WO2013115425A1 - Procédés de criblage d'un anticorps par une technique de panning sur un seul cycle - Google Patents
Procédés de criblage d'un anticorps par une technique de panning sur un seul cycle Download PDFInfo
- Publication number
- WO2013115425A1 WO2013115425A1 PCT/KR2012/000934 KR2012000934W WO2013115425A1 WO 2013115425 A1 WO2013115425 A1 WO 2013115425A1 KR 2012000934 W KR2012000934 W KR 2012000934W WO 2013115425 A1 WO2013115425 A1 WO 2013115425A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- polypeptide
- antibody
- cells
- group
- library
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 55
- 238000012216 screening Methods 0.000 title claims abstract description 10
- 238000004091 panning Methods 0.000 title description 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 48
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 47
- 239000000427 antigen Substances 0.000 claims abstract description 46
- 102000036639 antigens Human genes 0.000 claims abstract description 46
- 108091007433 antigens Proteins 0.000 claims abstract description 46
- 229920001184 polypeptide Polymers 0.000 claims abstract description 46
- 230000027455 binding Effects 0.000 claims abstract description 44
- 238000004519 manufacturing process Methods 0.000 claims abstract description 5
- 108020004414 DNA Proteins 0.000 claims description 54
- 210000004027 cell Anatomy 0.000 claims description 51
- 108090000623 proteins and genes Proteins 0.000 claims description 49
- 102000053602 DNA Human genes 0.000 claims description 40
- 102000004169 proteins and genes Human genes 0.000 claims description 31
- 241000894006 Bacteria Species 0.000 claims description 23
- 239000000523 sample Substances 0.000 claims description 20
- 238000012360 testing method Methods 0.000 claims description 16
- 238000012350 deep sequencing Methods 0.000 claims description 14
- 239000012634 fragment Substances 0.000 claims description 14
- 239000002773 nucleotide Substances 0.000 claims description 11
- 125000003729 nucleotide group Chemical group 0.000 claims description 11
- 238000012163 sequencing technique Methods 0.000 claims description 11
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 10
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 10
- 239000002245 particle Substances 0.000 claims description 8
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 108010003723 Single-Domain Antibodies Proteins 0.000 claims description 6
- 210000005253 yeast cell Anatomy 0.000 claims description 6
- 230000001131 transforming effect Effects 0.000 claims description 5
- 102000004889 Interleukin-6 Human genes 0.000 description 33
- 108090001005 Interleukin-6 Proteins 0.000 description 33
- 241000588724 Escherichia coli Species 0.000 description 30
- 101100112922 Candida albicans CDR3 gene Proteins 0.000 description 17
- 108010079246 OMPA outer membrane proteins Proteins 0.000 description 15
- 102100026018 Interleukin-1 receptor antagonist protein Human genes 0.000 description 14
- 101710144554 Interleukin-1 receptor antagonist protein Proteins 0.000 description 14
- 238000002965 ELISA Methods 0.000 description 13
- 150000001413 amino acids Chemical class 0.000 description 12
- 238000009396 hybridization Methods 0.000 description 12
- 238000011084 recovery Methods 0.000 description 11
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 10
- 229960003669 carbenicillin Drugs 0.000 description 10
- 150000007523 nucleic acids Chemical class 0.000 description 10
- 239000013598 vector Substances 0.000 description 10
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 9
- 229920001213 Polysorbate 20 Polymers 0.000 description 8
- 239000011324 bead Substances 0.000 description 8
- 102000039446 nucleic acids Human genes 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 8
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 8
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 230000009466 transformation Effects 0.000 description 8
- 238000000684 flow cytometry Methods 0.000 description 7
- 238000012175 pyrosequencing Methods 0.000 description 7
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 7
- 229960000268 spectinomycin Drugs 0.000 description 7
- 102000004127 Cytokines Human genes 0.000 description 6
- 108090000695 Cytokines Proteins 0.000 description 6
- 108010090804 Streptavidin Proteins 0.000 description 6
- 208000037065 Subacute sclerosing leukoencephalitis Diseases 0.000 description 6
- 206010042297 Subacute sclerosing panencephalitis Diseases 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 102000040430 polynucleotide Human genes 0.000 description 6
- 108091033319 polynucleotide Proteins 0.000 description 6
- 239000002157 polynucleotide Substances 0.000 description 6
- 108010004729 Phycoerythrin Proteins 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 238000013459 approach Methods 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000007480 sanger sequencing Methods 0.000 description 5
- 102100021942 C-C motif chemokine 28 Human genes 0.000 description 4
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 101000897477 Homo sapiens C-C motif chemokine 28 Proteins 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 239000000178 monomer Substances 0.000 description 4
- ZTOJFFHGPLIVKC-YAFCTCPESA-N (2e)-3-ethyl-2-[(z)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound S\1C2=CC(S(O)(=O)=O)=CC=C2N(CC)C/1=N/N=C1/SC2=CC(S(O)(=O)=O)=CC=C2N1CC ZTOJFFHGPLIVKC-YAFCTCPESA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 101001076408 Homo sapiens Interleukin-6 Proteins 0.000 description 3
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 3
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 3
- 101800001690 Transmembrane protein gp41 Proteins 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000003766 bioinformatics method Methods 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 102000052611 human IL6 Human genes 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000002823 phage display Methods 0.000 description 3
- 238000005096 rolling process Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000004304 visual acuity Effects 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 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 2
- 239000004471 Glycine Substances 0.000 description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 2
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 2
- 101710183187 Major outer membrane lipoprotein Proteins 0.000 description 2
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 108010006785 Taq Polymerase Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000001188 anti-phage Effects 0.000 description 2
- 239000012148 binding buffer Substances 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 210000004602 germ cell Anatomy 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 238000002887 multiple sequence alignment Methods 0.000 description 2
- 230000005298 paramagnetic effect Effects 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 230000003362 replicative effect Effects 0.000 description 2
- 238000002702 ribosome display Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- PZFZLRNAOHUQPH-DJBVYZKNSA-N (2r)-3-[(2r)-2,3-di(hexadecanoyloxy)propyl]sulfanyl-2-(hexadecanoylamino)propanoic acid Chemical compound CCCCCCCCCCCCCCCC(=O)N[C@H](C(O)=O)CSC[C@H](OC(=O)CCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCC PZFZLRNAOHUQPH-DJBVYZKNSA-N 0.000 description 1
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- ZTOJFFHGPLIVKC-UHFFFAOYSA-N 3-ethyl-2-[(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound S1C2=CC(S(O)(=O)=O)=CC=C2N(CC)C1=NN=C1SC2=CC(S(O)(=O)=O)=CC=C2N1CC ZTOJFFHGPLIVKC-UHFFFAOYSA-N 0.000 description 1
- HQFLTUZKIRYQSP-UHFFFAOYSA-N 3-ethyl-2h-1,3-benzothiazole-6-sulfonic acid Chemical compound OS(=O)(=O)C1=CC=C2N(CC)CSC2=C1 HQFLTUZKIRYQSP-UHFFFAOYSA-N 0.000 description 1
- 108091093088 Amplicon Proteins 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 108020004638 Circular DNA Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 101800001707 Spacer peptide Proteins 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 102100040247 Tumor necrosis factor Human genes 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000002819 bacterial display Methods 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000006957 competitive inhibition Effects 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000002998 immunogenetic effect Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 230000005291 magnetic effect Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1036—Retroviridae, e.g. leukemia viruses
- C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
- C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
-
- 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
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/244—Interleukins [IL]
- C07K16/248—IL-6
-
- 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
- C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
Definitions
- the present invention relates to a method for screening an antibody specifically binding to a target antigen and a method for producing a polypeptide specifically binding with a target polypeptide.
- nucleic acid sequencing An important goal of modern molecular biology is to link the genotypic information that comes from nucleic acid sequencing to the phenotype of normal and diseased organisms.
- the decoded nucleic acid information is directly linked to phenotype, because, in evolutionary terms, this is an endpoint analysis, and one assumes a high degree of fitness for any deduced protein.
- the information obtained from nucleic acid sequencing already relates to a highly selected phenotype of a functioning organism.
- phage systems have the potential to link phenotype to genotype
- their analysis is based on the process of selection which, when successful, necessarily results in the loss of information. For example, if molecules that bind less tightly but are otherwise related are discarded, one loses information concerning the evolutionary trajectory toward fitness.
- nucleic acid sequencing of libraries only gives information that without selection is, in the main, unrelated to phenotype.
- binding energy comes from a concert of interactions
- distant amino acid changes that were discarded along the evolutionary trajectory in favor of those at other positions that gave superior binding may be reinstated to give additive effects.
- irrelevant factors such as growth advantages may bias the selection.
- the present inventors have made an effort to develop an highly efficient and simple method for screening an antibody, and were able to select an antibody to a preselected epitope in the presence of a large number of irrelevant proteins on the E. coli surface. Namely, one begins with a large library of different proteins expressed in phage where only a very minor fraction of the members of the library exhibit a desired phenotype such as the binding to another protein or the surface of a cell. After an initial selection, the both noise and signal are existed. Next, the information content of the phenotypic pool is established by deep sequencing of its members. The nucleic acids encoding the molecules considered to be signal molecules are recovered by hybridization and converted back to phenotype by transformation of Escherichia coli , thereby completing the phenotype-information-phenotype-cycle.
- the method of the present invention can be generalized for selection of antibodies against targets that are present as minor components of complex systems.
- Fig. 1 shows the phenotype-information-phenotype cycle.
- the cycle starts with selections from a combinatorial antibody library displayed on the surface of phage.
- the phage library is selected against two populations of bacteria that either display the antigen or serve as control cells (phage specific for antigen are in the minority and are colored grey and other phage are indicated in black).
- bound phage are eluted and their phagemids (circles) are analyzed by deep sequencing.
- the sequencing results from paired samples were compared by a bioinformatics analysis and DNA representing sequences overrepresented in phage that bind to the antigen presenting E. coli are extracted.
- a biotinylated probe whose design is based on the VH CDR3 of interest is synthesized and hybridized in solution with singlestranded circular DNA isolated from phage particles.
- the ssDNA selected by hybridization is captured on magnetic streptavidin beads and released by heating.
- the ssDNA is converted to dsDNA before transformation of suitable host E. coli cells.
- Fig. 2 shows antigen display on bacterial surfaces.
- Structural models (A, Left) the Lpp leader sequence and first nine amino acids of the E. coli major outer membrane lipoprotein (OmpA) were used to attach a variety of proteins to the E. coli surface.
- the posttranslational tripalmitoyl-S-glycerylcysteine component of the lipoprotein anchors the complex by inserting into the E. coli surface membrane.
- IL-6 was fused to the C terminus of OmpA (amino acids 46-159) and displayed on the surface of E. coli cells (A, Right).
- IL-6 was fused to the C terminus of the MBP and the fusion product was anchored to the cell wall by linking it to the C terminus of OmpA.
- the view was generated using PyMOL.
- Protein Data Bank codes are 1ALU for IL-6, 3PGF for MBP, and 1QJP for OmpA.
- the IL-6 is represented as a black ribbon diagram; MBP is rendered in magenta; and OmpA is colored light grey.
- the grey and black slabs represent the exoplasmic and cytoplasmic surface, respectively.
- B Expression of Lpp-OmpA-cytokine fusions. Cytokines were fused directly to the C terminus of OmpA.
- the FLAG tag at the C terminus of fusion protein was detected by HRP-conjugated anti-FLAG antibody (black) and compared to a control antibody of the same isotype (grey).
- C Comparison of cytokine display level in the presence or absence of the MBP.
- IL-1, chemokine (C-C motif) ligand 28 (CCL28), and gp41 antigens were displayed in duplicate, either fused directly to OmpA or fused to MBP-OmpA.
- the cytokines on the cell surface were detected by cell-ELISA with HRP conjugated to anti-FLAG antibody (blak and white) and compared to a control antibody of the same isotype (light grey and dark grey).
- Fig. 3 shows schematic illustration of the three approaches used for scFv recovery based on the VH CDR3 DNA sequences.
- A Overlap PCR. The specific scFv were recovered by overlap PCR using primers (grey and white arrows) complementary to the VH CDR3 sequence as well as to vector sequences using phagemid pools as templates. The recovered scFv was ligated into the phagemid vector. After transformation into XL1-blue cells, the antibody genes present in randomly picked colonies were characterized by Sanger sequencing.
- B Rolling circle. Complementary primers based on selected VH CDR3 sequences were annealed to denatured phagemids followed by rolling circle amplification.
- Methylated and hemimethylated template DNA was removed by digestion with DpnI, leaving only the newly synthesized molecules which were then used to transform XL1-blue cells.
- the antibody genes present in the recovered colonies were characterized by Sanger sequencing.
- C Hybridization.
- the phage single-stranded phage DNA was recovered by hybridization to a probe selected for its specificity against the scFv antibody genes of interest.
- the ssDNA recovered by an affinity step using a biotinylated probe was converted to dsDNA prior to transformation of E. coli cells.
- Fig. 4 shows selection of IL-1RA antibodies from a spiked-in library.
- Phage encoding the IL-1RA binding scFv (H9) were spiked into a ScFv naive combinatorial antibody library containing 3.0 x 10 9 members at a ratio of one H9 encoding phage to 10 9 irrelevant phage.
- Two rounds of selection were carried out using a subtractive panning format in which at each round phage were first incubated with control bacteria and those that did not bind were next selected on bacteria displaying the IL-1RA antigen.
- the PCR-amplified VH repertoires from phage that bound to the paired samples at each round were sequenced using Roche 454 pyrosequencing.
- VH CDR3s were identified and the frequency of each unique VH CDR3 was determined for the paired samples from the same round of selection (control versus experimental) to obtain a ratio of frequencies.
- H1 selective appearance of another clone (H1).
- All CDR3s that had greater than 10 reads in at least one sample (ca. 0.3%) were ordered by similarity.
- the phylogenetic tree was constructed based on multiple sequence alignments via MAFFT and ClustalW2 tree generation methods (Left). The log2-fold change in frequency of the first round (black) and the second round (grey), and Z score of the first round (white) and the second round (light grey) are shown (Right).
- Fig. 5 shows epitope-directed antibody discovery.
- scFv displayed on phage were added to plates coated either with full-length or truncated human IL-6 (hIL-6) or BSA.
- the truncated IL-6 was prepared from HEK293F cells transfected with the truncated IL-6 overexpression vector and was purified on an anti-FLAGtag column. After 1 h incubation at 37 °C, the wells were washed five times with PBS, after which HRP-conjugated antiphage antibody was added. Incubation was for 1 h at 37 °C.
- the present invention relates to a one-cycle method for screening an antibody specifically binding to a target antigen, comprising following steps: (a) providing an antibody library of different proteins displayed on the surface of phages; (b) contacting the antibody library with (i) a group of cells which displays the target antigen (test group) and (ii) another group of cells which does not displays target antigen or displays less amounts of target antigen (control group); (c) removing the unbound phages and sequencing nucleotide of the bound phages; (d) comparing nucleotide sequences of the test group and control group; and (e) selecting nucleotide sequences the test group which are not common with the control group and identifying these sequences as sequences encoding the antibody specifically binding to the target antigen.
- the method for screening an antibody specificially binding with a target antigen may further comprise (f) extracting single-stranded DNA from phage particles and hybridizing to a tagged probe; and (g) converting the single-stranded DNA to double stranded DNA.
- the step (c) may be accomplished by deep sequencing.
- deep sequencing may refer to the "depth of coverage”, meaning how many times a single base is read during the sequencing process. Deep sequencing implies that you are sequencing to a depth that allows each base to be read hundreds of times. This allows identification of very rare sequence variants (mutations). Namely, deep sequencing may refer to the coverage, or depth, of the process that is many times larger than the length of the sequence under study.
- the deep sequencing method may be pyrosequencing but it is not limited thereto.
- the cells may be selected from the group consisting of bacteria, yeast and eukaryotic cells but it is not limited thereto.
- the antibody library may comprise single chain variable region fragments (scFvs) or single domain antibodies (dAbs) but it is not limited thereto.
- scFvs single chain variable region fragments
- dAbs single domain antibodies
- the antibody library may comprise a library of human antibodies but it is not limited thereto.
- the target antigen of step (b) may be an arfificial polypeptide or natural polypeptide but it is not limited thereto.
- antibody refers to a polypeptide chain(s) which exhibits a strong monovalent, bivalent or polyvalent binding to a given antigen, epitope or epitopes.
- Antibodies used in the invention can have sequences derived from any vertebrate, camelid, avian or pisces species. They can be generated using any suitable technology, e.g., hybridoma technology, ribosome display, phage display, gene shuffling libraries, semi-synthetic or fully synthetic libraries or combinations thereof.
- the term “antibody” as used in the present invention includes intact antibodies, antigen-binding polypeptide fragments and other designer antibodies that are described below or well known in the art (see, e.g., Serafini. J Nucl. Med. 34:533-6,1993).
- Antibodies to be used in the invention also include antibody fragments or antigen-binding fragments which contain the antigen-binding portions of an intact antibody that retain capacity to bind the cognate antigen.
- antibody fragments include (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and C H1 domains; (ii) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region: (iii) a Fd fragment consisting of the V H and C H1 domains; (iv) an Fv fragment consisting of the V L and V H domains of a single arm of an intact antibody; (v) disulfide stabilized Fvs (dsFvs) which have an interchain disulfide bond engineered between structurally conserved framework regions; (vi) a single domain antibody (dAb) which consists ofa V H domain (see, e.g., Ward et
- Antibodies suitable for practicing the present invention also encompass single chain antibodies.
- the term "single chain antibody” refers to a polypeptide comprising a V H domain and a V L domain in polypeptide linkage, generally linked via a spacer peptide, and which may comprise additional domains or amino acid sequences at the amino- and/or carboxyl-termini.
- a single-chain antibody may comprise a tether segment for linking to the encoding polynucleotide.
- a single chain variable region fragment (scFv) is a single-chain antibody. Compared to the V L and V H domains of the Fv fragment which arc coded for by separate genes, a scFv has the two domains joined (e.g. via recombinant methods) by a synthetic linker. This enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules.
- the various antibodies or antigen-binding fragments described herein can be produced by enzymatic or chemical modification of the intact antibodies or synthesized de novo using recombinant DNA methodologies or identified using phage display libraries. Methods for generating these antibodies or antigen-binding molecules are all well known in the art. For example, single chain antibodies can be generated using phage display libraries or ribosome display libraries, gene shuffled libraries. In particular, scFv antibodies can be obtained using methods described in, e.g., Bird et al., Science 242:423-426. 1988: and IIuston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988.
- Fv antibody tragments can be generated as described in Skerra and Pliickthun, Science 240:1038-41, 1988.
- Disulfide- stabilized fv fragments (dsfvs) can be made using methods described in. e.g.. Reiter et al., Int. J. Cancer 67:113-23. 1996.
- single domain antibodies (dAbs) can be produced by a variety of methods described in, e.g., Ward et al., Nature 341:544-546. 1989: and Cai and Garen, Proc. Natl. Acad. Sci. USA 93:6280-85. 1996.
- Polypeptides are polymer chains comprised of amino acid residue monomers which are joined together through amide bonds (peptide bonds).
- the amino acids may be thc L-optical isomer or thc D-optical isomer.
- polypeptides refer to long polymers of amino acid residues, e.g., those consisting of at least more than 10, 20. 50, lOO, 200, 500, or more amino acid residue monomers.
- polypeptide as used herein also encompass short peptides which typically contain two or more amino acid monomers, but usually not more than 10, 15, or 20 amino acid monomers.
- target refers to a molecule or biological cell of interest that is to be analyzed or detected, e.g., a nucleotide, an oligonucleotide, a polynucleotide, a polypeptide, a protein, or a blood cell.
- the target antigen of step (b) is an artificial polypetide or natural polypeptide, but it is not limited thereto.
- a cell has been "transformed" by an exogenous or heterologous polynucleotide when such polynueleotide has been introduced inside the cell.
- the transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.
- the transforming polynucleotide may be maintained on an episomal element such as a plasmid.
- a stably transformed cell is one in which the transforming polynucleotide has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication.
- a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
- Fig. 1 An exemplary scheme showing the strategy for the phenotype-information-phenotype cycle is illustrated in Fig. 1.
- the method of the present invention is designed for the selection of antibodies where the target molecule is a minor component of an otherwise complex mixture. Essentially, the method integrates phenotypic information derived from a binary selection format to a stringent affinity-based recovery of genotype.
- a phenotype-information-phenotype cycle (Fig. 1), one begins with a large library of different proteins expressed in phages where only a very minor fraction of the members of the library exhibit a desired phenotype such as binding to another protein or the surface of a cell. After an initial selection, the both noise and signal are existed.
- the information content of the phenotypic pool is established by deep sequencing of its members.
- the information of interest concerns the frequency distribution of antibody molecules that bind to the target relative to the control.
- the central concept is that, because the starting library is very large, a given sequence should not be seen multiple times unless it has been selected.
- one could derive many other relevant informational parameters such as protein homologies and/or predicted secondary and tertiary structures.
- a study of very large naive or synthetic antibody libraries allows one to determine what can happen.
- nucleic acids encoding the molecules considered to be signal are recovered by hybridization and converted back to phenotype by transformation of Escherichia coli thereby completing the phenotype-information-phenotype-cycle.
- we generated the cycle for antibodies it should work for any large collection of homologous or heterogeneous molecules that are genetically encoded or even for organic molecules contained in DNA-encoded combinatorial libraries.
- the important advantage of the cycle described here lies in its potential go beyond selection against protein singularities to find rare antibodies in which the target is a component of an otherwise complex mixture.
- the two main situations where this problem arises is in the selection of cognate antigen-antibody pairs in the library against library format and the selection of antibodies that bind to proteins in which, in each case, the target molecule may be only a minor component of a cell surface.
- these selections have been much more difficult than those against purified proteins.
- the present invention relates to a method for producing a polypeptide specifically binding to a target polypeptide, comprising one cycle of the following steps: (a) providing a library of different proteins displayed on the surface of phages; (b) contacting the library with (i) a group of cells which displays the target polypeptide (test group) and (ii) another group of cells which does not display the target polypeptide or displays less amounts of the target polypeptide (control group); (c) removing the unbound phages and sequencing the nucleotides of the bound phages; (d) comparing the nucleotide sequences of the test group and control group; (e) selecting nucleotide sequences from the test group which are not common with the control group and identifying these sequences as sequences encoding polypeptide specifically binding with the target polypeptide; (f) extracting single-stranded DNA from phage particles and hybridizing to a tagged probe; (g) converting the single-stranded DNA
- the step (c) may be accomplished by deep sequencing.
- the cells may be selected from the group consisting of bacteria, yeast and eukaryotic cells but it is not limited thereto.
- the library may comprise single chain variable region fragments (scFvs) or single domain antibodies (dAbs) but it is not limited thereto.
- scFvs single chain variable region fragments
- dAbs single domain antibodies
- the library may comprise a library of human polypeptides but it is not limited thereto.
- the target polypeptide of step (b) may be an arfificial polypeptide or natural polypeptide but it is not limited thereto.
- the signal peptide and first nine amino acids of the Escherichia coli major outer membrane lipoprotein and amino acids 46-159 of the outer membrane protein A was cloned into the pCGMT vector and the gene encoding the antigen was inserted directly behind OmpA.
- the gene encoding the maltose-binding protein was inserted between the OmpA and antigen to display an MBP antigen fusion protein.
- MBP maltose-binding protein
- a FLAG tag was added at the C terminus of the antigen.
- the plasmid was transformed into TOP10 F' bacteria.
- the bacteria were cultured overnight at 37 °C in super broth (SB) medium supplemented with 2% glucose and 50 ⁇ g/mL carbenicillin (CARB). The medium was changed to glucose-free SB medium and induced at 24 °C for 2 h without addition of IPTG.
- SB super broth
- CARB carbenicillin
- ABTS 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)
- Induced bacteria were incubated with 2 ng/mL phycoerythrin-labeled cytokine-specific antibodies at 22 °C for 1 h, washed twice with PBS and resuspended in PBS at 5 x 10 7 cells per mL, 10,000 E. coli events were measured with a Becton Dickinson flow cytometer, and data were analyzed with Flowjo software.
- the antibodies were obtained from the following vendors: anti-human(h)TNF, Biolegend 502908; antihCX3CL1, R&D Systems IC365P; anti-hIL-6 R&D Systems IC206, Biolegend, 501106; and anti-hIL-4, Biolegend 500703.
- Phage with a titer of 10 11 cfu were preincubated for 1 h with 5 x 10 8 of control bacteria. After centrifugation, the supernatant was transferred to freshly induced antigendisplaying bacteria and incubated for 1 h. Unbound phage were washed away by pelleting bacteria, resuspending them in PBS/3% BSA for five times, and transferring the bacteria to a new tube. Bound phages were eluted with 100 ⁇ l glycine elution buffer [200 mM glycine, 1 mgmL BSA, 0.05% Tween 20 (pH 2.2)].
- the buffer containing phage was neutralized with 7 ⁇ l 2 M Tris, after which XL1-blue cells infected with phage were plated and cultured at 30 °C overnight. Cells were scraped from the plate into 100 mL of SB with 2% glucose to OD 600 of 0.1. The culture was incubated at 37 °C until the OD 600 reached 0.8. Helper phage VCSM13 was added for 30 min at 37 °C without shaking, followed by shaking for 90 min at 37 °C at 300 rpm.
- Bar-coded primers were designed to amplify VH from phagemid pools of paired bacteria-bound phage. Phusion Hot Start High-Fidelity DNA Polymerase (New England Biolabs F-549) was used and 10 cycles of PCR were carried out to reduce bias incurred due to PCR amplification. The amplicons of VH were purified from 1% agarose gels and deep sequenced according to Roche 454 GS FLX instructions.
- Antibody sequences were analyzed by BLAST downloaded from the National Center for Biotechnology Information and compared to germ-line IGVH, IGDH, IGJH sequences obtained from the International Immunogenetics Information System database. Matching was done using the MEGABLASTalgorithm. Germ-line genes were assigned to each read using the MEGABLASTalgorithm. MEGABLAST was run requiring at least 80% sequence identity and using 11-bp word size setting to match IGVH genes. Reads were assigned to genes based upon a hierarchy of BLAST result parameters where the next parameter in the hierarchy was considered only if a read matched multiple genes with the previous parameter.
- the hierarchy was ordered to be more permissive to gaps in the BLAST alignments because of known homopolymer sequencing errors using 454 technology as follows: highest bit score, highest percent identity, longest alignment length, least number of mismatches, and, finally, least number of gaps.
- IGJH genes were assigned similarly, requiring 80% sequence identity, using a 7-bp word size, and requiring the read to span at least 23 bases of the J genes.
- CDR3 sequences were extracted by a more rigorous Smith-Waterman alignment of the identified IGVH and IGJH for each read in order to more precisely define the CDR3 boundary nucleotides.
- VH CDR3 variable heavy-chain complementarity determining region 3
- scFv flanking vector sequence-specific primers were used to amplify the scFv fragments with phagemid pools from round two as template.
- the two resulting DNA fragments were assembled by overlapping PCR, digested with SfiI, ligated into SfiI digested pcGMT3 phagemid and transformed into XL1-blue competent cell. Five to 20 colonies for each scFv were inoculated and minipreps of the overnight culture were sequenced by Sanger sequencing.
- Single-stranded DNA was purified from the phage particles selected in the second round with QIAprep Spin M13 kit (Catalog number 27704).
- a biotinylated probe corresponding to the most variable region of VH CDR3 was used to capture the ssDNA from the pool.
- Four micrograms of the total ssDNA library were mixed with 50 ng of the biotinylated probe in hybridization solution [5 x standard saline phosphate/EDTA (0.18 M NaCl/10 mM phosphate, pH 7.4/1 mM EDTA (SSPE), 0.1% Tween 20] and hybridized at 45 °C for 12 h.
- the hybrid was captured with streptavidin-coated paramagnetic beads (Dynabeads M-280 Streptavidin 112.05D) by incubation at 22 °C for 1 h in binding buffer (1 x SSPE, 0.1% Tween 20).
- the beads were collected with a magnet and washed in wash buffer (2 x SSPE, 0.1% Tween 20) six times.
- the beads were resuspended in water and heated at 90 °C for 1 min to release the captured DNA.
- the released ssDNA was annealed at 55 °C with a primer having the same sequence as the probe and the entire circle of ssDNA was replicated at 72 °C to form dsDNA and transformed into XL1-blue competent cell.
- the transformants were plated on CARB plates and 20 colonies were characterized with Sanger sequencing.
- ELISA plates were coated overnight at 4 °C with 50 ⁇ l of PBS containing 200-ng coating protein [IL-1 receptor antagonist (IL-1RA), IL-6, or truncated IL-6).
- Wells were washed twice with PBST and blocked with 50 ⁇ l of PBS/3% BSA for 1h at 37 °C.
- Different concentrations of phage in PBS Tween 20 (PBST)/3%BSA were added and incubated for 1 h at 37 °C.
- Wells were washed five times with PBST and then 50 ⁇ l of HRP-conjugated antiphage antibody in PBST/3% BSA was added and incubated for 1 h at 37 °C.
- Wells were again washed five times with PBST, and 50 ⁇ l of ABTS developer was added. After 15 min at 22 °C, the A 405 was measured.
- E. coli format to express the antigen was utilized so as to establish phenotype by a method that both might be generally useful and is representative of complex systems such as cell surfaces where a given antigen is but one component of an otherwise complex mixture.
- this system the interaction between antigens that are expressed on the surface of E. coli and antibodies expressed on the surface of phage are studied. Although the antigens were expressed on the surface of E. coli , other systems using yeast or eukaryotic cells could be implemented.
- the antibody protein is expressed on the surface of the phage and the DNA encoding its heavy and light chains is packaged in the interior of the phage, thereby linking genotype to phenotype in a way that will ultimately yield the information content of the system.
- This format allows for a stringent test of our method because the nonspecific binding of phage to the surface of E. coli and/or the binding to irrelevant molecules can be very high and, in our hands, is often difficult to circumvent.
- the binary selection format is critical because the proper control is not in doubt. Thus, one can compare phage that bind to the surface of E. coli or other cells that express antigen to those that do not.
- chemokine (C-C motif) ligand 28 (CCL28) and IL-1 ⁇ expressed best when fused to the MBP-OmpA fusion, whereas TNF- ⁇ and IL-6 could be efficiently displayed when directly fused to OmpA as determined by ELISA (Fig. 2 B and C) and flow cytometry (Fig. 2D).
- ELISA Fig. 2 B and C
- flow cytometry Fig. 2D
- the information of interest relates to frequency with which given nucleic acid sequences appear in the phenotypic pool and the ratio of their abundance in control versus experimental selections (frequency ratio). This information is obtained by pyrosequencing of the DNA contained in the selected phage populations followed by a bioinformatic analysis of the sequences. To return to phenotype, the DNA sequences of interest must be selectively recovered. Three different methods were tested for recovery of nucleic acid sequences considered to be signal (Fig 3).
- VH CDR3 variable heavy-chain complementarity determining region 3
- DH diverse heavy
- JH joining heavy
- a single-stranded phage vector encoding the scFv portion of an antibody that binds to IL-1RA as well as spectinomycin (SPEC) antibiotic resistance was added to a complex pool of vectors that encoded irrelevant antibody sequences and carbenicillin (CARB) antibiotic resistance (Fig 3 and Table 3).
- Selectivity was determined by measuring the ratio of SPEC- to CARB-resistant colonies after transformation of E. coli . After hybridization of the probe at either 45 or 65 °C, the hybrid complex was captured on streptavidin beads and the single-stranded phage genome was detached by melting at 90 °C for 1 min. E.
- coli was transformed with either the single-stranded phage DNA or double-stranded DNA that was generated by T4 or Taq DNA polymerase (Table 3). The best results were obtained when hybridization was at 45 °C and transformation was carried out using Taq DNA polymerase to generate doublestranded DNA (Table 3). Remarkably, under these conditions, selective recovery of the desired sequences approached 100%.
- ssDNA was purified from phage particles with QIAprep Spin M13 kit (catalog no. 27704).
- the ssDNA encoding scFv H9 and spectinomycin resistance (SPEC r ) was spiked into a pool of ssDNA encoding irrelevant scFVs and carbenicillin resistance (CARB r ) at a ratio of 1.100.
- a biotinylated probe corresponding to H9 VH CDR3 was used to capture the H9 encoding ssDNA from the pool.
- the released DNA was used to transform XL1-blue cells directly or converted to dsDNA before transformation.
- Two methods to convert ssDNA to dsDNA were compared.
- ssDNA was annealed at 55 °C with a primer having the same sequence as the probe that was phosphorylated at the 5 position and the entire circle of ssDNA was replicated at 72 °C to form a dsDNA with a nick in the newly synthesized strand.
- ssDNA was annealed with the same primer and converted to closed dsDNA with T4 DNA polymerase plus T4 DNA ligase at 22 °C.
- the transformants were plated on CARB plates and SPEC plates and the total colony number determined (CARB plate colony number plus SPEC plate colony number).
- the selectivity was determined by measuring the number of SPEC-resistant colonies as a function of the total number of colonies (SPEC plate colony numberthe total colony number).
- the present inventors tested the significance of the different frequency ratios by replicating six phage clones that had frequency ratios ranging between 1 and 57.7 (Table 6). Their binding to the two different IL-6 molecules expressed on the E. coli surface was determined by measuring the titer of bound phage. When the frequency ratio is above 10 (N27-1 and N27-2), the isolated phage clones show a strong binding preference for the cognate E. coli as determined by the titer of bound phage. However, no specific binding to the expressed target is observed when the ratio approximates one, even if the frequency is very high (N27-5). Antibodies that bind with a high frequency but do not discriminate could either bind to an irrelevant E.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- AIDS & HIV (AREA)
- Hematology (AREA)
- Oncology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
Cette invention concerne un procédé de criblage d'un anticorps se liant spécifiquement à un antigène cible et un procédé de production d'un polypeptide se liant spécifiquement à un polypeptide cible. Le procédé selon l'invention peut être appliqué pour sélectionner un anticorps ou un polypeptide parmi une importante collection de molécules homologues ou hétérogènes qui sont contenues dans des banques combinatoires.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201261592820P | 2012-01-31 | 2012-01-31 | |
US61/592,820 | 2012-01-31 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2013115425A1 true WO2013115425A1 (fr) | 2013-08-08 |
Family
ID=48905440
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/KR2012/000934 WO2013115425A1 (fr) | 2012-01-31 | 2012-02-08 | Procédés de criblage d'un anticorps par une technique de panning sur un seul cycle |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2013115425A1 (fr) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017214211A1 (fr) * | 2016-06-09 | 2017-12-14 | Igc Bio, Inc. | Procédés d'identification d'un anticorps de haute affinité |
WO2019230823A1 (fr) * | 2018-05-30 | 2019-12-05 | 株式会社Cоgnanо | Procédé d'obtention d'anticorps, procédé d'identification d'anticorps, procédé de production d'anticorps et anticorps |
EP4028584A4 (fr) * | 2019-09-13 | 2022-11-30 | Charles River Laboratories, Inc. | Procédés impliquant une méthode d'adhérence sur plastique d'anticorps dirigés contre des protéines cibles |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1803814A1 (fr) * | 2005-12-27 | 2007-07-04 | SIGMA-TAU Industrie Farmaceutiche Riunite S.p.A. | Procédé d'amélioration la capacité de sélection d'anticorps dans une bibliothèque d'affichage de phages |
US7811973B2 (en) * | 2005-07-07 | 2010-10-12 | Ribovax Biotechnologies Sa | Phage display technologies |
-
2012
- 2012-02-08 WO PCT/KR2012/000934 patent/WO2013115425A1/fr active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7811973B2 (en) * | 2005-07-07 | 2010-10-12 | Ribovax Biotechnologies Sa | Phage display technologies |
EP1803814A1 (fr) * | 2005-12-27 | 2007-07-04 | SIGMA-TAU Industrie Farmaceutiche Riunite S.p.A. | Procédé d'amélioration la capacité de sélection d'anticorps dans une bibliothèque d'affichage de phages |
Non-Patent Citations (3)
Title |
---|
HOOGENBOOM, H. R. ET AL.: "Antibody phage display technology and its applications", IMMUNOTECHNOLOGY, vol. 4, 1998, pages 1 - 20 * |
ZHANG, H. ET AL.: "Phenotype-information-phenotype cycle for deconvolution of combinatorial antibody libraries selected against complex systems", PNAS, vol. 108, no. 33, 16 August 2011 (2011-08-16), pages 13456 - 13461, XP055226217, DOI: doi:10.1073/pnas.1111218108 * |
ZHANG, H. ET AL.: "Selection of antibodies that regulate phenotype from intracellular combinatorial antibody libraries", PNAS, vol. 109, no. 39, 25 September 2012 (2012-09-25), pages 15728 - 15733, XP055183071, DOI: doi:10.1073/pnas.1214275109 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017214211A1 (fr) * | 2016-06-09 | 2017-12-14 | Igc Bio, Inc. | Procédés d'identification d'un anticorps de haute affinité |
WO2019230823A1 (fr) * | 2018-05-30 | 2019-12-05 | 株式会社Cоgnanо | Procédé d'obtention d'anticorps, procédé d'identification d'anticorps, procédé de production d'anticorps et anticorps |
CN112204142A (zh) * | 2018-05-30 | 2021-01-08 | 株式会社康格纳米 | 获得抗体的方法、抗体的确定方法、抗体的制造方法以及抗体 |
JPWO2019230823A1 (ja) * | 2018-05-30 | 2021-07-08 | 株式会社Cognano | 抗体を取得する方法、抗体の特定方法、抗体の製造方法、及び抗体 |
EP4028584A4 (fr) * | 2019-09-13 | 2022-11-30 | Charles River Laboratories, Inc. | Procédés impliquant une méthode d'adhérence sur plastique d'anticorps dirigés contre des protéines cibles |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6996821B2 (ja) | 抗体ファージディスプレイライブラリー | |
Rajan et al. | Simplified synthetic antibody libraries | |
KR101732552B1 (ko) | 안정화된 면역글로불린가변도메인 선별방법 및 선별된 도메인의 응용 | |
KR20110076906A (ko) | 개선된 rna 디스플레이 방법 | |
Azriel-Rosenfeld et al. | A human synthetic combinatorial library of arrayable single-chain antibodies based on shuffling in vivo formed CDRs into general framework regions | |
US11920258B2 (en) | Antibody libraries with maximized antibody developability characteristics | |
US20170044608A1 (en) | Methods of selecting antibodies and antibody fragments | |
WO2014139130A1 (fr) | Système intégré pour construction d'une bibliothèque, criblage de lieur par affinité et expression associée | |
WO2013115425A1 (fr) | Procédés de criblage d'un anticorps par une technique de panning sur un seul cycle | |
JP2019521642A (ja) | 免疫レパートリー発掘 | |
Sepulveda et al. | Design and testing of PCR primers for the construction of scFv libraries representing the immunoglobulin repertoire of rats | |
Siegel | Antibody affinity optimization using yeast cell surface display | |
WO2022045777A1 (fr) | Nouvelle méthode de préparation de banque d'anticorps et banque préparée par cette méthode | |
US20090318308A1 (en) | Highly diversified antibody libraries | |
WO2019078591A1 (fr) | Système d'affichage à protéine multimère utilisant la fluidité de la membrane cellulaire | |
US11001833B2 (en) | Method and kit for generating high affinity binding agents | |
EP1188771A1 (fr) | Bibliothèques de fragments variables des chaines lourdes humaines au format fonctionnel | |
WO2011054150A1 (fr) | Procédé de mutagenèse dirigée par pcr chevauchante et son utilisation dans le cadre du criblage d'anticorps monoclonaux | |
JP6293829B2 (ja) | 安定化された免疫グロブリン可変ドメイン選別方法及び選別されたドメインの応用 | |
Wang et al. | Construction and characterization of phage display library: recognition of mouse serologically detected male (SDM) antigen | |
US9938522B2 (en) | High throughput sequencing of end regions of long linear DNAs | |
WO2024049864A1 (fr) | Criblage d'anticorps | |
WO2023159221A9 (fr) | Banques d'anticorps à domaine unique ayant des caractéristiques de développement d'anticorps maximisées | |
CN115125236A (zh) | Benzonase免疫噬菌体展示抗体库的构建方法、单克隆抗体、编码基因和试剂盒 | |
Kieke | Directed evolution of T cell receptor antagonists |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 12867164 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 12867164 Country of ref document: EP Kind code of ref document: A1 |