA detection limit are ECLA-. Molecules that have an ECLA response equal to or greater than the ECLA detection limit are ECLA+. Detection limits can be raised or lowered to achieve a desired assay result.
The term "antibody" is used in the broadest sense and specifically includes single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, affinity-matured antibodies, humanized antibodies, chimeric antibodies, single chain antigen binding molecules such as monobodies, as well as antigen binding fragments or polypeptides (e.g., Fab, F(ab')2, scFv, and Fv) that exhibit a desired biological activity. An antibody can be natural or synthetic.
"Natural" or "naturally occurring" antibodies are derived from a nonsynthetic source, for example, from a differentiated antigen-specific B
cell obtained ex vivo, or its corresponding hybridoma cell line, or from the serum of an animal. These include antibodies generated in any type of immune response, either natural or otherwise induced. As used herein, natural antibodies differ from "synthetic antibodies", synthetic antibodies referring to antibody sequences that have been changed, for example, by the replacement, deletion, or addition of one or more amino acid, resulting in an antibody sequence that differs from the source antibody sequence.
The term "monoclonal antibody" as used herein refers to a natural or synthetic antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be made by the hybridoma method first described by Kohler et al., 1975, Nature, 256:495, or can be made by recombinant DNA
methods (see, a g., LT.S. Pat. No. 4,816,567). The monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson et al., 1991, Nature, 352:624-628 (1991) and Marks et al., 1991, J. Mol. Biol., 222:581-597, for example.
The term monoclonal antibodies specifically includes "chimeric" antibodies (irnmunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit a desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-6855).
"Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, specific framework region (FR) residues of the human immunoglobulin can be replaced by corresponding non-human residues.
Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs correspond to those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., 1986, Nature, 321:522-525; Reichmann et al., 1998, Nature, 332:323-329; and Presta et al., 1992, Curr. Op. Struct. Biol., 2:593-596. Heavy and light chain variable domains of a humanized antibody can also contain consensus framework regions as described, for example, in US Patent No. 6,054,297 to Carter.
An "Fv" fragment is an antibody fragment that contains a complete antigen recognition and binding site. This antibody fragment comprises a dimer of one heavy and one light chain variable domain in tight association that can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen.
A "Fab" fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain.
F(ab)'2 antibody fragments comprise a pair of Fab fragments that are generally covalently linked near their carboxy termini by hinge cysteines. Other chemical couplings of antibody fragments are also known.
"Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL
domains of antibody, where these domains are present in a single polypeptide chain.
Generally the Fv polypeptide further comprises a polypeptide linker between the VH
and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, 1994, In: The Pharmaeology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.
The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH
and VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., 1993, P~oc. Natl. Acad. Sci. USA, 90:6444-6448.
The expression "linear antibodies" refers to antibodies as described in ~apata et al., 1995, Protein Eng., 8(10):1057-1062. Briefly, these antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
The term "monobody" as used herein, refers to an antigen binding molecule with a heavy chain variable domain and no light chain variable domain. A
monobody can bind to an antigen in the absence of light chains and typically has three CDR regions designated CDRH1, CDRH2, and CDRH3. A heavy chain IgG
monobody has two heavy chain antigen binding molecules connected by a disulfide bond. The heavy chain variable domain comprises one or more CDR regions, preferably a CDRH3 region. A "VhH" or "VHH" refers to a variable domain of a heavy chain antibody such as a monobody.
"Enriching" a pool of analyte molecules, as used herein, refers to analytical means for generating a pool of analyte molecules that possess a desired characteristic from a larger pool of analyte molecules. By viewing the cross-screening affinity data according to the system and methods of the invention, analyte molecules lacking the desired characteristic are eliminated, resulting in a pool of analyte molecules enriched for analyte molecules having the desired characteristic.
For example, by viewing the cross-screening affinity data obtained from ELISA
and ECLA analysis of a pool of candidate anti-therapeutic antibodies as described in the Examples below, antibodies that demonstrate an ELISA-/ECLA+response form a pool of candidate antibodies enriched for candidate low affinity anti-therapeutic antibodies.
The term "library" refers to a plurality of polypeptide or polypeptide fragment sequences, the sequences being different in the combination of variant amino acids that are introduced into these sequences. In one embodiment, the polypeptide or polypeptide fragment sequences are antibody or antibody fragment sequences.
"Phage display" is a technique by which variant polypeptides are displayed as fusion proteins to a coat protein on the surface of phage, e.g., filamentous phage, particles. A utility of phage display lies in the fact that large libraries of randomized protein variants can be rapidly and eff ciently sorted for those sequences that bind to a target molecule with high affinity. Display of peptide and protein libraries on phages has been used for screening millions of polypeptides for ones with specific binding properties. Polyvalent phage display methods have been used for displaying small random peptides and small proteins through fusions to either gene III or gene VIII of filamentous phage. Wells and Lowman, 1992, Curr. Opif2. Struct. Biol., 3:355-362, and references cited therein. In monovalent phage display, a protein or peptide library is fused to a gene III or a portion thereof, and expressed at low levels in the presence of wild type gene III protein so that phage particles display one copy or none of the fusion proteins. Avidity effects are reduced relative to polyvalent phage so that sorting is on the basis of intrinsic ligand affinity. Lowman and Wells, 1991, Methods: A Companioy2 to Methods in Enzyrraology, 3:205-0216.
2H7, also known as PR070769, refers to a humanized monoclonal antibody that binds human CD20 antigen expressed on most B cells. 2H7 is currently being evaluated in clinical phase I/II trails for treatment of rheumatoid arthritis.
Monoclonal antibody 2H7 is commercially available, for example, from eBioscience, San Diego, CA.
Bevacizumab refers to a humanized monoclonal anti-VEGF antibody that inhibits angiogenesis. Bevacizumab is approved for treatment of metastatic cancer of the colon or rectum and is currently being evaluated in clinical phase III
trials for treatment of other types of cancers including pancreatic, renal, and breast cancers.
Bevacizumab is commercially available from Genentech Inc., South San Francisco, CA.
II. Methods for carrying out the invention The invention provides a cross-screening system and methods that analyze data generated in immunoassay (IA) and electrochemiluminescent assay (ECLA) methods to identify analyte molecules that have binding affinity for a target molecule. The cross-screening system and methods of the invention identify analyte molecules having binding affinities for a target molecule that are below detection limits of the individual immunoassay or ECLA.
In one aspect of the invention, the binding affinity of analyte molecules for a target molecule is cross-screened using immunoassay and ECLA methods. Analyte molecules that are IA~/ECLA+, IA-/ECLA+, or IA+/ECLA- are identified as analyte molecules that specifically bind the target molecule.
As shown in Figure 1, a pool of analyte molecules enriched for analyte molecules having a particular binding affinity for a target molecule can be generated from a large pool of analyte molecules using an embodiment of the cross-screening system and methods of the invention. The large pool of analyte molecules is screened with IA and ECLA. Optionally, the IA signal of the individual analyte molecules in the pool is plotted against the respective ECLA signal (Figure 1).
Analyte molecules in areas II (IA+/ECLA-) and III (IA+/ECLA~) form an enriched pool of candidate high affinity molecules (Figure 1). Analyte molecules in area IV
(IA-/ECLA+) form an enriched pool of candidate low affinity analyte molecules (Figure 1). Analyte molecules from the enriched pools of candidate low or high affinity analyte molecules can be confirmed as low or high affinity analyte molecules by determining the specific binding affinity of a selected analyte molecule, for example by surface plasmon resonance analysis. If the analyte molecules are monoclonal antibodies, the antibodies can be isotyped to identify monoclonal antibodies with different characteristics.
Analyte molecules that can be screened by the system and methods of the invention are typically small molecules, polypeptides, or polypeptide fragments, and can be, for example, antibodies, soluble receptors, or fragments thereof.
Antibodies can be monoclonal antibodies, typically produced by hybridoma cells.
Polypeptides, such as antibodies, soluble receptors, and fragments thereof, can also be expressed on phage. Therefore, a pool of analyte molecules can be a phage library.
A target molecule useful in the system and methods of the invention, is typically a small molecule, polypeptide, or polypeptide fragment. The target molecule can be, for example, an antigen if the analyte is an antibody, a receptor or antibody if the analyte is a small molecule or polypeptide, a polypeptide or small molecule if the analyte is a soluble receptor, or a phage expressing antibodies, soluble receptors, or fragments thereof if the analyte is a polypeptide or small molecule.
Preferably the target molecule is an antigen, and can be, for example, a polypeptide, polypeptide fragment, or small molecule. Examples of target molecules include, but are not limited to, renin; growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor;
parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;
insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone;
calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA);
bombesin;
thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta;
enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1-alpha); serum albumin such as human serum albumin; Muellerian-inhibiting substance; relaxin A-chain;
relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; microbial protein, such as beta-lactamase; DNase; IgE; cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); EG-VEGF; Bv~; receptors for hormones or growth factors; protein A or D; rheumatoid factors; neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or nerve growth factor; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II
(IGF-I
and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins such as CD3, CD4, CD8, CD 19 and CD20; erythropoietin;
osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10;
superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral antigen such as, for example, a portion of the AIDS
envelope; transport proteins; homing receptors; addressins; regulatory proteins;
integrins such as CD 11 a, CD 11 b, CD 11 c, CD 18, an ICAM, VLA-4 and VCAM;
tumor associated antigen such as HER2, HER3, or HER4 receptor; fragments of any of the above-listed polypeptides or specific epitopes thereof; and antibodies that bind any of these polypeptides.
Preferred target molecules for screening antibody analyte molecules include CD proteins such as CD3, CD4, CDB, CD19, CD20 and CD34; members of the ErbB receptor family such as the EGF receptor, HER2, HERS or HER4 receptor;
cell adhesion molecules such as LFA-1, Macl, p150,95, VLA-4, ICAM-1, VCAM
and a,,(33 integrin including either alpha or beta subunits thereof (e.g. anti-CD 11 a, anti-CD 18 or anti-CD 1 1b antibodies); growth factors such as VEGF; IgE;
blood group antigens; flk2/flt3 receptor; obesity (0B) receptor; mpl receptor; CTLA-4;
protein C; and antibodies that bind any of these polypeptides.
A target molecule can also be a polypeptide or antibody having therapeutic properties or activity. For example, a polypeptide that induces angiogenesis, such as for example VEGF, EG-VEGF, or BvB, can be used therapeutically to promote healing of a wound or surgical incision in a tissue. In an embodiment, the target molecule is an antigen, such as and anti-therapeutic monoclonal antibody.
Examples of anti-therapeutic monoclonal antibodies useful as target molecules in the invention include, but are not limited to, anti-VEGF antibodies such as bevacizumab and LUCENTISTM, anti-HER2 antibodies such as HERCEPTINO and OMNITARGTM, anti-CD20 antibodies such as RITUXAN~ and PR070769, anti-IgE antibodies such as XOLAIR~, and anti-CD 11 a antibodies such as RAPTIVA~. In an embodiment, the target molecule is the monoclonal antibody 2H7 and the analyte molecule to be screened is a pool of anti-2H7 antibodies or hybridoma supernatants of clones producing such anti-2H7 antibodies. In an embodiment, the target molecule is the monoclonal antibody bevacizumab and the analyte molecule to be screened is a pool of anti- bevacizumab antibodies or hybridoma supernatants of clones producing such anti- bevacizumab antibodies.
When the target molecule is a therapeutic antibody or therapeutic polypeptide, the cross-screening system and methods of the invention can be used to identify enriched pools of candidate anti-therapeutic antibodies for candidate anti-therapeutic antibodies having low binding affinity for the target molecule (therapeutic antibody), as described in the Examples below.
A. Immunoassay Conventional immunoassays can be used in the cross-screening system and methods of the invention. Examples of immunoassays useful in the invention include, but are not limited to, radioimmunoassay (RIA), fluoroluminescence assay (FLA), chemiluminescence assay (CA), and enzyme-linked immunosorbant assay (ELISA). See, for example, Johnstone and Thorpe, Inarnunochemistry in Practice, Blackwell, 3rd ed., 1996; Cu~~eht Protocols ih Molecular Biology, Ausbul et al.
eds., Wiley & Sons, 2003; Immuyaoassa~y Methods ahd Protocols, Ghindilis et al.
eds., Blackwell, 2003; U.S. 20030044865. The immunoassay can be a solid phase assay or liquid phase assay. Preferably the immunoassay is a solid phase assay such as, for example, ELISA.
Analyte molecules in a sample can be concentrated using microparticles.
The microparticles can be polymeric, including but not limited to, sepharose beads, Iatex beads, and shell-core particles. See, for example, U.S. Patent Nos.
4,305,925;
4,480,042; and 4,419,453. The microparticles can be magnetic to facilitate separation of the beads or microparticles from the sample. See, for example, U.S.
Patent Nos. 4,731,337; 4,777,145; and 4,115,535. Preferably, the magnetic beads are paramagnetic beads such as, for example, DYNABEADS (Dynal Biotech, Brown Deer, WI). When microparticles are used in the assay, a target molecule is conjugated to the beads. The target molecule can be conjugated to the microparticle by a non-covalent or covalent interaction or physical linkage as desired. For example, the microparticles can be coated with streptavidin to provide a binding surface for biotinylated target molecules. Techniques for attachment include those described in U.S. Pat. No. 4,376,110 and the references cited therein.
Preferably the immunoassay is a solid-phase ELISA or a capture ELISA. In a capture ELISA, immobilization of the target molecule to a solid phase is conventionally accomplished by insolubilizing a capture reagent either before the assay procedure, as by adsorption to a water-insoluble matrix or surface (LJ.S. Pat.
No. 3,720,760) or non-covalent or covalent coupling, for example, using glutaraldehyde or carbodiimide cross-linking, with or without prior activation of the support with, for example, nitric acid and a reducing agent as described in U.S. Pat.
No. 3,645,852 or in Rotmans et al., 1983, J. Immunol. Methods, 57:87-98, or after the assay procedure, for example, by immunoprecipitation. In an embodiment, the capture reagent is an antibody or a mixture of different antibodies against a target antigen or an antibody/antigen complex, where the bound antigen is available to bind an antibody from a sample. In a further embodiment, the capture reagent is an anti-isotype specific antibody complexed to a therapeutic antibody. For example, the capture reagent can be a goat anti-human IgG Fc specific antibody complexed to a humanized therapeutic IgG monoclonal antibody. In an embodiment, the humanized therapeutic IgG monoclonal antibody is an anti-2H7 antibody. In an embodiment, the humanized therapeutic IgG monoclonal antibody is an anti-bevacizumab antibody.
The solid phase used for immobilization can be any inert support or carrier that is essentially water insoluble and useful in immunoassays, including supports in the form of, for example, surfaces, particles, porous matrices, and the like.
Examples of commonly used supports include small sheets, Sephadex, polyvinyl chloride, plastic beads, microparticles, assay plates, or test tubes manufactured from polyethylene, polypropylene, polystyrene, and the like. Such supports include well microtiter plates, as well as particulate materials such as filter paper, agarose, cross-linked dextran, and other polysaccharides. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016;
4,195,128;
4,247,642; 4,229,537; and 4,330,440 are suitably employed for capture reagent immobilization. In an embodiment the immobilized capture reagent is coated on a microtiter plate. The preferred solid phase is a mufti-well microtiter plate that can be used to analyze several samples at one time.
The solid phase is coated with the capture reagent that can be linked by a non-covalent or covalent interaction or physical linkage, as desired.
Techniques for attachment include those described in U.S. Pat. No. 4,376,110 and the references cited therein. If covalent attachment of the capture reagent to the plate is utilized, the plate or other solid phase can be incubated with a cross-linking agent together with the capture reagent. Commonly used cross-linking agents for attaching the capture reagent to the solid phase substrate include, for example, l, l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates capable of forming cross-links in the presence of light.
If polystyrene or polypropylene plates are utilized, the wells in the plate are preferably coated with the capture reagent (typically diluted in a buffer such as 0.05 M sodium carbonate) by incubation for at least about 10 hours, more preferably at least overnight, at temperatures of about 4-20°C, more preferably about 4-8°C, and at a pH of about 8-12, more preferably about 9-10, and most preferably about 9.6. If shorter coating times (1-2 hours) are desired, the plate is coated at 37°C or plates with nitrocellulose filter bottoms such, as for example, Millipore MULTISCREENTM. The plates can be stacked and coated in advance of the assay, allowing for an immunoassay to be carried out simultaneously on several samples in a manual, semi-automatic, or automatic fashion, such as by using robotics.
The coated plates are typically treated with a blocking agent that binds non-specifically to, and saturates, the binding sites to prevent unwanted binding of free ligand to excess binding sites on the wells of the plate. Examples of appropriate blocking agents include, fox example, gelatin, bovine serum albumin, egg albumin, casein, and non-fat milk. The blocking treatment typically takes place under conditions of ambient temperatures for about 1-4 hours, preferably about 1.5 to 3 hours.
After coating and blocking, the sample to be analyzed is diluted as necessary and added to the immobilized phase. The preferred dilution rate is about 5-15%, preferably about 10%, by volume. Buffers that can be used for dilution include for example (a) phosphate buffered saline (PBS) containing 0.5% BSA, 0.05% TWEEN
20TM detergent (P20), 5 mM EDTA, 0.25% Chaps surfactant, 0.2% beta-gamma globulin, and 0.35M NaCI, pH 7.0; (b) PBS containing 0.5% BSA and 0.05% P20;
(c) PBS containing O.S% BSA, O.OS% P20, S mM EDTA, and 0.35 M NaCI, pH
6.35; (d) PBS containing O.S% BSA, 0.05% P20, S mM EDTA, 0.2% beta-gamma globulin, and 0.35 M NaCI; (e) PBS containing O.S% BSA, O.OS% P20, S mM
EDTA, 0.25% Chaps, and 0.35 M NaCI; and (f) PBS containing O.S% P20.
S For sufficient sensitivity, it is preferred that the immobilized capture reagent is in molar excess of the maximum molar concentration of the analyte anticipated in the sample after appropriate dilution. Depending on the analyte, the capture reagent can compete for binding sites with the detecting antibody yielding inaccurate results.
Therefore, the final concentration of the capture reagent will normally be determined empirically to maximize the sensitivity of the assay over the range of interest.
Conditions for incubation of sample and capture reagent are selected to maximize sensitivity of the assay and to minimize dissociation. Incubation time depends primarily on the temperature. Preferably, the incubation time is from about 0.5 to 3 hours, and more preferably 1.5-3 hours at 36-38°C. To maintain the 1 S sensitivity of the assay, incubation times greater than about 10 hours are avoided if possible. If the sample is a biological fluid, incubation times can be lengthened by adding a protease inhibitor to the sample to prevent proteases in the biological fluid from degrading the analyte.
The pH of the incubation buffer is chosen to maintain a significant level of specific binding of the capture reagent to the analyte being captured. The pH
of the incubation buffer is preferably about 6-9.5, more preferably about 6-7.
Various buffers can be employed to achieve and maintain the desired pH during this step, including borate, phosphate, carbonate, Tris-HCI or Tns-phosphate, acetate, barbital, and the like. The particular buffer employed is usually not critical, however, and in 2S individual assays one buffer may be preferred over another.
The sample is separated from the immobilized capture reagent with a wash solution to remove uncaptured analyte from the system. The wash solution is generally a buffer. The incubation buffers described above are suitable wash solutions. The pH of the wash solution is determined as described above for the incubation buffer. In an embodiment, the pH of the wash solution is about 6-9, more preferably about 6-7. Washes can be done one or more times. Minimizing the number of washes, however, to retain molecules that bind the target molecule with low aff nity increases the background noise of the assay. Preferably, the system is washed three times. The temperature of the wash solution is typically from about 0-40°C, more preferably about 4-30°C. An automated plate washer can be utilized. A
cross-linking agent or other suitable agent can be added to the wash solution to covalently attach the captured analyte to the capture reagent.
Following removal of uncaptured analyte molecules from the system, the captured analyte molecules are contacted with a detecting agent, such as an antibody, preferably at a temperature of about 20-40°C, more preferably about 36-38°C. When the analyte is humanized anti-therapeutic antibody, the detecting agent is an anti-isotype antibody from a different species. If the anti-therapeutic antibodies are human IgG, for example, the detecting agent can be a murine anti-human IgG antibody. In an embodiment, the analyte is murine monoclonal antibody and the detecting agent is sheep anti-mouse IgG.
The temperature and time for contacting the analyte molecule with the detecting agent is dependent primarily on the detection means employed. For example, when horseradish peroxidase (HRP) conjugated to sheep anti-mouse IgG
is 1 S used as the means for detection, the detecting agent is preferably incubated with the captured analyte for about 0.5-2 hours, more preferably about 1 hour. The system is washed as described above to remove unbound detecting agent from the system and developed by adding peroxidase substrate and incubating the plate for about 5 minutes at room temperature or until good color is visible.
In an embodiment, a molar excess of the detecting agent is added to the system after the unbound analyte has been washed from the system. The detecting agent can be a polyclonal or monoclonal antibody. In an embodiment, the antibody is a monoclonal antibody. In an embodiment, the monoclonal antibody is murine.
The detecting agent can be directly or indirectly detectable. If the detecting agent is an antibody that is not directly detectable, the detecting antibody is detected by addition of a molar excess of a second, labeled antibody directed against the isotype and animal species of the detecting antibody.
The affinity of the detecting agent must be sufficiently high such that small amounts of analyte can be detected. A fluorimetric or chemilimunescent label moiety has greater sensitivity in immunoassays compared to a conventional colorimetric label. The binding affinity of the selected detecting agent must be considered in view of the binding affinity of the capture agent, such that the detecting agent does not strip the analyte from the capture reagent.
The label moiety is any detectable functionality that does not interfere with the binding of the captured analyte to the detecting agent. Examples of suitable label moieties include moieties that can be detected directly, such as fluorochrome, chemiluminscent, and radioactive labels, as well as moieties, such as enzymes, that must be reacted or derivatized to be detected. Examples of such labels include the radioisotopes 32P, 14C, l2sh 3H, and 1311, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodainine and its derivatives, dansyl, umbelliferone, luceriferases, a g., firefly luciferase and bacterial luciferase (U.S. Pat.
No.
4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphiatase, (3-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HPP, lactoperoxidase, or microperoxidase, biotin/avidin, biotin/streptavidin, biotin/Streptavidin-(3-galactosidase with MUG, spin labels, bacteriophage labels, stable free radicals, and the like.
Conjugation of the label moiety to the detecting agent, such as for example an antibody, is a standard manipulative procedure in immunoassay techniques.
See, for example, O'Sullivan et al. "Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay," in Methods in Enzymology, ed. J. J.
Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), pp. 147-166. Conventional methods are available to bind the label moiety covalently to proteins or polypeptides. For example, coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized benzidine, and the like can be used to label antibodies with the above-described fluorescent, chemiluminescent, and enzyme labels. See, for example, U.S. Pat. Nos.
3,940,475 (fluorimetry) and 3,645,090 (enzymes); Hunter et al., 1962, Nature, 144:945;
David et al., 1974, Biochemistry, 13:1014-1021; Pain et al., 1981, J. Immunol Methods, 40:219-230; and Nygren J., 1982, Histoclzem. and Cytoclaem., 30:407-412.
Preferred labels herein are fluorescent or chemiluminescent to increase amplification and sensitivity to about 5-10 pglml. In an embodiment, the label moiety is HRP.
The amount of analyte bound to the capture reagent is determined by washing away unbound detecting agent from the immobilized phase and measuring the amount of detecting agent bound to the analyte using a detection method appropriate to the label. In an embodiment, the Iabel moiety is an enzyme. In the case of enzyme moieties, the amount of developed color is a direct measurement of the amount of captured analyte. For example, when HRP is the label moiety, color is detected by quantifying the optical density (0.D.) at 650 nm absorbance. In another embodiment, the quantity of analyte bound to the capture reagent is determined in-directly. The signal of an unlabeled detecting agent can be amplified for detection with an anti-detecting agent antibody conjugated to a label moiety. For example, the signal of an unlabeled mouse antibody that binds the target molecule can be amplified with a sheep anti-mouse IgG antibody labeled with HRP. The label moiety is detected using a detection method appropriate to the label. For example, HRP can be detected by reacting HRP with a colorimetric substrate and measuring the optical density of the reacted substrate at 650 nm absorbance.
The pH and/or temperature of the system can be varied to identify molecules . that bind the target molecule.
B. ECLA
Conventional methods for ECLA can be used in the cross-screening system and methods of the invention. See, for example, U.S. Patent Nos. 5,543,1 I2;
5,935,779; 6,316,607, and the patents referenced therein. In an embodiment, the capture reagent and detecting reagent are mixed with the analyte molecule and incubated at room temperature. In an embodiment, the capture reagent and detecting reagent are in molar excess of the maximum molar concentration of the analyte molecule anticipated in the sample. Depending on the analyte molecule, the capture reagent rnay compete for binding sites with the detecting reagent yielding inaccurate results. Therefore, the final concentration of the capture reagent will normally be determined empirically to maximize the sensitivity of the assay over the range of interest. In an embodiment, the capture reagent and detecting reagent are added to the sample in about a l :l ratio.
The capture reagent can be an antigen, receptor, antibody, or fragment thereof. Preferably the antibody is monoclonal. In an embodiment, the capture reagent is an antibody or a mixture of different antibodies against a target antigen.
In another embodiment, the capture reagent is a goat anti-human IgG antibody.
The detecting agent can be a receptor, antibody, or fragment thereof. In an embodiment, the antibody is monoclonal. The monoclonal antibody can be a marine monoclonal antibody. In an embodiment, the detecting reagent is an.antibody or a mixture of different antibodies against a target antigen. In another embodiment, the detecting agent is marine anti-human IgG antibody.
The incubation time can be from about 0.5 to 3 hours, more preferably 1.5-3 hours at 36-3~°C. The pH of the incubation buffer is chosen to maintain a significant level of specific binding of the capture reagent and detecting agent to the analyte. In an embodiment, the pH of the incubation buffer is about 6-9.5, more preferably about 6-7. Various buffers can be employed to achieve and maintain the desired pH during this step, including borate, phosphate, carbonate, Tris-HCI
or Tns-phosphate, acetate, barbital, and the like. The particular buffer employed is usually not critical, however, in individual assays one buffer may be preferred over another.
In an embodiment, the ECLA method utilizes a binding phase to immobilize the analyte complex, such as beads or microparticles. The beads or microparticles can have a diameter of 0.05 um to 200 um, more preferably 0.1 um to 100 um, more preferably 0.5 um to 10 um, and a surface component capable of binding the capture reagent. In an embodiment, the binding surface of the beads or microparticles is coated with streptavidin and the capture reagent is labeled with biotin. The microparticles can also be coated, for example, with glutathione, anti-IgG
antibody, or agglutinin. The capture reagent can be biotinylated with biotinylamidocaproic acid-N-Hydroxy-succinimide ester using standard amine chemistry at a ratio from about 1;1 to about 10:1 biotin to capture reagent, more preferably from about 2:1 to about 4: l biotin to capture reagent, more preferably about 2.5:1 biotin to capture reagent.
If the analyte is an anti-therapeutic antibody, the analyte, the capture reagent, and detecting reagent can be antibodies from the same species. In such instances, the same type of antibody can be separately utilized as both a capture reagent and detecting reagent. For example, separate batches of the same antibody are labeled, one with a component capable of binding the microparticle, such as biotin when the microparticle is coated with streptavidin, or another with or a label, such as Ori-Tag~. In an embodiment, the antibody is a therapeutic monoclonal antibody including, but are not limited to, anti-VEGF antibodies such as bevacizumab and LUCENTISTM, anti-HER2 antibodies such as HERCEPTIN~ and OMNITARGTM, anti-CD20 antibodies such as RITUXAN~ and PR070769, anti-IgE antibodies such as XOLAIR~, and anti-CDl la antibodies such as RAPTIVA~. In an embodiment, the capture reagent and detecting reagent are added to the analyte in about a I : I
ratio. The analyte must bind both a capture reagent and a detecting reagent to be detected by ECLA. Analytes that bind only capture reagent can bind to the microparticle but are not detectable.
After incubating the capture reagent and detecting reagent with the analyte molecule, microparticles capable of binding the capture reagent are added to the mixture and the mixture is incubated. In an embodiment, the microparticles are coated with a molecule that binds biotin, such as streptavidin. The incubation time can be from about 0.5 to 3 hours, preferably 1.5-3 hours at 36-3~°C.
The pH of the incubation buffer is chosen to maintain a significant level of specific binding of the capture reagent/detecting agent/analyte molecule complex to the microparticles.
The pH of the incubation buffer can be about 6-9.5, more preferably about 6-7.
The incubation buffer can include an electrolyte. The electrolyte can be one or more salts or other species. In an embodiment, the electrolyte is a sodium salt or potassium salt.
The microparticles are assayed with an apparatus that contains an electrode and a photodetector, such as an IGEN M3~4 analyzer (IGEN International Inc., Gaithersburg, MA). See, for example, U.S. Patent Nos. 5,543,112 and 5,935,779 describing apparatuses for measuring electrochemiluminescence. The label conjugated to the detecting agent is induced to emit electromagnetic radiation by stimulating the label into an excited state. Detection and/or quantitation of the analyte in a sample is typically made by comparing the luminescence of a sample to the luminescence emitted by a calibration standard developed with known amounts of the analyte and detecting agent. In an embodiment, the photodetector measures the light emitted by the label and software for analyzing data collected by the photodetector is used to calculate the concentration of analyte molecular or ECLA
response (in electrochemiluminescence units (ECLU)) of the analyte molecule.
In an embodiment, the label conjugated to the detecting reagent is a metal chelate that luminesces under the electrochemical conditions imposed by ECLA.
The metal can be, for example, a transition metal (such as a d-block transition metal) or a rare earth metal. In an embodiment, the metal is ruthenium, osmium, rhenium, iridium, rhodium, platinum, indium, palladium, molybdenum, technetium, copper, chromium, or tungsten. In an embodiment, the metal is ruthenium or osmium.
A ligand(s) linked to the metal of the chelate is usually heterocyclic or organic in nature, and plays a role in determining whether the metal chelate is soluble in an aqueous environment or in an organic or other nonaqueous environment. The ligands can be polydentate, and can be substituted.
Polydentate ligands include aromatic and aliphatic ligands. Suitable aromatic polydentate ligands include aromatic heterocyclic ligands. In an embodiment, the aromatic heterocyclic ligands are nitrogen-containing, such as, for example, bipyridyl, bipyrazyl, terpyridyl, and phenanthrolyl. Suitable substituents include for example, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxy, imino, hydroxycarbonyl, aminocarbonyl, amidine, guanidinium, ureide, sulfur-containing groups, phosphorus containing groups, and the carboxylate ester of N-hydroxysuccinimide. The chelate can have one or more monodentate ligands, a wide variety of which are known to the art. Suitable rnonodentate ligands include, for example, carbon monoxide, cyanides, isocyanides, halides, and aliphatic, aromatic and heterocyclic phosphines, amines, stilbenes, and arsines.
Examples of suitable chelates are bis [(4,4'-carbomethoxy)-2,2'-bipyridine]
2-[3-(4-methyl-2,2'-bipyridine-4-yl)propyl]-1,3-dioxolane ruthenium (II); bis (2,2'bipyridine) [4-(butan-1-al)-4'-methyl-2,2'-bipyridine] ruthenium (II);
bis (2,2'-bipyridine) [4-(4'methyl-2,2'-bipyridine-4'-yl)-butyric acid] ruthenium (II);
tris (2,2'bipyridine) ruthenium (II); (2,2'-bipyridine) [bis-bis(1,2-diphenylphosphino)ethylene] 2-[3-(4-methyl-2,2'-bipyridine-4'-yl)propyl]-1,3-dioxolane osmium (TI); bis (2,2'-bipyridine) [4-(4'-methyl-2,2'-bipyridine)-butylamine] ruthenium (II); bis (2,2'-bipyridine) [1-bromo-4(4'-methyl-2,2'-bipyridine-4-yl)butane] ruthenium (II); bis (2,2'-bipyridine)maleimidohexanoic acid, 4-methyl-2,2'-bipyridine-4'-butylamide ruthenium (II). Additional label moieties suitable for ECLA are described in U.S. Patent Nos. 5,591,51; 6,271,041;
6,316,607; and 6,451,225. In an embodiment, the label moiety is Ru(bpy)3z+ or ORI-TAGTM NHS ester (IGEN International Inc., Gaithersburg, MA).
The amount of label utilized is that amount which effectively results in the emission of a detectable, and if desired, quantifiable, emission of electromagnetic energy. In an embodiment, the detecting agent is conjugated with the label, using standard amine chemistry, at a ratio from about 1:1 to about 10:1 label to detecting reagent, more preferably from about 3:1 to about 7:1 label to detecting reagent, more preferably about 5:1 label to detecting reagent.
The pH and/or temperature of the system can be varied to identify molecules that bind the target molecule.
C. Detection limit The detection limit for the IA and ECLA is the minimum concentration of analyte that can be detected above the background level of the respective assay. The detection limit of IA and/or ECLA can be determined by conventional methods.
Below this detection limit, it is difficult to differentiate specific binding of the analyte from non-specific binding of the detecting agent. Molecules that are IA-/ECLA- are considered not to have specific binding affinity for the target molecule.
The detection limit can be determined by the amount of non-specific binding of the detecting agent in the system. The background level of the IA or ECLA
can be determined under the conditions of the respective assay in the absence of analyte.
For example, IA and ECLA can be used to detect an analyte, such as a low affinity antibody, in a homogenous mixture. The background level in the respective assay can be determined by quantifying the signal of the detecting antibody after the system has been incubated with incubation buffer containing no analyte. IA and ECLA can also be used to screen for an analyte, such as a low affinity binding anti-therapeutic, antibody, in a homogenous mixture, such as serum. The background level in the respective assay can be determined by quantifying the signal of the detecting agent after the system has been incubated with serum from one or more animals that were not administered the therapeutic.
The background level can also be determined using a control analyte that does not specifically bind the target molecule. In an embodiment, the detection limit is determined using a control antibody that does not specifically bind the target antigen. For example, IA and ECLA can be used to screen the supernatant of hybridoma clones for low affinity antibodies. The background level of the respective assay can be determined by quantifying the signal of the detecting antibody after the system is incubated with a supernatant sample containing control antibodies that do not specifically bind the target antigen. In an embodiment, the control antibodies are the same isotype as the antibodies being produced by the hybridoma clones of interest.
Reducing the background Ievel of the system reduces the detection limit thereby increasing the sensitivity of the assay. There are a number of ways the background level of the IA or ECLA can be reduced including, but not limited to, increasing the length of washes, adding additional washes, selecting a different wash buffer, selecting a solid phase of a different material, selecting a different blocking buffer, selecting a different detecting agent, selecting beads or microparticles with a lower autofluorescence level, reducing incubation times, changing the pH of one or more buffers, changing incubation temperature, or any combination thereof.
With respect to ECLA specifically, employing paramagnetic beads in association with a magnetic electrode can reduce the background.
The detection limit for IA and/or ECLA may require optimization to ensure the detection limit is not excluding analyte molecules with a desired characteristic, or to ensure that undesired molecules are excluded. For example, the binding affinities of randomly selected IA-IECLA- analyte molecules can be determined.
Randomly selected IA-/ECLA' analyte molecules found to have a binding affinity for the target molecule of about 10-2 may indicate the detection limit is set too high and that lowering the IA and/or ELISA detection limit may identify additional analyte molecules with the desired low binding affinity for the target molecule.
D. Determination of Binding Affinity Candidate low or high affinity analyte molecules are confirmed as low or high affinity analyte molecules based on their dissociation rate constant (Kd;sso~) for the target molecule or equilibrium dissociation constant (KD). Binding affinities of candidate molecules selected from the enriched pool of candidate molecules generated by the cross-screening system and methods of the invention can be confirmed by conventional equilibrium or kinetic methods. Examples include, but are not limited to, competitive ELISA, equilibrium dialysis, RIA, surface plasmon resonance such as BIACORE~ (Biacore Inc., Piscataway, N.J.), affinity chromatography, and ECLA. See, for example, Current Protocols in Molecular Biology, Ausbul et al. eds., Wiley & Sons, 2003; Current Protocols in Immunology, Bierer et al. eds, Wiley & Sons, 2003; and U.S. Patent Nos. 5,543,112;
5,935,779;
and 6,143,574: In an embodiment, the binding afEnity is determined by BIACORE~ analysis (see Example 3 below).
A high Kd;sso~ is indicative of low binding affinity. An analyte molecule with a Kd;sso~ greater then 10-6 for the target molecule or KD equal to or greater than 10-8 M, is identified as a molecule having low binding affinity for the target molecule. In an embodiment, the Kd;sso~ of the candidate low affinity analyte molecule for the target molecule is 10-5 or greater, more preferably 10-4 or greater, more preferably 10-3 or greater, and more preferably 10-2 or greater. In an embodiment, the Ko of the candidate low affinity analyte molecule for the target molecule is about 10-6 M, about 10-~ M, or about 10-8 M.
E. Isotyping The isotypes of the heavy and light chains of low affinity binding antibodies (analyte molecules) identified by the cross-screening system and methods of the invention can be determined by conventional methods, such as ELISA utilizing anti-isotype specific antibodies. See, for example, Current Protocols in Immunology, Bierer et al. eds, Wiley & Sons, 2003. In an embodiment, the antibodies are alkaline phosphatase or HRP-conjugated anti-mouse or anti-human antibodies. In an embodiment, a panel of anti-heavy chain isotype specific antibodies anti-isotype specific antibodies is employed. The panel of antibodies can include anti-heavy chain anti-isotype specific IgG, IgE, IgA, and IgM antibodies. In an embodiment, the panel of antibodies includes at least anti- IgGl, IgG2a, IgG2b, or IgG3 isotype specific antibodies.
F. iJses The cross-screening system and methods of the invention have many applications. The methods of the invention are particularly useful for identifying analyte molecules that bind a target molecule. For example, the methods are useful for identifying polypeptides or small molecules that bind a specific receptor, or antibodies that bind a specific antigen. In an embodiment, a receptor, antibody, or fragment thereof is expressed on a phage. In another embodiment, the antigen is a polypeptide or monoclonal antibody having therapeutic activity. For example, the monoclonal antibody can be anti-VEGF antibody such as bevacizumab and LUCENTISTM, anti-HER2 antibody such as HERCEPTIN~ and OMNITARGTM, anti-CD20 antibody such as RITUXAN~ and PR070769, anti-IgE antibody such as XOLAIR~, and anti-CD1 la antibody such as RAPTIVA~. In an embodiment, the target antigen is monoclonal antibody 2H7. In another embodiment, the target antigen is monoclonal antibody bevacizumab. The pH and/or temperature of the system can be varied to identify molecules that bind the target molecule.
2~
The cross-screening system and methods of the invention are particularly useful to detect small amounts of analyte molecule in a sample. Preferably the analyte molecule is an antibody. For example, the cross-screening system and methods of the invention can be used to identify a hybridoma producing antibodies that have high affinity for the target molecule but the concentration of antibodies in the supernatant is low. The concentration of antibodies in the supernatant can be below the detection limit of the individual IA or ECLA assay, but not the detection limit of the cross-screening system and methods of the invention.
The methods of the invention can be used to enrich a pool of analyte molecules for a desired characteristic. In an embodiment, the analyte molecules are antibodies. In an embodiment, the desired characteristic is binding affinity (IA-/ECLA+, IA+/ECLA+, and IA+/ECLA+) for the target molecule. In another embodiment, the desired characteristic is high binding affinity for a target molecule (IA+/ECLA+ or IA+/ECLA-). For example, a small molecule library can be screened for candidate library members that bind a target receptor with high affinity.
Similarly, a library of receptors, antibodies, or fragments thereof can be screened for candidate library members that bind a target polypeptide with high affinity.
In another embodiment, the desired characteristic is low binding affinity for a target molecule (IA-/ECLA+), such as for example, an antigen. The candidate molecules can be confirmed as high affinity or low affinity analyte molecules respectively, by determining a specific binding affinity of the analyte molecules for the target antigen.
Low affinity antibodies are needed, for example, in anti-therapeutic antibody assays required for regulatory approval of biological therapeutics. Immune responses are polyclonal; therefore, anti-therapeutic antibodies generated against a biological therapeutic can target different regions of the therapeutic or demonstrate different binding affinities and isotypes. In clinical trials, for example, a panel of anti-therapeutic antibodies that mimics the polyclonal nature of an immune response to the biological therapeutic undergoing clinical testing can be used to assess performance of an anti-therapeutic antibody assay. Low affinity antibodies identified by the cross-screening system and methods of the invention can be used to construct such a panel of antibodies. In an embodiment, the cross-screening system and methods of the invention are used to screen hybridoma clones for antibodies that bind a target antigen with low affinity. In an embodiment, the target antigen is a therapeutic monoclonal antibody.
In clinical trials for example, detection of antibodies to a biological therapeutic at an early stage in the trial is important for assessing the safety and efficacy of the therapeutic. In an embodiment, the methods of the invention are used to screen serum from a patient about to receive or who is receiving a biological therapeutic, such as a polypeptide or monoclonal antibody, for anti-therapeutic antibodies.
Low affinity antibodies are also useful in drug discovery methods. For example, it can be difficult to generate antibodies with high binding affinity for a therapeutic target. Low affinity antibodies can serve as a starting point for developing affinity matured antibodies. In an embodiment, antibodies are cross-screened utilizing the methods of the invention for analyte molecules exhibiting low bindixig affinity for the therapeutic target. Selected low affinity antibodies are affinity matured to produce therapeutic antibodies. In an embodiment, the antibodies are expressed on phage. In another embodiment, the antibodies are members of a phage library.
Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
All publications (including patents and patent applications) cited herein are hereby incorporated in their entirety by reference.
Example 1 Screening Hybridomas for Low Affinity Anti-2H7 Antibodies.
Hybridoma supernatants were screened for production of low affinity anti-2H7 antibodies using cross-screening methods employing ELISA and ECLA. A
workflow diagram of an embodiment of a hybridoma screening strategy is shown in Figure 1.
Hybridoma Production BALB/c mice were immunized and boosted 10 times with 0.5 ~g 2H7 resuspended in monophosphoryl lipid A/trehalose dicorynomycolate adjuvant (Corixa). The suspension was injected in each hind footpad at 3 to 4 days intervals.
Three days after final boost, poptileal lymph nodes were fused with cells of the myeloma cell line, P3X63Ag.U.1 (ATCC, Manassas, VA). Fused cells were selected by hypoxanthin-aminopterin-thymidine (HAT) medium selection.
ECLA Screening Supernatants from hybridoma cultures Were screened for low affinity anti-2H7 antibodies by plotting ECLA responses versus ELISA responses. ECLA was performed as described in Baker et al., 2002, Trends in BioteclZnol., 20:149-156.
Briefly, separate batches of 2H7 antibodies were labeled with biotin or Ori-tag (Igen International Inc, Gaithersburg, MD). 2H7 was biotinlylated with biotinylamiocaproic acid-N-hydroxy-succinimid ester (Organics Inc.) using standard amine based chemistry at target ratio of 2.5:1 biotin to 2H7. 2H7 was labeled with ORI-TAG NHS ester according to the manufacturer's instructions at a targeted ratio of 5:1 ORI-TAG to 2H7. A master working solution was prepared by mixing biotinylated 2H7 and Ori-tag labeled 2H7 in a 1:1 ratio. The final concentration of each labeled antigen in the master working solution was 1 ~.g/ml.
A panel of monoclonal antibodies was created by adding 50 ~1 of master working solution and 50 p.1 of supernatant from individual hybridoma clones to a 96-well round-bottom polypropylene plate. Each well in the plate contained supernatant from a single hybridoma clone. The plate was incubated at room temperature in the dark for two hours with a gentle agitation 10 ~,g of streptavidin coated magnetic beads in a volume of 100 ~,1 was added to each well. The plate was incubated for another one hour at room temperature in the dark with a gentle agitation. Post-incubation, the plates were read on an IGEN M384 analyzer using the following protocol parameters: bead type is set at 2.80 microns, aspiration volume of 200 ~,1, POP of 0 mv, gain of,l, wash volume of 700 ~l, clean cycle of 2, wash speed of 200 ml/sec. Data was collected and reported in electrochemiluminescence units (ECLU).
HAT medium was used a control. To be detected by ECLA, the analyte molecule must bind both a capture reagent and a detecting agent. Anti-2H7 antibodies (analyte molecules) from supernatant culture that formed a complex with both capture reagent and detecting agent were detected in the assay, (ECLA+).
Anti-2H7 antibodies from the supernatant culture that bound only capture reagent or only detecting reagent were not detected.
ELISA Screening ELISA was performed generally as described in Baker et al., 2002, Trends ira Biotechhol., 20:149-156. Briefly, a 384-well Greiner flat bottom plate was coated with 50 ~1 of goat anti-human IgG Fc specific (Cappel #55071) at a concentration of 2 ~g/ml in coating buffer (50 ~M carbonate buffer, pH 9.6). The plate was sealed and stored at 4 °C overnight. After removing the coating solution, 100 ~.l of blocking solution containing 2% of bovine serum albumin in PBS was added to each well. The plate was incubated at room temperature for one hour with agitation and then washed three times with PBS/0.05% Tween-20.
After the washing step, 50 ~,1 of antigen solution (0.4 ~,g/ml 2H7 in PBS
containing 0.5% bovine serum albumin) was added to each well and the plate was incubated at room temperature for one hour with agitation. The plated was washed three times with PBS/0.05% Tween-20. 35 ~,1 of supernatant from individual hybridoma clones was added such that each well in the plate contained supernatant from a single hybridoma clone. The plate was incubated for one hour at room temperature and washed three times with PBS/0.05% Tween-20.
After the washing step, 50 ~1 of a 1:1000 dilution of sheep anti-mouse IgG
HRP (no cross reactivity to human IgG, Cappel #55569) in PBS containing 0.5%
bovine serum albumin and 0.1 % Tween-20 was added to each well. The plate was incubated at room temperature for one hour with agitation, washed three times with PBS/0.05% Tween-20, rinsed with water, and shaken dry. The plate was developed by adding 40 ~l of TMB Microwell Peroxidase (tetramethylbenzidine) substrate (BioFX #TMBT-0100-O1) to each well in the plate and incubating the plate for 5 minutes at room temperature or until a good color was visible. Development was stopped by adding 40 ~l of the Stop solution (BioFX #BSTP-0100-O1) to each well, plates were read on a Sunrise plate reader (Tecan US, Research Triangle Park, NC) at 650 nm.
Identification of Low Affinity Antibodies Low affinity anti-2H7 antibodies were identified by plotting ECLA
responses (ECLU) against ELISA responses (0.D. at 650 nm) for each respective hybridoma supernatant (Figure 2). The detection limit for ELISA was set at O.D.
0.5. HAT medium was used as a control in ECLA to establish a detection limit of 250 ECLU.
Detection limits for ECLA and ELISA were used to establish a four quadrant grid on the ECLA:ELISA plot (Figure 2). Lines depicting detection limits form the boundaries of four quadrants. Antibodies in area I (ECLA-/ELISA-) represent antibodies that did not bind 2H7 or had binding that was not detected by either ELISA or ECLA. Antibodies in area III (ECLA+/ELISA~) represent candidate high affinity anti-2H7 antibodies. Antibodies in area II (ECLA-/ELISA+) represent candidate high affinity anti-2H7 antibodies that are believed to bind epitopes masked or altered by biotinlyation of 2H7 and/or labeling of 2H7 with Ori-Tag.
Antibodies in area IV (ECLA+/ELISA-) represent candidate low affinity anti-2H7 antibodies.
Antibodies in area IV represent a population of anti-2H7 antibodies not detected by ELISA. These antibodies may have been washed off the plate during the multiple wash steps in ELISA or had an ELISA response less than 0.5. Antibodies in area IV
presumptively produced low affinity antibodies. While Area IV is enriched in low affinity antibodies, it may also contain high affinity antibodies present in low concentration in the supernatant.
Biacore Analysis of Low Affinity Anti-2H7 Antibodies A number of hybridoma clones producing antibodies in quadrants I, II, III, or IV in Example 1 were selected for further characterization and confirmation of specific binding affinity. Dissociation rate constants (Kd;sso~) for antibodies produced by the selected hybridoma clones were determined by Biacore analysis.
The analysis was performed on a Biacore 3000 (USA Biacore, Inc., Piscataway, NJ). The monoclonal antibody 2H7 was immobilized on a CMS sensor chip in a flow cell. In brief, the flow cell was activated by injecting 35 p1 of a solution containing equal volumes of 11.5 mg/ml of N-hydroxysuccinimide (NHS).
and 7.5 mg/ml of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Following activation of the flow cell, 2H7 in sodium acetate (pH 5.0) was injected manually to reach a response of approximately 500 RU on the chip.
Thirty-five p.1 of 1 M ethanolamine hydrocholoride-NaOH (pH ~.0) was injected at a flow rate of 5 p,l/minute to block any un-reacted activated sites on the flow cell.
The final concentration of immobilized 2H7 after ethanolamine blocking was 556 RU. A
different flow cell was used as an in-line reference cell. The reference flow cell was activated as described above and immediately blocked with a 35 ~1 injection of ethanolamine hydrocholoride-NaOH (pH ~.0) at a flow rate of 5 ~l/minute.
After the sensor chip was prepared, 120 ~,l of supernatant from each hybridoma clone was injected over the flow cells at a flow rate of 30 ~1/minute.
Dissociation was allowed for 6 minutes. The chip was then regenerated with a consecutive injection of 50 ~1 each of 10 mM glycine, pH 2.0 and 10 mM
glycine, pH 2.5.
Dissociation rate constants were obtained using a separate kassoc/kdissoc f tting model with BIAevaluation 3.2 software provided by the manufacturer. The fitting model assumed 1:l binding.
I~;SSO~ calculated for each of the selected antibodies was plotted according to ECLA responses and ELISA responses described in Example 1 (Figure 2).
Antibodies in area II (ECLA-/ELISA+) were found to have a Kd;sso~ in the range of 10-3-10-5 1/sec (Table 1 and Figure 3). Antibodies in area III (ECLA~/ELISA+) were found to have a I~;SSO~ of 10-4 or less (Table 1 and Figure 3). Antibodies in area IV
(ECLA~/ELISA-) were found to have a Kd;sso~ in the range of 10-2-10-5 (Table 1 and Figure 3). Antibodies with a I~;SSO~ of 10-a were only found in area IV. The antibodies in area I (ECLA-/ELISA-) p~'esumably did not specifically bind 2H7.
Table 1 ECLA ELISA
Sample response response Positivekd;sso~Heavy ChainLight Number (ECLU) (0.D. in (1/s)a Iso a Chain at 650 nm) 1 269 0.653 Both 8.46E-04I G1/I G2b**ka a 2 277 0.572 Both * I G1 ka a 34 220 0.373 None 6.64E-03*
93 252 0.354 ECLA *
114 3695 1.175 Both 3.39E-05I G1 ka a 141 293 0.346 ECLA * I Gl ka a 205 197 0.776 ELISA 2.31E-04I G1 ka a 389 288 0.430 ECLA * I Gl ka a 421 201 0.582 ELISA * * *
425 199 0.534 ELISA 4.54E-04I G1 ka a 429 263 0.296 ECLA 0.030 * *
452 262 0.340 ECLA 2.19E-04 471 213 0.629 ELISA 8.99E-04I G1 lambda 492 214 0.584 ELISA 2.46E-03I G2b ka a 517 432 0.469 ECLA *
567 269 0.281 ECLA * * *
574 299 0.350 ECLA 1.37E-03*
634 296 0.359 ECLA * * *
664 205 0.561 ELISA 1.74E-03* *
705 260 0.384 ECLA * * *
729 206 0.689 ELISA 9.84E-04I G1 lambda 731 202 0.679 ELISA S.11E-03* *
740 261 0.273 ECLA 4.20E-05I G1 ka a 750 252 0.322 ECLA 1.37E-03* *
765 263 0.377 ECLA * I G3 ka a 770 253 0.344 ECLA * I G2a ka a 786 302 0.394 ECLA * * *
807 364 0.308 ECLA 1.01E-02* *
824 281 0.348 ECLA 1.93E-03*
876 290 0.300 ECLA 1.52E-02I G3 ka a 886 313 0.294 ECLA 1.33E-02* *
888 277 0.417 ECLA 1.64E-02* *
902 256 0.299 ECLA * *
911 267 0.304 ECLA * I G2b ka a 919 255 0.324 ECLA 0.017 * *
939 290 0.3I0 ECLA 7.91E-04IIgGl I *
a: measured by Biacore analysis *: not measurable due to a low concentration and/or exceeding assay limitation **: sample contained two different heavy chain isotypes, IgGl and IgG2b.
Antibodies that demonstrated a I~;SSO~ greater than about 10-5 were identified as low affinity antibodies. As shown in Table 1 and Figure 3, all but one of the identified low affinity antibodies were ECLA+/ELISA- or ECLA-/ELISA+. One antibody in area I (ECLA-/ELISA') was found to have a I~;SS°~ of 10-3, suggesting the detection limit for ECLA may have been set too high. Lowering the ECLA
detection limit may have identified additional low affinity anti-2H7 antibodies. One antibody in area IV was found to have a K.~;SS°~ of 10-5, suggesting the concentration of anti-2H7 antibody in the hybridoma supernatant producing this antibody was low.
Isotyping of Low Affinity Anti-2H7 Antibodies Isotypes were determined for antibodies produced by the hybridoma clones selected for further characterization in Example 2. An ELISA based antibody isotyping assay was performed Briefly, a polypropylene 96-well rnicrotiter plate was coated with 50 ~,l of isotype specific goat anti-mouse Ig (Southern Biotech, Pittsburgh, PA) and incubated overnight at 4°C.
The plate was washed with wash buffer (PBS with 0.05% Tween-20) and blocked with 200 ~1 of 2% BSA in PBS for one hour at room temperature. The plates were washed with wash buffer three times and 100 p,1 of hybridoma culture supernatant was added to the wells. The plate was incubated for 30 minutes at room temperature and washed three times. Fifty ~.1 of HRP goat anti-mouse IgG Fc specific (ICN) was added to each well and the plate was incubated for 30 minutes at room temperature. The plate was developed with HRP substrate as described for Example 1. Absorbance was measured as described for Example 1.
Heavy chain isotypes of antibodies produced by the selected hybridoma clones were plotted according to ECLA responses and ELISA responses as described for Example 1 (Figure 4). All antibodies tested showed a kappa light chain, except for two antibodies in area II (Table 1 ). These two antibodies showed a lambda light chain and are circled in Figure 4. Antibodies in area II (ECLA-/ELISA+) were found to have heavy chain isotypes of IgGl or IgG2b (Table 1 and Figure 4). Antibodies in area III (ECLA+/ELISA+) were found to have heavy chain isotypes of IgGl or IgG2b (Table 1 and Figure 4). Antibodies in area IV
(ECLA~/ELISA') were found to have heavy chain isotypes of IgG, IgG2a, IgG2b, or IgG3 (Table 1 and Figure 4).
Example 4 Screening Hybridomas for Low Affinity Anti-bevacizumab Antibodies Hybridoma supernatants were screened for production of low affinity anti-bevacizumab antibodies (Genentech Inc., South San Francisco, CA) using the ELISA/ECLA cross-screening method described in Example 1.
BALB/c mice were immunized and boosted with bevacizumab as described in Example 1. Three days after final boost, poptileal lymph nodes were fused with cells of the myeloma cell line, P3X63Ag.U.1 (ATCC, Manassas, VA). Fused cells were selected by hypoxanthin-aminopterin-thymidine (HAT) medium selection.
Supernatants from hybridoma cultures were screened for low affinity anti-bevacizumab antibodies by plotting ECLA responses versus ELISA responses.
ECLA screening and ELISA screening was performed as described in Example 1.
An enriched pool of candidate low affinity anti-bevacizumab antibodies was generated by plotting ECLA responses verses ELISA responses as described in Example 1. The detection limit for ELISA was set at O.D. 0.5. The detection limit for ECLA was set at 300 ECLU. As described in Example 1, lines depicting detection limits form the boundaries of four quadrants: area I (ELISA'/
ECLA'), area II (ELISA+/ ECLA'), area III (ELISA+/ ECLA+), and area IV (ELISA'/ ECLA+).
See, for example, Figure 1. Antibodies in area IV (ELISA'/ ECLA+) represent a population of candidate low affinity anti-bevacizumab monoclonal antibodies not detected by ELISA. Antibodies from two hybridoma clones (4B9 and 8F6) were identified as ELISA'/ ECLA+ (Table 2). These antibodies are candidate low affinity anti-bevacizumab monoclonal antibodies.
Table 2 Clone ELISA O.D. ECLA ECL
4B9 0.459 ELISA' 14101 ELCA+
4D7 0.967 ELISA''~ 22815 ELCA+
SE1. 0.987 ELISA+ 13334 ELCA+
6C2 1.005 ELISA+ 25270 ELCA+
6F11 0.930 ELISA+ 1834 ELCA+
8F6 0.474 ELISA' 3094 ELCA+
Specific binding affinities of candidate low affinity antibodies in area IV
were determined. Dissociation rate constants (I~;SSO~) for the antibodies were determined by Biacore analysis as described in Example 2. A fast off rate (Kd;sso~) typically correlates with low binding affinity. Antibodies that demonstrated a Kd;sso~
greater than about 10-S were identified as low affinity anti-bevacizumab antibodies.
Clones 4B9 and 8F6 were identified as producing low affinity anti-bevacizumab antibodies (Table 3).
While area IV (ELISA-/ ECLA+) is enriched in low affinity antibodies, it may also contain high affinity antibodies present in low concentration in the supernatant. To confirm identification of hybridoma clones producing low affinity antibodies, the concentration of monoclonal antibodies in the supernatant from the hybridoma clones was determined by Biacore analysis. Known concentrations of purified cynomologus monkey anti-bevacizumab polyclonal antibody were analyzed by Biacore as described in Example 2. A standard curve was generated by plotting the binding of the polyclonal antibody to bevacizumab in Biacore versus polyclonal antibody concentration and calculating the slope of the curve (Figure 5).
The concentration of monoclonal antibodies in supernatant from the individual hybridoma clones was calculated using the standard curve and this concentration was used to calculate dissociation rates constants (Kd;sso~), association rate constants (Kasso~), and equilibrium dissociation constants (KD) (Table 3). The equilibrium constants and rate constants were obtained using BIAevaluation 3.2 software provided by the manufacturer. Dissociation rate constants were obtained using a Kasso~ /Kd;sso~ fitting model with the BIAevaluation 3.2 software. The fitting model assumed 1:1 binding. Antibodies that demonstrated a KD equal to or greater than about 10-$ M were confirmed as low affinity anti-bevacizumab antibodies.
Clones 4B9 and 8F6 were confirmed as producing low affinity anti-bevacizumab antibodies (Table 3). .
Table 3 Estimated concentration in lone asso~ 1/Ms d;sso~ D the 1/s su ernatant* nM
4B9 6.40E+02 3.47E-05 5.42E-08 27.3 4D7 3.30E+05 8.32E-05 2.52E-10 98.1 5E1 2.96E+05 5.85E-05 1.98E-10 108.8 6C2 3.08E+05 8.88E-06 2.88E-11 59.8 6F11 8.04E+04 1.04E-04 1.29E-09 19.9 8F6 5.87E+03 3.65E-04 6.22E-08 4 * Concentrations were estimated using a standard curve generated with purified cynomologus monkey anti-bevacizumab polyclonal antibodies with Biacore (see Figure 5).
4,695,393; 4,698,302; and 4,554,088. Microparticles can be used in an amount ranging from about 1 to 10,000 ~g/ml, preferably 5 to 1.,000 ~,g/ml.
A "detection limit" for an analyte molecule in a particular assay is a minimum concentration of the analyte molecule that can be detected above background levels for that assay. For example, in IA and ECLA, the detection limit for an analyte molecule that specifically binds a target molecule can be the concentration at which the analyte molecule produces an IA signal or ECLA
signal above that produced by a control antibody that does not bind, or non-specifically binds, the target antigen. Molecules that have an IA response less than the IA
detection limit are IA-. Molecules that have an IA response equal to or greater than the IA detection limit are IA+. Molecules that have an ECLA response Iess than the ECLA detection limit are ECLA-. Molecules that have an ECLA response equal to or greater than the ECLA detection limit are ECLA+. Detection limits can be raised or lowered to achieve a desired assay result.
The term "antibody" is used in the broadest sense and specifically includes single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, affinity-matured antibodies, humanized antibodies, chimeric antibodies, single chain antigen binding molecules such as monobodies, as well as antigen binding fragments or polypeptides (e.g., Fab, F(ab')2, scFv, and Fv) that exhibit a desired biological activity. An antibody can be natural or synthetic.
"Natural" or "naturally occurring" antibodies are derived from a nonsynthetic source, for example, from a differentiated antigen-specific B
cell obtained ex vivo, or its corresponding hybridoma cell line, or from the serum of an animal. These include antibodies generated in any type of immune response, either natural or otherwise induced. As used herein, natural antibodies differ from "synthetic antibodies", synthetic antibodies referring to antibody sequences that have been changed, for example, by the replacement, deletion, or addition of one or more amino acid, resulting in an antibody sequence that differs from the source antibody sequence.
The term "monoclonal antibody" as used herein refers to a natural or synthetic antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be made by the hybridoma method first described by Kohler et al., 1975, Nature, 256:495, or can be made by recombinant DNA
methods (see, a g., LT.S. Pat. No. 4,816,567). The monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson et al., 1991, Nature, 352:624-628 (1991) and Marks et al., 1991, J. Mol. Biol., 222:581-597, for example.
The term monoclonal antibodies specifically includes "chimeric" antibodies (irnmunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit a desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-6855).
"Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, specific framework region (FR) residues of the human immunoglobulin can be replaced by corresponding non-human residues.
Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs correspond to those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., 1986, Nature, 321:522-525; Reichmann et al., 1998, Nature, 332:323-329; and Presta et al., 1992, Curr. Op. Struct. Biol., 2:593-596. Heavy and light chain variable domains of a humanized antibody can also contain consensus framework regions as described, for example, in US Patent No. 6,054,297 to Carter.
An "Fv" fragment is an antibody fragment that contains a complete antigen recognition and binding site. This antibody fragment comprises a dimer of one heavy and one light chain variable domain in tight association that can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen.
A "Fab" fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain.
F(ab)'2 antibody fragments comprise a pair of Fab fragments that are generally covalently linked near their carboxy termini by hinge cysteines. Other chemical couplings of antibody fragments are also known.
"Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL
domains of antibody, where these domains are present in a single polypeptide chain.
Generally the Fv polypeptide further comprises a polypeptide linker between the VH
and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, 1994, In: The Pharmaeology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.
The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH
and VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., 1993, P~oc. Natl. Acad. Sci. USA, 90:6444-6448.
The expression "linear antibodies" refers to antibodies as described in ~apata et al., 1995, Protein Eng., 8(10):1057-1062. Briefly, these antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
The term "monobody" as used herein, refers to an antigen binding molecule with a heavy chain variable domain and no light chain variable domain. A
monobody can bind to an antigen in the absence of light chains and typically has three CDR regions designated CDRH1, CDRH2, and CDRH3. A heavy chain IgG
monobody has two heavy chain antigen binding molecules connected by a disulfide bond. The heavy chain variable domain comprises one or more CDR regions, preferably a CDRH3 region. A "VhH" or "VHH" refers to a variable domain of a heavy chain antibody such as a monobody.
"Enriching" a pool of analyte molecules, as used herein, refers to analytical means for generating a pool of analyte molecules that possess a desired characteristic from a larger pool of analyte molecules. By viewing the cross-screening affinity data according to the system and methods of the invention, analyte molecules lacking the desired characteristic are eliminated, resulting in a pool of analyte molecules enriched for analyte molecules having the desired characteristic.
For example, by viewing the cross-screening affinity data obtained from ELISA
and ECLA analysis of a pool of candidate anti-therapeutic antibodies as described in the Examples below, antibodies that demonstrate an ELISA-/ECLA+response form a pool of candidate antibodies enriched for candidate low affinity anti-therapeutic antibodies.
The term "library" refers to a plurality of polypeptide or polypeptide fragment sequences, the sequences being different in the combination of variant amino acids that are introduced into these sequences. In one embodiment, the polypeptide or polypeptide fragment sequences are antibody or antibody fragment sequences.
"Phage display" is a technique by which variant polypeptides are displayed as fusion proteins to a coat protein on the surface of phage, e.g., filamentous phage, particles. A utility of phage display lies in the fact that large libraries of randomized protein variants can be rapidly and eff ciently sorted for those sequences that bind to a target molecule with high affinity. Display of peptide and protein libraries on phages has been used for screening millions of polypeptides for ones with specific binding properties. Polyvalent phage display methods have been used for displaying small random peptides and small proteins through fusions to either gene III or gene VIII of filamentous phage. Wells and Lowman, 1992, Curr. Opif2. Struct. Biol., 3:355-362, and references cited therein. In monovalent phage display, a protein or peptide library is fused to a gene III or a portion thereof, and expressed at low levels in the presence of wild type gene III protein so that phage particles display one copy or none of the fusion proteins. Avidity effects are reduced relative to polyvalent phage so that sorting is on the basis of intrinsic ligand affinity. Lowman and Wells, 1991, Methods: A Companioy2 to Methods in Enzyrraology, 3:205-0216.
2H7, also known as PR070769, refers to a humanized monoclonal antibody that binds human CD20 antigen expressed on most B cells. 2H7 is currently being evaluated in clinical phase I/II trails for treatment of rheumatoid arthritis.
Monoclonal antibody 2H7 is commercially available, for example, from eBioscience, San Diego, CA.
Bevacizumab refers to a humanized monoclonal anti-VEGF antibody that inhibits angiogenesis. Bevacizumab is approved for treatment of metastatic cancer of the colon or rectum and is currently being evaluated in clinical phase III
trials for treatment of other types of cancers including pancreatic, renal, and breast cancers.
Bevacizumab is commercially available from Genentech Inc., South San Francisco, CA.
II. Methods for carrying out the invention The invention provides a cross-screening system and methods that analyze data generated in immunoassay (IA) and electrochemiluminescent assay (ECLA) methods to identify analyte molecules that have binding affinity for a target molecule. The cross-screening system and methods of the invention identify analyte molecules having binding affinities for a target molecule that are below detection limits of the individual immunoassay or ECLA.
In one aspect of the invention, the binding affinity of analyte molecules for a target molecule is cross-screened using immunoassay and ECLA methods. Analyte molecules that are IA~/ECLA+, IA-/ECLA+, or IA+/ECLA- are identified as analyte molecules that specifically bind the target molecule.
As shown in Figure 1, a pool of analyte molecules enriched for analyte molecules having a particular binding affinity for a target molecule can be generated from a large pool of analyte molecules using an embodiment of the cross-screening system and methods of the invention. The large pool of analyte molecules is screened with IA and ECLA. Optionally, the IA signal of the individual analyte molecules in the pool is plotted against the respective ECLA signal (Figure 1).
Analyte molecules in areas II (IA+/ECLA-) and III (IA+/ECLA~) form an enriched pool of candidate high affinity molecules (Figure 1). Analyte molecules in area IV
(IA-/ECLA+) form an enriched pool of candidate low affinity analyte molecules (Figure 1). Analyte molecules from the enriched pools of candidate low or high affinity analyte molecules can be confirmed as low or high affinity analyte molecules by determining the specific binding affinity of a selected analyte molecule, for example by surface plasmon resonance analysis. If the analyte molecules are monoclonal antibodies, the antibodies can be isotyped to identify monoclonal antibodies with different characteristics.
Analyte molecules that can be screened by the system and methods of the invention are typically small molecules, polypeptides, or polypeptide fragments, and can be, for example, antibodies, soluble receptors, or fragments thereof.
Antibodies can be monoclonal antibodies, typically produced by hybridoma cells.
Polypeptides, such as antibodies, soluble receptors, and fragments thereof, can also be expressed on phage. Therefore, a pool of analyte molecules can be a phage library.
A target molecule useful in the system and methods of the invention, is typically a small molecule, polypeptide, or polypeptide fragment. The target molecule can be, for example, an antigen if the analyte is an antibody, a receptor or antibody if the analyte is a small molecule or polypeptide, a polypeptide or small molecule if the analyte is a soluble receptor, or a phage expressing antibodies, soluble receptors, or fragments thereof if the analyte is a polypeptide or small molecule.
Preferably the target molecule is an antigen, and can be, for example, a polypeptide, polypeptide fragment, or small molecule. Examples of target molecules include, but are not limited to, renin; growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor;
parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;
insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone;
calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA);
bombesin;
thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta;
enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1-alpha); serum albumin such as human serum albumin; Muellerian-inhibiting substance; relaxin A-chain;
relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; microbial protein, such as beta-lactamase; DNase; IgE; cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); EG-VEGF; Bv~; receptors for hormones or growth factors; protein A or D; rheumatoid factors; neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or nerve growth factor; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II
(IGF-I
and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins such as CD3, CD4, CD8, CD 19 and CD20; erythropoietin;
osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10;
superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral antigen such as, for example, a portion of the AIDS
envelope; transport proteins; homing receptors; addressins; regulatory proteins;
integrins such as CD 11 a, CD 11 b, CD 11 c, CD 18, an ICAM, VLA-4 and VCAM;
tumor associated antigen such as HER2, HER3, or HER4 receptor; fragments of any of the above-listed polypeptides or specific epitopes thereof; and antibodies that bind any of these polypeptides.
Preferred target molecules for screening antibody analyte molecules include CD proteins such as CD3, CD4, CDB, CD19, CD20 and CD34; members of the ErbB receptor family such as the EGF receptor, HER2, HERS or HER4 receptor;
cell adhesion molecules such as LFA-1, Macl, p150,95, VLA-4, ICAM-1, VCAM
and a,,(33 integrin including either alpha or beta subunits thereof (e.g. anti-CD 11 a, anti-CD 18 or anti-CD 1 1b antibodies); growth factors such as VEGF; IgE;
blood group antigens; flk2/flt3 receptor; obesity (0B) receptor; mpl receptor; CTLA-4;
protein C; and antibodies that bind any of these polypeptides.
A target molecule can also be a polypeptide or antibody having therapeutic properties or activity. For example, a polypeptide that induces angiogenesis, such as for example VEGF, EG-VEGF, or BvB, can be used therapeutically to promote healing of a wound or surgical incision in a tissue. In an embodiment, the target molecule is an antigen, such as and anti-therapeutic monoclonal antibody.
Examples of anti-therapeutic monoclonal antibodies useful as target molecules in the invention include, but are not limited to, anti-VEGF antibodies such as bevacizumab and LUCENTISTM, anti-HER2 antibodies such as HERCEPTINO and OMNITARGTM, anti-CD20 antibodies such as RITUXAN~ and PR070769, anti-IgE antibodies such as XOLAIR~, and anti-CD 11 a antibodies such as RAPTIVA~. In an embodiment, the target molecule is the monoclonal antibody 2H7 and the analyte molecule to be screened is a pool of anti-2H7 antibodies or hybridoma supernatants of clones producing such anti-2H7 antibodies. In an embodiment, the target molecule is the monoclonal antibody bevacizumab and the analyte molecule to be screened is a pool of anti- bevacizumab antibodies or hybridoma supernatants of clones producing such anti- bevacizumab antibodies.
When the target molecule is a therapeutic antibody or therapeutic polypeptide, the cross-screening system and methods of the invention can be used to identify enriched pools of candidate anti-therapeutic antibodies for candidate anti-therapeutic antibodies having low binding affinity for the target molecule (therapeutic antibody), as described in the Examples below.
A. Immunoassay Conventional immunoassays can be used in the cross-screening system and methods of the invention. Examples of immunoassays useful in the invention include, but are not limited to, radioimmunoassay (RIA), fluoroluminescence assay (FLA), chemiluminescence assay (CA), and enzyme-linked immunosorbant assay (ELISA). See, for example, Johnstone and Thorpe, Inarnunochemistry in Practice, Blackwell, 3rd ed., 1996; Cu~~eht Protocols ih Molecular Biology, Ausbul et al.
eds., Wiley & Sons, 2003; Immuyaoassa~y Methods ahd Protocols, Ghindilis et al.
eds., Blackwell, 2003; U.S. 20030044865. The immunoassay can be a solid phase assay or liquid phase assay. Preferably the immunoassay is a solid phase assay such as, for example, ELISA.
Analyte molecules in a sample can be concentrated using microparticles.
The microparticles can be polymeric, including but not limited to, sepharose beads, Iatex beads, and shell-core particles. See, for example, U.S. Patent Nos.
4,305,925;
4,480,042; and 4,419,453. The microparticles can be magnetic to facilitate separation of the beads or microparticles from the sample. See, for example, U.S.
Patent Nos. 4,731,337; 4,777,145; and 4,115,535. Preferably, the magnetic beads are paramagnetic beads such as, for example, DYNABEADS (Dynal Biotech, Brown Deer, WI). When microparticles are used in the assay, a target molecule is conjugated to the beads. The target molecule can be conjugated to the microparticle by a non-covalent or covalent interaction or physical linkage as desired. For example, the microparticles can be coated with streptavidin to provide a binding surface for biotinylated target molecules. Techniques for attachment include those described in U.S. Pat. No. 4,376,110 and the references cited therein.
Preferably the immunoassay is a solid-phase ELISA or a capture ELISA. In a capture ELISA, immobilization of the target molecule to a solid phase is conventionally accomplished by insolubilizing a capture reagent either before the assay procedure, as by adsorption to a water-insoluble matrix or surface (LJ.S. Pat.
No. 3,720,760) or non-covalent or covalent coupling, for example, using glutaraldehyde or carbodiimide cross-linking, with or without prior activation of the support with, for example, nitric acid and a reducing agent as described in U.S. Pat.
No. 3,645,852 or in Rotmans et al., 1983, J. Immunol. Methods, 57:87-98, or after the assay procedure, for example, by immunoprecipitation. In an embodiment, the capture reagent is an antibody or a mixture of different antibodies against a target antigen or an antibody/antigen complex, where the bound antigen is available to bind an antibody from a sample. In a further embodiment, the capture reagent is an anti-isotype specific antibody complexed to a therapeutic antibody. For example, the capture reagent can be a goat anti-human IgG Fc specific antibody complexed to a humanized therapeutic IgG monoclonal antibody. In an embodiment, the humanized therapeutic IgG monoclonal antibody is an anti-2H7 antibody. In an embodiment, the humanized therapeutic IgG monoclonal antibody is an anti-bevacizumab antibody.
The solid phase used for immobilization can be any inert support or carrier that is essentially water insoluble and useful in immunoassays, including supports in the form of, for example, surfaces, particles, porous matrices, and the like.
Examples of commonly used supports include small sheets, Sephadex, polyvinyl chloride, plastic beads, microparticles, assay plates, or test tubes manufactured from polyethylene, polypropylene, polystyrene, and the like. Such supports include well microtiter plates, as well as particulate materials such as filter paper, agarose, cross-linked dextran, and other polysaccharides. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016;
4,195,128;
4,247,642; 4,229,537; and 4,330,440 are suitably employed for capture reagent immobilization. In an embodiment the immobilized capture reagent is coated on a microtiter plate. The preferred solid phase is a mufti-well microtiter plate that can be used to analyze several samples at one time.
The solid phase is coated with the capture reagent that can be linked by a non-covalent or covalent interaction or physical linkage, as desired.
Techniques for attachment include those described in U.S. Pat. No. 4,376,110 and the references cited therein. If covalent attachment of the capture reagent to the plate is utilized, the plate or other solid phase can be incubated with a cross-linking agent together with the capture reagent. Commonly used cross-linking agents for attaching the capture reagent to the solid phase substrate include, for example, l, l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates capable of forming cross-links in the presence of light.
If polystyrene or polypropylene plates are utilized, the wells in the plate are preferably coated with the capture reagent (typically diluted in a buffer such as 0.05 M sodium carbonate) by incubation for at least about 10 hours, more preferably at least overnight, at temperatures of about 4-20°C, more preferably about 4-8°C, and at a pH of about 8-12, more preferably about 9-10, and most preferably about 9.6. If shorter coating times (1-2 hours) are desired, the plate is coated at 37°C or plates with nitrocellulose filter bottoms such, as for example, Millipore MULTISCREENTM. The plates can be stacked and coated in advance of the assay, allowing for an immunoassay to be carried out simultaneously on several samples in a manual, semi-automatic, or automatic fashion, such as by using robotics.
The coated plates are typically treated with a blocking agent that binds non-specifically to, and saturates, the binding sites to prevent unwanted binding of free ligand to excess binding sites on the wells of the plate. Examples of appropriate blocking agents include, fox example, gelatin, bovine serum albumin, egg albumin, casein, and non-fat milk. The blocking treatment typically takes place under conditions of ambient temperatures for about 1-4 hours, preferably about 1.5 to 3 hours.
After coating and blocking, the sample to be analyzed is diluted as necessary and added to the immobilized phase. The preferred dilution rate is about 5-15%, preferably about 10%, by volume. Buffers that can be used for dilution include for example (a) phosphate buffered saline (PBS) containing 0.5% BSA, 0.05% TWEEN
20TM detergent (P20), 5 mM EDTA, 0.25% Chaps surfactant, 0.2% beta-gamma globulin, and 0.35M NaCI, pH 7.0; (b) PBS containing 0.5% BSA and 0.05% P20;
(c) PBS containing O.S% BSA, O.OS% P20, S mM EDTA, and 0.35 M NaCI, pH
6.35; (d) PBS containing O.S% BSA, 0.05% P20, S mM EDTA, 0.2% beta-gamma globulin, and 0.35 M NaCI; (e) PBS containing O.S% BSA, O.OS% P20, S mM
EDTA, 0.25% Chaps, and 0.35 M NaCI; and (f) PBS containing O.S% P20.
S For sufficient sensitivity, it is preferred that the immobilized capture reagent is in molar excess of the maximum molar concentration of the analyte anticipated in the sample after appropriate dilution. Depending on the analyte, the capture reagent can compete for binding sites with the detecting antibody yielding inaccurate results.
Therefore, the final concentration of the capture reagent will normally be determined empirically to maximize the sensitivity of the assay over the range of interest.
Conditions for incubation of sample and capture reagent are selected to maximize sensitivity of the assay and to minimize dissociation. Incubation time depends primarily on the temperature. Preferably, the incubation time is from about 0.5 to 3 hours, and more preferably 1.5-3 hours at 36-38°C. To maintain the 1 S sensitivity of the assay, incubation times greater than about 10 hours are avoided if possible. If the sample is a biological fluid, incubation times can be lengthened by adding a protease inhibitor to the sample to prevent proteases in the biological fluid from degrading the analyte.
The pH of the incubation buffer is chosen to maintain a significant level of specific binding of the capture reagent to the analyte being captured. The pH
of the incubation buffer is preferably about 6-9.5, more preferably about 6-7.
Various buffers can be employed to achieve and maintain the desired pH during this step, including borate, phosphate, carbonate, Tris-HCI or Tns-phosphate, acetate, barbital, and the like. The particular buffer employed is usually not critical, however, and in 2S individual assays one buffer may be preferred over another.
The sample is separated from the immobilized capture reagent with a wash solution to remove uncaptured analyte from the system. The wash solution is generally a buffer. The incubation buffers described above are suitable wash solutions. The pH of the wash solution is determined as described above for the incubation buffer. In an embodiment, the pH of the wash solution is about 6-9, more preferably about 6-7. Washes can be done one or more times. Minimizing the number of washes, however, to retain molecules that bind the target molecule with low aff nity increases the background noise of the assay. Preferably, the system is washed three times. The temperature of the wash solution is typically from about 0-40°C, more preferably about 4-30°C. An automated plate washer can be utilized. A
cross-linking agent or other suitable agent can be added to the wash solution to covalently attach the captured analyte to the capture reagent.
Following removal of uncaptured analyte molecules from the system, the captured analyte molecules are contacted with a detecting agent, such as an antibody, preferably at a temperature of about 20-40°C, more preferably about 36-38°C. When the analyte is humanized anti-therapeutic antibody, the detecting agent is an anti-isotype antibody from a different species. If the anti-therapeutic antibodies are human IgG, for example, the detecting agent can be a murine anti-human IgG antibody. In an embodiment, the analyte is murine monoclonal antibody and the detecting agent is sheep anti-mouse IgG.
The temperature and time for contacting the analyte molecule with the detecting agent is dependent primarily on the detection means employed. For example, when horseradish peroxidase (HRP) conjugated to sheep anti-mouse IgG
is 1 S used as the means for detection, the detecting agent is preferably incubated with the captured analyte for about 0.5-2 hours, more preferably about 1 hour. The system is washed as described above to remove unbound detecting agent from the system and developed by adding peroxidase substrate and incubating the plate for about 5 minutes at room temperature or until good color is visible.
In an embodiment, a molar excess of the detecting agent is added to the system after the unbound analyte has been washed from the system. The detecting agent can be a polyclonal or monoclonal antibody. In an embodiment, the antibody is a monoclonal antibody. In an embodiment, the monoclonal antibody is murine.
The detecting agent can be directly or indirectly detectable. If the detecting agent is an antibody that is not directly detectable, the detecting antibody is detected by addition of a molar excess of a second, labeled antibody directed against the isotype and animal species of the detecting antibody.
The affinity of the detecting agent must be sufficiently high such that small amounts of analyte can be detected. A fluorimetric or chemilimunescent label moiety has greater sensitivity in immunoassays compared to a conventional colorimetric label. The binding affinity of the selected detecting agent must be considered in view of the binding affinity of the capture agent, such that the detecting agent does not strip the analyte from the capture reagent.
The label moiety is any detectable functionality that does not interfere with the binding of the captured analyte to the detecting agent. Examples of suitable label moieties include moieties that can be detected directly, such as fluorochrome, chemiluminscent, and radioactive labels, as well as moieties, such as enzymes, that must be reacted or derivatized to be detected. Examples of such labels include the radioisotopes 32P, 14C, l2sh 3H, and 1311, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodainine and its derivatives, dansyl, umbelliferone, luceriferases, a g., firefly luciferase and bacterial luciferase (U.S. Pat.
No.
4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphiatase, (3-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HPP, lactoperoxidase, or microperoxidase, biotin/avidin, biotin/streptavidin, biotin/Streptavidin-(3-galactosidase with MUG, spin labels, bacteriophage labels, stable free radicals, and the like.
Conjugation of the label moiety to the detecting agent, such as for example an antibody, is a standard manipulative procedure in immunoassay techniques.
See, for example, O'Sullivan et al. "Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay," in Methods in Enzymology, ed. J. J.
Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), pp. 147-166. Conventional methods are available to bind the label moiety covalently to proteins or polypeptides. For example, coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized benzidine, and the like can be used to label antibodies with the above-described fluorescent, chemiluminescent, and enzyme labels. See, for example, U.S. Pat. Nos.
3,940,475 (fluorimetry) and 3,645,090 (enzymes); Hunter et al., 1962, Nature, 144:945;
David et al., 1974, Biochemistry, 13:1014-1021; Pain et al., 1981, J. Immunol Methods, 40:219-230; and Nygren J., 1982, Histoclzem. and Cytoclaem., 30:407-412.
Preferred labels herein are fluorescent or chemiluminescent to increase amplification and sensitivity to about 5-10 pglml. In an embodiment, the label moiety is HRP.
The amount of analyte bound to the capture reagent is determined by washing away unbound detecting agent from the immobilized phase and measuring the amount of detecting agent bound to the analyte using a detection method appropriate to the label. In an embodiment, the Iabel moiety is an enzyme. In the case of enzyme moieties, the amount of developed color is a direct measurement of the amount of captured analyte. For example, when HRP is the label moiety, color is detected by quantifying the optical density (0.D.) at 650 nm absorbance. In another embodiment, the quantity of analyte bound to the capture reagent is determined in-directly. The signal of an unlabeled detecting agent can be amplified for detection with an anti-detecting agent antibody conjugated to a label moiety. For example, the signal of an unlabeled mouse antibody that binds the target molecule can be amplified with a sheep anti-mouse IgG antibody labeled with HRP. The label moiety is detected using a detection method appropriate to the label. For example, HRP can be detected by reacting HRP with a colorimetric substrate and measuring the optical density of the reacted substrate at 650 nm absorbance.
The pH and/or temperature of the system can be varied to identify molecules . that bind the target molecule.
B. ECLA
Conventional methods for ECLA can be used in the cross-screening system and methods of the invention. See, for example, U.S. Patent Nos. 5,543,1 I2;
5,935,779; 6,316,607, and the patents referenced therein. In an embodiment, the capture reagent and detecting reagent are mixed with the analyte molecule and incubated at room temperature. In an embodiment, the capture reagent and detecting reagent are in molar excess of the maximum molar concentration of the analyte molecule anticipated in the sample. Depending on the analyte molecule, the capture reagent rnay compete for binding sites with the detecting reagent yielding inaccurate results. Therefore, the final concentration of the capture reagent will normally be determined empirically to maximize the sensitivity of the assay over the range of interest. In an embodiment, the capture reagent and detecting reagent are added to the sample in about a l :l ratio.
The capture reagent can be an antigen, receptor, antibody, or fragment thereof. Preferably the antibody is monoclonal. In an embodiment, the capture reagent is an antibody or a mixture of different antibodies against a target antigen.
In another embodiment, the capture reagent is a goat anti-human IgG antibody.
The detecting agent can be a receptor, antibody, or fragment thereof. In an embodiment, the antibody is monoclonal. The monoclonal antibody can be a marine monoclonal antibody. In an embodiment, the detecting reagent is an.antibody or a mixture of different antibodies against a target antigen. In another embodiment, the detecting agent is marine anti-human IgG antibody.
The incubation time can be from about 0.5 to 3 hours, more preferably 1.5-3 hours at 36-3~°C. The pH of the incubation buffer is chosen to maintain a significant level of specific binding of the capture reagent and detecting agent to the analyte. In an embodiment, the pH of the incubation buffer is about 6-9.5, more preferably about 6-7. Various buffers can be employed to achieve and maintain the desired pH during this step, including borate, phosphate, carbonate, Tris-HCI
or Tns-phosphate, acetate, barbital, and the like. The particular buffer employed is usually not critical, however, in individual assays one buffer may be preferred over another.
In an embodiment, the ECLA method utilizes a binding phase to immobilize the analyte complex, such as beads or microparticles. The beads or microparticles can have a diameter of 0.05 um to 200 um, more preferably 0.1 um to 100 um, more preferably 0.5 um to 10 um, and a surface component capable of binding the capture reagent. In an embodiment, the binding surface of the beads or microparticles is coated with streptavidin and the capture reagent is labeled with biotin. The microparticles can also be coated, for example, with glutathione, anti-IgG
antibody, or agglutinin. The capture reagent can be biotinylated with biotinylamidocaproic acid-N-Hydroxy-succinimide ester using standard amine chemistry at a ratio from about 1;1 to about 10:1 biotin to capture reagent, more preferably from about 2:1 to about 4: l biotin to capture reagent, more preferably about 2.5:1 biotin to capture reagent.
If the analyte is an anti-therapeutic antibody, the analyte, the capture reagent, and detecting reagent can be antibodies from the same species. In such instances, the same type of antibody can be separately utilized as both a capture reagent and detecting reagent. For example, separate batches of the same antibody are labeled, one with a component capable of binding the microparticle, such as biotin when the microparticle is coated with streptavidin, or another with or a label, such as Ori-Tag~. In an embodiment, the antibody is a therapeutic monoclonal antibody including, but are not limited to, anti-VEGF antibodies such as bevacizumab and LUCENTISTM, anti-HER2 antibodies such as HERCEPTIN~ and OMNITARGTM, anti-CD20 antibodies such as RITUXAN~ and PR070769, anti-IgE antibodies such as XOLAIR~, and anti-CDl la antibodies such as RAPTIVA~. In an embodiment, the capture reagent and detecting reagent are added to the analyte in about a I : I
ratio. The analyte must bind both a capture reagent and a detecting reagent to be detected by ECLA. Analytes that bind only capture reagent can bind to the microparticle but are not detectable.
After incubating the capture reagent and detecting reagent with the analyte molecule, microparticles capable of binding the capture reagent are added to the mixture and the mixture is incubated. In an embodiment, the microparticles are coated with a molecule that binds biotin, such as streptavidin. The incubation time can be from about 0.5 to 3 hours, preferably 1.5-3 hours at 36-3~°C.
The pH of the incubation buffer is chosen to maintain a significant level of specific binding of the capture reagent/detecting agent/analyte molecule complex to the microparticles.
The pH of the incubation buffer can be about 6-9.5, more preferably about 6-7.
The incubation buffer can include an electrolyte. The electrolyte can be one or more salts or other species. In an embodiment, the electrolyte is a sodium salt or potassium salt.
The microparticles are assayed with an apparatus that contains an electrode and a photodetector, such as an IGEN M3~4 analyzer (IGEN International Inc., Gaithersburg, MA). See, for example, U.S. Patent Nos. 5,543,112 and 5,935,779 describing apparatuses for measuring electrochemiluminescence. The label conjugated to the detecting agent is induced to emit electromagnetic radiation by stimulating the label into an excited state. Detection and/or quantitation of the analyte in a sample is typically made by comparing the luminescence of a sample to the luminescence emitted by a calibration standard developed with known amounts of the analyte and detecting agent. In an embodiment, the photodetector measures the light emitted by the label and software for analyzing data collected by the photodetector is used to calculate the concentration of analyte molecular or ECLA
response (in electrochemiluminescence units (ECLU)) of the analyte molecule.
In an embodiment, the label conjugated to the detecting reagent is a metal chelate that luminesces under the electrochemical conditions imposed by ECLA.
The metal can be, for example, a transition metal (such as a d-block transition metal) or a rare earth metal. In an embodiment, the metal is ruthenium, osmium, rhenium, iridium, rhodium, platinum, indium, palladium, molybdenum, technetium, copper, chromium, or tungsten. In an embodiment, the metal is ruthenium or osmium.
A ligand(s) linked to the metal of the chelate is usually heterocyclic or organic in nature, and plays a role in determining whether the metal chelate is soluble in an aqueous environment or in an organic or other nonaqueous environment. The ligands can be polydentate, and can be substituted.
Polydentate ligands include aromatic and aliphatic ligands. Suitable aromatic polydentate ligands include aromatic heterocyclic ligands. In an embodiment, the aromatic heterocyclic ligands are nitrogen-containing, such as, for example, bipyridyl, bipyrazyl, terpyridyl, and phenanthrolyl. Suitable substituents include for example, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxy, imino, hydroxycarbonyl, aminocarbonyl, amidine, guanidinium, ureide, sulfur-containing groups, phosphorus containing groups, and the carboxylate ester of N-hydroxysuccinimide. The chelate can have one or more monodentate ligands, a wide variety of which are known to the art. Suitable rnonodentate ligands include, for example, carbon monoxide, cyanides, isocyanides, halides, and aliphatic, aromatic and heterocyclic phosphines, amines, stilbenes, and arsines.
Examples of suitable chelates are bis [(4,4'-carbomethoxy)-2,2'-bipyridine]
2-[3-(4-methyl-2,2'-bipyridine-4-yl)propyl]-1,3-dioxolane ruthenium (II); bis (2,2'bipyridine) [4-(butan-1-al)-4'-methyl-2,2'-bipyridine] ruthenium (II);
bis (2,2'-bipyridine) [4-(4'methyl-2,2'-bipyridine-4'-yl)-butyric acid] ruthenium (II);
tris (2,2'bipyridine) ruthenium (II); (2,2'-bipyridine) [bis-bis(1,2-diphenylphosphino)ethylene] 2-[3-(4-methyl-2,2'-bipyridine-4'-yl)propyl]-1,3-dioxolane osmium (TI); bis (2,2'-bipyridine) [4-(4'-methyl-2,2'-bipyridine)-butylamine] ruthenium (II); bis (2,2'-bipyridine) [1-bromo-4(4'-methyl-2,2'-bipyridine-4-yl)butane] ruthenium (II); bis (2,2'-bipyridine)maleimidohexanoic acid, 4-methyl-2,2'-bipyridine-4'-butylamide ruthenium (II). Additional label moieties suitable for ECLA are described in U.S. Patent Nos. 5,591,51; 6,271,041;
6,316,607; and 6,451,225. In an embodiment, the label moiety is Ru(bpy)3z+ or ORI-TAGTM NHS ester (IGEN International Inc., Gaithersburg, MA).
The amount of label utilized is that amount which effectively results in the emission of a detectable, and if desired, quantifiable, emission of electromagnetic energy. In an embodiment, the detecting agent is conjugated with the label, using standard amine chemistry, at a ratio from about 1:1 to about 10:1 label to detecting reagent, more preferably from about 3:1 to about 7:1 label to detecting reagent, more preferably about 5:1 label to detecting reagent.
The pH and/or temperature of the system can be varied to identify molecules that bind the target molecule.
C. Detection limit The detection limit for the IA and ECLA is the minimum concentration of analyte that can be detected above the background level of the respective assay. The detection limit of IA and/or ECLA can be determined by conventional methods.
Below this detection limit, it is difficult to differentiate specific binding of the analyte from non-specific binding of the detecting agent. Molecules that are IA-/ECLA- are considered not to have specific binding affinity for the target molecule.
The detection limit can be determined by the amount of non-specific binding of the detecting agent in the system. The background level of the IA or ECLA
can be determined under the conditions of the respective assay in the absence of analyte.
For example, IA and ECLA can be used to detect an analyte, such as a low affinity antibody, in a homogenous mixture. The background level in the respective assay can be determined by quantifying the signal of the detecting antibody after the system has been incubated with incubation buffer containing no analyte. IA and ECLA can also be used to screen for an analyte, such as a low affinity binding anti-therapeutic, antibody, in a homogenous mixture, such as serum. The background level in the respective assay can be determined by quantifying the signal of the detecting agent after the system has been incubated with serum from one or more animals that were not administered the therapeutic.
The background level can also be determined using a control analyte that does not specifically bind the target molecule. In an embodiment, the detection limit is determined using a control antibody that does not specifically bind the target antigen. For example, IA and ECLA can be used to screen the supernatant of hybridoma clones for low affinity antibodies. The background level of the respective assay can be determined by quantifying the signal of the detecting antibody after the system is incubated with a supernatant sample containing control antibodies that do not specifically bind the target antigen. In an embodiment, the control antibodies are the same isotype as the antibodies being produced by the hybridoma clones of interest.
Reducing the background Ievel of the system reduces the detection limit thereby increasing the sensitivity of the assay. There are a number of ways the background level of the IA or ECLA can be reduced including, but not limited to, increasing the length of washes, adding additional washes, selecting a different wash buffer, selecting a solid phase of a different material, selecting a different blocking buffer, selecting a different detecting agent, selecting beads or microparticles with a lower autofluorescence level, reducing incubation times, changing the pH of one or more buffers, changing incubation temperature, or any combination thereof.
With respect to ECLA specifically, employing paramagnetic beads in association with a magnetic electrode can reduce the background.
The detection limit for IA and/or ECLA may require optimization to ensure the detection limit is not excluding analyte molecules with a desired characteristic, or to ensure that undesired molecules are excluded. For example, the binding affinities of randomly selected IA-IECLA- analyte molecules can be determined.
Randomly selected IA-/ECLA' analyte molecules found to have a binding affinity for the target molecule of about 10-2 may indicate the detection limit is set too high and that lowering the IA and/or ELISA detection limit may identify additional analyte molecules with the desired low binding affinity for the target molecule.
D. Determination of Binding Affinity Candidate low or high affinity analyte molecules are confirmed as low or high affinity analyte molecules based on their dissociation rate constant (Kd;sso~) for the target molecule or equilibrium dissociation constant (KD). Binding affinities of candidate molecules selected from the enriched pool of candidate molecules generated by the cross-screening system and methods of the invention can be confirmed by conventional equilibrium or kinetic methods. Examples include, but are not limited to, competitive ELISA, equilibrium dialysis, RIA, surface plasmon resonance such as BIACORE~ (Biacore Inc., Piscataway, N.J.), affinity chromatography, and ECLA. See, for example, Current Protocols in Molecular Biology, Ausbul et al. eds., Wiley & Sons, 2003; Current Protocols in Immunology, Bierer et al. eds, Wiley & Sons, 2003; and U.S. Patent Nos. 5,543,112;
5,935,779;
and 6,143,574: In an embodiment, the binding afEnity is determined by BIACORE~ analysis (see Example 3 below).
A high Kd;sso~ is indicative of low binding affinity. An analyte molecule with a Kd;sso~ greater then 10-6 for the target molecule or KD equal to or greater than 10-8 M, is identified as a molecule having low binding affinity for the target molecule. In an embodiment, the Kd;sso~ of the candidate low affinity analyte molecule for the target molecule is 10-5 or greater, more preferably 10-4 or greater, more preferably 10-3 or greater, and more preferably 10-2 or greater. In an embodiment, the Ko of the candidate low affinity analyte molecule for the target molecule is about 10-6 M, about 10-~ M, or about 10-8 M.
E. Isotyping The isotypes of the heavy and light chains of low affinity binding antibodies (analyte molecules) identified by the cross-screening system and methods of the invention can be determined by conventional methods, such as ELISA utilizing anti-isotype specific antibodies. See, for example, Current Protocols in Immunology, Bierer et al. eds, Wiley & Sons, 2003. In an embodiment, the antibodies are alkaline phosphatase or HRP-conjugated anti-mouse or anti-human antibodies. In an embodiment, a panel of anti-heavy chain isotype specific antibodies anti-isotype specific antibodies is employed. The panel of antibodies can include anti-heavy chain anti-isotype specific IgG, IgE, IgA, and IgM antibodies. In an embodiment, the panel of antibodies includes at least anti- IgGl, IgG2a, IgG2b, or IgG3 isotype specific antibodies.
F. iJses The cross-screening system and methods of the invention have many applications. The methods of the invention are particularly useful for identifying analyte molecules that bind a target molecule. For example, the methods are useful for identifying polypeptides or small molecules that bind a specific receptor, or antibodies that bind a specific antigen. In an embodiment, a receptor, antibody, or fragment thereof is expressed on a phage. In another embodiment, the antigen is a polypeptide or monoclonal antibody having therapeutic activity. For example, the monoclonal antibody can be anti-VEGF antibody such as bevacizumab and LUCENTISTM, anti-HER2 antibody such as HERCEPTIN~ and OMNITARGTM, anti-CD20 antibody such as RITUXAN~ and PR070769, anti-IgE antibody such as XOLAIR~, and anti-CD1 la antibody such as RAPTIVA~. In an embodiment, the target antigen is monoclonal antibody 2H7. In another embodiment, the target antigen is monoclonal antibody bevacizumab. The pH and/or temperature of the system can be varied to identify molecules that bind the target molecule.
2~
The cross-screening system and methods of the invention are particularly useful to detect small amounts of analyte molecule in a sample. Preferably the analyte molecule is an antibody. For example, the cross-screening system and methods of the invention can be used to identify a hybridoma producing antibodies that have high affinity for the target molecule but the concentration of antibodies in the supernatant is low. The concentration of antibodies in the supernatant can be below the detection limit of the individual IA or ECLA assay, but not the detection limit of the cross-screening system and methods of the invention.
The methods of the invention can be used to enrich a pool of analyte molecules for a desired characteristic. In an embodiment, the analyte molecules are antibodies. In an embodiment, the desired characteristic is binding affinity (IA-/ECLA+, IA+/ECLA+, and IA+/ECLA+) for the target molecule. In another embodiment, the desired characteristic is high binding affinity for a target molecule (IA+/ECLA+ or IA+/ECLA-). For example, a small molecule library can be screened for candidate library members that bind a target receptor with high affinity.
Similarly, a library of receptors, antibodies, or fragments thereof can be screened for candidate library members that bind a target polypeptide with high affinity.
In another embodiment, the desired characteristic is low binding affinity for a target molecule (IA-/ECLA+), such as for example, an antigen. The candidate molecules can be confirmed as high affinity or low affinity analyte molecules respectively, by determining a specific binding affinity of the analyte molecules for the target antigen.
Low affinity antibodies are needed, for example, in anti-therapeutic antibody assays required for regulatory approval of biological therapeutics. Immune responses are polyclonal; therefore, anti-therapeutic antibodies generated against a biological therapeutic can target different regions of the therapeutic or demonstrate different binding affinities and isotypes. In clinical trials, for example, a panel of anti-therapeutic antibodies that mimics the polyclonal nature of an immune response to the biological therapeutic undergoing clinical testing can be used to assess performance of an anti-therapeutic antibody assay. Low affinity antibodies identified by the cross-screening system and methods of the invention can be used to construct such a panel of antibodies. In an embodiment, the cross-screening system and methods of the invention are used to screen hybridoma clones for antibodies that bind a target antigen with low affinity. In an embodiment, the target antigen is a therapeutic monoclonal antibody.
In clinical trials for example, detection of antibodies to a biological therapeutic at an early stage in the trial is important for assessing the safety and efficacy of the therapeutic. In an embodiment, the methods of the invention are used to screen serum from a patient about to receive or who is receiving a biological therapeutic, such as a polypeptide or monoclonal antibody, for anti-therapeutic antibodies.
Low affinity antibodies are also useful in drug discovery methods. For example, it can be difficult to generate antibodies with high binding affinity for a therapeutic target. Low affinity antibodies can serve as a starting point for developing affinity matured antibodies. In an embodiment, antibodies are cross-screened utilizing the methods of the invention for analyte molecules exhibiting low bindixig affinity for the therapeutic target. Selected low affinity antibodies are affinity matured to produce therapeutic antibodies. In an embodiment, the antibodies are expressed on phage. In another embodiment, the antibodies are members of a phage library.
Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
All publications (including patents and patent applications) cited herein are hereby incorporated in their entirety by reference.
Example 1 Screening Hybridomas for Low Affinity Anti-2H7 Antibodies.
Hybridoma supernatants were screened for production of low affinity anti-2H7 antibodies using cross-screening methods employing ELISA and ECLA. A
workflow diagram of an embodiment of a hybridoma screening strategy is shown in Figure 1.
Hybridoma Production BALB/c mice were immunized and boosted 10 times with 0.5 ~g 2H7 resuspended in monophosphoryl lipid A/trehalose dicorynomycolate adjuvant (Corixa). The suspension was injected in each hind footpad at 3 to 4 days intervals.
Three days after final boost, poptileal lymph nodes were fused with cells of the myeloma cell line, P3X63Ag.U.1 (ATCC, Manassas, VA). Fused cells were selected by hypoxanthin-aminopterin-thymidine (HAT) medium selection.
ECLA Screening Supernatants from hybridoma cultures Were screened for low affinity anti-2H7 antibodies by plotting ECLA responses versus ELISA responses. ECLA was performed as described in Baker et al., 2002, Trends in BioteclZnol., 20:149-156.
Briefly, separate batches of 2H7 antibodies were labeled with biotin or Ori-tag (Igen International Inc, Gaithersburg, MD). 2H7 was biotinlylated with biotinylamiocaproic acid-N-hydroxy-succinimid ester (Organics Inc.) using standard amine based chemistry at target ratio of 2.5:1 biotin to 2H7. 2H7 was labeled with ORI-TAG NHS ester according to the manufacturer's instructions at a targeted ratio of 5:1 ORI-TAG to 2H7. A master working solution was prepared by mixing biotinylated 2H7 and Ori-tag labeled 2H7 in a 1:1 ratio. The final concentration of each labeled antigen in the master working solution was 1 ~.g/ml.
A panel of monoclonal antibodies was created by adding 50 ~1 of master working solution and 50 p.1 of supernatant from individual hybridoma clones to a 96-well round-bottom polypropylene plate. Each well in the plate contained supernatant from a single hybridoma clone. The plate was incubated at room temperature in the dark for two hours with a gentle agitation 10 ~,g of streptavidin coated magnetic beads in a volume of 100 ~,1 was added to each well. The plate was incubated for another one hour at room temperature in the dark with a gentle agitation. Post-incubation, the plates were read on an IGEN M384 analyzer using the following protocol parameters: bead type is set at 2.80 microns, aspiration volume of 200 ~,1, POP of 0 mv, gain of,l, wash volume of 700 ~l, clean cycle of 2, wash speed of 200 ml/sec. Data was collected and reported in electrochemiluminescence units (ECLU).
HAT medium was used a control. To be detected by ECLA, the analyte molecule must bind both a capture reagent and a detecting agent. Anti-2H7 antibodies (analyte molecules) from supernatant culture that formed a complex with both capture reagent and detecting agent were detected in the assay, (ECLA+).
Anti-2H7 antibodies from the supernatant culture that bound only capture reagent or only detecting reagent were not detected.
ELISA Screening ELISA was performed generally as described in Baker et al., 2002, Trends ira Biotechhol., 20:149-156. Briefly, a 384-well Greiner flat bottom plate was coated with 50 ~1 of goat anti-human IgG Fc specific (Cappel #55071) at a concentration of 2 ~g/ml in coating buffer (50 ~M carbonate buffer, pH 9.6). The plate was sealed and stored at 4 °C overnight. After removing the coating solution, 100 ~.l of blocking solution containing 2% of bovine serum albumin in PBS was added to each well. The plate was incubated at room temperature for one hour with agitation and then washed three times with PBS/0.05% Tween-20.
After the washing step, 50 ~,1 of antigen solution (0.4 ~,g/ml 2H7 in PBS
containing 0.5% bovine serum albumin) was added to each well and the plate was incubated at room temperature for one hour with agitation. The plated was washed three times with PBS/0.05% Tween-20. 35 ~,1 of supernatant from individual hybridoma clones was added such that each well in the plate contained supernatant from a single hybridoma clone. The plate was incubated for one hour at room temperature and washed three times with PBS/0.05% Tween-20.
After the washing step, 50 ~1 of a 1:1000 dilution of sheep anti-mouse IgG
HRP (no cross reactivity to human IgG, Cappel #55569) in PBS containing 0.5%
bovine serum albumin and 0.1 % Tween-20 was added to each well. The plate was incubated at room temperature for one hour with agitation, washed three times with PBS/0.05% Tween-20, rinsed with water, and shaken dry. The plate was developed by adding 40 ~l of TMB Microwell Peroxidase (tetramethylbenzidine) substrate (BioFX #TMBT-0100-O1) to each well in the plate and incubating the plate for 5 minutes at room temperature or until a good color was visible. Development was stopped by adding 40 ~l of the Stop solution (BioFX #BSTP-0100-O1) to each well, plates were read on a Sunrise plate reader (Tecan US, Research Triangle Park, NC) at 650 nm.
Identification of Low Affinity Antibodies Low affinity anti-2H7 antibodies were identified by plotting ECLA
responses (ECLU) against ELISA responses (0.D. at 650 nm) for each respective hybridoma supernatant (Figure 2). The detection limit for ELISA was set at O.D.
0.5. HAT medium was used as a control in ECLA to establish a detection limit of 250 ECLU.
Detection limits for ECLA and ELISA were used to establish a four quadrant grid on the ECLA:ELISA plot (Figure 2). Lines depicting detection limits form the boundaries of four quadrants. Antibodies in area I (ECLA-/ELISA-) represent antibodies that did not bind 2H7 or had binding that was not detected by either ELISA or ECLA. Antibodies in area III (ECLA+/ELISA~) represent candidate high affinity anti-2H7 antibodies. Antibodies in area II (ECLA-/ELISA+) represent candidate high affinity anti-2H7 antibodies that are believed to bind epitopes masked or altered by biotinlyation of 2H7 and/or labeling of 2H7 with Ori-Tag.
Antibodies in area IV (ECLA+/ELISA-) represent candidate low affinity anti-2H7 antibodies.
Antibodies in area IV represent a population of anti-2H7 antibodies not detected by ELISA. These antibodies may have been washed off the plate during the multiple wash steps in ELISA or had an ELISA response less than 0.5. Antibodies in area IV
presumptively produced low affinity antibodies. While Area IV is enriched in low affinity antibodies, it may also contain high affinity antibodies present in low concentration in the supernatant.
Biacore Analysis of Low Affinity Anti-2H7 Antibodies A number of hybridoma clones producing antibodies in quadrants I, II, III, or IV in Example 1 were selected for further characterization and confirmation of specific binding affinity. Dissociation rate constants (Kd;sso~) for antibodies produced by the selected hybridoma clones were determined by Biacore analysis.
The analysis was performed on a Biacore 3000 (USA Biacore, Inc., Piscataway, NJ). The monoclonal antibody 2H7 was immobilized on a CMS sensor chip in a flow cell. In brief, the flow cell was activated by injecting 35 p1 of a solution containing equal volumes of 11.5 mg/ml of N-hydroxysuccinimide (NHS).
and 7.5 mg/ml of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Following activation of the flow cell, 2H7 in sodium acetate (pH 5.0) was injected manually to reach a response of approximately 500 RU on the chip.
Thirty-five p.1 of 1 M ethanolamine hydrocholoride-NaOH (pH ~.0) was injected at a flow rate of 5 p,l/minute to block any un-reacted activated sites on the flow cell.
The final concentration of immobilized 2H7 after ethanolamine blocking was 556 RU. A
different flow cell was used as an in-line reference cell. The reference flow cell was activated as described above and immediately blocked with a 35 ~1 injection of ethanolamine hydrocholoride-NaOH (pH ~.0) at a flow rate of 5 ~l/minute.
After the sensor chip was prepared, 120 ~,l of supernatant from each hybridoma clone was injected over the flow cells at a flow rate of 30 ~1/minute.
Dissociation was allowed for 6 minutes. The chip was then regenerated with a consecutive injection of 50 ~1 each of 10 mM glycine, pH 2.0 and 10 mM
glycine, pH 2.5.
Dissociation rate constants were obtained using a separate kassoc/kdissoc f tting model with BIAevaluation 3.2 software provided by the manufacturer. The fitting model assumed 1:l binding.
I~;SSO~ calculated for each of the selected antibodies was plotted according to ECLA responses and ELISA responses described in Example 1 (Figure 2).
Antibodies in area II (ECLA-/ELISA+) were found to have a Kd;sso~ in the range of 10-3-10-5 1/sec (Table 1 and Figure 3). Antibodies in area III (ECLA~/ELISA+) were found to have a I~;SSO~ of 10-4 or less (Table 1 and Figure 3). Antibodies in area IV
(ECLA~/ELISA-) were found to have a Kd;sso~ in the range of 10-2-10-5 (Table 1 and Figure 3). Antibodies with a I~;SSO~ of 10-a were only found in area IV. The antibodies in area I (ECLA-/ELISA-) p~'esumably did not specifically bind 2H7.
Table 1 ECLA ELISA
Sample response response Positivekd;sso~Heavy ChainLight Number (ECLU) (0.D. in (1/s)a Iso a Chain at 650 nm) 1 269 0.653 Both 8.46E-04I G1/I G2b**ka a 2 277 0.572 Both * I G1 ka a 34 220 0.373 None 6.64E-03*
93 252 0.354 ECLA *
114 3695 1.175 Both 3.39E-05I G1 ka a 141 293 0.346 ECLA * I Gl ka a 205 197 0.776 ELISA 2.31E-04I G1 ka a 389 288 0.430 ECLA * I Gl ka a 421 201 0.582 ELISA * * *
425 199 0.534 ELISA 4.54E-04I G1 ka a 429 263 0.296 ECLA 0.030 * *
452 262 0.340 ECLA 2.19E-04 471 213 0.629 ELISA 8.99E-04I G1 lambda 492 214 0.584 ELISA 2.46E-03I G2b ka a 517 432 0.469 ECLA *
567 269 0.281 ECLA * * *
574 299 0.350 ECLA 1.37E-03*
634 296 0.359 ECLA * * *
664 205 0.561 ELISA 1.74E-03* *
705 260 0.384 ECLA * * *
729 206 0.689 ELISA 9.84E-04I G1 lambda 731 202 0.679 ELISA S.11E-03* *
740 261 0.273 ECLA 4.20E-05I G1 ka a 750 252 0.322 ECLA 1.37E-03* *
765 263 0.377 ECLA * I G3 ka a 770 253 0.344 ECLA * I G2a ka a 786 302 0.394 ECLA * * *
807 364 0.308 ECLA 1.01E-02* *
824 281 0.348 ECLA 1.93E-03*
876 290 0.300 ECLA 1.52E-02I G3 ka a 886 313 0.294 ECLA 1.33E-02* *
888 277 0.417 ECLA 1.64E-02* *
902 256 0.299 ECLA * *
911 267 0.304 ECLA * I G2b ka a 919 255 0.324 ECLA 0.017 * *
939 290 0.3I0 ECLA 7.91E-04IIgGl I *
a: measured by Biacore analysis *: not measurable due to a low concentration and/or exceeding assay limitation **: sample contained two different heavy chain isotypes, IgGl and IgG2b.
Antibodies that demonstrated a I~;SSO~ greater than about 10-5 were identified as low affinity antibodies. As shown in Table 1 and Figure 3, all but one of the identified low affinity antibodies were ECLA+/ELISA- or ECLA-/ELISA+. One antibody in area I (ECLA-/ELISA') was found to have a I~;SS°~ of 10-3, suggesting the detection limit for ECLA may have been set too high. Lowering the ECLA
detection limit may have identified additional low affinity anti-2H7 antibodies. One antibody in area IV was found to have a K.~;SS°~ of 10-5, suggesting the concentration of anti-2H7 antibody in the hybridoma supernatant producing this antibody was low.
Isotyping of Low Affinity Anti-2H7 Antibodies Isotypes were determined for antibodies produced by the hybridoma clones selected for further characterization in Example 2. An ELISA based antibody isotyping assay was performed Briefly, a polypropylene 96-well rnicrotiter plate was coated with 50 ~,l of isotype specific goat anti-mouse Ig (Southern Biotech, Pittsburgh, PA) and incubated overnight at 4°C.
The plate was washed with wash buffer (PBS with 0.05% Tween-20) and blocked with 200 ~1 of 2% BSA in PBS for one hour at room temperature. The plates were washed with wash buffer three times and 100 p,1 of hybridoma culture supernatant was added to the wells. The plate was incubated for 30 minutes at room temperature and washed three times. Fifty ~.1 of HRP goat anti-mouse IgG Fc specific (ICN) was added to each well and the plate was incubated for 30 minutes at room temperature. The plate was developed with HRP substrate as described for Example 1. Absorbance was measured as described for Example 1.
Heavy chain isotypes of antibodies produced by the selected hybridoma clones were plotted according to ECLA responses and ELISA responses as described for Example 1 (Figure 4). All antibodies tested showed a kappa light chain, except for two antibodies in area II (Table 1 ). These two antibodies showed a lambda light chain and are circled in Figure 4. Antibodies in area II (ECLA-/ELISA+) were found to have heavy chain isotypes of IgGl or IgG2b (Table 1 and Figure 4). Antibodies in area III (ECLA+/ELISA+) were found to have heavy chain isotypes of IgGl or IgG2b (Table 1 and Figure 4). Antibodies in area IV
(ECLA~/ELISA') were found to have heavy chain isotypes of IgG, IgG2a, IgG2b, or IgG3 (Table 1 and Figure 4).
Example 4 Screening Hybridomas for Low Affinity Anti-bevacizumab Antibodies Hybridoma supernatants were screened for production of low affinity anti-bevacizumab antibodies (Genentech Inc., South San Francisco, CA) using the ELISA/ECLA cross-screening method described in Example 1.
BALB/c mice were immunized and boosted with bevacizumab as described in Example 1. Three days after final boost, poptileal lymph nodes were fused with cells of the myeloma cell line, P3X63Ag.U.1 (ATCC, Manassas, VA). Fused cells were selected by hypoxanthin-aminopterin-thymidine (HAT) medium selection.
Supernatants from hybridoma cultures were screened for low affinity anti-bevacizumab antibodies by plotting ECLA responses versus ELISA responses.
ECLA screening and ELISA screening was performed as described in Example 1.
An enriched pool of candidate low affinity anti-bevacizumab antibodies was generated by plotting ECLA responses verses ELISA responses as described in Example 1. The detection limit for ELISA was set at O.D. 0.5. The detection limit for ECLA was set at 300 ECLU. As described in Example 1, lines depicting detection limits form the boundaries of four quadrants: area I (ELISA'/
ECLA'), area II (ELISA+/ ECLA'), area III (ELISA+/ ECLA+), and area IV (ELISA'/ ECLA+).
See, for example, Figure 1. Antibodies in area IV (ELISA'/ ECLA+) represent a population of candidate low affinity anti-bevacizumab monoclonal antibodies not detected by ELISA. Antibodies from two hybridoma clones (4B9 and 8F6) were identified as ELISA'/ ECLA+ (Table 2). These antibodies are candidate low affinity anti-bevacizumab monoclonal antibodies.
Table 2 Clone ELISA O.D. ECLA ECL
4B9 0.459 ELISA' 14101 ELCA+
4D7 0.967 ELISA''~ 22815 ELCA+
SE1. 0.987 ELISA+ 13334 ELCA+
6C2 1.005 ELISA+ 25270 ELCA+
6F11 0.930 ELISA+ 1834 ELCA+
8F6 0.474 ELISA' 3094 ELCA+
Specific binding affinities of candidate low affinity antibodies in area IV
were determined. Dissociation rate constants (I~;SSO~) for the antibodies were determined by Biacore analysis as described in Example 2. A fast off rate (Kd;sso~) typically correlates with low binding affinity. Antibodies that demonstrated a Kd;sso~
greater than about 10-S were identified as low affinity anti-bevacizumab antibodies.
Clones 4B9 and 8F6 were identified as producing low affinity anti-bevacizumab antibodies (Table 3).
While area IV (ELISA-/ ECLA+) is enriched in low affinity antibodies, it may also contain high affinity antibodies present in low concentration in the supernatant. To confirm identification of hybridoma clones producing low affinity antibodies, the concentration of monoclonal antibodies in the supernatant from the hybridoma clones was determined by Biacore analysis. Known concentrations of purified cynomologus monkey anti-bevacizumab polyclonal antibody were analyzed by Biacore as described in Example 2. A standard curve was generated by plotting the binding of the polyclonal antibody to bevacizumab in Biacore versus polyclonal antibody concentration and calculating the slope of the curve (Figure 5).
The concentration of monoclonal antibodies in supernatant from the individual hybridoma clones was calculated using the standard curve and this concentration was used to calculate dissociation rates constants (Kd;sso~), association rate constants (Kasso~), and equilibrium dissociation constants (KD) (Table 3). The equilibrium constants and rate constants were obtained using BIAevaluation 3.2 software provided by the manufacturer. Dissociation rate constants were obtained using a Kasso~ /Kd;sso~ fitting model with the BIAevaluation 3.2 software. The fitting model assumed 1:1 binding. Antibodies that demonstrated a KD equal to or greater than about 10-$ M were confirmed as low affinity anti-bevacizumab antibodies.
Clones 4B9 and 8F6 were confirmed as producing low affinity anti-bevacizumab antibodies (Table 3). .
Table 3 Estimated concentration in lone asso~ 1/Ms d;sso~ D the 1/s su ernatant* nM
4B9 6.40E+02 3.47E-05 5.42E-08 27.3 4D7 3.30E+05 8.32E-05 2.52E-10 98.1 5E1 2.96E+05 5.85E-05 1.98E-10 108.8 6C2 3.08E+05 8.88E-06 2.88E-11 59.8 6F11 8.04E+04 1.04E-04 1.29E-09 19.9 8F6 5.87E+03 3.65E-04 6.22E-08 4 * Concentrations were estimated using a standard curve generated with purified cynomologus monkey anti-bevacizumab polyclonal antibodies with Biacore (see Figure 5).
Claims (38)
1, A method of enriching a pool of analyte molecules with candidate analyte molecules that selectively bind a target molecule, comprising:
(a) determining ECLA responses for individual members of a pool of analyte molecules binding a target molecule;
(b) determining IA responses for individual members of the pool of analyte molecules binding the target molecule; and (c) generating a pool of candidate analyte molecules comprising:
i) IA-/ECLA+, and enriched for low affinity analyte molecules;
ii) IA+/ECLA+ or IA+/ECLA-, and enriched for high affinity analyte molecules; or iii) IA+/ECLA-, and enriched for analyte molecules that bind the target molecule at a binding site not recognized by ECLA.
(a) determining ECLA responses for individual members of a pool of analyte molecules binding a target molecule;
(b) determining IA responses for individual members of the pool of analyte molecules binding the target molecule; and (c) generating a pool of candidate analyte molecules comprising:
i) IA-/ECLA+, and enriched for low affinity analyte molecules;
ii) IA+/ECLA+ or IA+/ECLA-, and enriched for high affinity analyte molecules; or iii) IA+/ECLA-, and enriched for analyte molecules that bind the target molecule at a binding site not recognized by ECLA.
2. The method of claim 1, wherein the pool of candidate analyze molecules is IA-/ECLA+, and enriched for low affinity analyte molecules.
3. The method of claim 2, wherein the pool of candidate analyte molecule is IA+/ECLA+ or IA+/ECLA-, and enriched for high amity analyte molecules.
4. The method of claim 2, wherein the pool of candidate analyte molecules is IA+/ECLA-, and enriched for analyze molecules that bind the target molecule at a binding site not recognized by ECLA.
5. A method of identifying candidate low affinity analyte molecules from a pool of analyte molecules, comprising:
(a) determining ECLA responses for individual members of the pool of analyte molecules binding a target molecule; and (b) determining IA responses for individual members of the pool of analyte molecules binding the target molecule;
wherein analyte molecules that are IA-/ECLA+ are identified as candidate low affinity molecules.
(a) determining ECLA responses for individual members of the pool of analyte molecules binding a target molecule; and (b) determining IA responses for individual members of the pool of analyte molecules binding the target molecule;
wherein analyte molecules that are IA-/ECLA+ are identified as candidate low affinity molecules.
6. The method of any one of claims 1-5, further comprising:
(a) applying a detection limit to the analysis of the ECLA response, wherein an ECLA response equal to or greater than the ECLA detection limit identifies an ECLA+ analyte molecule and an ECLA response less than the ECLA
detection identifies an ECLA- analyte molecule; and (b) applying a detection limit to the analysis of the ELISA response, wherein an ELISA response equal to or greater than the ELISA detection limit is ELISA+ and an ELISA response less than the ELISA detection limit is ELISA-.
(a) applying a detection limit to the analysis of the ECLA response, wherein an ECLA response equal to or greater than the ECLA detection limit identifies an ECLA+ analyte molecule and an ECLA response less than the ECLA
detection identifies an ECLA- analyte molecule; and (b) applying a detection limit to the analysis of the ELISA response, wherein an ELISA response equal to or greater than the ELISA detection limit is ELISA+ and an ELISA response less than the ELISA detection limit is ELISA-.
7. The method of claim 6, wherein the ELISA detection limit is 0.5 O.D. at 650 nm.
8. The method of claim 6, wherein the detection limit for the ECLA response is 250 ECLU.
9. The method of any one of claims 1-8, further comprising confirming specific binding affinity of an analyte molecule selected from the enriched pool of candidate analyte molecules.
10. The method of claim 9, wherein a K dissoc of about 10 -6 1/sec or less identifies a high affinity analyte molecule.
11. The method of claim 9, wherein a K dissoc greater than about 10 -6 1/sec identifies a low affinity analyte molecule.
12, The method of claim 9, wherein a K dissoc greater or equal to about 10 -5 1/sec identifies a low affinity analyte molecule.
13. The method of claim 9, wherein a K dissoc greater or equal to about 10 -3 1/sec identifies a low affinity analyze molecule.
14. The method of claim 9, wherein a K D equal to or greater than about 10 -8 M
identifies a low affinity analyte molecule.
identifies a low affinity analyte molecule.
15. The method of claim 9, wherein a K D of about 10 -6 M to about 10 -8 M
identifies a low affinity analyte molecule.
identifies a low affinity analyte molecule.
16. The method of any one of claims 1-15, wherein the analyte molecules are antibodies or antigen binding portions thereof.
17, The method of claim 16, wherein the antibodies are anti-therapeutic antibodies.
18. The method of any one of claims 1-17, wherein the target molecule is an antigen.
19. The method of claim 18, wherein the antigen is an antibody or antigen binding portion thereof.
20. The method of any one of claims 1-17, wherein the target molecule is an antibody or antigen binding fragment thereof.
21. The method of claim 19 or 20, wherein the antibody is a therapeutic antibody.
22. The method of claim 21, wherein the antibody binds CD20.
23. The method of claim 21, wherein the antibody binds VEGF.
24. The method of any one of claims 1-23, wherein the analyte or target antibodies arc monoclonal.
25. The method of any one of claims 1-24, further comprising isotyping the analyte antibodies.
26. The method of any one of claims 1-25, wherein the analyte antibodies are IgG.
27, An antibody having a K dissoc in the range of 10 -2 to 10 -6 selected by the method of any one of claims 1-26, wherein the target molecule is an anti-VEGF
antibody, anti-HER2 antibody, anti-CD20 antibody, anti-IgE antibody, anti-CD11a antibody, or antigen binding fragment thereof.
antibody, anti-HER2 antibody, anti-CD20 antibody, anti-IgE antibody, anti-CD11a antibody, or antigen binding fragment thereof.
28. An antibody having a K D in the range of 10 -6 M to 10 -8 M selected by the method of any one of claims 1-26, wherein the target molecule is an anti-VEGF
antibody, anti-HER2 antibody, anti-CD20 antibody, anti-IgE antibody, anti-CD11a antibody, or antigen binding fragment thereof.
antibody, anti-HER2 antibody, anti-CD20 antibody, anti-IgE antibody, anti-CD11a antibody, or antigen binding fragment thereof.
29. The antibody of claim 27 or 28, wherein the target molecule is 2H7 or bevacizumab.
30. Use of one or more antibody having a K dissoc in the range of 10 -2 to 10 -6 for a target molecule, the one or more antibody selected by the method of any one of claims 1-26, in an assay for detecting an immune response to the target molecule.
31. Use of one or more antibody having a K D in the range of 10 -6 M to 10 -8 M for a target molecule, the one or more antibody selected by the method of any one of claims 1-26, in an assay for detecting an immune response to the target molecule.
32. The use according to claim 30 or 31, wherein the target molecule is an anti-VEGF antibody, anti-HER2 antibody, anti-CD20 antibody, anti-IgE antibody, anti-CD11a antibody, or antigen binding fragment thereof.
33. The use according to claim 30, wherein the target molecule is 2H7 or bevacizumab,
34. The use according to claim 31, wherein the target molecule is bevacizumab.
35. A method for producing a high affinity antibody to a target molecule, comprising subjecting a low affinity antibody selected by the method of any one of claims 1-26 to affinity maturation, thereby producing an affinity-matured antibody having high affinity for the target molecule.
36. The method of claim 35, wherein the low affinity antibody has a K dissoc in the range of 10 -2 to 10 -6.
37. The method of claim 35, wherein the low affinity antibody has a K D in the range of 10 -6 M to 10 -8 M.
38. The method of any one of claims 35-37, wherein the high affinity antibody has a K dissoc of about 10 -6 1/sec or less.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US57115704P | 2004-05-15 | 2004-05-15 | |
US60/571,157 | 2004-05-15 | ||
US62682704P | 2004-11-09 | 2004-11-09 | |
US60/626,827 | 2004-11-09 | ||
PCT/US2005/016770 WO2005114218A2 (en) | 2004-05-15 | 2005-05-13 | Cross-screening system and methods for detecting a molecule having binding affinity for a target molecule |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2562800A1 true CA2562800A1 (en) | 2005-12-01 |
Family
ID=35311822
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002562800A Abandoned CA2562800A1 (en) | 2004-05-15 | 2005-05-13 | Cross-screening system and methods for detecting a molecule having binding affinity for a target molecule |
Country Status (6)
Country | Link |
---|---|
US (1) | US7514223B2 (en) |
EP (1) | EP1769243A2 (en) |
JP (1) | JP2007538258A (en) |
AU (1) | AU2005246289A1 (en) |
CA (1) | CA2562800A1 (en) |
WO (1) | WO2005114218A2 (en) |
Families Citing this family (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1637160A3 (en) | 1999-05-07 | 2006-05-03 | Genentech, Inc. | Treatment of autoimmune diseases with antagonists which bind to B cell surface markers |
US8858434B2 (en) | 2004-07-13 | 2014-10-14 | Dexcom, Inc. | Transcutaneous analyte sensor |
US7920906B2 (en) | 2005-03-10 | 2011-04-05 | Dexcom, Inc. | System and methods for processing analyte sensor data for sensor calibration |
PL1692182T3 (en) * | 2003-11-05 | 2010-09-30 | Roche Glycart Ag | Cd20 antibodies with increased fc receptor binding affinity and effector function |
US9247900B2 (en) | 2004-07-13 | 2016-02-02 | Dexcom, Inc. | Analyte sensor |
AU2005246289A1 (en) | 2004-05-15 | 2005-12-01 | Genentech, Inc. | Cross-screening system and methods for detecting a molecule having binding affinity for a target molecule |
US20060270922A1 (en) | 2004-07-13 | 2006-11-30 | Brauker James H | Analyte sensor |
US20100041063A1 (en) | 2006-09-12 | 2010-02-18 | Ulrich Essig | Anti-drug antibody assay |
TW201014605A (en) | 2008-09-16 | 2010-04-16 | Genentech Inc | Methods for treating progressive multiple sclerosis |
WO2010075249A2 (en) | 2008-12-22 | 2010-07-01 | Genentech, Inc. | A method for treating rheumatoid arthritis with b-cell antagonists |
AR078161A1 (en) | 2009-09-11 | 2011-10-19 | Hoffmann La Roche | VERY CONCENTRATED PHARMACEUTICAL FORMULATIONS OF AN ANTIBODY ANTI CD20. USE OF THE FORMULATION. TREATMENT METHOD |
WO2011065912A1 (en) * | 2009-11-30 | 2011-06-03 | Ge Healthcare Bio-Sciences Ab | Method and system for interaction analysis |
CN102667447B (en) * | 2009-11-30 | 2016-03-23 | 通用电气健康护理生物科学股份公司 | For the method and system that bonding behavior is analyzed |
JP5841072B2 (en) | 2010-02-10 | 2016-01-06 | イミュノジェン・インコーポレーテッド | CD20 antibody and use thereof |
US8956859B1 (en) | 2010-08-13 | 2015-02-17 | Aviex Technologies Llc | Compositions and methods for determining successful immunization by one or more vaccines |
US9435743B2 (en) * | 2012-05-07 | 2016-09-06 | Alexey Gennadievich Zdanovsky | Methods using a modified bacteriophage for the detection of target molecules |
JP6389868B2 (en) | 2013-03-15 | 2018-09-12 | ジーピービー デット ホールディングス トゥー エルエルシー | Apparatus and related methods for performing luminescence and fluorescence measurements of samples |
Family Cites Families (51)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
SE337223B (en) * | 1967-05-23 | 1971-08-02 | Pharmacia Ab | |
US3720760A (en) * | 1968-09-06 | 1973-03-13 | Pharmacia Ab | Method for determining the presence of reagin-immunoglobulins(reagin-ig)directed against certain allergens,in aqueous samples |
US3654090A (en) * | 1968-09-24 | 1972-04-04 | Organon | Method for the determination of antigens and antibodies |
US3691016A (en) * | 1970-04-17 | 1972-09-12 | Monsanto Co | Process for the preparation of insoluble enzymes |
US3940475A (en) * | 1970-06-11 | 1976-02-24 | Biological Developments, Inc. | Radioimmune method of assaying quantitatively for a hapten |
CA1023287A (en) * | 1972-12-08 | 1977-12-27 | Boehringer Mannheim G.M.B.H. | Process for the preparation of carrier-bound proteins |
US4195128A (en) * | 1976-05-03 | 1980-03-25 | Bayer Aktiengesellschaft | Polymeric carrier bound ligands |
IL49685A (en) * | 1976-05-31 | 1978-10-31 | Technion Res & Dev Foundation | Specific binding assay method for determining the concentration of materials and reagent means therefor |
US4330440A (en) * | 1977-02-08 | 1982-05-18 | Development Finance Corporation Of New Zealand | Activated matrix and method of activation |
CA1093991A (en) * | 1977-02-17 | 1981-01-20 | Hideo Hirohara | Enzyme immobilization with pullulan gel |
US4115535A (en) * | 1977-06-22 | 1978-09-19 | General Electric Company | Diagnostic method employing a mixture of normally separable protein-coated particles |
US4229537A (en) * | 1978-02-09 | 1980-10-21 | New York University | Preparation of trichloro-s-triazine activated supports for coupling ligands |
US4277437A (en) * | 1978-04-05 | 1981-07-07 | Syva Company | Kit for carrying out chemically induced fluorescence immunoassay |
US4238195A (en) * | 1979-01-18 | 1980-12-09 | Miles Laboratories, Inc. | Fluorescer-labeled specific binding assays |
DE2918342A1 (en) * | 1979-05-07 | 1980-11-20 | Behringwerke Ag | LATEX REAGENT |
US4280815A (en) * | 1979-06-18 | 1981-07-28 | Technicon Instruments Corporation | Electrochemiluminescent immunoassay and apparatus therefor |
US4378344A (en) * | 1979-09-28 | 1983-03-29 | Ventrex Laboratories, Inc. | Method and apparatus for performing multiple, simultaneous in vitro diagnostic tests using a solid phase system |
US4293310A (en) * | 1980-03-14 | 1981-10-06 | University Of Pittsburgh | Photoelectrochemical immunoassay |
US4376110A (en) * | 1980-08-04 | 1983-03-08 | Hybritech, Incorporated | Immunometric assays using monoclonal antibodies |
US4711955A (en) * | 1981-04-17 | 1987-12-08 | Yale University | Modified nucleotides and methods of preparing and using same |
US4419453A (en) * | 1981-09-28 | 1983-12-06 | The Dow Chemical Company | Immunological agglutination assays with dyed or colored latex and kits |
US4459360A (en) * | 1981-10-05 | 1984-07-10 | Mast Medical Industries, Ltd. | Multiple-component binding assay system and method of making and using it |
US4480042A (en) * | 1981-10-28 | 1984-10-30 | E. I. Du Pont De Nemours And Company | Covalently bonded high refractive index particle reagents and their use in light scattering immunoassays |
US4514508A (en) * | 1982-07-06 | 1985-04-30 | Biond Inc. | Assaying for a multiplicity of antigens or antibodies with a detection compound |
US4816567A (en) | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
US4628037A (en) * | 1983-05-12 | 1986-12-09 | Advanced Magnetics, Inc. | Binding assays employing magnetic particles |
US4695393A (en) * | 1983-05-12 | 1987-09-22 | Advanced Magnetics Inc. | Magnetic particles for use in separations |
US4698302A (en) * | 1983-05-12 | 1987-10-06 | Advanced Magnetics, Inc. | Enzymatic reactions using magnetic particles |
US4554088A (en) * | 1983-05-12 | 1985-11-19 | Advanced Magnetics Inc. | Magnetic particles for use in separations |
US4687732A (en) * | 1983-06-10 | 1987-08-18 | Yale University | Visualization polymers and their application to diagnostic medicine |
FI842992A0 (en) * | 1984-07-26 | 1984-07-26 | Labsystems Oy | IMMUNOLOGISKT DEFINITIONSFOERFARANDE. |
DK365785A (en) * | 1984-09-17 | 1986-03-18 | Hoffmann La Roche | metal complex |
FI844027A (en) * | 1984-10-12 | 1986-04-13 | Labsystems Oy | IMMUNOLOGISKT BESTAEMNINGSFOERFARANDE. |
US5221605A (en) * | 1984-10-31 | 1993-06-22 | Igen, Inc. | Luminescent metal chelate labels and means for detection |
US5310687A (en) * | 1984-10-31 | 1994-05-10 | Igen, Inc. | Luminescent metal chelate labels and means for detection |
US5238808A (en) * | 1984-10-31 | 1993-08-24 | Igen, Inc. | Luminescent metal chelate labels and means for detection |
US4737456A (en) * | 1985-05-09 | 1988-04-12 | Syntex (U.S.A.) Inc. | Reducing interference in ligand-receptor binding assays |
US6451225B1 (en) * | 1986-04-30 | 2002-09-17 | Igen International, Inc. | Electrochemiluminescent reaction utilizing amine-derived reductant |
US6271041B1 (en) * | 1986-04-30 | 2001-08-07 | Igen International, Inc. | Electrochemiluminescent reaction utilizing amine-derived reductant |
US5591581A (en) * | 1986-04-30 | 1997-01-07 | Igen, Inc. | Electrochemiluminescent rhenium moieties and methods for their use |
US6316607B1 (en) * | 1986-04-30 | 2001-11-13 | Igen International, Inc. | Electrochemiluminescent assays |
US4965392A (en) * | 1987-03-26 | 1990-10-23 | Neorx Corporation | Chelating compounds for metal-radionuclide labeled proteins |
US5935779A (en) * | 1988-11-03 | 1999-08-10 | Igen International Inc. | Methods for improved particle electrochemiluminescence assay |
DE3920358A1 (en) | 1989-06-22 | 1991-01-17 | Behringwerke Ag | BISPECIFIC AND OLIGO-SPECIFIC, MONO- AND OLIGOVALENT ANTI-BODY CONSTRUCTS, THEIR PRODUCTION AND USE |
WO1994004679A1 (en) * | 1991-06-14 | 1994-03-03 | Genentech, Inc. | Method for making humanized antibodies |
EP0617706B1 (en) | 1991-11-25 | 2001-10-17 | Enzon, Inc. | Multivalent antigen-binding proteins |
US5466416A (en) * | 1993-05-14 | 1995-11-14 | Ghaed; Ali | Apparatus and methods for carrying out electrochemiluminescence test measurements |
US5527710A (en) * | 1994-12-02 | 1996-06-18 | Igen, Inc. | Rate measurements of biomolecular reactions using electrochemiluminescence |
SE9504046D0 (en) * | 1995-11-14 | 1995-11-14 | Pharmacia Ab | Method of determining affinity and kinetic properties |
ATE315789T1 (en) * | 1999-11-16 | 2006-02-15 | Genentech Inc | ELISA FOR VEGF |
AU2005246289A1 (en) | 2004-05-15 | 2005-12-01 | Genentech, Inc. | Cross-screening system and methods for detecting a molecule having binding affinity for a target molecule |
-
2005
- 2005-05-13 AU AU2005246289A patent/AU2005246289A1/en not_active Abandoned
- 2005-05-13 WO PCT/US2005/016770 patent/WO2005114218A2/en active Application Filing
- 2005-05-13 CA CA002562800A patent/CA2562800A1/en not_active Abandoned
- 2005-05-13 JP JP2007527314A patent/JP2007538258A/en active Pending
- 2005-05-13 US US11/128,981 patent/US7514223B2/en active Active
- 2005-05-13 EP EP05779248A patent/EP1769243A2/en not_active Withdrawn
Also Published As
Publication number | Publication date |
---|---|
WO2005114218A3 (en) | 2006-02-16 |
AU2005246289A1 (en) | 2005-12-01 |
JP2007538258A (en) | 2007-12-27 |
US7514223B2 (en) | 2009-04-07 |
EP1769243A2 (en) | 2007-04-04 |
US20050255527A1 (en) | 2005-11-17 |
WO2005114218A2 (en) | 2005-12-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7514223B2 (en) | Cross-screening system and methods for detecting a molecule having binding affinity for a target molecule | |
US20060172357A1 (en) | Detecting human antibodies in non-human serum | |
CA2662416C (en) | Anti-drug antibody assay | |
US20110229908A1 (en) | Methods and products for measuring free immunoglobulin light chain molecules | |
US20110020840A1 (en) | Method and kits for detecting antibodies against therapeutic antibodies | |
Karlsson et al. | Comparison of surface plasmon resonance binding curves for characterization of protein interactions and analysis of screening data | |
US20200018770A1 (en) | Methods for mitigating drug target interference in an anti-drug antibody (ada) immunoassay | |
TW200829601A (en) | Conjugate and its use as a standard in an immunoassay | |
Nahshol et al. | Parallel kinetic analysis and affinity determination of hundreds of monoclonal antibodies using the ProteOn XPR36 | |
US20070161089A1 (en) | Method of Producing Pan-Specific Antibodies | |
Na et al. | Colloidal gold-based immunochromatographic strip assay for the rapid detection of bacitracin zinc in milk | |
CN113785203A (en) | Improved competitive ligand binding assays | |
Kuhne et al. | Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA | |
Miller et al. | Epitope binning of murine monoclonal antibodies by a multiplexed pairing assay | |
KR102653734B1 (en) | Antibodies and testing devices for detecting Campylobacter genus bacteria | |
Clark et al. | Biomarkers for non-human primate type-I hypersensitivity: antigen-specific immunoglobulin E assays | |
EP4157870A2 (en) | Rabbit antibodies to human immunoglobulins g | |
CN107505459A (en) | Quantitatively detect people H FABP time-resolved fluoroimmunoassay chromatograph test strip, kit and preparation method thereof | |
EP1200826B1 (en) | Internally referenced immunoassay and test device | |
US20230393125A1 (en) | Method for determining the free antigen of an antibody in a sample | |
Gronski et al. | Indications of neutralising anti-idiotypic antibodies and selective proteolytic fragmentation in polyclonal anti-D IgG preparations | |
JPH01223349A (en) | Reagent for judging anti d blood type | |
JP2003344411A (en) | Immobilization method of receptor, and receptor composition manufactured by the method |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FZDE | Dead |